Poloxamer 407/188 Binary Thermosensitive Gel as a Moxidectin Delivery System: In Vitro Release and In Vivo Evaluation.

Moxidectin (MXD) is an antiparasitic drug used extensively in veterinary clinics. In this study, to develop a new formulation of MXD, a thermosensitive gel of MXD (MXD-TG) was prepared based on poloxamer 407/188. Furthermore, the gelation temperature, the stability, in vitro release kinetics and in vivo pharmacokinetics of MXD-TG were evaluated. The results showed that the gelation temperature was approximately 27 °C. MXD-TG was physically stable and can be released continuously for more than 96 h in vitro. The Korsmeyer−Peppas model provided the best fit to the release kinetics, and the release mechanism followed a diffusive erosion style. MXD-TG was released persistently for over 70 days in sheep. Part of pharmacokinetic parameters had a difference in female and male sheep (p < 0.05). It was concluded that MXD-TG had a good stability, and its release followed the characteristics of a diffusive erosion style in vitro and a sustained release pattern in vivo.

Maduramicin induces cardiotoxicity via Rac1 signaling-independent methuosis in H9c2 cells.

Maduramicin frequently induces severe cardiotoxicity in target and nontarget animals in clinic. Apoptotic and non-apoptotic cell death mediate its cardiotoxicity; however, the underlying non-apoptotic cell death induced by maduramicin remains unclear. In current study, a recently described non-apoptotic cell death "methuosis" caused by maduramicin was defined in mammalian cells. Rat myocardial cell H9c2 was used as an in vitro model, showing excessively cytoplasmic vacuolization upon maduramicin (0.0625-5 µg/mL) exposure for 24 h. Maduramicin-induced reversible cytoplasmic vacuolization of H9c2 cells in a time- and concentration-dependent manner. The vacuoles induced by maduramicin were phase lucent with single membrane and were not derived from the swelling of organelles such as mitochondria, endoplasmic reticulum, lysosome, and Golgi apparatus. Furthermore, maduramicin-induced cytoplasmic vacuoles are generated from micropinocytosis, which was demonstrated by internalization of extracellular fluid-phase marker Dextran-Alexa Fluor 488 into H9c2 cells. Intriguingly, these cytoplasmic vacuoles acquired some characteristics of late endosomes and lysosomes rather than early endosomes and autophagosomes. Vacuolar H+ -ATPase inhibitor bafilomycin A1 efficiently prevented the generation of cytoplasmic vacuoles and decreased the cytotoxicity of H9c2 cells triggered by maduramicin. Mechanism studying indicated that maduramicin activated H-Ras-Rac1 signaling pathway at both mRNA and protein levels. However, the pharmacological inhibition and siRNA knockdown of Rac1 rescued maduramicin-induced cytotoxicity of H9c2 cells but did not alleviate cytoplasmic vacuolization. Based on these findings, maduramicin induces methuosis in H9c2 cells via Rac-1 signaling-independent seriously cytoplasmic vacuolization.

Effect of maduramicin on crayfish (Procambius clarkii): Hematological parameters, oxidative stress, histopathological changes and stress response.

Maduramicin, an extensively used anticoccidial drug, has been introduced into environment due to poorly absorbed in the intestine of broiler chicken. To understand the potential ecological toxicity of maduramicin on aquatic organisms, acute and subacute toxicity, hemolymph biochemistry, histopathology and the expressions of drug metabolism and stress response genes of crayfish (Procambius clarkii) were investigated in this study. For the first time, the 96 h median lethal concentration (LC50) of maduramicin on crayfish was 67.03 mgL-1 with a 95% confidence interval (54.06-81.32 mgL-1). Then, the crayfish were exposed to 0.7 mgL-1 (1/100 LC50), 3.5 mgL-1 (1/20 LC50) and 7.0 mgL-1 (1/10 LC50) maduramicin for 28 days. Maduramicin significantly altered biochemical parameters including AST, ALT, CK, LDH and ALP of hemolymph in crayfish at several time points. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) of crayfish gills, hepatopancreas and abdominal muscle were significantly decreased or elevated by different concentrations of maduramicin treatment at varying time points. Furthermore, histopathological damage of crayfish gills, hepatopancreas and abdominal muscle were observed in a concentration-dependent manner. The expressions of metabolic and stress response genes (CYP450, GST, COX1, COX2, HSP70 and MT) in hepatopancreas of crayfish were significantly up-regulated by maduramicin (7.0 mgL-1) treatment for 8 h to 7 d, and returned to normal levels after the removal of maduramicin for 3-7 days. In conclusion, our findings demonstrated that environmental exposure of maduramicin threaten to the health of crayfish living in the areas nearby livestock farms or pharmaceutical factory. Crayfish exhibited resistance to the stress of maduramicin via activating drug metabolite and detoxification pathways.

Development of PLGA-PEG-PLGA Hydrogel Delivery System for Enhanced Immunoreaction and Efficacy of Newcastle Disease Virus DNA Vaccine.

The highly contagious Newcastle disease virus (NDV) continues to threaten poultry all over the world. The NDV DNA vaccine is a promising solution to the current Newcastle disease (ND) challenges, and thus an efficient delivery system should be developed to facilitate the efficacy of DNA vaccines. In this study, we developed a DNA vaccine delivery system consisting of a triblock copolymer of poly(lactide co-glycolide acid) and polyethylene glycol (PLGA-PEG-PLGA) hydrogel in which the recombinant NDV hemagglutinin-neuraminidase (HN) plasmid was encapsulated. Its characteristics, security, immune responses, and efficacy against highly virulent NDV were detected. The results showed that the plasmids were gradually released in a sustained manner from the hydrogel, which improved the biological stability of the plasmids and demonstrated a high biocompatibility. The plasmids, when they were incorporated into the hydrogel delivery system, enhanced immune activation and provided 100% protection against the highly virulent NDV strain. Furthermore, we proved that this NDV DNA hydrogel vaccine could improve the lymphocyte proliferation and increase the immunological cytokine production via the PI3K/Akt pathway. These results indicate that the PLGA-PEG-PLGA thermosensitive hydrogel could be a promising delivery system for the NDV DNA vaccine in order to achieve a sustained supply of plasmids and induce potent immune responses.

Synthesis of Tilmicosin Nanostructured Lipid Carriers for Improved Oral Delivery in Broilers: Physiochemical Characterization and Cellular Permeation.

This study aimed to develop nanostructured lipid carriers (NLCs) for improved oral absorption of tilmicosin (TMS) in broilers. Thus, palmitic acid, lauric acid, and stearic acid were selected as solid lipids to formulate TMS-pNLCs, TMS-lNLCs, and TMS-sNLCs, respectively. They showed similar physicochemical properties and meanwhile possessed excellent storage and gastrointestinal stability. The TMS interacted with the lipid matrix and was encapsulated efficiently in NLCs in an amorphous structure. NLCs could enhance oral absorption of TMS compared to 10% tilmicosin phosphate solution in broilers, among which the TMS-sNLCs were the most efficient drug delivery carriers, with a relative oral bioavailability of 203.55%. NLCs could inhibit the efflux of P-glycoprotein (P-pg) toward TMS, which may be involved with improved oral absorption. Taken together, these types of solid lipids influenced the enhanced level of NLCs toward oral bioavailability of TMS, and the sNLCs proved to be the most promising oral delivery carriers of TMS.

Effects of maduramicin on adult zebrafish (Danio rerio): Acute toxicity, tissue damage and oxidative stress.

Maduramicin, a potent polyether ionophore antibiotic, has been widely used to control coccidiosis in the poultry production. Nevertheless, incomplete metabolism of maduramicin in chicken may result in its accumulation in the aquatic environment, while maduramicin's threat to fish remains largely unknown. In the present study, we focused on acute toxicity, histopathological lesion and oxidative stress damage of maduramicin in adult zebrafish. Primarily, we obtained that the 96-h median lethal concentration (96 h LC50) of adult zebrafish exposure to maduramicin was 13.568 mg/L. On basis of that, adult zebrafish were separately exposed to 0.1 mg/L (1/125 LC50), 0.5 mg/L (1/25 LC50) and 2.5 mg/L (1/5 LC50) maduramicin for 14 days. On day 3, 0.1 mg/L maduramicin significantly increased the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione s-transferase (GST) in the liver of zebrafish, while the activities of these antioxidant enzymes in the liver were significantly inhibited by 2.5 mg/L maduramicin. Moreover, the contents of malondialdehyde (MDA) in the liver of different dose groups were all significantly promoted after 14 days of exposure. For the gill of zebrafish, the increase in MDA contents was found after only 3 days of exposure to maduramicin. Furthermore, maduramicin treatment significantly up-regulated the mRNA levels of genes (sod1, gpx1a, gstr, nrf2 and keap1) in the liver of zebrafish after 3 days of exposure. On days 6, 9 and 14, maduramicin treatment significantly down-regulated the mRNA levels of these genes in the liver of zebrafish. Meanwhile, maduramicin significantly down-regulated the mRNA levels of genes (sod1, cat, gpx1a, gstr, nrf2 and keap1) in the gill of zebrafish during the 14-day of exposure. In addition, a dose-dependent induction in histopathological lesion was observed in multiple organs after 14 days of exposure, including lamellar fusion, epithelial lifting in the gill and vacuole formation in the liver as well as the fracture of intestinal villus in the intestine. Taken together, our findings demonstrated that waterborne maduramicin (2.5 mg/L) exposure can induce severe oxidative stress and tissue damage in adult zebrafish while this damage was not enough to kill them after 14 days of waterborne exposure.

Transcriptome profile analysis reveals cardiotoxicity of maduramicin in primary chicken myocardial cells.

Maduramicin, an excellent ionophore antibiotic, is extensively used to control coccidiosis in poultry. Numerous maduramicin intoxications have been reported in farm animal and human due to its relatively narrow safety range, with necrosis or degeneration of cardiac and skeletal muscles as hallmark. To date, the mechanisms of maduramicin-induced cardiotoxicity remain unclear in chicken and other animals. Maduramicin (5 µg/mL)-treated primary chicken myocardial cells were used for RNA sequencing (RNA-Seq) and bioinformatics analysis in this study. A total of 1442 differential expressed genes were identified. 810 genes were up-regulated and the rest 632 genes were down-regulated. Transcriptome analysis revealed that the cytokine-cytokine receptor interaction, apoptosis, calcium signal and cytoplasmic vacuolization pathways were significantly affected. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed that gene expression patterns were consistent with RNA-Seq analysis. Pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8), apoptosis ratios, cleaved caspase-3, intracellular calcium level and Ca2+-ATPase activity were elevated after maduramicin (0.05, 0.5 and 5 µg/mL) treatment. Massive vacuole formation was found in the cytoplasm by morphology and transmission electron microscopy observation. Taken together, the results suggested that maduramicin exerted its cardiotoxicity by multiple molecular mechanisms in primary chicken myocardial cells.

Using halofuginone-silver thermosensitive nanohydrogels with antibacterial and anti-inflammatory properties for healing wounds infected with Staphylococcus aureus.

Contamination by pathogens, such as bacteria, can irritate a wound and prevent its healing, which may affect the physical fitness of the infected person. As such, the development of more novel nano-biomaterials able to cope with the inflammatory reaction to bacterial infection during the wound healing process to accelerate wound healing is required. Herein, a halofuginone­silver nano thermosensitive hydrogel (HTPM&AgNPs-gel) was prepared via a physical swelling method. HTPM&AgNPs-gel was characterized based on thermogravimetric analysis, differential scanning calorimetry, morphology, injectability, and rheological mechanics that reflected its exemplary nature. Moreover, HTPM&AgNPs-gel was further tested for its ability to facilitate healing of skin fibroblasts and exert antibacterial activity. Finally, HTPM&AgNPs-gel was tested for its capacity to accelerate general wound healing and treat bacterially induced wound damage. HTPM&AgNPs-gel appeared spherical under a transmission electron microscope and showed a grid structure under a scanning electron microscope. Additionally, HTPM&AgNPs-gel demonstrated excellent properties, including injectability, temperature-dependent swelling behavior, low loss at high temperatures, and appropriate rheological properties. Further, HTPM&AgNPs-gel was found to effectively promote healing of skin fibroblasts and inhibit the proliferation of Escherichia coli and Staphylococcus aureus. An evaluation of the wound healing efficacy demonstrated that HTPM&AgNPs-gel had a more pronounced ability to facilitate wound repair and antibacterial effects than HTPM-gel or AgNPs-gel alone, and exhibited ideal biocompatibility. Notably, HTPM&AgNPs-gel also inhibited inflammatory responses in the healing process. HTPM&AgNPs-gel exhibited antibacterial, anti-inflammatory, and scar repair features, which remarkably promoted wound healing. These findings indicated that HTPM&AgNPs-gel holds great clinical potential as a promising and valuable wound healing treatment.

Injectable nano-in situ-thermosensitive-hydrogels based on halofuginone and silver for postoperative treatment against triple-negative breast cancer.

Postoperative distant metastasis and high recurrence rate causes a dilemma in treating triple-negative breast cancer (TNBC) owing to its unforeseeable invasion into various organs or tissues. The wealth of nutrition provided by vascular may facilitate the proliferation and angiogenesis of cancer cells, which further enhance the rates of postoperative metastasis and recurrence. Chemotherapy, as a systemic postoperative adjuvant therapy, is generally applied to diminish recurrence and metastasis of TNBC. Herein, an halofuginone-silver nano thermosensitive hydrogel (HTPM&AgNPs-gel) was prepared via a physical swelling method. The in vitro anticancer efficacy of HTPM&AgNPs-gel was analyzed by investigating cell proliferation, migration, invasion, and angiogenesis capacity. Furthermore, the in vivo anti-cancer activity of HTPM&AgNPs-gel was further appraised through the tumor suppression, anti-metastatic, anti-angiogenic, and anti-inflammatory ability. The optimized HTPM&AgNPs-gel, a thermosensitive hydrogel, showed excellent properties, including syringeability, swelling behavior, and a sustained release effect without hemolysis. In addition, HTPM&AgNPs-gel was confirmed to effectively inhibit the proliferation, migration, invasion, and angiogenesis of MDA-MB-231 cells. An evaluation of the in vivo anti-tumor efficacy demonstrated that HTPM&AgNPs-gel showed a stronger tumor inhibition rate (68.17%) than did HTPM-gel or AgNPs-gel used alone and exhibited outstanding biocompatibility. Notably, HTPM&AgNPs-gel also inhibited lung metastasis induced by residual tumor tissue after surgery and further blocked angiogenesis-related inflammatory responses. Taken together, the suppression of inflammation by interdicting the blood vessels adjoining the tumor and inhibiting angiogenesis is a potential strategy to attenuate the recurrence and metastasis of TNBC. HTPM&AgNPs-gel is a promising anticancer agent for TNBC as a local postoperative treatment.

Antibiofilm activity of polyethylene glycol-quercetin nanoparticles-loaded gelatin-N,O-carboxymethyl chitosan composite nanogels against Staphylococcus epidermidis.

BACKGROUND: Biofilms, such as those from Staphylococcus epidermidis, are generally insensitive to traditional antimicrobial agents, making it difficult to inhibit their formation. Although quercetin has excellent antibiofilm effects, its clinical applications are limited by the lack of sustained and targeted release at the site of S. epidermidis infection. OBJECTIVES: Polyethylene glycol-quercetin nanoparticles (PQ-NPs)-loaded gelatin-N,O-carboxymethyl chitosan (N,O-CMCS) composite nanogels were prepared and assessed for the on-demand release potential for reducing S. epidermidis biofilm formation. METHODS: The formation mechanism, physicochemical characterization, and antibiofilm activity of PQ-nanogels against S. epidermidis were studied. RESULTS: Physicochemical characterization confirmed that PQ-nanogels had been prepared by the electrostatic interactions between gelatin and N,O-CMCS with sodium tripolyphosphate. The PQ-nanogels exhibited obvious pH and gelatinase-responsive to achieve on-demand release in the micro-environment (pH 5.5 and gelatinase) of S. epidermidis. In addition, PQ-nanogels had excellent antibiofilm activity, and the potential antibiofilm mechanism may enhance its antibiofilm activity by reducing its relative biofilm formation, surface hydrophobicity, exopolysaccharides production, and eDNA production. CONCLUSIONS: This study will guide the development of the dual responsiveness (pH and gelatinase) of nanogels to achieve on-demand release for reducing S. epidermidis biofilm formation.

Maduramicin-guided nanotherapy: A polymeric micelles for targeted drug delivery in canine mammary tumors.

Canine mammary tumors (CMT) can severely compromise the life quality of the affected dogs through local recurrence, distant metastases and ultimately succumb to death. Recently, more attention has been given to the potential antimetastatic effect of maduramicin (MAD) on breast cancer. However, its poor aqueous solubility and toxicity to normal tissues limit its clinical application. Therefore, to address the drawbacks of MAD and enhance its anticancer and antimetastatic effects, MAD-loaded TPGS polymeric micelles (MAD-TPGS) were prepared by a thin-film hydration technique. The optimized MAD-TPGS exhibited excellent size distribution, stability and improved water solubility. Cellular uptake assays showed that TPGS polymer micelles could enhance drug internalization. Moreover, TPGS synergistically improved the cytotoxicity of MAD by targeting mitochondrial organelles, improving reactive oxygen species levels and reducing the mitochondrial transmembrane potential. More importantly, MAD-TPGS significantly impeded the metastasis of tumor cells. In vivo results further confirmed that, in addition to exhibiting excellent biocompatibility, MAD-TPGS exhibited greater antitumor efficacy than free MAD. Interestingly, MAD-TPGS displayed superior suppression of CMT metastasis via tail vein injection compared to oral administration, indicating its suitability for intravenous delivery. Overall, MAD-TPGS could be applied as a potential antimetastatic cancer agent for CMT.

Enhanced In Vitro Antiviral Activity of Ivermectin-Loaded Nanostructured Lipid Carriers against Porcine Epidemic Diarrhea Virus via Improved Intracellular Delivery.

Porcine epidemic diarrhea virus (PEDV) is an acute enteric coronavirus, inducing watery diarrhea and high mortality in piglets, leading to huge economic losses in global pig industry. Ivermectin (IVM), an FDA-approved antiparasitic agent, is characterized by high efficacy and wide applicability. However, the poor bioavailability limits its application. Since the virus is parasitized inside the host cells, increasing the intracellular drug uptake can improve antiviral efficacy. Hence, we aimed to develop nanostructured lipid carriers (NLCs) to enhance the antiviral efficacy of IVM. The findings first revealed the capacity of IVM to inhibit the infectivity of PEDV by reducing viral replication with a certain direct inactivation effect. The as-prepared IVM-NLCs possessed hydrodynamic diameter of 153.5 nm with a zeta potential of -31.5 mV and high encapsulation efficiency (95.72%) and drug loading (11.17%). IVM interacted with lipids and was enveloped in lipid carriers with an amorphous state. Furthermore, its encapsulation in NLCs could enhance drug internalization. Meanwhile, IVM-NLCs inhibited PEDV proliferation by up to three orders of magnitude in terms of viral RNA copies, impeding the accumulation of reactive oxygen species and mitigating the mitochondrial dysfunction caused by PEDV infection. Moreover, IVM-NLCs markedly decreased the apoptosis rate of PEDV-induced Vero cells. Hence, IVM-NLCs showed superior inhibitory effect against PEDV compared to free IVM. Together, these results implied that NLCs is an efficient delivery system for IVM to improve its antiviral efficacy against PEDV via enhanced intracellular uptake.

Primary culture and endocrine functional analysis of Leydig cells in ducks (Anas platyrhynchos).

Testicular Leydig cells (LCs) are the primary known source of testosterone, which is necessary for maintaining spermatogenesis and male fertility. However, the isolation, identification, and functional analysis of testosterone in duck LCs are still ambiguous. The aim of the present study was to establish a feasible method for isolating highly purified primary duck LCs. The highly purified primary duck LCs were isolated from the fresh testes of 2-month-old ducks via the digestion of collagenase IV and Percoll density gradient centrifugation; hematoxylin and eosin (H&E), immunohistochemistry (IHC) staining, ELISA, and radioimmunoassay were performed. Results revealed that the LCs were prominently noticeable in the testicular interstitium of 2-month-old ducks as compared to 6-month-old and 1-year-old ducks. Furthermore, IHC demonstrated that the cultured LCs occupied 90% area of the petri dish and highly expressed 3ß-HSD 24 h after culture (hac) as compared to 48 and 72 hac. Additionally, ELISA and radioimmunoassay indicate that the testosterone level in cellular supernatant was highly expressed in 24 and 48 hac, whereas the testosterone level gradually decreased in 72 and 96 hac, indicating the primary duck LCs secrete testosterone at an early stage. Based on the above results, the present study has effectively developed a technique for isolating highly purified primary duck LCs and identified its biological function in synthesizing testosterone.

Application of Silver Nanoparticles in Parasite Treatment.

Silver nanoparticles (AgNPs) are ultra-small silver particles with a size from 1 to 100 nanometers. Unlike bulk silver, they have unique physical and chemical properties. Numerous studies have shown that AgNPs have beneficial biological effects on various diseases, including antibacterial, anti-inflammatory, antioxidant, antiparasitic, and antiviruses. One of the most well-known applications is in the field of antibacterial applications, where AgNPs have strong abilities to kill multi-drug resistant bacteria, making them a potential candidate as an antibacterial drug. Recently, AgNPs synthesized from plant extracts have exhibited outstanding antiparasitic effects, with a shorter duration of use and enhanced ability to inhibit parasite multiplication compared to traditional antiparasitic drugs. This review summarizes the types, characteristics, and the mechanism of action of AgNPs in anti-parasitism, mainly focusing on their effects in leishmaniasis, flukes, cryptosporidiosis, toxoplasmosis, Haemonchus, Blastocystis hominis, and Strongylides. The aim is to provide a reference for the application of AgNPs in the prevention and control of parasitic diseases.

V-ATPase V0 subunit activation mediates maduramicin-induced methuosis through blocking endolysosomal trafficking in vitro and in vivo.

Methuosis, a novel cell death phenotype, is characterized by accumulation of cytoplasmic vacuolization upon external stimulus. Methuosis plays a critical role in maduramicin-induced cardiotoxicity despite the underlying mechanism is largely unknown. Herein, we aimed to investigate the origin and intracellular trafficking of cytoplasmic vacuoles, as well as the molecular mechanism of methuosis caused by maduramicin (1 µg/mL) in myocardial cells. H9c2 cells and broiler chicken were used and were exposed to maduramicin at doses of 1 µg/mL in vitro and 5 ppm-30 ppm in vivo. Morphological observation and dextran-Alexa Fluor 488 tracer experiment showed that endosomal compartments swelling and excessive macropinocytosis contributed to madurdamcin-induced methuosis. Cell counting kit-8 assay and morphology indicated pharmacological inhibition of macropinocytosis largely prevent H9c2 cells from maduramicin-triggered methuosis. In addition, late endosomal marker Rab7 and lysosomal associated membrane protein 1 (LAMP1) increased in a time-dependent manner after maduramicin treatment, and the recycling endosome marker Rab11 and ADP-ribosylation factor 6 (Arf6) were decreased by maduramicin. Vacuolar-H+-ATPase (V-ATPase) was activated by maduramicin, and pharmacological inhibition and genetic knockdown V0 subunit of V-ATPase restore endosomal-lysosomal trafficking and prevent H9c2 cells methuosis. Animal experiment showed that severe cardiac injury included the increase of creatine kinase (CK) and creatine kinase-MB (CK-MB), and vacuolar degeneration resembled methuosis in vivo after maduramicin treatment. Taken together, these findings demonstrate that targeting the inhibition of V-ATPase V0 subunit will prevent myocardial cells methuosis by restoring endosomal-lysosomal trafficking.

Silver nanoparticles combat Salmonella Typhimurium: Suppressing intracellular infection and activating dendritic cells.

Salmonella Typhimurium (ST) can hide inside cells, avoid antibiotic therapy and being killed by host's immune system to cause persistent infection in humans and animals. Metal nanoparticles are regarded as an alternative to overcome the above limitations, silver nanoparticles especially have been applied in combating drug-resistant bacteria. However, the therapeutic effects of silver nanoparticles against intracellular infection and their impacts on host immunity remain an area of further investigation. In this work, we synthesized Ganoderma extract-capped silver nanoparticles (Ag@Ge) and explored the therapeutic potential and immune adjuvant effects of Ag@Ge against intracellular ST. Firstly, Ag@Ge had a small particle size of 35.52±7.46 nm, good stability, and biocompatibility. Then, Ag@Ge effectively entered RAW 264.7 cells, suppressed intracellular ST infection. Furthermore, Ag@Ge activated mouse dendritic cells (DCs) in vitro, evidenced by increased phenotypic markers (CD80/CD86/CD40/major compatibility complex II (MHCII)) expression and cytokine and chemokine (interleukin-6 (IL-6), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), chemokine (C-C motif) ligand 2 (CCL-2), and chemokine (C-C motif) receptor-7 (CCR-7)) transcription. More notably, the combination of Ag@Ge with inactivated ST recruited intestinal DCs to mitigate ST infection in mice, evidenced by decreased body weight loss and bacterial loads in the tissues (liver, jejunum, and colon), and improved platelets count. The above findings indicate that Ag@Ge has the potential as an alternative nano-antibiotic against intracellular ST infection.

Safety considerations of chloroquine in the treatment of patients with diabetes and COVID-19.

Patients with underlying diseases and coronavirus disease 2019 (COVID-19) are at increased risk of death. Using the recommended anti-COVID-19 drug, chloroquine phosphate (CQ), to treat patients with severe cases and type 2 diabetes (T2D) could potentially cause harm. We aimed to understand the safety of CQ in patients with T2D by administrating the recommended dose (63 mg/kg twice daily for 7 days) and a high dose (126 mg/kg twice daily for 7 days) of CQ in T2D rats. We found that CQ increased the total mortality of the T2D rats from 27.3% to 72.7% in the recommended and high-dose groups during the whole period. CQ also induced hematotoxicity of T2D rats in the high-dose group; the hepatic enzymes in T2D rats were significantly elevated. CQ also changed the electrocardiograms, prolonged the QTc intervals, and produced urinary leukocytes and proteins in the T2D rats. Histopathological observations revealed that CQ caused severe damage to the rats' heart, jejunum, liver, kidneys, spleen, and retinas. Furthermore, CQ significantly decreased the serum IL-1ß and IL-6 levels. In conclusion, the CQ dosage and regimen used to treat COVID-19 induced adverse effects in diabetic rats, suggesting the need to reevaluate the effective dose of CQ in humans.

Orally Administered Halofuginone-Loaded TPGS Polymeric Micelles Against Triple-Negative Breast Cancer: Enhanced Absorption and Efficacy with Reduced Toxicity and Metastasis.

Background: Halofuginone (HF)-loaded TPGS polymeric micelles (HTPM) were successfully fabricated using the thin-film hydration technique. HTPM via intravenous injection have been demonstrated to exert an excellent anticancer effect against triple-negative breast cancer (TNBC) cells and subcutaneous xenografts. In the present study, we further explored the potential treatment effect and mechanism of orally administered HTPM alone and in combination with surgical therapy on TNBC in subcutaneous and orthotopic mouse models. Methods: Herein, the stability and in vitro release behavior of HTPM were first evaluated in the simulated gastrointestinal fluids. Caco-2 cell monolayers were then used to investigate the absorption and transport patterns of HF with/without encapsulation in TPGS polymeric micelles. Subsequently, the therapeutic effect of orally administered HTPM was checked on subcutaneous xenografts of TNBC in nude mice. Ultimately, orally administered HTPM, combined with surgical therapy, were utilized to treat orthotopic TNBC in nude mice. Results: Our data confirmed that HTPM exhibited good stability and sustained release in the simulated gastrointestinal fluids. HF was authenticated to be a substrate of P-glycoprotein (P-gp), and its permeability across Caco-2 cell monolayers was markedly enhanced via heightening intracellular absorption and inhibiting P-gp efflux due to encapsulation in TPGS polymeric micelles. Compared with HF alone, HTPM showed stronger tumor-suppressing effects in subcutaneous xenografts of MDA-MB-231 cells when orally administered. Moreover, compared with HTPM or surgical therapy alone, peroral HTPM combined with partial surgical excision synergistically retarded the growth of orthotopic TNBC. Fundamentally, HTPM orally administered at the therapeutic dose did not cause any pathological injury, while HF alone led to weight loss and jejunal bleeding in the investigated mice. Conclusion: Taken together, HTPM could be applied as a potential anticancer agent for TNBC by oral administration.

Optimization of Maduramicin Ammonium-Loaded Nanostructured Lipid Carriers Using Box-Behnken Design for Enhanced Anticoccidial Effect against Eimeria tenella in Broiler Chickens.

Maduramicin ammonium (MAD) is one of the most frequently used anticoccidial agents in broiler chickens. However, the high toxicity and low solubility of MAD limit its clinical application. In this study, MAD-loaded nanostructured lipid carriers (MAD-NLCs) were prepared to overcome the defects of MAD by using highly soluble nanostructured lipid carriers (NLCs). The formulation was optimized via a three-level, three-factor Box-Behnken response surface method. Then, the optimal MAD-NLCs were evaluated according to their hydrodynamic diameter (HD), zeta potential (ZP), crystal structure, encapsulation efficiency (EE), drug loading (DL), in vitro release, and anticoccidial effect. The optimal MAD-NLCs had an HD of 153.6 ± 3.044 nm and a ZP of -41.4 ± 1.10 mV. The X-ray diffraction and Fourier-transform infrared spectroscopy results indicated that the MAD was encapsulated in the NLCs in an amorphous state. The EE and DL were 90.49 ± 1.05% and 2.34 ± 0.04%, respectively, which indicated that the MAD was efficiently encapsulated in the NLCs. In the in vitro study, the MAD-NLCs demonstrated a slow and sustained drug release behavior. Notably, MAD-NLCs had an excellent anticoccidial effect against Eimeria tenella in broiler chickens. In summary, MAD-NLCs have huge potential to form a new preparation administered via drinking water with a powerful anticoccidial effect.

Repurposing maduramicin as a novel anticancer and anti-metastasis agent for triple-negative breast cancer as enhanced by nanoemulsion.

Triple-negative breast cancer (TNBC) is featured by aggression and metastasis and remains an unmet medical challenge due to high death rate. We aimed to repurpose maduramicin (MAD) as an effective drug against TNBC, and develop a nanoemulsion system to enhance anticancer efficacy of MAD. MDA-MB-231 and 4 T1 cells were used as in vitro model, and cell viability was determined by performing cell counting kit-8 and a colony-formation assay. Furthermore, MAD loaded nanoemulsion (MAD-NEs) was manufactured and characterized by a series of tests. The anticancer and anti-metastasis mechanism of MAD-NEs were assessed by performing cell cycle, apoptosis, wound-healing, transwell assay and Western blotting assays. Herein, MAD was firstly demonstrated to be an effective agent to suppress growth of TNBC cells. Subsequently, the optimized MAD-NEs were shown to have stability and high encapsulation efficiency, and could arrested cells in G0/G1 phase and induced apoptosis in TNBC cells. More importantly, MAD-NEs significantly impeded the metastasis of tumor cells, which was further demonstrated by the significant altered expression of epithelial-mesenchymal transition and extracellular matrix markers in vitro and in vivo. Moreover, compared to MAD, MAD-NEs exhibited higher efficacy in shrinking breast tumor size and repressing liver and lung metastasis in vivo, and showed excellent biocompatibility in tumor-bearing mice. The successfully prepared MAD-NEs are expected to be harnessed to suppress tumor growth, invasion and metastasis in the battle against malignant TNBC.