Rapid method to screen biomaterial angiogenesis in vivo using fluorescence imaging in mice.

Effective vascularization is crucial for repairing and enhancing the longevity of engineered tissues and organs. As the field advances, there is a vital need for efficient and reliable methods for assessing vascularization in real-time. The integration and performance of constructed biomaterials in living organisms rely on angiogenesis and vascularization, making it essential to evaluate vascular development and networks within biomaterials. Current histology-based methods are limited and labor-intensive. On the other hand, fluorescence imaging offers promise for efficient, real-time evaluation of angiogenesis, reducing the time needed for screening many compounds and offering a high-throughput alternative to histology-based methods. Here, we investigated a novel, non-invasive method for quick and repeated analysis of the angiogenic and vascularization process in biomaterials via fluorescence IVIS imaging. Multi-domain peptides (MDPs), self-assembling peptide hydrogels that can possess pro-angiogenic properties depending on their primary sequence, were synthesized and utilized as angiogenic biomaterials and screened with a fluorescence IVIS probe to demonstrate real-time rapid angiogenesis in vivo. The fluorescence-based imaging showed the influence of the peptide chemistry, volume, and concentration on angiogenesis, with one particular MDP, SLanc, promoting robust angiogenesis after one week at 2 w/v%. Through this method, we were able to identify the optimal peptide for rapid and sustained angiogenesis. This approach enables real-time monitoring of angiogenic responses and vascularization processes in the same living subject. It promotes the development of new biomaterials that facilitate vascularization and validates an advanced in vivo screening technique for angiogenesis.

Heterotrimeric collagen helix with high specificity of assembly results in a rapid rate of folding.

The most abundant natural collagens form heterotrimeric triple helices. Synthetic mimics of collagen heterotrimers have been found to fold slowly, even compared to the already slow rates of homotrimeric helices. These prolonged folding rates are not understood. Here we compare the stabilities, specificities and folding rates of three heterotrimeric collagen mimics designed through a computationally assisted approach. The crystal structure of one ABC-type heterotrimer verified a well-controlled composition and register and elucidated the geometry of pairwise cation-π and axial and lateral salt bridges in the assembly. This collagen heterotrimer folds much faster (hours versus days) than comparable, well-designed systems. Circular dichroism and NMR data suggest the folding is frustrated by unproductive, competing heterotrimer species and these species must unwind before refolding into the thermodynamically favoured assembly. The heterotrimeric collagen folding rate is inhibited by the introduction of preformed competing triple-helical assemblies, which suggests that slow heterotrimer folding kinetics are dominated by the frustration of the energy landscape caused by competing triple helices.

Red light-activated depletion of drug-refractory glioblastoma stem cells and chemosensitization of an acquired-resistant mesenchymal phenotype.

Glioblastoma stem cells (GSCs) are potent tumor initiators resistant to radiochemotherapy, and this subpopulation is hypothesized to re-populate the tumor milieu due to selection following conventional therapies. Here, we show that 5-aminolevulinic acid (ALA) treatment-a pro-fluorophore used for fluorescence-guided cancer surgery-leads to elevated levels of fluorophore conversion in patient-derived GSC cultures, and subsequent red light-activation induces apoptosis in both intrinsically temozolomide chemotherapy-sensitive and -resistant GSC phenotypes. Red light irradiation of ALA-treated cultures also exhibits the ability to target mesenchymal GSCs (Mes-GSCs) with induced temozolomide resistance. Furthermore, sub-lethal light doses restore Mes-GSC sensitivity to temozolomide, abrogating GSC-acquired chemoresistance. These results suggest that ALA is not only useful for fluorescence-guided glioblastoma tumor resection, but that it also facilitates a GSC drug-resistance agnostic, red light-activated modality to mop up the surgical margins and prime subsequent chemotherapy.

Tunable Macroscopic Alignment of Self-Assembling Peptide Nanofibers.

Progress in the design and synthesis of nanostructured self-assembling systems has facilitated the realization of numerous nanoscale geometries, including fibers, ribbons, and sheets. A key challenge has been achieving control across multiple length scales and creating macroscopic structures with nanoscale organization. Here, we present a facile extrusion-based fabrication method to produce anisotropic, nanofibrous hydrogels using self-assembling peptides. The application of shear force coinciding with ion-triggered gelation is used to kinetically trap supramolecular nanofibers into aligned, hierarchical macrostructures. Further, we demonstrate the ability to tune the nanostructure of macroscopic hydrogels through modulating phosphate buffer concentration during peptide self-assembly. In addition, increases in the nanostructural anisotropy of fabricated hydrogels are found to enhance their strength and stiffness under hydrated conditions. To demonstrate their utility as an extracellular matrix-mimetic biomaterial, aligned nanofibrous hydrogels are used to guide directional spreading of multiple cell types, but strikingly, increased matrix alignment is not always correlated with increased cellular alignment. Nanoscale observations reveal differences in cell-matrix interactions between variably aligned scaffolds and implicate the need for mechanical coupling for cells to understand nanofibrous alignment cues. In total, innovations in the supramolecular engineering of self-assembling peptides allow us to decouple nanostructure from macrostructure and generate a gradient of anisotropic nanofibrous hydrogels. We anticipate that control of architecture at multiple length scales will be critical for a variety of applications, including the bottom-up tissue engineering explored here.

Tunable Macroscopic Alignment of Self-Assembling Peptide Nanofibers.

Fibrous proteins that comprise the extracellular matrix (ECM) guide cellular growth and tissue organization. A lack of synthetic strategies able to generate aligned, ECM-mimetic biomaterials has hampered bottom-up tissue engineering of anisotropic tissues and led to a limited understanding of cell-matrix interactions. Here, we present a facile extrusion-based fabrication method to produce anisotropic, nanofibrous hydrogels using self-assembling peptides. The application of shear force coinciding with ion-triggered gelation is used to kinetically trap supramolecular nanofibers into aligned, hierarchical structures. We establish how modest changes in phosphate buffer concentration during peptide self-assembly can be used to tune their alignment and packing. In addition, increases in the nanostructural anisotropy of fabricated hydrogels are found to enhance their strength and stiffness under hydrated conditions. To demonstrate their utility as an ECM-mimetic biomaterial, aligned nanofibrous hydrogels are used to guide directional spreading of multiple cell types, but strikingly, increased matrix alignment is not always correlated with increased cellular alignment. Nanoscale observations reveal differences in cell-matrix interactions between variably aligned scaffolds and implicate the need for mechanical coupling for cells to understand nanofibrous alignment cues. In total, innovations in the supramolecular engineering of self-assembling peptides allow us to generate a gradient of anisotropic nanofibrous hydrogels, which are used to better understand directed cell growth.

COVID-19: From emerging variants to vaccination.

The vigorous spread of SARS-CoV-2 resulted in the rapid infection of millions of people worldwide and devastation of not only public healthcare, but also social, educational, and economic infrastructures. The evolution of SARS-CoV-2 over time is due to the mutations that occurred in the genome during each replication. These mutated forms of SARS-CoV-2, otherwise known as variants, were categorized as variants of interest (VOI) or variants of concern (VOC) based on the increased risk of transmissibility, disease severity, immune escape, decreased effectiveness of current social measures, and available vaccines and therapeutics. The swift development of COVID-19 vaccines has been a great success for biomedical research, and billions of vaccine doses, including boosters, have been administered worldwide. BNT162b2 vaccine (Pfizer-BioNTech), mRNA-1273 (Moderna), ChAdOx1 nCoV-19 (AstraZeneca), and Janssen (Johnson & Johnson) are the four major COVID-19 vaccines that received early regulatory authorization based on their efficacy. However, some SARS-CoV-2 variants resulted in higher resistance to available vaccines or treatments. It has been four years since the first reported infection of SARS-CoV-2, yet the Omicron variant and its subvariants are still infecting people worldwide. Despite this, COVID-19 vaccines are still expected to be effective at preventing severe disease, hospitalization, and death from COVID. In this review, we provide a comprehensive overview of the COVID-19 pandemic focused on evolution of VOC and vaccination strategies against them.

Orthogonal Self-Assembly of Amphiphilic Peptide Hydrogels and Liposomes Results in Composite Materials with Tunable Release Profiles.

Self-assembled nanomaterials are promising candidates for drug delivery by providing a higher degree of spatiotemporal control compared to free drugs. However, challenges such as burst release, inadequate targeting, and drug-nanomaterial incompatibility leave room for improvement. The combination of orthogonal self-assembling systems can result in more useful materials that improve upon these weaknesses. In this work, we investigate an orthogonal self-assembling system of nanofibrous MultiDomain Peptide (MDP) hydrogels encapsulating liposomes. Both positively charged and negatively charged MDPs were prepared and mixed with positively charged, negatively charged, or zwitterionic liposomes for a total of six composites. We demonstrate that, despite both systems being amphiphilic, they are able to mix while retaining their independent identities. We show that changing the charge of either liposomes or MDPs does not hinder the self-assembly of either system or significantly affect their rheological properties. In all six cases, small molecules encapsulated in liposome-MDP composites resulted in slower release than was possible in MDP hydrogels alone. However, in one case, positively charged MDPs destabilized negatively charged liposomes and resulted in a unique release profile. Finally, we show that MDP hydrogels substantially decrease the release of chemotherapeutic doxorubicin from its liposomal formulation, Doxil, for 24 h. This work demonstrates the chemical compatibility of amphiphilic, orthogonally self-assembled systems and the range of their drug-delivering capabilities.

Thermogelling hydrogel charge and lower critical solution temperature influence cellular infiltration and tissue integration in an ex vivo cartilage explant model.

Thermogelling hydrogels based on poly(N-isopropyl acrylamide) (p[NiPAAm]) and crosslinked with a peptide-bearing macromer poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol) (PdBT) were fabricated to assess the role of hydrogel charge and lower critical solution temperature (LCST) over time in influencing cellular infiltration and tissue integration in an ex vivo cartilage explant model over 21 days. The p(NiPAAm)-based thermogelling polymer was synthesized to possess 0, 5, and 10 mol% dimethyl-γ-butyrolactone acrylate (DBA) to raise the LCST over time as the lactone rings hydrolyzed. Further, three peptides were designed to impart charge into the hydrogels via conjugation to the PdBT crosslinker. The positively, neutrally, and negatively charged peptides K4 (+), zwitterionic K2E2 (0), and E4 (-), respectively, were conjugated to the modular PdBT crosslinker and the hydrogels were evaluated for their thermogelation behavior in vitro before injection into the cartilage explant models. Samples were collected at days 0 and 21, and tissue integration and cellular infiltration were assessed via mechanical pushout testing and histology. Negatively charged hydrogels whose LCST changed over time (10 mol% DBA) were demonstrated to promote the greatest tissue integration when compared to the positive and neutral gels of the same thermogelling polymer formulation due to increased transport and diffusion across the hydrogel-tissue interface. Indeed, the negatively charged thermogelling polymer groups containing 5 and 10 mol% DBA demonstrated cellular infiltration and cartilage-like matrix deposition via histology. This study demonstrates the important role that material physicochemical properties play in dictating cell and tissue behavior and can inform future cartilage tissue engineering strategies.

Remediating Desmoplasia with EGFR-Targeted Photoactivable Multi-Inhibitor Liposomes Doubles Overall Survival in Pancreatic Cancer.

Desmoplasia is characteristic of pancreatic ductal adenocarcinoma (PDAC), which exhibits 5-year survival rates of 3%. Desmoplasia presents physical and biochemical barriers that contribute to treatment resistance, yet depleting the stroma alone is unsuccessful and even detrimental to patient outcomes. This study is the first demonstration of targeted photoactivable multi-inhibitor liposomes (TPMILs) that induce both photodynamic and chemotherapeutic tumor insult, while simultaneously remediating desmoplasia in orthotopic PDAC. TPMILs targeted with cetuximab (anti-EGFR mAb) contain lipidated benzoporphyrin derivative (BPD-PC) photosensitizer and irinotecan. The desmoplastic tumors comprise human PDAC cells and patient-derived cancer-associated fibroblasts. Upon photoactivation, the TPMILs induce 90% tumor growth inhibition at only 8.1% of the patient equivalent dose of nanoliposomal irinotecan (nal-IRI). Without EGFR targeting, PMIL photoactivation is ineffective. TPMIL photoactivation is also sixfold more effective at inhibiting tumor growth than a cocktail of Visudyne-photodynamic therapy (PDT) and nal-IRI, and also doubles survival and extends progression-free survival by greater than fivefold. Second harmonic generation imaging reveals that TPMIL photoactivation reduces collagen density by >90% and increases collagen nonalignment by >103 -fold. Collagen nonalignment correlates with a reduction in tumor burden and survival. This single-construct phototoxic, chemotherapeutic, and desmoplasia-remediating regimen offers unprecedented opportunities to substantially extend survival in patients with otherwise dismal prognoses.

A Combined Conduit-Bioactive Hydrogel Approach for Regeneration of Transected Sciatic Nerves.

Transected peripheral nerve injury (PNI) affects the quality of life of patients, which leads to socioeconomic burden. Despite the existence of autografts and commercially available nerve guidance conduits (NGCs), the complexity of peripheral nerve regeneration requires further research in bioengineered NGCs to improve surgical outcomes. In this work, we introduce multidomain peptide (MDP) hydrogels, as intraluminal fillers, into electrospun poly(ε-caprolactone) (PCL) conduits to bridge 10 mm rat sciatic nerve defects. The efficacy of treatment groups was evaluated by electromyography and gait analysis to determine their electrical and motor recovery. We then studied the samples' histomorphometry with immunofluorescence staining and automatic axon counting/measurement software. Comparison with negative control group shows that PCL conduits filled with an anionic MDP may improve functional recovery 16 weeks postoperation, displaying higher amplitude of compound muscle action potential, greater gastrocnemius muscle weight retention, and earlier occurrence of flexion contracture. In contrast, PCL conduits filled with a cationic MDP showed the least degree of myelination and poor functional recovery. This phenomenon may be attributed to MDPs' difference in degradation time. Electrospun PCL conduits filled with an anionic MDP may become an attractive tissue engineering strategy for treating transected PNI when supplemented with other bioactive modifications.

Evaluating the physicochemical effects of conjugating peptides into thermogelling hydrogels for regenerative biomaterials applications.

Thermogelling hydrogels, such as poly(N-isopropylacrylamide) [P(NiPAAm)], provide tunable constructs leveraged in many regenerative biomaterial applications. Recently, our lab developed the crosslinker poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol), which crosslinks P(NiPAAm-co-glycidyl methacrylate) via thiol-epoxy reaction and can be functionalized with azide-terminated peptides via alkyne-azide click chemistry. This study's aim was to evaluate the impact of peptides on the physicochemical properties of the hydrogels. The physicochemical properties of the hydrogels including the lower critical solution temperature, crosslinking times, swelling, degradation, peptide release and cytocompatibility were evaluated. The gels bearing peptides increased equilibrium swelling indicating hydrophilicity of the hydrogel components. Comparable sol fractions were found for all groups, indicating that inclusion of peptides does not impact crosslinking. Moreover, the inclusion of a matrix metalloproteinase-sensitive peptide allowed elucidation of whether release of peptides from the network was driven by hydrolysis or enzymatic cleavage. The hydrophilicity of the network determined by the swelling behavior was demonstrated to be the most important factor in dictating hydrogel behavior over time. This study demonstrates the importance of characterizing the impact of additives on the physicochemical properties of hydrogels. These characteristics are key in determining design considerations for future in vitro and in vivo studies for tissue regeneration.

One-Step Detection and Classification of Bacterial Carbapenemases in 10 Minutes Using Fluorescence Identification of ß-Lactamase Activity.

Rapid and accurate diagnosis of bacterial carbapenemases remains a major challenge for clinical laboratories. A novel assay was developed here using fluorescence identification of ß-lactamase activity (FIBA) to permit rapid detection and classification of bacterial carbapenemases. By mixing a fluorogenic ß-lactamase substrate, ß-LEAF (ß-lactamase enzyme-activated fluorophore), with bacterial isolates plus the respective inhibitor (imipenem for noncarbapenemase ß-lactamases, clavulanic acid for type A carbapenemases, and EDTA for type B carbapenemases), objective results with 95% to 100% sensitivity and specificity were generated in 10 min. FIBA is ≈$1/test and consists of only a single mixing step. Given the combination of rapidity, accuracy, low cost, and simplicity, this novel carbapenemase detection and classification assay is well positioned to be applied in clinical microbiology laboratories to provide guidance for the choice of proper treatment and control of globally prevalent carbapenemase-positive infections.

NIR Photodynamic Destruction of PDAC and HNSCC Nodules Using Triple-Receptor-Targeted Photoimmuno-Nanoconjugates: Targeting Heterogeneity in Cancer.

Receptor heterogeneity in cancer is a major limitation of molecular targeting for cancer therapeutics. Single-receptor-targeted treatment exerts selection pressures that result in treatment escape for low-receptor-expressing tumor subpopulations. To overcome this potential for heterogeneity-driven resistance to molecular targeted photodynamic therapy (PDT), we present for the first time a triple-receptor-targeted photoimmuno-nanoconjugate (TR-PIN) platform. TR-PIN functionalization with cetuximab, holo-transferrin, and trastuzumab conferred specificity for epidermal growth factor receptor (EGFR), transferrin receptor (TfR), and human epidermal growth factor receptor 2 (HER-2), respectively. The TR-PINs exhibited up to a 24-fold improvement in cancer cell binding compared with EGFR-specific cetuximab-targeted PINs (Cet-PINs) in low-EGFR-expressing cell lines. Photodestruction using TR-PINs was significantly higher than the monotargeted Cet-PINs in heterocellular 3D in vitro models of heterogeneous pancreatic ductal adenocarcinoma (PDAC; MIA PaCa-2 cells) and heterogeneous head and neck squamous cell carcinoma (HNSCC, SCC9 cells) containing low-EGFR-expressing T47D (high TfR) or SKOV-3 (high HER-2) cells. Through their capacity for multiple tumor target recognition, TR-PINs can serve as a unique and amenable platform for the effective photodynamic eradication of diverse tumor subpopulations in heterogeneous cancers to mitigate escape for more complete and durable treatment responses.

Photoimmunotherapy of Ovarian Cancer: A Unique Niche in the Management of Advanced Disease.

Ovarian cancer (OvCa) is the leading cause of gynecological cancer-related deaths in the United States, with five-year survival rates of 15-20% for stage III cancers and 5% for stage IV cancers. The standard of care for advanced OvCa involves surgical debulking of disseminated disease in the peritoneum followed by chemotherapy. Despite advances in treatment efficacy, the prognosis for advanced stage OvCa patients remains poor and the emergence of chemoresistant disease localized to the peritoneum is the primary cause of death. Therefore, a complementary modality that is agnostic to typical chemo- and radio-resistance mechanisms is urgently needed. Photodynamic therapy (PDT), a photochemistry-based process, is an ideal complement to standard treatments for residual disease. The confinement of the disease in the peritoneal cavity makes it amenable for regionally localized treatment with PDT. PDT involves photochemical generation of cytotoxic reactive molecular species (RMS) by non-toxic photosensitizers (PSs) following exposure to non-harmful visible light, leading to localized cell death. However, due to the complex topology of sensitive organs in the peritoneum, diffuse intra-abdominal PDT induces dose-limiting toxicities due to non-selective accumulation of PSs in both healthy and diseased tissue. In an effort to achieve selective damage to tumorous nodules, targeted PS formulations have shown promise to make PDT a feasible treatment modality in this setting. This targeted strategy involves chemical conjugation of PSs to antibodies, referred to as photoimmunoconjugates (PICs), to target OvCa specific molecular markers leading to enhanced therapeutic outcomes while reducing off-target toxicity. In light of promising results of pilot clinical studies and recent preclinical advances, this review provides the rationale and methodologies for PIC-based PDT, or photo-immunotherapy (PIT), in the context of OvCa management.