Conjugation of Amphiphilic Proteins to Hydrophobic Ligands in Organic Solvent.

Protein-ligand conjugations are usually carried out in aqueous media in order to mimic the environment within which the conjugates will be used. In this work, we focus on the conjugation of amphiphilic variants of elastin-like polypeptide (ELP), short elastin (sEL), to poorly water-soluble compounds like OPPVs ( p-phenylenevinylene oligomers), triarylamines, and polypyridine-metal complexes. These conjugations are problematic when carried out in aqueous phase because hydrophobic ligands tend to avoid exposure to water, which in turn causes the ligand to self-aggregate and/or interact noncovalently with hydrophobic regions of the amphiphile. Ultimately, this behavior leads to low conjugation efficiency and contamination with strong noncovalent "conjugates". After exploring the solubility of sEL in various organic solvents, we have established an efficient conjugation methodology for obtaining covalent conjugates virtually free of contaminating noncovalent complexes. When conjugating carboxylated ligands to the amphiphile amines, we demonstrate that even when only one amine (the N-terminus) is present, its derivatization is 98% efficient. When conjugating amine moieties to the amphiphile carboxyls (a problematic configuration), protein multimerization is avoided, 98-100% of the protein is conjugated, and the unreacted ligand is recovered in pure form. Our syntheses occur in "one pot", and our purification procedure is a simple workup utilizing a combination of water and organic solvent extractions. This conjugation methodology might provide a solution to problems arising from solubility mismatch of protein and ligand, and it is likely to be widely applied for modification of recombinant amphiphiles used for drug delivery (PEG-antibodies, polymer-enzymes, food proteins), cell adhesion (collagen, hydrophobins), synthesis of nanostructures (peptides), and engineering of biocompatible optoelectronics (biological polymers), to cite a few.

Macromolecular Cargo Encapsulation via In Vitro Assembly of Two-Component Protein Nanoparticles.

Self-assembling protein nanoparticles are a promising class of materials for targeted drug delivery. Here, the use of a computationally designed, two-component, icosahedral protein nanoparticle is reported to encapsulate multiple macromolecular cargoes via simple and controlled self-assembly in vitro. Single-stranded RNA molecules between 200 and 2500 nucleotides in length are encapsulated and protected from enzymatic degradation for up to a month with length-dependent decay rates. Immunogenicity studies of nanoparticles packaging synthetic polymers carrying a small-molecule TLR7/8 agonist show that co-delivery of antigen and adjuvant results in a more than 20-fold increase in humoral immune responses while minimizing systemic cytokine secretion associated with free adjuvant. Coupled with the precise control over nanoparticle structure offered by computational design, robust and versatile encapsulation via in vitro assembly opens the door to a new generation of cargo-loaded protein nanoparticles that can combine the therapeutic effects of multiple drug classes.

Arming Immune Cell Therapeutics with Polymeric Prodrugs.

Engineered immune cells are an exciting therapeutic modality, which survey and attack tumors. Backpacking strategies exploit cell targeting capabilities for delivery of drugs to combat tumors and their immune-suppressive environments. Here, a new platform for arming cell therapeutics through dual receptor and polymeric prodrug engineering is developed. Macrophage and T cell therapeutics are engineered to express a bioorthogonal single chain variable fragment receptor. The receptor binds a fluorescein ligand that directs cell loading with ligand-tagged polymeric prodrugs, termed "drugamers." The fluorescein ligand facilitates stable binding of drugamer to engineered macrophages over 10 days with 80% surface retention. Drugamers also incorporate prodrug monomers of the phosphoinositide-3-kinase inhibitor, PI-103. The extended release of PI-103 from the drugamer sustains antiproliferative activity against a glioblastoma cell line compared to the parent drug. The versatility and modularity of this cell arming system is demonstrated by loading T cells with a second fluorescein-drugamer. This drugamer incorporates a small molecule estrogen analog, CMP8, which stabilizes a degron-tagged transgene to provide temporal regulation of protein activity in engineered T cells. These results demonstrate that this bioorthogonal receptor and drugamer system can be used to arm multiple immune cell classes with both antitumor and transgene-activating small molecule prodrugs.

Fully synthetic injectable depots with high drug content and tunable pharmacokinetics for long-acting drug delivery.

Clinical studies have validated that antiretroviral (ARV) drugs can serve as an HIV pre-exposure prophylactic (PrEP) strategy. Dosing adherence remains a crucial factor determining the final efficacy outcomes, and both long-acting implants and injectable depot systems are being developed to improve patient adherence. Here, we describe an injectable depot platform that exploits a new mechanism for both formation and controlled release. The depot is a polymeric prodrug synthesized from monomers that incorporate an ARV drug tenofovir alafenamide (TAF) with degradable linkers that can be designed to control release rates. The prodrug monomers are synthetically incorporated into homopolymer or block designs that exhibit high drug weight percent (wt%) and also are hydrophobized in these prodrug segments to drive depot formation upon injection. Drug release converts those monomers to more hydrophilic pendant groups via linker cleavage, and as this drug release proceeds, the polymer chains losing hydrophobicity are then disassociated from the depot and released over time to provide a depot dissolution mechanism. We show that long-acting TAF depots can be designed as block copolymers or as homopolymers. They can also be designed with different linkers, for example with faster or slower degrading p-hydroxybenzyloxycarbonyl (Benzyl) and ethyloxycarbonyl (Alkyl) linkers, respectively. Diblock designs of p(glycerol monomethacrylate)-b-p(Alkyl-TAF-methacrylate) and p(glycerol monomethacrylate)-b-p(Benzyl-TAF-methacrylate) were first characterized in a mouse subcutaneous injection model. The alkylcarbamate linker design (TAF 51 wt%) showed excellent sustained release profiles of the key metabolite tenofovir (TFV) in skin and plasma over a 50-day period. Next, the homopolymer design with a high TAF drug wt% of 73% was characterized in the same model. The homopolymer depots with p(Alkyl-TAFMA) exhibited sustained TFV and TAF release profiles in skin and blood over 60 days, and TFV-DP concentrations in peripheral blood mononuclear cells (PBMC) were found to be at least 10-fold higher than the clinically suggested minimally EC90 protective concentration of 24 fmol/106 cells. These are the first reports of sustained parent TAF dosing observed in mouse and TFV-DP in mouse PBMC. IVIS imaging of rhodamine labeled homopolymer depots showed that degradation and release of the depot coincided with the sustained TAF release. Finally, these polymers showed excellent stability in accelerated stability studies over a six-month time period, and exceptional solubility of over 700 mg/mL in the DMSO formulation solvent. The homopolymer designs have a drug reservoir potential of well over a year at mg/day dosing and may not require cold chain storage for global health and developed world long-acting drug delivery applications.