stimuli-responsive hybrid nanoparticles for controlled chemical delivery co-investigators: hamid...
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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:
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)