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.

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.

Development of Potent Dual BET/HDAC Inhibitors via Pharmacophore Merging and Structure-Guided Optimization.

Bromodomain and extra-terminal domain (BET) proteins and histone deacetylases (HDACs) are prime targets in cancer therapy. Recent research has particularly focused on the development of dual BET/HDAC inhibitors for hard-to-treat tumors, such as pancreatic cancer. Here, we developed a new series of potent dual BET/HDAC inhibitors by choosing starting scaffolds that enabled us to optimally merge the two functionalities into a single compound. Systematic structure-guided modification of both warheads then led to optimized binders that were superior in potency to both parent compounds, with the best molecules of this series binding to both BRD4 bromodomains as well as HDAC1/2 with EC50 values in the 100 nM range in cellular NanoBRET target engagement assays. For one of our lead molecules, we could also show the selective inhibition of HDAC1/2 over all other zinc-dependent HDACs. Importantly, this on-target activity translated into promising efficacy in pancreatic cancer and NUT midline carcinoma cells. Our lead molecules effectively blocked histone H3 deacetylation in pancreatic cancer cells and upregulated the tumor suppressor HEXIM1 and proapoptotic p57, both markers of BET inhibition. In addition, they have the potential to downregulate the oncogenic drivers of NUT midline carcinoma, as demonstrated for MYC and TP63 mRNA levels. Overall, this study expands the portfolio of available dual BET/class I HDAC inhibitors for future translational studies in different cancer models.

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.

Combined imputation of HLA genotype and self-identified race leads to better donor-recipient matching.

Allogeneic Hematopoietic Cell Transplantation (HCT) is a curative therapy for hematologic disorders and often requires human leukocyte antigen (HLA)-matched donors. Donor registries have recruited donors utilizing evolving technologies of HLA genotyping methods. This necessitates in-silico ambiguity resolution and statistical imputation based on haplotype frequencies estimated from donor data stratified by self-identified race and ethnicity (SIRE). However, SIRE has limited genetic validity and presents a challenge for individuals with unknown or mixed SIRE. We present MR-GRIMM "Multi-Race Graph IMputation and Matching" that simultaneously imputes the race/ethnic category and HLA genotype using a SIRE based prior. Additionally, we propose a novel method to impute HLA typing inconsistent with current haplotype frequencies. The performance of MR-GRIMM was validated using a dataset of 170,000 donor-recipient pairs. MR-GRIMM has an average 20 % lower matching error (1-AUC) than single-race imputation. The recall metric (sensitivity) of the race/ethnic category imputation from HLA was measured by comparing the imputed donor race with the donor-provided SIRE. Accuracies of 0.74 and 0.55 were obtained for the prediction of 5 broad and 21 detailed US population groups respectively. The operational implementation of this algorithm in a registry search could help improve match predictions and access to HLA-matched donors.

The genotype list string code syntax for exchanging nomenclature-level genotyping results in clinical and research data management and analysis systems.

The nomenclatures used to describe HLA and killer-cell immunoglobulin-like receptor (KIR) alleles distinguish unique nucleotide and peptide sequences, and patterns of expression, but are insufficient for describing genotyping results, as description of ambiguities and relations across loci require terminology beyond allele names. The genotype list (GL) String grammar describes genotyping results for genetic systems with defined nomenclatures, like HLA and KIR, documenting what is known and unknown about a given genotyping result. However, the accuracy of a GL String is dependent on the reference database version under which it was generated. Here, we describe the GL string code (GLSC) system, which associates each GL String with meta-data describing the specific reference context in which the GL String was created, and in which it should be interpreted. GLSC is a defined syntax for exchanging GL Strings in the context of a specific gene-family namespace, allele-name code-system, and pertinent reference database version. GLSC allows HLA and KIR genotyping data to be transmitted, parsed and interpreted in the appropriate context, in an unambiguous manner, on modern data-systems, including Health Level 7 Fast Healthcare Interoperability Resource systems. Technical specification for GLSC can be found at https://glstring.org.

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.

Genotype List String 1.1: Extending the Genotype List String grammar for describing HLA and Killer-cell Immunoglobulin-like Receptor genotypes.

The Genotype List (GL) String grammar for reporting HLA and Killer-cell Immunoglobulin-like Receptor (KIR) genotypes in a text string was described in 2013. Since this initial description, GL Strings have been used to describe HLA and KIR genotypes for more than 40 million subjects, allowing these data to be recorded, stored and transmitted in an easily parsed, text-based format. After a decade of working with HLA and KIR data in GL String format, with advances in HLA and KIR genotyping technologies that have fostered the generation of full-gene sequence data, the need for an extension of the GL String system has become clear. Here, we introduce the new GL String delimiter "?," which addresses the need to describe ambiguity in assigning a gene sequence to gene paralogs. GL Strings that do not include a "?" delimiter continue to be interpreted as originally described. This extension represents version 1.1 of the GL String grammar.

Assessment of systemic AAV-microdystrophin gene therapy in the GRMD model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by the absence of dystrophin, a membrane-stabilizing protein encoded by the DMD gene. Although mouse models of DMD provide insight into the potential of a corrective therapy, data from genetically homologous large animals, such as the dystrophin-deficient golden retriever muscular dystrophy (GRMD) model, may more readily translate to humans. To evaluate the clinical translatability of an adeno-associated virus serotype 9 vector (AAV9)-microdystrophin (µDys5) construct, we performed a blinded, placebo-controlled study in which 12 GRMD dogs were divided among four dose groups [control, 1 × 1013 vector genomes per kilogram (vg/kg), 1 × 1014 vg/kg, and 2 × 1014 vg/kg; n = 3 each], treated intravenously at 3 months of age with a canine codon-optimized microdystrophin construct, rAAV9-CK8e-c-µDys5, and followed for 90 days after dosing. All dogs received prednisone (1 milligram/kilogram) for a total of 5 weeks from day -7 through day 28. We observed dose-dependent increases in tissue vector genome copy numbers; µDys5 protein in multiple appendicular muscles, the diaphragm, and heart; limb and respiratory muscle functional improvement; and reduction of histopathologic lesions. As expected, given that a truncated dystrophin protein was generated, phenotypic test results and histopathologic lesions did not fully normalize. All administrations were well tolerated, and adverse events were not seen. These data suggest that systemically administered AAV-microdystrophin may be dosed safely and could provide therapeutic benefit for patients with DMD.

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).

Electrical resistance of the current collector controls lithium morphology.

The electrodeposition of low surface area lithium is critical to successful adoption of lithium metal batteries. Here, we discover the dependence of lithium metal morphology on electrical resistance of substrates, enabling us to design an alternative strategy for controlling lithium morphology and improving electrochemical performance. By modifying the current collector with atomic layer deposited conductive (ZnO, SnO2) and resistive (Al2O3) nanofilms, we show that conductive films promote the formation of high surface area lithium deposits, whereas highly resistive films promote the formation of lithium clusters of low surface area. We reveal an electrodeposition mechanism in which radial diffusion of electroactive species is promoted on resistive substrates, resulting in lateral growth of large (150 µm in diameter) planar lithium deposits. Using resistive substrates, similar lithium morphologies are formed in three distinct classes of electrolytes, resulting in up to ten-fold improvement in battery performance. Ultimately, we report anode-free pouch cells using the Al2O3-modified copper that maintain 60 % of their initial discharge capacity after 100 cycles, displaying the benefits of resistive substrates for controlling lithium electrodeposition.

The Importance of Decarbonylation Mechanisms in the Atomic Layer Deposition of High-Quality Ru Films by Zero-Oxidation State Ru(DMBD)(CO)3.

Achieving facile nucleation of noble metal films through atomic layer deposition (ALD) is extremely challenging. To this end, η4 -2,3-dimethylbutadiene ruthenium(0) tricarbonyl (Ru(DMBD)(CO)3 ), a zero-valent complex, has recently been reported to achieve good nucleation by ALD at relatively low temperatures and mild reaction conditions. The authors study the growth mechanism of this precursor by in situ quartz-crystal microbalance and quadrupole mass spectrometry during Ru ALD, complemented by ex situ film characterization and kinetic modeling. These studies reveal that Ru(DMBD)(CO)3 produces high-quality Ru films with excellent nucleation properties. This results in smooth, coalesced films even at low film thicknesses, all important traits for device applications. However, Ru deposition follows a kinetically limited decarbonylation reaction scheme, akin to typical chemical vapor deposition processes, with a strong dependence on both temperature and reaction timescale. The non-self-limiting nature of the kinetically driven mechanism presents both challenges for ALD implementation and opportunities for process tuning. By surveying reports of similar precursors, it is suggested that the findings can be generalized to the broader class of zero-oxidation state carbonyl-based precursors used in thermal ALD, with insight into the design of effective saturation studies.

1-Year Outcomes of Blinded Physiological Assessment of Residual Ischemia After Successful PCI: DEFINE PCI Trial.

OBJECTIVES: The aim of this study was to identify the post-percutaneous coronary intervention (PCI) target value of instantaneous wave-free ratio (iFR) that would best discriminate clinical events at 1 year in the DEFINE PCI (Physiologic Assessment of Coronary Stenosis Following PCI) study. BACKGROUND: The impact of residual ischemia detected by iFR post-PCI on clinical and symptom-related outcomes is unknown. METHODS: Blinded iFR pull back was performed after successful stent implantation in 500 patients. The primary endpoint was the rate of residual ischemia, defined as iFR ≤0.89, after operator-assessed angiographically successful PCI. Secondary endpoints included clinical events at 1 year and change in Seattle Angina Questionnaire angina frequency (SAQ-AF) score during follow-up. RESULTS: As reported, 24.0% of patients had residual ischemia (iFR ≤0.89) after successful PCI, with 81.6% of cases attributable to angiographically inapparent focal lesions. Post-PCI iFR ≥0.95 (present in 182 cases [39%]) was associated with a significant reduction in the composite of cardiac death, spontaneous myocardial infarction, or clinically driven target vessel revascularization compared with post-PCI iFR <0.95 (1.8% vs 5.7%; P = 0.04). Baseline SAQ-AF score was 73.3 ± 22.8. For highly symptomatic patients (baseline SAQ-AF score ≤60), SAQ-AF score increased by ≥10 points more frequently in patients with versus without post-PCI iFR ≥0.95 (100.0% vs 88.5%; P = 0.01). CONCLUSIONS: In DEFINE PCI, despite angiographically successful PCI, highly symptomatic patients at baseline without residual ischemia by post-PCI iFR had greater reductions in anginal symptoms at 1 year compared with patients with residual ischemia. Achieving post-PCI iFR ≥0.95 was also associated with improved 1-year event-free survival. (Physiologic Assessment of Coronary Stenosis Following PCI [DEFINE PCI]; NCT03084367).

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.

Failed Acellular Nerve Allografts: A Critical Review.

BACKGROUND: Acellular nerve allograft (ANA) occupies an increasingly prominent role in the treatment of peripheral nerve reconstruction. There is demonstrable efficacy; however, some grafts fail to support axonal regrowth and the reasons for this are unclear. This study examines the ANA experience in a specialized peripheral nerve surgery department to discuss the clinical and histological findings in failed cases. METHOD: Failed ANA grafts were identified from a prospective database using Medical Research Council Classification (MRCC) S3 and M3 as thresholds for success. Cases in which ANA grafting was indicated for nerve related pain and dysesthesia but where no subjective improvement in symptoms occurred were also included. Patients requiring revision surgery after ANA grafting were also considered failures. Cases were then examined in conjunction with a literature review to identify possible mechanisms of failure, including detailed histological analysis in 2 cases. RESULTS: Eight failed procedures were identified from a database of 99 separate allograft records on 74 patients. This included procedures for 2 tibial nerves, 2 superficial radial nerves, 2 median nerves, 1 digital nerve and a lateral cord brachial plexus injury (male/female, 5:3; age range, 24-54 years). Allograft length range 25 to 120 mm. One postoperative infection was identified. Histological findings in 2 cases included adequate vascularization of allograft material without subsequent axonal regeneration, a reduction of large myelinated fibers proximal to a tibial nerve allograft in the setting of a chronic injury, and a preference for small rather than large fiber regeneration. CONCLUSIONS: This article reports instances of ANA graft failure in a variety of contexts, for which the primary reasons for failure remain unclear. The etiology is likely to be multifactorial with both patient, graft and surgeon factors contributing to failure. Further clinical and histological analysis of ANA failures will improve our understanding of the mechanisms of graft failure.

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.