Stimuli-Responsive Hybrid Nanoparticles for Controlled Chemical Delivery Co-Investigators: Hamid Ghandehari 1,2*, Philip DeShong 1,3, Douglas English 1,3, Michael R. Zachariah 1,3,4 1 Center for Nanomedicine & Cellular Delivery, 2 Dept of Pharmaceutical Sciences, University of Maryland Baltimore; 3 Dept of Chemistry & Biochemistry, 4 Dept 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 Active 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 polymers 1. 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 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) Fig 2. Fabrication of porous nanoparticles by controlled chemical evaporation 2. 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). 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) Boc-VPG-COOH + HCl*H 2 N  XG  OEt + DIPEA, BOP / EtOAc  Boc-VPGXG-COOH  H 2 N  VPGXG  COOH X = Val, His H 2 N  VPGXG  COOH + 2-isocyanatoethyl methacrylate  methacrylate-functionalized VPGXG [ MA-VPGXG] X = Val, His m  MA  VPGVG + n  MA  VPGHG + 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 analogues 4.  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. SYNTHESIS OF STIMULI-SENSITIVE POLYMERSBIOLOGICAL 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 luminescence 5. 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. Time (s) REFERENCES 1.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 , 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)