Kishore Raghupathi

Surface charge generation in nanogels for activated cellular uptake at tumor-relevant pH

Nanocarriers that can be effectively transported across cellular membranes have potential in a variety of biomedical applications. Among these, materials that are capable of changing their surface properties and thus gain entry into a cell, in response to a specific tissue environment, are of particular interest. In this manuscript we report a facile route to prepare nanogels, which generate surface charge with pH as stimulus. This is achieved by designing a polymeric nanogel containing 2-diisopropylamino (DPA) moieties. The choice of DPA nanogel is based on its pKb, which causes this functional group to be rapidly protonated upon change in pH. It is noteworthy that the pH change at which the surface charge is generated in the nanogel corresponds to the slightly acidic conditions observed in the extracellular environment of solid tumor. We show that the pH at which the charge is generated, i.e. the isoelectric point (pI) of the nanogel, can be adjusted by varying the percentage of DPA units in the nanogel, its preparation process and crosslinking density. Intracellular delivery of these nanogels was greatly enhanced in an acidic pH environment due to the surface charge generation. This study demonstrates the versatile nature of the nanogels to introduce specific functionalities with relative ease to achieve desired functional behavior.

Composite supramolecular nanoassemblies with independent stimulus sensitivities

Nanoscale assemblies with stimuli-sensitive features have attracted significant attention due to implications in a variety of areas ranging from materials to biology. Recently, there have been excellent developments in obtaining nanoscale structures that are concurrently sensitive to multiple stimuli. Such nanostructures are primarily focused on a single nanostructure containing an appropriate combination of functional groups within the nanostructure. In this work, we outline a simple approach to bring together two disparate supramolecular assemblies that exhibit very different stimuli-sensitive characteristics. These composite nanostructures comprise a block copolymer micelle core and nanogel shell, both of which can preserve their respective morphology and stimulus sensitivities. The block copolymer is based on poly(2-(diisopropylamino)ethylmethacrylate-b-2-aminoethylmethacrylate hydrochloride), which contains a pH-sensitive hydrophobic block. Similarly, the redox-sensitive nanogel is derived from a poly(oligoethyleneglycolmonomethylethermethacrylate-co-glycidylmethacrylate-co-pyridyldisulfide ethylmethacrylate) based random copolymer. In addition to the independent pH-response of the micellar core and redox-sensitivity of the nanogel shell in the composite nanostructures, the synergy between the micelles and the nanogels have been demonstrated through a robust charge generation in the nanogels during the disassembly of the micelles. The supramolecular assembly and disassembly have been characterized using transmission electron microscopy, dynamic light scattering, zeta potential measurements, fluorescence spectroscopy and cellular uptake.

Influence of backbone conformational rigidity in temperature-sensitive amphiphilic supramolecular assemblies.

Molecular design features that endow amphiphilic supramolecular assemblies with a unique temperature-sensitive transition have been investigated. We find that conformational rigidity in the backbone is an important feature for eliciting this feature. We also find that intramolecular hydrogen-bonding can induce such rigidity in amphiphile backbone. Guest encapsulation stability of these assemblies was found to be significantly altered within a narrow temperature window, which correlates with the temperature-sensitive size transition of the molecular assembly. Molecular design principles demonstrated here could have broad implications in developing future temperature-responsive systems.

pH responsive soft nanoclusters with size and charge variation features

Size and surface properties of a drug delivery vehicle play a very crucial role in determining its biological fate. The success of nanotherapeutic systems depends on several features such as deeper tumor tissue penetration, good retention and rapid cellular uptake. These properties are strongly influenced by the size and surface properties of the delivery vehicles. In this study, we demonstrate a polymeric nanoparticle that exhibits the size and surface charge variation features in response to slight changes in pH, which are biologically relevant (pH 7.4 to 6.5). These features have been demonstrated using a pH sensitive interparticle crosslinking by dynamic covalent imine bond formation between nanogels. The pH sensitive interparticle crosslinking is demonstrated using DLS, TEM, SEC, fluorescamine assay and cell uptake study.

Utilizing Inverse Emulsion Polymerization To Generate Responsive Nanogels for Cytosolic Protein Delivery

Therapeutic biologics have various advantages over synthetic drugs in terms of selectivity, their catalytic nature, and, thus, therapeutic efficacy. These properties offer the potential for more effective treatments that may also overcome the undesirable side effects observed due to off-target toxicities of small molecule drugs. Unfortunately, systemic administration of biologics is challenging due to cellular penetration, renal clearance, and enzymatic degradation difficulties. A delivery vehicle that can overcome these challenges and deliver biologics to specific cellular populations has the potential for significant therapeutic impact. In this work, we describe a redox-responsive nanoparticle platform, which can encapsulate hydrophilic proteins and release them only in the presence of a reducing stimulus. We have formulated these nanoparticles using an inverse emulsion polymerization (IEP) methodology, yielding inverse nanoemulsions, or nanogels. We have demonstrated our ability to overcome the liabilities that contribute to activity loss by delivering a highly challenging cargo, functionally active caspase-3, a cysteine protease susceptible to oxidative and self-proteolytic insults, to the cytosol of HeLa cells by encapsulation inside a redox-responsive nanogel.

Biodistribution Analysis of NIR-Labeled Nanogels using In Vivo FMT Imaging in Triple Negative Human Mammary Carcinoma Models

The purpose of this study is to evaluate the biodistribution properties of random-copolymer-based core-cross-linked nanogels of various sizes and surface poly(ethylene glycol) composition. Systematic variations of near-IR labeled nanogels, comprising varying particle sizes (28-135 nm), PEG corona quantity (0-50 mol %), and PEG length (PEG Mn 1000, 2000, and 5000), were prepared and injected in mice that had been subcutaneously implanted with MDA-MB-231-luc-D3H2LN human mammary carcinoma. In vivo biodistribution was obtained using fluorescence molecular tomography imaging at 0, 6, 24, 48, and 72 h postinjection. Retention of total body probe and percentages of total injected dose in the tumor, liver, spleen, lungs, heart, intestines, and kidneys were obtained. Smaller nanogels (∼30-40 nm) with a high PEG conjugation (∼43-46 mol %) of Mn 2000 on their coronas achieved the highest tumor specificity with peak maximum 27% ID/g, a statistically significant propensity toward accumulation with 16.5% ID/g increase from 0 to 72 h of imaging, which constitutes a 1.5-fold increase. Nanogels with greater tumor localization also had greater retention of total body probe over 72 h. Nanogels without extensive PEGylation were rapidly excreted, even at similar sizes to PEGylated nanogels exhibiting whole body retention. Of all tissues, the liver had the highest % ID, however, like other tissues, it displayed a monotonic decrease over time, suggesting nanogel clearance by hepatic metabolism. Ex vivo quantification of individual tissues from gross necropsy at 72 h postinjection generally correlated with the FMT analysis, providing confidence in tissue signal segmentation in vivo. The parameters determined to most significantly direct a nanogel to the desired tumor target can lead to improve effectiveness for nanogels as therapeutic delivery vehicles.

Nano-Armoring of Enzymes

The formulation in which therapeutic proteins are administered plays a key role in retaining their biological activity. Enzyme wrapping, using synthetic polymers, is a strategy employed to provide enzymes with lower immunogenicity, longer circulation times, and better targeting capabilities. Protein-polymer complexation methods, involving covalent, noncovalent, and electrostatic interactions, that can provide means to develop formulations for retaining enzyme stability are discussed in this chapter. Amphiphilic self-cross-linkable polymer was used to encapsulate capsase-3 enzyme in the nanogel, while inverse emulsion polymerization method was used to entrap α-glucosidase enzyme in the nanogel. These nanogels were characterized by dynamic light scattering, transmission electron microscopy, and gel electrophoresis. Upon release of caspase-3 enzyme from polymeric nanogel, it retained nearly 86% of its original activity. Similarly, α-glucosidase that was encased in the acid cleavable polymeric nanogel exhibited substantial activity after release under acidic conditions (pH 5, 48h). Nano-armoring of the enzymes were nearly complete and provided high yields of the encased enzyme.

Nano-Armoring of Enzymes: Rational Design of Polymer-Wrapped Enzymes

The formulation in which therapeutic proteins are administered plays a key role in retaining their biological activity. Enzyme wrapping, using synthetic polymers, is a strategy employed to provide enzymes with lower immunogenicity, longer circulation times, and better targeting capabilities. Protein-polymer complexation methods, involving covalent, noncovalent, and electrostatic interactions, that can provide means to develop formulations for retaining enzyme stability are discussed in this chapter. Amphiphilic self-cross-linkable polymer was used to encapsulate capsase-3 enzyme in the nanogel, while inverse emulsion polymerization method was used to entrap α-glucosidase enzyme in the nanogel. These nanogels were characterized by dynamic light scattering, transmission electron microscopy, and gel electrophoresis. Upon release of caspase-3 enzyme from polymeric nanogel, it retained nearly 86% of its original activity. Similarly, α-glucosidase that was encased in the acid cleavable polymeric nanogel exhibited substantial activity after release under acidic conditions (pH 5, 48h). Nano-armoring of the enzymes were nearly complete and provided high yields of the encased enzyme.

Chapter Sixteen – Nano-Armoring of Enzymes: Rational Design of Polymer-Wrapped Enzymes

The formulation in which therapeutic proteins are administered plays a key role in retaining their biological activity. Enzyme wrapping, using synthetic polymers, is a strategy employed to provide enzymes with lower immunogenicity, longer circulation times, and better targeting capabilities. Protein-polymer complexation methods, involving covalent, noncovalent, and electrostatic interactions, that can provide means to develop formulations for retaining enzyme stability are discussed in this chapter. Amphiphilic self-cross-linkable polymer was used to encapsulate capsase-3 enzyme in the nanogel, while inverse emulsion polymerization method was used to entrap α-glucosidase enzyme in the nanogel. These nanogels were characterized by dynamic light scattering, transmission electron microscopy, and gel electrophoresis. Upon release of caspase-3 enzyme from polymeric nanogel, it retained nearly 86% of its original activity. Similarly, α-glucosidase that was encased in the acid cleavable polymeric nanogel exhibited substantial activity after release under acidic conditions (pH 5, 48h). Nano-armoring of the enzymes were nearly complete and provided high yields of the encased enzyme.