Kinetically Controlled Polyelectrolyte Complex Assembly of microRNA-Peptide Nanoparticles toward Treating Mesothelioma.

Broad size distributions and poor long-term colloidal stability of microRNA-carrying nanoparticles, especially those formed by polyelectrolyte complexation, represent major hurdles in realizing their clinical translation. Herein, peptide design is used alongside optimized flash nanocomplexation (FNC) to produce uniform peptide-based miRNA particles of exceptional stability that display anticancer activity against mesothelioma in vitro and in vivo. Modulating the content and display of lysine-based charge from small intrinsically disordered peptides used to complex miRNA proves essential in achieving stable colloids. FNC facilitates kinetic isolation of the mechanistic steps involved in particle formation to allow the preparation of particles of discrete size in a highly reproducible, scalable, and continuous manner, facilitating pre-clinical studies. To the best of the authors knowledge, this work represents the first example of employing FNC to prepare polyelectrolyte complexes of miRNA and peptide. Encapsulation of these particles into an injectable hydrogel matrix allows for their localized in vivo delivery by syringe. A one-time injection of a gel containing particles composed of miRNA-215-5p and the peptide PKM1 limits tumor progression in a xenograft model of mesothelioma.

The Design of a Participatory Peptide Nucleic Acid Duplex Crosslinker to Enhance the Stiffness of Self-Assembled Peptide Gels.

Herein, peptide nucleic acids (PNAs) are employed in the design of a participatory duplex PNA-peptide crosslinking agent. Biophysical and mechanical studies show that crosslinkers present during peptide assembly leading to hydrogelation participate in the formation of fibrils while simultaneously installing crosslinks into the higher-order network that constitutes the peptide gel. The addition of 2 mol % crosslinker into the assembling system results in a ~100 % increase in mechanical stiffness without affecting the rate of peptide assembly or the local morphology of fibrils within the gel network. Stiffness enhancement is realized by only affecting change in the elastic component of the viscoelastic gel. A synthesis of the PNA-peptide duplex crosslinkers is provided that allows facile variation in peptide composition and addresses the notorious hydrophobic content of PNAs. This crosslinking system represents a new tool for modulating the mechanical properties of peptide-based hydrogels.

De Novo Design of a Versatile Peptide-Based Coating to Impart Targeted Functionality at the Surface of Native Polystyrene.

Peptide sequence periodicity is a simple design tool that can be used to generate functional peptide-based surface coatings. De novo-designed peptide N3-PEG-VK16 is characterized by a hydrophobic periodicity of two that avidly binds to native polystyrene priming its surface for subsequent targeted functionalization via chemical ligation. The peptidic portion of N3-PEG-VK16 is responsible for surface binding, converting polystyrene's hydrophobic surface into a wettable and electrostatically charged environment that facilitates cell attachment. Native polystyrene surfaces are coated by simple peptide adsorption from an aqueous buffered solution, and the resulting primed surface is easily functionalized by cycloaddition chemistry. Herein, we show that ligating a vitronectin-derived peptide to primed polystyrene surfaces enables adhesion, expansion, long-term culture, and phenotype maintenance of human induced pluripotent stem cells. To demonstrate scope, we also show that additional functional ligands can be used, for example, nerve growth factor protein, to control neurite outgrowth.

Supramolecular Filament Hydrogel as a Universal Immunomodulator Carrier for Immunotherapy Combinations.

A major challenge of cancer immunotherapy is to develop delivery strategies that can effectively and safely augment the immune system's antitumor response. Here, we report on the design and synthesis of a peptide-based supramolecular filament (SF) hydrogel as a universal carrier for localized delivery of three immunomodulating agents of distinct action mechanisms and different molecular weights, including an aPD1 antibody, an IL15 cytokine, and a STING agonist (CDA). We show that in situ hydrogelation can be triggered to occur upon intratumoral injection of SF solutions containing each of aPD1, IL15, or CDA. The formed hydrogel serves as a scaffold depot for sustained and MMP-2-responsive release of immunotherapeutic agents, achieving enhanced antitumor activities and reduced side effects. When administered in combination, the aPD1/IL15 or aPD1/CDA hydrogel led to substantially increased T-cell infiltration and prevented the development of adaptive immune resistance induced by IL15 or CDA alone. These immunotherapy combinations resulted in complete regression of established large GL-261 tumors in all mice and elicited a protective long-acting and systemic antitumor immunity to prevent tumor recurrence while eradicating distant tumors. We believe this SF hydrogel offers a simple yet generalizable strategy for local delivery of diverse immunomodulators for enhanced antitumoral response and improved treatment outcomes.

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.

Supramolecular filaments for concurrent ACE2 docking and enzymatic activity silencing enable coronavirus capture and infection prevention.

Coronaviruses have historically precipitated global pandemics of severe acute respiratory syndrome (SARS) into devastating public health crises. Despite the virus's rapid rate of mutation, all SARS coronavirus 2 (SARS-CoV-2) variants are known to gain entry into host cells primarily through complexation with angiotensin-converting enzyme 2 (ACE2). Although ACE2 has potential as a druggable decoy to block viral entry, its clinical use is complicated by its essential biological role as a carboxypeptidase and hindered by its structural and chemical instability. Here we designed supramolecular filaments, called fACE2, that can silence ACE2's enzymatic activity and immobilize ACE2 to their surface through enzyme-substrate complexation. This docking strategy enables ACE2 to be effectively delivered in inhalable aerosols and improves its structural stability and functional preservation. fACE2 exhibits enhanced and prolonged inhibition of viral entry compared with ACE2 alone while mitigating lung injury in vivo.

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).

Collagen-Binding Peptide-Enabled Supramolecular Hydrogel Design for Improved Organ Adhesion and Sprayable Therapeutic Delivery.

Spraying serves as an attractive, minimally invasive means of administering hydrogels for localized delivery, particularly due to high-throughput deposition of therapeutic depots over an entire target site of uneven surfaces. However, it remains a great challenge to design systems capable of rapid gelation after shear-thinning during spraying and adhering to coated tissues in wet, physiological environments. We report here on the use of a collagen-binding peptide to enable a supramolecular design of a biocompatible, bioadhesive, and sprayable hydrogel for sustained release of therapeutics. After spraying, the designed peptide amphiphile-based supramolecular filaments exhibit fast, physical cross-linking under physiological conditions. Our ex vivo studies suggest that the hydrogelator strongly adheres to the wet surfaces of multiple organs, and the extent of binding to collagen influences release kinetics from the gel. We envision that the sprayable organ-adhesive hydrogel can serve to enhance the efficacy of incorporated therapeutics for many biomedical applications.

Therapeutic supramolecular tubustecan hydrogel combined with checkpoint inhibitor elicits immunity to combat cancer.

The clinical benefit of PD-1/PD-L1 blockade immunotherapy is substantially restricted by insufficient infiltration of T lymphocytes into tumors and compromised therapeutic effects due to immune-related adverse events following systemic administration. Some chemotherapeutic agents have been reported to trigger tumor-associated T cell responses, providing a promising strategy to achieve potent immune activation in a synergistic manner with PD-1 blockade immunotherapy. In light of this, a localized chemoimmunotherapy system was developed using an anti-cancer drug-based supramolecular polymer (SP) hydrogel to "re-edit" the host's immune system to combat cancer. This in situ forming injectable aPD1/TT6 SP hydrogel serves as a drug-delivery depot for sustained release of bioactive camptothecin (CPT) and aPD1 into the tumor microenvironment, priming the tumor for robust infiltration of tumor-associated T cells and subsequently prompting a response to the immune checkpoint blockade. Our in vivo results demonstrate that this chemoimmunotherapy hydrogel provokes a long-term and systemic anticancer T cell immune response, which elicits tumor regression while also inhibiting tumor recurrence and potential metastasis.

Propagation-Instigated Self-Limiting Polymerization of Multiarmed Amphiphiles into Finite Supramolecular Polymers.

A fundamental goal in the noncovalent synthesis of ordered supramolecular polymers (SPs) is to achieve precise control over their size and size distribution; however, the reversible nature of noncovalent interactions often results in formation of living SPs with high dispersity in length. We report here on the self-limiting supramolecular polymerization (SPZ) of a series of multiarmed amphiphiles with propagation-attenuated reactivities that can automatically terminate the polymerization process, enabling effective control in both lengths and polydispersity. Through incorporating multiarmed oligoethylene-glycol (OEG) onto a quadratic aromatic segment, the lengths of the resultant SPs can be tuned from ∼1 µm to 130 and 50 nm with a polydispersity index of ∼1.2 for the last two SPs. We believe that the level of chain frustration of the multiarmed OEG segments, determined by both the number of arms and the degree of polymerization, poses physical and entropic constrains for supramolecular propagation to exceed a threshold length.

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.

Tumour sensitization via the extended intratumoural release of a STING agonist and camptothecin from a self-assembled hydrogel.

Tumours with an immunosuppressive microenvironment respond poorly to therapy. Activation of the stimulator of interferon genes (STING) pathway can enhance intratumoural immune activation, but STING agonists are associated with high toxicity and degrade prematurely, which limits their effectiveness. Here, we show that the extended intratumoural release of the STING agonist cyclic di-AMP transforms the tumour microenvironment from immunosuppressive to immunostimulatory, increasing the efficacy of antitumour therapies. The STING agonist was electrostatically complexed with nanotubes comprising a peptide-drug conjugate (a peptide that binds to the protein neuropilin-1, which is highly expressed in tumours, and the chemotherapeutic agent camptothecin) that self-assemble in situ into a supramolecular hydrogel. In multiple mouse models of murine tumours, a single low dose of the STING agonist led to tumour regression and increased animal survival, and to long-term immunological memory and systemic immune surveillance, which protected the mice against tumour recurrence and the formation of metastases. Locally delivered STING agonists could help to reduce tumour immunosuppression and enhance the efficacy of a wide range of cancer therapies.

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.

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.

Inhalable nanotherapeutics to improve treatment efficacy for common lung diseases.

Respiratory illnesses are prevalent around the world, and inhalation-based therapies provide an attractive, noninvasive means of directly delivering therapeutic agents to their site of action to improve treatment efficacy and limit adverse systemic side effects. Recent trends in medicine and nanoscience have prompted the development of inhalable nanomedicines to further enhance effectiveness, patient compliance, and quality of life for people suffering from lung cancer, chronic pulmonary diseases, and tuberculosis. Herein, we discuss recent advancements in the development of inhalable nanomaterial-based drug delivery systems and analyze several representative systems to illustrate their key design principles that can translate to improved therapeutic efficacy for prevalent respiratory diseases. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Respiratory Disease.

Interface-Enrichment-Induced Instability and Drug-Loading-Enhanced Stability in Inhalable Delivery of Supramolecular Filaments.

Filamentous microorganisms traveling in aerosol particles display enhanced deposition and retention in the lungs. Inspired by this shape-related biological effect, we report here on the use of supramolecular filaments as potential inhalable drug carriers within aerosols via jet nebulization. We found that the peptide design and supramolecular stability play a crucial role in the interfacial stability and aerosolization properties of the supramolecular filaments. Monomeric units with a positively charged C-terminus produced filaments with reduced aerosol stability, promoting morphological changes after nebulization. Conversely, having a neutral or negatively charged terminus yielded filaments with enhanced stability, where supramolecular integrity is maintained with only reduced length. Our results suggest that molecular enrichment at the air-liquid interface during nebulization is the primary factor to deplete the monomeric peptide amphiphiles in solution, accounting for the observed morphological disruption/transitions. Importantly, encapsulation of drugs and dyes within filaments notably stabilize their supramolecular structure during nebulization, and the loaded filaments exhibit a linear release profile from a nebulizer device. We envision the use of this supramolecular carrier system as an effective platform for the inhalation-based treatment of many lung diseases.

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.

Peptide-based nanoprobes for molecular imaging and disease diagnostics.

Pathological changes in a diseased site are often accompanied by abnormal activities of various biomolecules in and around the involved cells. Identifying the location and expression levels of these biomolecules could enable early-stage diagnosis of the related disease, the design of an appropriate treatment strategy, and the accurate assessment of the treatment outcomes. Over the past two decades, a great diversity of peptide-based nanoprobes (PBNs) have been developed, aiming to improve the in vitro and in vivo performances of water-soluble molecular probes through engineering of their primary chemical structures as well as the physicochemical properties of their resultant assemblies. In this review, we introduce strategies and approaches adopted for the identification of functional peptides in the context of molecular imaging and disease diagnostics, and then focus our discussion on the design and construction of PBNs capable of navigating through physiological barriers for targeted delivery and improved specificity and sensitivity in recognizing target biomolecules. We highlight the biological and structural roles that low-molecular-weight peptides play in PBN design and provide our perspectives on the future development of PBNs for clinical translation.

Role of cytoskeletal mechanics and cell membrane fluidity in the intracellular delivery of molecules mediated by laser-activated carbon nanoparticles.

Exposure of cells and nanoparticles to near-infrared nanosecond pulsed laser light can lead to efficient intracellular delivery of molecules while maintaining high cell viability by a photoacoustic phenomenon known as transient nanoparticle energy transduction (TNET). Here, we examined the influence of cytoskeletal mechanics and plasma membrane fluidity on intracellular uptake of molecules and loss of cell viability due to TNET. We found that destabilization of actin filaments using latrunculin A led to greater uptake of molecules and less viability loss caused by TNET. Stabilization of actin filaments using jasplakinolide had no significant effect on uptake or viability loss caused by TNET. To study the role of plasma membrane fluidity, we increased fluidity by depletion of membrane cholesterol using methyl-ß-cyclodextrin and decreased fluidity by enrichment of the membrane with cholesterol using water-soluble cholesterol. Neither of these membrane fluidity changes significantly altered cellular uptake or viability loss caused by TNET. We conclude that weakening mechanical integrity of the cytoskeleton can increase intracellular uptake and decrease loss of cell viability, while plasma membrane fluidity does not appear to play a significant role in uptake or viability loss caused by TNET. The positive effects of cytoskeletal weakening may be due to an enhanced ability of the cell to recover from the effects of TNET and maintain viability. Biotechnol. Bioeng. 2017;114: 2390-2399. © 2017 Wiley Periodicals, Inc.

Protease-Sensitive Nanomaterials for Cancer Therapeutics and Imaging.

Many diseases can be characterized by the abnormal activity exhibited by various biomolecules, the targeting of which can provide therapeutic and diagnostic utility. Recent trends in medicine and nanotechnology have prompted the development of protease-sensitive nanomaterials systems for therapeutic, diagnostic, and theranostic applications. These systems can act specifically in response to the target enzyme and its associated disease conditions, thus enabling personalized treatment and improved prognosis. In this Review, we discuss recent advancements in the development of protease-responsive materials for imaging and drug delivery and analyze several representative systems to illustrate their key design principles.