Jonathan K. Pokorski

Stealth filaments: Polymer chain length and conformation affect the in vivo fate of PEGylated potato virus X.

Nanoparticles hold great promise for delivering medical cargos to cancerous tissues to enhance contrast and sensitivity of imaging agents or to increase specificity and efficacy of therapeutics. A growing body of data suggests that nanoparticle shape, in combination with surface chemistry, affects their in vivo fates, with elongated filaments showing enhanced tumor targeting and tissue penetration, while promoting immune evasion. The synthesis of high aspect ratio filamentous materials at the nanoscale remains challenging using synthetic routes; therefore we turned toward natures materials, developing and studying the filamentous structures formed by the plant virus potato virus X (PVX). We recently demonstrated that PVX shows enhanced tumor homing in various preclinical models. Like other nanoparticle systems, the proteinaceous platform is cleared from circulation and tissues by the mononuclear phagocyte system (MPS). To increase bioavailability we set out to develop PEGylated stealth filaments and evaluate the effects of PEG chain length and conformation on pharmacokinetics, biodistribution, as well as potential immune and inflammatory responses. We demonstrate that PEGylation effectively reduces immune recognition while increasing pharmacokinetic profiles. Stealth filaments show reduced interaction with cells of the MPS; the protein:polymer hybrids are cleared from the body tissues within hours to days indicating biodegradability and biocompatibility. Tissue compatibility is indicated with no apparent inflammatory signaling in vivo. Tailoring PEG chain length and conformation (brush vs. mushroom) allows tuning of the pharmacokinetics, yielding long-circulating stealth filaments for applications in nanomedicine.

Surface Modification of Melt Extruded Poly(ε-caprolactone) Nanofibers: Toward a New Scalable Biomaterial Scaffold

A photochemical modification of melt-extruded polymeric nanofibers is described. A bioorthogonal functional group is used to decorate fibers made exclusively from commodity polymers, covalently attach fluorophores and peptides, and direct cell growth. Our process begins by using a layered coextrusion method, where poly(ε-caprolactone) (PCL) nanofibers are incorporated within a macroscopic poly(ethylene oxide) (PEO) tape through a series of die multipliers within the extrusion line. The PEO layer is then removed with a water wash to yield rectangular PCL nanofibers with controlled cross-sectional dimensions. The fibers can be subsequently modified using photochemistry to yield a “clickable” handle for performing the copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction on their surface. We have attached fluorophores, which exhibit dense surface coverage when using ligand-accelerated CuAAC reaction conditions. In addition, an RGD peptide motif was coupled to the surface of the fibers. Subsequent cell-based studies have shown that the RGD peptide is biologically accessible at the surface, leading to increased cellular adhesion and spreading versus PCL control surfaces. This functionalized coextruded fiber has the advantages of modularity and scalability, opening a potentially new avenue for biomaterials fabrication.

Proteins as substrates for controlled radical polymerization

This review describes fundamental contributions in the area of proteins as macroinitiators and macro-chain transfer agents (CTA) for controlled radical polymerization (CRP). The review specifically highlights the concept of ‘grafting-from’ proteins, as new and efficient chemistry has been developed to polymerize directly from protein substrates in aqueous media. As the past ten years have shown, CRP has proven a powerful tool in the functionalization of proteins. This review considers the methods used to install protein based initiators and CTAs, the principle examples of ATRP and RAFT as polymerization methods, and finishes with more advanced methodologies such as the combination of genetic modifications and polymer chemistry, proteins as nanoparticles for drug delivery, and unnatural amino acid initiators.

3D Printing Biocompatible Polyurethane/Poly(lactic acid)/Graphene Oxide Nanocomposites: Anisotropic Properties

Blending thermoplastic polyurethane (TPU) with poly(lactic acid) (PLA) is a proven method to achieve a much more mechanically robust material, whereas the addition of graphene oxide (GO) is increasingly applied in polymer nanocomposites to tailor further their properties. On the other hand, additive manufacturing has high flexibility of structure design which can significantly expand the application of materials in many fields. This study demonstrates the fused deposition modeling (FDM) 3D printing of TPU/PLA/GO nanocomposites and its potential application as biocompatible materials. Nanocomposites are prepared by solvent-based mixing process and extruded into filaments for FDM printing. The addition of GO largely enhanced the mechanical property and thermal stability of the nanocomposites. Interestingly, we found that the mechanical response is highly dependent on printing orientation. Furthermore, the 3D printed nanocomposites exhibit good biocompatibility with NIH3T3 cells, indicating promise as biomaterials scaffold for tissue engineering applications.

Coextruded, Aligned, and Gradient-Modified Poly(ε-caprolactone) Fibers as Platforms for Neural Growth

Polymeric fibers are of increasing interest to regenerative medicine, as materials made from these fibers are porous, allowing for cell infiltration, influx of nutrients, and efflux of waste products. Recently, multilayered coextrusion has emerged as a scalable and rapid fabrication method to yield microscale to submicron fibers. In this report, we describe the multilayered coextrusion of aligned poly(ε-caprolactone) (PCL) fibers, followed by a simple photochemical patterning to create surface-immobilized gradients onto the polymer fibers. PCL fibers were photochemically decorated with a linear gradient of propargyl benzophenone using a gradient photomask to control light source intensity. The pendant alkynes were then able to undergo the copper-catalyzed azide-alkyne cycloaddition reaction with an azide-modified IKVAV peptide to further functionalize the surface. Gradient-modified IKVAV fibers were evaluated for neural cell adhesion and neural differentiation, using PC-12 cells cultured onto the fibers. The aligned gradient fibers provided directional cues for neurite outgrowth and alignment of neural cells, as observed by cellular elongation, neurite differentiation, and orientation. The work presented herein describes a scalable fiber system combined with simple chemical patterning to generate aligned fibers with controlled surface gradients as cell-seeding scaffolds.

Photodynamic activity of viral nanoparticles conjugated with C60

The development of viral nanoparticles (VNP) displaying multiple copies of the buckyball (C(60)) and their photodynamic activity is described. VNP-C(60) conjugates were assembled using click chemistry. Cell uptake and cell killing using white light therapy and a prostate cancer cell line is demonstrated.

Multifunctional and Spatially Controlled Bioconjugation to Melt Coextruded Nanofibers

Polymeric fibers have drawn recent interest for uses in biomedical technologies that span drug delivery, regenerative medicine, and wound-healing patches, amongst others. We have recently reported a new class of fibrous biomaterials fabricated using coextrusion and a photochemical modification procedure to introduce functional groups onto the fibers. In this report, we extend our methodology to control surface modification density, describe methods to synthesize multifunctional fibers, and provide methods to spatially control functional group modification. Several different functional fibers are reported for bioconjugation, including propargyl, alkene, alkoxyamine, and ketone modified fibers. The modification scheme allows for control over surface density and provides a handle for downstream functionalization with appropriate bioconjugation chemistries. Through the use of multiple orthogonal chemistries, fiber chemistry could be differentially controlled to append multiple modifications. Spatial control on the fiber surface was also realized, leading to reverse gradients of small molecule dyes. One application is demonstrated for pH-responsive drug delivery of an anti-cancer therapeutics. Finally, the introduction of orthogonal chemical modifications onto these fibers allowed for modification with multiple cell-responsive peptides providing a substrate for osteoblast differentiation.

Polymer Structure and Conformation Alter the Antigenicity of Virus-like Particle–Polymer Conjugates

Covalent conjugation of water-soluble polymers to proteins is critical for evading immune surveillance in the field of biopharmaceuticals. The most common and long-standing polymer modification is the attachment of methoxypoly(ethylene glycol) (mPEG), termed PEGylation, which has led to several clinically approved pharmaceuticals. Recent data indicate that brush-type polymers significantly enhance in vitro and in vivo properties. Herein, the polymer conformation of poly(ethylene glycol) is detailed and compared with those of water-soluble polyacrylate and polynorbornene (PNB) when attached to icosahedral virus-like particles. Small-angle neutron scattering reveals vastly different polymer conformations of the multivalent conjugates. Immune recognition of conjugated particles was evaluated versus PEGylated particles, and PNB conjugation demonstrated the most effective shielding from antibody recognition.

“Graft-to” Protein/Polymer Conjugates Using Polynorbornene Block Copolymers

A series of water-soluble polynorbornene block copolymers prepared via Ring-Opening Metathesis Polymerization (ROMP) were grafted to proteins to form ROMP-derived bioconjugates. ROMP afforded low-dispersity polymers and allowed for strict control over polymer molecular weight and architecture. The polymers consisted of a large block of PEGylated monoester norbornene and were capped with a short block of norbornene dicarboxylic anhydride. This cap served as a reactive linker that facilitated attachment of the polymer to lysine residues under mildly alkaline conditions. The generality of this approach was shown by synthesizing multivalent polynorbornene-modified viral nanoparticles derived from bacteriophage Qβ, a protein nanoparticle used extensively for nanomedicine. The conjugated nanoparticles showed no cytotoxicity to NIH 3T3 murine fibroblast cells. These findings establish protein bioconjugation with functionalized polynorbornenes as an effective alternative to conventional protein/polymer modification strategies and further expand the toolbox for protein bioconjugates.

PEGylation to Improve Protein Stability During Melt Processing.

Biopharmaceuticals are some of the most effective drugs on the market, however, delivery remains a challenge. Melt processing is a viable protein encapsulation method because it is solvent free, is high throughput, and yields very high encapsulation efficiencies. Problematically, proteins can lose activity during melt processing due to high heat and shear forces. Covalent attachment of poly(ethylene glycol), or PEGylation, has been widely used to increase thermal stability and prevent aggregation in solution. This study explored the effect of PEGylation on protein stability during melt processing using lysozyme and PLGA. The results indicate that PEGylation increases the retained activity of lysozyme, increases dispersion in the melt, and reduces the biphasic release profile in melt processed systems.