X-ray imageable, drug-loaded hydrogel that forms at body temperature for image-guided, needle- based locoregional drug delivery.

Liver cancer ranks as the fifth leading cause of cancer-related death globally. Direct intratumoral injections of anti-cancer therapeutics may improve therapeutic efficacy and mitigate adverse effects compared to intravenous injections. Some challenges of intratumoral injections are that the liquid drug formulation may not remain localized and have unpredictable volumetric distribution. Thus, drug delivery varies widely, highly-dependent upon technique. An x-ray imageable poloxamer 407 (POL)-based drug delivery gel was developed and characterized, enabling real-time feedback. Utilizing three needle devices, POL or a control iodinated contrast solution were injected into an ex vivo bovine liver. The 3D distribution was assessed with cone beam computed tomography (CBCT). The 3D distribution of POL gels demonstrated localized spherical morphologies regardless of the injection rate. In addition, the gel 3D conformal distribution could be intentionally altered, depending on the injection technique. When doxorubicin (DOX) was loaded into the POL and injected, DOX distribution on optical imaging matched iodine distribution on CBCT suggesting spatial alignment of DOX and iodine localization in tissue. The controllability and localized deposition of this formulation may ultimately reduce the dependence on operator technique, reduce systemic side effects, and facilitate reproducibility across treatments, through more predictable standardized delivery.

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.

Hydrophilic Azide-Containing Amino Acid to Enhance the Solubility of Peptides for SPAAC Reactions.

We report a new positively charged azidoamino acid for strain-promoted azide-alkyne cycloaddition (SPAAC) applications that overcomes possible solubility limitations of commonly used azidolysine, especially in systems with numerous ligation sites. The residue is easily synthesized, is compatible with Fmoc-based solid-phase peptide synthesis employing a range of coupling conditions, and offers efficient second-order rate constants in SPAAC ligations employing DBCO (0.34 M-1 s-1) and BCN (0.28 M-1 s-1).

Peptide hydrogels for affinity-controlled release of therapeutic cargo: Current and potential strategies.

The development of devices for the precise and controlled delivery of therapeutics has grown rapidly over the last few decades. Drug delivery materials must provide a depot with delivery profiles that satisfy pharmacodynamic and pharmacokinetic requirements resulting in clinical benefit. Therapeutic efficacy can be limited due to short half-life and poor stability. Thus, to compensate for this, frequent administration and high doses are often required to achieve therapeutic effect, which in turn increases potential side effects and systemic toxicity. This can potentially be mitigated by using materials that can deliver drugs at controlled rates, and material design principles that allow this are continuously evolving. Affinity-based release strategies incorporate a myriad of reversible interactions into a gel network, which have affinities for the therapeutic of interest. Reversible binding to the gel network impacts the release profile of the drug. Such affinity-based interactions can be modulated to control the release profile to meet pharmacokinetic benchmarks. Much work has been done developing affinity-based control in the context of polymer-based materials. However, this strategy has not been widely implemented in peptide-based hydrogels. Herein, we present recent advances in the use of affinity-controlled peptide gel release systems and their associated mechanisms for applications in drug delivery.

INSTIs and NNRTIs Potently Inhibit HIV-1 Polypurine Tract Mutants in a Single Round Infection Assay.

Integrase strand transfer inhibitors (INSTIs) are a class of antiretroviral compounds that prevent the insertion of a DNA copy of the viral genome into the host genome by targeting the viral enzyme integrase (IN). Dolutegravir (DTG) is a leading INSTI that is given, usually in combination with nucleoside reverse transcriptase inhibitors (NRTIs), to treat HIV-1 infections. The emergence of resistance to DTG and other leading INSTIs is rare. However, there are recent reports suggesting that drug resistance mutations can occur at positions outside the integrase gene either in the HIV-1 polypurine tract (PPT) or in the envelope gene (env). Here, we used single round infectivity assays to measure the antiviral potencies of several FDA-approved INSTIs and non-nucleoside reverse transcriptase inhibitors (NNRTIs) against a panel of HIV-1 PPT mutants. We also tested several of our promising INSTIs and NNRTIs in these assays. No measurable loss in potency was observed for either INSTIs or NNRTIs against the HIV-1 PPT mutants. This suggests that HIV-1 PPT mutants are not able, by themselves, to confer resistance to INSTIs or NNRTIs.

Surface-fill hydrogel attenuates the oncogenic signature of complex anatomical surface cancer in a single application.

Tumours growing in a sheet-like manner on the surface of organs and tissues with complex topologies represent a difficult-to-treat clinical scenario. Their complete surgical resection is difficult due to the complicated anatomy of the diseased tissue. Residual cancer often responds poorly to systemic therapy and locoregional treatment is hindered by the limited accessibility to microscopic tumour foci. Here we engineered a peptide-based surface-fill hydrogel (SFH) that can be syringe- or spray-delivered to surface cancers during surgery or used as a primary therapy. Once applied, SFH can shape change in response to alterations in tissue morphology that may occur during surgery. Implanted SFH releases nanoparticles composed of microRNA and intrinsically disordered peptides that enter cancer cells attenuating their oncogenic signature. With a single application, SFH shows efficacy in four preclinical models of mesothelioma, demonstrating the therapeutic impact of the local application of tumour-specific microRNA, which might change the treatment paradigm for mesothelioma and possibly other surface cancers.

Antibacterial Gel Coatings Inspired by the Cryptic Function of a Mussel Byssal Peptide.

Although the adhesive and cohesive nature of mussel byssal proteins have long served to inspire the design of materials embodying these properties, their characteristic amino acid compositions suggest that they might also serve to inspire an unrelated material function not yet associated with this class of protein. Herein, it is demonstrated that a peptide derived from mussel foot protein-5, a key protein in mussel adhesion, displays antibacterial properties, a yet unreported activity. This cryptic function serves as inspiration for the design of a new class of peptide-based antibacterial adhesive hydrogels prepared via self-assembly, which are active against drug-resistant Gram-positive bacteria. The gels exert two mechanisms of action, surface-contact membrane disruption and oxidative killing affected by material-produced H2 O2 . Detailed studies relating amino acid composition and sequence to material mechanical adhesion/cohesion and antibacterial activity affords the MIKA2 adhesive gel, a material with a superior activity that is shown to inhibit colonization of titanium implants in mice.

From structure to application: Progress and opportunities in peptide materials development.

For over 20 years, peptide materials in their hydrogel or soluble fibril form have been used for biomedical applications such as drug delivery, cell culture, vaccines, and tissue regeneration. To facilitate the translation of these materials, key areas of research still need to be addressed. Their structural characterization lags compared to amyloid proteins. Many of the structural features designed to guide materials formation are primarily being characterized by their observation in atomic resolution structures of amyloid assemblies. Herein, these motifs are examined in relation to peptide designs identifying common interactions that drive assembly and provide structural specificity. Current efforts to design complex structures, as reviewed here, highlight the need to extend the structural revolution of amyloid proteins to peptide assemblies to validate design principles. With respect to clinical applications, the fundamental interactions and responses of proteins, cells, and the immune system to peptide materials are still not well understood. Only a few trends are just now emerging for peptide materials interactions with biological systems. Understanding how peptide material properties influence these interactions will enable the translation of materials towards current and emerging applications.

Defining the Landscape of the Pauling-Corey Rippled Sheet: An Orphaned Motif Finding New Homes.

When peptides are mixed with their mirror images in an equimolar ratio, two-dimensional periodic structural folds can form, in which extended peptide strands are arrayed with alternating chirality. The resultant topography class, termed the rippled ß-sheet, was introduced as a theoretical concept by Pauling and Corey in 1953. Unlike other fundamental protein structural motifs identified around that time, including the α-helix and the pleated ß-sheet, it took several decades before conclusive experimental data supporting the proposed rippled ß-sheet motif were gained. Much of the key experimental evidence was provided over the course of the past decade through the concurrent efforts of our three laboratories. Studies that focused on developing new self-assembling hydrogel materials have shown that certain amphiphilic peptides form fibrils and hydrogel networks that are more rigid and have a higher thermodynamic stability when made from racemic peptide mixtures as opposed to pure enantiomers. Related interrogation of assemblies composed of mixtures of l- and d-amphiphilic peptides confirmed that the resulting fibrils were composed of alternating l/d peptides consistent with rippled ß-sheets. It was also demonstrated that mirror-image amyloid beta (Aß) could act as a molecular chaperone to promote oligomer-to-fibril conversion of the natural Aß enantiomer, which was found to reduce Aß neurotoxicity against different neuronal cell models. With a cross-disciplinary approach that combines experiment and theory, our three laboratories have demonstrated the unique biophysical, biochemical, and biological properties that arise upon mixing of peptide enantiomers, in consequence of rippled ß-sheet formation. In this Account, we give an overview of the early history of the rippled ß-sheet and provide a detailed structural description/definition of this motif relative to the pleated ß-sheet. We then summarize the key findings, obtained on three unique sets of aggregating mirror-image peptide pairs through independent efforts of our three laboratories, and use these results to delineate the landscape of the rippled ß-sheet structural motif to inspire future studies. Peptide sequence parameters that favor rippled ß-sheet assembly are described, along with the accompanying kinetic and thermodynamic properties, as well as the resulting emergent physical properties of the assemblies. The Account then concludes with a brief overview of some key unresolved challenges in this nascent field. There is much potential for future applications of this unique supramolecular motif in the realm of materials design and biomedical research. We hope this Account will stimulate much-needed discussion of this fascinating structural class to eventually produce a fully quantitative, rational framework for the molecular engineering of rippled ß-sheets in the future.

Dopamine Self-Polymerization as a Simple and Powerful Tool to Modulate the Viscoelastic Mechanical Properties of Peptide-Based Gels.

Dopamine is a small versatile molecule used for various biotechnological and biomedical applications. This neurotransmitter, in addition to its biological role, can undergo oxidative self-polymerization to yield polydopamine, a robust universal coating material. Herein, we harness dopamine self-polymerization to modulate the viscoelastic mechanical properties of peptide-based gels, expanding their ever-growing application potential. By combining rapid peptide assembly with slower dopamine auto-polymerization, a double network gel is formed, where the fibrillar peptide gel network serves as a scaffold for polydopamine deposition, allowing polydopamine to interpenetrate the gel network as well as establishing crosslinks within the matrix. We have shown that triggering the assembly of a lysine-rich peptide gelator in the presence of dopamine can increase the mechanical rigidity of the resultant gel by a factor of 90 in some cases, while retaining the gel's shear thin-recovery behavior. We further investigate how factors such as polymerization time, dopamine concentration and peptide concentration alter the mechanical properties of the resultant gel. The hybrid peptide-dopamine gel systems were characterized using rheological measurements, circular dichroism spectroscopy and transmission electron microscopy. Overall, triggering peptide gelation in the presence of dopamine represents a simple yet powerful approach to modulate the viscoelastic mechanical properties of peptide-based gels.

Serum Protein Adsorption Modulates the Toxicity of Highly Positively Charged Hydrogel Surfaces.

Hydrogels formed from peptide self-assembly are a class of materials that are being explored for their utility in tissue engineering, drug and cell delivery, two- and three-dimensional cell culture, and as adjuvants in surgical procedures. Most self-assembled peptide gels can be syringe-injected in vivo to facilitate the local delivery of payloads, including cells, directly to the targeted tissue. Herein, we report that highly positively charged peptide gels are inherently toxic to cells, which would seem to limit their utility. However, adding media containing fetal bovine serum, a common culture supplement, directly transforms these toxic gels into cytocompatible materials capable of sustaining cell viability even in the absence of added nutrients. Multistage mass spectrometry showed that at least 40 serum proteins can absorb to a gel's surface through electrostatic attraction ameliorating its toxicity. Further, cell-based studies employing model gels having only bovine serum albumin, fetuin-A, or vitronectin absorbed to the gel surface showed that single protein additives can also be effective depending on the identity of the cell line. Separate studies employing these model gels showed that the mechanism(s) responsible for mitigating apoptosis involve both the pacification of gel surface charge and adsorbed protein-mediated cell signaling events that activate both the PI3/Akt and MAPK/ERK pathways which are known to facilitate resistance to stress-induced apoptosis and overall cell survival.

Structure-based non-nucleoside inhibitor design: Developing inhibitors that are effective against resistant mutants.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) inhibit reverse transcription and block the replication of HIV-1. Currently, NNRTIs are usually used as part of a three-drug combination given to patients as antiretroviral therapy. These combinations involve other classes of anti-HIV-1 drugs, commonly nucleoside reverse transcriptase inhibitors (NRTIs). However, attempts are being made to develop two-drug maintenance therapies, some of which involve an NNRTI and an integrase strand transfer inhibitor. This has led to a renewed interest in developing novel NNRTIs, with a major emphasis on designing compounds that can effectively inhibit the known NNRTI-resistant mutants. We have generated and tested novel rilpivirine (RPV) analogs. The new compounds were designed to exploit a small opening in the upper right periphery of the NNRTI-binding pocket. The best of the new compounds, 12, was a more potent inhibitor of the NNRTI-resistant mutants we tested than either doravirine or efavirenz but was inferior to RPV. We describe the limitations on the modifications that can be appended to the "upper right side" of the RPV core and the effects of substituting other cores for the central pyrimidine core of RPV and make suggestions about how this information can be used in NNRTI design.

Uncoupling the Folding-Function Paradigm of Lytic Peptides to Deliver Impermeable Inhibitors of Intracellular Protein-Protein Interactions.

Here, we describe the use of peptide backbone N-methylation as a new strategy to transform membrane-lytic peptides (MLPs) into cytocompatible intracellular delivery vehicles. The ability of lytic peptides to engage with cell membranes has been exploited for drug delivery to carry impermeable cargo into cells, but their inherent toxicity results in narrow therapeutic windows that limit their clinical translation. For most linear MLPs, a prerequisite for membrane activity is their folding at cell surfaces. Modification of their backbone with N-methyl amides inhibits folding, which directly correlates to a reduction in lytic potential but only minimally affects cell entry. We synthesized a library of N-methylated peptides derived from MLPs and conducted structure-activity studies that demonstrated the broad utility of this approach across different secondary structures, including both ß-sheet and helix-forming peptides. Our strategy is highlighted by the delivery of a notoriously difficult class of protein-protein interaction inhibitors that displayed on-target activity within cells.

Multiphase Assembly of Small Molecule Microcrystalline Peptide Hydrogel Allows Immunomodulatory Combination Therapy for Long-Term Heart Transplant Survival.

Combination therapies that target multiple pathways involved in immune rejection of transplants hold promise for patients in need of restorative surgery. Herein, a noninteracting multiphase molecular assembly approach is developed to crystallize tofacitinib, a potent JAK1/3 inhibitor, within a shear-thinning self-assembled fibrillar peptide hydrogel network. The resulting microcrystalline tofacitinib hydrogel (MTH) can be syringe-injected directly to the grafting site during surgery to locally deliver the small molecule. The rate of drug delivered from MTH is largely controlled by the dissolution of the encapsulated microcrystals. A single application of MTH, in combination with systemically delivered CTLA4-Ig, a co-stimulation inhibitor, affords significant graft survival in mice receiving heterotopic heart transplants. Locoregional studies indicate that the local delivery of tofacitinib at the graft site enabled by MTH is required for the observed enhanced graft survival.

National Cancer Institute (NCI) Program for Natural Products Discovery: Rapid Isolation and Identification of Biologically Active Natural Products from the NCI Prefractionated Library.

An automated, high-capacity, and high-throughput procedure for the rapid isolation and identification of biologically active natural products from a prefractionated library is presented. The semipreparative HPLC method uses 1 mg of the primary hit fraction and produces 22 subfractions in an assay-ready format. Following screening, all active fractions are analyzed by NMR, LCMS, and FTIR, and the active principle structural classes are elucidated. In the proof-of-concept study, we show the processes involved in generating the subfractions, the throughput of the structural elucidation work, as well as the ability to rapidly isolate and identify new and biologically active natural products. Overall, the rapid second-stage purification conserves extract mass, requires much less chemist time, and introduces knowledge of structure early in the isolation workflow.

Utilizing Frémy's Salt to Increase the Mechanical Rigidity of Supramolecular Peptide-Based Gel Networks.

Peptide-based supramolecular gels are an important class of biomaterials that can be used for biomedical applications ranging from drug delivery to tissue engineering. Methodology that allows one to readily modulate the mechanical properties of these gels will allow yet even a broader range of applications. Frémy's salt is an inorganic salt and long-lived free radical that is known to oxidize phenols. Herein, we show that Frémy's salt can be used to dramatically increase the mechanical rigidity of hydrogels formed by tyrosine-containing self-assembling ß-hairpin peptides. When Frémy's salt is added to pre-formed gels, it converts tyrosine residues to o-quinones that can subsequently react with amines present within the lysine side chains of the assembled peptide. This results in the installation of chemical crosslinks that reinforce the gel matrix. We characterized the unoxidized and oxidized gel systems using UV-Vis, transmission electron microscopy and rheological measurements and show that Frémy's salt increases the gel rigidity by nearly one order of magnitude, while retaining the gel's shear-thin/recovery behavior. Thus, Frémy's salt represents an on-demand method to modulate the mechanical rigidity of peptide-based self-assembled gels.

The effect of turn residues on the folding and cell-penetrating activity of ß-hairpin peptides and applications toward protein delivery.

Cell-penetrating peptides (CPPs) are useful tools for the delivery of a wide variety of cargo into cells. Our lab has developed two classes of CPPs based on ß-hairpin sequences, one that folds at the surface of cell membranes and the other that is intrinsically disordered. Although these peptides can effectively deliver different types of cargo, their use in protein delivery has been hindered due to the presence of non-natural D-proline within the central turn region of both sequences, which prohibits functionalizing proteins with the CPPs via standard expression protocols. In this work, we describe new CPPs that replace the non-natural turn region with natural turn motifs amenable to protein expression. We first investigate how these changes within the turn affect various CPP-related properties in the absence of protein cargo, and then generate protein fusions for intracellular delivery.

New anti-IL-7Rα monoclonal antibodies show efficacy against T cell acute lymphoblastic leukemia in pre-clinical models.

Pediatric T cell acute lymphoblastic leukemia (T-ALL) cells frequently contain mutations in the interleukin-7 (IL-7) receptor pathway or respond to IL-7 itself. To target the IL-7 receptor on T-ALL cells, murine monoclonal antibodies (MAbs) were developed against the human IL-7Rα chain and chimerized with human IgG1 constant regions. Crystal structures demonstrate that the two MAbs bound different IL-7Rα epitopes. The MAbs mediated antibody-dependent cell-mediated cytotoxicity (ADCC) against patient-derived xenograft (PDX) T-ALL cells, which was improved by combining two MAbs. In vivo, the MAbs showed therapeutic efficacy via ADCC-dependent and independent mechanisms in minimal residual and established disease. PDX T-ALL cells that relapsed following a course of chemotherapy displayed elevated IL-7Rα, and MAb treatment is effective against relapsing disease, suggesting the use of anti-IL7Rα MAbs in relapsed T-ALL patients or patients that do not respond to chemotherapy.