Protein charge parameters that influence stability and cellular internalization of polyelectrolyte complex micelles

Proteins are an important class of biologics, but there are several recurring challenges to address when designing protein-based therapeutics. These challenges include: the propensity of proteins to aggregate during formulation, relatively low loading in traditional hydrophobic delivery vehicles, and inefficient cellular uptake. This last criterion is particularly challenging for anionic proteins as they cannot cross the anionic plasma membrane. Here we investigated the complex coacervation of anionic proteins with a block copolymer of opposite charge to form polyelectrolyte complex (PEC) micelles for use as a protein delivery vehicle. Using genetically modified variants of the model protein green fluorescent protein (GFP), we evaluated the role of protein charge and charge localization in the formation and stability of PEC micelles. A neutral-cationic block copolymer, POEGMA79-b-qP4VP175, was prepared via RAFT polymerization for complexation and microphase separation with the panel of engineered anionic GFPs. We found that isotropically supercharged proteins formed micelles at higher ionic strength relative to protein variants with charge localized to a polypeptide tag. We then studied GFP delivery by PEC micelles and found that they effectively delivered the protein cargo to mammalian cells. However, cellular delivery varied as a function of protein charge and charge distribution and we found an inverse relationship between the PEC micelle critical salt concentration and delivery efficiency. This model system has highlighted the potential of polyelectrolyte-complexes to deliver anionic proteins intracellularly as well as the importance of correlating solution structure and desired functional activity.

Carbohydrate/protein hydrogels as responsive scaffolds in controlling inorganic crystallization

<p>The strategies that both invertebrate and vertebrate organism use to produce organic-inorganic composite materials for different purposes such as mechanical support and protection for the body are fascinating. While extensive research has been done on understanding the basic principles of biomineral formation, mimicking the critical principles of the mechanisms of biomineralization in vitro and fully capturing the structural information and characteristics remain challenging issues for scientists. Calcium is an essential element in biological systems. It plays a central role in the mineralization and maintenance of the skeleton as well as in fundamental physiological processes including growth and development in vertebrates. Within a biological organism calcium ions are stored, delivered, or released in the presence of different anions such as phosphate, carbonate and citrate. Competition between the different anions which interact with calcium ions in different hydrogel matrices leads to manipulation of the various composite materials produced such as bone and nacre. Soluble anionic acidic macromolecules associated with biominerals play a vital role in modulating the mineral morphology and hierarchy of the organized composite. Understanding the interaction between the constituent ions and the organic matrix is crucial if we are to make synthetic materials, the structure and properties of which replicate those of native biominerals, or materials that have the storage and/or release characteristics of foods, for example. Carbohydrate-based hydrogels versus protein-based hydrogels are used here as scaffolds for the synthesis of calcium carbonate and calcium phosphate biominerals. Water soluble acidic additives are used to modulate the nucleation and growth of the minerals. In particular chitosan and gelatin hydrogel templates were used as the mineralization scaffolds. Three different mineralization methods were used: the Kitano, alternate soaking and McGrath methods. Monomeric vs. polymeric additives (acrylic acid, glutamic acid, aspartic acid and their corresponding polymers) were introduced into all systems in order to control the nucleation and growth of the so-formed minerals. The morphology, crystallinity, polymorphism and composition of the synthesized organic-inorganic composites were investigated. Analyses were carried out using a number of techniques including Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), X-ray Diffraction (XRD), micro-Raman spectroscopy and solid-sate NMR. In the case of calcium carbonate the morphology and crystallinity were more affected by the use of polymeric additives compared with their monomeric equivalents. In particular the calcium carbonate preferentially grew laterally within and on the periphery of the chitosan or gelatin hydrogel scaffold. This results from the formation of a polyelectrolyte complex between the chitosan or gelatin hydrogels and the added polymer. The presence of the polyelectrolyte complex modifies the nucleation of the mineral. Nanoparticles are preferentially formed which then aggregate together maintaining a lateral perspective with the scaffold. In the case of calcium phosphate mostly spherical and platelet-shaped morphologies composed of amorphous calcium phosphate and poorly crystalline hydroxylapatite respectively were always formed within both chitosan and gelatin hydrogel scaffolds. pH was also found to be a key factor in controlling which polymorph of calcium phosphate precipitates. The crystallinity is influenced by the presence of additives for chitosan scaffold. For systems with added aspartic or polyaspartic acid platelet-shaped CaP forms. These crystals are more highly crystalline compared to those where predominately the porous spherical calcium phosphate morphology is observed which form when L-glutamic acid is added. In the presence of polymeric additives nanoparticles form which then aggregate to yield larger crystals. Such aggregation was preferentially observed for gelatin scaffolds. Citrate anions are particularly important in calcium phosphate precipitation in bone. Biomimetic hydroxylapatite-chitosan and hydroxylapatite-gelatin nanocomposite were synthesized where citrate ions were used to control the size and crystallinity of the hydroxylapatite crystals. TEM data show that the size of the hydroxylapatite crystals decreases upon introducing citrate ions into the systems. Solid-state NMR dipolar dephasing data indicate the hydroxylapatite precipitation can be stabilized with 2.5 wt% sodium citrate with respect to the chitosan and gelatin mass. The data included within this thesis illustrate that both gelatin and chitosan hydrogel scaffolds display similar ability in modulating calcium carbonate or calcium phosphate crystallization in the absence and presence of additives. The role of soluble acidic additives is significant in the formation of biominerals. These results reveal therefore the possibility that carbohydrate-based systems, which have many advantages over protein-based systems, could be used to provide more options for fabricating new implantable materials for humans and animals. The results from the combination of techniques used including XRD, SSNMR and TEM showed the possibility of in vitro synthesis of a bio-nanocomposite material in the presence of citrate similar to that of natural bone (in terms of composition and morphology). The achievement of this work demonstrates that new advanced materials with various composite structures and morphologies can be synthesized through a biomimetic biomineralization mechanism under ambient conditions similar to natural materials such as bone and nacre. These advancements have potential application in biomedical research and more specifically in fabrication of implantable materials.</p>

Carbohydrate/protein hydrogels as responsive scaffolds in controlling inorganic crystallization

<p>The strategies that both invertebrate and vertebrate organism use to produce organic-inorganic composite materials for different purposes such as mechanical support and protection for the body are fascinating. While extensive research has been done on understanding the basic principles of biomineral formation, mimicking the critical principles of the mechanisms of biomineralization in vitro and fully capturing the structural information and characteristics remain challenging issues for scientists. Calcium is an essential element in biological systems. It plays a central role in the mineralization and maintenance of the skeleton as well as in fundamental physiological processes including growth and development in vertebrates. Within a biological organism calcium ions are stored, delivered, or released in the presence of different anions such as phosphate, carbonate and citrate. Competition between the different anions which interact with calcium ions in different hydrogel matrices leads to manipulation of the various composite materials produced such as bone and nacre. Soluble anionic acidic macromolecules associated with biominerals play a vital role in modulating the mineral morphology and hierarchy of the organized composite. Understanding the interaction between the constituent ions and the organic matrix is crucial if we are to make synthetic materials, the structure and properties of which replicate those of native biominerals, or materials that have the storage and/or release characteristics of foods, for example. Carbohydrate-based hydrogels versus protein-based hydrogels are used here as scaffolds for the synthesis of calcium carbonate and calcium phosphate biominerals. Water soluble acidic additives are used to modulate the nucleation and growth of the minerals. In particular chitosan and gelatin hydrogel templates were used as the mineralization scaffolds. Three different mineralization methods were used: the Kitano, alternate soaking and McGrath methods. Monomeric vs. polymeric additives (acrylic acid, glutamic acid, aspartic acid and their corresponding polymers) were introduced into all systems in order to control the nucleation and growth of the so-formed minerals. The morphology, crystallinity, polymorphism and composition of the synthesized organic-inorganic composites were investigated. Analyses were carried out using a number of techniques including Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), X-ray Diffraction (XRD), micro-Raman spectroscopy and solid-sate NMR. In the case of calcium carbonate the morphology and crystallinity were more affected by the use of polymeric additives compared with their monomeric equivalents. In particular the calcium carbonate preferentially grew laterally within and on the periphery of the chitosan or gelatin hydrogel scaffold. This results from the formation of a polyelectrolyte complex between the chitosan or gelatin hydrogels and the added polymer. The presence of the polyelectrolyte complex modifies the nucleation of the mineral. Nanoparticles are preferentially formed which then aggregate together maintaining a lateral perspective with the scaffold. In the case of calcium phosphate mostly spherical and platelet-shaped morphologies composed of amorphous calcium phosphate and poorly crystalline hydroxylapatite respectively were always formed within both chitosan and gelatin hydrogel scaffolds. pH was also found to be a key factor in controlling which polymorph of calcium phosphate precipitates. The crystallinity is influenced by the presence of additives for chitosan scaffold. For systems with added aspartic or polyaspartic acid platelet-shaped CaP forms. These crystals are more highly crystalline compared to those where predominately the porous spherical calcium phosphate morphology is observed which form when L-glutamic acid is added. In the presence of polymeric additives nanoparticles form which then aggregate to yield larger crystals. Such aggregation was preferentially observed for gelatin scaffolds. Citrate anions are particularly important in calcium phosphate precipitation in bone. Biomimetic hydroxylapatite-chitosan and hydroxylapatite-gelatin nanocomposite were synthesized where citrate ions were used to control the size and crystallinity of the hydroxylapatite crystals. TEM data show that the size of the hydroxylapatite crystals decreases upon introducing citrate ions into the systems. Solid-state NMR dipolar dephasing data indicate the hydroxylapatite precipitation can be stabilized with 2.5 wt% sodium citrate with respect to the chitosan and gelatin mass. The data included within this thesis illustrate that both gelatin and chitosan hydrogel scaffolds display similar ability in modulating calcium carbonate or calcium phosphate crystallization in the absence and presence of additives. The role of soluble acidic additives is significant in the formation of biominerals. These results reveal therefore the possibility that carbohydrate-based systems, which have many advantages over protein-based systems, could be used to provide more options for fabricating new implantable materials for humans and animals. The results from the combination of techniques used including XRD, SSNMR and TEM showed the possibility of in vitro synthesis of a bio-nanocomposite material in the presence of citrate similar to that of natural bone (in terms of composition and morphology). The achievement of this work demonstrates that new advanced materials with various composite structures and morphologies can be synthesized through a biomimetic biomineralization mechanism under ambient conditions similar to natural materials such as bone and nacre. These advancements have potential application in biomedical research and more specifically in fabrication of implantable materials.</p>

Targeted polyelectrolyte complex micelles treat vascular complications in vivo

Vascular disease is a leading cause of morbidity and mortality in the United States and globally. Pathological vascular remodeling, such as atherosclerosis and stenosis, largely develop at arterial sites of curvature, branching, and bifurcation, where disturbed blood flow activates vascular endothelium. Current pharmacological treatments of vascular complications principally target systemic risk factors. Improvements are needed. We previously devised a targeted polyelectrolyte complex micelle to deliver therapeutic nucleotides to inflamed endothelium in vitro by displaying the peptide VHPKQHR targeting vascular cell adhesion molecule 1 (VCAM-1) on the periphery of the micelle. This paper explores whether this targeted nanomedicine strategy effectively treats vascular complications in vivo. Disturbed flow-induced microRNA-92a (miR-92a) has been linked to endothelial dysfunction. We have engineered a transgenic line (miR-92aEC-TG/Apoe−/−) establishing that selective miR-92a overexpression in adult vascular endothelium causally promotes atherosclerosis in Apoe−/− mice. We tested the therapeutic effectiveness of the VCAM-1–targeting polyelectrolyte complex micelles to deliver miR-92a inhibitors and treat pathological vascular remodeling in vivo. VCAM-1–targeting micelles preferentially delivered miRNA inhibitors to inflamed endothelial cells in vitro and in vivo. The therapeutic effectiveness of anti–miR-92a therapy in treating atherosclerosis and stenosis in Apoe−/− mice is markedly enhanced by the VCAM-1–targeting polyelectrolyte complex micelles. These results demonstrate a proof of concept to devise polyelectrolyte complex micelle-based targeted nanomedicine approaches treating vascular complications in vivo.