Core-shell particles with glycopolymer shell and polynucleoside core via RAFT: From micelles to rods

Core-Shell Particles with Glycopolymer Shell andPolynucleoside Core via RAFT: From Micelles to Rods

SAMUEL PEARSON, NATHAN ALLEN, MARTINA H. STENZEL

Centre for Advanced Macromolecular Design, School of Chemical Sciences and Engineering,The University of New South Wales, Sydney, New South Wales 2052, Australia

Received 16 November 2008; accepted 5 January 2009DOI: 10.1002/pola.23275Published online in Wiley InterScience (www.interscience.wiley.com).

ABSTRACT: Amphiphilic block copolymers were synthesized via the reversible addi-tion fragmentation chain transfer (RAFT) copolymerisation of 2-methacrylamido glu-copyranose (MAG) and 50-O-methacryloyl uridine (MAU). Homopolymerisations ofboth monomers using (4-cyanopentanoic acid)-4-dithiobenzoate (CPADB) proceededwith pseudo first order kinetics in a living fashion, displaying linear evolution of mo-lecular weight with conversion and low PDIs. A bimodal molecular weight distribu-tion was observed for PMAU at low conversions courtesy of hybrid behavior betweenliving and conventional free radical polymerization. This effect was more pronouncedwhen a PMAG macroRAFT agent was chain extended with MAU, however, in bothcases, good control was attained once the main RAFT equilibrium was established. Astability study on PMAU found that its hydrolysis is diffusion controlled, and isaccelerated at physiological pH compared with neutral conditions. Self-assembly offour block copolymers with increasing hydrophobic (PMAU) block lengths producedmicelles, which demonstrated an increased tendency to form rods as the PMAU blocklength increased. Interestingly, none of the block copolymers were surface-active. Aninitial assessment of PMAU’s ability to bind the nucleoside adenosine through basepairing was highly promising, with DSC measurements indicating that adenosine isfully miscible in the PMAU matrix. VVC 2009 Wiley Periodicals, Inc. J Polym Sci Part A:

Polym Chem 47: 1706–1723, 2009

Keywords: core-shell polymers; diblock copolymers; living polymerization;nanoparticles; reversible addition fragmentation chain transfer (RAFT)

INTRODUCTION

Core-shell nano-objects such as micelles, rods,and vesicles, which are formed from the solutionself-assembly of amphiphilic block copolymers,show great promise as drug and gene deliveryvehicles, and possess several clear advantagesover viral gene delivery systems, including easier

and cheaper preparation on a large scale andlower risk of an immunogenic response.1 Thechoice of monomers from which to synthesizeblock copolymers is vast, and allows great flexibil-ity in controlling the size, stability, gene and drugloading capacity and biological interactions of theresulting micelles.2–5 Although much workfocuses on the preparation of spherical particlessuch as micelles, it has recently been suggestedthat rod-like structures can enhance the circula-tion time of nanoparticles in the bloodstream.6

Of particular interest for drug and gene deliv-ery systems is the incorporation of stimuli-respon-sive groups or targeting groups onto the shell of

Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 47, 1706–1723 (2009)VVC 2009 Wiley Periodicals, Inc.

Additional Supporting Information may be found in theonline version of this article.

Correspondence to: M. H. Stenzel (E-mail: [email protected])

1706

the core-shell nanoparticles. Targeting ligandssuch as peptides,7 antibodies,8 and carbohy-drates9 can greatly improve the efficiency of thecarriers by encouraging adhesion to the mem-branes of specific cells and cellular uptake viaendocytosis.3,10 Indeed, block copolymer micellespossessing a hydrophilic glycopolymer shell basedon galactose exhibit superior targeting of livercancer cells compared with nonglycosylated ana-logues, which is attributed to enhanced binding tothe asialoglycoprotein receptor.11

Incorporation of carbohydrate moieties aseither pendant or terminal groups to a polymerchain leads to the effective synthesis of glycopoly-mers.12–15 Various polymerization techniqueshave been used to generate well-defined glycopoly-mers such as living ionic polymerization,16,17

ring opening polymerization,18,19 ring openingmetathesis polymerization,20,21 click chemis-try,22,23 cyanoxyl-mediated free radical polymer-ization,24–26 ATRP,27–29 and NMP.30 Nevertheless,these techniques exhibit limitations such assevere reaction conditions for ionic polymeriza-tion, the need for the removal of metal catalyst orthe recourse to protecting and deprotecting chem-istry. Reversible addition fragmentation chaintransfer (RAFT) polymerization31–35 holds greatpromise as a versatile polymerization techniquefor the synthesis of glycopolymers without theneed for protective chemistry. Polymerizationscan be carried out in water in the presence of freehydroxyl groups as demonstrated for the firsttime using methacryloxyethyl glucoside.36 Subse-quent polymerizations of unprotected glycomono-mers in water or protic solvents using a range ofsugars further demonstrated the versatility of theRAFT process for glycopolymer synthesis.37–45

Glycopolymers are typically water soluble if nolong hydrophobic spacer has been introduced.Amphiphilic block copolymers based on glycopoly-mers will therefore form self-assembled struc-tures with a glycopolymer shell in aqueous solu-tion. The core-forming blocks of reported micellesare typically based on hydrophobic and neutralmonomers. In recent years, polymers containingadditional recognition elements such as supramo-lecular features have received increasing atten-tion. Mimicking the highly specific hydrogenbonds of the base pairs thymine/adenine and gua-nine/cytosine in DNA, several concepts to buildup polymer architectures incorporating H-bondshave been explored. Examples are the formationof block copolymers by joining polymers carryingcomplementary hydrogen bonding units.46–49

The interactive forces between two complemen-tary species could be explored in miceller systemsby incorporating one type of H-bonding unit aspendant groups and loading the micelle with acompound or polymer containing the complemen-tary unit. This concept could be used to developan alternative carrier for antisense oligonucleo-tides (ASONs) which show great promise in thetreatment of many infectious and genetic dis-eases. To date, micellar systems for ASON encap-sulation have almost exclusively used a cationichydrophobic block such as poly(L-lysine) (PLL),50

polyethylene imine (PEI),51 poly(N,N-dimethyla-minoethyl methacrylate) (PDMAEMA),52 andpoly(N-isopropyl-acrylamide) (PNiPAAm),53 whichcan electrostatically bind the negatively chargedoligonucleotide. However, toxicity problems havebeen associated with the use of cationic polymersfor gene delivery,54 providing impetus to developa more benign polymer for gene binding.

In the present research, we investigated thesynthesis of block copolymers with the water-solu-ble block carrying pendant glucose and the hydro-phobic block comprising of a polynucleoside basedon uridine (Scheme 1). Chemo-enzymatic synthe-sis provided efficient and highly regioselectiveaccess to the uridine monomer through acylationat the primary hydroxyl group of uridine usingvinyl methacrylate.55 CAL435, a lipase derivedfrom Candida antarctica, was used to catalyzethe reaction in acetone.

Glycomonomers can also be synthesized usingan enzymatic approach to regioselectively func-tionalise the primary hydroxyl in the 6-position.However, introducing the vinyl functionality inthis position has been found to eliminate the bio-logical activity of the resulting glycopolymer.56 Avariety of procedures have been developed to ster-eoselectively attach vinyl groups to sugars inother positions, many of which are multi-stepsequences requiring the use of toxic organic sol-vents, heavy metal catalysts, and protectinggroup strategies.57,58 In this work, we used gluco-samine to perform a single step procedure withoutcatalysis in aqueous solution to generate themonomer 2-methacrylamido glucopyranose.

Both monomers were polymerized via RAFTpolymerization to generate amphiphilic blockcopolymers (Scheme 1). The water-soluble poly(2-methacrylamido glucopyranose) block was pre-pared first to have the thiocarbonylthio group(RAFT group) located at the end of the hydro-phobic poly(50-O-methacryloyl uridine) block.This allows crosslinking of the self-assembled

CORE-SHELL PARTICLES 1707

Journal of Polymer Science: Part A: Polymer ChemistryDOI 10.1002/pola

structure in solution to achieve further stabiliza-tion similar to the process described elsewhere.45

EXPERIMENTAL

Materials

Unless otherwise specified, all chemicals werereagent grade and used as received. Glucosamine

hydrochloride (Sigma, 99%), sodium carbonate(Ajax Finechemicals, 99.8%), sodium nitrite (AjaxFinechem, 98%), uridine (Sigma, 99%), 2,6-di-tert-butyl-4-methylphenol (BHT, Sigma-Aldrich),methacryloyl chloride (Lancaster, 97%), vinylmethacrylate (Aldrich, 98%), N,N0-dimethylaceta-mide (DMAc, Sigma, HPLC grade 99.9%), metha-nol (MeOH, Aldrich, HPLC grade 99.9%), cyclo-hexane (Ajax Finechemicals, 99%), ethanol

Scheme 1. Synthetic approach to block copolymers.

1708 PEARSON, ALLEN, AND STENZEL


(EtOH, Ajax Finechemicals, 99%), ethyl acetate(EtOAc, Ajax Finechemicals, 99.5%), deuteriumoxide (D2O, Aldrich, 99.9 atom % D), deuterateddimethylsulfoxide (d6-DMSO, Cambridge IsotopeLaboratories, 99.9 atom % D) were all used asreceived. The Novozym 435V

R

enzyme was kindlydonated by Novozymes A/S. Acetone (Aldrich,HPLC grade) was dried for 24 h over 3 A molecu-lar sieves activated at 200 �C for 12 h. 2,20-azobisi-sobutyronitrile (AIBN, Fluka, 98%) was purifiedby recrystallization from methanol. The RAFTagent (4-cyanopentanoic acid)-4-dithiobenzoate(CPADB) was synthesized according to litera-ture59,60 and recrystallised from toluene.

Analyses

Size exclusion chromatography (SEC) was per-formed using a Shimandzu modular system con-taining a DGU-12A degasser, a LC-10AT pump, aSIL-10AD automatic injector, a CTO-10A columnoven, and a RID-10A refractive index detector. A50 � 7.8 mm guard column and four 300 � 7.8mm linear columns (500, 103, 104, and 105 A poresize, 5 lm particle size) were used for the analy-ses. N,N0-dimethylacetamide (HPLC grade, 0.05%w/v BHT, 0.03% w/v LiBr) with a flow rate of 1mL min�1 was used as the mobile phase with aninjection volume of 50 lL. The samples werefiltered through 0.45 lm filters. The unit wascalibrated using commercially available linearpolystyrene standards (0.5–1000 kDa, PolymerLaboratories). Chromatograms were processesusing Cirrus 2.0 software (Polymer Laboratories).

Nuclear magnetic resonance (NMR) analyseswere performed using a Bruker DPX-300 with a1H/X inverse broadband z gradient BBI probeusing 16 scans as default.

Liquid chromatography–mass spectrometry(LC–MS) analysis was carried out using aThermo-MAT high pressure LC system consistingof a SCM1000 solvent degasser, a P4000 quater-nary pump, an AS3000 auto-injector, a UV2000dual-wavelength UV detector, and a C8 LunaV

R

reverse phase column (Phenomenex, 150 � 4.6mm, 100 A pore size, 5 lm particle size). The mo-bile phase was a gradient program of acetonitrileand water buffered with 1 mM acetic acid. Thechromatography system was coupled to a ThermoFinnigan LCQ Deca ion-trap mass spectrometerequipped with an atmospheric pressure ionizationsource operated in nebulizer assisted electro-spray mode (ESI). Calibration was performed

with caffeine (Aldrich), MRFA (tetrapeptide,Thermo Finnigan), and Ultramark 1621 (Lancas-ter) with a mass range of 195–3822 Da. Spectrawere obtained over the mass to charge range of150–2000 Da applying a spray voltage of 5 kV,capillary voltage of 39 V, and capillary tempera-ture of 275 �C. Nitrogen was used as the sheathgas with a flow rate of 0.5 L min�1 and heliumused as the damping gas.

UV/Vis analysis was performed on a Cary 300spectrometer. A solution of 3.2 � 10�6 mol L�1

CPADB in DMAc/H2O (9:1 v/v) was prepared. Asimilar solution also containing 0.8 M of MAG,which had been purified by column chromatogra-phy but not recrystallised served as the test system.The pH was �6.5 in both systems. No deoxygena-tion was performed. Each solution was heated to70 �C in a stoppered quartz cuvette in the UV/Visspectrometer and scanned from 200 to 900 nm atregular intervals up to 3.5 h. The baseline absorb-ance at 720 nm (an arbitrary wavelength whoseabsorbance was always approximately zero givenno components are active above about 650 nm) wassubtracted from the RAFT agent peak at 515 nm togive the net absorbance of the RAFTagent.

Transmission electron microscopy (TEM)analyses were performed using a JEOL1400microscope at 80 kV beam voltage. Samples wereprepared by placing a droplet of solution on form-amide- and graphite-coated copper grids anddraining the excess using filter paper. Once com-pletely dry, the grids were immersed in a dropletof uranyl acetate (2% aqueous solution) for 20 s,and the excess solution drained.

Dynamic light scattering (DLS) measurementswere performed Brookhaven ZetePlus ParticleSizer with a dust-cutoff of 40. The polymer solu-tions were made up to 1 mg mL�1 and werefiltered through 0.45 lm filters.

Surface tension measurements were preformedon a NIMA DST 9005 computer controlled surfacetensiometer with a Pt/Ir du Nouy ring.

Perkin–Elmer differential scanning calorimeter(DSC 7) and thermal analysis controller (TAC71DX) were used in conjunction, to measure glasstransition temperatures and melting points. Sam-ples were analyzed in 50 lL pans, over a tempera-ture range of 25–250 �C with scanning rate of20 �C min�1. Concentrated solutions in DMF withknown concentrations were prepared and pouredinto the pan. Slow evaporation of the solventensured an even coverage of the bottom of thepan. The samples were then dried under highvacuum to remove traces of solvent.



Synthesis of 2-Methacrylamido Glucopyranose

Glucosamine hydrochloride (8.67 g, 40.3 mmol),sodium carbonate (6.40 g, 60.4 mmol), and sodiumnitrite (0.174 g, 2.52 mmol) as inhibitor wereadded to 25 mL of water and the mixture wascooled to �10 �C in an ice/salt bath. Methacryloylchloride (3.98 mL, 40.3 mmol) was added drop-wise with vigorous stirring. The solution wasstirred for 2 h while maintaining the temperatureat �10 �C then stirred for a further 20 h at roomtemperature. The crude solution was added drop-wise to 200 mL of rapidly stirred methanol. Theprecipitated salt was removed by suction filtrationand washed with methanol. The volume of thecombined washings was reduced to �100 mLusing the rotary evaporator. Silica gel (30 g) wasadded and the remaining solvent removed undervacuum. The crude product dispersed on the silicawas purified by column chromatography using anethyl acetate/methanol mixture as eluent (8:2 v/v). The product solutions (Rf ¼ 0.44) were evapo-rated to dryness to give the desired product (as amixture of anomers), which was recrystallisedfrom the same solvent mixture to yield fine whiteneedles (32%, 98% purity) characterized by 1Hand 13C NMR spectroscopy (Scheme 2).

1H NMR (300 MHz, D2O) (a and b anomers): d(ppm) 5.63 (s, 1H, H9), 5.40 (s, 1H, H8), 5.16–5.15(d, 0.52H, H1 a, J ¼ 3.41 Hz), 4.73–4.70 (d, 0.48H,H1 b, J ¼ 8.34 Hz), 3.35–3.95 (m, 6H, H2, H3, H4,H5, H6, H60), 1.87 (s, 3H, H7).

13C NMR (300 MHz, D2O) (a and b anomers): d(ppm): 172.83, 172.44 (Cg a þ b), 139.22, 138.98(Ch a þ b), 121.07, 120.93 (Cj a þ b), 94.75 (Ca b),90.74 (Ca a), 75.86 (Ce b), 73.63 (Ce a), 71.48,70.43, 70.04, 69.85 (Cc a þ b, Cd a þ b), 60.65,60.49 (Cf a þ b), 56.77, 54.18 (Cb a þ b), 17.64,17.61 (Ci a þ b). See Figure A and Figure B insupplementary information for full spectra.

Synthesis of 50-O-Methacryloyl Uridine

Uridine (15.64 g, 64.1 mmol), Novozyme 435enzyme beads (6.41 g), vinyl methacrylate (9.24mL, 76.9 mmol), anhydrous acetone (50 mL), and2,6-di-tert-butyl-4-methylphenol (BHT, 50 mg) asinhibitor and were added to a 250 mL round bot-tom flask. The flask was stoppered with a rubberseptum, sealed with paraffin film and copperwire, and placed in an oscillating water bath for 6days at 150 rpm and 50 �C. Aliquots (50 lL) werewithdrawn from the reaction solution at regularintervals and diluted in acetonitrile (containing1 mmol L�1 acetic acid) for analysis by HPLC. Atthe completion of the reaction, the enzyme beadsand unreacted uridine were removed by vacuumfiltration and washed with acetone (100 mL). Theacetone washing was combined with the originalfiltrate, and 60 lm silica (40 g) added before evap-orating to dryness. The product (adsorbed onsilica) was purified by flash chromatographyusing an EtOAc/cyclohexane/EtOH mixture(6.5:2.5:1 v/v) as eluant. The collected fractionswere examined by TLC under a UV lamp andthose containing the product (Rf ¼ 0.50) werecombined and evaporated to dryness. The verypale yellow sticky solid was dissolved in minimalwater (�30 mL) and freeze dried to obtain whitepowder (11.62 g, 58%), which was analyzed by 1Hand 13C NMR spectroscopy (Scheme 2) and ESI-MS.

1H NMR (300 MHz, d6-DMSO) d (ppm) 11.12–11.58 (br s, 1 H, H1), 7.61–7.74 (d, 1 H, H3, J ¼8.28 Hz), 6.03 (s, 1 H, H13), 5.68–5.79 (m, 2 H, H4

and H12), 5.58–5.63 (d, 1 H, H2, J ¼ 8.28 Hz),5.35–5.58 (br s, 1 H, H5), 5.12–5.35 (br s, 1 H, H7),4.20–4.39 (m, 2 H, H10), 3.94–4.11 (m, 3 H, H6,H8, H9), 1.88 (s, 3 H, H11).

13C NMR (300 MHz, d6-DMSO) d (ppm): 18.38(Cm), 64.42 (Ci), 70.04 (Cg), 73.13 (Cf), 81.35 (Ch),

Scheme 2. Structures of 2-methacrylamido glucopyranose and 50-O-methacryloylur-idine with 1H and 13C NMR peak assignments.



89.31 (Ce), 102.34 (Cc), 126.57 (Ck), 135.99 (Cl),141.08 (Cd), 150.91 (Ca), 163.36 (Cb), 166.72 (Cj).See Figure C and Figure D in supplementaryinformation for full spectra.

ESI–MS: calcd for C13H15N2O7, 311.27; found311.1 (M-Hþ); calcd for C13H16N2NaO7, 335.27;found 335.1 (M þ Naþ).

Polymerization of 2-MethacrylamidoGlucopyranose

2-Methacrylamido glucopyranose (MAG, 0.250 g,1.01 � 10�3 mol), (4-cyanopentanoic acid)-4-dithiobenzoate (CPADB, 2.82 � 10�3 g, 1.01 �10�5 mol) as RAFT agent, and 2,20-azo-bisisobutyronitrile (AIBN, 8.30 � 10�4 g, 5.06 �10�6 mol) as initiator were dissolved in a DMAc/H2O mixture (9:1 v/v, 1264 lL) in a 10 mLSchlenk tube to give [MAG]:[CPADB]:[AIBN] ¼100:1:0.5 and [MAG] ¼ 0.80 mol L�1. The solutionwas thoroughly deoxygenated using four consecu-tive freeze-pump-thaw cycles followed by 1 h ofnitrogen purging. The solution was placed in aconstant temperature water bath at 60 �C. Ali-quots of the solution were withdrawn at regularintervals using a gas-tight glass syringe pre-purged with nitrogen, and plunged immediatelyinto ice at the time of withdrawal to cease thepolymerization. The final solution (530 lL aftersample withdrawal) was precipitated into rapidlystirred chilled methanol. The precipitate was iso-lated by centrifugation and dried to give poly(2-methacrylamido glucopyranose) (PMAG) as apink powder (57 mg, 54%). Total reaction time ¼ 6h, XNMR ¼ 73%, XSEC ¼ 76%, Mn,SEC ¼ 29 300 gmol�1, PDI ¼ 1.23.

A portion of the 6 h PMAG (12.5 mg) was dis-solved in DMAc (1250 lL) and appropriate vol-ume aliquots used to prepare four solutions withconcentrations of 2.5 mg mL�1, 5.0 mg mL�1, 7.5mg mL�1, and 10.0 mg mL�1. These standard sol-utions and the series of time-dependant sampleswere analyzed consecutively by SEC.

Conversions were calculated from the SECchromatograms by plotting the peak area A (mVs�1) versus polymer concentration [P] (mg mL�1)for the standard solutions and finding the gradi-ent m (mV s�1 mL mg�1) of the linear leastsquares regression model (which passed throughthe origin) given by eq 1:

A ¼ m:½P� (1)

From this correlation, the concentration andtherefore the mass of polymer MP in each time-

dependant sample was determined. The ratio ofthe calculated mass of polymer to the known com-bined mass of monomer MM and polymer MP

(based on the volume of the aliquot taken fromthe polymerization solution and the initial mono-mer concentration) gave the conversion, XSEC

(eq 2):

XSEC ¼ MP

ðMM þMPÞ (2)

This calculation assumes that the response ofthe refractive index detector is proportional to thenumber of monomer repeat units in the mobilephase at any given instant, and is therefore inde-pendent of the molecular weight of the polymer towhich they belong.61

The conversion at each sample time was alsoestimated using NMR spectroscopy by comparingthe integral of the H9 vinyl proton IH9 at 5.63ppm to that of the CH2 peak of the polymer back-bone ICH2

from 0.70 to 1.30 ppm (eq 3):

XNMR ¼ ðICH2=2Þ

ðICH2=2Þ þ IH9

(3)

The theoretical molecular weight Mn,theor ofeach polymer sample was determined using eq 4:

Mn;theor ¼ X �MWM � ½M�0½RAFT�0

þMWRAFT (4)

where X is the conversion, MWM and MWRAFT arethe molecular weights of monomer and RAFTagent respectively, and [M]0 and [RAFT]0 are theinitial concentrations of monomer and RAFTagent, respectively. Note that an average of theconversion obtained by NMR and SEC was used.The measured molecular weight (Mn,SEC) and thepolydispersity index (PDI) of each sample wereobtained from the SEC chromatograms.

Polymerization of 50-O-Methacryloyl Uridine

In a typical experiment, 50-O-methacryloyl uri-dine (MAU, 0.468 g, 1.53 � 10�3 mol), (4-cyano-pentanoic acid)-4-dithiobenzoate (CPADB, 5.05 �10�3 g, 1.81 � 10�5 mol) as RAFT agent, and 2,20-azobisisobutyronitrile (AIBN, 7.43 � 10�4 g,4.51 � 10�6 mol) as initiator were dissolved indimethyl sulfoxide (DMSO, 2000 lL) in a 10 mLSchlenk tube to give [MAU]:[CPADB]:[AIBN] ¼1000:12:3 and [MAU] ¼ 0.75 mol L�1. The tubewas stoppered with a rubber septum and sealedwith paraffin film and copper wire. The solution



was thoroughly deoxygenated by four consecutivefreeze-evacuate-thaw cycles followed by 1 h ofnitrogen purging then placed in a constant tem-perature water bath at 70 �C. Aliquots of the solu-tion were withdrawn at regular intervals using a100 lL gas-tight glass syringe prepurged withnitrogen. Each sample was quenched in ice imme-diately after withdrawal to cease the polymeriza-tion. The final reaction solution (1470 lL aftersample withdrawal) was precipitated into cold,rapidly stirred methanol, isolated by centrifuga-tion and dried on the Schlenk line to give a palepink powder (259 mg, 55%). Total reaction time ¼180 min, XNMR ¼ 74%, XSEC ¼ 71%, Mn,SEC ¼31,300 g mol�1, PDI ¼ 1.20.

A stock solution containing 7.7 mg of PMAU in1250 lL of DMAc was used to prepare a series of500 lL solutions with concentrations of 1.54 mgmL�1, 3.08 mg mL�1, 4.62 mg mL�1, and 6.16 mgmL�1. These standard solutions and the time-dependant solutions were analyzed consecutivelyby SEC and the conversions calculated from thepeak areas using eq 1 and eq 2. The measuredmolecular weights and PDIs were obtaineddirectly from the SEC chromatograms.

To the remaining volumes of each time-depend-ant sample was added d6-DMSO and the conver-sions estimated by 1H NMR (300 MHz) spectros-copy by comparing the integral of the H13 vinylproton IH13 at 6.03 ppm to that of the H1 diimideproton IH1 at 11.33 ppm.

XNMR ¼ 1� IH13

IH1

� �(5)

The theoretical molecular weight was calcu-lated from eq 4 using the average of XNMR andXSEC for X.

Hydrolysis of Poly(50-O-Methacryloyl Uridine)

Five solutions were prepared containing high mo-lecular weight PMAU (Mn [ 1000,000 g mol�1,100 mg) in 10 mL of water, water/deuterium oxide(1:1), deuterium oxide, phosphate-buffered saline(PBS) pH 7.4, and PBS pH 7.6. The solutionswere then incubated at 37 �C with gentle shakingat 40 rpm.

Samples (1 mL) were taken at regular intervalsand filtered and the sampled volume replacedwith the appropriate solution to make the volumeback up to 10 mL. The hydrolyzed uridine concen-tration was determined by HPLC after accounting

for the previous dilutions. High molecular weightPMAU is essentially insoluble and uridine ishighly water-soluble, which validates the adop-tion of this approach since the reaction is notdependant upon an equilibrium.

Chain Extension of Poly(2-MethacrylamidoGlucopyranose) with 50-O-Methacryloyl Uridine

A batch of PMAG (108 mg) was synthesized tar-geting a theoretical molecular weight of 15,000 gmol�1 by polymerising MAG for 4 h 15 min usingCPADB under identical conditions as thosedescribed previously (XNMR ¼ 59%, Mn,theor ¼14,800 g mol�1, Mn,SEC ¼ 23,000 g mol�1, PDI ¼1.21). MAU (0.176 g, 5.63 � 10�4 mol), PMAG(Mn,theor ¼ 14,800 g mol�1, PDI 1.21, 0.100 g,6.78 � 10�6 mol) as macroRAFT agent, and AIBN(2.83 � 10�4 g, 1.69 � 10�6 mol) were dissolved inDMSO (750 lL) in a 10 mL Schlenk tube to give[MAU]:[PMAG]:[AIBN] ¼ 1000:12:3 and [MAU] ¼0.75 mol L�1. The tube was stoppered with a rub-ber septum and sealed with paraffin film and cop-per wire. The solution was thoroughly deoxygen-ated using four consecutive freeze-evacuate-thawcycles followed by 1 h of nitrogen purging thenplaced in a constant temperature water bath at70 �C. Aliquots of the solution were withdrawn ev-ery 10 min up to 70 min using a 100 lL gas-tightglass syringe prepurged with nitrogen. Each sam-ple was quenched in ice immediately after with-drawal.

The 70 min polymer solution (420 lL after sam-ple withdrawal) was precipitated into cold, rapidlystirred methanol, isolated by centrifugation, anddried on the Schlenk line to give a pale pink pow-der (112 mg, 73%). Total reaction time ¼ 70 min,XNMR ¼ 81%, Mn,theor ¼ 35,700 g mol�1, Mn,SEC ¼50,000 g mol�1, PDI ¼ 1.23. The conversion ofeach time-dependent sample was found from itsNMR spectrum using eq 5, and the theoreticalmolecular weight was calculated using eq 4.

Three of the time-dependant samples (20 min,40 min, and 70 min) were dialysed against waterusing a cellulose dialysis membrane (MW cut-off3400 g mol�1) and made up to 1.0 mg mL�1 usingdistilled water. The resulting solutions were ana-lyzed by DLS, TEM, and tensiometry. Note thatthe dialysis removed the remaining monomer andinitiator but not the very small amount of homo-polymer, which is inevitably produced in RAFTpolymerizations.

A similar block copolymer with a longer PMAUblock was synthesized by chain extending a



PMAG macroRAFT agent (Mn,theor ¼ 13,800 gmol�1, Mn,SEC ¼ 19,200 g mol�1, PDI ¼ 1.21)with MAU in DMSO at 70 �C using a [MAU]:[PMAG]:[AIBN] ratio of 1000:9:3 and a monomerconcentration of 0.75 mol L�1. After 70 min, thepolymerization was ceased and the polymer pre-cipitated into MeOH and dried under vacuum togive PMAG-b-PMAU (157 mg, 78%). Total reactiontime ¼ 70 min, XNMR ¼ 87%, Mn,theor ¼ 43,100 gmol�1, Mn,SEC ¼ 66,600 g mol�1, PDI ¼ 1.61. A1.0 mg mL�1 aqueous solution of the block copoly-mer was prepared by dialysis as described above,and analyzed by DLS, TEM and tensiometry.

RESULTS AND DISCUSSION

Synthesis of 2-Methacrylamido Glucopyranose

MAG was synthesized in a one step procedure byacylating glucosamine with methacryloyl chloridein aqueous solution (Scheme 1). Excess base (1.5mol of sodium carbonate per mole of glucosamine)ensured that the amino group remained deproto-nated and therefore nucleophilic throughout thereaction. A low reaction temperature, very slowdrop-wise addition of methacryloyl chloride, andthe use of a stoichiometric quantity (rather thanan excess) of methacryloyl chloride favored highconversion to MAG, and purification by columnchromatography was aided by adsorption of thecrude product onto silica before loading onto thecolumn. Existing literature procedures for thissynthesis are performed at 0 �C using an excess ofacylating agent, and do not adsorb the crude prod-uct on silica prior to column chromatography.62,63

Synthesis of 50-O-Methacryloyl Uridine

MAU was synthesized by enzymatic trans-esterifi-cation of uridine with vinyl methacrylate in dryacetone at 50 �C for 6 days (Scheme 1). Theenzyme used as the catalyst was immobilized C.Antarctica lipase 435 (CAL435), commerciallyavailable as Novozyme 435V

R

. Vinyl methacrylatewas chosen as the acylating agent because thevinyl alcohol by-product rapidly tautomerises toacetaldehyde and is effectively removed from theequilibrium. 1H NMR (300 MHz, CDCl3) indicatedthat the major impurity in the crude solution wasmethacrylic acid; d (ppm) 10.42 (br s, 1H), 6.24 (s,1H), 5.67 (s, 1H), and 1.95 (s, 3H), highlightingthe importance of performing the reaction underanhydrous conditions.

The concentration of MAU throughout thereaction was monitored by HPLC and was foundto plateau at around 70% conversion after 3 to 4days (Fig. 1), which is attributed to the deactiva-tion of the enzyme.64 The temperature of 50 �C forthe synthesis was chosen as it provides for a rea-sonably high enzyme activity while minimizingthis deactivation. The final conversion of uridineto MAU was estimated by 1H NMR and was usedto convert the HPLC intensity values into conver-sions. A small quantity of uridine remainedunreacted at the completion of the reaction.

The structure was elucidated and the acylationat the 50-O- position on the sugar ring was con-firmed by 1D and 2D NMR. 1H NMR demon-strated the product is mono-substituted whereasthe HMBC correlation showed coupling betweenthe sugar ring and the methacrylate moiety at the50-O- position (H10 coupled to Cj). MAU was foundto be highly hygroscopic and prone to spontaneouspolymerization during the solvent removal steps.

Polymerization of 2-MethacrylamidoGlucopyranose

MAG was polymerized at 60 �C using the[MAG]:[CPADB]:[AIBN] ratio of 100:1:0.5 and amonomer concentration of 0.80 mol L�1 in DMAc/H2O (9:1 v/v). The relatively high [AIBN]:[CPADB] ratio is justified given that at 60 �C thehalf-life of AIBN is �21 h, which means only asmall fraction of the initiator decomposed over the6 h of polymerization and the requirement for alow radical concentration relative to RAFT agentwas still met. A similar polymerization using halfthe initiator concentration (and keeping all other

Figure 1. Conversion of uridine to MAU as deter-mined by HPLC.



parameters unchanged) proceeded prohibitivelyslowly, reaching only 16% conversion after 6 h.

The conversions of the methacrylamide mono-mer were calculated using 1H NMR spectroscopyand SEC (see experimental section). Both valuesare in excellent agreement (Fig. 2), confirming thevalidity of each technique in calculating homopo-lymerisation conversions. An inhibition period ofaround 1 h 20 min is observed, beyond which thepolymerization proceeds with pseudo first orderkinetics indicative of a constant radical concentra-tion to reach a conversion of �70% in 6 h.

The SEC chromatograms in Figure 3 clearlydepict both the increase in conversion (from thepeak area) and the corresponding shift of the poly-mer peaks towards higher molecular weight. Thetheoretical and measured molecular weights andthe PDI are shown as a function of conversion inFigure 4. The measured molecular weightincreased approximately linearly with conversionand the PDI remained low (\1.23) throughout thepolymerization, indicating that the polymeriza-tion proceeded with good control. The differencein hydrodynamic volume between PMAG andpolystyrene, for which the SEC is calibrated,accounts for the deviation between the measuredand theoretical molecular weights. Slight tailingon the low molecular weight side of the SEC chro-matograms indicates the production of a smallamount of dead polymer, but good control was stillmaintained up to high conversions.

The living behavior of a RAFT polymerizationcan be hampered by the presence of primaryamines or other basic functionalities. Amino-groups are known to cleave the thiocarbonyl func-tionality,65 which results in a loss of color andbroadening of the molecular weight distribution.A color change from pink to orange/pink wasobserved for several preliminary MAG polymer-izations, and was accompanied by relatively largePDIs ([1.3). Monomer purity was suspected as apotential cause of the apparent RAFT agent deg-radation, and accordingly a UV/Vis experimentwas conducted to verify the effect. One samplewas prepared containing 8.0 � 10�3 mol L�1

CPADB in DMAc/H2O (9:1 v/v); an identical

Figure 3. SEC chromatograms from the polymeriza-tion of MAG; conditions as per Figure 2.

Figure 2. Pseudo first order kinetic plot for thepolymerization of MAG at 60 �C; [MAG] ¼ 0.80 molL�1, [CPADB] ¼ 8.0 � 10�3 mol L�1, [AIBN] ¼ 4.0 �10�3 mol L�1 in DMAc/H2O (9:1 v/v).

Figure 4. Molecular weight and PDI versus timefor the polymerization of MAG; conditions as perFigure 2.



RAFT concentration and solvent mixture to thatof the MAG polymerization. A similar solutionalso containing 0.8 M of MAG, which had beenpurified by column chromatography but notrecrystallised served as the test system. The pHwas �6.5 in both systems. Each solution washeated to 70 �C up to 3.5 h monitoring the peakintensity at 515 nm. The peak at 515 nm corre-sponds to the HOMO to LUMO electronic transi-tion of the RAFT agent’s thiocarbonyl group, andwas expected to diminish in the event of hydroly-sis or aminolysis.66 Assuming that the net peakheight is approximately proportional to the RAFTagent concentration, the relative RAFT agent con-centration was plotted as a function of time forboth the control and test systems (Fig. 5). Clearly,RAFT agent degradation is more pronouncedwhen monomer is present, with the peak intensitydeclining 14% over 3.5 h compared with only 6%in the control system. The result was verifiedqualitatively, with an obvious color changeobserved in the monomer system compared withno noticeable color change in the control. We at-tribute the degradation to traces of unreacted glu-cosamine in the MAG, because the pure monomershould not be capable of producing such an effect;numerous glycomonomers have been polymerizedvia RAFT and no detrimental effects on the RAFTagent have been reported. We therefore recrystal-lised the monomer and achieved much more reli-able polymerization results without pronouncedchanges in color, highlighting the importance ofworking with high purity monomers, particularlywhen the monomer precursor is a primary amine.

Polymerization of 50-O-Methacryloyl Uridine andChain Extension of Poly(2-MethacrylamidoGlucopyranose) with 50-O-Methacryloyl Uridine

MAU was polymerized at 70 �C in DMSO using a[MAU]:[CPADB]:[AIBN] ratio of 1000:12:3 and amonomer concentration of 0.75 mol L�1. Both 1HNMR spectroscopy and SEC were used to calcu-late the conversion at each time point.

The pseudo first order kinetic plot (Fig. 6) forthe homopolymerisation of MAU is approximatelylinear, indicating that the radical flux remainedconstant throughout. In addition, it is clear thatthe SEC and NMR conversion measurements arein close agreement, again validating the use ofeither technique in calculating homopolymerisa-tion conversions. The polymerization exhibits aninhibition period of �65 min, a feature which hasbeen observed before in methacrylate systems.67

The theoretical molecular weights and themeasured molecular weights and PDIs from theMAU homopolymerisation (and the subsequentchain extension) are presented in Figure 7. Themolecular weight increases approximately line-arly with conversion and the PDI remains low(\1.28) throughout the polymerization. In allcases, the measured molecular weights usingpolystyrene standards are higher than the theo-retical values (bottom straight line in Fig. 7).It should, however, be pointed out that themolecular weight in the early stages of the

Figure 5. CPADB degradation at 70 �C both withand without monomer as measured by UV/Vis spec-trometry; [CPADB] ¼ 8.0 � 10�3 mol L�1 in DMAc/H2O (9:1 v/v), pH ¼ 6.5.

Figure 6. Pseudo first order plot for the homopoly-merisation of MAU using CPADB and the chainextension of a PMAG macroRAFT agent with MAU at70 �C; [MAU] ¼ 0.75 mol L�1, [CPADB] (homopoly-merisation) and [PMAG] (chain extension) ¼ 9.04 �10�3 mol L�1, [AIBN] ¼ 2.26 � 10�3 mol L�1 inDMSO.



polymerization is slightly higher than expectedeven taking into consideration that the hydrody-namic volume of the polymer does not match thehydrodynamic volume of the SEC’s calibrationstandard. In addition, the PDI reaches a peakvalue of 1.28 at 12% conversion then declines to amuch lower value of 1.20 at 73% conversion.Inspection of the SEC chromatograms (Fig. 8)reveals that the slightly elevated molecularweights and PDIs in the early stages are associ-ated with bimodal molecular weight distributions,which become unimodal as the conversionincreases. This observation can be attributed to‘‘hybrid behavior’’ between conventional and con-trolled polymerization.68 Such hybrid behavioroccurs if kb (the fragmentation rate coefficient ofthe RAFT-centered radicals in the pre-equilib-rium) is too low relative to the propagation ratecoefficient,69 resulting in a rapid increase in mo-lecular weight with conversion in the early stagesof the polymerization, primarily through a con-ventional free radical polymerization mechanism.The molecular weight distribution is broad or bi-modal under these conditions because only a frac-tion of the propagating radicals have undergonereversible chain transfer with a RAFT agent.However, once all the RAFT species have beenattacked by a radical and have fragmented torelease their R group, the main RAFT equilibriumpredominates. The fragmentation chain transferprocess becomes more efficient because the radi-cals on each side of the equilibrium have approxi-mately equal stability, promoting more uniformchain growth and consequently narrowing themolecular weight distribution.

Block copolymers were synthesized by thechain extension of PMAG with MAU. The reac-tion was carried out in DMSO at 70 �C using iden-tical concentrations to those chosen for the MAUhomopolymerisation.

The pseudo first order kinetic plot (Fig. 6)shows that the chain extension proceeds morerapidly than the homopolymerisation and doesnot display an inhibition period despite employingidentical concentrations and reaction conditions;the sole difference was the use of the PMAGmacroRAFT agent rather than CPADB. The ab-sence of inhibition indicates that the addition ofPMAU oligoradicals to the PMAG macroRAFTagent in the pre-equilibrium was followed rapidlyby forward fragmentation of the RAFT-centeredradicals and rapid re-initiation of the releasedPMAG macroradicals.70 That is, the relative sta-bility of each leaving group seems to favor frag-mentation to the PMAG macroradical (Scheme 3).Note that this rapid fragmentation does not implythat the addition of the propagating PMAU oligor-adicals to the PMAG macroRAFT agent wasrapid; in fact, the addition to RAFT agent was notas efficient as in the homopolymerisation as indi-cated by the presence of the macroRAFT agent inthe SEC curves until higher conversions wereattained (Fig. 9).

The pseudo first order kinetic plot is approxi-mately linear until high conversions, indicatingthat the radical flux remained constant duringthis period. The deviation from linearity beyond�75% conversion is likely to be a result of a morepronounced cage effect,71 in which the rising

Figure 7. Molecular weight evolution and PDI forthe homopolymerisation of MAU and chain extensionof PMAG with MAU; conditions as per Figure 6.

Figure 8. SEC chromatograms from the polymeriza-tion of MAU; conditions as per Figure 6.



viscosity suppressed initiation by hindering theseparation of the initiator-derived radicals.

The molecular weight distributions obtainedfrom the SEC chromatograms are shown in Fig-ure 9. The curves were height-normalized to moreclearly demonstrate the evolution of molecularweight with time. Successful chain extension isconfirmed by the progression of the polymer peakto larger molecular weights with increasing time.Similarly to the homopolymerisation of MAU, thechain extension exhibits hybrid behavior, withbimodal molecular weight distributions observedat lower conversions. However, the peaks becomenarrower and more symmetrical with increasingtime, indicating that effective control over the po-lymerization was achieved once the main RAFTequilibrium was established.

The theoretical molecular weights, and themeasured molecular weights and PDIs from theSEC chromatograms, are presented in Figure 7.The increase in molecular weight with conversionis approximately linear, which is consistent with awell controlled chain extension. The PDI alsoremains low throughout the polymerization, witha slight decline at higher conversions to a value of1.23 at 81% conversion.

Stability of Poly(50-O-Methacryloyl Uridine)

Before the self-assembly study in aqueous solu-tion, the stability of the polymers was tested inaqueous solution. Although the MAG with its am-ide group is expected to be very stable against hy-drolysis, the ester functionality of MAU was sus-pected to be prone to cleavage. Therefore, PMAUwas suspended in a range of aqueous solutions(note: PMAU is not soluble in water). The threewater and deuterium oxide solutions wereintended to provide information about the hydro-lysis mechanism72 whereas the PBS solutionswere used to simulate the pH conditions found in

biological systems. Samples were taken over 33days and the amount of hydrolyzed (‘‘free’’) uri-dine determined.

Figure 10 shows hydrolysis occurs more rapidlyin the PBS solutions with pH ¼ 7.4 and 7.6,respectively, confirming that the hydrolysis isbase-catalyzed. Upon ester hydrolysis, metha-crylic acid repeating units appear along the poly-mer chain resulting in a negatively charged poly-mer.

Of particular interest is the difference in hydro-lysis rate between D2O and H2O. To cleave theester, water must first diffuse into the lightlyswollen PMAU. Figure 10 shows that the acid cat-alyzed hydrolysis is almost independent of thedegree of deuteration of the solvent, confirmingthat the process is diffusion controlled, and there-fore not influenced by the kinetic isotope effect.

The outcome of this study has implications onthe preparation of micelles in solution. Dependingon the pH value, significant hydrolysis can occur.

Scheme 3. Block copolymer formation by the addition of PMAU macroradicals ontoPMAG macroRAFT agent.

Figure 9. Height-normalized SEC chromatogramsfor the chain extension of PMAG with MAU; condi-tions as per Figure 6.



Storage of aqueous solutions can, in the worstcase scenario, result in the complete degradationof the ester bonds to give free uridine and poly(methacrylic acid) and should therefore beavoided.

Self-Assembly of PMAG-b-PMAU

An investigation into the solution behavior of thePMAG-b-PMAU was commenced by dissolvingfour dry PMAG-b-PMAU samples of differenthydrophobic block lengths (PMAG59-b-PMAU36,PMAG59-b-PMAU55, PMAG59-b-PMAU67, andPMAG55-b-PMAU97) in DMAc and dialyzingagainst water (24 h, MW cut-off ¼ 3 400 g mol�1)to encourage the slow self-assembly of the amphi-philic polymer chains. The dialysed mixtureswere made up to 1 mg mL�1 with distilled waterand analyzed by DLS, TEM, and tensiometry.

Initial confirmation for the formation of core-shell structures was obtained using NMR studies.The intensity of the NMR signal of the block co-polymer in DMSO—which is a good solvent forboth blocks—represents actual molar ratios. Inwater, however, the signal intensity of uridine issignificantly suppressed indicative of a prolongedNMR relaxation time, which can be found in poly-mers with restricted mobility such as polymerforming the core of a micelle. (see Figure E, sup-plementary information)

Each sample’s particle size distribution wasobtained by automatically compiling the datafrom four DLS runs, each 2 min in duration. Fig-ure 11 shows a progressive increase in the particlesize with increasing hydrophobic block length, atrend which is mirrored by the TEM analyses

(Fig. 12). It is interesting to note that the increasein particle diameter as measured by DLS isaccompanied by an increasing tendency for theblock copolymers to form rod-like structures astheir hydrophobic block length increases. The sizeof the self-assembled sphere is known to bedependent on the length of both blocks. TheHalperin model predicts that the radius (r, innanometres) of a micelle will obey the empiricalrelationship r ¼ N0:6

A N0:26B , where NA and NB are

the number of repeat units in the hydrophilicblock and the hydrophobic block, respectively.73

However, this model applies only to star-likemicelles whose outer blocks are larger than theircore blocks; therefore it is more appropriate toexplain the different morphologies of our micellesin terms of chain stretching. With a decreasing ra-tio of hydrophilic block (here PMAG) to hydropho-bic block (PMAU), repulsion between the chainsof the outer block increases. To avoid furtherstretching of chains, the aggregation numberincreases resulting in larger micelles. Simultane-ously, the core of the micelle becomes larger, forc-ing more and more chains to stretch and take ona fully extended conformation. Entropically, thisis an unfavorable event. Consequently, in a sys-tem containing even longer hydrophobic chains,further stretching of the core-forming block isimpossible and the transition to rods occurs.74

Finally, the surface tension as a function ofblock copolymer concentration was measured bytensiometry, and showed no deviation from that ofpure water (72 mN m�1) over a concentrationrange of 2 � 10�4 mg mL�1 to 0.35 mg mL�1 forall four block copolymers. The introduction of a

Figure 11. Particle size distributions (DLS) of themicelles formed by self-assembly of PMAG-b-PMAU.

Figure 10. Uridine concentration versus time forthe hydrolysis of PMAU as measured by HPLC.



neutral amphiphilic block copolymer to water isgenerally accompanied by adsorption of moleculesto the air-water interface to form the Gibbs mono-layer. The excess of block copolymer molecules onthe surface compared with in the bulk solutionresults in a decrease in surface tension as thepolymer concentration increases, until the criticalmicelle concentration (cmc) is reached. At thispoint the surface is ‘‘saturated’’ and all polymermolecules added thereafter form micelles in thebulk solution and do not further reduce thesurface tension.

It has been recently reported that amphiphilicdiblock copolymers containing an ionic hydro-

philic block do not exhibit any significant surfaceactivity despite their ability to form micelles in so-lution.75–78 This interesting effect is attributed to‘‘image charge repulsion,’’ in which the polymerchains are forbidden to localize on the air-waterinterface due to an electrostatic repulsive forceonly effective at the surface.76 However, the imagecharge repulsion effect is not a satisfactory expla-nation for the lack of surface activity in our sys-tem because the hydrophilic block contains pend-ant glucose linked through highly stable amidebonds to the backbone, therefore there is nopossibility that this block is charged as a re-sult of hydrolytic degradation during sample

Figure 12. Transmission electron microscope images of micelles formed from: (a)PMAG59-b-PMAU36; (b) PMAG59-b-PMAU55; (c) PMAG59-b-PMAU67; (d) PMAG55-b-PMAU97.



preparation. We can therefore tentatively con-clude that either the block copolymer does notadsorb on the surface, or that adsorption doesoccur but not to the extent that the surface con-centration exceeds that of the bulk. These scenar-ios are both possible given that our block copoly-mer is not a strongly amphiphilic. 1H NMR of theblock copolymer shows that a significant decreasein the size of the uridine proton peaks (relative tothe glucose proton peaks) occurs when the solventis changed from pure d6-DMSO to D2O/d6-DMSO(v/v), confirming the formation of a core-shellstructure (see Figure E in supplementary infor-mation). The contrast between the hydrophilicityof the PMAG block and the hydrophobicity of thePMAU block is therefore sufficiently large to pro-mote self-assembly when a solvent for both blocksis replaced by water, but is apparently not signifi-cant enough to observe true surfactant behavior.

Thermal Properties of Polymer Blendswith Adenosine

The ability of the pendant uridine groups to bindwith adenosine was tested using DSC. Since uri-dine and adenosine start decomposing beyondtheir melting points, it was necessary to preparethe samples such that meaningful results couldbe obtained from the first heating curve. There-fore, each sample was prepared from a DMF solu-tion, which was poured into a measuring pan.Evaporation of the solvent formed a thin polymerfilm whose uniform thickness ensured constantheat transfer through the sample. The results aresummarized in Table 1. The melting point ofadenosine decreased from 234 �C to 211 �C whenmixed with a stochiometric amount of uridine,which is approximately midway between themelting points of uridine (170 �C) and adenosine.The H-bonding of this base pair lead to the forma-

tion of one single melting point as expected forsuch a strong interaction (Scheme 4).

In a subsequent step, PMAU was mixed withadenosine to again obtain equimolar quanitities ofuridine and adenosine units. The glass transitiontemperature (Tg) of PMAU alone was found to be151 �C, whereas the adenosine-loaded polymerhas a Tg of 136 �C. Adenosine dissolved in thepolymer obviously acts as a plasticizer. Of impor-tance, however, is the complete disappearance ofthe melting point of adenosine. Adenosine istherefore fully miscible in the polymer matrix.

As a control experiment, adenosine was mixedwith the glycopolymer PMAG to produce a mix-ture in which base pairing is not possible. The Tg

of PMAG shifted from 100 �C to 81 �C with the

Table 1. Glass Transition Temperatures Tg and Melting Points Tm Measured Using DSC (20 K min�1)Including the Amount of Adenosine (A) and Polymer (PMAU, PMAG) or Uridine (U) Usedfor the Sample Preparation

Sample mSample (mg) madenosine (mg) Tg (�C) Dcp (J g�1 K�1) Tm (�C) DH (J g�1)

A – 5 – – 234 244U-A 2.38 2.62 – – 211 63U 5 – – – 170 126PMAU 5 – 151 0.84 – –PMAU-A 2.7 2.3 136 0.54 – –PMAG 5 – 100 0.062 – –PMAG-A 2.39 2.61 81 0.055 176 190

Scheme 4. H-bonding between adenosine andPMAU.



addition of adenosine. Again, adenosine acts asplasticizer, increasing the free volume andthereby decreasing the Tg. However, in contrastto the PMAU-adenosine mixture, the meltingpoint of adenosine was still clearly visible, whichindicates the presence of adenosine crystals in thepolymer matrix.

The strong interaction between PMAU andadenosine is a very promising indication of thepotential of this block copolymer system forencapsulating therapeutic agents capable ofstrong hydrogen bonding such as short ASONs.

CONCLUSIONS

The two monomers 2-methacrylamido glucopyra-nose (MAG) and 50-O-methacryloyl uridine (MAU)were polymerized in a controlled fashion using theRAFT technique. A bimodal molecular weight pro-file at low conversions was an interesting feature oftheMAU polymerization, which can be attributed to‘‘hybrid behavior’’ between controlled and conven-tional free radical polymerization. Chain extensionof a PMAGmacroRAFTagent withMAU also exhib-ited this hybrid behavior but was well controlledonce the main RAFT equilibrium was fully estab-lished, and allowed the generation of a series ofblock copolymers with varying hydrophobic (PMAU)block lengths and low polydispersities. The self-assembly of four such block copolymers showed astructural transition from micelles when the hydro-phobic block was relatively short to rods as thehydrophobic block became longer. Interestingly, de-spite this ability to form well-defined structures insolution, none of the four samples showed any sur-face activity, which we believe is a result of the blockcopolymer’s relatively weak amphiphilic character.

Our block copolymer, with a glycopolymer shelland a polynucleoside core, was designed with theultimate aim of developing a new carrier for shortASONs, which uses base pairing rather than elec-trostatic interactions to bind the gene in the core.The preliminary investigation of the polyuridine’sability to bind free adenosine (uridine’s comple-mentary base in RNA) was highly promising,with strong interaction between the two speciesobserved by DSC. Considering that recent reportssuggest cellular uptake of rods is superior to thatof spherical micelles, we foresee great potentialfor this system to fulfil its intended aim, and con-sequently its capability for encapsulating ASONsthrough base pairing is the focus of currentexperiments.

REFERENCES AND NOTES

1. Noureddini, S. C.; Krendelshchikov, A.; Simo-nenko, V.; Hedley, S. J.; Douglas, J. T.; Curiel, D.T.; Korokhov, N. Virus Res 2006, 116, 185–195.

2. Boyd, B. Expert Opin Drug Deliv 2008, 5, 69–85.3. Katizawa, Y.; Kataoka, K. Adv Drug Deliv Rev

2002, 54, 203–222.4. Mahmud, A.; Xiong, X.-B.; Aliabadi, H. M.; Lava-

sanifar, A. J Drug Target 2007, 15, 553–584.5. Tong, R.; Cheng, J. Polymer Rev 2007, 47, 345–

381.6. Geng, Y.; Dalhaimer, P.; Cai, S.; Tsai, R.; Tewari,

M.; Minko, T.; Discher, D. E. Nat Nano 2007, 2,249–255.

7. Nah, J.-W.; Yu, L.; Han, S.-O.; Ahn, C.-H.; Kim,S. W. J Control Release 2002, 78, 273–284.

8. Merdan, T.; Callahan, J.; Peterson, H.; Kunath,K.; Bakowsky, U.; Kopeckova, P. Bioconjug Chem2003, 14, 989–996.

9. Wakebayashi, D.; Nishiyama, N.; Yamasaki, Y.;Ikata, K.; Kanayama, N.; Harada, A. J ControllRelease 2004, 95, 653–664.

10. Miura, Y. J Polym Sci Part A: Polymer Chemistry2007, 45, 5031–5036.

11. Hashida, M.; Takemura, S.; Nishikawa, M.; Taka-kura, Y. J Control Release 1998, 53, 301.

12. Okada, M. Prog Polym Sci 2001, 26, 67–104.13. Thoma, G.; Patton, J. T.; Magnani, J. L.; Ernst,

B.; Ohrlein, R.; Duthaler, R. O. J Am Chem Soc1999, 121, 5919–5929.

14. Spain, S. G.; Gibson, M. I.; Cameron, N. R. JPolym Sci Part A: Polymer Chemistry 2007, 45,2059–2072.

15. Dupayage, L.; Save, M.; Dellacherie, E.; Nouvel,C.; Six, J.-L. J Polym Sci Part A: Polymer Chem-istry 2008, 46, 7606–7620.

16. Loykulnant, S.; Hirao, A. Macromolecules 2000,33, 4757–4764.

17. Bernard, J.; Schappacher, M.; Deffieux, A.; Viville,P.; Lazzaroni, R.; Charles, M. H.; Charreyre, M.T.; Delair, T. Bioconjug Chem 2006, 17, 6–14.

18. Aoi, K.; Tsutsumiuchi, K.; Okada, M. Macromole-cules 1994, 27, 875–877.

19. Tsutsumiuchi, K.; Aoi, K.; Okada, M. Macromole-cules 1997, 30, 4013–4017.

20. Fraser, C.; Grubbs, R. H. Macromolecules 1995,28, 7248.

21. Mortell, K. H.; Gingras, M.; Kiessling, L. L. J AmChem Soc 1994, 116, 12053.

22. Ladmiral, V.; Mantovani, G.; Clarkson, G. J.;Cauet, S.; Irwin, J. L.; Haddleton, D. M. J AmChem Soc 2006, 128, 4823–4830.

23. Ting, S. R. S.; Granville, A. M.; Quemener, D.;Davis, T. P.; Stenzel, M. H.; Barner-Kowollik, C.Aust J Chem 2007, 60, 405–409.

24. Grande, D.; Baskaran, S.; Baskaran, C.; Gnanou,Y.; Chaikof, E. L. Macromolecules 2000, 33, 1123–1125.



25. Hou, S.; Sun, X. L.; Dong, C. M.; Chaikof, E. L.Bioconjug Chem 2004, 15, 954–959.

26. Sun, X.-L.; Faucher, K. M.; Houston, M.; Grande,D.; Chaikof, E. L. J Am Chem Soc 2002, 124,7258–7259.

27. Matyjaszewski, K.; Xia, J. Chem Rev 2001, 101,2921–2990.

28. Narain, R.; Armes, S. P. Macromolecules 2003, 36,4675–4678.

29. Narain, R.; Armes, S. P. Biomacromolecules 2003,4, 1746–1758.

30. Hawker, C. J.; Bosman, A. W.; Harth, E. ChemRev 2001, 101, 3661–3688.

31. Barner, L.; Davis, T. P.; Stenzel, M. H.; Barner-Kowollik, C. Macromol Rapid Commun 2007, 28,539–559.

32. Barner-Kowollik, C.; Davis, T. P.; Heuts, J. P. A.;Stenzel, M. H.; Vana, P.; Whittaker, M. J PolymSci Part A: Polymer Chemistry 2003, 41, 365–375.

33. Favier, A.; Charreyre, M.-T. Macromol RapidCommun 2006, 27, 653–692.

34. Moad, G.; Rizzardo, E.; Thang, S. H. Aust J Chem2005, 58, 379–410.

35. Perrier, S.; Takolpuckdee, P. J Polym Sci Part A:Polymer Chemistry 2005, 43, 5347–5393.

36. Lowe, A. B.; Sumerlin, B. S.; McCormick, C. L.Polymer 2003, 44, 6761–6765.

37. Albertin, L.; Cameron, N. R. Macromolecules2007, 40, 6082–6093.

38. Albertin, L.; Kohlert, C.; Stenzel, M.; Foster, J.R.; Davis, T. P. Biomacromolecules 2004, 5, 255–260.

39. Albertin, L.; Stenzel, M.; Barner-Kowollik, C.;Foster, L. J. R.; Davis, T. P. Macromolecules 2004,37, 7530–7537.

40. Albertin, L.; Stenzel, M. H.; Barner-Kowollik, C.;Foster, L. J. R.; Davis, T. P. Macromolecules 2005,38, 9075–9084.

41. Guo, T.-Y.; Liu, P.; Zhu, J.-W.; Song, M.-D.; Zhang,B.-H. Biomacromolecules 2006, 7, 1196–1202.

42. Spain, S. G.; Albertin, L.; Cameron, N. R. ChemCommun 2006, 40, 4198–4200.

43. Al-Bagoury, M.; Buchholz, K.; Yaacoub, E.-J. Poly-mer Adv Tech 2007, 18, 313–322.

44. Bernard, J.; Favier, A.; Zhang, L.; Nilasaroya, A.;Davis, T. P.; Barner-Kowollik, C.; Stenzel, M. H.Macromolecules 2005, 38, 5475–5484.

45. Zhang, L.; Bernard, J.; Davis, T. P.; Barner-Kowollik, C.; Stenzel, M. H. Macromol RapidCommun 2008, 29, 123–129.

46. Binder, W. H.; Kunz, M. J.; Kluger, C.; Hayn, G.;Saf, R. Macromolecules 2004, 37, 1749–1759.

47. Celiz, A. D.; Scherman, O. A. Macromolecules2008, 41, 4115–4119.

48. Feldman, K. E.; Kade, M. J.; De Greef, T. F. A.;Meijer, E. W.; Kramer, E. J.; Hawker, C. J. Macro-molecules 2008, 41, 4694–4700.

49. Yang, X.; Hua, F.; Yamato, K.; Ruckenstein, E.;Gong, B.; Kim, W.; Ryu, C. Y. Angew Chem IntEd 2004, 43, 6471–6474.

50. Katizawa, Y.; Harada, A.; Kataoka, K. Biomacro-molecules 2001, 2, 491–497.

51. Peterson, H.; Fechner, A. L.; Martin, A. L.;Kunath, K.; Stolnik, S.; Roberts, C. J.; Fischer,D.; Davies, M. C.; Kissel, T. Bioconjug Chem2002, 13, 845–854.

52. Zhang, L.; Nguyen, T. L. U.; Bernard, J.; Davis,T. P.; Barner-Kowollik, C.; Stenzel, M. H. Bioma-cromolecules 2007, 8, 2890–2901.

53. Mao, Z.; Ma, L.; Yan, J.; Yan, M.; Gao, C.; Shen,J. Biomaterials 2007, 28, 4488–4500.

54. Lv, H.; Zhang, S.; Wang, B.; Cui, S.; Yan, J.J Control Release 2006, 114, 100–109.

55. Fan, H.; Kintagawa, M.; Raku, T.; Tokiwa, Y.Biochem Eng J 2004, 21, 279–283.

56. Granville, A. M.; Quemener, D.; Davis, T. P.;Barner-Kowollik, C.; Stenzel, M. H. MacromolSymp 2007, 255, 81–89.

57. Ambrosi, M.; Batsanov, A. S.; Cameron, N. R.;Davis, B. G.; Howard, J. A. K.; Hunter, R. JChem Soc [Perkin 1] 2002, 1, 45–52.

58. Kitazawa, S.; Okumura, M.; Sakakibara, K. ChemLett 1990, 1733–1736.

59. Mitsukami, Y.; Donovan, M. S.; Lowe, A. B.;McCormick, C. L. Macromolecules 2001, 34,2248–2256.

60. Oae, S.; Yagihara, T.; Okabe, T. Tetrahedron 1972,28, 3203–3216.

61. Mori, S.; Barth, H. G. Size Exclusion Chromatog-raphy; Springer: New York, 1999.

62. Bernard, J.; Hao, X.; Davis, T. P.; Barner-Kowol-lik, C.; Stenzel, M. H. Biomacromolecules 2006, 7,232–238.

63. Klein, J.; Herzog, D.; Hajibegli, A. MacromolChem Rapid Commun 1985, 6, 675–678.

64. Cao, L.; Bornscheuer, U. T.; Schmid, R. D. J MolCatal B: Enzym 1999, 6, 279–285.

65. Thomas, D. B.; Convertine, A. J.; Hester, R. D.;Lowe, A. B.; McCormick, C. L. Macromolecules2004, 37, 1735–1741.

66. Li, M.; De, P.; Gondi, S. R.; Sumerlin, B. S. JPolym Sci Part A: Polymer Chemistry 2008, 46,5093–5100.

67. Saricilar, S.; Knott, R.; Barner-Kowollik, C.;Davis, T. P.; Heuts, J. P. A. Polymer 2003, 44,5169–5176.

68. Theis, A.; Feldermann, A.; Charton, N.; Stenzel,M. H.; Davis, T. P.; Barner-Kowollik, C. Macromo-lecules 2005, 38, 2595–2605.

69. Barner-Kowollik, C.; Quinn, J. F.; Nguyen, T. L.U.; Heuts, J. P. A.; Davis, T. P. Macromolecules2001, 34, 7849–7857.

70. Perrier, S.; Barner-Kowollik, C.; Quinn, J. F.;Vana, P.; Davis, T. P. Macromolecules 2002, 35,8300–8306.



71. Charton, N.; Feldermann, A.; Theis, A.; Stenzel, M.H.; Davis, T. P.; Barner-Kowollik, C. J Polym SciPart A: Polymer Chemistry 2004, 42, 5170–5179.

72. Hurrell, S.; Milroy, G. E.; Cameron, R. E. Polymer2003, 44, 1421–1424.

73. Halperin, A. Macromolecules 1987, 20, 2943–2946.

74. Cameron, N. S.; Corbierre, M. K.; Eisenberg, A.Can J Chem 1999, 77, 1311–1326.

75. Iddon, P. D.; Robinson, K. L.; Armes, S. P. Poly-mer 2004, 45, 759–768.

76. Kaewsaiha, P.; Matsumoto, K.; Matsuoka, H.Langmuir 2005, 21, 9938–9945.

77. Matsuoka, H.; Maeda, S. I.; Kaewsaiha, P.;Matsumoto, K. Langmuir 2004, 20, 7412–7421.

78. Matsuoka, H.; Matsutani, M.; Mouri, E.;Matsumoto, K. Macromolecules 2003, 36, 5321–5330.



Core-shell particles with glycopolymer shell and polynucleoside core via RAFT: From micelles to rods

Documents

Transcript of Core-shell particles with glycopolymer shell and polynucleoside core via RAFT: From micelles to rods