Synthesis and Solution Self-Assembly of Poly(L …...The self-assembly of these amphiphilic block...

Synthesis and Solution Self-Assembly of Poly(L-Lactide)-Based Amphiphilic Block Copolymers

by

Amy Petretic

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Chemistry University of Toronto

© Copyright by Amy Petretic 2015

ii

Synthesis and Solution Self-Assembly of Poly(L-Lactide)-Based Amphiphilic Block Copolymers

Amy Petretic

Master of Science

Graduate Department of Chemistry University of Toronto

2015

Abstract

Nanostructures based on biocompatible and biodegradable polymers are desirable for

numerous biomedical applications. An amphiphilic block copolymer, poly(L-lactide)-b-

poly(acrylic acid) (PLLA-b-PAA), was synthesized by combination of ring-opening

polymerization (ROP) of L-lactide and reversible-addition fragmentation chain transfer

(RAFT) polymerization of tert-butyl acrylate, a protected acrylic acid. Methoxy-terminated

poly(ethylene glycol)-amine, a protein-repellent polymer, was conjugated to a portion of the

PAA block to prepare a brush copolymer, PLLA-b-P(AA-r-PEGAm). The self-assembly of

these amphiphilic block copolymers was explored in dilute aqueous solution using a solvent-

switch method. A number of morphologies were observed when the self-assembly conditions

(e.g., solvent, concentration, temperature) were varied. These structures included fibers,

lozenges, and intermediate fiber-lozenge structures. The most intriguing result was that

PLLA-b-PAA formed well-defined lozenge-shaped structures when the self-assembly was

performed at 55 °C. The –COOH groups on the PAA corona of the micelles were used to

coordinate to cisplatin, demonstrating the potential of the micelles to be employed as

nanocarriers for drug delivery.

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Dedication

To my mother, a truly inspirational role model.

iv

Acknowledgements

I would like to thank my supervisor, Professor Mitchell A. Winnik, for his valuable advice

and direction throughout the project. I would also like to extend my gratitude to Dr. Yijie Lu

for the direction he gave me during the start of my research, and Dr. Lin Jia for his guidance

and the many discussions we shared. I would like to thank all of the members of the Winnik

group for the numerous invaluable discussions and for making this a rewarding experience. I

am grateful to Kris Kim and Ilya Gourevich for the collaborations we shared.

Most importantly, I would like to thank my family for their continuous love, encouragement,

and endless support. In particular, I thank my parents who have been a source of strength and

inspiration throughout my academic career. I am grateful to RPN for his love, support, and

motivational speeches.

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Table of Contents

Acknowledgements .................................................................................................................... iv

Table of Contents ........................................................................................................................ v

List of Tables .......................................................................................................................... viii

List of Figures ............................................................................................................................ ix

1 Introduction ............................................................................................................................ 1

1.1 Rod-Like Micelles as Nanocarriers for Cancer Therapy ................................................ 1

1.2 Solution Self-Assembly of Amphiphilic Block Copolymers.......................................... 4

1.3 Research Objectives ...................................................................................................... 10

1.4 Thesis Outline ............................................................................................................... 11

2 Synthesis and Characterization of Poly(L-Lactide)-Based Block Copolymers ................... 12

2.1 Introduction ................................................................................................................... 12

2.2 Results and Discussion ................................................................................................. 17

2.2.1 Synthesis of 1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexyl-thiourea .......... 17

2.2.2 Synthesis of dodecyl 4-(hydroxymethyl) benzyl carbonotrithioate .................. 18

2.2.3 Ring-Opening Polymerization of L-Lactide ..................................................... 19

2.2.4 RAFT Polymerization of tert-Butyl Acrylate ................................................... 25

2.2.5 Deprotection of tert-Butyl Ester to form PLLA-b-PAA ................................... 28

2.2.6 Amide Coupling with mPEG-NH2 to form PLLA-b-P(AA-r-PEGAm) .......... 30

2.3 Conclusion .................................................................................................................... 34

2.4 Experimental Section .................................................................................................... 35

2.4.1 Materials ........................................................................................................... 35

2.4.2 Instrumentation ................................................................................................. 35

2.4.3 Synthesis of 1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexyl-thiourea

(2.1) ................................................................................................................... 36

vi

2.4.4 Synthesis of dodecyl 4-(hydroxymethyl) benzyl carbonotrithioate (2.2) ......... 36

2.4.5 Synthesis of Poly(L-Lactide) Macro CTA (2.3) ............................................... 37

2.4.6 Synthesis of Poly(L-Lactide)-b-Poly(tert-Butyl Acrylate) (2.4) ...................... 38

2.4.7 Deprotection of tert-Butyl Ester to form PLLA-b-PAA (2.5) .......................... 39

2.4.8 Amide Coupling with mPEG-NH2 to form PLLA-b-P(AA-r-PEGAm)

(2.6) ................................................................................................................... 39

3 Solution Self-Assembly of PLLA-b-PAA ........................................................................... 41

3.1 Introduction ................................................................................................................... 41


3.2.1 Solution Self-Assembly of PLLA-b-PAA ........................................................ 44

3.2.2 Treating Micelles with Cisplatin ....................................................................... 54

3.3 Conclusion .................................................................................................................... 57


3.4.1 Materials ........................................................................................................... 58


3.4.3 Solution Self-Assembly: Solvent Effect ........................................................... 60

3.4.4 Solution Self-Assembly: Concentration Effect ................................................. 60

3.4.5 Solution Self-Assembly: Temperature Effect ................................................... 61


4 Solution Self-Assembly of PLLA-b-P(AA-r-PEGAm) ....................................................... 62

4.1 Introduction ................................................................................................................... 62




4.3 Conclusion .................................................................................................................... 71

vii


4.4.1 Materials ........................................................................................................... 72




5 Future Work ......................................................................................................................... 74

References ................................................................................................................................. 76

Appendix A ............................................................................................................................... 82

Appendix B ............................................................................................................................... 89

viii

List of Tables

Table 2.1 Summary of the degree of polymerization (DPn), number-average molecular

weight (Mn), and molecular weight distribution (Đ) of the PLLA macro CTA (2.3).

................................................................................................................................. 25

Table 2.2 Summary of the degree of polymerization (DPn), number-average molecular

weight (Mn), and molecular weight distribution (Đ) of PLLA53-b-PtBA88 (2.4). ... 27

Table 2.3 Summary of the number-average degree of polymerization (DPn), molecular

weight (Mn), and molecular weight distribution (Đ) of PLLA-b-P(AA-r-PEGAm)

(2.6–2.8). ................................................................................................................. 33

Table 2.4 Summary of the PLLA-b-P(AA-r-PEGAm) polymers synthesized. ................... 40

Table 3.1 Summary of the sample numbers assigned to the micelle solutions prepared by

addition of water to PLLA53-b-PAA88 (2.5) in acetone, THF, or dioxane.............. 44


addition of water to PLLA53-b-PAA88 (2.5) at various concentrations in dioxane. 46


addition of water to a solution of PLLA53-b-PAA88 (2.5) in dioxane

maintained at the specified temperatures. ........................................................... 50

Table 3.4 Summary of the samples prepared by staining with cisplatin. ................................. 61


addition of water to a solution of PLLA53-b-P(AA48-r-PEGAm40) (2.8) in

dioxane maintained at the specified temperatures. ............................................. 64

ix

List of Figures

Figure 1.1 Representation of polymeric nanomedicines that have been used in drug

delivery: polymer-drug conjugates, polymeric micelles, dendrimers, and

polymersomes. Polymer-drug conjugates are hydrophilic polymers bound to

hydrophobic drug molecules. Polymeric micelles are core-shell nanostructures

with a hydrophobic core that can be used to encapsulate hydrophobic drug

molecules. Dendrimers are highly-branched macromolecules that can

conjugate drugs to the periphery or encapsulate drugs inside. Polymersomes

are spherical nanostructures that enclose a solution and can carry hydrophilic

therapeutic agents. The concept of this figure is from Reference 2. ...................... 2

Figure 1.2 Micellar nanostructures formed by amphiphilic block copolymers in solution. The

packing parameter can be used to predict the morphology of the structure based

on the size of the hydrophilic and hydrophobic blocks. The packing parameter (p)

is determined through p = v/aolc, where v is the volume of the hydrophobic

segment, ao is the area of the hydrophilic head group, and lc is the length of the

hydrophobic segment. The concept of this figure is from Reference 1. ................... 6

Figure 2.1 The target polymer, poly(L-lactide)-b-poly(acrylic acid) (PLLAn-b-PAAm) (n ≈ 50

units, m ≈ 100 units). .............................................................................................. 13

Figure 2.2 Synthetic scheme for the preparation of the target polymer, PLLA-b-PAA.

Poly(L-lactide) (2.3) was first prepared by the ring-opening polymerization of

L-lactide (L-LA) initiated by dodecyl 4-(hydroxymethyl) benzyl

carbonotrithioate (2.2) in dichloromethane (DCM). The PLLA macro CTA (2.3)

was then used in the RAFT polymerization of tert-butyl acrylate (tBA) to

obtain the block copolymer, PLLA-b-PtBA (2.4). The tert-butyl ester was

deprotected with trifluoroacetic acid (TFA) in DCM to afford PLLA-b-PAA

(2.5). ........................................................................................................................ 13

x

Figure 2.3 Synthesis of 1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexyl-thiourea (2.1) by

the addition of cyclohexylamine to a solution of bis(trifluoromethyl)phenyl

isothiocyanate in THF. ............................................................................................ 14

Figure 2.4 Synthesis of dodecyl 4-(hydroxymethyl) benzyl carbonotrithioate (2.2) by

addition of 4-(chloromethyl)benzyl alcohol to a solution of dodecanethiol,

potassium phosphate (K3PO4), and carbon disulfide (CS2) in acetone. .................. 14

Figure 2.5 Synthesis of PLLA (2.3) through the ring-opening polymerization of L-lactide

(L-LA) initiated by the bifunctional ROP-RAFT agent (2.2) in DCM. The

polymerization was catalyzed by a thiourea (2.1) and tertiary amine

(TU/DMAP) organocatalytic system. .................................................................. 15

Figure 2.6 Synthesis of PLLA-b-PtBA (2.4) through the RAFT polymerization of tert-

butyl acrylate (tBA) using the PLLA macro CTA (2.3) in chloroform. ........... 15

Figure 2.7 Synthesis of PLLA-b-PAA (2.5) by treatment of PLLA-b-PtBA (2.4) with

trifluoroacetic acid (TFA) in DCM. ..................................................................... 16

Figure 2.8 Synthesis of PLLA-b-P(AA-r-PEGAm) (2.6–2.8) via DMTMM-activated

amidation of PLLA-b-PAA (2.5) with mPEG-NH2 (560 Da or 2 kDa) in THF. .... 16

Figure 2.9 1H NMR spectrum of 1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexyl-thiourea

(2.1) in benzene-d6. The peaks are assigned according to the labelled protons of

the structure. The aromatic proton (a) was used as the reference and the integral

was set to 1.00. ........................................................................................................ 18

Figure 2.10 1H NMR spectrum of dodecyl 4-(hydroxymethyl) benzyl carbonotrithioate (2.2)

in dichloromethane-d2. The peaks are assigned according to the labelled protons

of the structure. The aromatic protons (a) were used as the reference and the

integral was set to 4.00............................................................................................ 19

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Figure 2.11 1H NMR spectrum of the PLLA macro CTA (2.3) in dichloromethane-d2.

The ring-opening polymerization of L-lactide was initiated by the bifunctional

ROP/RAFT agent and catalyzed by a thiourea/DMAP system. The monomer-

to-initiator ratio was 50. The aromatic protons (a) were used as a reference

(integral set to 4.00) to determine the degree of polymerization and molecular

weight of the polymer. .......................................................................................... 21

Figure 2.12 1H NMR spectra of the methine region of poly(L-lactide) (2.3) in THF-d8

(600 MHz). (a) poly(L-lactide), and (b) homonuclearly decoupled poly(L-

lactide). The homonuclear decoupled spectrum shows a single peak indicative

of an isotactic polymer. 4 transients were acquired for each spectrum with a

relaxation delay of 1.0 s and an acquisition time of 4.5 s. ...................................... 22

Figure 2.13 Molecular weight distribution of PLLA53 (2.3, Mn = 11.6 kDa, Đ = 1.2) in

THF containing tetrabutylammonium bromide (TBAB) as monitored by UV

detector (set to 309 nm). ....................................................................................... 23

Figure 2.14 UV-VIS spectrum and calibration curve of dodecyl 4-(hydroxymethyl) benzyl

carbonotrithioate in chloroform which shows a maximum absorbance at 309

nm and an extinction coefficient (ε) of 18,700 ± 490 M-1

cm-1

. ......................... 23

Figure 2.15 1H NMR spectrum of PLLA-b-PtBA (2.4) in THF-d8. The RAFT polymerization

of tBA was performed at 65 °C in the presence of the PLLA macro CTA and

AMBN as the initiator. The molar ratio of tBA:CTA:AMBN was 120:1:0.1. The

main peaks are assigned according to the labelled protons of the structure drawn

in the figure. The aromatic protons (a) are used as a reference (integral set to 4.00)

to determine the degree of polymerization of the PtBA block. .............................. 26

Figure 2.16 Molecular weight distribution of PLLA53 (2.3, Mn = 11.6 kDa, Đ = 1.2) and

PLLA53-b-PtBA88 (2.4, Mn = 25.9 kDa, Đ = 1.1) in THF containing TBAB as

monitored by UV detector (set to 309 nm). ......................................................... 27

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Figure 2.17 1H NMR spectra of (a) PLLA53-b-PtBA88 (2.4) and (b) PLLA53-b-PAA88 (2.5) in

THF-d8. After treatment with trifluoroacetic acid, the signal from the tert-butyl

ester (d) disappeared and the signal from the polyacrylate backbone downfield

shifted (b). ............................................................................................................... 29

Figure 2.18 Homonuclear decoupled 1H NMR spectra of the methine region of (a)

PLLA53 (2.3), and (b) PLLA53-b-PAA88 (2.5) in THF-d8 (600 MHz). 16

transients were acquired for each spectrum with a relaxation delay of 1.0 s and an

acquisition time of 4.5 s. ......................................................................................... 30

Figure 2.19 Coupling of mPEG-NH2 (560 Da or 2 kDa) to the PAA groups of PLLA53-b-

PAA88 (2.5) in THF to obtain PLLA53-b-P(AAo-r-PEGAml) (2.6–2.8). ................ 31

Figure 2.20 1H NMR spectrum of PLLA53-b-P(AA35-r-PEGAm53) (2.6) in dichloromethane-

d2. The coupling reaction was performed at room temperature in the presence of

DMTMM and mPEG45-NH2. The molar ratio PLLA53-b-PAA88:mPEG-

NH2:DMTMM was 1:54:54. The main peaks are assigned according to the

labelled protons of the structure. The methine protons of the PLLA backbone (a)

were used as a reference (integral was set to 106.00) to determine the acrylamide

content. .................................................................................................................... 32

Figure 2.21 Molecular weight distribution of PLLA53 (2.3, Mn = 11.6 kDa, Đ = 1.2),

PLLA53-b-PtBA88 (2.4, Mn = 25.9 kDa, Đ = 1.1) and PLLA53-b-P(AA35-r-

PEGAm53) (2.6, Mn = 239 kDa, Đ = 1.1) in THF containing TBAB as

monitored by UV detector (set to 309 nm). ......................................................... 33

Figure 3.1 TEM images of assemblies prepared by addition of water to PLLA53-b-PAA88 in

one of the following organic solvents: (a,d) acetone (3.1), (b,e) THF (3.2), and (c,f)

dioxane (3.3). The polymer was dissolved in the organic solvent and maintained

at room temperature during the addition of water (1 mL) over 16 hours (rate =

0.06 mL/h). The samples were stained with uranyl acetate before imaging........... 45

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dioxane at the following copolymer concentration: (a,d) 0.2 mg/mL (3.4), (b,e)

0.5 mg/mL (3.5), and (c,f) 1 mg/mL (3.6). The polymer was dissolved in dioxane

and maintained at room temperature during the addition of water (1 mL) over 16

hours (rate = 0.06 mL/h). The samples were stained with uranyl acetate before

imaging. .................................................................................................................. 48

Figure 3.3 TEM images of (a) PEO45-b-PCL18 and (b) PEO45-b-PCL24 micellar structures

with varying PCL10 content. As the PCL weight percent was increased, rods (2 wt%

PCL10), thin strips (6 wt% PCL10), and wide lamellae (10 wt% PCL10) were

observed. Scale bar: 2 μm. The figure is reproduced with permission from

Reference 48 © 2014 John Wiley & Sons. ............................................................. 49


dioxane maintained at the following temperatures: (a,d) RT (3.7), (b,e) 40 °C

(3.8), and (c,f) 55 °C (3.9). The polymer (0.5 mg) was dissolved in dioxane (1

mL) and maintained at one of these temperatures during the addition of water (1

mL) over 8 hours (rate = 0.12 mL/h). The samples were stained with uranyl

acetate before imaging. ........................................................................................... 51

Figure 3.5 The length versus the width of the lozenges (3.9) as measured by TEM. The

linearity of the plot indicates that the aspect ratio remains the same regardless

of the size of the lozenge. ..................................................................................... 52

Figure 3.6 AFM height image of a PLLA53-b-PAA88 lozenge (3.9) and the corresponding

height profile. .......................................................................................................... 53

Figure 3.7 TEM images of PLLA53-b-PAA88 (a,c) fiber-like micelles (3.6-CDDP), and (b,d)

lozenges (3.9-CDDP) stained with cisplatin. .......................................................... 55

xiv

Figure 3.8 (a) SEM image of PLLA53-b-PAA88 fiber-like micelles bound to cisplatin (3.6-

CDDP). The line indicates the defined region in which the EDX collected the data.

EDX line scans from carbon, oxygen, and platinum are indicated in (b), (c), and

(d), respectively. The signal from platinum is much weaker than that of carbon

and oxygen. ............................................................................................................. 56

Figure 3.9 (a) SEM image of PLLA53-b-PAA88 lozenges bound to cisplatin (3.9-CDDP). The

line indicates the defined region in which the EDX collected the data. EDX line

scans from carbon, oxygen, and platinum are indicated in (b), (c), and (d),

respectively. The signal from platinum is much weaker than that of carbon and

oxygen. .................................................................................................................... 56

Figure 3.10 The Tygon tubing used to add water to the block copolymer solutions. The

Tygon tube was connected to a 1 mL syringe on one end and a syringe needle on

the other end. ........................................................................................................... 58

Figure 3.11 The glass vial containing the BCP solution was fitted with a rubber septum. The

syringe needle attached to the Tygon tube was inserted with the tip of the needle

above the surface of the liquid. ............................................................................... 60

Figure 4.1 DMTMM-activated amidation of PLLA53-b-PAA88 (2.5) with mPEG12-NH2 (560

Da) in THF to afford PLLA53-b-P(AA48-r-PEGAm40) (2.8). ................................. 63

Figure 4.2 TEM images of assemblies prepared by addition of water to PLLA53-b-

P(AA48-r-PEGAm40) in dioxane maintained at the following temperatures:

(a,d) RT (4.1), (b,e) 40 °C (4.2), and (c,f) 55 °C (4.3). The polymer (0.5 mg)

was dissolved in dioxane (1 mL) and maintained at these temperatures during

the addition of water (1 mL) over 8 hours (rate = 0.12 mL/h). The samples

were stained with uranyl acetate before imaging. ............................................... 66

Figure 4.3 Number-average length and width of PLLA53-b-P(AA48-r-PEGAm40) lozenges

as a function of temperature. ................................................................................... 67

xv

Figure 4.4 AFM height image of a PLLA53-b-P(AA48-r-PEGAm40) lozenge (4.3) and the

corresponding height profile. .................................................................................. 68

Figure 4.5 (a,b) TEM images of PLLA53-b-P(AA48-r-PEGAm40) lozenges (4.1-CDDP)

stained with cisplatin............................................................................................... 69

Figure 4.6 a) SEM image of a PLLA53-b-P(AA48-r-PEGAm40) lozenge bound to cisplatin

(4.1-CDDP). The line indicates the defined region in which the EDX collected the

data. EDX line scans from carbon, oxygen, and platinum are indicated in (b), (c),

and (d), respectively. The signal from platinum is much weaker than that of

carbon and oxygen. ................................................................................................. 69

Figure A-1 13

C NMR spectrum of 1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexyl-thiourea

(2.1) in benzene-d6. The peaks are assigned according to the labelled protons of

the structure. ............................................................................................................ 82

Figure A-2 13

C NMR spectrum of dodecyl 4-(hydroxymethyl) benzyl carbonotrithioate (2.2)

in dichloromethane-d2. The peaks are assigned according to the labelled protons

of the structure. ....................................................................................................... 83

Figure A-3 1

H NMR spectrum of poly(L-lactide) in dichloromethane-d2 before treatment

with trifluoroacetic acid. The aromatic protons (a) were used as a reference

and the integration was set to 4.00. The peaks are assigned according to the

structure in the figure. ........................................................................................... 84

Figure A-4 1H NMR spectrum of poly(L-lactide) in dichloromethane-d2 after treatment

with trifluoroacetic acid. The aromatic protons (a) were used as a reference

and the integration was set to 4.00. The peaks are assigned according to the

structure in the figure. ........................................................................................... 85

xvi

Figure A-5 Molecular weight distribution of PLLA and PLLA+TFA in THF containing

TBAB. The dispersity of the polymer was unchanged after treatment with

TFA, demonstrating that the polymer had not degraded under the acidic

conditions. .............................................................................................................. 85

Figure A-6 1H NMR spectrum of PLLA53-b-P(AA68-r-PEGAm20) (2.7) in

dichloromethane-d2. The coupling reaction was performed at room temperature in

the presence of DMTMM and mPEG45-NH2. The molar ratio PLLA53-b-

PAA88:mPEG-NH2:DMTMM was 1:25:25. The main peaks are assigned

according to the labelled protons of the structure. The methine protons of the

PLLA backbone (a) were used as a reference to determine the acrylamide content.86

Figure A-7 1H NMR spectrum of PLLA53-b-P(AA48-r-PEGAm40) (2.8) in

dichloromethane-d2. The coupling reaction was performed at room temperature in

the presence of DMTMM and mPEG12-NH2. The molar ratio PLLA53-b-

PAA88:mPEG-NH2:DMTMM was 1:44:44. The main peaks are assigned

according to the labelled protons of the structure. The methine protons of the

PLLA backbone (a) were used as a reference to determine the acrylamide content.87

Figure A-8 Molecular weight distribution of PLLA53 (2.3) (Mn = 11.6 kDa, Đ = 1.2),

PLLA53-b-PtBA88 (2.4) (Mn = 25.9 kDa, Đ = 1.1), PLLA53-b-P(AA35-r-

PEGAm53) (2.6) (Mn = 239 kDa, Đ = 1.1), PLLA53-b-P(AA68-r-PEGAm20)

(2.7) (Mn = 117 kDa, Đ = 1.1), and PLLA53-b-P(AA48-r-PEGAm40) (2.8) (Mn

= 110 kDa, Đ = 1.1) in THF containing TBAB as monitored by UV detector

(set to 309 nm). ...................................................................................................... 88

Figure B-1 (a-d) TEM images of assemblies prepared by addition of water to PLLA53-b-

PAA88 in dioxane (0.5 mg/mL, 3.5). The polymer (0.5 mg) was dissolved in

dioxane (1 mL) and maintained at room temperature during the addition of water

(1 mL) over 16 hours (rate = 0.06 mL/h). The samples were stained with uranyl

acetate before imaging. ........................................................................................... 89

xvii


PAA88 in dioxane (1 mg/mL, 3.6). The polymer (1 mg) was dissolved in dioxane

(1 mL) and maintained at room temperature during the addition of water (1 mL)

over 16 hours (rate = 0.06 mL/h). The samples were stained with uranyl acetate

before imaging. ....................................................................................................... 90


PAA88 in dioxane maintained at RT (3.7). The polymer (0.5 mg) was dissolved in

dioxane (1 mL) and maintained at RT during the addition of water (1 mL) over 8

hours (rate = 0.12 mL/h). The samples were stained with uranyl acetate before

imaging. .................................................................................................................. 91


PAA88 in dioxane maintained at 40 °C (3.8). The polymer (0.5 mg) was dissolved

in dioxane (1 mL) and maintained at 40 °C during the addition of water (1 mL)


before imaging. ....................................................................................................... 92

Figure B-5 (a,b,d,e) TEM images of assemblies prepared by addition of water to PLLA53-b-

PAA88 in dioxane maintained at 55 °C (3.9). The polymer (0.5 mg) was dissolved

in dioxane (1 mL) and maintained at 55 °C during the addition of water (1 mL)


before imaging. (c) Length distribution histogram and (f) width distribution

histogram................................................................................................................. 93

Figure B-6 AFM height images of PLLA53-b-PAA88 lozenges (3.9) and the corresponding

height profiles. ........................................................................................................ 94

Figure B-7 TEM images of PLLA53-b-PAA88 fiber-like micelles (a) before (3.6) and (c) after

staining with cisplatin (3.6-CDDP). TEM images of PLLA53-b-PAA88 lozenges (b)

before (3.9) and (d) after staining with cisplatin (3.9-CDDP). The morphology of

the micelles remained the same after coupling to cisplatin. ................................... 95

xviii


P(AA48-r-PEGAm40) in dioxane maintained at RT (4.1). The polymer (0.5 mg)

was dissolved in dioxane (1 mL) and maintained at RT during the addition of

water (1 mL) over 8 hours (rate = 0.12 mL/h). The samples were stained with

uranyl acetate before imaging. ................................................................................ 95

Figure B-9 (a,b) TEM images of lozenges prepared by addition of water to PLLA53-b-PAA88

in dioxane maintained at RT (4.1). The lozenges had a tendency to stack,

preventing accurate measurement of the length and width of the structures. ......... 96


P(AA48-r-PEGAm40) in dioxane maintained at 40 °C (4.2). The polymer (0.5 mg)

was dissolved in dioxane (1 mL) and maintained at 40 °C during the addition of




P(AA48-r-PEGAm40) in dioxane maintained at 55 °C (4.3). The polymer (0.5 mg)

was dissolved in dioxane (1 mL) and maintained at 55 °C during the addition of



Figure B-12 AFM height image of a PLLA53-b-P(AA48-r-PEGAm40) lozenge (4.3) and the

corresponding height profile. .................................................................................. 97

Figure B-13 The length versus the width of the lozenges prepared at (a) RT (4.1), (b)

40 °C (4.2), and (c) 55 °C (4.3) as measured by TEM. The linearity of the plot

indicates that the aspect ratio remains the same regardless of the size of the

lozenge. .................................................................................................................. 98

Figure B-14 TEM images of PLLA53-b-P(AA48-r-PEGAm40) lozenges (a) before (4.1) and

(b) after staining with cisplatin (4.1-CDDP). The morphology of the micelles

remained the same after coupling to cisplatin......................................................... 98

1

Chapter 1

1 Introduction

1.1 Rod-Like Micelles as Nanocarriers for Cancer Therapy

Cancer is one of the leading causes of death worldwide, and the development of

efficient nanomedicines in the treatment of cancer remains a challenge.1 The term

nanomedicine refers to nanostructures that contain therapeutic or diagnostic agents.2 These

nanomedicines are used to treat cancer by delivering drug molecules or radioactive atoms to

the tumour site. The radioactive atoms can also enable imaging; for example, 64

Cu is a

positron-emitting radionuclide used for positron emission tomography (PET), and 111

In emits

-rays for single photon emission computed tomography (SPECT).3 A number of polymeric

nanocarriers for cancer therapy have been developed and evaluated in clinical studies,

including polymer-drug conjugates, polymeric micelles, dendrimers, and polymersomes

(Figure 1.1).2

These delivery vehicles have been proposed to address the many barriers to drug

delivery, such as circulation time and cellular uptake; however, obtaining an optimal

nanocarrier remains a challenge. An ideal nanocarrier would be biocompatible and

biodegradable, with a long blood circulation time, high drug loading capacity and cellular

uptake, and a targeted delivery.1,2

Amphiphilic block copolymers self-assemble to form

polymeric nanostructures that are ideal for encapsulating or conjugating to drug molecules. A

range of morphologies are accessible with polymeric nanostructures which allows for the

exploration of a relatively new design element in drug delivery: nanocarrier shape. The

performance of a polymeric nanocarrier is influenced by a number of properties, namely the

polymer composition, nanostructure shape and size, and its targeting capabilities.1

2

Figure 1.1 Representation of polymeric nanomedicines that have been used in drug delivery:

polymer-drug conjugates, polymeric micelles, dendrimers, and polymersomes. Polymer-drug

conjugates are hydrophilic polymers bound to hydrophobic drug molecules. Polymeric micelles

are core-shell nanostructures with a hydrophobic core that can be used to encapsulate

hydrophobic drug molecules. Dendrimers are highly-branched macromolecules that can

conjugate drugs to the periphery or encapsulate drugs inside. Polymersomes are spherical

nanostructures that enclose a solution and can carry hydrophilic therapeutic agents. The concept

of this figure is from Reference 2.

The chemistry of the block copolymer affects the circulation time of the nanocarrier

and the incorporation of drug molecules. For biomedical applications, it is important that the

amphiphilic block copolymers are biocompatible.1 Biocompatible polymers that are typically

applied as the hydrophilic block include poly(ethylene glycol) (PEG) and N-(2-

hydroxypropyl)methacrylamide (HPMA). PEG is widely used as the hydrophilic block due

to its flexible backbone, low tissue binding, and hydrophilic properties that improve the

stealthiness of the nanocarrier.1,2

HPMA has a greater number of pendent hydroxyl groups

for conjugation to small molecules and has often been used as an alternative to the more

traditional PEG. For the hydrophobic block, polyesters, such as poly(ε-caprolactone) (PCL)

or polylactide (PLA), are most commonly applied due to their nontoxic degradation products

that can be easily removed from the body.1,2

Nanocarriers based on these biocompatible

polymers are suitable for applications in cancer therapy.

The performance of a nanocarrier is in large part affected by the shape of the

nanoparticle. Over the past decade, a large number of drug delivery systems based on

spherical micelles have been described in the literature.4 Influenced by the work of Discher et

al., the attention has recently shifted from traditional spherical micelles to flexible, worm-like

nanocarriers (“filomicelles”) that were shown to have longer circulation times and improved

cellular uptake.4–7

In reference to biological systems, viruses and bacteria have developed

into non-spherical forms (filaments or cylinders) to evade an immune response, supporting

3

the idea that non-spherical nanocarriers may show improved properties over spherical

particles.4

Discher and coworkers prepared filomicelles consisting of a hydrophilic PEG corona

and hydrophobic core of inert polyethylethylene (PEE) or PCL through careful control of the

relative lengths of the core- and corona-forming blocks. Depending on the molecular weight

of the polymer, they obtained filomicelles with diameters between 22 and 60 nm and lengths

between 1 and 8 μm.5 The flexible filomicelles circulated in vivo for up to a week in mice,

while PEGylated vesicles at the same dose were eliminated in two days. Interestingly, they

found that longer filomicelles, ~8 μm in length, had the longest blood circulation time in a

mouse model. This study emphasizes the correlation between nanoparticle shape and

circulation time.5

To understand how filomicelles interact with phagocytic cells, these authors studied

the flow effects in vitro by comparing the uptake of spherical vesicles and cylindrical

micelles as they flowed past phagocytes.5 Generally, nanocarriers are tagged as foreign

bodies and rapidly cleared by phagocytic cells.1 In the flow studies, when the spherical

vesicles contacted the phagocytic cell, they were taken up. Filomicelles, on the other hand,

were pulled off of the phagocytes by the hydrodynamic shear that flow-aligns the cylindrical

micelles. In the authors’ view, this hydrodynamic shear allowed the worm-like micelles to

circulate for extended periods of time, while spherical vesicles were eliminated by

phagocytes.5

The biodistribution of nanocarriers is an essential factor in the performance of a

therapeutic agent.4 Nanocarriers accumulate in tumours due to the high permeability of their

vasculatures and poor lymphatic drainage, a process known as the enhanced permeation and

retention (EPR) effect.2 Nanocarriers that circulate for longer periods of time have a greater

chance of accumulating in the tumour via the EPR effect. The flexible shape of filomicelles

allows them to move around obstacles and accumulate in tumours through the vasculatures.

In contrast, spherical particles of the same volume cannot enter the tumour through the small

pores without distortion of the particle.4 With a higher concentration of worm-like micelles

accumulating in tumours, the administered dose can be reduced and side-effects resulting

4

from multiple administrations can be minimized.4 Discher and coworkers showed that drug-

loaded filomicelles were successful in delivering paclitaxel, an anticancer drug, to tumour

cells in mice, resulting in tumour shrinkage and cell apoptosis.5 A number of reports on

filomicelles of varying composition indicate similar results.5,6,8–10

To enhance the efficiency of the therapeutic agent, nanocarriers must be delivered to

the tumour site while minimizing distribution to healthy cells.4 Beyond the EPR effect, the

accumulation of nanocarriers in the tumour site can be further improved by introducing

targeting moieties.1 In order to selectively target the desired area, antibodies, peptides,

carbohydrates, and other small organic molecules have been incorporated to the nanocarriers.

The targeting moieties can be introduced through the functionalization of the hydrophilic

block before formation of the micelles, which allows for easy purification and

characterization.1 Discher and coworkers showed that biotinylated worm-like micelles loaded

with paclitaxel were more effective in targeting and killing cells than worm-like micelles

without biotin.8 Overall, worm-like micelles show exceptional promise as nanocarriers in

cancer therapy due to their non-spherical shape that allows for long circulation times and

high accumulation in tumours.

1.2 Solution Self-Assembly of Amphiphilic Block Copolymers

Amphiphilic block copolymers (BCPs) have been explored as drug delivery vehicles

for many years. The core-shell micellar nanostructures they form offer a hydrophobic region

for encapsulation of drugs and a hydrophilic shell for functionalization. Essentially, when the

amphiphilic BCPs are placed in water, the hydrophobic block phase separates from water to

form a core and the hydrophilic block surrounds the hydrophobic regions to form a shell.2

The formation of these core-shell micelles is governed by the free energy of the system.11

The factors that contribute to the free energy include the degree of stretching of the core-

forming block, the core-solvent interfacial energy, and the repulsive interactions between the

solvent-swollen corona-forming chains. Many of these factors are influenced by variations in

the block copolymer composition, copolymer concentration, water content, and the nature of

the organic solvent.11

5

The high molecular weight and/or high glass transition temperatures of amphiphilic

BCPs often prevents their direct dissolution in water.12

Alternatively, these BCPs can be first

dissolved in a water-miscible solvent through an approach known as the solvent-switch (co-

solvent) method. Here, the amphiphilic BCP is dissolved in a common solvent for both

blocks followed by the slow addition of a selective solvent (typically water, a non-solvent for

the hydrophobic block). An excess of water is then added to freeze the aggregate morphology

and the common solvent is removed by dialysis against water.11

This method is particularly

relevant to my research as the self-assembly studies described in this thesis follow the

solvent-switch approach.

Most BCP micelles consist of a spherical core surrounded by a solvent-swollen

corona. By adjusting the molecular weight of the hydrophilic and/or hydrophobic blocks, the

micelles can also assemble into a number of non-spherical morphologies, including

cylindrical micelles and polymersomes (Figure 1.2).1 The morphology of amphiphilic BCPs

in water can be predicted by the packing parameter (p) that is related to the size of the

hydrophilic and hydrophobic blocks. The packing parameter can be determined through p =

v/aolc, where v is the volume of the hydrophobic segment, ao is the area of the hydrophilic

head group, and lc is the length of the hydrophobic segment. Generally, block copolymers

with a large hydrophilic block will assemble into spherical micelles (p ≤ 1/3). As the size of

the hydrophobic block is increased, cylindrical micelles (1/3 ≤ p ≤ 1/2) and polymersomes

(1/2 ≤ p ≤ 1) are formed.1 Evident from the packing parameter of cylindrical micelles, the

range of compositions which allow for the formation of cylindrical or rod-like micelles is

quite narrow.

6

Figure 1.2 Micellar nanostructures formed by amphiphilic block copolymers in solution. The packing

parameter can be used to predict the morphology of the structure based on the size of the hydrophilic

and hydrophobic blocks. The packing parameter (p) is determined through p = v/aolc, where v is the

volume of the hydrophobic segment, ao is the area of the hydrophilic head group, and lc is the length

of the hydrophobic segment. The concept of this figure is from Reference 1.

Previously, studies have predominantly focused on the solution self-assembly of

block copolymers consisting of amorphous core-forming blocks. Since the 1960s, when Lotz

and Keller13

first reported the crystallization behaviour of polystyrene-b-poly(ethylene oxide)

(PS-b-PEO) in PS-selective solvents, there has been a growing interest in block copolymers

with crystalline core-forming blocks. These authors found that PS-b-PEO formed square-

shaped lamellar platelets by solution crystallization in ethyl benzene, a selective solvent for

PS. More recent reports have determined that after drying the samples, the PS-b-PEO

lamellar structures consist of PEO single crystals covered by two glassy PS layers.14–16

Winnik, Manners, and coworkers study the crystallization-driven self-assembly

(CDSA) of block copolymers consisting of crystalline poly(ferrocenyldimethylsilane) (PFS).

In some of their early work on poly(isoprene-b-ferrocenyldimethylsilane) (PI-b-PFS), they

found that cylindrical micelles or platelet structures formed depending on the ratio of the

length of the crystallizable PFS core-forming block to that of the amorphous corona-forming

block.17

They found that using a short PI block resulted in the formation of tape-like

structures, whereas a long PI block resulted in the formation of cylindrical micelles. In a later

study, they found that by adding more block copolymer to pre-formed cylindrical micelles,

the length of the cylinders can be extended by epitaxial growth.18

This allowed for cylinders

to be prepared with narrow length distributions. To date, there are a number of examples of

PFS-based block copolymers with different corona-forming blocks, some of which include

poly(ferrocenyldimethylsilane)-b-poly(2-vinylpyridine) (PFS-b-P2VP)19,20

,

poly(ferrocenyldimethylsilane)-b-poly(dimethylsiloxane) (PFS-b-PDMS)21,22

, polystyrene-b-

7

poly(ferrocenyldimethylsilane) (PS-b-PFS)23

and poly(ethylene oxide)-b-

poly(ferrocenyldimethylsilane) (PEO-b-PFS)24

. A compelling property of PFS-based block

copolymers, such as PI-b-PFS and PFS-b-PDMS, is that they can form rod-like micelles

under a broad range of compositions.18

These PFS-based block copolymers are valuable

systems for studying the CDSA of block copolymers in solution.

Block copolymers consisting of biocompatible core-forming blocks, such as semi-

crystalline PCL or PLA, are attractive materials for biomedical applications. Obtaining

micelles with controlled geometries is particularly important as nanocarriers are required to

have uniform shapes and sizes for drug delivery applications. A number of research groups

have studied the CDSA of PCL- or PLA-based block copolymers, including (PCL-b-PLLA)-

b-mPEG25

, PCL-b-PEO5,6,10,26

, and PLLA-b-PAA27,28

. More interesting to us is the

morphology obtained as the nature of the polymer or copolymer composition is varied. For

example, Tu and coworkers examined the effect of the crystallinity of the core-forming block

on the micellar morphology of (PCL-b-PLA)-b-mPEG. Here they found that crystalline

(PCL-b-PLLA)-b-mPEG formed cylindrical micelles, whereas amorphous (PCL-b-P(D,L-

LA))-b-mPEG formed spherical micelles.25

In the authors’ view, the crystallization of the

core-forming block was the driving force for the formation of the cylindrical morphology.

Fan and coworkers studied the solution self-assembly of PCL-b-PEO block copolymers with

varying PCL lengths.26

As they increased the PCL content, they found that the micellar

morphology changed from spherical, rod-like, and worm-like micelles, to band-like lamellae.

Recently, Rachel O’Reilly and coworkers examined the solution self-assembly of

BCPs in which PLA was the core-forming block. Their aim was to obtain biorelevant

cylindrical micelles of uniform size and shape, similar to the controlled geometries obtained

by Winnik, Manners, and coworkers with PFS-based block copolymers.27

This study had a

strong influence on my own project in which I aimed to prepare similar micelles with

modified corona chains for use in drug delivery or radioimmunotherapy applications. Here I

will describe the experiments performed by O’Reilly and coworkers in more detail, outlining

the synthesis of PLA-containing block copolymers and their solution self-assembly

behaviour.

8

The polylactide-based BCPs were prepared through a combination of ring-opening

polymerization (ROP) and reversible addition-fragmentation chain transfer (RAFT)

polymerization using a bifunctional initiator/chain transfer agent (CTA).27

The bifunctional

initiator/CTA (ROP-RAFT agent) consisted of a benzylic hydroxyl group for the ROP and a

trithiocarbonate group for RAFT polymerization. Trithiocarbonate end-functionalized PLA

was prepared through the ROP of L-lactide, D-lactide, and D,L-lactide using a

thiourea/tertiary amine organocatalytic system in dichloromethane.27

Traditionally, metal-

based catalysts are used for the ROP of lactide; however, organocatalytic systems are more

suitable for biomedical applications and offer isotactic enrichment.29–31

RAFT

polymerization was then employed in the chain extension of PLA with tetrahydropyranyl-

acrylate (THPA) in chloroform to yield a block copolymer.27

Following the RAFT

polymerization, poly(acrylic acid) (PAA) was obtained by deprotection of the THPA esters

with a mild acid.

The self-assembly of the PLA-based amphiphilic BCPs (P(L-LA)32-b-PAA265, P(D-

LA)42-b-PAA220, P(DL-LA)33-b-PAA240) was performed by heating the BCP samples in water

at 65 °C for one hour followed by rapid cooling. They found that the BCPs with an

enantiopure PLA block formed cylindrical structures with average lengths of 200 nm for

PLLA32-b-PAA265 and 180 nm for PDLA42-b-PAA220. The BCP with an atactic PLA block,

however, was unable to crystallize and formed spherical micelles 80 nm in diameter.27

The

authors then explored the effect of dissolution time on the BCPs with an enantiopure PLA

block. Here, they observed a linear relationship between the time they heated the sample and

the cylinder lengths they obtained.

In the self-assembly studies of PLA-b-PAA, O’Reilly and coworkers found that it

was necessary for the BCP solution to be heated above the glass transition temperature (Tg)

of PLA for the cylindrical micelles to form. In the authors’ view, above the Tg of PLA (~55–

60 °C), the chains orient within the core and induce the CDSA of the BCP.27

Moreover, the

authors concluded that the length of the cylinders could be kinetically controlled similar to

the epitaxial micelle growth observed with PFS-based BCPs. These cylindrical micelles,

based on a biocompatible and crystallizable polylactide block, show potential for biomedical

applications.

9

In addition to O’Reilly’s study of PLA-b-PAA, also relevant to my research is the

work of Stephen Cheng32

, who obtained lozenge-shaped planar structures by solution

crystallization of PLLA-b-PS. These studies are more reminiscent of the work on PS-b-PEO

described earlier, where square platelets were formed. It has been known for many years that

some block copolymers with a crystalline core-forming block form lamellar single crystals

with well-defined shapes in dilute solution.13–16,32–35

At the beginning of this research project,

I was unaware of Cheng and coworkers’ studies on PLLA-b-PS single crystals. As will be

described in the following chapters, I obtained lozenge-shaped structures during my self-

assembly studies of PLLA-b-PAA. Cheng and coworkers’ study on PLLA-b-PS is important

to my research, and I will therefore describe their experiments in more detail.

Cheng and coworkers32

prepared single crystals of PLLA138-b-PS88 from a dilute

solution in amyl acetate. Using a self-seeding technique, the solution was first heated to

130 °C for 15 minutes and left at room temperature overnight. The sample was then heated to

the self-seeding temperature (Ts = 110 °C) for 15 minutes and transferred into another oil

bath at 72 °C, where it was allowed to anneal for up to one day. The authors observed PLLA-

b-PS lozenge-shaped lamellar single crystals. Analogous to the PS-b-PEO platelets, after

drying the sample, these lamellar structures consisted of a single crystalline layer sandwiched

between two glassy layers.

10

1.3 Research Objectives

The objective of my M. Sc. research project is twofold: to synthesize and characterize

polylactide-based amphiphilic block copolymers, and to study their self-assembly behaviour

in solution. My first goal was to synthesize the amphiphilic block copolymer, poly(L-

lactide)-b-poly(acrylic acid), through a combination of ring-opening polymerization of L-

lactide and RAFT polymerization of tert-butyl acrylate (a protected acrylic acid). I then

focused my study on the self-assembly behaviour of the block copolymers under a range of

conditions. I was particularly interested in finding conditions in which these amphiphilic

block copolymers might form rod-like structures. The corona-forming chains were designed

to serve either of two purposes:

1) To carry the anticancer drug, cisplatin. These micelles would function as drug delivery

vehicles by sequestering cisplatin until the micelle reaches the tumour through the EPR

effect.

2) To carry 111

In. These micelles would carry 111

In to the tumour site, killing malignant cells

by the emission of Auger electrons while enabling gamma ray imaging.

While my polymer synthesis worked well, obtaining well-defined self-assembled

nanostructures in water proved to be much more challenging than originally anticipated.

Thus most of my effort was devoted to examining how the assembly of these block

copolymers was affected by the sample preparation protocol.

11

1.4 Thesis Outline

Chapter 1 is a general introduction to this work, which includes the application of worm-like

micelles in drug delivery and the solution self-assembly of amphiphilic block copolymers.

Chapter 2 outlines the synthesis and characterization of the poly(L-lactide)-based

amphiphilic block copolymers through a combination of ring-opening polymerization and

reversible addition-fragmentation chain transfer polymerization.

Chapter 3 explores the solution self-assembly of PLLA-b-PAA through a solvent-switch

method. Here, the effect of the solvent, copolymer concentration, and temperature on the

morphology of the aggregates was investigated. Cisplatin was then coupled to the micelles

using the PAA groups of the block copolymer.

Chapter 4 describes the solution self-assembly of the PEGylated polymer, PLLA-b-P(AA-r-

PEGAm), through a solvent-switch method. The effect of temperature on the micelle

morphology was explored. Cisplatin was then coupled to the micelles using the residual PAA

groups of the brush copolymer.

Chapter 5 outlines the potential future directions of this project.

12

Chapter 2

2 Synthesis and Characterization of Poly(L-Lactide)-Based Block Copolymers

2.1 Introduction

Over the past few decades, there has been a growing interest in the development of

biodegradable polymers.36

Aliphatic polyesters are the most researched biodegradable

materials owing to their ease of synthesis, degradability, and nontoxic nature.37

Polylactide

(PLA) is a particularly attractive polyester known for its good processibility,

biocompatibility, and nontoxic biodegradation products. To date, PLA and its copolymers

have been employed in a range of biomedical applications such as drug delivery systems,

tissue engineering, and the development of biocomposite materials, prostheses, and

sutures.36,37

For drug delivery systems, PLA copolymers of low molecular weight are desired

since they have shorter degradation times than high molecular weight PLA. The

aforementioned properties of PLA and its copolymers make these materials attractive as drug

delivery platforms. While a wide range of PLA-based block copolymers have been

developed, the BCP of interest in the present work is poly(L-lactide)-b-poly(acrylic acid)

(PLLA-b-PAA). The target polymer contains semi-crystalline PLLA as the hydrophobic

block (n ≈ 50 units), and a hydrophilic PAA block (m ≈ 100 units) that can be prepared by

the deprotection of poly(tert-butyl acrylate) (Figure 2.1). The PAA block was desired for

future incorporation of PEG, anticancer drug molecules, and radionuclides.

http://en.wikipedia.org/wiki/%E2%89%88


13

Figure 2.1 The target polymer, poly(L-lactide)-b-poly(acrylic acid) (PLLAn-b-PAAm) (n ≈ 50 units,

m ≈ 100 units).

The sequence in which the reactions were performed is shown in Figure 2.2. Briefly,

the poly(L-lactide) block (2.3) was prepared by the ROP of L-lactide (L-LA) initiated by the

bifunctional ROP-RAFT agent (2.2) and catalyzed by a thiourea (2.1)/DMAP organocatalytic

system. The PLLA macro CTA was then used in the RAFT polymerization of tert-butyl

acrylate (tBA) to yield the block copolymer, PLLA-b-PtBA (2.4). Finally, deprotection of the

tert-butyl ester by treatment with trifluoroacetic acid (TFA) afforded the target amphiphilic

block copolymer, PLLA-b-PAA (2.5). This experimental design was inspired by the work of

O’Reilly and coworkers.27

Figure 2.2 Synthetic scheme for the preparation of the target polymer, PLLA-b-PAA. Poly(L-lactide)

(2.3) was first prepared by the ring-opening polymerization of L-lactide (L-LA) initiated by

dodecyl 4-(hydroxymethyl) benzyl carbonotrithioate (2.2) in dichloromethane (DCM). The PLLA

macro CTA (2.3) was then used in the RAFT polymerization of tert-butyl acrylate (tBA) to

obtain the block copolymer, PLLA-b-PtBA (2.4). The tert-butyl ester was deprotected with

trifluoroacetic acid (TFA) in DCM to afford PLLA-b-PAA (2.5).



14

Following the synthetic scheme, the first step was to synthesize the small molecules

required for the ROP of L-lactide, i.e., the thiourea and bifunctional ROP-RAFT agent.

O’Reilly and coworkers synthesized the thiourea (2.1) by the dropwise addition of

cyclohexylamine to bis(trifluoromethyl)phenyl isothiocyanate in THF (Figure 2.3).

Figure 2.3 Synthesis of 1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexyl-thiourea (2.1) by the

addition of cyclohexylamine to a solution of bis(trifluoromethyl)phenyl isothiocyanate in THF.

The bifunctional ROP-RAFT agent (2.2) was then prepared by the addition of 4-

(chloromethyl)benzyl alcohol to a solution of dodecanethiol and carbon disulfide in acetone

(Figure 2.4). The important feature of this ROP-RAFT agent is that it contains a hydroxyl

group for initiating the ROP of L-lactide and a trithiocarbonate end group to serve as the

chain transfer agent during the RAFT polymerization.

Figure 2.4 Synthesis of dodecyl 4-(hydroxymethyl) benzyl carbonotrithioate (2.2) by addition of 4-

(chloromethyl)benzyl alcohol to a solution of dodecanethiol, potassium phosphate (K3PO4), and

carbon disulfide (CS2) in acetone.

O’Reilly and coworkers then prepared PLLA by the ROP of L-lactide initiated by the

ROP-RAFT agent and catalyzed by thiourea/(–)-sparteine. Due to the poor availability of (–)-

sparteine, I chose to use 4-dimethylaminopyridine (DMAP) as the tertiary amine. Following

a procedure described by Hedrick et al.31

, PLLA (2.3) was synthesized by the ROP of L-

lactide using a thiourea/DMAP organocatalytic system (Figure 2.5).

15

Figure 2.5 Synthesis of PLLA (2.3) through the ring-opening polymerization of L-lactide (L-LA)

initiated by the bifunctional ROP-RAFT agent (2.2) in DCM. The polymerization was catalyzed

by a thiourea (2.1) and tertiary amine (TU/DMAP) organocatalytic system.

The PLLA homopolymer was then employed as the macro CTA in the RAFT

polymerization of tetrahydropyranyl-acrylate (THPA) in chloroform. O’Reilly and coworkers

synthesized the monomer THPA as a precursor to poly(acrylic acid).27

I chose to use the

monomer, tert-butyl acrylate (tBA), as an alternative to THPA, since it is commercially

available and can be easily deprotected to yield poly(acrylic acid). The PLLA macro CTA

was used in the RAFT polymerization of tert-butyl acrylate in chloroform, yielding PLLA-b-

PtBA (2.4) (Figure 2.6).38

Figure 2.6 Synthesis of PLLA-b-PtBA (2.4) through the RAFT polymerization of tert-butyl

acrylate (tBA) using the PLLA macro CTA (2.3) in chloroform.

The poly(acrylic acid) block was synthesized by deprotection of the tert-butyl ester.

This was achieved by treating PLLA-b-PtBA (2.4) with trifluoroacetic acid (TFA) to afford

the target polymer, PLLA-b-PAA (2.5) (Figure 2.7).38

16

Figure 2.7 Synthesis of PLLA-b-PAA (2.5) by treatment of PLLA-b-PtBA (2.4) with

trifluoroacetic acid (TFA) in DCM.

In addition to synthesizing the target polymer, I was interested in modifying the PAA

block of PLLA-b-PAA by coupling to PEG. This was achieved by the DMTMM-activated

amidation of the PAA groups with monoamine-functionalized, methoxy-terminated

poly(ethylene glycol) (mPEG-NH2). Following a procedure described by Kunishima and

coworkers39

, PLLA-b-PAA (2.5), mPEG-NH2 (560 Da or 2 kDa), and DMTMM were mixed

in THF overnight (Figure 2.8). The objective was to randomly incorporate PEG while

retaining a portion of the PAA block for future coupling to cisplatin, an anticancer drug. By

adjusting the molar ratio between mPEG-NH2 and the PAA block, I prepared three

PEGylated polymers with different grafting densities of PEG (2.6–2.8).

Figure 2.8 Synthesis of PLLA-b-P(AA-r-PEGAm) (2.6–2.8) via DMTMM-activated amidation of

PLLA-b-PAA (2.5) with mPEG-NH2 (560 Da or 2 kDa) in THF.

17

2.2 Results and Discussion

2.2.1 Synthesis of 1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexyl-

thiourea

1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexyl-thiourea (TU, 2.1) was prepared

following a procedure published by Hedrick et al.31

The thiourea was synthesized by the

dropwise addition of cyclohexylamine to bis(trifluoromethyl)phenyl isothiocyanate in THF

(Figure 2.3). The thiourea (2.1) was characterized through 1H NMR spectroscopy in

deuterated benzene. The 1H NMR spectrum is shown in Figure 2.9. The assigned peaks

correspond to the labelled protons of the structure shown in the figure. The peak at 7.43 ppm

is ascribed to the aromatic proton (a); this peak was used as a reference and the integral was

set to 1.00. The peak at 7.29 ppm corresponds to the two aromatic protons (b). The broad

peaks at 6.67 and 5.15 ppm correspond to the protons of the thiourea (c and d). The peaks at

4.27 ppm and 2.01–0.72 ppm correspond to the 11 protons of the cyclohexyl group (e and f).

The peak at 0.44 ppm appears due to the water present in the deuterated benzene. The 13

C

NMR spectrum can be found in Figure A-1.

18

Figure 2.9 1H NMR spectrum of 1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexyl-thiourea (2.1) in

benzene-d6. The peaks are assigned according to the labelled protons of the structure. The aromatic

proton (a) was used as the reference and the integral was set to 1.00.

2.2.2 Synthesis of dodecyl 4-(hydroxymethyl) benzyl carbonotrithioate

The bifunctional ROP-RAFT agent, dodecyl 4-(hydroxymethyl) benzyl

carbonotrithioate, was synthesized following a protocol published by O’Reilly and

coworkers.27

Potassium phosphate and carbon disulfide were added to dodecanethiol in

acetone and stirred for 2 hours. 4-(chloromethyl)benzyl alcohol was then added and the

reaction was left for 72 hours (Figure 2.4). The crude product was then dissolved in DCM

and washed with dilute HCl, water, and a saturated brine solution. The product was further

purified by flash chromatography using a hexane/ethyl acetate (3:2 v/v) mixture as the eluent.

The collected fractions were monitored by thin layer chromatography (TLC, Rf = 0.56) to

locate the purified product. After removal of the solvent, dodecyl 4-(hydroxymethyl) benzyl

carbonotrithioate (2.2) was characterized by 1H NMR spectroscopy. The

1H NMR spectrum

in Figure 2.10 shows the assigned peaks and corresponding labelled structure. The peak at

19

7.33 ppm represents the four aromatic protons (a); the integral of this peak was set to 4.00.

The peaks at 4.66 and 4.61 ppm correspond to the protons of the methylene groups adjacent

to the aromatic ring (b and c). The peak at 3.38 ppm represents the methylene protons

adjacent to the trithiocarbonate (d). The peaks at 1.71 ppm, 1.41 ppm, and 1.28 ppm are

ascribed to the 20 protons of the alkyl chain (e, f, and g). The peak at 0.89 ppm corresponds

to the methyl end group (h). The 13

C NMR spectrum can be found in Figure A-2.

Figure 2.10 1H NMR spectrum of dodecyl 4-(hydroxymethyl) benzyl carbonotrithioate (2.2) in

dichloromethane-d2. The peaks are assigned according to the labelled protons of the structure. The

aromatic protons (a) were used as the reference and the integral was set to 4.00.

2.2.3 Ring-Opening Polymerization of L-Lactide

The ring-opening polymerization of L-lactide was initiated by the bifunctional ROP-

RAFT agent and catalyzed by TU/DMAP in anhydrous DCM (Figure 2.5). The PLLA macro

CTA was first characterized through 1H NMR spectroscopy. The

1H NMR spectrum is

shown in Figure 2.11. The peak at 7.33 ppm corresponds to the four aromatic protons (a);

this peak was used as a reference and the integral was set to 4.00. The peak at 5.18 ppm

20

corresponds to the methine of the PLLA backbone and methylene adjacent to the aromatic

ring (b + d). The peak at 4.62 ppm is ascribed to the methylene group adjacent to the

aromatic ring (e). The peak at 4.33 ppm corresponds to the methine adjacent to the hydroxyl

end group (f). The peak at 3.39 ppm corresponds to the methylene protons adjacent to the

trithiocarbonate (g). The peak at 1.53 ppm corresponds to the methyl protons of the PLLA

backbone (c). The peaks at 1.71, 1.29, and 0.88 ppm correspond to the remaining protons of

the alkyl chain (h, i, and j).

End group analysis through 1H NMR spectroscopy allowed for the determination of

the number-average degree of polymerization (DPn) and molecular weight (Mn) of the

polymer. The DPn was determined by the ratio of the integrals of peaks b, c, and f (methine

and methyl protons of the polymer backbone) over the total number of protons using

Equation 2.1. The integral of peak b (Ib) can be determined by assuming that the integral of

peak d is 2.0, therefore Ib is 104.91.

DPn = Ib+Ic+If

nb+nc nb + nc = 8 (2.1)

In this equation, Ix and nx are the integral and number of protons of peak x,

respectively. The DPn was determined to be 53 which is in agreement with the monomer-to-

initiator feed ratio used ([M]/[I] = 50). Using the molecular weight of the repeating unit and

end groups, the absolute molecular weight of the polymer was calculated to be 8.1 kDa.

21

Figure 2.11 1H NMR spectrum of the PLLA macro CTA (2.3) in dichloromethane-d2. The ring-

opening polymerization of L-lactide was initiated by the bifunctional ROP/RAFT agent and

catalyzed by a thiourea/DMAP system. The monomer-to-initiator ratio was 50. The aromatic

protons (a) were used as a reference (integral set to 4.00) to determine the degree of

polymerization and molecular weight of the polymer.

To determine the degree of stereoregularity, a homonuclear decoupling 1H NMR

experiment was performed. This experiment involves setting the decoupler offset frequency

to the methyl protons (1.5 ppm) which causes the quartet in the methine region (5.1–5.2

ppm) to collapse to a singlet. If epimerization does not occur during the polymerization, a

single peak (iii resonance) will be present in the methine region of the homonuclear

decoupled 1H NMR spectrum.

40 This indicates that the polymer is isotactic and adjacent

carbons have the same configuration. In Figure 2.12, the 1H NMR spectra of the methine

regions of PLLA and the homonuclearly decoupled PLLA are shown. The quartet collapses

into a singlet in the homonuclear decoupled spectrum, validating that the organocatalytic

system employed prevents epimerization during the polymerization, providing a

stereoregular polymer.

22

Figure 2.12 1H NMR spectra of the methine region of poly(L-lactide) (2.3) in THF-d8 (600

MHz). (a) poly(L-lactide), and (b) homonuclearly decoupled poly(L-lactide). The homonuclear

decoupled spectrum shows a single peak indicative of an isotactic polymer. 4 transients were

acquired for each spectrum with a relaxation delay of 1.0 s and an acquisition time of 4.5 s.

Gel permeation chromatography (GPC) was employed to determine the molecular

weight and dispersity of the polymer. The GPC trace (Figure 2.13) demonstrates a unimodal

molecular weight distribution (MWD) in the UV response with a dispersity of 1.2 and

nominal number-average molecular weight of 11.6 kDa. The molecular weight obtained by

GPC is not an accurate value because it is determined from a calibration curve based on

poly(methyl methacrylate) (PMMA) standards. Nevertheless, the GPC results demonstrate

the narrow molecular weight distribution of the polymer, confirming the living

characteristics of the ROP of L-lactide.

23

Figure 2.13 Molecular weight distribution of PLLA53 (2.3, Mn = 11.6 kDa, Đ = 1.2) in THF

containing tetrabutylammonium bromide (TBAB) as monitored by UV detector (set to 309 nm).

The PLLA macro CTA was then characterized by UV-VIS spectroscopy since the

trithiocarbonate end group is UV-active (λmax = 309 nm)27

. A calibration curve was first

prepared with solutions of dodecyl 4-(hydroxymethyl) benzyl carbonotrithioate in

chloroform ranging in concentration from 15.4 to 74.5 μM (Figure 2.14). Using Beer’s Law

(A = εlc), the slope of the calibration curve (A/c = 9370 M-1

), and the path length (l = 0.5 cm),

the extinction coefficient was calculated. The extinction coefficient () at 309 nm was 18,700

± 490 M-1

cm-1

.

Figure 2.14 UV-VIS spectrum and calibration curve of dodecyl 4-(hydroxymethyl) benzyl

carbonotrithioate in chloroform which shows a maximum absorbance at 309 nm and an extinction

coefficient (ε) of 18,700 ± 490 M-1

cm-1

.

24

The UV-VIS absorbance of a solution of PLLA (2.3) in chloroform (0.09 mg/mL) at

309 nm was then recorded. The molar concentration of trithiocarbonate (cCTA) in the polymer

solution was determined by plotting the absorbance of the polymer at 309 nm on the

calibration curve. The molar amount of trithiocarbonate (nCTA) was then calculated using the

molar concentration of trithiocarbonate (cCTA = 1.16×10-5

mol/L) and volume (V = 2.27 mL)

of the polymer solution in Equation 2.2. Using Equation 2.3, the mass of PLLA (mPLLA =

0.207 mg) in the polymer solution and the molecular weight of LA (MWLA = 144 g/mol)

were used to calculate the molar amount of LA (nLA). The degree of polymerization (DPn)

was determined by using Equation 2.4. The degree of polymerization, however, had to be

corrected for the total molecular weight of the polymer, taking into account the end groups.

Equation 2.5 defines the molecular weight of the polymer (MWPLLA = DPn×MWLA) including

the molecular weight of the end groups (MWend groups = 399 g/mol). By rearranging this

equation, Equation 2.6 allowed for the determination of the actual DPn (DPn(actual) = 52). The

molecular weight was then calculated to be 7.9 kDa.

nCTA = (cCTA)(V) (2.2)

nLA = (mPLLA)/(MWLA) (2.3)

DPn = (nLA)/(nCTA) (2.4)

MWPLLA = (DPn(actual))(MWLA) + MWend groups (2.5)

DPn(actual) = MWPLLA−MWend groups

MWLA (2.6)

The degree of polymerization and molecular weight obtained through 1H NMR and

UV-VIS analysis agree with the theoretical values, as demonstrated by the results in Table

2.1. The theoretical monomer-to-initiator ratio was 50 and I was able to obtain a polymer

with 53 repeat units and a molecular weight of 8.1 kDa. Moreover, the polymer had a narrow

molecular weight distribution of 1.2. These results are indicative of a living polymerization

with a controlled molecular weight, narrow dispersity, and high end group control.

25

Table 2.1 Summary of the degree of polymerization (DPn), number-average molecular weight (Mn),

and molecular weight distribution (Đ) of the PLLA macro CTA (2.3).

NMRa

GPCb

UVc

DPnNMR

MnNMR

(kDa) MnGPC

(kDa) Đ DPnUV

MnUV

(kDa)

PLLA53 (2.3) 53 8.1 11.6 1.2 52 7.9 a 1

H NMR in CD2Cl2 b GPC in THF (containing 0.25 g/L tetrabutylammonium bromide) as the eluent and PMMA standards

c UV-VIS in chloroform

2.2.4 RAFT Polymerization of tert-Butyl Acrylate

The PLLA macro CTA was then used in the RAFT polymerization of tert-butyl

acrylate in chloroform (Figure 2.6). The block copolymer was characterized through 1H

NMR spectroscopy in deuterated THF to determine the number-average degree of

polymerization and molecular weight. The 1H NMR spectrum is shown in Figure 2.15 with

the assigned peaks corresponding to the labelled structure. The peak at 7.22 ppm corresponds

to the four aromatic protons (a); this peak was used as a reference and the integral was set to

4.00. The peak at 5.14 ppm is ascribed to the methine protons of the PLLA backbone (b).

The peak at 2.28 ppm corresponds to the methine proton of the PtBA backbone (c). The peak

at 1.66–1.50 ppm corresponds to the methyl protons of the PLLA backbone (d). The peak at

1.50–1.38 ppm corresponds to the tert-butyl protons (e).

26

Figure 2.15 1H NMR spectrum of PLLA-b-PtBA (2.4) in THF-d8. The RAFT polymerization of tBA

was performed at 65 °C in the presence of the PLLA macro CTA and AMBN as the initiator. The

molar ratio of tBA:CTA:AMBN was 120:1:0.1. The main peaks are assigned according to the

labelled protons of the structure drawn in the figure. The aromatic protons (a) are used as a reference

(integral set to 4.00) to determine the degree of polymerization of the PtBA block.

Through 1H NMR analysis, the DPn of the PtBA block and the molecular weight of

the block copolymer were determined. The ratio between the end group (a) and PLLA

backbone (b) was unchanged after the polymerization, indicating that the DPn of PLLA

remained constant (DPn = 53). The signal from the PtBA backbone (c) was used to determine

the degree of polymerization (DPn = 88) and absolute molecular weight of the block

copolymer (Mn = 19.3 kDa).

27

Figure 2.16 Molecular weight distribution of PLLA53 (2.3, Mn = 11.6 kDa, Đ = 1.2) and PLLA53-

b-PtBA88 (2.4, Mn = 25.9 kDa, Đ = 1.1) in THF containing TBAB as monitored by UV detector

(set to 309 nm).

The block copolymer was characterized by GPC to determine the molecular weight

distribution of the polymer. The GPC trace (Figure 2.16) of PLLA53-b-PtBA88 (the subscript

refers to the number-average degree of polymerization) demonstrates a unimodal molecular

weight distribution (MWD) in the UV response with a dispersity of 1.1 and nominal number-

average molecular weight of 25.9 kDa. The GPC results confirm the formation of a higher

molecular weight polymer. In addition, the different retention volumes of PLLA53 (2.3) and

PLLA53-b-PtBA88 (2.4) indicates that the block copolymer does not contain unreacted PLLA.

The NMR and GPC results of the block copolymer are summarized in Table 2.2.

Table 2.2 Summary of the degree of polymerization (DPn), number-average molecular weight (Mn),

and molecular weight distribution (Đ) of PLLA53-b-PtBA88 (2.4).

NMRa

GPCb

DPn (PLLA) DPn (PtBA) Mn (kDa) Mn (kDa) Đ

PLLA53-b-PtBA88 (2.4) 53 88 19.3 25.9 1.1

a 1H NMR in THF-d8

b GPC in THF (containing 0.25 g/L TBAB) as the eluent and PMMA standards

28

2.2.5 Deprotection of tert-Butyl Ester to form PLLA-b-PAA

The following step involved the deprotection of the tert-butyl ester to obtain

poly(acrylic acid) (Figure 2.7). It was important to first consider the stability of PLLA under

the acidic conditions required for the deprotection given that the ester backbone is

susceptible to hydrolysis. To determine whether degradation of PLLA occurs under these

conditions, a control experiment was conducted by applying the deprotection conditions to

the PLLA homopolymer and characterizing the polymer through 1H NMR spectroscopy and

GPC. The 1H NMR spectra before and after treatment with TFA are shown in Figure A-3 and

Figure A-4, respectively. Here, there is no noticeable difference between the integrals of the

backbone methine and methyl proton signals, demonstrating that the degree of

polymerization of PLLA was unaltered. Furthermore, the molecular weight distribution

before and after TFA treatment (Figure A-5) remained constant at 1.15. These results proved

that PLLA is quite robust under the anhydrous acidic conditions employed in the

deprotection of the block copolymer.

The deprotection conditions were then extended to the block copolymer, PLLA53-b-

PtBA88. The tert-butyl ester was deprotected by treatment with trifluoroacetic acid in DCM

(TFA/DCM = 1:6 v/v) to afford the amphiphilic block copolymer, PLLA53-b-PAA88. The

deprotection was characterized by a comparison of the 1H NMR spectra of PLLA53-b-PtBA88

(2.4) and PLLA53-b-PAA88 (2.5) (Figure 2.17). The peak at 5.2 ppm is ascribed to the

methine proton of the PLLA backbone (a). The peak at 2–2.5 ppm corresponds to the

methine proton of the PtBA/PAA backbone (b). The peak at 1.6 ppm corresponds to the

methyl protons of the PLLA backbone (c). The peak at 1.4 ppm corresponds to the tert-butyl

protons (d). The deprotection of the tert-butyl ester can be recognized through 1) the

disappearance of the tert-butyl proton signal and 2) the downfield shifting of the polyacrylate

backbone signal.41

Evident from the 1H NMR spectra in Figure 2.17, after treatment with

TFA, the tert-butyl signal (d) at 1.44 ppm disappeared and the backbone signal (b) at 2.28

ppm downfield shifted to 2.46 ppm. Most importantly, the DPn of PLLA and the PLLA/PAA

block ratio were unchanged after treatment with TFA, validating that the deprotection

occurred without degradation of the PLLA block.

29

Figure 2.17 1H NMR spectra of (a) PLLA53-b-PtBA88 (2.4) and (b) PLLA53-b-PAA88 (2.5) in THF-d8.

After treatment with trifluoroacetic acid, the signal from the tert-butyl ester (d) disappeared and the

signal from the polyacrylate backbone downfield shifted (b).

Homonuclear decoupled 1H NMR spectroscopy was performed to evaluate whether

epimerization of PLLA occurred after treatment with TFA. The homodecoupled 1H NMR

spectra of PLLA53 (2.3) and PLLA53-b-PAA88 (2.5) are shown in Figure 2.18. There does not

appear to be any noticeable changes between the methine regions of the PLLA homopolymer

and block copolymer. This suggests that epimerization did not occur and the stereoregularity

of the PLLA block was retained after treatment with TFA.27

30

Figure 2.18 Homonuclear decoupled 1H NMR spectra of the methine region of (a) PLLA53 (2.3),

and (b) PLLA53-b-PAA88 (2.5) in THF-d8 (600 MHz). 16 transients were acquired for each

spectrum with a relaxation delay of 1.0 s and an acquisition time of 4.5 s.


The final step in the polymer synthesis was the DMTMM-activated amide coupling

between methoxy PEG-amine and the PAA groups of the BCP (Figure 2.19). This was

achieved by addition of DMTMM to a solution of PLLA53-b-PAA88 (2.5) and mPEG-NH2

(560 Da or 2 kDa) in THF at room temperature. By adjusting the molar ratio between mPEG-

NH2 and the PAA block, the grafting density of PEG was varied. I first used 54 equivalents

of mPEG45-NH2 (2 kDa) to prepare the brush copolymer, PLLA-b-P(AA-r-PEGAm) (2.6).

31

Figure 2.19 Coupling of mPEG-NH2 (560 Da or 2 kDa) to the PAA groups of PLLA53-b-PAA88 (2.5)

in THF to obtain PLLA53-b-P(AAo-r-PEGAml) (2.6–2.8).

The brush copolymer (2.6) was characterized by 1H NMR spectroscopy in deuterated

DCM. The 1H NMR spectrum in Figure 2.20 shows the labelled peaks corresponding to the

structure in the figure. The peak at 5.20–5.12 ppm is ascribed to the methine proton of the

PLLA backbone (a); this peak was used as the reference and the integral was set to 106. The

peak at 3.36–3.32 ppm corresponds to the methyl end group of PEG (c). The broad peak

from 2–3 ppm corresponds to the PAA backbone. The peak at 1.60–1.52 ppm is ascribed to

the methyl protons of the PLLA backbone (b). The peaks at 1.26 and 0.88 ppm correspond to

the end group protons (d and e). The number of PEG acrylamide (PEGAm) groups was

determined by the ratio of the integral of peak c (methyl end group of PEG) to the number of

protons (n = 3). The DPn of PEGAm was determined to be 53, leaving 35 PAA groups

unmodified. The absolute molecular weight of the block copolymer was 122 kDa.

32

Figure 2.20 1H NMR spectrum of PLLA53-b-P(AA35-r-PEGAm53) (2.6) in dichloromethane-d2. The

coupling reaction was performed at room temperature in the presence of DMTMM and mPEG45-NH2.

The molar ratio PLLA53-b-PAA88:mPEG-NH2:DMTMM was 1:54:54. The main peaks are assigned

according to the labelled protons of the structure. The methine protons of the PLLA backbone (a)

were used as a reference (integral was set to 106.00) to determine the acrylamide content.

The PEGylated polymer was further characterized by GPC to determine the

molecular weight distribution. The GPC traces of PLLA53 (2.3), PLLA53-b-PtBA88 (2.4), and

PLLA53-b-P(AA35-r-PEGAm53) (2.6) are collected in Figure 2.21. The GPC trace of 2.6

shows a unimodal MWD with a dispersity of 1.1 and a nominal molecular weight of 239

kDa. From the GPC trace, it is evident that the UV response of PLLA53-b-P(AA35-r-

PEGAm53) is weaker than its precursors. Due to the high molecular weight of this polymer

and the presence of PAA, a dilute sample was injected into the GPC instrument as a

precautionary measure. This GPC trace shows that the molecular weight of PLLA53-b-

P(AA35-r-PEGAm53) is much greater than the precursors, indicated by its faster elution time.

The GPC results demonstrate that a portion of the PAA block had indeed been modified by

mPEG-NH2 through the amide coupling reaction.

33

Figure 2.21 Molecular weight distribution of PLLA53 (2.3, Mn = 11.6 kDa, Đ = 1.2), PLLA53-b-

PtBA88 (2.4, Mn = 25.9 kDa, Đ = 1.1) and PLLA53-b-P(AA35-r-PEGAm53) (2.6, Mn = 239 kDa, Đ

= 1.1) in THF containing TBAB as monitored by UV detector (set to 309 nm).

In addition to PLLA53-b-P(AA35-r-PEGAm53), I prepared two other PEGylated

polymers with different grafting densities of PEG (2.7 and 2.8). This was achieved by

adjusting the molar ratio between mPEG-NH2 and the PAA block. The NMR and GPC

results for the three PEGylated polymers are collected in Table 2.3. The 1H NMR spectra and

GPC traces of the individual polymers can be found in Appendix A.

Table 2.3 Summary of the number-average degree of polymerization (DPn), molecular weight (Mn),

and molecular weight distribution (Đ) of PLLA-b-P(AA-r-PEGAm) (2.6–2.8).

NMRa

GPCb

DPn (PLLA) DPn (PAA) DPn (PEGAm) Mn (kDa) Mn (kDa) Đ

2.6 53 35 53 121.8 239 1.1

2.7 53 68 20 54.6 117 1.1

2.8 53 48 40 35.2 110 1.1 a 1

H NMR in DCM-d2 b GPC in THF (containing 0.25 g/L TBAB) as the eluent and PMMA standards

34

2.3 Conclusion

A poly(L-lactide) macro chain transfer agent with a high degree of stereoregularity

and a short PLLA block was prepared through the ring-opening polymerization of L-lactide.

Using a thiourea/tertiary amine organocatalytic system, the ring-opening polymerization

proceeded with retention of stereochemistry as determined by homonuclear decoupled 1H

NMR experiments. The number-average degree of polymerization of PLLA was determined

to be 53 by 1H NMR analysis.

The diblock copolymer was prepared by chain extension of the PLLA macro CTA by

RAFT polymerization of tert-butyl acrylate. The degree of polymerization of the PtBA block

was 88 as determined by 1H NMR analysis. The tert-butyl ester was subsequently

deprotected by treatment with trifluoroacetic acid. Full conversion of the PtBA block to PAA

was obtained while retaining the stereoregularity of the PLLA block, as confirmed by 1H

NMR spectroscopy. Finally, three PEGylated polymers with different grafting densities of

PEG were obtained through the amide coupling of methoxy PEG-amine to a portion of the

PAA block. The solution self-assembly of the amphiphilic block copolymers will be

introduced in the following chapter.

35

2.4 Experimental Section

2.4.1 Materials

All reagents and solvents were purchased from Sigma-Aldrich and used without further

purification unless otherwise indicated. The reagents include: bis(trifluoromethyl)phenyl

isothiocyanate (98%), cyclohexylamine (≥99%), dodecanethiol (≥98%), potassium phosphate

tribasic (≥98%), 4-(chloromethyl)benzyl alcohol (99%), 4-dimethylaminopyridine (DMAP)

(≥99%), triethylamine (TEA) (≥99%), and trifluoroacetic acid (TFA) (99%). L-lactide (L-

LA) (98%, Aldrich) was recrystallized three times from anhydrous toluene (99.8%, Aldrich)

before use. Carbon disulfide (≥99.9%, Aldrich) was dried over 4Å molecular sieves. Tert-

butyl acrylate (tBA) (98%, Aldrich) was passed through alumina (activated neutral, Aldrich)

to remove the inhibitor before use. 2,2′-Azobis(2-methylpropionitrile) (AIBN) (98%,

Aldrich) was recrystallized twice from diethyl ether prior to polymerization. 2,2'-azobis-2-

methylbutyronitrile (AMBN) was purchased from Dupont, USA. Benzoic acid (99.5%) was

purchased from ACP Chemicals Inc., Canada. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-

methylmorpholinium chloride (DMTMM) (>98%) was purchased from Tokyo Chemical

Industry Co., Ltd. Methoxy PEG amine (mPEG45-NH2, MW 2000), HCl salt was purchased

from JenKem Technology, USA. Methoxy PEG amine (mPEG12-NH2, MW 560, ≥95%) was

purchased from ChemPep Inc., USA. Anhydrous THF was obtained by passing the solvent

through a Pure Solv Solvent Purification System (Innovative Technology, Inc.). Water was

purified through a Milli-Q water purification system (10 Mcm). The 15 mL 10 kDa

MWCO Millipore Amicon spin filters were purchased from Fisher Science, Canada.

2.4.2 Instrumentation

The nominal molecular weights and dispersities (Đ = Mw/Mn) of the polymers were

measured with a Viscotek GPC Max gel permeation chromatography (GPC) system equipped

with VE 3580 RI and UV 2500 (set to 309 nm) detectors and a Waters Styragel 5 micrometer

HR (7.8×300 mm) column. The GPC traces were collected at room temperature with THF

containing 0.25 g/L tetrabutylammonium bromide (TBAB) as the eluent. The flow rate was

http://www.google.ca/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CB0QFjAA&url=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Faldrich%2F441090&ei=jT9eVNG6OoOmyATEloKAAw&usg=AFQjCNEV7yjaus1U4ccNOJaPl_mW2IOBIw&bvm=bv.79189006,d.aWw

36

maintained at 0.6 mL/min using a Waters 515 HPLC Pump. The column was calibrated with

poly(methyl methacrylate) (PMMA) standards.

1H NMR and

13C NMR (400 MHz) spectra were recorded with a Varian Mercury 400

spectrometer.

UV-VIS spectra of the PLLA homopolymer in chloroform were collected with a Perkin

Elmer Lambda 35 UV/VIS spectrometer. The extinction coefficient ( at 309 nm was

measured using solutions of dodecyl 4-(hydroxymethyl) benzyl carbonotrithioate in

chloroform.

2.4.3 Synthesis of 1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexyl-

thiourea (2.1)

The synthesis of 1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexyl-thiourea (2.1) was

performed following a procedure published by Hedrick et al.31

The TU was synthesized by

dropwise addition of cyclohexylamine (1.3 mL, 11.2 mmol) to a 35 mL glass vial containing

bis(trifluoromethyl)phenyl isothiocyanate (2.0 mL, 11.1 mmol) in THF (12 mL) while

stirring. The solution was stirred for 4 hours at room temperature. The solvent was

evaporated using a rotary evaporator and the crude product was recrystallized from

chloroform three times. The product was vacuum dried to obtain a white powder. Yield:

3.089 g (75%) 1

H NMR (benzene-d6): (ppm), 7.46–7.39 (s, 1H, ArH, integration = 1.00),

7.34–7.24 (s, 2H, ArH, integration = 1.96), 6.77–6.57 (s, 1H, ArNH, integration = 0.96),

5.33–4.97 (s, 1H, CyNH, integration = 0.96), 4.43–4.11 (br m, 1H, NCyH, integration =

0.97), 2.01–0.72 (m, 10H, CyH, integration = 10.20). 13

C NMR (benzene-d6): (ppm), 179.1

(C=S), 139.4 (Caryl), 132.3 (d, JC,F = 33 Hz, CF3), 124.4 (Caryl), 122.4 (Caryl), 117.6 (Caryl),

53.4 (Calkyl), 32.1 (Calkyl), 25.2 (Calkyl), 24.5 (Calkyl).

2.4.4 Synthesis of dodecyl 4-(hydroxymethyl) benzyl carbonotrithioate

(2.2)

The synthesis of dodecyl 4-(hydroxymethyl) benzyl carbonotrithioate (2.2) was performed

following a procedure published by O’Reilly et al.27

Potassium phosphate tribasic (0.71 g,

37

3.4 mmol) and carbon disulfide (0.55 mL, 9.1 mmol) were added to a 20 mL scintillation vial

containing dodecanethiol (0.73 mL, 3.0 mmol) in acetone (4.8 mL). After stirring at room

temperature for 2 hours, 4-(chloromethyl)benzyl alcohol (0.48 g, 3.0 mmol) was added to the

yellow solution and allowed to mix for 72 hours. The solvent was removed using a rotary

evaporator. The solid was then dissolved in DCM (50 mL) and washed with 0.1 M HCl (50

mL), Milli-Q water (50 mL×3), and a saturated brine solution (50 mL) using a 125 mL

separatory funnel. The organic phase was dried over magnesium sulfate and concentrated

before purification by flash chromatography (Rf = 0.56 in 3:2 hexane:ethyl acetate eluent).

The collected fractions were combined and the solvent was evaporated using a rotary

evaporator. The purified product was vacuum dried to recover a yellow solid. Yield: 0.834 g

(69%) 1H NMR (CD2Cl2): (ppm, integrated peak areas are based on C6H4 = 4H as the

reference), 7.38–7.28 (q, 4H, ArH, integration = 4.00), 4.68–4.63 (d, 2H, ArCH2OH,

integration = 2.00), 4.63–4.59 (s, 2H, ArCH2S, integration = 2.13), 3.41–3.34 (t, 2H,

CH2SCS, integration = 2.07), 1.77–1.65 (m, 2H, CH2CH2SCS, integration = 2.84), 1.46–1.36

(m, 2H, CH3CH2, integration = 1.59), 1.36–1.19 (s, 16H, CH3CH2(CH2)8CH2CH2, integration

= 15.63), 0.93–0.84 (t, 3H, CH3(CH2)10CH2, integration = 2.88). 13

C NMR (CD2Cl2): (ppm),

223.8 (C=S), 140.8 (Caryl), 134.5 (Caryl), 129.3 (Caryl), 127.1 (Caryl), 64.6 (CH2OH), 40.9

(SCH2Ar), 37.1 (Calkyl), 31.9 (Calkyl), 29.6 (Calkyl), 29.6 (Calkyl), 29.5 (Calkyl), 29.4 (Calkyl), 29.3

(Calkyl), 29.1 (Calkyl), 28.8 (Calkyl), 27.9 (Calkyl), 22.7 (Calkyl), 13.8 (-CH3).

2.4.5 Synthesis of Poly(L-Lactide) Macro CTA (2.3)

The ROP of L-lactide was performed following a procedure published by Hedrick et al.31

In

a 20 mL scintillation vial fitted with a magnetic stir bar and rubber septum, L-LA (2.02 g,

14.0 mmol) was dissolved in anhydrous DCM (16 mL). A solution of DMAP (0.22 g, 1.8

mmol, 10 mol%), 2.1 (1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexyl-thiourea, 0.67 g, 1.8

mmol, 10 mol%), and 2.2 (dodecyl 4-(hydroxymethyl) benzyl carbonotrithioate, 0.11 g, 0.28

mmol, [M]/[I] = 50) in anhydrous DCM (4 mL) was added to the scintillation vial and purged

with Ar for 10 minutes. After stirring for 48 hours at room temperature, the catalyst was

neutralized by benzoic acid (20 mg) in ethyl acetate (0.4 mL). The crude polymer was

precipitated in cold methanol three times, dissolving the solid in a minimal amount of DCM

each time. The polymer was vacuum dried to recover a pale yellow solid. Yield: 1.77 g

38

(88%) 1H NMR (DCM-d2): (ppm, integrated peak areas are based on C6H4 = 4H as the

reference) 7.39–7.27 (q, 4H, ArH, integration = 4.00), 5.24–5.11 (q, 1H, backbone methine

and ArCH2O, integration = 106.91), 4.66–4.57 (SCH2Ar, 2H, integration = 1.94), 4.36–4.29

(q, 1H, CHOH, integration = 1.30), 3.43–3.34 (CH3(CH2)9CH2CH2S, 2H, integration = 2.11),

1.75–1.67 (CH3(CH2)9CH2CH2S, 2H, integration = 2.66), 1.67–1.38 (m, 3H, backbone

methyl, integration = 320.44), 1.37–1.21 (s, 18H, CH3(CH2)9CH2CH2, integration = 17.82),

0.93–0.83 (t, 3H, CH3(CH2)9CH2CH2, integration = 3.42). The degree of polymerization was

calculated by comparing the integration of the 1H NMR signals at 5.24–5.11, 4.36–4.29, and

1.67–1.38 ppm (backbone methine and methyl) to that at 7.39–7.27 ppm (C6H4 end group)

Mn = 8.1 kDa, DPn = 53; GPC (THF, RI) Mn = 11.6 kDa, Mw/Mn = 1.2; UV (CHCl3) Mn =

7.9 kDa, DPn = 52 calculated using Equations 2.2–2.6.

2.4.6 Synthesis of Poly(L-Lactide)-b-Poly(tert-Butyl Acrylate) (2.4)

The RAFT polymerization of tert-butyl acrylate was performed following published

procedures.27,38

The polymerization was carried out in a 20 mL scintillation vial containing a

magnetic stir bar and fitted with a rubber septum. The monomer, tBA (1.5 mL, 10.4 mmol),

was added to the vial containing the PLLA macro CTA (2.3) (0.70 g, 0.09 mmol, tBA/CTA =

120) in chloroform (1.5 mL). The initiator, AMBN (0.0017 g, 9 μmol, AMBN/CTA = 0.10),

was finally added and Ar was bubbled through the solution for 10 minutes as the degassing

method. After stirring for 3 hours in a 65 °C oil bath, the solution appeared to be viscous and

was quenched by freezing the solution. An aliquot was removed and dissolved in CDCl3 to

determine the monomer conversion through 1H NMR analysis [monomer conversion = 87%

by comparison of the 1H NMR signals of the vinylic monomer (6.29, 6.04, and 5.72 ppm,

3H, integration = 1.00) to the methine polymer backbone signal (2.36–2.10 ppm, 1H,

integration = 6.60)]. The viscous solution was dissolved in DCM and precipitated into a cold

methanol/water (1:1 v/v) mixture three times and vacuum dried. Yield: 1.34 g (66%) 1H

NMR (THF-d8): (ppm, integrated peak areas are based on C6H4 = 4H as the reference)

7.31–7.12 (q, 4H, ArH, integration = 4.00), 5.25–5.07 (q, 1H, PLLA backbone methine,

integration = 106.66), 2.38–2.19 (br, 1H, PtBA backbone methine, integration = 88.20),

1.92–1.78 (br, end group, integration = 29.52), 1.66–1.50 (m, PLLA backbone methyl,

integration = 394.18), 1.50–1.38 (s, tert-butyl ester, integration = 867.65), 1.38–1.24 (br, end

39

group, integration = 43.54). The degree of polymerization was calculated by comparing the

integration of the 1H NMR signals at 5.25–5.07 (PLLA backbone) to that at 2.38–2.19 ppm

(PtBA backbone) Mn = 19.3 kDa, DPPLLA = 53, and DPPtBA = 88; GPC (THF, RI) Mn = 25.9

kDa, Mw/Mn = 1.1.

2.4.7 Deprotection of tert-Butyl Ester to form PLLA-b-PAA (2.5)

Trifluoroacetic acid (5 mL) was added to a solution of PLLA53-b-PtBA88 (2.4, 500 mg) in

DCM (30 mL) and stirred at room temperature for 24 h. The solution was precipitated into

cold diethyl ether three times and vacuum dried to recover PLLA53-b-PAA88 (2.5). Yield:

321 mg (86%) 1H NMR (THF-d8): (ppm, integrated peak areas are based on C6H4 = 4H as

the reference), 7.30–7.16 (q, 4H, ArH, integration = 4.00), 5.24–5.10 (q, 1H, PLLA backbone

methine, integration = 106.34), 2.57–2.34 (br, 1H, PtBA backbone methine, integration =

87.04), 2.02–1.85 (br, end group, integration = 28.79), 1.62–1.42 (m, PLLA backbone

methyl, integration = 370.10), 1.42–1.26 (m, end group, integration = 21.93). The degree of

polymerization was calculated by comparing the integration of the 1H NMR signals at 5.20–

5.12 (PLLA backbone) and 2.58–2.33 ppm (PAA backbone) Mn = 14.3 kDa, DPPLLA = 53,

and DPPAA = 88.


(2.6)

The amide coupling reaction was performed following a published procedure.39

TEA (21 μL,

0.15 mmol) was added to a solution of mPEG45-NH2 (MW 2000), HCl salt (307 mg, 0.15

mmol, 54 eq.) in THF (2 mL) and stirred for 1 hour. The cloudy mixture was filtered with a

0.2 μm PTFE syringe filter and added to a solution of 2.5 (40.6 mg, 2.8 μmol, 1 eq.) in THF

(2 mL). After stirring for 10 minutes, a suspension of DMTMM (42.5 mg, 0.15 mmol, 54

eq.) in THF (1 mL) was added to the reaction mixture and allowed to stir overnight at room

temperature. The polymer was precipitated in water (15 mL) and THF was removed by

rotary evaporator. The remaining solution was spin-filtered (MWCO 10,000) seven times

followed by freeze-drying overnight to recover the PEGylated polymer. Yield: 0.253 g (74%)

1H NMR (DCM-d2): (ppm, integrated peak areas are based on the PLLA backbone methine

40

proton, CH = 106 H as the reference), 5.20–5.12 (q, 1H, PLLA backbone methine,

integration = 106.00), 3.36–3.32 (s, 3H, CH3 PEG, integration = 157.52), 1.60–1.52 (d, 3H,

PLLA backbone methyl, integration = 345.84). The acrylamide content was calculated by

comparing the integration of the 1H NMR signals at 5.20–5.12 ppm (PLLA backbone

methine) and 3.36–3.32 ppm (CH3 PEG) Mn = 121.8 kDa, DPPEGAm = 53, DPPAA = 35; GPC

(THF, RI) Mn = 239 kDa, Mw/Mn = 1.1.

PLLA-b-P(AA-r-PEGAm) (2.7). Yield: 0.032 g (44%). 1H NMR (DCM-d2): (ppm,

integrated peak areas are based on the PLLA backbone methine proton, CH = 106 H as the

reference), 5.22–5.12 (q, 1H, PLLA backbone methine, integration = 106.00), 3.37–3.32 (s,

3H, CH3 PEG, integration = 60.31), 1.68–1.52 (d, 3H, PLLA backbone methyl, integration =

324.87). The acrylamide content was calculated by comparing the integration of the 1H NMR

signals at 5.22–5.12 ppm (PLLA backbone methine) and 3.37–3.32 ppm (CH3 PEG) Mn =

54.6 kDa, DPPEGAm = 20, DPPAA = 68; GPC (THF, RI) Mn = 117 kDa, Mw/Mn = 1.1.

PLLA-b-P(AA-r-PEGAm) (2.8). Yield: 0.038 g (62%). 1H NMR (DCM-d2): (ppm,

integrated peak areas are based on the PLLA backbone methine proton, CH = 106 H as the

reference), 5.22–5.11 (q, 1H, PLLA backbone methine, integration = 106.00), 3.37–3.30 (s,

3H, CH3 PEG, integration = 119.58), 1.62–1.51 (d, 3H, PLLA backbone methyl, integration

= 306.48). The acrylamide content was calculated by comparing the integration of the 1H

NMR signals at 5.22–5.11 ppm (PLLA backbone methine) and 3.37–3.30 ppm (CH3 PEG)

Mn = 35.2 kDa, DPPEGAm = 40, DPPAA = 48; GPC (THF, RI) Mn = 110 kDa, Mw/Mn = 1.1.

Table 2.4 Summary of the PLLA-b-P(AA-r-PEGAm) polymers synthesized.

2.6 2.7 2.8

PLLA53-b-PAA88 (2.5) 40 mg, 2.8 μmol, 1 eq. 19 mg, 1.3 μmol, 1 eq. 25 mg, 1.7 μmol, 1 eq.

mPEG45-NH2, HCl 307 mg, 154 μmol, 54 eq. 66 mg, 33 μmol, 25 eq. –

mPEG12-NH2 – – 43 mg, 76 μmol, 44 eq.

TEA 21 μL, 154 μmol 4.6 μL, 33 μmol –

DMTMM 43 mg, 154 μmol 9.1 mg, 33 μmol 21 mg, 76 μmol

THF 5 mL 3 mL 3 mL

41

Chapter 3

3 Solution Self-Assembly of PLLA-b-PAA

3.1 Introduction

Currently, amphiphilic block copolymers are the subject of extensive research for

their application as polymer-based nanocarriers in cancer therapy. In a selective solvent,

amphiphilic block copolymers (BCPs) self-assemble to form micellar nanostructures

consisting of an insoluble core surrounded by a solvent-swollen corona.42

These structures

are particularly attractive as nanocarriers because the core offers a hydrophobic region to

carry drug molecules, while the corona increases the water solubility and stealthiness of the

nanostructure.2 Depending on the size of the hydrophobic and hydrophilic blocks, the

micelles can assemble into a number of morphologies, including spheres, cylinders, and

vesicles.1 The shape of the nanostructures has been shown to have a considerable effect on

the performance of the nanocarrier.4–6

In addition, for these polymeric micelles to be suitable

for application in vivo, it is important that the BCPs are based on biocompatible blocks.

Polylactide is often employed as the hydrophobic block owing to its biocompatibility and

nontoxic biodegradation products.36,43

The cylindrical micelles prepared by O’Reilly and coworkers using polylactide-based

BCPs (PLLA32-b-PAA265) were of interest to us due to their shape and size (15 nm wide and

~200 nm long).27

The cylindrical shape of these polylactide-based micelles satisfies Discher

and coworkers’ suggested morphology to enhance circulation times and cellular uptake in

vivo.5 In O’Reilly’s study, cylindrical micelles were obtained by heating PLA-based

amphiphilic BCPs (P(L-LA)32-b-PAA265 and P(D-LA)42-b-PAA220) in water at 65 °C for one

hour followed by rapid cooling. They found that it was necessary for the BCP solution to be

heated above the glass transition temperature (Tg) of PLA (~55–60 °C) for the cylindrical

micelles to form.27

Above the Tg of PLA, the chains orient within the core and induce the

42

crystallization-driven self-assembly of the BCP. In contrast, the BCP with an atactic PLA

block (P(DL-LA)33-b-PAA240) was unable to crystallize and formed spherical micelles 80 nm

in diameter. As they increased the dissolution time of the enantiopure BCPs from 10 to 60

minutes, they found a linear correlation between the cylinder length and assembly time. The

authors concluded that the cylinder length could be kinetically controlled similar to the

epitaxial micelle growth observed with PFS-based BCPs.27

Further exploring the effect of temperature on the formation of cylindrical micelles,

the authors employed the solvent-switch method at room temperature in the assembly of

PLLA32-b-PAA265. The BCP (10 mg) was dissolved in acetone (10 mL) and added dropwise

to water (30 mL). The solution was left overnight without a lid in order to remove acetone.

Through TEM analysis, well-defined spherical particles with an average diameter of 99 nm

were obtained. O’Reilly and coworkers concluded that temperature played a vital role in the

formation of cylindrical micelles.27

In addition to spherical and cylindrical micelles, the self-assembly of polylactide-

based block copolymers has been reported to form lozenge-shaped structures.32,34

For

example, Cheng and coworkers32

used a self-seeding technique to grow lozenge-shaped

single crystals of PLLA138-b-PS88. The block copolymer was heated to 130 °C in amyl

acetate for 15 minutes to form a homogeneous solution and then left at room temperature

overnight to allow for crystallization of the BCP. Next, the sample was heated to the self-

seeding temperature (Ts = 110 °C) for 15 minutes and then transferred into another oil bath at

72 °C for up to a day. In TEM images, lozenge-shaped lamellar single crystals (~8 μm in

length) were observed. These PLLA-b-PS lamellar structures consisted of a PLLA single

crystal covered by two glassy PS layers.32

The self-assembly studies described by O’Reilly et al.27

and Cheng et al.32

offer

insight to our self-assembly system. The vastly different morphologies obtained in these

studies occur not only due to the self-assembly conditions, but presumably due to the ratio

between the crystalline and amorphous blocks. In the O’Reilly study, cylindrical micelles

were obtained by heating PLLA32-b-PAA265 in water at 65 °C.27

Cheng and coworkers, on the

other hand, obtained lozenge-shaped structures by heating PLLA138-b-PS88 in amyl acetate.32

43

In both studies, the solutions were heated above the Tg of PLA, emphasizing the role of

temperature in the crystallization of PLLA-based BCPs.

In this chapter, I describe my experiments to study the solution self-assembly of the

amphiphilic block copolymer, PLLA53-b-PAA88, whose synthesis and characterization is

described in Chapter 2. In my preliminary experiments, the self-assembly of the BCP was

carried out by direct dissolution and by solvent-switch methods using similar conditions as

O’Reilly and coworkers27

. In addition to the conditions outlined by O’Reilly et al., I wanted

to explore the effect of various parameters on the self-assembly of the diblock copolymer,

including the common solvent, copolymer concentration, and temperature. Considering that

O’Reilly and Cheng’s studies revealed the importance of temperature on the self-assembly of

polylactide containing BCPs, I was particularly interested in observing changes in the

morphology at various temperatures. The PLLA53-b-PAA88 assemblies were characterized by

transmission electron microscopy (TEM) and atomic force microscopy (AFM).

With plans to apply the PLLA-based block copolymer micelles as nanocarriers of

radionuclides or platinum-based anticancer drugs, I wanted to explore their potential to

incorporate cisplatin. Cisplatin (cis-[PtCl2(NH3)2], CDDP) is a well-known platinum

anticancer drug that can be complexed to block copolymers with carboxylic groups through

substitution of the anionic ligands (Cl2).44,45

Typically, a weak base is added to deprotonate

the carboxylic acid groups and accelerate the substitution process. Kataoka and coworkers46

coupled cisplatin to poly(ethylene glycol)-b-poly(glutamic acid) block copolymers in water

after mixing for 72 hours. Inspired by this study, I wanted to use the PAA groups of the

PLLA53-b-PAA88 block copolymer micelles to couple with cisplatin through a similar

procedure. The cisplatin-complexed micelles were characterized by TEM and energy-

dispersive X-ray (EDX) spectroscopy.

44


3.2.1 Solution Self-Assembly of PLLA-b-PAA

3.2.1.1 Solvent Effect

PLLA53-b-PAA88 (2.5) was synthesized through a combination of the ROP of L-

lactide and RAFT polymerization of tert-butyl acrylate as described in the previous chapter.

Following the self-assembly studies by O’Reilly and coworkers, I first attempted to induce

the self-assembly of the BCP by direct dissolution in water. When the BCP (0.25 mg) was

placed in water (1 mL), I found that it was insoluble at both room temperature and 65 °C.

Next, the self-assembly of PLLA53-b-PAA88 was carried out using the solvent-switch method.

The amphiphilic BCP was dissolved in a common solvent for both blocks (acetone, THF, or

dioxane) to prepare a homogeneous solution at a concentration of 0.5 mg/mL. Water was

then gradually added over a 16 hour period. The common solvent was removed by dialysis

against water and an aliquot of each micelle solution was taken for TEM analysis using

uranyl acetate as the stain (Figure 3.1). In Table 3.1, I assign sample numbers to the micelle

solutions prepared by the addition of water to the BCP in each of the organic solvents.

Table 3.1 Summary of the sample numbers assigned to the micelle solutions prepared by addition of

water to PLLA53-b-PAA88 (2.5) in acetone, THF, or dioxane.

Solvent Acetone THF Dioxane

Sample 3.1 3.2 3.3

Samples 3.1–3.3 were prepared by dissolving PLLA53-b-

PAA88 (0.5 mg) in one of the organic solvents followed by

the slow addition of water (1 mL) over 16 hours.

The TEM images of the micelles prepared by addition of water to acetone solutions

(3.1) are shown in Figure 3.1 (a) and (d) at different magnifications. In these images, one can

see somewhat irregular diamond-shaped structures, or lozenges, and free unassembled

polymer in the background. By measuring 30 lozenges, I calculated a number-average length

(Ln) of 813 nm (Lw/Ln = 1.0) and a number-average width (Wn) of 491 nm (Lw/Ln = 1.0). The

TEM images of the micelles prepared by addition of water to THF solutions (3.2) are shown

45

in Figure 3.1 (b) and (e). The staining of the structures in these images makes it difficult to

view the structures clearly, however, long fiber-like micelles can be observed. On close

inspection of Figure 3.1 (b), it is evident that the fibers have a tendency to bundle and form

aggregates. The TEM images of the micelles prepared by addition of water to dioxane

solutions (3.3) are shown in Figure 3.1 (c) and (f). Here it is evident that the BCP formed

long fiber-like micelles with uniform widths (Wn = 16.6 nm, Ww/Wn = 1.1). The outer region

of the fibers (PAA corona) appears to be darker than the PLLA core due to the positive

staining of PAA by uranyl acetate.47

In the low magnification image (c), one can see that the

fibers have a tendency to align side by side. In contrast to the previous samples, we observe

less free polymer in the background of this sample.

Figure 3.1 TEM images of assemblies prepared by addition of water to PLLA53-b-PAA88 in one of

the following organic solvents: (a,d) acetone (3.1), (b,e) THF (3.2), and (c,f) dioxane (3.3). The

polymer was dissolved in the organic solvent and maintained at room temperature during the addition

of water (1 mL) over 16 hours (rate = 0.06 mL/h). The samples were stained with uranyl acetate

before imaging.

The self-assembly behaviour of our BCP was quite different than that described by


. Through the direct dissolution method in water, they were able to

46

obtain well-defined cylindrical micelles. The self-assembly of our BCP (PLLA53-b-PAA88)

by direct dissolution was unsuccessful since PLLA53-b-PAA88 was insoluble in water at both

room temperature and 65 °C. In comparison to our BCP (PLLA53-b-PAA88), the BCP

employed by O’Reilly and coworkers (PLLA32-b-PAA265) was more water soluble due to its

longer PAA block. Through the solvent-switch method, O’Reilly and coworkers obtained

spherical micelles. Interestingly, when PLLA53-b-PAA88 was self-assembled by solvent-

switch, I obtained lozenges or fibers depending on the organic solvent employed. The reason

for these contrasting results is presumably due to differences in the ratio of the organic

solvent and water I used (50:50), and the block ratio of PLLA53-b-PAA88.

3.2.1.2 Concentration Effect

I next examined the solution self-assembly of the amphiphilic BCP by varying the

copolymer concentration. In Table 3.2, I assign sample numbers to the micelle solutions

prepared at each concentration. PLLA53-b-PAA88 (2.5) was dissolved in dioxane, yielding a

homogeneous solution at a concentration of 0.2 mg/mL. Micelles were formed by the slow

addition of water (1 mL) at room temperature over 16 hours. Residual dioxane was then

removed by dialysis against water. An aliquot of the micelle solution was taken for TEM

analysis using uranyl acetate as the stain (Figure 3.2 (a) and (d)). In these images, it is

difficult to clearly see the structures due to the poor staining, however, large lozenge-like

structures can be observed. On close inspection of Figure 3.2 (a), the lozenges tend to

aggregate and overlap in some areas, evident from the clusters of lozenges. While the

overlapping structures make it difficult to determine the size of the lozenges, they do appear

to vary in size. In Figure 3.2 (d), a single lozenge is observed. The dark points in the

background of the images are most likely due to the stain, uranyl acetate.


water to PLLA53-b-PAA88 (2.5) at various concentrations in dioxane.

Concentration (mg/mL) 0.2 0.5 1.0

Sample 3.4 3.5 3.6

Samples 3.4–3.6 were prepared by dissolving PLLA53-b-PAA88 (0.2,

0.5, or 1 mg) in dioxane followed by the slow addition of water

(1 mL) over 16 hours.

47

Next, PLLA53-b-PAA88 (2.5) was dissolved in dioxane, yielding a homogeneous

solution at a concentration of 0.5 mg/mL. Micelles were formed by the slow addition of

water at room temperature over 16 hours. Dioxane was removed by dialysis against water

and an aliquot of the micelle solution was taken for TEM analysis (Figure 3.2 (b) and (e),

Figure B-1). Previously, we observed long fibers under these same conditions. In this case,

two populations of morphologies are observed. Some BCP assemblies appear to be bundles

of fibers while others are intermediate structures of fibers and lozenges. In the bundled fibers,

individual fibers can be observed throughout the structure. The intermediate fiber-lozenge

structures, however, are thin sheets with relatively smooth sides and fibers on the ends. These

structures tend to overlap and aggregate as shown in Figure 3.2 (b) and (e), which prevented

the size distribution from being determined. Although the lozenge-shaped structures appear

to vary in size, most structures are around 2 μm in length.

Finally, PLLA53-b-PAA88 (2.5) was dissolved in dioxane, yielding a homogeneous

solution at a concentration of 1 mg/mL. Micelles were formed by the slow addition of water

at room temperature over 16 hours. Dioxane was removed by dialysis against water and an

aliquot of the micelle solution was taken for TEM analysis (Figure 3.2 (c) and (f), Figure B-

2). The predominant morphology formed in this sample is shown in Figure 3.2 (c), where

intermediate fiber-lozenge structures are observed. These intermediate structures coexist with

bundles of fibers, shown in Figure 3.2 (f). The intermediate fiber-lozenge structures had a

tendency to overlap, preventing the measurement of the length and widths of the structures.

Although the structures vary in size, most appear to be larger than those prepared at 0.5

mg/mL and are approximately 3 μm in length. Varying the copolymer concentration proved

to have an interesting effect on the self-assembly morphology of PLLA53-b-PAA88. At low

copolymer concentration (0.2 mg/mL), I observed lozenge-shaped structures. As I increased

the copolymer concentration to 0.5 mg/mL and 1 mg/mL, I observed bundles of fibers and

intermediate fiber-lozenge structures.

48

Figure 3.2 TEM images of assemblies prepared by addition of water to PLLA53-b-PAA88 in dioxane

at the following copolymer concentration: (a,d) 0.2 mg/mL (3.4), (b,e) 0.5 mg/mL (3.5), and (c,f) 1

mg/mL (3.6). The polymer was dissolved in dioxane and maintained at room temperature during the

addition of water (1 mL) over 16 hours (rate = 0.06 mL/h). The samples were stained with uranyl

acetate before imaging.

The most intriguing result I obtained when exploring the effect of the copolymer

concentration on the micellar morphology was the formation of intermediate fiber-lozenge

structures. These fiber-lozenge structures resemble the fringed lamellae described recently by

van de Ven and coworkers48

in self-assembly studies of poly(ethylene oxide)-b-

polycaprolactone (PEO-b-PCL). In their work, PEO45-b-PCL18 or PEO45-b-PCL24 was co-

dissolved with PCL10 homopolymer (2–10 wt%) in dioxane followed by the dropwise

addition of water at a rate of 1 mL/min. They then diluted the micelle solution by dropwise

addition into water before characterization of the micelles. The authors initially observed

micelles that were spherical in shape with a diameter of 20–30 nm. As the micelle solutions

were aged, the spheres formed higher-order structures. Depending on the PCL10 content, they

observed rods (2 wt% PCL10), thin strips (6 wt% PCL10), and wide lamellae (10 wt% PCL10)

(Figure 3.3). The thin strips and lamellae had smooth longer edges with rods emerging from

the narrow edges. In many of their samples, they observed these lamellae co-existing with

49

free and bundled rods, suggesting that growth was initiated at different times. Van de Ven

and coworkers proposed that the lamellae assembled when rods of similar length come

together and fuse, forming a “raft”. They proposed that the raft then grew by inclusion of

rods along the edges.48

Figure 3.3 TEM images of (a) PEO45-b-PCL18 and (b) PEO45-b-PCL24 micellar structures with

varying PCL10 content. As the PCL weight percent was increased, rods (2 wt% PCL10), thin strips (6

wt% PCL10), and wide lamellae (10 wt% PCL10) were observed. Scale bar: 2 μm. The figure is

reproduced with permission from Reference 48 © 2014 John Wiley & Sons.

3.2.1.3 Temperature Effect

The next step involved maintaining the polymer solution at different temperatures

during the addition of water. In Table 3.3, I assign sample numbers to the micelle solutions

prepared by addition of water to BCP solutions maintained at certain temperatures. PLLA53-

b-PAA88 (2.5) was dissolved in dioxane (0.5 mg/mL) and maintained at room temperature

during the slow addition of water over 8 hours. At this point, I chose to increase the rate of

water addition since it had no impact on the self-assembly of the BCPs, as no noticeable

difference was found between micelles prepared by adding water over 8 or 16 hours. The

micelle solutions were dialyzed against water and an aliquot was taken for TEM analysis

(Figure 3.4 (a) and (d), Figure B-3). Here, one can see free fibers, bundled fibers, and

intermediate fiber-lozenge structures coexisting. The fibers can be seen in the background of

the TEM images and are variable in length. Fibers of roughly the same length tend to align

50

and form bundles. In Figure 3.4 (a), an intermediate fiber-lozenge structure, which is a

lozenge-shaped sheet with fibers at the ends, is observed. Also noticeable in this TEM image

are three separate bundles of fibers with free fibers in the background. In Figure 3.4 (d),

bundles of fibers are observed, where one of the bundles in the center appears to be sheet-like

with fibers at the ends.


water to a solution of PLLA53-b-PAA88 (2.5) in dioxane maintained at the specified temperatures.

Temperature RT 40 °C 55 °C

Sample 3.7 3.8 3.9

Samples 3.7-3.9 were prepared by maintaining a solution of

PLLA53-b-PAA88 (0.5 mg) in dioxane at RT, 40 °C or 55 °C

during the slow addition of water (1 mL) over 8 hours.

Next, the BCP solution in dioxane was heated during the addition of water. PLLA53-

b-PAA88 (2.5) was dissolved in dioxane (0.5 mg/mL) and maintained at 40 °C during the

addition of water over 8 hours. The micelle solutions were dialyzed against water and an

aliquot was taken for TEM analysis using uranyl acetate as the stain (Figure 3.4 (b) and (e),

Figure B-4). In Figure 3.4 (b), one can see free fibers, irregular-shaped structures, and

lozenges coexisting. Although a number of morphologies coexist in the TEM images, I

primarily observed lozenges of varying size similar to those found in Figure 3.4 (e).

Measuring 100 lozenges, I calculated a number-average length of 2.0 μm (Lw/Ln = 1.2) and a

number-average width of 1.2 μm (Ww/Wn = 1.2). In both Figure 3.4 (b) and (e), the edges of

the lozenges appear to have a greater contrast than the center of the structures.

51

Figure 3.4 TEM images of assemblies prepared by addition of water to PLLA53-b-PAA88 in dioxane

maintained at the following temperatures: (a,d) RT (3.7), (b,e) 40 °C (3.8), and (c,f) 55 °C (3.9). The

polymer (0.5 mg) was dissolved in dioxane (1 mL) and maintained at one of these temperatures

during the addition of water (1 mL) over 8 hours (rate = 0.12 mL/h). The samples were stained with

uranyl acetate before imaging.

Next, I maintained the temperature of the BCP solution at 55 °C during the addition

of water. PLLA53-b-PAA88 (2.5) was dissolved in dioxane (0.5 mg/mL) and maintained at

55 °C during the slow addition of water over 8 hours. The micelle solutions were dialyzed

against water and an aliquot was taken for TEM analysis using uranyl acetate as the stain

(Figure 3.4 (c) and (f), Figure B-5). In Figure 3.4 (c) and (f), a mixture of large and small

lozenges is observed. Under these self-assembly conditions, I obtained well-defined lozenge

structures and did not observe intermediate fiber-lozenge structures or free fibers. Measuring

100 small and large lozenges, I calculated a number-average length of 2.2 μm (Lw/Ln = 1.4)

and a number-average width of 1.2 μm (Ww/Wn = 1.4). Evident from the TEM images and

size distribution measurements, the lozenges, although uniform in morphology, have a broad

size distribution. In Figure 3.5, I plot the lozenge length against lozenge width. The linearity

of this plot indicates that the aspect ratio remains the same regardless of the size of the

lozenge.

52

Figure 3.5 The length versus the width of the lozenges (3.9) as measured by TEM. The linearity

of the plot indicates that the aspect ratio remains the same regardless of the size of the lozenge.

Atomic force microscopy (AFM, performed by Kris Kim) was then employed to

determine the thickness of the lozenges prepared at 55 °C. An aliquot of the micelle solution

(3.9) was dried on a silicon wafer for AFM analysis (Figure 3.6 and Figure B-6). Figure 3.6

shows a height image and the corresponding height profiles of a single lozenge. In the height

image, the bright areas correspond to higher domains and the dark areas correspond to lower

domains. Although the height profile is irregularly shaped, two distinct heights are evident.

The AFM height image indicates that the thickness of the lozenge ranges from 6 to 33 nm,

where the inner domain is ~12 nm lower than the surrounding edges. Referring to the cross-

section, the highest domains are the edges (18–20 nm thick) and the center (25–33 nm thick),

while the lower domain is 6–10 nm thick.

53

Figure 3.6 AFM height image of a PLLA53-b-PAA88 lozenge (3.9) and the corresponding height

profile.

After obtaining these lozenge-shaped structures, I reviewed the literature in search of

similar reports to better understand their structure and formation. Here I found that my

studies on PLLA-b-PAA lozenges were similar to the work of Cheng and coworkers32

and

He and coworkers34

, who prepared lozenge-shaped PLLA-b-PS single crystals through

solution crystallization. Cheng and coworkers32

obtained single crystals from PLLA138-b-

PS88 through a self-seeding technique in amyl acetate. In TEM images, the lozenge-shaped

lamellar single crystals were observed to be ~8 μm in length. He and coworkers34

used a

similar self-seeding technique in p-xylene to obtain nearly monodisperse single crystals from

PLLA135-b-PS202. These lozenge-shaped single crystals (~2.5 μm in length) were smaller than

those obtained by Cheng and coworkers. The different sized single crystals obtained are

presumably due the ratio between the crystalline and amorphous blocks. After drying, the

PLLA-b-PS lamellar crystals consisted of a PLLA single crystal sandwiched between two

glassy PS layers. The PLLA-b-PS single crystals in both studies were prepared above the Tg

of PLLA. Similarly, I was able to obtain lozenge-shaped structures when the BCP solution

was heated at 55 °C (near the Tg of PLLA, ~55–60 °C27) during the addition of water. Under

these conditions, a mixture of large and small lozenges with a number-average length of 2.2

μm and width of 1.2 μm were obtained. Based on our own self-assembly studies and those

conducted by O’Reilly, Cheng, and He, I have found that temperature is a key factor in

obtaining PLLA-b-PAA structures of uniform shape.

54

3.2.2 Treating Micelles with Cisplatin

In this section I describe the experiments involved in the preparation of cisplatin-

complexed PLLA53-b-PAA88 micelles. The idea here was to add cisplatin (CDDP) to pre-

formed micelles and examine the incorporation of cisplatin by TEM and EDX spectroscopy.

Since I observed a number of micellar morphologies during my self-assembly studies, I

chose to use two micelle solutions with different morphologies to examine whether either of

these micelles was effective in coupling to cisplatin. The cisplatin-coupling was carried out

using fiber-like micelles (3.6) and lozenges (3.9) described in the previous sections of this

chapter.

Following an approach described by Kataoka and coworkers46

, an aliquot of the fiber-

like micelles (3.6, 0.5 mL), triethylamine (0.16 mg), and CDDP (0.05 mg) was gently shaken

for 72 hours. After removal of excess CDDP by dialysis against water, an aliquot of the

cisplatin-complexed fibers, which I refer to as 3.6-CDDP, was taken for analysis by TEM.

Figure 3.7 (a) and (c) show the TEM images of fibers bound to cisplatin (3.6-CDDP) at

different magnifications. Here one can see fibers of varying lengths with a significant amount

of contrast due to the bound platinum.

Cisplatin-complexed lozenges were prepared through an analogous procedure. An

aliquot of the lozenges (3.9, 1 mL), triethylamine (0.16 mg), and CDDP (0.05 mg) was

gently shaken for 72 hours. Excess CDDP was then removed by dialysis against water. I refer

to these cisplatin-complexed lozenges as 3.9-CDDP. Figure 3.7 (b) and (d) show the TEM

images of the lozenges bound to cisplatin (3.9-CDDP). In these images, we observe well-

defined lozenges with dark edges.

55

Figure 3.7 TEM images of PLLA53-b-PAA88 (a,c) fiber-like micelles (3.6-CDDP), and (b,d) lozenges

(3.9-CDDP) stained with cisplatin.

To further characterize the cisplatin-complexed PLLA53-b-PAA88 micelles, energy

dispersive X-ray (EDX, performed by Ilya Gourevich) spectroscopy was employed. EDX

line scans provide an elemental analysis of a defined region of the micelles. For the cisplatin-

complexed fibers (3.6-CDDP), our attempts to scan a single fiber were not effective as the

instrument was not sensitive enough to detect such a low concentration of platinum ions. We

therefore chose to scan a group of fibers where the platinum ions were more concentrated, as

shown in Figure 3.8. The micelles show high intensity carbon and oxygen signals while the

platinum signal is relatively low. Figure 3.9 shows EDX line scans of the cisplatin-

complexed lozenges (3.9-CDDP). The line scans show strong signals of carbon and oxygen

in comparison to platinum. The platinum line scan shows that the platinum signal is strongest

along the edges of the lozenge.

56

Figure 3.8 (a) SEM image of PLLA53-b-PAA88 fiber-like micelles bound to cisplatin (3.6-CDDP).

The line indicates the defined region in which the EDX collected the data. EDX line scans from

carbon, oxygen, and platinum are indicated in (b), (c), and (d), respectively. The signal from platinum

is much weaker than that of carbon and oxygen.

Figure 3.9 (a) SEM image of PLLA53-b-PAA88 lozenges bound to cisplatin (3.9-CDDP). The line

indicates the defined region in which the EDX collected the data. EDX line scans from carbon,

oxygen, and platinum are indicated in (b), (c), and (d), respectively. The signal from platinum is

much weaker than that of carbon and oxygen.

57

The TEM images of the cisplatin-complexed micelles indicate that both the fiber-like

micelles and lozenges retain their shape after complexation with cisplatin. The EDX results

demonstrate that cisplatin is present in the micelles, however, the platinum signal is very

weak. Considering that the polymer is mostly composed of carbon and oxygen, the relatively

weaker platinum signal is a reasonable result. Although time did not permit, further

characterization of these micelles by inductively coupled plasma mass spectrometry (ICP-

MS) will allow for a quantitative determination of the amount of CDDP complexed to the

micelles.

3.3 Conclusion

I have described the self-assembly of PLLA53-b-PAA88 in solvent-water mixtures by

the solvent-switch method. In my preliminary self-assembly studies, I explored the effect of

the common solvent, copolymer concentration, and temperature on the morphology of the

BCP in solution. The biggest challenge in these studies was obtaining structures of a single

morphology. I showed that maintaining the BCP solution at 55 °C during the assembly of

PLLA53-b-PAA88 was a key factor in obtaining uniformly shaped structures. I also

demonstrated that the PAA groups of the micelles can be used to carry cisplatin. In the next

chapter, I will examine the effect of temperature on the solution self-assembly of the

PEGylated polymer, PLLA53-b-P(AA48-r-PEGAm40).

58


3.4.1 Materials

All reagents and solvents were purchased from Sigma Aldrich and used without further

purification. Anhydrous THF was obtained by passing the solvent through a Pure Solv

Solvent Purification System (Innovative Technology, Inc.). Spectra/Por 3 dialysis tubing

3.5K MWCO, 18 mm flat width, 50 foot length was purchased from Spectrumlabs.com.

Water was purified with a Milli-Q water purification system (10 Mcm). Lacey

Formvar/Carbon, 200 mesh Copper TEM grids were purchased from Ted Pella, USA.

Solutions of uranyl acetate (98%, Electron microscopy sciences) in water (2 wt%) were used

as the staining agent for microscopy. Cis-Diammineplatinum(II) dichloride was purchased

from Sigma-Aldrich (cisplatin, CDDP) (crystalline).


The following setup was used for the slow addition of water to solutions of the block

copolymer in an organic solvent. A syringe needle was fastened to one end of a 2 foot long

Tygon tube (1/16″ × 1/8″ × 1/32″) using Parafilm. A 1 mL syringe containing water was

attached to this end and fixed to a syringe pump (KD Scientific). To connect the tubing to a

syringe needle, a plastic coupler was fastened to the other end of the tube using Parafilm, and

a syringe needle was attached (Figure 3.10).

Figure 3.10 The Tygon tubing used to add water to the block copolymer solutions. The Tygon tube

was connected to a 1 mL syringe on one end and a syringe needle on the other end.

59

Transmission electron microscopy (TEM) was performed using a Hitachi H-7000 electron

microscope operated at an acceleration voltage of 100 kV. The samples for TEM experiments

were prepared by adding 5 μL of the micelle solution onto formvar/carbon grids (Ted Pella)

and removing excess solution by touching the edge of the droplet with filter paper. The grids

were then stained with 2 wt% uranyl acetate in water and dried at room temperature. The

micelles from the TEM images were traced using the software ImageJ (NIH, US). The

number-average lengths (Ln) and weight-average lengths (Lw) of the micelles were calculated

using the following equations:

1n

1

N

i i

iN

i

i

N L

L

N

(3.1)

2

1w

1

N

i i

iN

i i

i

N L

L

N L

(3.2)

where Ni is the number of micelles of length Li, and N is the total number of micelles. The

distribution of the micelle length is determined by Lw/Ln.

Atomic force microscopy (AFM) was performed using a Dimension 5000 AFM (Digital

Instruments, Santa Barbara) in tapping mode to determine the micelle thickness. The

topographic and phase images were obtained using NCH rectangular shape silicon probes

(Nanoworld, Switzerland) with resonance frequencies between 280–320 kHz and a spring

constant of 40 N/m. The samples for AFM experiments were prepared by adding 5 μL of the

micelle solution onto silicon wafers and vacuum dried at room temperature.

Energy-dispersive X-ray (EDX) spectroscopy was performed using an EDX attachment

(INCA, Oxford Instruments) on a Hitachi S-5200 scanning electron microscopy (SEM)

instrument. The accelerating voltage for EDX measurements was 20 kV. Composition-based

line scans and elemental mapping were carried out on the micelles to determine the platinum

content in the micelles.

60

3.4.3 Solution Self-Assembly: Solvent Effect

In a 1 dram glass vial, PLLA53-b-PAA88 (2.5, 0.5 mg) was dissolved in one of three organic

solvents: acetone, THF, or dioxane (1 mL) to obtain a homogeneous solution. The glass vial

was fitted with a rubber septum and the syringe needle attached to the Tygon tubing was

inserted with the tip of the needle above the surface of the liquid (Figure 3.11). The polymer

solution was lowered into an oil bath at room temperature and mixing was promoted by

gentle agitation caused by stirring of the oil bath. The syringe on the other end of the tubing

was fixed to the syringe pump and 1 mL of water was added dropwise over a period of 16

hours (Rate = 0.06 mL/h). Following the addition of water, solutions were transferred to

dialysis tubes (MWCO 3,500) and dialyzed against water at room temperature for 2 days to

remove the organic solvent. The dialysis medium (water) was changed approximately 3 times

each day for a total of 6 times. An aliquot of each micelle solution was taken for analysis by

TEM.

Figure 3.11 The glass vial containing the BCP solution was fitted with a rubber septum. The syringe

needle attached to the Tygon tube was inserted with the tip of the needle above the surface of the

liquid.

3.4.4 Solution Self-Assembly: Concentration Effect

In a 1 dram glass vial, PLLA53-b-PAA88 (2.5) (0.2, 0.5, or 1.0 mg) was dissolved in dioxane

(1 mL) to obtain a homogeneous solution. Using the same setup as previously described, the

polymer solution was lowered into an oil bath at room temperature. 1 mL of water was added

dropwise over a period of 16 hours using a syringe pump (Rate = 0.06 mL/h). Following the

addition of water, solutions were dialyzed against water at room temperature for 2 days to

remove residual dioxane. The dialysis medium was changed approximately 3 times each day

for a total of 6 times. An aliquot of each micelle solution was taken for analysis by TEM.

61

3.4.5 Solution Self-Assembly: Temperature Effect

In a 1 dram glass vial, PLLA53-b-PAA88 (2.5, 0.5 mg) was dissolved in dioxane (1 mL) to

obtain a homogeneous solution. Using the same setup as previously introduced, the polymer

solution was gently swirled in an oil bath maintained at RT, 40 °C, or 55 °C during the

addition of water. Using a syringe pump, 1 mL of water was added dropwise over a period of

8 hours (Rate = 0.12 mL/h). Following the addition of water, solutions were dialyzed against

water at room temperature for 2 days to remove residual dioxane. The dialysis medium

(water) was changed approximately 3 times each day for a total of 6 times. An aliquot of

each micelle solution was taken for analysis by TEM.


The incorporation of cisplatin was performed following a procedure published by Kataoka

and coworkers.46

In a 1 dram glass vial, CDDP (0.05 mg, 0.17 μmol, (CDDP)/(3.6) = 10), 3.6

(0.5 mL, 0.017 μmol), and TEA (0.16 mg) were dissolved in Milli-Q water (0.5 mL). The

mixture was gently shaken for 72 hours using a vortex mixer. The solution was then

transferred to dialysis tubing (MWCO 3,500) and dialyzed against water for 2 days to

remove excess cisplatin. The dialysis medium (water) was changed approximately 3 times

each day for a total of 6 times. This procedure was also performed with PLLA-b-PAA

lozenges (3.9) as outlined in Table 3.4.

Table 3.4 Summary of the samples prepared by staining with cisplatin.

Cisplatin-complexed fibers

(3.6-CDDP)

Cisplatin-complexed lozenges

(3.9-CDDP)

Fibers (3.6)a

0.5 mL, 0.017 μmol –

Lozenges (3.9)b

– 1.0 mL, 0.017 μmol

CDDP 0.05 mg, 0.17 μmol 0.05 mg, 0.17 μmol

TEA 0.16 mg, 1.5 μmol 0.16 mg, 1.5 μmol

water 0.5 mL – aSample 3.6 was prepared by dissolving PLLA53-b-PAA88 (1 mg) in dioxane followed by the slow

addition of water (1 mL) over 16 hours. bSample 3.9 was prepared by maintaining a solution of PLLA53-b-PAA88 (0.5 mg) in dioxane at

55 °C during the slow addition of water (1 mL) over 8 hours.

62

Chapter 4

4 Solution Self-Assembly of PLLA-b-P(AA-r-PEGAm)

4.1 Introduction

Micelles based on biocompatible polymers have shown to be successful nanocarriers

in cancer therapy. The treatment of solid tumours has been hindered, however, by the fast

circulation times of nanocarriers.1,2

Among the many design elements of a successful

nanocarrier, the polymer composition is of particular importance for avoiding rapid

clearance. PEG is a widely used biocompatible polymer known to improve the stealthiness of

block copolymer micelles and to extend blood circulation times.2,4

Additionally, by

incorporation of PEG, the molecular weight of the BCP can be increased above the threshold

of renal filtration, which, depending on the chemistry, shape, and flexibility of the polymer,

ranges from 30 to 50 kDa.49

Polymers with molecular weights above this threshold have

longer circulation times. I therefore chose to couple PEG to PLLA-b-PAA in order to

increase the water solubility and molecular weight of the block copolymer.

In Chapter 2, I described the DMTMM-activated conjugation of monoamine-

functionalized, methoxy-terminated poly(ethylene glycol) to the PAA groups of the diblock

copolymer (Figure 4.1). The objective was to randomly incorporate PEG, while retaining a

portion of the PAA block for coupling with cisplatin. In this way, the molecular weight of the

BCP can be modified by adjusting the molar ratio between the PEG chains and the PAA

groups.

63

Figure 4.1 DMTMM-activated amidation of PLLA53-b-PAA88 (2.5) with mPEG12-NH2 (560 Da) in

THF to afford PLLA53-b-P(AA48-r-PEGAm40) (2.8).

In this chapter, I describe the solution self-assembly of the amphiphilic block

copolymer, PLLA53-b-P(AA48-r-PEGAm40) (2.8). I was interested in investigating the self-

assembly morphology of the PEGylated amphiphilic block copolymer compared to that of

PLLA53-b-PAA88 described in the previous chapter. Results from earlier self-assembly

studies suggested that temperature has an effect on the assembly of the PLLA-based block

copolymers. Using the solvent-switch method, I explored the effect of temperature on the

morphology of PLLA53-b-P(AA48-r-PEGAm40) assemblies. The micelles were characterized

by TEM and AFM. The pre-formed micelles were then coupled with cisplatin and

characterized by TEM and EDX spectroscopy.



All of the self-assembly studies described in this chapter were carried out using

PLLA53-b-P(AA48-r-PEGAm40) (2.8). This brush copolymer was synthesized by a simple

amidation of the diblock copolymer, PLLA53-b-PAA88, as described in Chapter 2. By

adjusting the molar ratio between mPEG-NH2 and PLLA53-b-PAA88, the polymer was

designed to contain 44 PAA groups and 44 PEGAm groups. This was achieved by mixing

PLLA53-b-PAA88 (1 eq.), mPEG12-NH2 (560 Da, 44 eq.), and DMTMM (44 eq.) in THF

overnight. I obtained a brush copolymer, PLLA53-b-P(AA48-r-PEGAm40) (2.8), with a

molecular weight of 35.2 kDa as determined by 1H NMR. The self-assembly studies of the

64

brush copolymer allowed us to evaluate if, in the presence of PEG, the amphiphilic polymer

formed lozenges similar to those reported in Chapter 3.

One of the challenges I faced in the preliminary self-assembly studies with the

diblock copolymer (PLLA53-b-PAA88) was obtaining structures of uniform morphology.

With PLLA53-b-PAA88, I found that it was essential to perform the solvent-switch at a

temperature near the Tg of PLA in order to obtain lozenges, instead of a mixture of

morphologies. The self-assembly of PLLA53-b-P(AA48-r-PEGAm40) (2.8) was performed

by the solvent-switch method under the optimized conditions described in the previous

chapter. In Table 4.1, I assign sample numbers to the micelle solutions prepared by addition

of water to the BCP solutions maintained at specific temperatures. First, PLLA53-b-P(AA48-r-

PEGAm40) (2.8) was dissolved in dioxane to prepare a homogeneous solution at a

concentration of 0.5 mg/mL. The BCP solution was maintained at room temperature during

the addition of water over 8 hours. Residual dioxane was removed by dialysis against water

and an aliquot of the micelle solution was taken for TEM analysis (Figure 4.2 (a) and (d),

Figure B-8).


water to a solution of PLLA53-b-P(AA48-r-PEGAm40) (2.8) in dioxane maintained at the

specified temperatures.

Temperature RT 40 °C 55 °C

Sample 4.1 4.2 4.3

Samples 4.1–4.3 were prepared by maintaining a solution of

PLLA53-b-P(AA48-r-PEGAm40) (0.5 mg) in dioxane at RT, 40 °C

or 55 °C during the slow addition of water (1 mL) over 8 hours.

In the TEM images (Figure 4.2 (a) and (d)), one can see a mixture of lozenges of

varying size and thin sheets or strips. On close inspection of Figure 4.2 (a), a large lozenge is

observed with an overlapping smaller lozenge. In the background of the TEM image, there

are thin strips of assembled polymer. In Figure 4.2 (d), a single lozenge is observed with thin

rectangular sheets in the background. Taking a closer look, the thin sheets can be seen to

overlap with the lozenge. From the TEM images, it was difficult to determine the

predominant morphology as I observed large populations of both lozenges and thin sheets.

65

Throughout many of the TEM images, I observed damaged lozenges with frayed

edges or ripped corners. By measuring 30 lozenges, I calculated a number-average length of

4.9 μm (Lw/Ln = 1.2) and a number-average width of 2.6 μm (Ww/Wn = 1.2). However, these

values may not accurately represent the entire population of lozenges since many of the

lozenges were stacked or overlapped and could not be measured. It is worth noting that when

several lozenges were stacked within larger lozenges, I measured the outermost lozenge. It

was difficult to measure the inner lozenges because the corners could not be clearly identified.

I show examples of these stacked lozenges in Figure B-9. I also plot the length against the

width of the lozenges to demonstrate that the aspect ratio remains the same regardless of the

size of the lozenge (Figure B-13 (a)). The thin sheets are observed throughout many of the

TEM images and have considerably different lengths. The widths, on the other hand, appear

to be quite uniform and by measuring 50 sheets, I calculated a number-average width of 125

nm (Ww/Wn = 1.1).

Next, the BCP solution in dioxane was heated during the addition of water. PLLA53-

b-P(AA48-r-PEGAm40) (2.8) was dissolved in dioxane to prepare a 0.5 mg/mL solution. This

BCP solution was maintained at 40 °C during the slow addition of water over 8 hours.

Dioxane was then removed by dialysis against water and an aliquot of the micelle solution

was taken for TEM analysis (Figure 4.2 (b) and (e), Figure B-10). The TEM images show a

mixture of lozenges and long, thin sheets. Figure 4.2 (b) shows several stacked lozenges of

varying size. Although it is difficult to clearly see each lozenge, smaller lozenges can be

faintly seen in the center of the large lozenge. Due to the overlapping lozenges, I was only

able to accurately measure the lengths and widths of 25 lozenges. I calculated a number-

average length of 7.5 μm (Lw/Ln = 1.2) and a number-average width of 4.2 μm (Ww/Wn =

1.2). Similar to the previous sample, I plotted the lozenge length against width in Figure B-

13 (b). The linear trend indicates that the length and width are proportional. In addition to

lozenges, I also observed thin sheets. Figure 4.2 (e) shows stacked and overlapping thin

sheets of varying lengths. By measuring 40 sheets, I determined that the number-average

width was 265 nm (Ww/Wn = 1.1). Although I observed a large population of these thin strips,

their origin and mechanism of formation is unclear.

66

Figure 4.2 TEM images of assemblies prepared by addition of water to PLLA53-b-P(AA48-r-

PEGAm40) in dioxane maintained at the following temperatures: (a,d) RT (4.1), (b,e) 40 °C (4.2),

and (c,f) 55 °C (4.3). The polymer (0.5 mg) was dissolved in dioxane (1 mL) and maintained at

these temperatures during the addition of water (1 mL) over 8 hours (rate = 0.12 mL/h). The

samples were stained with uranyl acetate before imaging.

I then maintained the BCP solution at 55 °C during the addition of water. PLLA53-b-

P(AA48-r-PEGAm40) (2.8) was dissolved in dioxane (0.5 mg/mL) and maintained at 55 °C

during the addition of water over 8 hours. Dioxane was then removed by dialysis against

water and an aliquot of the solution was taken for TEM analysis (Figure 4.2 (c) and (f),

Figure B-11). Under these self-assembly conditions, I no longer observed a mixture of

morphologies; instead, I observed only lozenges. Figure 4.2 (c) shows a lozenge with uneven

or jagged edges. Similarly, in Figure 4.2 (f), a large lozenge with jagged edges is observed

with some small fragments of the lozenge visible in the background. The small black points

in the background are from the stain, uranyl acetate. In many of the TEM images, I observed

torn lozenges or small fragments of lozenges. By measuring 20 lozenges, I calculated a

number-average length of 9.9 μm (Lw/Ln = 1.3) and a number-average width of 5.4 μm

67

(Ww/Wn = 1.3). In Figure B-13 (c), I plot the lozenge length against width. The linearity

indicates that the length and width of the lozenges are proportional.

Combining the size measurements of the lozenges from the self-assembly studies

performed at all three temperatures, I plot the temperature against the number-average length

and width of the lozenges in Figure 4.3. Interestingly, even though the size distribution of the

lozenges is broad, the average lozenge size increases with temperature. This provides us with

greater insight into the role of temperature in the self-assembly of the PLA-based polymers;

particularly, into the behaviour of the lozenges as a function of temperature.

Figure 4.3 Number-average length and width of PLLA53-b-P(AA48-r-PEGAm40) lozenges as a

function of temperature.

Atomic force microscopy (AFM, performed by Kris Kim) was then employed to

determine the thickness of the lozenges. An aliquot of the micelle solution (4.3), prepared

through the solvent-switch method at 55 °C, was dried on a silicon wafer for AFM analysis

(Figure 4.4 and Figure B-12). Figure 4.4 shows the height image and corresponding height

profiles of a single lozenge. The AFM height image indicates that the thickness of the

lozenge ranges from about 5 to 10 nm. Referring to the cross-section, the highest domain is

at the center (8–10 nm thick), while the remainder of the lozenge is 5–7 nm thick.

68

Figure 4.4 AFM height image of a PLLA53-b-P(AA48-r-PEGAm40) lozenge (4.3) and the

corresponding height profile.

In the present self-assembly study of PLLA53-b-P(AA48-r-PEGAm40), I found that

the effect of temperature was important for obtaining structures of uniform shape. At

temperatures below the Tg of PLA (i.e. RT and 40 °C), I observed a mixture of lozenges and

thin sheets. When the solvent-switch was performed near the Tg of PLA (i.e. 55 °C), I

observed only lozenges. The lozenges prepared from PLLA53-b-P(AA48-r-PEGAm40) were

generally larger in size than those formed by PLLA53-b-PAA88. The PEGylated lozenges,

however, were not as well-defined as those obtained with the diblock copolymer, and I often

found that the lozenges were damaged. Their tendency to tear may be related to the thickness

of the lozenges. The PLLA53-b-P(AA48-r-PEGAm40) lozenges were only 5–10 nm thick,

while the PLLA53-b-PAA88 lozenges were 6-33 nm thick. Unlike the PLLA53-b-PAA88

lozenges, the PLLA53-b-P(AA48-r-PEGAm40) lozenges did not have thicker edges.


In this section I describe the preparation of cisplatin-complexed lozenges by

coordination of cisplatin to the residual carboxylates of the PEGylated polymer. An aliquot

of the micelle solution (4.1, 1 mL), triethylamine (0.03 mg), and CDDP (0.05 mg) was gently

shaken for 72 hours. After removal of excess CDDP by dialysis against water, an aliquot of

the solution was taken for analysis by TEM and EDX. I refer to these cisplatin-complexed

lozenges as 4.1-CDDP. Figure 4.5 (a) and (b) show TEM images of the lozenges bound to

cisplatin (4.1-CDDP). In these TEM images, the lozenges can be observed only faintly due

to poor contrast.

69

Figure 4.5 (a,b) TEM images of PLLA53-b-P(AA48-r-PEGAm40) lozenges (4.1-CDDP) stained with

cisplatin.

To further characterize the cisplatin-complexed PLLA53-b-P(AA48-r-PEGAm40)

lozenges (4.1-CDDP), energy dispersive X-ray (EDX, performed by Ilya Gourevich)

spectroscopy was employed. Figure 4.6 shows EDX line scans of the cisplatin-complexed

lozenges. The line scans show high intensity carbon and oxygen signals, while the platinum

signal is very weak.

Figure 4.6 a) SEM image of a PLLA53-b-P(AA48-r-PEGAm40) lozenge bound to cisplatin (4.1-

CDDP). The line indicates the defined region in which the EDX collected the data. EDX line scans

from carbon, oxygen, and platinum are indicated in (b), (c), and (d), respectively. The signal from

platinum is much weaker than that of carbon and oxygen.

70

The TEM and EDX results indicate that the cisplatin-complexed lozenges

contained only a small amount of cisplatin. The lozenges could only be faintly observed

in the TEM images, suggesting that platinum was present in low levels. In addition, the

EDX line scans indicated that the platinum signal was very weak. The platinum signal

from the PLLA53-b-P(AA48-r-PEGAm40) lozenges was much weaker than that observed

with the cisplatin-complexed PLLA53-b-PAA88 lozenges. In comparison to the diblock

copolymer, the PEGylated polymer contains half as many PAA groups, limiting the

amount of cisplatin that can couple to the micelles. Further characterization of these

micelles by ICP-MS is required to determine the amount of CDDP complexed to the

lozenges.

71

4.3 Conclusion

I have described the solution self-assembly of PLLA53-b-P(AA48-r-PEGAm40) in

dioxane-water mixtures via the solvent-switch method. Through my self-assembly studies, I

found that when the BCP solution was maintained at temperatures below the Tg of PLA

during the addition of water (i.e. RT and 40 °C), a mixture of lozenges and thin sheets were

obtained. When the BCP solution was maintained at 55 °C during the addition of water,

instead of a mixture of morphologies, only lozenges were observed. The lozenges, however,

had a tendency to break apart, which may be related to the fact that they are only 5–10 nm

thick, as determined by AFM. Combining the results at all three temperatures, I showed that

the size of the lozenges varied as a function of temperature. I also demonstrated that the

residual PAA groups of the PEGylated lozenges can be used to carry cisplatin.

The self-assembly studies of PLLA53-b-PAA88 described in Chapter 3 demonstrated

that well-defined lozenge-shaped structures could be obtained when the temperature of the

BCP solution was maintained at 55 °C during the solvent-switch. These lozenges had a broad

size distribution with a number-average length of 2.2 μm and width of 1.2 μm. In

comparison, the PLLA53-b-P(AA48-r-PEGAm40) lozenges prepared by solvent-switch at

55 °C tended to be larger than the PLLA-b-PAA lozenges, with a number-average length of

9.9 μm and width of 5.4 μm. Unlike the well-defined PLLA-b-PAA lozenges, the PEGylated

lozenges had frayed edges and were easily damaged. The thickness of the PLLA-b-PAA

lozenges ranged from 6–33 nm, while the PEGylated lozenges were much thinner with a

thickness of 5–10 nm. With both types of lozenges, I showed that the length and width were

proportional regardless of the broad size distribution. The next step would be to modify the

brush copolymer and self-assembly conditions to obtain lozenges that are less susceptible to

breakage.

72


4.4.1 Materials

All reagents and solvents were purchased from Sigma Aldrich and used without further

purification. Spectra/Por 3 dialysis tubing 3.5K MWCO, 18 mm flat width, 50 foot length

was purchased from Spectrumlabs.com. Water was purified with a Milli-Q water purification

system (10 Mcm). Lacey Formvar/Carbon, 200 mesh Copper TEM grids were purchased

from Ted Pella, USA. Solutions of uranyl acetate (98%, Electron microscopy sciences) in

water (2 wt%) were used as the staining agent for microscopy. Cis-Diammineplatinum(II)

dichloride was purchased from Sigma-Aldrich (cisplatin, CDDP) (crystalline).


The setup used to add water to the polymer solutions was described in Chapter 3.

The TEM, AFM, and EDX instrumentation, sample preparation, and analysis of the images

were described in Chapter 3.


In a 1 dram glass vial, PLLA53-b-P(AA48-r-PEGAm40) (2.8, 0.5 mg) was dissolved in

dioxane (1 mL) to obtain a homogeneous solution. The glass vial was fitted with a rubber

septum and the syringe needle attached to the Tygon tubing was inserted with the tip of the

needle above the surface of the liquid. The polymer solution was lowered into an oil bath

maintained at RT, 40 °C, or 55 °C and the solutions were gently swirled. The syringe on the

other end of the tubing was fixed to the syringe pump and 1 mL of water was added dropwise

over a period of 8 hours (Rate = 0.12 mL/h). Following the addition of water, solutions were

transferred to dialysis tubes (MWCO 3,500) and dialyzed against water for 2 days to remove

residual dioxane. The dialysis medium (water) was changed approximately 3 times each day

for a total of 6 times. An aliquot of each micelle solution was taken for analysis by TEM.

73


The cisplatin coupling was performed following a procedure published by Kataoka and

coworkers.46

In a 1 dram glass vial, CDDP (0.05 mg, 0.17 μmol, (CDDP)/(4.1) = 25) and

TEA (0.03 mg) were added to 4.1 (1.0 mL, 0.007 μmol). The mixture was gently shaken for

72 hours using a vortex mixer. The solution was then transferred to dialysis tubing (MWCO

3,500) and dialyzed against water for 2 days to remove excess cisplatin. The dialysis medium

(water) was changed approximately 3 times each day for a total of 6 times.

74

Chapter 5

5 Future Work

In this chapter, I will outline two general directions in which the work presented in

this thesis might be expanded upon in the future. The first avenue of research would be to

synthesize a broader library of block copolymers with a higher ratio of corona to core-

forming block. The second avenue of research would involve further studying the self-

assembly system under a variety of conditions.

The diblock copolymer (PLLA53-b-PAA88) that I synthesized only occasionally

assembled into fiber-like micelles. My hypothesis is that by increasing the length of the

corona-forming block, one could enhance the tendency to form rod-like micelles. O’Reilly

and coworkers27

showed that cylindrical micelles could be obtained with PLLA32-b-PAA265,

suggesting that a longer corona-forming block may favour the formation of cylindrical

micelles. For this reason, it would be interesting to synthesize multiple block copolymers

with varying PAA block lengths in order to investigate their self-assembly behaviour.

The self-assembly of PLLA-based block copolymers explored in this thesis is only in

its beginning stages. There are many opportunities for the further development of the self-

assembly system. In particular, performing the self-assembly under a range of conditions

could provide better insight into the factors that promote the formation of rod-like micelles.

This would include exploring the solution self-assembly in alcohols as well as water, through

both the direct dissolution and solvent-switch methods. One point that merits further

investigation is the apparent relationship between temperature and shape of the PLLA-based

BCP micelles. This relationship can be explored by performing the solvent-switch method at

additional temperatures above 55 °C in order to observe changes in the shape and size of the

structures. The temperature effect can also be examined in an alcohol solvent, such as

methanol, ethanol, or 2-propanol. In this way, the BCP solution can be heated above the Tg

75

of PLA and cooled to form the BCP aggregates. I imagine that these suggested experiments

would provide greater insight into the self-assembly system.


studied the semi-crystalline nature of the PLA core of their

micelles using wide-angle X-ray diffraction (WAXD). With this technique, they

demonstrated the difference in the crystallinity between micelles consisting of regioregular

PLLA and racemic P(DL-LA) cores. It would be valuable to perform a similar experiment to

determine the crystallinity of the PLLA in our micelles. This would enable one to compare

the degree of crystallinity of the fiber-like micelles to that of the lozenge structures and

determine whether there are differences in the way they crystallize.

The PEGylated polymer (PLLA53-b-P(AA48-r-PEGAm40)) that I synthesized formed

lozenge-shaped structures through the solvent-switch method. Unlike the lozenges formed by

PLLA-b-PAA, the PEGylated lozenges were fragile with jagged edges. Instead of modifying

the diblock copolymer before self-assembly, I can imagine coupling mPEG-NH2 to pre-

formed PLLA-b-PAA fibers or lozenges. In this way, the PLLA-b-PAA fibers or lozenges

serve as the foundation of the nanostructure, allowing the PAA groups on the surface of the

micelles to be modified by coupling with mPEG-NH2 in water.

76

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82

Appendix A

Figure A-1 13

C NMR spectrum of 1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexyl-thiourea (2.1) in

benzene-d6. The peaks are assigned according to the labelled protons of the structure.

83

Figure A-2 13

C NMR spectrum of dodecyl 4-(hydroxymethyl) benzyl carbonotrithioate (2.2) in

dichloromethane-d2. The peaks are assigned according to the labelled protons of the structure.

84

Figure A-3 1

H NMR spectrum of poly(L-lactide) in dichloromethane-d2 before treatment with

trifluoroacetic acid. The aromatic protons (a) were used as a reference and the integration was

set to 4.00. The peaks are assigned according to the structure in the figure.

85

Figure A-4 1H NMR spectrum of poly(L-lactide) in dichloromethane-d2 after treatment with

trifluoroacetic acid. The aromatic protons (a) were used as a reference and the integration was

set to 4.00. The peaks are assigned according to the structure in the figure.

Figure A-5 Molecular weight distribution of PLLA and PLLA+TFA in THF containing TBAB.

The dispersity of the polymer was unchanged after treatment with TFA, demonstrating that the

polymer had not degraded under the acidic conditions.

86

Figure A-6 1H NMR spectrum of PLLA53-b-P(AA68-r-PEGAm20) (2.7) in dichloromethane-d2.

The coupling reaction was performed at room temperature in the presence of DMTMM and mPEG45-

NH2. The molar ratio PLLA53-b-PAA88:mPEG-NH2:DMTMM was 1:25:25. The main peaks are

assigned according to the labelled protons of the structure. The methine protons of the PLLA

backbone (a) were used as a reference to determine the acrylamide content.

87

Figure A-7 1H NMR spectrum of PLLA53-b-P(AA48-r-PEGAm40) (2.8) in dichloromethane-d2.

The coupling reaction was performed at room temperature in the presence of DMTMM and mPEG12-

NH2. The molar ratio PLLA53-b-PAA88:mPEG-NH2:DMTMM was 1:44:44. The main peaks are

assigned according to the labelled protons of the structure. The methine protons of the PLLA

backbone (a) were used as a reference to determine the acrylamide content.

88

Figure A-8 Molecular weight distribution of PLLA53 (2.3) (Mn = 11.6 kDa, Đ = 1.2), PLLA53-b-

PtBA88 (2.4) (Mn = 25.9 kDa, Đ = 1.1), PLLA53-b-P(AA35-r-PEGAm53) (2.6) (Mn = 239 kDa, Đ

= 1.1), PLLA53-b-P(AA68-r-PEGAm20) (2.7) (Mn = 117 kDa, Đ = 1.1), and PLLA53-b-P(AA48-r-

PEGAm40) (2.8) (Mn = 110 kDa, Đ = 1.1) in THF containing TBAB as monitored by UV

detector (set to 309 nm).

89

Appendix B

Figure B-1 (a-d) TEM images of assemblies prepared by addition of water to PLLA53-b-PAA88 in

dioxane (0.5 mg/mL, 3.5). The polymer (0.5 mg) was dissolved in dioxane (1 mL) and maintained at

room temperature during the addition of water (1 mL) over 16 hours (rate = 0.06 mL/h). The samples

were stained with uranyl acetate before imaging.

90


dioxane (1 mg/mL, 3.6). The polymer (1 mg) was dissolved in dioxane (1 mL) and maintained at

room temperature during the addition of water (1 mL) over 16 hours (rate = 0.06 mL/h). The samples


91


dioxane maintained at RT (3.7). The polymer (0.5 mg) was dissolved in dioxane (1 mL) and

maintained at RT during the addition of water (1 mL) over 8 hours (rate = 0.12 mL/h). The samples


92


dioxane maintained at 40 °C (3.8). The polymer (0.5 mg) was dissolved in dioxane (1 mL) and

maintained at 40 °C during the addition of water (1 mL) over 8 hours (rate = 0.12 mL/h). The


93

Figure B-5 (a,b,d,e) TEM images of assemblies prepared by addition of water to PLLA53-b-PAA88 in

dioxane maintained at 55 °C (3.9). The polymer (0.5 mg) was dissolved in dioxane (1 mL) and

maintained at 55 °C during the addition of water (1 mL) over 8 hours (rate = 0.12 mL/h). The

samples were stained with uranyl acetate before imaging. (c) Length distribution histogram and (f)

width distribution histogram.

94

Figure B-6 AFM height images of PLLA53-b-PAA88 lozenges (3.9) and the corresponding height

profiles.

95

Figure B-7 TEM images of PLLA53-b-PAA88 fiber-like micelles (a) before (3.6) and (c) after staining

with cisplatin (3.6-CDDP). TEM images of PLLA53-b-PAA88 lozenges (b) before (3.9) and (d) after

staining with cisplatin (3.9-CDDP). The morphology of the micelles remained the same after

coupling to cisplatin.

Figure B-8 (a-d) TEM images of assemblies prepared by addition of water to PLLA53-b-P(AA48-r-

PEGAm40) in dioxane maintained at RT (4.1). The polymer (0.5 mg) was dissolved in dioxane (1 mL)

and maintained at RT during the addition of water (1 mL) over 8 hours (rate = 0.12 mL/h). The


96

Figure B-9 (a,b) TEM images of lozenges prepared by addition of water to PLLA53-b-PAA88 in

dioxane maintained at RT (4.1). The lozenges had a tendency to stack, preventing accurate

measurement of the length and width of the structures.


PEGAm40) in dioxane maintained at 40 °C (4.2). The polymer (0.5 mg) was dissolved in dioxane (1

mL) and maintained at 40 °C during the addition of water (1 mL) over 8 hours (rate = 0.12 mL/h).

The samples were stained with uranyl acetate before imaging.

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PEGAm40) in dioxane maintained at 55 °C (4.3). The polymer (0.5 mg) was dissolved in dioxane (1

mL) and maintained at 55 °C during the addition of water (1 mL) over 8 hours (rate = 0.12 mL/h).

The samples were stained with uranyl acetate before imaging.

Figure B-12 AFM height image of a PLLA53-b-P(AA48-r-PEGAm40) lozenge (4.3) and the

corresponding height profile.

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Figure B-13 The length versus the width of the lozenges prepared at (a) RT (4.1), (b) 40 °C (4.2),

and (c) 55 °C (4.3) as measured by TEM. The linearity of the plot indicates that the aspect ratio

remains the same regardless of the size of the lozenge.

Figure B-14 TEM images of PLLA53-b-P(AA48-r-PEGAm40) lozenges (a) before (4.1) and (b) after

staining with cisplatin (4.1-CDDP). The morphology of the micelles remained the same after

coupling to cisplatin.

Synthesis and Solution Self-Assembly of Poly(L …...The self-assembly of these amphiphilic block...

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