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Erythrocytes have been extensively investigated as drug delivery vehicles. However, many existing methods for loading therapeutic biomolecules into erythrocytes perturb the cell membrane, resulting in a loss of erythrocyte structural integrity and recognition by the host immune system. To overcome this challenge, we developed a novel nanoparticle carrier for transporting proteins into intact erythrocytes. We designed and characterized a nanoparticle where a poly-L-lysine-grafted-poly(ethylene) glycol copolymer was used to encapsulate a model protein, bovine serum albumin. The nanoparticle was functionalized with either an erythrocyte targeting peptide ERY1, the cell-penetrating peptide low molecular weight protamine, or a combination of the two peptides. The three resulting constructs, ENP, LNP, and ELNP, had a core/shell structure and, depending on functionalization, were approximately 30–90 nm in diameter and had negative surface charge. Esterase-like activity of the encapsulated protein was not affected by encapsulation. Under physiological conditions, nanoparticles caused no significant hemolysis and were resistant to dissociation by surfactants. The three nanoparticle constructs were evaluated using confocal microscopy and flow cytometry for their ability to bind and internalize within intact erythrocytes. LNPs showed significant binding and internalization into intact erythrocytes, with ~90% of erythrocytes containing particles after 24 h of co-incubation. Internalization occurred without adversely affecting erythrocyte membrane structure. Together, these results demonstrate that low molecular weight protamine–functionalized nanoparticles may serve as a universal platform to deliver protein therapeutics into erythrocytes. [ABSTRACT FROM AUTHOR]

Advancement of therapeutic protein therapies can be hindered by their poor stability and short in vivo half‐life. There is emerging evidence that biocompatible zwitterionic materials can prevent nonspecific interactions within proteins systems that contribute to protein instability. Here, zwitterionic hydrogel beads are synthesized from poly(sulfobetaine methyl methacrylate), pSB, using an inverse emulsion, free radical polymerization reaction technique. The transport properties within the zwitterionic hydrogels were characterized using 1H NMR diffusometry. Equilibrium water content as high as 0.90 was measured for the synthesized hydrogels. Our study revealed that the pSB hydrogels are nontoxic, ion responsive, and their swelling is temperature dependent. The zwitterionic hydrogel beads were capable of undergoing lyophilization without aggregation. Hydrogel beads were loaded with a model protein, bovine serum albumin (BSA), using a postfabrication loading technique. The protein loading was studied using confocal laser microscopy, indicating homogenous protein dispersion of up to 40 μg BSA/mg hydrogel within the beads. Furthermore, the release rate of the protein from the synthesized hydrogel was studied at different crosslinker to monomer ratios. The protein encapsulated within the zwitterionic hydrogel had slower rates of thermal aggregation compared to nonencapsulated protein in solution. Furthermore, the protein‐loaded inside the zwitterionic hydrogel better maintained its bioactivity. [ABSTRACT FROM AUTHOR]

The aim of the present study was to evaluate a library of poly- l -lysine (PLL)-graft (g)-polyethylene glycol (PEG) copolymers for the ability to encapsulate effectively a model protein, bovine serum albumin (BSA), and to characterize the stability and protein function of the resulting nanoparticle. A library of nine grafted copolymers was produced by varying PLL molecular weight and PEG grafting ratio. Electrostatic self-assembly of the protein and the grafted copolymer drove encapsulation. The formation of protein/polymer nanoparticles with a core/shell structure was confirmed using PAGE, dynamic light scattering, and electron microscopy. Encapsulation of the BSA into nanoparticles was strongly dependent on the copolymer-to-protein mass ratio, PEG grafting ratio, and PLL molecular weight. A copolymer-to-protein mass ratio of 7:1 and higher was generally required for high levels of encapsulation, and under these conditions, no loss of protein activity was observed. Copolymer characteristics also influenced nanoparticle resistance to polyanions and protease degradation. The results indicate that a copolymer of 15–30 kDa PLL, with a PEG grafting ratio of 10:1, is most promising for protein delivery. [ABSTRACT FROM AUTHOR]

Stability of therapeutic proteins is one of the most important obstacles in their development. We studied the effects of the zwitterionic amphiphile cocamidopropyl betaine (CAPB) on protein stability against elevated temperatures and shear. Bovine serum albumin (BSA) was selected as a model protein to study the interaction with CAPB. Techniques such as gel electrophoresis, fluorimetry, and surface tensiometry were utilized to study protein aggregation and protein/CAPB complex formation. Our results indicate that CAPB effectively decreased the concentration of BSA at the air/water interface and subsequently prevented aggregation of the protein at the interface. Furthermore, complexes formed by the association of BSA and CAPB were more stable against both temperature and shear. The increased stability was achieved without loss of enzymatic activity of the protein. The effects of zwitterionic CAPB on protein enzymatic activity and conformational (tertiary structure) stability were also compared with polysorbate 20 and sodium dodecyl sulfate. • CAPB prevents protein aggregation and denaturation at elevated temperatures. • The enzymatic activity/conformational stability of the protein is not diminished. • Zwitterionic CAPB associates with BSA, to form stable, less surface active complexes. [ABSTRACT FROM AUTHOR]

The interfacial adsorption and denaturation of proteins is a significant concern for the loss of protein structure and function during protein processing or storage. Surfactants can be used to prevent interfacial damage to proteins. Zwitterionic surfactants have shown intriguing effects on stability and activity of proteins. The aim of this research was to study the effect of cocamidopropyl betaine (CAPB), a non-denaturing zwitterionic carboxy betaine containing surfactant, on the air/water interfacial adsorption of a model protein bovine serum albumin (BSA). Dynamic and equilibrium surface tensions were measured using the pendant drop method. The results of surface tension measurements were coupled with a thermodynamic analysis, based on the interface and bulk chemical potentials, to estimate the surface coverage of the components and to calculate surface adsorption. Our results indicated that CAPB can effectively associate with the protein at different concentrations, even at concentrations lower than the critical micelle concentration (CMC). Furthermore, the addition of the protein significantly increased the CMC of the CAPB form 45 ppm to 407 ppm. Most importantly, CAPB prevented the adsorption of the protein at the interface. The interface of aqueous solution of CAPB+BSA was also compared with the Triton X-100 + BSA. Results of this study will help us to understand surfactant+protein interactions at interfaces. Unlabelled Image • Interfacial behavior of zwitterionic surfactant+protein is experimentally investigated. • Protein and surfactant surface coverages are estimated using thermodynamic modeling. • Spontaneous self-assembly of zwitterionic surfactant-albumin reduced system surface activity. • Zwitterionic surfactant interacts with the protein more strongly than Triton X-100. [ABSTRACT FROM AUTHOR]