Elucidation of the Role of TRPV1, VEGF-A, TXA2, Redox Homeostasis, and Inflammatory Cascades in Protection against Cold Injuries by Herbosomal-Loaded PEG-Poloxamer Topical Formulation.

High-altitude regions, cold deserts, permafrost regions, and the polar region have some of the severest cold conditions on earth and pose immense perils of cold injuries to exposed individuals. Accidental and unintended exposures to severe cold, either unintentionally or due to occupational risks, can greatly increase the risk of serious conditions including hypothermia, trench foot, and cold injuries like frostbite. Cold-induced vasoconstriction and intracellular/intravascular ice crystal formation lead to hypoxic conditions at the cellular level. The condition is exacerbated in individuals having inadequate and proper covering and layering, particularly when large area of the body are exposed to extremely cold environments. There is a paucity of preventive and therapeutic pharmacological modalities that have been explored for managing and treating cold injuries. Given this, an efficient modality that can potentiate the healing of frostbite was investigated by studying various complex pathophysiological changes that occur during severe cold injuries. In the current research, we report the effectiveness and healing properties of a standardized formulation, i.e., a herbosomal-loaded PEG-poloxamer topical formulation (n-HPTF), on frostbite. The intricate mechanistic pathways modulated by the novel formulation have been elucidated by studying the pathophysiological sequelae that occur following severe cold exposures leading to frostbite. The results indicate that n-HPTF ameliorates the outcome of frostbite, as it activates positive sensory nerves widely distributed in the epidermis transient receptor potential vanilloid 1 (TRPV1), significantly (p < 0.05) upregulates cytokeratin-14, promotes angiogenesis (VEGF-A), prominently represses the expression of thromboxane formation (TXA2), and significantly (p < 0.05) restores levels of enzymatic (glutathione reductase, superoxide dismutase, and catalase) and nonenzymatic antioxidants (glutathione). Additionally, n-HPTF attenuates oxidative stress and the expression of inflammatory proteins PGF-2α, NFκB-p65, TNF-α, IL-6, IL-1ß, malondialdehyde (MDA), advanced oxidative protein products (AOPP), and protein carbonylation (PCO). Masson's Trichrome staining showed that n-HPTF stimulates cellular proliferation, and increases collagen fiber deposition, which significantly (p < 0.05) promotes the healing of frostbitten tissue, as compared to control. We conclude that protection against severe cold injuries by n-HPTF is mediated via modulation of pathways involving TRPV1, VEGF-A, TXA2, redox homeostasis, and inflammatory cascades. The study is likely to have widespread implications for the prophylaxis and management of moderate-to-severe frostbite conditions.

Improving material properties of a poloxamer P407 hydrogel-based hydroxyapatite bone substitute material by adding silica-A comparative in vivo study.

The structure and handling properties of a P407 hydrogel-based bone substitute material (BSM) might be affected by different poloxamer P407 and silicon dioxide (SiO2) concentrations. The study aimed to compare the mechanical properties and biological parameters (bone remodeling, BSM degradation) of a hydroxyapatite: silica (HA)-based BSM with various P407 hydrogels in vitro and in an in vivo rat model. Rheological analyses for mechanical properties were performed on one BSM with an SiO2-enriched hydrogel (SPH25) as well on two BSMs with unaltered hydrogels in different gel concentrations (PH25 and PH30). Furthermore, the solubility of all BSMs were tested. In addition, 30 male Wistar rats underwent surgical creation of a well-defined bone defect in the tibia. Defects were filled randomly with PH30 (n = 15) or SPH25 (n = 15). Animals were sacrificed after 12 (n = 5 each), 21 (n = 5 each), and 63 days (n = 5 each). Histological evaluation and histomorphometrical quantification of new bone formation (NB;%), residual BSM (rBSM;%), and soft tissue (ST;%) was conducted. Rheological tests showed an increased viscosity and lower solubility of SPH when compared with the other hydrogels. Histomorphometric analyses in cancellous bone showed a decrease of ST in PH30 (p = .003) and an increase of NB (PH30: p = .001; SPH: p = .014) over time. A comparison of both BSMs revealed no significant differences. The addition of SiO2 to a P407 hydrogel-based hydroxyapatite BSM improves its mechanical stability (viscosity, solubility) while showing similar in vivo healing properties compared to PH30. Additionally, the SiO2-enrichment allows a reduction of poloxamer ratio in the hydrogel without impairing the material properties.

Dynamic RGD ligands derived from highly mobile cyclodextrins regulate spreading and proliferation of endothelial cells to promote vasculogenesis.

A thiolated RGD was incorporated into the threaded allyl-ß-cyclodextrins (Allyl-ß-CDs) of the polyrotaxane (PR) through a thiol-ene click reaction, resulting in the formation of dynamic RGD ligands on the PR surface (dRGD-PR). When maintaining consistent RGD density and other physical properties, endothelial cells (ECs) cultured on dRGD-PR exhibited significantly increased cell proliferation and a larger cell spreading area compared to those on the non-dynamic RGD (nRGD-PCL). Furthermore, ECs on dRGD-PR demonstrated elevated expression levels of FAK, p-FAK, and p-AKT, along with a larger population of cells in the G2/M stage during cell cycle analysis, in contrast to cells on nRGD-PCL. These findings suggest that the movement of the RGD ligands may exert additional beneficial effects in promoting EC spreading and proliferation, beyond their essential adhesion and proliferation-promoting capabilities, possibly mediated by the RGD-integrin-FAK-AKT pathway. Moreover, in vitro vasculogenesis tests were conducted using two methods, revealing that ECs cultured on dRGD-PR exhibited much better vasculogenesis than nRGD-PCL in vitro. In vivo testing further demonstrated an increased presence of CD31-positive tissues on dRGD-PR. In conclusion, the enhanced EC spreading and proliferation resulting from the dynamic RGD ligands may contribute to improved in vitro vasculogenesis and in vivo vascularization.

Analysis of the impact of pluronic acid on the thermal stability and infectivity of AAV6.2FF.

BACKGROUND: The advancement of AAV vectors into clinical testing has accelerated rapidly over the past two decades. While many of the AAV vectors being utilized in clinical trials are derived from natural serotypes, engineered serotypes are progressing toward clinical translation due to their enhanced tissue tropism and immune evasive properties. However, novel AAV vectors require formulation and stability testing to determine optimal storage conditions prior to their use in a clinical setting. RESULTS: Here, we evaluated the thermal stability of AAV6.2FF, a rationally engineered capsid with strong tropism for lung and muscle, in two different buffer formulations; phosphate buffered saline (PBS), or PBS supplemented with 0.001% non-ionic surfactant Pluronic F68 (PF-68). Aliquots of AAV6.2FF vector encoding the firefly luciferase reporter gene (AAV6.2FF-ffLuc) were incubated at temperatures ranging from -20°C to 55°C for varying periods of time and the impact on infectivity and particle integrity evaluated. Additionally, the impact of several rounds of freeze-thaw treatments on the infectivity of AAV6.2FF was investigated. Vector infectivity was measured by quantifying firefly luciferase expression in HEK 293 cells and AAV particle integrity was measured by qPCR quantification of encapsidated viral DNA. CONCLUSIONS: Our data demonstrate that formulating AAV6.2FF in PBS containing 0.001% PF-68 leads to increased stability and particle integrity at temperatures between -20℃ to 21℃ and protection against the destructive effects of freeze-thaw. Finally, AAV6.2FF-GFP formulated in PBS supplemented with 0.001% PF-68 displayed higher transduction efficiency in vivo in murine lung epithelial cells following intranasal administration than vector buffered in PBS alone further demonstrating the beneficial properties of PF-68.

Tannic acid-poloxamer self-assembled nanoparticles for advanced atherosclerosis therapy by regulation of macrophage polarization.

Atherosclerosis (AS) is a significant contributor to cardiovascular events. Advanced AS is particularly concerning, as it leads to the formation of high-risk vulnerable plaques. Current treatments for AS focus on antithrombotic and lipid-lowering interventions, which are effective in treating early-stage AS. Recent studies have shown that macrophage polarization plays a crucial role in the development of AS. This study presents a new biomedical application of natural tannic acid as an anti-inflammatory nanoplatform for advanced AS. Tannic acid-poloxamer nanoparticles (TPNP) are fabricated through self-assembly of tannic acid (TA) and poloxamer. TPNP has the potential to provide effective treatment for advanced AS. According to in vitro studies, TPNP has been found to suppress the inflammatory response in lipopolysaccharide-stimulated macrophages by scavenging reactive oxygen species (ROS), downregulating the expression levels of inflammatory cytokines (such as interleukin-10 and tumor necrosis factor-α) and regulating polarization of macrophages. In vivo studies further reveal that TPNP can retard the development of advanced atherosclerotic plaques by reducing ROS production and promoting M2 macrophage polarization in the aorta of ApoE-/- mice. Overall, these findings suggest that TPNP could be used to develop natural multifunctional nanoplatforms for molecular therapy of AS and other inflammation-related diseases.

Harnessing Mechanical Stress with Viscoelastic Biomaterials for Periodontal Ligament Regeneration.

The viscoelasticity of mechanically sensitive tissues such as periodontal ligaments (PDLs) is key in maintaining mechanical homeostasis. Unfortunately, PDLs easily lose viscoelasticity (e.g., stress relaxation) during periodontitis or dental trauma, which disrupt cell-extracellular matrix (ECM) interactions and accelerates tissue damage. Here, Pluronic F127 diacrylate (F127DA) hydrogels with PDL-matched stress relaxation rates and high elastic moduli are developed. The hydrogel viscoelasticity is modulated without chemical cross-linking by controlling precursor concentrations. Under cytomechanical loading, F127DA hydrogels with fast relaxation rates significantly improved the fibrogenic differentiation potential of PDL stem cells (PDLSCs), while cells cultured on F127DA hydrogels with various stress relaxation rates exhibited similar fibrogenic differentiation potentials with limited cell spreading and traction forces under static conditions. Mechanically, faster-relaxing F127DA hydrogels leveraged cytomechanical loading to activate PDLSC mechanotransduction by upregulating integrin-focal adhesion kinase pathway and thus cytoskeletal rearrangement, reinforcing cell-ECM interactions. In vivo experiments confirm that faster-relaxing F127DA hydrogels significantly promoted PDL repair and reduced abnormal healing (e.g., root resorption and ankyloses) in delayed replantation of avulsed teeth. This study firstly investigated how matrix nonlinear viscoelasticity influences the fibrogenesis of PDLSCs under mechanical stimuli, and it reveals the underlying mechanobiology, which suggests novel strategies for PDL regeneration.

Antimicrobial potential of a biosurfactant gel for the prevention of mixed biofilms formed by fluconazole-resistant C. albicans and methicillin-resistant S. aureus in catheters.

Dual-species biofilms formed by Candida albicans and Staphylococcus aureus have high virulence and drug resistance. In this context, biosurfactants produced by Pseudomonas aeruginosa have been widely studied, of which a new derivative (RLmix_Arg) stands out for possible application in formulations. The objective of this study was to evaluate the antibiofilm activity of RLmix_Arg, both alone and incorporated in a gel prepared with Pluronic F-127, against dual-species biofilms of fluconazole-resistant C. albicans (FRCA) and methicillin-resistant S. aureus (MRSA) in impregnated catheters. Broth microdilution tests, MTT reduction assays of mature biofilms, impregnation of RLmix_Arg and its gel in peripheral venous catheters, durability tests and scanning electron microscopy (SEM) were performed. RLmix_Arg showed antimicrobial activity against Candida spp. and S. aureus, by reducing the cell viability of mixed biofilms of FRCA and MRSA, and preventing their formation in a peripheral venous catheter. The incorporation of this biosurfactant in the Pluronic F-127 gel considerably enhanced its antibiofilm activity. Thus, RLmix_Arg has potential application in gels for impregnation in peripheral venous catheters, helping to prevent development of dual-species biofilms of FRCA and MRSA.

Treatment using vanillin-derived synthetic molecules incorporated into polymeric micelles is effective against infection caused by Leishmania amazonensis species.

Treatment against leishmaniasis presents problems, mainly due to the toxicity of the drugs, high cost, and the emergence of resistant strains. A previous study showed that two vanillin-derived synthetic molecules, 3s [4-(2-hydroxy-3-(4-octyl-1H-1,2,3-triazol-1-yl)propoxy)-3-methoxybenzaldehyde] and 3t [4-(3-(4-decyl-1H-1,2,3-triazol-1-yl)-2-hydroxypropoxy)-3-methoxybenzaldehyde], presented antileishmanial activity against Leishmania infantum, L. amazonensis, and L. braziliensis species. In the present work, 3s and 3t were evaluated to treat L. amazonensis-infected mice. Molecules were used pure or incorporated into Poloxamer 407-based micelles. In addition, amphotericin B (AmpB) and its liposomal formulation, Ambisome®, were used as control. Animals received the treatment and, one and 30 days after, they were euthanized to evaluate immunological, parasitological, and biochemical parameters. Results showed that the micellar compositions (3s/Mic and 3t/Mic) induced significant reductions in the lesion mean diameter and parasite load in the infected tissue and distinct organs, as well as a specific and significant antileishmanial Th1-type immune response, which was based on significantly higher levels of IFN-γ, IL-12, nitrite, and IgG2a isotype antibodies. Drug controls showed also antileishmanial action; although 3s/Mic and 3t/Mic have presented better and more significant parasitological and immunological data, which were based on significantly higher IFN-γ production and lower parasite burden in treated animals. In addition, significantly lower levels of urea, creatinine, alanine transaminase, and aspartate transaminase were found in mice treated with 3s/Mic and 3t/Mic, when compared to the others. In conclusion, results suggest that 3s/Mic and 3t/Mic could be considered as therapeutic candidates to treat against L. amazonensis infection.

Modulation of macrophages by a phillyrin-loaded thermosensitive hydrogel promotes skin wound healing in mice.

BACKGROUND: Impaired wound healing in traumatic skin injuries remains a severe clinical challenge due to impaired re-vascularization, harmful bacteria infection, and inflammation dysregulation. Macrophages are recognized as prominent immune cells in tissue regeneration and wound healing. Consequently, the modulation of macrophages provides a promising therapeutic target for wound healing disorders. Here, we aimed to explore whether a novel constructed combination of thermosensitive hydrogel Pluronic F-127 (PF-127) and phillyrin (PH, the main active compound of forsythia suspensa) could improve skin wound healing. METHODS: Firstly, the biological effects of pH on the phenotype and inflammation of macrophages were assessed by flow cytometry and ELISA. The biocompatibility of the PF-127 plus PH combination was investigated on keratinocytes and red blood cells. The biological effect of PF-127/PH hydrogel on the migratory ability of keratinocytes in vitro was evaluated using the scratch and transwell migration assays. In addition,S. aureusandE. coliwere employed to test the antibacterial properties of the PF-127 plus PH combination. Finally, PF-127 plus PH scaffold was appliedto the full-thickness skin defect in mice. Histomorphological evaluation and immunochemistry were performed to explore the wound-healing activity of PF-127/PH hydrogel. RESULTS: PH can promote the polarization of macrophages from the M1 (pro-inflammatory) phenotype to the M2 (anti-inflammatory) phenotype. The PF-127/PH hydrogel was highly biocompatible and showed a potent stimulative effect on the migration of keratinocytesin vitro. The combination of PF-127 and PH exerted a pronounced antibacterial activity onS. aureusandE. coli in vitro.PF-127/PH hydrogel potently accelerates the healing of full-thickness skin defects by promoting skin cell proliferation, accelerating angiogenesis, and inhibiting inflammation. CONCLUSIONS: Our study suggests that PF-127/PH hydrogel has excellent potential for treating traumatic skin defects.

Effect of anti-adhesive supplement doses on sperm motility characteristics in the European whitefish (Coregonus lavaretus).

Sperm adhering to glass slides is one of the main problems during fish sperm motility analyses with CASA systems. To mitigate this, albumin is the supplement added most frequently to activating solutions. However, there is no data on the use of supplements other than albumin (in various concentrations) in analyses of European whitefish (Coregonus lavaretus) sperm motility. This issue was investigated in the presented research using three anti-adhesive supplements (albumin, casein, Pluronic F-127) that were added to Billard solution (BS: 20 mM Tris, 1 mM CaCl2, 154 mM NaCl, 30 mM glycine at pH 9.0) at different concentrations (0.0; 0.1; 0.2; 0.5; 1.0; 2.0%). It was noted that the addition of the lowest concentration (0.1%) of albumin, casein, or the pluronic to BS had a significant effect on the motility and kinetic parameters of whitefish sperm compared to pure BS. BS supplemented with 0.2-0.5% albumin was the most appropriate variant used for whitefish sperm motility activation in the present experiment. BS supplemented with the pluronic at 1.0-2.0% concentrations resulted in significantly higher values of almost all CASA parameters compared to casein at the same concentrations. Moreover, CASA parameters determined in this variant of the pluronic (1.0-2.0%) were similar to those when BS was supplemented with the same albumin concentrations. This indicated that instead of albumin, the pluronic at higher concentrations in BS might be used to analyze whitefish sperm motility.

A Poloxamer 407/chitosan-based thermosensitive hydrogel dressing for diabetic wound healing via oxygen production and dihydromyricetin release.

The current clinical treatment of diabetic wounds is still based on oxygen therapy, and the slow healing of skin wounds due to hypoxia has always been a key problem in the repair of chronic skin injuries. To overcome this problem, the oxygen-producing matrix CaO2NPS based on the temperature-sensitive dihydromyricetin-loaded hydrogel was prepared. In vitro activity showed that the dihydromyricetin (DHM) oxygen-releasing temperature-sensitive hydrogel composite (DHM-OTH) not only provided a suitable oxygen environment for cells around the wound to survive but also had good biocompatibility and various biological activities. By constructing a T2D wound model, we further investigated the repairing effect of DHM-OTH on chronic diabetic skin wounds and the mechanisms involved. DHM-OTH was able to reduce inflammatory cells and collagen deposition and promote angiogenesis and cell proliferation for diabetic wound healing. These in vitro and in vivo data suggest that DHM-OTH accelerates diabetic wound repair as a novel method to efficiently deliver oxygen to wound tissue, providing a promising strategy to improve diabetic wound healing.

Lysosome-targeted ruthenium(II) complex encapsulated with pluronic® F-127 induces oncosis in A549 cells.

Transition metal complexes with characteristics of unique packaging in nanoparticles and remarkable cancer cell cytotoxicity have emerged as potential alternatives to platinum-based antitumor drugs. Here we report the synthesis, characterization, and antitumor activities of three new Ruthenium complexes that introduce 5-fluorouracil-derived ligands. Notably, encapsulation of one such metal complex, Ru3, within pluronic® F-127 micelles (Ru3-M) significantly enhanced Ru3 cytotoxicity toward A549 cells by a factor of four. To determine the mechanisms underlying Ru3-M cytotoxicity, additional in vitro experiments were conducted that revealed A549 cell treatment with lysosome-targeting Ru3-M triggered oxidative stress, induced mitochondrial membrane potential depolarization, and drastically reduced intracellular ATP levels. Taken together, these results demonstrated that Ru3-M killed cells mainly via a non-apoptotic pathway known as oncosis, as evidenced by observed Ru3-M-induced cellular morphological changes including cytosolic flushing, cell swelling, and cytoplasmic vacuolation. In turn, these changes together caused cytoskeletal collapse and activation of porimin and calpain1 proteins with known oncotic functions that distinguished this oncotic process from other cell death processes. In summary, Ru3-M is a potential anticancer agent that kills A549 cells via a novel mechanism involving Ru(II) complex triggering of cell death via oncosis.

The effect of supplementing the calcium phosphate cement containing poloxamer 407 on cellular activities.

Calcium phosphate cement (CPC) is generally used for bone repair and augmentation. Poloxamers are tri-block copolymers that are used as surfactants but have applications in drug and antibiotic delivery. However, their biological effects on bone regeneration systems remain unelucidated. Here, we aimed to understand how supplementing the prototype CPC with poloxamer would impact cellular activity and its function as a bone-grafting material. A novel CPC, modified beta-tricalcium phosphate (mß-TCP) powder, was developed through a planetary ball-milling process using a beta-tricalcium phosphate (ß-TCP). The mß-TCP dissolves rapidly and accelerates hydroxyapatite precipitation; successfully shortening the cement setting time and enhancing the strength. Furthermore, the addition of poloxamer 407 to mß-TCP could reduce the risk of leakage from bone defects and improve fracture toughness while maintaining mechanical properties. In this study, the poloxamer addition effects (0.05 and 0.1 g/mL) on the cellular activities of MC3T3-E1 cells cultured in vitro were investigated. The cell viability of mß-TCP containing poloxamer 407 was similar to that of mß-TCP. All specimens showed effective cell attachment and healthy polygonal extension of the cytoplasm firmly attached to hydroxyapatite (HA) crystals. Therefore, even with the addition of poloxamer to mß-TCP, it does not have a negative effect to osteoblast growth. These data demonstrated that the addition of poloxamer 407 to mß-TCP might be considered a potential therapeutic application for the repair and regeneration of bone defects.

Novel CO2-encapsulated Pluronic F127 hydrogel for the treatment of Achilles tendon injury.

Nonsurgical treatment and surgical repairment of injured Achilles tendons seldom restore the wounded tendon to its original elasticity and stiffness. Therefore, we hypothesized that the surgically repaired Achilles tendon can achieve satisfactory regeneration by applying multi-drug encapsulated hydrogels. In this study, a novel bupivacaine-eluting carbon dioxide-encapsulated Pluronic F127 hydrogel (BC-hydrogel) was developed for the treatment of Achilles tendon injuries. The rheological properties of BC-hydrogel were measured. A high-performance liquid chromatography assay was used to assess the release characteristics of bupivacaine in both in vitro and in vivo settings. Furthermore, the effectiveness of BC-hydrogel in treating torn tendons was examined in a rat model, and histological analyses were conducted. Evidently, the degradable hydrogels continuously eluted bupivacaine for more than 14 days. The animal study results revealed that the BC-hydrogel improved the post-surgery mobility of the animals compared with pristine hydrogels. Histological assay results demonstrated a significant reaction to high vascular endothelial growth factor in the surrounding tissues and expression of collagen I within the repaired tendon. This demonstrates the potential of this novel BC-hydrogel as an effective treatment method for Achilles tendon injuries.

Pluronic F-127 Hydrogel Loaded with Human Adipose-Derived Stem Cell-Derived Exosomes Improve Fat Graft Survival via HIF-1α-Mediated Enhancement of Angiogenesis.

Purpose: Autologous fat grafting is playing an increasingly important role in plastic surgery. However, high absorption and low survival of autologous fat grafts limit their clinical application. This study aimed to investigate whether human adipose-derived stem cell-derived exosomes (hASC-Exos) encapsulated in a PF-127 hydrogel can improve the survival of autologous fat grafts and to elucidate the underlying mechanisms. Patients and Methods: Exosomes were isolated from hASCs and identified using transmission electron microscopy, nanoparticle tracking analysis and Western blotting. We performed functional assays in vitro to assess the effect of hASC-Exos on proliferation, migration, and tube formation as well as their regulatory role in the HIF-1α/VEGF signaling pathway. hASC-Exos encapsulated in the PF-127 hydrogel were used as an in vivo autologous fat graft model. The effects of the PF-127 hydrogel/hASC-Exos and the role of the HIF-1α/VEGF signaling pathway in promoting angiogenesis in an autologous fat grafting model were assessed. Results: hASC-Exos were taken up by human umbilical vein endothelial cells and enhanced their proliferation, migration, and tubule formation in vitro. The effects of hASC-Exos on promoting angiogenesis were mediated by the HIF-1α/VEGF signaling pathway. Moreover, we fabricated a PF-127 hydrogel for the sustained release of hASC-Exos, and in vivo results showed that hASC-Exos encapsulated in PF-127 hydrogel improved the survival of autologous fat grafts. Conclusion: Our findings indicated that hASC-Exos encapsulated in PF-127 hydrogel serve as a key regulator of angiogenesis by activating the HIF-1α/VEGF signaling pathway and provide a promising strategy for autologous fat grafting treatment.

Formulation and evaluation of simvastatin cubosomal nanoparticles for assessing its wound healing effect.

Wound healing is one of the most challenging medical circumstances for patients. Pathogens can infect wounds, resulting in tissue damage, inflammation, and disruption of the healing process. Simvastatin was investigated recently, as a wound healing agent that may supersede the present therapies for wounds. Our goal in this paper is to focus on formulation of simvastatin cubosomes for topical delivery, as a potential approach to improve simvastatin skin permeation. By this technique its wound healing effect could be improved. Cubosomes were prepared using the top-down method and the prepared cubosomes were characterized by several techniques. The most optimal simvastatin cubosomal formulation was then included in a cubogel dosage form using different gelling agents. The results showed that the average particle size of the prepared cubosomes was 113.90 ± 0.58 nm, the entrapment efficiency was 93.95 ± 0.49% and a sustained simvastatin release was achieved. The optimized formula of simvastatin cubogel displayed pseudoplastic rheological behavior. This same formula achieved enhancement in drug permeation through excised rat skin compared to free simvastatin hydrogel with flux values of 46.18 ± 2.12 mcg cm-2 h-1 and 25.92 ± 3.45 mcg cm-2 h-1 respectively. Based on the in-vivo rat studies results, this study proved a promising potential of simvastatin cubosomes as wound healing remedy.

Highly reproducible and cost-effective one-pot organoid differentiation using a novel platform based on PF-127 triggered spheroid assembly.

Organoid technology offers sophisticatedin vitrohuman models for basic research and drug development. However, low batch-to-batch reproducibility and high cost due to laborious procedures and materials prevent organoid culture standardization for automation and high-throughput applications. Here, using a novel platform based on the findings that Pluronic F-127 (PF-127) could trigger highly uniform spheroid assembly through a mechanism different from plate coating, we develop a one-pot organoid differentiation strategy. Using our strategy, we successfully generate cortical, nephron, hepatic, and lung organoids with improved reproducibility compared to previous methods while reducing the original costs by 80%-95%. In addition, we adapt our platform to microfluidic chips allowing automated culture. We showcase that our platform can be applied to tissue-specific screening, such as drug toxicity and transfection reagents testing. Finally, we generateNEAT1knockout tissue-specific organoids and showNEAT1modulates multiple signaling pathways fine-tuning the differentiation of nephron and hepatic organoids and suppresses immune responses in cortical organoids. In summary, our strategy provides a powerful platform for advancing organoid research and studying human development and diseases.

Evaluation of Antibacterial Efficacy of Two Commercially Available Probiotics as Intracanal Medicament against Enterococcus faecalis: An In Vitro Study.

AIM: This study was performed to evaluate the antibacterial efficacy of two commercially available probiotics (BIFILAC and VSL 3) as intracanal medicament against Enterococcus faecalis in endodontic therapy. MATERIALS AND METHODS: Microorganisms from commercially available probiotics (BIFILAC and VSL 3) were extracted via the manufacturer's recommendations and mixed by weight. About 30 microliters were then placed on sterile discs. The pathogenic test organism was E. faecalis set to a 1 McFarland standard challenge. A two-probiotic disc template on blood agar plates was inoculated with E. faecalis and incubated at 37°C for 48 hours and 1 week respectively. Phase-1 of the study was conducted by a disc diffusion assay test to evaluate zones of inhibition (ZOI) in millimeters (mm). Phase-2 was conducted by mixing 9 mL of 30% poloxamer 407 and MRS broth in a test tube, together with the two probiotic mixtures and E. faecalis, set at a 2 McFarland standard. Serial dilutions up to 108 were done and the mixture was placed inside root canals and incubated at 37ºC for 36 hours and evaluated for colony-forming unit (CFU)/mL counts. RESULTS: The results of phase-1 showed that probiotics Lactobacillus rhamnosus and Bifidobacterium species are effective in fighting against E. faecalis with the acceptable zone of inhibition. The results of phase-2 showed that both the probiotics are effective against E. faecalis with a reduction in the number of CFU after probiotic usage. CONCLUSION: Commercially available probiotics can be used effectively as an intracanal medicament to fight against E. faecalis, Poloxamer 407 is a promising vehicle for delivering probiotics inside the root canal system. Further in vitro and in vivo studies are needed to determine the full potential of "Bacteriotherapy" with an application of probiotics. CLINICAL SIGNIFICANCE: If probiotics are proved to be an effective intracanal medicament against E.faecalis they can be used as an alternative to calcium hydroxide as intracanal medicament with no side effects to the host.

Surfactants reduce aggregation of monoclonal antibodies in cell culture medium with improvement in performance of mammalian cell culture.

Therapeutic monoclonal antibodies (mAbs) are biologics produced using mammalian cells and represent an important class of biotherapeutics. Aggregation in mAbs is a major challenge that can be mitigated by rigorous and reproducible upstream and downstream approaches. The impact of frequently used surfactants, like polysorbate 20, polysorbate 80, poloxamer 188, and 2-hydroxypropyl-beta-cyclodextrin, on aggregation of mAbs during cell culture was investigated in this study. Their impact on cell proliferation, viability, and mAb titer was also investigated. Polysorbate 20 and polysorbate 80 at the concentration of 0.01 g/L and poloxamer 188 at the concentration of 5 g/L were found to be effective in reducing aggregate formation in cell culture medium, without affecting the cell growth or viability. Furthermore, their presence in culture media resulted in increased cell proliferation as compared to the control group. Addition of these surfactants at the specified concentrations increased monomer production while decreasing high molecular weight species in the medium. After mAbs were separated, using protein "A" chromatography, flasks with surfactant exhibited improved antibody stability, when analyzed by DLS. Thus, while producing aggregation-prone mAbs via mammalian cell culture, these excipients may be employed as cell culture medium supplements to enhance the quality and yield of functional mAbs.

Modulating and optimizing Pluronic F-68 concentrations and feeding for intensified perfusion Chinese hamster ovary cell cultures.

Perfusion culture is often performed with micro-sparger to fulfill the high oxygen demand from the densified cells. Protective additive Pluronic F-68 (PF-68) is widely used to mitigate the adverse effect in cell viability from micro-sparging. In this study, different PF-68 retention ratio in alternating tangential filtration (ATF) columns was found to be crucial for cell performance of different perfusion culture modes. The PF-68 in the perfusion medium was found retained inside the bioreactor when exchanged through ATF hollow fibers with a small pore size (50 kD). The accumulated PF-68 could provide sufficient protection for cells under micro-sparging. On the other hand, with large-pore-size (0.2 µm) hollow fibers, PF-68 could pass through the ATF filtration membranes with little retention, and consequently led to compromised cell growth. To overcome the defect, a PF-68 feeding strategy was designed and successfully verified on promoting cell growth with different Chinese hamster ovary (CHO) cell lines. With PF-68 feeding, enhancements were observed in both viable cell densities (20%-30%) and productivity (~30%). A threshold PF-68 concentration of 5 g/L for high-density cell culture (up to 100 × 106 cells/mL) was also proposed and verified. The additional PF-68 feeding was not observed to affect product qualities. By designing the PF-68 concentration of perfusion medium to or higher than the threshold level, a similar cell growth enhancement was also achieved. This study systematically investigated the protecting role of PF-68 in intensified CHO cell cultures, shedding a light on the optimization of perfusion cultures through the control of protective additives.