Poloxamer-188 Exacerbates Brain Amyloidosis, Presynaptic Dystrophies, and Pathogenic Microglial Activation in 5XFAD Mice.

BACKGROUND: Alzheimer's disease (AD) is initiated by aberrant accumulation of amyloid beta (Aß) protein in the brain parenchyma. The microenvironment surrounding amyloid plaques is characterized by the swelling of presynaptic terminals (dystrophic neurites) associated with lysosomal dysfunction, microtubule disruption, and impaired axonal transport. Aß-induced plasma membrane damage and calcium influx could be potential mechanisms underlying dystrophic neurite formation. OBJECTIVE: We tested whether promoting membrane integrity by brain administration of a safe FDA approved surfactant molecule poloxamer-188 (P188) could attenuate AD pathology in vivo. METHODS: Three-month-old 5XFAD male mice were administered several concentrations of P188 in the brain for 42 days with mini-osmotic pumps. After 42 days, mice were euthanized and assessed for amyloid pathology, dystrophic neurites, pathogenic microglia activation, tau phosphorylation, and lysosomal / vesicular trafficking markers in the brain. RESULTS: P188 was lethal at the highest concentration of 10mM. Lower concentrations of P188 (1.2, 12, and 120µM) were well tolerated. P188 increased brain Aß burden, potentially through activation of the γ-secretase pathway. Dystrophic neurite pathology was exacerbated in P188 treated mice as indicated by increased LAMP1 accumulation around Aß deposits. Pathogenic microglial activation was increased by P188. Total tau levels were decreased by P188. Lysosomal enzyme cathepsin D and calciumdependent vesicular trafficking regulator synaptotagmin-7 (SYT7) were dysregulated upon P188 administration. CONCLUSION: P188 brain delivery exacerbated amyloid pathology, dystrophic neurites, and pathogenic microglial activation in 5XFAD mice. These effects correlated with lysosomal dysfunction and dysregulation of plasma membrane vesicular trafficking. P188 is not a promising therapeutic strategy against AD pathogenesis.

Injectable Micelle-Incorporated Hydrogels for the Localized Chemo-Immunotherapy of Breast Tumors.

Although immune checkpoint blockade (ICB) holds potential for the treatment of various tumors, a considerable proportion of patients show a limited response to ICB therapy due to the low immunogenicity of a variety of tumors. It has been shown that some chemotherapeutics can turn low-immunogenic tumors into immunogenic phenotypes by inducing a cascade of immune responses. In this paper, we synthesized an injectable micelle-incorporated hydrogel, which was able to sequentially release the chemotherapeutic gemcitabine (GEM) and the hydrophobic indoleamine 2, 3-dioxygenase inhibitor, d-1-methyltryptophan (d-1MT) at tumor sites. The hydrogel was formed via the thiol-ene click reaction between the thiolated chondroitin sulfate and the micelle formed by amphiphilic methacrylated Pluronic F127, in which hydrophobic d-1MT was encapsulated in the core of the F127 micelles and the hydrophilic GEM was dispersed in the hydrogel network. The successive release of chemotherapeutics and immune checkpoint inhibitors at tumor tissues will first promote the infiltration of cytotoxic T lymphocytes and subsequently induce a robust antitumor immune response, ultimately exerting a synergetic therapeutic efficacy. In a 4T1 tumor-bearing mice model, our results showed that the combination of chemotherapy and immunotherapy through the micelle-incorporated hydrogel triggered an effective antitumor immune response and inhibited tumor metastasis to the lung. Our results highlight the potential of the injectable micelle-incorporated hydrogel for the localized chemo-immunotherapy in the treatment of breast tumors.

Comprehensive and comparative studies on nanocytotoxicity of glyceryl monooleate- and phytantriol-based lipid liquid crystalline nanoparticles.

BACKGROUND: Lipid liquid crystalline nanoparticles (LLCNPs) emerge as a suitable system for drug and contrast agent delivery. In this regard due to their unique properties, they offer a solubility of a variety of active pharmaceutics with different polarities increasing their stability and the possibility of controlled delivery. Nevertheless, the most crucial aspect underlying the application of LLCNPs for drug or contrast agent delivery is the unequivocal assessment of their biocompatibility, including cytotoxicity, genotoxicity, and related aspects. Although studies regarding the cytotoxicity of LLCNPs prepared from various lipids and surfactants were conducted, the actual mechanism and its impact on the cells (both cancer and normal) are not entirely comprehended. Therefore, in this study, LLCNPs colloidal formulations were prepared from two most popular structure-forming lipids, i.e., glyceryl monooleate (GMO) and phytantriol (PHT) with different lipid content of 2 and 20 w/w%, and the surfactant Pluronic F-127 using the top-down approach for further comparison of their properties. Prepared formulations were subjected to physicochemical characterization and followed with in-depth biological characterization, which included cyto- and genotoxicity towards cervical cancer cells (HeLa) and human fibroblast cells (MSU 1.1), the evaluation of cytoskeleton integrity, intracellular reactive oxygen species (ROS) generation upon treatment with prepared LLCNPs and finally the identification of internalization pathways. RESULTS: Results denote the higher cytotoxicity of PHT-based nanoparticles on both cell lines on monolayers as well as cellular spheroids, what is in accordance with evaluation of ROS activity level and cytoskeleton integrity. Detected level of ROS in cells upon the treatment with LLCNPs indicates their insignificant contribution to the cellular redox balance for most concentrations, however distinct for GMO- and PHT-based LLCNPs. The disintegration of cytoskeleton after administration of LLCNPs implies the relation between LLCNPs and F-actin filaments. Additionally, the expression of four genes involved in DNA damage and important metabolic processes was analyzed, indicating concentration-dependent differences between PHT- and GMO-based LLCNPs. CONCLUSIONS: Overall, GMO-based LLCNPs emerge as potentially more viable candidates for drug delivery systems as their impact on cells is not as deleterious as PHT-based as well as they were efficiently internalized by cell monolayers and 3D spheroids.

Polypseudorotaxane and polydopamine linkage-based hyaluronic acid hydrogel network with a single syringe injection for sustained drug delivery.

Polypseudorotaxane structure and polydopamine bond-based crosslinked hyaluronic acid (HA) hydrogels including donepezil-loaded microspheres were developed for subcutaneous injection. Both dopamine and polyethylene glycol (PEG) were covalently bonded to the HA polymer for catechol polymerization and inclusion complexation with alpha-cyclodextrin (α-CD), respectively. A PEG chain of HA-dopamine-PEG (HD-PEG) conjugate was threaded with α-CD to make a polypseudorotaxane structure and its pH was adjusted to 8.5 for dopamine polymerization. Poly(lactic-co-glycolic acid) (PLGA)/donepezil microsphere (PDM) was embedded into the HD-PEG network for its sustained release. The HD-PEG/α-CD/PDM 8.5 hydrogel system exhibited an immediate gelation pattern, injectability through single syringe, self-healing ability, and shear-thinning behavior. Donepezil was released from the HD-PEG/α-CD/PDM 8.5 hydrogel in a sustained pattern. Following subcutaneous injection, the weight of excised HD-PEG/α-CD/PDM 8.5 hydrogel was higher than the other groups on day 14. These findings support the clinical feasibility of the HD-PEG/α-CD/PDM 8.5 hydrogel for subcutaneous injection.

Antibiotic Cross-linked Micelles with Reduced Toxicity for Multidrug-Resistant Bacterial Sepsis Treatment.

One potential approach to address the rising threat of antibiotic resistance is through novel formulations of established drugs. We designed antibiotic cross-linked micelles (ABC-micelles) by cross-linking the Pluronic F127 block copolymers with an antibiotic itself, via a novel one-pot synthesis in aqueous solution. ABC-micelles enhanced antibiotic encapsulation while also reducing systemic toxicity in mice. Using colistin, a hydrophilic, potent ″last-resort" antibiotic, ABC-micelle encapsulation yield was 80%, with good storage stability. ABC-micelles exhibited an improved safety profile, with a maximum tolerated dose of over 100 mg/kg colistin in mice, at least 16 times higher than the free drug. Colistin-induced nephrotoxicity and neurotoxicity were reduced in ABC-micelles by 10-50-fold. Despite reduced toxicity, ABC-micelles preserved bactericidal activity, and the clinically relevant combination of colistin and rifampicin (co-loaded in the micelles) showed a synergistic antimicrobial effect against antibiotic-resistant strains of Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. In a mouse model of sepsis, colistin ABC-micelles showed equivalent efficacy as free colistin but with a substantially higher therapeutic index. Microscopic single-cell imaging of bacteria revealed that ABC-micelles could kill bacteria in a more rapid manner with distinct cell membrane disruption, possibly reflecting a different antimicrobial mechanism from free colistin. This work shows the potential of drug cross-linked micelles as a new class of biomaterials formed from existing antibiotics and represents a new and generalized approach for formulating amine-containing drugs.

Synthesis, Characterization, and Toxicity Assessment of Pluronic F127-Functionalized Graphene Oxide on the Embryonic Development of Zebrafish (Danio rerio).

BACKGROUND: In the current literature, there are ongoing debates on the toxicity of graphene oxide (GO) that demonstrate contradictory findings regarding its toxicity profile. As a potential drug carrier, these findings are very concerning due to the safety concerns in humans, as well as the dramatic rise of GO being excreted into the environment. Therefore, there is an imperative need to mitigate the potential toxicity of GO to allow for a safer application in the future. PURPOSE: The present study aims to address this issue by functionalizing GO with Pluronic F127 (PF) as a means to mitigate toxicity and resolve the biocompatibility of GO. Although results from previous studies generally indicated that Pluronic functionalized GO exhibits relatively low toxicity to living organisms, reports that emphasize on its toxicity, particularly during embryonic developmental stage, are still scarce. METHODS: In the present study, two different sizes of native GO samples, GO and NanoGO, as well as PF-functionalized GO, GO-PF and NanoGO-PF, were prepared and characterized using DLS, UV-Vis, Raman spectroscopy, FTIR, and FESEM analyses. Toxicological assessment of all GO samples (0-100 µg/mL) on zebrafish embryonic developmental stages (survival, hatching and heart rates, and morphological changes) was recorded daily for up to 96 hours post-fertilization (hpf). RESULTS: The toxicity effects of each GO sample were observed to be higher at increasing concentrations and upon prolonged exposure. NanoGO demonstrated lower toxicity effects compared to GO. GO-PF and NanoGO-PF were also found to have lower toxicity effects compared to native GO samples. GO-PF showed the lowest toxicity response on zebrafish embryo. CONCLUSION: These findings highlight that toxicity is dependent on the concentration, size, and exposure period of GO. Functionalization of GO with PF through surface coating could potentially mitigate the toxicity effects of GO in embryonic developmental stages, but further investigation is warranted for broader future applications.

Enhancing the Hypolipidemic Effect of Simvastatin in Poloxamer-Induced Hyperlipidemic Rats via Liquisolid Approach: Pharmacokinetic and Pharmacodynamic Evaluation.

This study aimed to enhance the dissolution of simvastatin (SMV) through its formulation in liquisolid tablets (LSTs) to improve its bioavailability and hypolipidemic activity after oral administration. SMV-LSTs were optimized using Box-Behnken design to maximize the rate and extent of SMV dissolution. The optimized SMV-LST was evaluated for pharmacokinetic parameters and potential hypolipidemic activity on induced hyperlipidemic rats. The dissolution parameters revealed a shortening of mean dissolution time from 10.99 to 6.82 min, increasing of dissolution rate during the first 10 min from 1253.15 to 1667.31 µg/min, and enhancing of dissolution efficiency after 60 min from 71.92 to 86.93% for SMV-LSTs versus the commercial SMV tablets. The obtained data reflected an improvement in the relative bioavailability of SMV with 148.232% which was confirmed by the significant reduction of the levels of circulating total cholesterol, triglycerides that reached the normal level after 12 h. In particular, the optimized SMV-LSTs reduced serum low-density lipoproteins (LDL) by 44.6% which was significantly different from the commercial SMV tablets. In contrast, the level of serum high-density lipoprotein (HDL) was significantly augmented after 4 h in rats treated with the optimized SMV-LSTs by 47.6%. Finally, the optimized SMV-LSTs showed a significant lower atherosclerotic index value which could maximize its potential in decreasing the risk of coronary disease and atherosclerosis. Overall enhancement in pharmacokinetics and pharmacodynamics in comparison with the commercial tablets confers the potential of the liquisolid approach as a promising alternative for improved oral bioavailability, hypolipidemic, and cardioprotective effects of SMV. Graphical abstract.

Performance and toxicity of different absorption enhancers used in the preparation of Poloxamer thermosensitive in situ gels for ketamine nasal administration.

The purpose of this study was to investigate the nasal absorption rate and nasal mucosal toxicity of thermosensitive ketamine in situ gels containing various absorption enhancers. The optimal composition ratio for the gel matrix was determined to be 17.2% Poloxamer 407 and 2% Poloxamer 188, as this combination resulted in solutions with a gelation point within the range found in the nasal cavity. Ketamine gels containing the tested enhancers, namely, ethylenediaminetetraacetic acid disodium salt, hydroxypropyl-ß-cyclodextrin, propylene glycol, or Tween-80, were compared with enhancer-free counterparts to determine the absorption of the drug, in vivo by measuring its plasma levels in rats and in vitro using a Franz diffusion cell. Moreover, the toxicity of each gel type was assessed by microscopic observation of the morphology of rat nasal mucosa as well as by determining the mobility of the mucosal cilia using an established toad model. The results showed that gels containing hydroxypropyl-ß-cyclodextrin could promote the absorption of ketamine without added toxicity compared to enhancer-free gels. Thus, we consider hydroxypropyl-ß-cyclodextrin as the most promising absorption enhancer for the nasal administration of ketamine using in situ gels.

Therapeutic efficacy of thermosensitive Pluronic hydrogel for codelivery of resveratrol microspheres and cisplatin in the treatment of liver cancer ascites.

Ascites constitutes the most frequent decompensating event in patients with advanced liver cancer and is associated with poor quality of life and high mortality. Intraperitoneal chemotherapy appears to be a reliable treatment strategy for advanced liver cancer ascites. However, the rapid metabolism of drugs and ascites dilution limits the efficacy of chemotherapeutics. Therefore, the present study aimed to develop a novel thermosensitive hydrogel drug system for targeted therapy of advanced hepatocellular carcinoma (HCC) ascites through intraperitoneal administration. The system was prepared by blending resveratrol (RES) microspheres and cisplatin (DDP) into thermosensitive Pluronic F127 hydrogel. The in vitro anti-tumor activity against H22 cells indicated that the prepared drug system could initiate apoptosis and induce cell cycle arrest at the G1 phase. The mice model of ascites with advanced HCC was established to validate the therapeutic potential of the F127 hydrogel drug system in vivo. The results revealed that intraperitoneal administration of F127 hydrogel drug could significantly inhibit the number of ascites, the proliferation of tumor cells, micro-angiogenesis, and prolong the survival of mice, thus, augmenting the efficacy of intraperitoneal chemotherapy. Moreover, immunohistochemical staining revealed that the F127 hydrogel drug system was safe and presented low toxicity to major vital organs. Collectively, this study highlights the clinical application potential of the F127 hydrogel drug delivery system.

Hyaluronic acid on the urokinase sustained release with a hydrogel system composed of poloxamer 407: HA/P407 hydrogel system for drug delivery.

Pleural empyema is an inflammatory condition characterized by accumulation of pus inside the pleural cavity, which is usually followed by bacterial pneumonia. During the disease process, the pro-inflammatory and pro-fibrotic cytokines in the purulent pleural effusion cause proliferation of fibroblasts and deposition of extracellular matrix, which lead to fibrin deposition and fibrothorax. Urokinase instillation therapy through a chest drainage tube is frequently used for fibrinolysis in patients with empyema. However, urokinase treatment requires multiple instillation (2-3 times per day, for 4-8 days) and easily flows out from the chest drainage tube due to its high water solubility. In this in vitro study, we developed a thermo-responsive hydrogel based on poloxamer 407 (P407) combined with hyaluronic acid (HA) for optimal loading and release of urokinase. Our results show that the addition of HA to poloxamer gels provides a significantly more compact microstructure, with smaller pore sizes (**p < 0.001). The differential scanning calorimetry (DSC) profile revealed no influence on the micellization intensity of poloxamer gel by HA. The 25% poloxamer-based gel was significantly superior to the 23% poloxamer-based gel, with slower gel erosion when comparing the 16th hour residual gel weight of both gels (*p < 0.05; **p < 0.001). The 25% poloxamer-HA gel also exhibited a superior urokinase release profile and longer release time. A Fourier-transform infrared spectroscopy (FT-IR) study of the P407/HA hydrogel showed no chemical interactions between P407 and HA in the hydrogel system. The thermoresponsive P407/HA hydrogel may have a promising potential in the loading and delivery of hydrophilic drugs. On top of that, in vitro toxicity test of this combination demonstrates a lower toxicity.

Intranasal delivery system of bacterial antigen using thermosensitive hydrogels based on a Pluronic-Gantrez conjugate.

Thermosensitive hydrogels have been studied as feasible needle-avoidance alternative to vaccine delivery. In this work, we report the development of a new thermal-sensitive hydrogel for intranasal vaccine delivery. This delivery system was formulated with a combination of the polymer Gantrez® AN119 and the surfactant Pluronic® F127 (PF127), with a high biocompatibility, biodegradability and immunoadjuvant properties. Shigella flexneri outer membrane vesicles were used as the antigen model. A stable and easy-to-produce thermosensitive hydrogel which allowed the incorporation of the OMV-antigenic complex was successfully synthetized. A rapid gel formation was achieved at body temperature, which prolonged the OMV-antigens residence time in the nasal cavity of BALB/c mice when compared to intranasal delivery of free-OMVs. In addition, the bacterial antigens showed a fast release profile from the hydrogel in vitro, with a peak at 30 min of incubation at 37 °C. Hydrogels appeared to be non-cytotoxic in the human epithelial HeLa cell line and nose epithelium as well, as indicated by the absence of histopathological features. Immunohistochemical studies revealed that after intranasal administration the OMVs reached the nasal associated lymphoid tissue. These results support the use of here described thermosensitive hydrogels as a potential platform for intranasal vaccination.

Soft liquid metal nanoparticles achieve reduced crystal nucleation and ultrarapid rewarming for human bone marrow stromal cell and blood vessel cryopreservation.

High warming rates during cryopreservation are crucial and essential for successful vitrification. However, realizing a faster warming rate in low-concentration cryoprotective agents appears to be challenging for conventional warming process through convective heat transfer. Herein, we developed a liquid metal (LM) nanosystem that can act as a spatial source to significantly enhance the warming rates with near-infrared laser irradiation during the warming process. The synthetic Pluronic F127-liquid metal nanoparticles (PLM NPs) displayed multiple performances with uniform particle size, superior photothermal conversion efficiency (52%), repeatable photothermal stability, and low cytotoxicity. Particularly, it is more difficult for the liquid PLM NPs with less surface free energy to form crystal nucleation than other solid NPs such as gold and Fe3O4, which is beneficial for the cooling process during cryopreservation. The viability of human bone marrow-derived mesenchymal stem cells postcryopreservation reached 78±3%, which is threefold higher than that obtained by the conventional warming method (25±6%). Additionally, the cells postcryopreservation maintained their normal attachment, proliferation, surface marker expression, and intact multilineage differentiation properties. Moreover, the results of mouse tails including blood vessel cryopreservation showed a relatively improved intact structure when using PLM NP rewarming compared with the results of conventional warming. The new LM nanosystem provides a universal platform for cryopreservation that is expected to have potential for widespread applications including bioengineering, cell-based medicine, and clinical translation. STATEMENT OF SIGNIFICANCE: In this study, we fabricated soft liquid metal nanoparticles with high photothermal conversion efficiency, repeatable photothermal stability, and low cytotoxicity. Particularly, soft liquid metal nanoparticles with less surface free energy and suppression effects of ice formation were first introduced to mediate cryopreservation. Superior ice-crystallization inhibition is achieved as a result of less crystal nucleation and ultrarapid rewarming during the freezing and warming processes of cryopreservation, respectively. Collectively, cryopreservation of human bone marrow stromal cells (HBMSCs) and mouse tails including blood vessels can be successfully performed using this new nanoplatform, showing great potential in the application of soft nanoparticles in cryopreservation.

Development of nanodispersion-based sildenafil metered-dose inhalers stabilized by poloxamer 188: a potential candidate for the treatment of pulmonary arterial hypertension.

Objectives: This study aims to formulate nanodispersion-based sildenafil metered-dose inhalers (MDIs) by using poloxamer 188 (P188) as a stabilizer; to evaluate their stability, aerosol characteristics, cytotoxicity, and inflammatory effects; and to investigate the effects of P188 on stability and aerosol characteristics of the MDIs. Methods: The stability and uniformity of the formulations were evaluated by high-performance liquid chromatography method. The aerosol characteristics were evaluated by the Next Generation Impactor. The cytotoxicity and inflammatory effects on respiratory epithelial cells and alveolar macrophages were evaluated by MTT assay and TNF-α, IL-1ß, and NO assay, respectively. Results: The optimal formulation was stable and well-uniform after 6 months. The fine particle fraction and mass median aerodynamic diameter (MMAD) of the formulation were 61.9% ± 2.5% and 1.69 ± 0.06 µm, respectively. The formulation was found to be nontoxic to respiratory epithelial cells and did not induce the inflammatory responses of alveolar macrophages. A positive correlation between P188 concentration and MMAD of the MDIs was observed. P188 possesses an ability to prevent the growth of sildenafil citrate monohydrate crystals in the formulations. Conclusions: The findings provided a basis for the development of sildenafil MDI as a potential candidate for the treatment of pulmonary arterial hypertension.

Local Toxicity of Topically Administrated Thermoresponsive Systems: In Vitro Studies with In Vivo Correlation.

Thermoresponsive materials have the ability to respond to a small change in temperature-a property that makes them useful in a wide range of applications and medical devices. Although very promising, there is only little conclusive data about the cytotoxicity and tissue toxicity of these materials. This work studied the biocompatibility of three Food and Drug Administration approved thermoresponsive polymers: poly( N-isopropyl acrylamide), poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) tri-block copolymer, and poly(lactic acid-co-glycolic acid) and poly(ethylene glycol) tri-block copolymer. Fibroblast NIH 3T3 and HaCaT keratinocyte cells were used for the cytotoxicity testing and a mouse model for the in vivo evaluation. In vivo results generally showed similar trends as the results seen in vitro, with all tested materials presenting a satisfactory biocompatibility in vivo. pNIPAM, however, showed the highest toxicity both in vitro and in vivo, which was explained by the release of harmful monomers and impurities. More data focusing on the biocompatibility of novel thermoresponsive biomaterials will facilitate the use of existing and future medical devices.

Influence of magnesium particles and Pluronic F127 on compressive strength and cytocompatibility of nanocomposite injectable and moldable beads for bone regeneration.

A novel one-step preparation of magnesium particles and Pluronic F127 incorporated with calcium sulfate hemihydrate (CSH) and nano-hydroxyapatite (nHA) ready to use injectable or moldable beads was developed for bone tissue regeneration applications. The nanocomposite showed setting time less than 15 min, very good injectability (75-85%) and good mechanical strength (52-80 MPa). Samples immersed in SBF showed controlled degradation (40-45% reduction in weight) in 28 days. The nanocomposite bone graft was cytocompatible against MG63 osteosarcoma cells and increased the osteogenic gene expression by 2-3 folds. These results indicate that it can be a potential defect filling biomaterial for bone tissue regeneration at the fracture site.

Reprogramming axial ligands facilitates the self-assembly of a platinum(iv) prodrug: overcoming drug resistance and safer in vivo delivery of cisplatin.

We herein reprogrammed axial ligands of platinum(iv) prodrugs, conferring the constructed prodrug entities with the ability to self-assemble in aqueous solution. Further cloaking of the PEG chain with Pluronic surfactant makes this nanoassembly (termed Pt(iv)-NP) suitable for systemic injection. Notably, Pt(iv)-NP significantly alleviated the toxicity of the parent cisplatin drug in vivo while preserving the pharmacological activity.

Mechanisms Underlying Early-Stage Changes in Visual Performance and Retina Function After Experimental Induction of Sustained Dyslipidemia.

Visual and retinal function was measured in a mouse model of chemically induced, sustained dyslipidemia to determine the contribution of dyslipidemia to the pathogenesis of retinopathy in the context of metabolic syndrome. Fifteen male C57BL/6Crl mice were divided into three groups. Poloxamer 407 (P-407), 14.5% w/w was delivered at a rate of 6 µl/day by implanted osmotic mini-pumps either subcutaneously (P-407 SQ) or intraperitoneally (P-407 IP) to P-407-treated mice, whereas saline was administered at the same rate to control mice using only the subcutaneous route of administration. Total cholesterol (TC) and true triglyceride (TG) levels were quantified from plasma. Optomotor responses to stimuli of varying spatial frequency or contrast were used to measure visual acuity and contrast sensitivity. Retinal function was determined using Ganzfeld flash electroretinography (ERG). At 32 days, TC for the P-407 IP group was significantly elevated compared to saline controls (169.4 ± 16.5 mg/dl, 0.001 < P < 0.01). TG levels for both the P-407 SQ (59.3 ± 22.4 mg/dl, 0.01 < P < 0.05) and P-407 IP groups (67.7 ± 18.0 mg/dl, 0.001 < P < 0.01) were significantly elevated relative to controls. Electroretinography demonstrated a very significant decline in the b/a ratio (1.80 ± 0.11, P < 0.01) for the P-407 IP group. The b/a ratio exhibited a moderate, significant correlation with TC levels (r = - 0.4425, P = 0.0392) and a strong, very significant correlation with TG levels (r = - 0.6190, P = 0.0021). Delivery of P-407 via osmotic mini-pump resulted in the sustained, significant elevation of plasma TC and TG levels. This elevation in plasma lipid levels was correlated with a decline in inner retinal function.

Characterization of rabies pDNA nanoparticulate vaccine in poloxamer 407 gel.

Plasmid DNA (pDNA) vaccines have the potential for protection against a wide range of diseases including rabies but are rapid in degradation and poor in uptake by antigen-presenting cells. To overcome the limitations, we fabricated a pDNA nanoparticulate vaccine. The negatively charged pDNA was adsorbed onto the surface of cationic PLGA (poly (d, l-lactide-co-glycolide))-chitosan nanoparticles and were used as a delivery vehicle. To create a hydrogel for sustainable vaccine release, we dispersed the pDNA nanoparticles in poloxamer 407 gel which is liquid at 4 °C and turns into soft gels at 37 °C, providing ease of administration and preventing burst release of pDNA. Complete immobilization of pDNA to cationic nanoparticles was achieved at a pDNA to nanoparticles ratio (P/N) of 1/50. Cellular uptake of nanoparticles was both time and concentration dependent and followed a saturation kinetics with Vmax of 11.389 µg/mL h and Km of 139.48 µg/mL. The in vitro release studies showed the nanoparticulate vaccine has a sustained release for up to 24 days. In summary, pDNA PLGA-chitosan nanoparticles were non-cytotoxic, their buffering capacity and cell uptake were enhanced, and sustained the release of pDNA. We expect our pDNA vaccine's potency will be greatly improved in the animal studies.

Quantitation of cell-associated carbon nanotubes: selective binding and accumulation of carboxylated carbon nanotubes by macrophages.

To understand the influence of carboxylation on the interaction of carbon nanotubes with cells, the amount of pristine multi-walled carbon nanotubes (P-MWNTs) or carboxylated multi-walled carbon nanotubes (C-MWNTs) coated with Pluronic® F-108 that were accumulated by macrophages was measured by quantifying CNTs extracted from cells. Mouse RAW 264.7 macrophages and differentiated human THP-1 (dTHP-1) macrophages accumulated 80-100 times more C-MWNTs than P-MWNTs during a 24-h exposure at 37 °C. The accumulation of C-MWNTs by RAW 264.7 cells was not lethal; however, phagocytosis was impaired as subsequent uptake of polystyrene beads was reduced after a 20-h exposure to C-MWNTs. The selective accumulation of C-MWNTs suggested that there might be receptors on macrophages that bind C-MWNTs. The binding of C-MWNTs to macrophages was measured as a function of concentration at 4 °C in the absence of serum to minimize the potential interference by serum proteins or temperature-dependent uptake processes. The result was that the cells bound 8.7 times more C-MWNTs than P-MWNTs, consistent with the selective accumulation of C-MWNTs at 37 °C. In addition, serum strongly antagonized the binding of C-MWTS to macrophages, suggesting that serum contained inhibitors of binding. Moreover, inhibitors of class A scavenger receptor (SR-As) reduced the binding of C-MWNTs by about 50%, suggesting that SR-As contribute to the binding and endocytosis of C-MWNTs in macrophages but that other receptors may also be involved. Altogether, the evidence supports the hypothesis that macrophages contain binding sites selective for C-MWNTs that facilitate the high accumulation of C-MWNTs compared to P-MWNTs.

Electroacupuncture ameliorates poloxamer 407-induced hyperlipidemia through suppressing hepatic SREBP-2 expression in rats.

AIMS: Acupuncture, particularly electroacupuncture (EA) has been shown to have the lipid-lowering effects, but not completely investigated. The present study was aimed to examine whether EA could attenuate poloxamer-407 (P-407)-induced hyperlipidemia in the rats and to investigate its potential mechanisms. MAIN METHODS: Rats received P-407 (0.4 g/kg, i.p.) to induce hyperlipidemia. EA was performed at ST36 and ST40 acupoints a total of three times with 12 h-interval starting 1 h before the P-407 injection at 0.6 mA intensity and 2 Hz frequency for 10 min. KEY FINDINGS: In P-407-induced hyperlipidemic rats, EA stimulation at ST36 and ST40 acupoints significantly lowered the serum levels of triglycerides, total cholesterol, LDL-cholesterol and atherogenic index, while markedly increasing the serum HDL-cholesterol levels. Meanwhile, hyperlipidemic rats had significantly higher expression of sterol regulatory element-binding protein (SREBP)-2, without any difference in SREBP-1 expression in the liver, as compared with normal ones. EA significantly attenuated the expression of SREBP-2 with a subsequent decrease in 3-hydroxy-3-methylglutaryl coenzyme A reductase and an increase in low-density lipoprotein receptor at both mRNA and protein levels in the liver of hyperlipidemic rats. These changes did not occur after electrical stimulation at a non-acupoint. SIGNIFICANCE: Taken together, our findings indicate that EA stimulation to P-407-induced hyperlipidemic rats improves the lipid abnormalities, which may be associated with regulation of the expression of key enzymes of cholesterol synthesis in the liver through modulation of SREBP-2.