Harnessing Peptide Binding to Capture and Reclaim Phosphate.

With rising consumer demands, society is tapping into wastewater as an innovative source to recycle depleting resources. Novel reclamation technologies have been recently explored for this purpose, including several that optimize natural biological processes for targeted reclamation. However, this emerging field has a noticeable dearth of synthetic material technologies that are programmed to capture, release, and recycle specified targets; and of the novel materials that do exist, synthetic platforms incorporating biologically inspired mechanisms are rare. We present here a prototype of a materials platform utilizing peptide amphiphiles that has been molecularly engineered to sequester, release, and reclaim phosphate through a stimuli-responsive pH trigger, exploiting a protein-inspired binding mechanism that is incorporated directly into the self-assembled material network. This material is able to harvest and controllably release phosphate for multiple cycles of reuse, and it is selective over nitrate and nitrite. We have determined by simulations that the binding conformation of the peptide becomes constrained in the dense micelle corona at high pH such that phosphate is expelled when it otherwise would be preferentially bound. However, at neutral pH, this dense structure conversely employs multichain binding to further stabilize phosphate when it would otherwise be unbound, opening opportunities for higher-order conformational binding design to be engineered into this controllably packed corona. With this work, we are pioneering a new platform to be readily altered to capture other valuable targets, presenting a new class of capture and release materials for recycling resources on the nanoscale.

CD44-Targeted Polymer-Paclitaxel Conjugates to Control the Spread and Growth of Metastatic Tumors.

One of the greatest challenges in cancer therapy is to control metastatic spread, seeding, and growth of tumors in distant organs. Recently, we reported on the design of a novel "drug-free" therapeutic copolymer bearing the antimigratory A5G27 peptide, designated P-(A5G27)-FITC, that shows excellent specificity to cancer cells overexpressing CD44v3 and CD44v6 and inhibits cancer cell migration and invasion. We demonstrated that P-(A5G27)-FITC accumulated preferentially in subcutaneous (sc) implanted 4T1 tumors following parenteral administration. Moreover, we showed that pretreatment of mice with P-(A5G27)-FITC prior to 4T1 cell inoculation inhibited colonization of circulating 4T1 cells in the lungs. In this study, we designed a new polymer-peptide-drug conjugate to inhibit vigorously growing primary tumors and control invasive behavior of cancer cells. To this end, the antimitotic drug (paclitaxel, PTX) was conjugated to P-(A5G27)-FITC. The targeted polymer-drug conjugate (P-(A5G27)-PTX) was significantly more toxic toward CD44-overexpressing cancer cells than the nontargeted copolymer. In vivo, a single iv injection of P-(A5G27)-PTX prolonged the survival of C57BL/6 mice with established B16-F10 lung metastases. When injected intraperitoneally into BALB/c mice implanted sc with 4T1 tumors, P-(A5G27)-PTX significantly decreased the rate of primary tumor growth, increased the median survival of mice, and reduced the number of 4T1 metastases in the lungs when compared to nontargeted copolymer. Most interestingly, the CD44-targeted "drug-free" copolymer P-(A5G27) (without PTX) significantly inhibited the rate of tumor growth and further prolonged the median survival time of mice to the same extent as the PTX-containing formulations (P-(A5G27)-PTX or free PTX). Overall, this study highlights the therapeutic potential of the HPMA copolymer-A5G27 conjugates ("drug-free" and PTX-bearing copolymers) to control the metastatic spread of cancer.

Multimodal characterization of the collagen hydrogel structure and properties in response to physiologically relevant pH fluctuations.

pH fluctuations within the extracellular matrix (ECM) and its principal constituent collagen, particularly in solid tumors and chronic wounds, may influence its structure and function. Whereas previous research examined the impact of pH on collagen fibrillogenesis, this study focuses on determining how pH fluctuations affect collagen hydrogels that mimic the physiological ECM. Utilizing a type I collagen hydrogel, we examined the influence of pH fluctuations on its structure, properties, and function while keeping the collagen hydrated. We show that collagen's secondary structure remains unaltered during pathologically relevant microenvironmental pH changes. By employing cryo scanning electron microscopy and artificial intelligence-assisted image analysis, we show that at physiological pH, collagen hydrogel presents densely packed, aligned, and elongated fibrils, which upon a decrease to pH 6.5, are transformed into shorter, sparser, and disoriented fibrils. The collagen possesses a higher storage modulus yet a lower permeability at pH 7 and 7.8 compared with pH 6.5 and 7.4. Exposing acidified collagen to a basic buffer reinstates its native structure and viscoelastic properties. Our study offers an innovative approach to analyze and characterize perturbations in hydrated collagen-based systems with potential implications for better understanding and combating disease progression. STATEMENT OF SIGNIFICANCE: As the main component of the extracellular matrix, collagen undergoes conformational changes associated with pH changes during disease. We analyze the impact of pH on pre-formed collagen fibers mimicking healthy tissues subjected to disease, and do not focus on the more studied fibrillogenesis process. Using cryogenic SEM, which allowed imaging close to the native state, we show that even minor fluctuations in the pH affect the collagen thickness, length, fiber alignment, and rheological properties. Following exposure to acidic pH, the collagen had short fibers, lacked orientation, and had low mechanical strength. This acidic collagen restored its original properties after returning to a neutral pH. These findings can help determine how pH changes can be modulated to restore healthy collagen properties.

Inhibition of CD44v3 and CD44v6 function blocks tumor invasion and metastatic colonization.

The prevention of cancer cell dissemination and secondary tumor formation are major goals of cancer therapy. Here, we report on the development of a new CD44-targeted copolymer carrying multiple copies of the A5G27 peptide, known for its ability to bind specifically to CD44v3 and CD44v6 on cancer cells and inhibit tumor cell migration, invasion, and angiogenesis. We hypothesized that conjugation of A5G27 to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer would enhance tumor tissue accumulation, promote selective binding to cancer cells, with concomitant increased inhibition of cancer cell invasiveness and migration. Fluorescein-5-isothiocyanate or the near-infrared fluorophore IR783 were attached to the copolymer backbone through a non-cleavable linkage to assess in vitro binding to cancer cells and biodistribution of the polymer in 4T1 murine mammary adenocarcinoma-bearing mice, respectively. The anti-migratory activity was evaluated both in vitro and in vivo. The binding of the targeted copolymer to cancer cells correlated well with the level of CD44 expression, with the polymer being internalized more efficiently by cancer cells. Pre-treatment of mice with polymer-bound A5G27 significantly inhibited lung colonization of migrating 4T1 cells in vivo, with the targeted copolymer accumulating preferentially in subcutaneous 4T1 tumors, when compared to a non-targeted system. As such, the HPMA copolymer-A5G27 conjugate is a promising candidate for inhibiting cancer cell migration and can also be used as a drug or imaging probe carrier for detection and treatment of cancer.

Receptor Protein Tyrosine Phosphatase Sigma (RPTP-σ) Increases pro-MMP Activity in a Trabecular Meshwork Cell Line Following Oxidative Stress Conditions.

PURPOSE: To elucidate the role of phosphatases in the eye drainage system by overexpressing the receptor tyrosine phosphatase sigma (RPTP-σ) in a human normal trabecular meshwork (NTM) cell line. METHODS: The efficacy, expression, and location of RPTP-σ were evaluated following its transfection in NTM cells (NTM(T)) and in NTM control cells. The cells were also analyzed for viability, matrix metalloproteinase (MMP) activity, and phosphatase activity following oxidative stress conditions. Assays were conducted in the presence or absence of a specific RPTP-σ inhibitor. RESULTS: Transfection efficacy measurements revealed that RPTP-σ expression measured via GFP fluorescence was significantly higher (×3.8) in NTM(T) cells than in control cells. Western blot analyses showed that RPTP-σ expression was significantly higher (×2.25) in NTM(T) cells than in control cells. No significant differences were observed in cell viability between NTM(T) and control cells after oxidative stress. We found that pro-MMP-2 and pro-MMP-9 showed a significantly higher activity (×2.18 and ×1.9; respectively) in NTM(T) cells than in control cells. Serine/threonine phosphatase activity in NTM(T) cells was significantly increased following oxidative stress. The specific phosphatase inhibitor PTP-IV inhibited 15% of the RPTP-σ expression in NTM cells and 31% in NTMT cells. The activity of pro-MMP-9, pro-MMP-2, and MMP-9 was significantly inhibited (48%, 35%, and 78% respectively). CONCLUSIONS: The findings indicate that RPTP-σ is expressed constituently in NTM cells and that oxidative stress changes the general phosphatase balance in NTM cells. In addition, the results show that expression levels of RPTP-σ affect the activity of various forms of MMP.