Mukesh K. Gupta

Current Progress in Reactive Oxygen Species (ROS)-Responsive Materials for Biomedical Applications

Recently, significant progress has been made in developing “stimuli-sensitive” biomaterials as a new therapeutic approach to interact with dynamic physiological conditions. Reactive oxygen species (ROS) production has been implicated in important pathophysiological events, such as atherosclerosis,aging, and cancer. ROS are often overproduced locally in diseased cells and tissues, and they individually and synchronously contribute to many of the abnormalities associated with local pathogenesis. Therefore, the advantages of developing ROS-responsive materials extend beyond site-specific targeting of therapeutic delivery, and potentially include navigating,sensing, and repairing the cellular damages via programmed changes in material properties. Here we review the mechanism and development of biomaterials with ROS-induced solubility switch or degradation, as well as their performance and potential for future biomedical applications.

Poly(PS-b-DMA) micelles for reactive oxygen species triggered drug release

A new micelle drug carrier that consists of a diblock polymer of propylene sulfide (PS) and N,N-dimethylacrylamide (poly(PS₇₄-b-DMA₃₁₀)) has been synthesized and characterized for site-specific release of hydrophobic drugs to sites of inflammation. Propylene sulfide was first polymerized using a thioacyl group transfer (TAGT) method with the RAFT chain transfer agent (CTA) 4-cyano-4-(ethylsulfanylthiocarbonylsulfanyl) pentanoic acid (CEP), and the resultant poly(PS₇₄-CEP) macro-CTA was used to polymerize a second polymer block of DMA using reversible addition-fragmentation chain transfer (RAFT). The formation of the poly(PS₇₄-b-DMA₃₁₀) diblock polymer was confirmed by ¹H NMR spectra and gel permeation chromatography (GPC). Poly(PS₇₄-b-DMA₃₁₀) formed 100 nm micelles in aqueous media as confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Micelles loaded with the model drugs Nile red and DiO were used to demonstrate the ROS-dependent drug release mechanism of these micelles following treatment with hydrogen peroxide (H₂O₂), 3-morpholinosydnonimine (SIN-1), and peroxynitrite. These oxidants were found to oxidize the micelle PPS core, making it more hydrophilic and triggering micelle disassembly and cargo release. Delivery of poly(PS₇₄-b-DMA₃₁₀) micelles dual-loaded with the Förster Resonance Energy Transfer (FRET) fluorophore pair DiI and DiO was used to prove that endogenous oxidants generated by lipopolysaccharide (LPS)-treated RAW 264.7 macrophages significantly increased release of nanocarrier contents relative to macrophages that were not activated. In vitro studies also demonstrated that the poly(PS₇₄-b-DMA₃₁₀) micelles were cytocompatible across a broad range of concentrations. These combined data suggest that the poly(PS₇₄-b-DMA₃₁₀) micelles synthesized using a combination of TAGT and RAFT have significant potential for site-specific drug delivery to tissues with high levels of oxidative stress.

Tunable delivery of siRNA from a biodegradable scaffold to promote angiogenesis in vivo.

A system has been engineered for temporally controlled delivery of siRNA from biodegradable tissue regenerative scaffolds. Therapeutic application of this approach to silence prolyl hydroxylase domain 2 promoted expression of pro-angiogenic genes controlled by HIF1α and enhanced scaffold vascularization in vivo. This technology provides a new standard for efficient and controllable gene silencing to modulate host response within regenerative biomaterials.

Sustained local delivery of siRNA from an injectable scaffold

Controlled gene silencing technologies have significant, unrealized potential for use in tissue regeneration applications. The design described herein provides a means to package and protect siRNA within pH-responsive, endosomolytic micellar nanoparticles (si-NPs) that can be incorporated into nontoxic, biodegradable, and injectable polyurethane (PUR) tissue scaffolds. The si-NPs were homogeneously incorporated throughout the porous PUR scaffolds, and they were shown to be released via a diffusion-based mechanism for over three weeks. The siRNA-loaded micelles were larger but retained nanoparticulate morphology of approximately 100 nm diameter following incorporation into and release from the scaffolds. PUR scaffold releasate collected in vitro in PBS at 37 °C for 1-4 days was able to achieve dose-dependent siRNA-mediated silencing with approximately 50% silencing achieved of the model gene GAPDH in NIH3T3 mouse fibroblasts. This promising platform technology provides both a research tool capable of probing the effects of local gene silencing and a potentially high-impact therapeutic approach for sustained, local silencing of deleterious genes within tissue defects.

Tuning PEGylation of mixed micelles to overcome intracellular and systemic siRNA delivery barriers.

A series of endosomolytic mixed micelles was synthesized from two diblock polymers, poly[ethylene glycol-b-(dimethylaminoethyl methacrylate-co-propylacrylic acid-co-butyl methacrylate)] (PEG-b-pDPB) and poly[dimethylaminoethyl methacrylate-b-(dimethylaminoethyl methacrylate-co-propylacrylic acid-co-butyl methacrylate)] (pD-b-pDPB), and used to determine the impact of both surface PEG density and PEG molecular weight on overcoming both intracellular and systemic siRNA delivery barriers. As expected, the percent PEG composition and PEG molecular weight in the corona had an inverse relationship with mixed micelle zeta potential and rate of cellular internalization. Although mixed micelles were internalized more slowly, they generally produced similar gene silencing bioactivity (∼ 80% or greater) in MDA-MB-231 breast cancer cells as the micelles containing no PEG (100 D/no PEG). The mechanistic explanation for the potent bioactivity of the promising 50 mol% PEG-b-DPB/50 mol% pD-b-pDPB (50 D) mixed micelle formulation, despite its relatively low rate of cellular internalization, was further investigated as a function of PEG molecular weight (5 k, 10 k, or 20 k PEG). Results indicated that, although larger molecular weight PEG decreased cellular internalization, it improved cytoplasmic bioavailability due to increased intracellular unpackaging (quantitatively measured via FRET) and endosomal release. When delivered intravenously in vivo, 50 D mixed micelles with a larger molecular weight PEG in the corona also demonstrated significantly improved blood circulation half-life (17.8 min for 20 k PEG micelles vs. 4.6 min for 5 kDa PEG micelles) and a 4-fold decrease in lung accumulation. These studies provide new mechanistic insights into the functional effects of mixed micelle-based approaches to nanocarrier surface PEGylation. Furthermore, the ideal mixed micelle formulation identified (50 D/20 k PEG) demonstrated desirable intracellular and systemic pharmacokinetics and thus has strong potential for in vivo therapeutic use.

Combinatorial Polymer Electrospun Matrices Promote Physiologically-Relevant Cardiomyogenic Stem Cell Differentiation

Myocardial infarction results in extensive cardiomyocyte death which can lead to fatal arrhythmias or congestive heart failure. Delivery of stem cells to repopulate damaged cardiac tissue may be an attractive and innovative solution for repairing the damaged heart. Instructive polymer scaffolds with a wide range of properties have been used extensively to direct the differentiation of stem cells. In this study, we have optimized the chemical and mechanical properties of an electrospun polymer mesh for directed differentiation of embryonic stem cells (ESCs) towards a cardiomyogenic lineage. A combinatorial polymer library was prepared by copolymerizing three distinct subunits at varying molar ratios to tune the physicochemical properties of the resulting polymer: hydrophilic polyethylene glycol (PEG), hydrophobic poly(ε-caprolactone) (PCL), and negatively-charged, carboxylated PCL (CPCL). Murine ESCs were cultured on electrospun polymeric scaffolds and their differentiation to cardiomyocytes was assessed through measurements of viability, intracellular reactive oxygen species (ROS), α-myosin heavy chain expression (α-MHC), and intracellular Ca2+ signaling dynamics. Interestingly, ESCs on the most compliant substrate, 4%PEG-86%PCL-10%CPCL, exhibited the highest α-MHC expression as well as the most mature Ca2+ signaling dynamics. To investigate the role of scaffold modulus in ESC differentiation, the scaffold fiber density was reduced by altering the electrospinning parameters. The reduced modulus was found to enhance α-MHC gene expression, and promote maturation of myocyte Ca2+ handling. These data indicate that ESC-derived cardiomyocyte differentiation and maturation can be promoted by tuning the mechanical and chemical properties of polymer scaffold via copolymerization and electrospinning techniques.

Dual MMP7-Proximity-Activated and Folate Receptor-Targeted Nanoparticles for siRNA Delivery

A dual-targeted siRNA nanocarrier has been synthesized and validated that is selectively activated in environments where there is colocalization of two breast cancer hallmarks, elevated matrix metalloproteinase (MMP) activity and folate receptor overexpression. This siRNA nanocarrier is self-assembled from two polymers containing the same pH-responsive, endosomolytic core-forming block but varying hydrophilic, corona-forming blocks. The corona block of one polymer consists of a 2 kDa PEG attached to a terminal folic acid (FA); the second polymer contains a larger (Y-shaped, 20 kDa) PEG attached to the core block by a proximity-activated targeting (PAT), MMP7-cleavable peptide. In mixed micelle smart polymer nanoparticles (SPNs) formed from the FA- and PAT-based polymers, the proteolytically removable PEG on the PAT polymers shields nonspecific SPN interactions with cells or proteins. When the PAT element is cleaved within an MMP-rich environment, the PEG shielding is removed, exposing the underlying FA and making it accessible for folate receptor-mediated SPN uptake. Characterization of mixed micelles prepared from these two polymers revealed that uptake and siRNA knockdown bioactivity of a 50% FA/50% PAT formulation was dependent on both proteolytic activation and FA receptor engagement. MMP activation and delivery of this formulation to breast cancer cells expressing the FA receptor achieved greater than 50% protein-level knockdown of a model gene with undetectable cytotoxicity. This modular nanoparticle design represents a new paradigm in cell-selective siRNA delivery and allows for stoichiometric tuning of dual-targeting components to achieve superior targeting specificity.

ROS-cleavable proline oligomer crosslinking of polycaprolactone for pro-angiogenic host response

A reactive oxygen species (ROS)-degradable scaffold is fabricated by crosslinking biocompatible, hydrolytically-degradable poly(ε-caprolactone) (PCL) with a ROS-degradable oligoproline peptide, KP7K. The ROS-mediated degradability triggers favorable host responses of the scaffold including improved cell infiltration and angiogenesis in vivo, indicating its unique advantages for tissue engineering applications.

Pendant allyl crosslinking as a tunable shape memory actuator for vascular applications

UNLABELLED Thermo-responsive shape memory polymers (SMPs) can be programmed to fit into small-bore incisions and recover their functional shape upon deployment in the body. This property is of significant interest for developing the next generation of minimally-invasive medical devices. To be used in such applications, SMPs should exhibit adequate mechanical strengths that minimize adverse compliance mismatch-induced host responses (e.g. thrombosis, hyperplasia), be biodegradable, and demonstrate switch-like shape recovery near body temperature with favorable biocompatibility. Combinatorial approaches are essential in optimizing SMP material properties for a particular application. In this study, a new class of thermo-responsive SMPs with pendant, photocrosslinkable allyl groups, x%poly(ε-caprolactone)-co-y%(α-allyl carboxylate ε-caprolactone) (x%PCL-y%ACPCL), are created in a robust, facile manner with readily tunable material properties. Thermomechanical and shape memory properties can be drastically altered through subtle changes in allyl composition. Molecular weight and gel content can also be altered in this combinatorial format to fine-tune material properties. Materials exhibit highly elastic, switch-like shape recovery near 37°C. Endothelial compatibility is comparable to tissue culture polystyrene (TCPS) and 100%PCL in vitro and vascular compatibility is demonstrated in vivo in a murine model of hindlimb ischemia, indicating promising suitability for vascular applications. STATEMENT OF SIGNIFICANCE With the ongoing thrust to make surgeries minimally-invasive, it is prudent to develop new biomaterials that are highly compatible and effective in this workflow. Thermo-responsive shape memory polymers (SMPs) have great potential for minimally-invasive applications because SMP medical devices (e.g. stents, grafts) can fit into small-bore minimally-invasive surgical devices and recover their functional shape when deployed in the body. To realize their potential, it is imperative to devise combinatorial approaches that enable optimization of mechanical, SM, and cellular responses for a particular application. In this study, a new class of thermo-responsive SMPs is created in a robust, facile manner with readily tunable material properties. Materials exhibit excellent, switch-like shape recovery near body temperature and promising biocompatibility for minimally-invasive vascular applications.

Modular Polymer Design to Regulate Phenotype and Oxidative Response of Human Coronary Artery Cells for Potential Stent Coating Applications

Polymer properties can be tailored by copolymerizing subunits with specific physico-chemical characteristics. Vascular stent materials require biocompatibility, mechanical strength, and prevention of restenosis. Here we copolymerized poly(ε-caprolactone) (PCL), poly(ethylene glycol) (PEG), and carboxyl-PCL (cPCL) at varying molar ratios and characterized the resulting material properties. We then performed a short-term evaluation of these polymers for their applicability as potential coronary stent coating materials with two primary human coronary artery cell types: smooth muscle cells (HCASMC) and endothelial cells (HCAEC). Changes in proliferation and phenotype were dependent upon intracellular reactive oxygen species (ROS) levels, and 4%PEG-96%PCL-0%cPCL was identified as the most appropriate coating material for this application. After 3days on this substrate HCASMC maintained a healthy contractile phenotype and HCAEC exhibited a physiologically relevant proliferation rate and a balanced redox state. Other test substrates promoted a pathological, synthetic phenotype of HCASMC and/or hyperproliferation of HCAEC. Phenotypic changes of HCASMC appeared to be modulated by the Youngs modulus and surface charge of the test substrates, indicating a structure-function relationship that can be exploited for intricate control over vascular cell functions. These data indicate that tailored copolymer properties can direct vascular cell behavior and provide insights for further development of biologically instructive stent coating materials.