Constructing Antiretroviral Supramolecular Polymers as Long-Acting Injectables through Rational Design of Drug Amphiphiles with Alternating Antiretroviral-Based and Hydrophobic Residues.

One of the main challenges in the development of long-acting injectables for HIV treatment is the limited duration of drug release, which results in the need for frequent dosing and reduced patient adherence. In this context, we leverage the intrinsic reversible features of supramolecular polymers and their unique ability to form a three-dimensional network under physiological conditions to design a class of self-assembling drug amphiphiles (DAs) based upon lamivudine, a water-soluble antiretroviral (ARV) agent and nucleoside reverse transcriptase inhibitor. The designed ARV DAs contain three pairs of alternating hydrophobic valine (V) and hydrophilic lamivudine-modified lysine (K3TC) residues with a varying number of glutamic acids (E) placed on the C-terminus. Upon dissolution in deionized water, all three ARV DAs were found to spontaneously associate into supramolecular filaments of several micrometers in length, with varying levels of lateral stacking. Addition of 1× PBS triggered immediate gelation of the two ARV DAs with 2 or 3 E residues, and upon dilution in an in vitro setting, the dissociation from the supramolecular state to the monomeric state enabled a long-acting linear release of the ARV DAs. In vivo studies further confirmed their injectability, rapid in situ hydrogel formation, enhanced local retention, and long-acting therapeutic release over a month. Importantly, our pharmacokinetic studies suggest that the injected ARV supramolecular polymeric hydrogel was able to maintain a plasma concentration of lamivudine above its IC50 value for more than 40 days in mice and showed minimal systemic immunogenicity. We believe that these results shed important light on the rational design of long-acting injectables using the drug-based molecular assembly strategy, and the reported ARV supramolecular hydrogels hold great promise for improving HIV treatment outcomes.

The role of critical micellization concentration in efficacy and toxicity of supramolecular polymers.

The inception and development of supramolecular chemistry have provided a vast library of supramolecular structures and materials for improved practice of medicine. In the context of therapeutic delivery, while supramolecular nanostructures offer a wide variety of morphologies as drug carriers for optimized targeting and controlled release, concerns are often raised as to how their morphological stability and structural integrity impact their in vivo performance. After intravenous (i.v.) administration, the intrinsic reversible and dynamic feature of supramolecular assemblies may lead them to dissociate upon plasma dilution to a concentration below their critical micellization concentration (CMC). As such, CMC represents an important characteristic for supramolecular biomaterials design, but its pharmaceutical role remains elusive. Here, we report the design of a series of self-assembling prodrugs (SAPDs) that spontaneously associate in aqueous solution into supramolecular polymers (SPs) with varying CMCs. Two hydrophobic camptothecin (CPT) molecules were conjugated onto oligoethylene-glycol (OEG)-decorated segments with various OEG repeat numbers (2, 4, 6, 8). Our studies show that the lower the CMC, the lower the maximum tolerated dose (MTD) in rodents. When administrated at the same dosage of 10 mg/kg (CPT equivalent), SAPD 1, the one with the lowest CMC, shows the best efficacy in tumor suppression. These observations can be explained by the circulation and dissociation of SAPD SPs and the difference in molecular and supramolecular distribution between excretion and organ uptake. We believe these findings offer important insight into the role of supramolecular stability in determining their therapeutic index and in vivo efficacy.

Selective Capture and Recovery of Monoclonal Antibodies by Self-Assembling Supramolecular Polymers of High Affinity for Protein Binding.

The separation and purification of therapeutic proteins from their biological resources pose a great limitation for industrial manufacturing of biologics in an efficient and cost-effective manner. We report here a supramolecular polymeric system that can undergo multiple reversible processes for efficient capture, precipitation, and recovery of monoclonal antibodies (mAbs). These supramolecular polymers, namely immunofibers (IFs), are formed by coassembly of a mAb-binding peptide amphiphile with a rationally designed filler molecule of varying stoichiometric ratios. Under the optimized conditions, IFs can specifically capture mAbs with a precipitation yield greater than 99%, leading to an overall mAb recovery yield of 94%. We also demonstrated the feasibility of capturing and recovering two mAbs from clarified cell culture harvest. These results showcase the promising potential of peptide-based supramolecular polymers as reversible affinity precipitants for mAb purification.

Supramolecular Design of Unsymmetric Reverse Bolaamphiphiles for Cell-Sensitive Hydrogel Degradation and Drug Release.

Self-assembly of peptide-based building units into supramolecular nanostructures creates an important class of biomaterials with robust mechanical properties and improved resistance to premature degradation. Yet, upon aggregation, substrate-enzyme interactions are often compromised because of the limited access of macromolecular proteins to the peptide substrate, leading to either a reduction or loss of responsiveness to biomolecular cues. Reported here is the supramolecular design of unsymmetric reverse bolaamphiphiles (RBA) capable of exposing a matrix metalloproteinase (MMP) substrate on the surface of their filamentous assemblies. Upon addition of MMP-2, these filaments rapidly break into fragments prior to reassembling into spherical micelles. Using 3D cell culture, it is shown that drug release is commensurate with cell density, revealing more effective cell killing when more cancer cells are present. This design platform could serve as a cell-responsive therapeutic depot for local chemotherapy.

Macrocyclization of a Class of Camptothecin Analogues into Tubular Supramolecular Polymers.

Nanostructured supramolecular polymers (SPs) are filamentous assemblies possessing a high degree of internal order and have important uses in regenerative medicine, drug delivery, and soft matter electronics. Despite recent advances in functional SPs, a challenging topic is the development of robust assembly protocols enabling the incorporation of various functional units without altering its supramolecular architecture. We report here the robust tubular assembly of camptothecin (CPT) analogues into functional SPs. Covalent linkage of two CPT moieties to various short hydrophilic segments (e.g., nonionic, cationic, anionic, and zwitterionic) leads to a class of CPT analogues that self-assemble in water into tubular SPs. Systemic administration of nonionic SPs effectively suppresses tumor growth. Furthermore, these tubular SPs act as universal dispersing agents in water for low-molecular-weight hydrophobes.

Self-healable, tough and highly stretchable ionic nanocomposite physical hydrogels.

We present a facile strategy to synthesize self-healable tough and highly stretchable hydrogels. Our design rationale for the creation of ionic cross-linked hydrogels is to graft an acrylic acid monomer on the surface of vinyl hybrid silica nanoparticles (VSNPs) for the growth of poly(acrylic) acid (PAA), and the obtained VSNP-PAA nanobrush can be used as a gelator. Physical cross-linking through hydrogen bonding and ferric ion-mediated ionic interactions between PAA polymer chains of the gelators yielded ionic nanocomposite physical hydrogels with excellent and balanced mechanical properties (tensile strength 860 kPa, elongation at break ∼2300%), and the ability to self-repair (tensile strength ∼560 kPa, elongation at break ∼1800%). The toughness and stretchability arise from the reversible cross-linking interactions between the polymer chains that help dissipate energy through stress (deformation) triggered dynamic processes. These unique properties will enable greater application of these hydrogel materials, especially in tissue engineering.

Rational design of MMP degradable peptide-based supramolecular filaments.

One-dimensional nanostructures formed by self-assembly of small molecule peptides have been extensively explored for use as biomaterials in various biomedical contexts. However, unlike individual peptides that can be designed to be specifically degradable by enzymes/proteases of interest, their self-assembled nanostructures, particularly those rich in ß-sheets, are generally resistant to enzymatic degradation because the specific cleavage sites are often embedded inside the nanostructures. We report here on the rational design of ß-sheet rich supramolecular filaments that can specifically dissociate into less stable micellar assemblies and monomers upon treatment with matrix metalloproteases-2 (MMP-2). Through linkage of an oligoproline segment to an amyloid-derived peptide sequence, we first synthesized an amphiphilic peptide that can undergo a rapid morphological transition in response to pH variations. We then used MMP-2 specific peptide substrates as multivalent cross-linkers to covalently fix the amyloid-like filaments in the self-assembled state at pH 4.5. Our results show that the cross-linked filaments are stable at pH 7.5 but gradually break down into much shorter filaments upon cleavage of the peptidic cross-linkers by MMP-2. We believe that the reported work presents a new design platform for the creation of amyloid-like supramolecular filaments responsive to enzymatic degradation.

Multimeric disintegrin protein polymer fusions that target tumor vasculature.

Recombinant protein therapeutics have increased in number and frequency since the introduction of human insulin, 25 years ago. Presently, proteins and peptides are commonly used in the clinic. However, the incorporation of peptides into clinically approved nanomedicines has been limited. Reasons for this include the challenges of decorating pharmaceutical-grade nanoparticles with proteins by a process that is robust, scalable, and cost-effective. As an alternative to covalent bioconjugation between a protein and nanoparticle, we report that biologically active proteins may themselves mediate the formation of small multimers through steric stabilization by large protein polymers. Unlike multistep purification and bioconjugation, this approach is completed during biosynthesis. As proof-of-principle, the disintegrin protein called vicrostatin (VCN) was fused to an elastin-like polypeptide (A192). A significant fraction of fusion proteins self-assembled into multimers with a hydrodynamic radius of 15.9 nm. The A192-VCN fusion proteins compete specifically for cell-surface integrins on human umbilical vein endothelial cells (HUVECs) and two breast cancer cell lines, MDA-MB-231 and MDA-MB-435. Confocal microscopy revealed that, unlike linear RGD-containing protein polymers, the disintegrin fusion protein undergoes rapid cellular internalization. To explore their potential clinical applications, fusion proteins were characterized using small animal positron emission tomography (microPET). Passive tumor accumulation was observed for control protein polymers; however, the tumor accumulation of A192-VCN was saturable, which is consistent with integrin-mediated binding. The fusion of a protein polymer and disintegrin results in a higher intratumoral contrast compared to free VCN or A192 alone. Given the diversity of disintegrin proteins with specificity for various cell-surface integrins, disintegrin fusions are a new source of biomaterials with potential diagnostic and therapeutic applications.

Antiviral supramolecular polymeric hydrogels by self-assembly of tenofovir-bearing peptide amphiphiles.

The development of long-acting antiviral therapeutic delivery systems is crucial to improve the current treatment and prevention of HIV and chronic HBV. We report here on the conjugation of tenofovir (TFV), an FDA approved nucleotide reverse transcriptase inhibitor (NRTI), to rationally designed peptide amphiphiles (PAs), to construct antiviral prodrug hydrogelators (TFV-PAs). The resultant conjugates can self-assemble into one-dimensional nanostructures in aqueous environments and consequently undergo rapid gelation upon injection into 1× PBS solution to create a drug depot. The TFV-PA designs containing two or three valines could attain instantaneous gelation, with one displaying sustained release for more than 28 days in vitro. Our studies suggest that minor changes in peptide design can result in differences in supramolecular morphology and structural stability, which impacted in vitro gelation and release. We envision the use of this system as an important delivery platform for the sustained, linear release of TFV at rates that can be precisely tuned to attain therapeutically relevant TFV plasma concentrations.

[Distribution specificity of human fucosyltransferase 5 and its expression and localization in spermatids].

OBJECTIVE: To investigate distribution specificity of human fucosyltransferase 5 (FUT5) as well as its expression and localization in spermatids. METHODS: Human semen, vaginal swab, saliva and venous blood from healthy individuals were collected. The spermatids were isolated and the spermatid membrane protein was then extracted. Expression levels of FUT5 from human spermatid membrane, seminal plasma, vaginal fluid, saliva and serum were detected by immunoblotting technique. The expression and localization of FUT5 in spermatids were analyzed by immunofluorescent method. RESULTS: Immunoblotting technique showed that FUT5 was expressed on spermatid membranes and in serum, but not in seminal plasma, vaginal fluid and saliva. The expressed FUT5 on spermatids was mostly localized on head of spermatids by fluorescent microscopy, suggesting that there was certain amount of FUT5 on human spermatid membrane, and the spermatids might be isolated from mixed stains with vaginal fluid by antigen-antibody reaction. CONCLUSION: Human FUT5 shows a characteristic distribution specificity, and this feature may be used for identification of mixed stain involved in criminal sexual offence in future forensic practice.

Discovery of Y-Shaped Supramolecular Polymers in a Self-Assembling Peptide Amphiphile System.

Supramolecular polymers (SPs) formed by self-assembly of peptide-based molecular units assume a variety of interesting one-dimensional (1D) morphologies. While the morphological complexity and phase behavior of self-assembling peptide conjugates bear some resemblance to those of low-molecular-weight and macromolecular surfactants, Y-junctions, or three-way connected constructs, a topological defect observed in traditional surfactants has not been identified, likely due to the intolerance of defective packing by the strong, associative interactions afforded by the peptide segments. Here we report our discovery of branched SPs with Y-junctions and occasionally enlarged spherical end-caps formed by micellization of a ferrocene-based peptide amphiphile in water. Our results suggest that the incorporation of two ferrocenes into the amphiphile design is key to ensure the formation of branched SPs. We hypothesize that the complex interplay of internal interactions limits the effective propagation of hydrogen bonding within the assemblies and, consequently, creates fragmented ß-sheets that are more tolerant for supramolecular branching. Given the redox sensitivity of the ferrocene units, sequential addition of reductants and oxidants to the solution led the assemblies to reversibly transform between branched SPs and spherical aggregates.

Leveraging the therapeutic, biological, and self-assembling potential of peptides for the treatment of viral infections.

Peptides and peptide-based materials have an increasing role in the treatment of viral infections through their use as active pharmaceutical ingredients, targeting moieties, excipients, carriers, or structural components in drug delivery systems. The discovery of peptide-based therapeutic compounds, coupled with the development of new stabilization and formulation strategies, has led to a resurgence of antiviral peptide therapeutics over the past two decades. The ability of peptides to bind cell receptors and to facilitate membrane penetration and subsequent intracellular trafficking enables their use in various antiviral systems for improved targeting efficiency and treatment efficacy. Importantly, the self-assembly of peptides into well-defined nanostructures provides a vast library of discrete constructs and supramolecular biomaterials for systemic and local delivery of antiviral agents. We review here the recent progress in exploiting the therapeutic, biological, and self-assembling potential of peptides, peptide conjugates, and their supramolecular assemblies in treating human viral infections, with an emphasis on the treatment strategies for Human Immunodeficiency Virus (HIV).

Multicompartment polymer nanostructures with ratiometric dual-emission pH-sensitivity.

Pyrazine-labeled multicompartment nanostructures are shown to exhibit enhanced pH-responsive blue-shifted fluorescence emission intensities compared to their simpler core-shell spherical analogs. An amphiphilic linear triblock terpolymer of ethylene oxide, N-acryloxysuccinimide, and styrene, PEO(45)-b-PNAS(105)-b-PS(45), which lacks significant incompatibility for the hydrophobic block segments and undergoes gradual hydrolysis of the NAS units, underwent supramolecular assembly in mixtures of organic solvent and water to afford multicompartment micelles (MCMs) with a narrow size distribution. The assembly process was followed over time and found to evolve from individual polymer nanodroplets containing internally phase segregated domains, of increasing definition, and ultimately to dissociate into discrete micelles. Upon covalent cross-linking of the MCMs with pH-insensitive pyrazine-based diamino cross-linkers, pH-responsive, photonic multicompartment nanostructures (MCNs) were produced. These MCNs exhibited significant enhancement of overall structural stability, in comparison with the MCMs, and internal structural tunability through the cross-linking chemistry. Meanwhile, the complex compartmentalized morphology exerted unique pH-responsive fluorescence dual-emission properties, indicating promise in ratiometric pH-sensing applications.

Theranostic supramolecular polymers formed by the self-assembly of a metal-chelating prodrug.

Therapeutic constructs with imaging modalities hold great promise for improving the treatment efficacy for cancer and many other diseases. We report here the design and synthesis of a self-assembling prodrug (SAPD) by the direct linkage of camptothecin (CPT), an anticancer drug, to a metal-chelating agent, DOTA. We found that under physiological conditions the DOTA-conjugated CPT prodrug can self-assemble into tubular supramolecular polymers (SPs) with a length of several micrometers. Our studies also suggest that the resultant assemblies were stable in biological environments and exhibited a fast drug release rate in the presence of intracellular glutathione. Furthermore, the SAPD exhibited remarkable in vitro efficacy against various cancer cell lines and effectively inhibited the growth of tumor spheroids. We believe that the design and optimization of self-assembling theranostic conjugates could provide a robust yet simple platform for the development of new imaging-guided drug delivery systems.

Crafting Polymeric and Peptidic Hydrogels for Improved Wound Healing.

Wound healing is a multifaceted biological process involving the replacement of damaged tissues and cellular structures, restoring the skin barrier's function, and maintaining internal homeostasis. Over the past two decades, numerous approaches are undertaken to improve the quality and healing rate of complex acute and chronic wounds, including synthetic and natural polymeric scaffolds, skin grafts, and supramolecular hydrogels. In this context, this review assesses the advantages and drawbacks of various types of supramolecular hydrogels including both polymeric and peptide-based hydrogels for wound healing applications. The molecular design features of natural and synthetic polymers are examined, as well as therapeutic-based and drug-free peptide hydrogels, and the strategies for each system are analyzed to integrate key elements such as biocompatibility, bioactivity, stimuli-responsiveness, site specificity, biodegradability, and clearance.

Emerging biomaterials for downstream manufacturing of therapeutic proteins.

Downstream processing is considered one of the most challenging phases of industrial manufacturing of therapeutic proteins, accounting for a large portion of the total production costs. The growing demand for therapeutic proteins in the biopharmaceutical market in addition to a significant rise in upstream titers have placed an increasing burden on the downstream purification process, which is often limited by high cost and insufficient capacities. To achieve efficient production and reduced costs, a variety of biomaterials have been exploited to improve the current techniques and also to develop superior alternatives. In this work, we discuss the significance of utilizing traditional biomaterials in downstream processing and review the recent progress in the development of new biomaterials for use in protein separation and purification. Several representative methods will be highlighted and discussed in detail, including affinity chromatography, non-affinity chromatography, membrane separations, magnetic separations, and precipitation/phase separations. STATEMENT OF SIGNIFICANCE: Nowadays, downstream processing of therapeutic proteins is facing great challenges created by the rapid increase of the market size and upstream titers, starving for significant improvements or innovations in current downstream unit operations. Biomaterials have been widely used in downstream manufacturing of proteins and efforts have been continuously devoted to developing more advanced biomaterials for the implementation of more efficient and economical purification methods. This review covers recent advances in the development and application of biomaterials specifically exploited for various chromatographic and non-chromatographic techniques, highlighting several promising alternative strategies.

Kinetic Control in Assembly of Plasmid DNA/Polycation Complex Nanoparticles.

Polyelectrolyte complex (PEC) nanoparticles assembled from plasmid DNA (pDNA) and polycations such as linear polyethylenimine (lPEI) represent a major nonviral delivery vehicle for gene therapy tested thus far. Efforts to control the size, shape, and surface properties of pDNA/polycation nanoparticles have been primarily focused on fine-tuning the molecular structures of the polycationic carriers and on assembly conditions such as medium polarity, pH, and temperature. However, reproducible production of these nanoparticles hinges on the ability to control the assembly kinetics, given the nonequilibrium nature of the assembly process and nanoparticle composition. Here we adopt a kinetically controlled mixing process, termed flash nanocomplexation (FNC), that accelerates the mixing of pDNA solution with polycation lPEI solution to match the PEC assembly kinetics through turbulent mixing in a microchamber. This achieves explicit control of the kinetic conditions for pDNA/lPEI nanoparticle assembly, as demonstrated by the tunability of nanoparticle size, composition, and pDNA payload. Through a combined experimental and simulation approach, we prepared pDNA/lPEI nanoparticles having an average of 1.3 to 21.8 copies of pDNA per nanoparticle and average size of 35 to 130 nm in a more uniform and scalable manner than bulk mixing methods. Using these nanoparticles with defined compositions and sizes, we showed the correlation of pDNA payload and nanoparticle formulation composition with the transfection efficiencies and toxicity in vivo. These nanoparticles exhibited long-term stability at -20 °C for at least 9 months in a lyophilized formulation, validating scalable manufacture of an off-the-shelf nanoparticle product with well-defined characteristics as a gene medicine.

Preclinical development of drug delivery systems for paclitaxel-based cancer chemotherapy.

Paclitaxel (PTX) is one of the most successful drugs ever used in cancer chemotherapy, acting against a variety of cancer types. Formulating PTX with Cremophor EL and ethanol (Taxol®) realized its clinical potential, but the formulation falls short of expectations due to side effects such as peripheral neuropathy, hypotension, and hypersensitivity. Abraxane®, the albumin bound PTX, represents a superior replacement of Taxol® that mitigates the side effects associated with Cremophor EL. While Abraxane® is now considered a gold standard in chemotherapy, its 21% response rate leaves much room for further improvement. The quest for safer and more effective cancer treatments has led to the development of a plethora of innovative PTX formulations, many of which are currently undergoing clinical trials. In this context, we review recent development of PTX drug delivery systems and analyze the design principles underpinning each delivery strategy. We chose several representative examples to highlight the opportunities and challenges of polymeric systems, lipid-based formulations, as well as prodrug strategies.

Isomeric control of the mechanical properties of supramolecular filament hydrogels.

Supramolecular filament hydrogels are an emerging class of biomaterials that hold great promise for regenerative medicine, tissue engineering, and drug delivery. However, fine-tuning of their bulk mechanical properties at the molecular level without altering their network structures remains a significant challenge. Here we report an isomeric strategy to construct amphiphilic peptides through the conjugation of isomeric hydrocarbons to influence the local viscoelastic properties of their resulting supramolecular hydrogels. In this case, the packing requirements of the chosen isomeric hydrocarbons within the supramolecular filaments are dictated by their atomic arrangements at the molecular and intermolecular levels. Atomistic molecular dynamics simulations suggest that this design strategy can subtly alter the molecular packing at the interface between the peptide domain and the hydrophobic core of the supramolecular assemblies, without changing both the filament width and morphology. Our results from wide-angle X-ray scattering and molecular simulations further confirm that alterations to the intermolecular packing at the interface impact the strength and degree of hydrogen bonding within the peptide domains. This subtle difference in the isomeric hydrocarbon design and their consequent packing difference led to variations in the persistence length of the individual supramolecular filaments. Microrheological analysis reveals that this difference in filament stiffness enables the fine-tuning of the mechanical properties of the hydrogel at the macroscopic scale. We believe that this isomeric platform provides an innovative method to tune the local viscoelastic properties of supramolecular polymeric hydrogels without necessarily altering their network structures.

Molecularly engineered self-assembling membranes for cell-mediated degradation.

The use of peptide engineering to develop self-assembling membranes that are responsive to cellular enzyme activities is reported. The membranes are obtained by combining hyaluronan (HA) and a rationally designed peptide amphiphile (PA) containing a proteolytic domain (GPQGIWGQ octapeptide) sensitive to matrix metalloproteinase-1 (MMP-1). Insertion of an octapeptide in a typical PA structure does not disturb its self-assembly into fibrillar nanostructures neither the ability to form membranes with HA. In vitro enzymatic degradation with hyaluronidase and MMP-1 shows that membranes containing the MMP-1 substrate exhibit enhanced enzymatic degradation, compared with control membranes (absence of MMP-1 cleavable peptide or containing a MMP-1 insensitive sequence), being completely degraded after 7 days. Cell viability and proliferation is minimally affected by the enzymatically cleavable functionality of the membrane, but the presence of MMP-1 cleavable sequence does stimulate the secretion of MMP-1 by fibroblasts and interfere with matrix deposition, particularly the deposition of collagen. By showing cell-responsiveness to biochemical signals presented on self-assembling membranes, this study highlights the ability of modulating certain cellular activities through matrix engineering. This concept can be further explored to understand the cellular remodeling process and as a strategy to develop artificial matrices with more biomimetic degradation for tissue engineering applications.