Role of Oligoethylene Glycol Side Chain Length in Responsive Polymeric Nanoassemblies.

An oft-desired feature of a responsive nanomaterial is that it should undergo disassembly or morphological change upon application of a specific stimulus. The extent of response has been found to depend on factors such as the nature and the number of responsive functionalities incorporated into these particles. In this work, the length of oligoethylene glycol (OEG) side chains associated with the polymers has been shown to greatly influence the responsive behavior of polymeric nanoparticles. The integrity of these OEG-based polymeric assemblies was found to depend not only on the chemical cross-links but also on the physical cross-links in these aggregates in cases where the polymer chains bear long OEG side chains. The physical cross-linking in longer OEG side chain containing polymeric nanogels is present in the form of crystalline domains. Our results here highlight that these ethylene glycol-based hydrophilic units are not to be ignored as spectator units with water-solubilization characteristics but must be analyzed in the context of assembly stabilization and triggerability with the targeted stimulus.

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.

Hyaluronic Acid Microgels as Intracellular Endosomolysis Reagents.

Hyaluronic acid (HA) microgels were investigated as biocompatible and biodegradable reagents for facilitating endosomolysis in human cells. Employing inverse emulsion templates, HA microgels were prepared by cross-linking aqueous sodium hyaluronate droplets with divinyl sulfone (DVS). Introduction of ether sulfone cross-links was confirmed by infrared (IR) spectroscopy and elemental analysis. The degree of cross-linking of the microgels was estimated using high performance liquid chromatography (HPLC). The size distribution of the water-dispersible HA microgels was studied by laser diffraction analysis, and the gel morphology was investigated using scanning electron microscopy (SEM). Aqueous microgel suspensions were found to be well-tolerated in human cells at concentrations of up to 100 µg/mL. Endosome-rupturing properties of the HA microgels were evaluated in vitro using calcein internalization and Cre protein delivery assays. The results of this study serve as a proof-of-principle for the utility of cross-linked HA microgels as a new class of biocompatible and biodegradable endosomolytic reagents.

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.

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.

Dual Site Phosphorylation of Caspase-7 by PAK2 Blocks Apoptotic Activity by Two Distinct Mechanisms.

Caspases, the cysteine proteases that execute apoptosis, are tightly regulated via phosphorylation by a series of kinases. Although all apoptotic caspases work in concert to promote apoptosis, different kinases regulate individual caspases. Several sites of caspase-7 phosphorylation have been reported, but without knowing the molecular details, it has been impossible to exploit or control these complex interactions, which normally prevent unwanted proliferation. During dysregulation, PAK2 kinase plays an alternative anti-apoptotic role, phosphorylating caspase-7 and promoting unfettered cell growth and chemotherapeutic resistance. PAK2 phosphorylates caspase-7 at two sites, inhibiting activity using two different molecular mechanisms, before and during apoptosis. Phosphorylation of caspase-7 S30 allosterically obstructs its interaction with caspase-9, preventing intersubunit linker processing, slowing or preventing caspase-7 activation. S239 phosphorylation renders active caspase-7 incapable of binding substrate, blocking later events in apoptosis. Each of these mechanisms is novel, representing new opportunities for synergistic control of caspases and their counterpart kinases.

Reactive Self-Assembly of Polymers and Proteins to Reversibly Silence a Killer Protein.

Conjugation of biologically active proteins to polymeric materials is of great interest in the treatment of cancer and other diseases of protein deficiency. The conjugation of such biomacromolecules is challenging both due to their hydrophilicity and propensity to denature under non-native conditions. We describe a novel reactive self-assembly approach to "wrap" a protein with polymers, simultaneously protecting its delicate folded state and silencing its enzymatic activity. This approach has been demonstrated using caspase-3, an apoptosis-inducing protein, as the first case study. The protein-polymer conjugation is designed to be reversed under the native conditions for caspase-3, that is, the reducing environment found in the cytosol. The current strategy allowed release and recovery of up to 86% of caspase activity and nanogel-caspase-3 conjugates induced 70-80% apoptotic cell death shortly thereafter. This approach is widely generalizable and should be applicable to the intracellular delivery of a wide range of therapeutic proteins for treatment of complex and genetic diseases.

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.

Self-assembly of random copolymers.

Self-assembly of random copolymers has attracted considerable attention recently. In this feature article, we highlight the use of random copolymers to prepare nanostructures with different morphologies and to prepare nanomaterials that are responsive to single or multiple stimuli. The synthesis of single-chain nanoparticles from random copolymers and their potential applications are also discussed in some detail. We aim to draw more attention to these easily accessible copolymers, which are likely to play an important role in translational polymer research.