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Rapid clearance of thrombolytics from blood following intravenous injection is a major clinical challenge in cardiovascular medicine. To overcome this barrier, nanoparticle (NP) based drug delivery systems have been reported. Although superior than conventional therapy, a large proportion of the injected NP is still cleared by the reticuloendothelial system. Previously, we and others showed that ex vivo attachment of bioscavengers, thrombolytics, and nanoparticles (NPs) to glycophorin A receptors on red blood cells (RBCs) improved the blood half-life. This is promising, but ex-vivo approaches are cumbersome and challenging to translate clinically. Here, we developed a novel Ter119-polymeric NP containing tissue plasminogen activator for on-demand targeting of GPA receptors in vivo. Upon intravenous injection, the Ter119-NPs achieved remarkable RBC labeling efficiencies (>95%), resulting in marked enhancement of blood residence time of tPA from minutes to several days without any morphological, hematological, and histological complications. Our approach of RBC labeling with the NPs also prevented reticuloendothelial detections and the activations of innate and adaptive immune system. Data suggest that real-time targeting of therapeutics to RBC with NPs can potentially improve outcomes and reduce complications against a variety chronic disease., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Therapeutic proteins are utilized in a variety of clinical applications, but side effects and rapid in vivo clearance still present hurdles. An approach that addresses both drawbacks is protein encapsulation within in a polymeric nanoparticle, which is effective but introduces the additional challenge of destabilizing the nanoparticle shell in clinically relevant locations. This study examined the effects of crosslinking self-assembled poly(l-lysine)-grafted-poly(ethylene glycol) nanoparticles with redox-responsive 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) to achieve nanoparticle destabilization in a reductive environment. The polymer-protein nanoparticles (DTSSP NPs) were formed through electrostatic self-assembly and crosslinked with DTSSP, which contains a glutathione-reducible disulfide. As glutathione is upregulated in various cancers, DTSSP NPs could display destabilization within cancer cells. A library of DTSSP NPs was formed with varying copolymer to protein (C:P) and crosslinker to protein (X:P) mass ratios and characterized by size and encapsulation efficiency. DTSSP NPs with a 7:1 C:P ratio and 2:1 X:P ratio were further characterized by stability in the presence proteases and reducing agents. DTSSP NPs fully encapsulated the model protein and displayed 81% protein release when incubated with 5 mM dithiothreitol for 12 hr. This study contributes to understanding stimulus-responsive crosslinking of polymeric nanoparticles and could be foundational to clinical administration of therapeutic proteins., (© 2020 Wiley Periodicals LLC.)

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.

Purpose: Nanoparticle (NP) attachment to biocompatible secondary carriers such as red blood cell (RBC) can prolong blood residence time of drug molecules and help create next-generation nanotherapeutics. However, little is known about the impact of RBC-targeted NPs on erythrocyte function., Methods: The objectives of this study were to develop and characterize in vitro a novel poly-L-lysine (PLL) and polyethylene glycol (PEG) copolymer-based NP containing fluorescent-tagged bovine serum albumin (BSA), and conjugated with ERY1, a 12 amino acid peptide with high affinity for the RBC membrane protein glycophorin A (ENP)., Results: Confocal and flow cytometry data suggest that ENPs efficiently and irreversibly bind to RBC, with approximately 70% of erythrocytes bound after 24 h in a physiologic flow loop model compared to 10% binding of NPs without ERY1. Under these conditions, synthesized ENPs were not toxic to the RBCs. The rheological parameters at the applied shear. (0-15 Pa) were not influenced by ENP attachment to the RBCs. However, at high concentration, the strong affinity of ENPs to the glycophorin-A reduced the deformability of the RBC., Conclusions: 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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Nearly 30 years 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. [ABSTRACT FROM AUTHOR]