Stimuli-Responsive Hybrid Nanoparticles for Controlled Chemical DeliveryCo-Investigators: Hamid Ghandehari1,2*, Philip DeShong1,3, Douglas English1,3, Michael R. Zachariah1,3,4

1Center for Nanomedicine & Cellular Delivery, 2Dept of Pharmaceutical Sciences, University of Maryland Baltimore; 3Dept of Chemistry & Biochemistry, 4Dept of Mechanical Engineering, University of Maryland College Park

*Present Address: Departments of Pharmaceutics & Pharmaceutical Chemistry and Bioengineering, University of Utah

University of Maryland Baltimore

Hamid Ghandehari (PI)

Anjan Nan, Res. Asst Professor

Vladimir Seregin, Post Doc

Mathew Dowling, Grad Student

Jake Mitchell, NSF REU Summer Fellow

For more information contact:

hamid.ghandehari@pharm.utah.edu

University of Maryland College Park

Philip DeShong (PI)

Michael Zachariah (PI)

Douglas English (PI)

Daniel C. Stein, Professor

Chip Luckett, Grad Student

Sara Lioi, Grad Student

Xiang Wang, Grad Student

Juhee Park, Grad Student

ACKNOWLEDGEMENTS

National Science Foundation Grant 0608906Active Nanostructures and Nanosystems

Nanoscience Interdisciplinary Research Team

The overall objective of this interdisciplinary research is to construct hybrid stimuli-responsive nanoparticles for controlled delivery of bioactive agents. The focus is on the fabrication of hybrid inorganic porous nanoparticles with “on-off” pore caps on their surface made of thermal and pH - responsive recombinant polymers1. These hybrid nanostructures provide the benefit of robust inorganic cores (gold, silica or iron-oxide) on one hand where their size and porosity do not change in biological environments, and the flexible surface grafted polymers on the other allowing controlled release of bioactive agents (Fig 1). The educational component of this project will facilitate training of new generations of students and scientists able to work at the interface of mechanical engineering, chemistry, material science and pharmaceutics designing novel nanoconstructs for use in biomedical applications.

INTRODUCTION

offoff

offon

onon

Blood stream(pH 7.4)

Tumor extracellular space

(pH 6.0-6.8)

Tumor intracellular space (endosomes)

(pH 5.0-6.0)

Non pH sensitive soluble polymer bearing targeting moiety

pH sensitive polymer which is collapsed (insoluble) at body temperature and neutral pH of the blood stream

Targetable receptor on tumor cell surface

Tumor cell

Porous nanoparticle loaded with biomolecule and bearing targeting moiety and pH sensitive polymers. In the neutral pH of blood stream the pH sensitive polymers are collapsed on the pores of the particles forming closed trap doors (off).

Nanoparticles are recognized by receptors on tumor cell surface. In the tumor extracellular space the reduced pH causes phase transition of pH sensitive polymers leading to solubilization and partial opening of trap doors (on). As a result there is controlled diffusion of biomolecules at the tumor site.

Nanoparticles are internalized and in the intracellular endosomal compartment reduction of pH leads to complete solubilization of polymer and opening of all trap doors. As a result additional controlled diffusion of biomolecules occurs.

offoff

offon

offon

onon onon

Blood stream(pH 7.4)

Tumor extracellular space

(pH 6.0-6.8)

Tumor intracellular space (endosomes)

(pH 5.0-6.0)

Non pH sensitive soluble polymer bearing targeting moiety

pH sensitive polymer which is collapsed (insoluble) at body temperature and neutral pH of the blood stream

Targetable receptor on tumor cell surface

Tumor cell

Porous nanoparticle loaded with biomolecule and bearing targeting moiety and pH sensitive polymers. In the neutral pH of blood stream the pH sensitive polymers are collapsed on the pores of the particles forming closed trap doors (off).

Nanoparticles are recognized by receptors on tumor cell surface. In the tumor extracellular space the reduced pH causes phase transition of pH sensitive polymers leading to solubilization and partial opening of trap doors (on). As a result there is controlled diffusion of biomolecules at the tumor site.

Nanoparticles are internalized and in the intracellular endosomal compartment reduction of pH leads to complete solubilization of polymer and opening of all trap doors. As a result additional controlled diffusion of biomolecules occurs.

Fig 1. Typical rationale of active nanoparticles for controlled chemical delivery

CORE NANOPARTICLE SYNTHESIS AND CHARACTERIZATION

Fig 3. Transmission electron microscopy images of (A) Porous and (B) Hollow silica nanoparticles. (C) Confocal microscopy image of hollow silica nanoparticles filled with a model compound Doxorubicin

(A) (B) (C)

Solvent+ A + B+ C

A=TEOSB=SurfactantC= Salt filler

Removefiller by calcining and

washing

Porous particle

Controlled evaporation

Controlled evaporation

Create microstructure

HEAT

Lock Microstructure

Form SilicaSolvent+ A + B+ C

A=TEOSB=SurfactantC= Salt filler

Removefiller by calcining and

washing

Porous particle

Controlled evaporation

Controlled evaporation

Create microstructure

HEAT

Lock Microstructure

Form Silica

Fig 2. Fabrication of porous nanoparticles by controlled chemical evaporation2. The evaporation process results in increased concentration of reactants. Precipitation is basically a collision (coagulation process) which varies as the viscosity of the solvent increases due to solute precipitation.

EXPERIMENTS & RESULTS

SURFACE MODIFICATION OF NANOPARTICLES

Fig 5. Strategy for derivatization of nanoparticle surfaces using glucose as a model. This strategy will be similarly adapted to attach the stimuli sensitive polymers via the terminal lysine group to form a peptide (CONH) linkage. The other end of the chelator will permit modification of silica surfaces via the siloxane group (2) or gold surfaces via disulfide group (3). (inset) Example of a gold nanoparticle conjugated to a model peptide arginine-glycine-aspartic acid (RGD).

O

OAc

AcOAcO

AcON3

1) PPh3, reflux

2)

N S

O

1) Me3P, rt

O

OH

HOHO

HN

O

HO

O

OH

HOHO

HO HN

O

N S

O2)

3) NaOMe

3) NaOMe 12

Rh (I) catalyst

O

OH

HOHO

HN

O

HO

SiR 2(OEt)

3

H SiR

R

OEt

S S SS

• Recombinant polymer synthesis

Linear stimuli sensitive elastin like polymers (ELPs)3

[(GVGVP)m–(GXGVP)n]Z X = His

Factors which influence pH / temperature-sensitive phase transition of ELPs:

- Length of elastin units (m)

- Polymer molecular weight (z)

- Presence of ionizable groups (n)

Histidine (pKa 6.0) is introduced into the polymer sequence to lower the pKa of the ELPs and shift their phase transition to lower pH values.

Fig 4. Synthesis of comonomers and polymerization of stimuli sensitive elastin based polymers.

• Chemical polymer synthesis

Elastin-based side-chain polymers (EBPs)

OO

C

NHO

Val

CH3

Pro

Gly

Val

Gly

COOH

n

OO

C

NHO

Val

CH3

Pro

Gly

His

Gly

COOH

m

Boc Pro COOH + HCl*H2N Gly OEt

Boc Pro Gly OEt

DIPEA, BOP / EtOAc

HCl / MeOH

HCl*H2N Pro Gly OEt

+ Boc-Val-COOH, DIPEA, BOP / EtOAc

Boc Val Pro Gly OEt

NaOH, H2O / dioxane

Boc Val Pro Gly COOH

Boc Pro COOH + HCl*H2N Gly OEtBoc Pro COOH + HCl*H2N Gly OEt

Boc Pro Gly OEt

DIPEA, BOP / EtOAc

HCl / MeOH

HCl*H2N Pro Gly OEt

+ Boc-Val-COOH, DIPEA, BOP / EtOAc

Boc Val Pro Gly OEt

NaOH, H2O / dioxane

Boc Val Pro Gly COOH

Boc Val COOH + HCl*H2N Gly OEt

Boc Val Gly OEt

DIPEA, BOP / EtOAc

HCl / MeOH

HCl*H2N Val Gly OEt

Boc Val COOH + HCl*H2N Gly OEtBoc Val COOH + HCl*H2N Gly OEt

Boc Val Gly OEt

DIPEA, BOP / EtOAc

HCl / MeOH

HCl*H2N Val Gly OEt

Boc His COOH + HCl*H2N Gly OEt

Boc His Gly OEt

DIPEA, BOP / EtOAc, MeOH

HCl / MeOH

HCl*H2N His Gly OEt

Boc His COOH + HCl*H2N Gly OEtBoc His COOH + HCl*H2N Gly OEt

Boc His Gly OEt

DIPEA, BOP / EtOAc, MeOH

HCl / MeOH

HCl*H2N His Gly OEt

Boc-VPG-COOH + HCl*H2NXGOEt + DIPEA, BOP / EtOAc Boc-VPGXG-COOH H2NVPGXGCOOH X = Val, His

H2NVPGXGCOOH + 2-isocyanatoethyl methacrylate methacrylate-functionalized VPGXG [ MA-VPGXG] X = Val, His

mMAVPGVG + nMAVPGHG + EBIB, CuCl, bipy / DMSO –atom transfer radical polymerization Poly[(MA-VPGVG)m(MA-VPGHG)n]

Synthetic polymers with pendant VPGVG peptide sequences are readily accessible ELP analogues4.

Stimuli sensitivity of EBP polymers is affected by the same parameters as linear ELPs.

EBPs are more responsive to pH changes than ELPs due the large number of pendant carboxylic acid groups of the terminal glycines.

HN

NH

HN

NH

HN

O

O

O

O

HN

H2N NH

N

O

OH

OHO

O

OH

O

S S

4.5 nm

SYNTHESIS OF STIMULI-SENSITIVE POLYMERS BIOLOGICAL EVALUATION OF NANOPARTICLE CONJUGATES

CELLULAR TARGETING

Fig 8. Binding of lactose functionalized gold nanoparticles to cell surface lactose receptors of Neisseria gonorrhoeae, a biological pathogen, leads to dramatically enhanced luminescence5. Fluorescence images under broadband UV irradiation with red and green filters. Lane 1: autofluorescence of cells with no additives. Lane 2: cells containing citrate coated 3 nm gold nanoparticles. Lane 3: cells containing glucose coated gold nanoparticles. Lane 4: cells containing lactose coated gold nanoparticles.

BIOCOMPATIBILITY

Fig 6. Growth inhibition assay of gold nanoparticle-RGD peptide on model endothelial cells demonstrating biocompatibility of nanoparticle conjugates.

IN VITRO BIOMOLECULE RELEASE

Fig 7. Doxorubicin release from porous model alumina particles (pore diameter ~11 nm and particle diameter < 20 m) at 37 °C (red) and 20 °C (black). A significant increase in both the rate and amount of release is observed at 37 °C. These results show that biomolecule release is a thermally activated process with a significant entropic component.

20

15

10

5

0

% R

ele

ase

16001400120010008006004002000Time (min)Time (s)

REFERENCES1. Dandu,R, & Ghandehari,H, Progress in Polymer Science 32, 1008 (2007)2. Kim,SH, Liu,BYH, & Zachariah,MR, Chem Mater 14, 2899 (2002)3. Urry,DW et al., in Controlled drug delivery: challenges and strategies. ed. Park,K.

405-437, American Chemical Society, Washington, D.C. (1997)4. Ferna´ndez-Trillo,F et al., Macromolecules, 40, 6094 (2007)5. DeShong,P et al., U.S.Patent LS-2004-052 (2004)