Dexamethasone-loaded, injectable pullulan-poly(ethylene glycol) hydrogels for bone tissue regeneration in chronic inflammatory conditions.

Chronic inflammation, infection, and fixation stability disrupts bone tissue regeneration by implants. The elevated levels of inflammatory markers and reactive oxygen species (ROS) damage tissues, inhibit osteoblastic differentiation, and promote bone resorption. Activation of local and chronic inflammatory responses due to the implantable biomaterial poses a high risk of implant failure and compromised bone repair in several pathological conditions. Not much progress has been made in the development of biomaterials that can counter inflammation and ROS along with inducing osteogenic activities for managing bone defects/injuries. We have developed, for the first time, injectable polymeric hydrogels by crosslinking oxidized pullulan (OP, 1% w/v) and 8-arm PEG hydrazine (PEG-HY, 10% w/v) using pH-sensitive and dynamic hydrazone linkages at 37 °C in buffer. The hydrogels were loaded with dexamethasone (Dex), an anti-inflammatory corticosteroid and osteogenic inducer, by covalently linking it to PEG-HY by hydrazone linkages, and their morphological, injectability, viscoelastic, self-healing, swelling, and drug-release properties were investigated. The hydrogels provided a pH-sensitive sustained release of PEG-Dex conjugate (3.62 wt%, 9.22 × 10-5 mol of Dex/gram) for 28 days, with 74.54 and 55.15% PEG-Dex conjugate being released at pH 6.5 and 7.4. ABTS assay showed that hydrogels inhibited 68% radicals within 1 h, and treatment with hydrogel releasates inhibited the pro-inflammatory markers, IL-6 and IL-1ß, and elevated the anti-inflammatory marker, TGF-ß, in murine osteoblast precursor cells (MC3T3-E1). The hydrogels were found suitable for cell encapsulation and they exhibited 110% viability on treatment with releasates. Finally, the osteogenic activities of hydrogels were ascertained by alkaline phosphatase (ALP) activities, alizarin red S staining, and osteogenic gene expressions- RUNX2, Col-I, OPN, and IBSP. Overall, PEG-Dex conjugate released from hydrogels improved the cell viability and proliferation, and induced the osteoblastic differentiation. The hydrogels with their promising antioxidant and anti-inflammatory properties along with the osteogenic activities show a strong potential as an injectable, extracellular matrix (ECM)-mimicking implantable drug-depot for bone repair applications in chronic inflammatory conditions.

Discovery of boronic acid-based potent activators of tumor pyruvate kinase M2 and development of gastroretentive nanoformulation for oral dosing.

Several studies have established that cancer cells explicitly over-express the less active isoform of pyruvate kinase M2 (PKM2) is critical for tumorigenesis. The activation of PKM2 towards tetramer formation may increase affinity towards phosphoenolpyruvate (PEP) and avoidance of the Warburg effect. Herein, we describe the design, synthesis, and development of boronic acid-based molecules as activators of PKM2. The designed molecules were inspired by existing anticancer scaffolds and several fragments were assembled in the derivatives. 6a-6d were synthesized using a multi-step synthetic strategy in 55-70% yields, starting from cheap and readily available materials. The compounds were selectively cytotoxic to kill the cancerous cells at 80 nM, while they were non-toxic to the normal cells. The kinetic studies established the compounds as novel activators of PKM2 and (E/Z)-(4-(3-(2-((4-chlorophenyl)amino)-4-(dimethylamino)thiazol-5-yl)-2-(ethoxycarbonyl)-3-oxoprop-1-en-1-yl) phenyl)boronic acid (6c) emerged as the most potent derivative. 6c was further evaluated using various in silico tools to understand the molecular mechanism of tetramer formation. Docking studies revealed that 6c binds to the PKM2 dimer at the dimeric interface. Further to ascertain the binding site and mechanism of action, rigorous MD (molecular dynamics) simulations were undertaken, which led to the conclusion that 6c stabilizes the center of the dimeric interface that possibly promotes tetramer formation. We further planned to make a tablet of the developed molecule for oral delivery, but it was seriously impeded owing to poor aqueous solubility of 6c. To improve aqueous solubility and retain 6c at the lower gastrointestinal tract, thiolated chitosan-based nanoparticles (TCNPs) were prepared and further developed as tablet dosage form to retain anticancer potency in the excised goat colon. Our findings may provide a valuable pharmacological mechanism for understanding metabolic underpinnings that may aid in the clinical development of new anticancer agents targeting PKM2.

Self-Assembled Fmoc-Arg-Phe-Phe Peptide Gels with Highly Potent Bactericidal Activities.

The emergence of antibiotic resistance and the increasing rate of bacterial infections have motivated scientists to explore novel antibacterial materials and strategies to circumvent this challenge. Gels fabricated from ultrashort self-assembled peptides have turned out to be the most promising bactericidal materials. Self-assembled Fmoc-Phe-Phe gels have been extensively investigated earlier, and it has been shown that these gels possess potent bactericidal properties but suffer from disadvantages, such as poor proteolytic stabilities. In the present work, we report the highly potent bactericidal activities and proteolytic stability of gels fabricated from Fmoc-l-Arg-d-Phe-d-Phe-CONH2 (RFF) peptide, which are best in class. We fabricated and characterized self-assembled gels (1-2% w/v) from Fmoc-d-Phe-d-Phe-CONH2 (FF), Fmoc-l-His-d-Phe-d-Phe-CONH2 (HFF), and Fmoc-l-Arg-d-Phe-d-Phe-CONH2 (RFF) in aq dimethyl sulfoxide (35% v/v). The gels were characterized for their surface morphology, viscoelastic, self-healing, and stability characteristics. On incubation with proteolytic enzymes, FF gels did not show statistically significant degradation, and HFF and RFF gels showed only 43 and 32% degradation within 72 h at 37 °C, which is much better than gels reported earlier. The RFF gels (2%) exhibited more than 90% inhibition against Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) within 6 h, and the activities were sustained for up to 72 h. The high-resolution transmission electron microscopy studies indicated electrostatic interactions between the gel and bacterial membrane components, leading to cell lysis and death, which was further confirmed by the bacterial cell Live/Dead assay. MTT assay showed that the gels were not toxic to mammalian cells (L929). The bactericidal characteristics of RFF gels have not been reported so far. The RFF gels show strong potential for treating device-related infections caused by antimicrobial-resistant bacteria.

Retroperitoneal Single Port Versus Transperitoneal Multiport Donor Nephrectomy: A Prospective Randomized Control Trial.

BACKGROUND: Laparoscopic donor nephrectomy (LDN) converted a retroperitoneal (RP) procedure into a transperitoneal (TP) operation with reports of bowel and solid organ injuries leading to mortality in occasional cases. Laparoscopic RP donor nephrectomy can reduce these risks but never became popular because of the muscle cutting approach. Lumbotomy incision can be used to approach retroperitoneum by incising fascial planes, eliminating disadvantages of the RP approach. This report compares the outcomes of the standard multiport TP LDN with translumbar laparoendoscopic single-site donor nephrectomy (LESS-DN). METHODS: Between January 2016 and June 2017, 50 voluntary kidney donors out of 267 donors were randomized to undergo LESS-DN vs LDN. Donors with body mass index ≥30 kg/m2, multiple renal arteries, and right-sided nephrectomy were excluded from the study. Postoperative pain, duration of surgery, length of graft vessels and ureter, warm ischemia time, intraoperative blood loss, incision length, convalescence period, duration of hospital stay, and recipients' creatinine at discharge were compared among both the groups. Pain assessment was done using visual analogue scale (VAS). RESULTS: The RP group experienced lesser pain (VAS score 0.3 ± 0.3 vs 1.1 ± 0.0, p = 0.000), lesser analgesic requirement (186 ± 51.07 mg vs 254 ± 62.7 mg, p = 0.000), and faster convalescence (7.0 ± 3.0 days vs 10.7 ± 3.3 days, p = 0.00) related to smaller cumulative incision (7.8 ± 0.8 cm vs12.4 ± 2.0 cm, p = 0.00), and had reduced operative time (142 ± 26.2 minutes vs 170.8 ± 34.75 minutes, p = 0.001) and blood loss. Other recorded parameters were similar in both the groups. CONCLUSIONS: The single port RP approach significantly reduced postoperative pain and hastened recovery when compared with the TP approach. Converting to a RP approach presents an opportunity for surgeons to further reduce morbidity associated with the donor nephrectomy.

Alternatively-spliced extra domain A of fibronectin promotes acute inflammation and brain injury after cerebral ischemia in mice.

BACKGROUND AND PURPOSE: The fibronectin isoform containing the alternatively spliced extra domain A (EDA(+)-FN) is normally absent from the circulation, but plasma levels of EDA(+)-FN can become markedly elevated in several human pathological conditions associated with inflammation including ischemic stroke. It remains unknown whether EDA(+)-FN contributes to stroke pathogenesis or is simply an associative marker. Several in vitro studies suggest that EDA(+)-FN can activate Toll-like receptor 4, an innate immune receptor that triggers proinflammatory responses. We undertook a genetic approach in mice to investigate the ability of EDA(+)-FN to mediate inflammatory brain damage in a focal cerebral ischemia/reperfusion injury model. METHODS: We used genetically modified EDA(+/+) mice, which constitutively express EDA(+)-FN. Extent of injury, neurological outcome, and inflammatory mechanisms were assessed after 1-hour cerebral ischemia/23-hour reperfusion injury and compared with wild-type mice. RESULTS: We found that EDA(+/+) mice developed significantly larger infarcts and severe neurological deficits that were associated with significant increased neutrophil and macrophage infiltration as quantitated by immunohistochemistry. Additionally, we found upregulation of nuclear factor-κB, cyclo-oxygenase-2, and inflammatory cytokines tumor necrosis factor-α, interleukin-1ß, and interleukin-6 in the EDA(+/+) mice compared with wild-type mice. Interestingly, increased brain injury and neurological deficits were largely abrogated in EDA(+/+) mice by treatment with a specific Toll-like receptor 4 inhibitor. CONCLUSIONS: These findings provide the first evidence that EDA(+)-FN promotes inflammatory brain injury after ischemic stroke and suggest that the elevated levels of plasma EDA(+)-FN observed in chronic inflammatory conditions could worsen injury and outcome in patients after acute stroke.