Nearly 30years ago, certain small, relatively nontoxic peptides were discovered to be capable of traversing the cell membrane. These cell-penetrating peptides, as they are now called, have been shown to not only be capable of crossing the cell membrane themselves but can also carry many different therapeutic agents into cells, including small molecules, plasmid DNA, siRNA, therapeutic proteins, viruses, imaging agents, and other various nanoparticles. Many cell-penetrating peptides have been derived from natural proteins, but several other cell-penetrating peptides have been developed that are either chimeric or completely synthetic. How cell-penetrating peptides are internalized into cells has been a topic of debate, with some peptides seemingly entering cells through an endocytic mechanism and others by directly penetrating the cell membrane. Although the entry mechanism is still not entirely understood, it seems to be dependent on the peptide type, the peptide concentration, the cargo the peptide transports, and the cell type tested. With new intracellular disease targets being discovered, cell-penetrating peptides offer an exciting approach for delivering drugs to these intracellular targets. There are hundreds of cell-penetrating peptides being studied for drug delivery, and ongoing studies are demonstrating their success both in vitro and in vivo.
ENPs can be efficiently attached to the RBCs without adversely affecting cellular function, and this may potentially enhance circulatory half-life of drug molecules.
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.
Effective use of exogenous human BChE as a bioscavenger for organophosphorus toxicants (OPs) is hindered by its limited availability and rapid clearance. Complexes made from recombinant human BChE (rhBChE) and copolymers may be useful in addressing these problems. We used in vitro approaches to compare enzyme activity, sensitivity to inhibition, stability and bioscavenging capacity of free enzyme and copolymer-rhBChE complexes (C-BCs) based on one of nine different copolymers, from combinations of three molecular weights (MW) of poly-L-lysine (PLL; high MW, 30-70 kDa; medium MW, 15-30 kDa; low MW, 4-15 kDa) and three grafting ratios of poly(ethylene glycol) (PEG; 2:1, 10:1, 20:1). Retarded protein migration into acrylamide gels stained for BChE activity was noted with all copolymers as the copolymer-to-protein ratio was increased. BChE activity of C-BCs was lower relative to free enzyme, with the 2:1 grafting ratio showing generally greater reduction. Free enzyme and C-BCs showed relatively similar in vitro sensitivity to inhibition by paraoxon, but use of the 20:1 grafting ratio led to lower potencies. Through these screening assays we selected three C-BCs (high, medium and low MW; 10:1 grafting) for further characterizations. BChE activity was higher in C-BCs made with the medium and low compared to high MW-based copolymer. C-BCs generally showed higher stability than free enzyme when maintained for long periods at 37 °C or following incubation with chymotrypsin. Free enzyme and C-BCs were similarly effective at inactivating paraoxon in vitro. While these results are promising for further development, additional studies are needed to evaluate in vivo performance.
Red blood cells (RBCs) express a variety of immunomodulatory markers that enable the body to recognize them as self. We have shown that RBC membrane glycophorin A (GPA) receptor can mediate membrane attachment of protein therapeutics. A critical knowledge gap is whether attaching drug-encapsulated nanoparticles (NPs) to GPA and modification with cell-penetrating peptide (CPP) will impact binding, oxygenation, and the induction of cellular stress. The objective of this study was to formulate copolymer-based NPs containing model fluorescent-tagged bovine serum albumin (BSA) with GPA-specific targeting ligands such as ERY1 (ENPs), single-chain variable antibody (scFv TER-119, SNPs), and low-molecular-weight protamine-based CPP (LNPs) and to determine their biocompatibility using a variety of complementary high-throughput in vitro assays. Experiments were conducted by coincubating NPs with RBCs at body temperature, and biocompatibility was evaluated by Raman spectroscopy, hemolysis, complement lysis, and oxidative stress assays. Data suggested that LNPs effectively targeted RBCs, conferring 2-fold greater uptake in RBCs compared to ENPs and SNPs. Raman spectroscopy results indicated no adverse effect of NP attachment or internalization on the oxygenation status of RBCs. Cellular stress markers such as glutathione, malondialdehyde, and catalase were within normal limits, and complement-mediated lysis due to NPs was negligible in RBCs. Under the conditions tested, our data demonstrates that molecular targeting of the RBC membrane is a feasible translational strategy for improving drug pharmacokinetics and that the proposed high-throughput assays can prescreen diverse NPs for preclinical and clinical biocompatibility.
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