Naodesheng Pills Ameliorate Cerebral Ischemia Reperfusion-Induced Ferroptosis via Inhibition of the ERK1/2 Signaling Pathway.

Background: Ferroptosis plays a crucial role in the occurrence and development of cerebral ischemia-reperfusion (I/R) injury and is regulated by mitogen-activated protein kinase 1/2 (ERK1/2). In China, Naodesheng Pills (NDSP) are prescribed to prevent and treat cerebrosclerosis and stroke. However, the protective effects and mechanism of action of NDSP against cerebral I/R-induced ferroptosis remain unclear. We investigated whether NDSP exerts its protective effects against I/R injury by regulating ferroptosis and aimed to elucidate the underlying mechanisms. Methods: The efficacy of NDSP was evaluated using a Sprague-Dawley rat model of middle cerebral artery occlusion and an in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) model. Brain injury was assessed using 2,3,5-triphenyltetrazolium chloride (TTC), hematoxylin and eosin staining, Nissl staining, and neurological scoring. Western blotting was performed to determine the expression levels of glutathione peroxidase 4 (GPX4), divalent metal-ion transporter-1 (DMT1), solute carrier family 7 member 11 (SLC7A11), and transferrin receptor 1 (TFR1). Iron levels, oxidative stress, and mitochondrial morphology were also evaluated. Network pharmacology was used to assess the associated mechanisms. Results: NDSP (1.08 g/kg) significantly improved cerebral infarct area, cerebral water content, neurological scores, and cerebral tissue damage. Furthermore, NDSP inhibited I/R- and OGD/R-induced ferroptosis, as evidenced by the increased protein expression of GPX4 and SLC7A11, suppression of TFR1 and DMT1, and an overall reduction in oxidative stress and Fe2+ levels. The protective effects of NDSP in vitro were abolished by the GPX4 inhibitor RSL3. Network pharmacology analysis revealed that ERK1/2 was the core target gene and that NDSP reduced the amount of phosphorylated ERK1/2. Conclusion: NDSP exerts its protective effects against I/R by inhibiting cerebral I/R-induced ferroptosis, and this mechanism is associated with the regulation of ferroptosis via the ERK1/2 signaling pathway.

Biomimetic nanodecoys deliver cholesterol-modified heteroduplex oligonucleotide to target dopaminergic neurons for the treatment of Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by the accumulation of α-synuclein (α-syn) aggregates called Lewy bodies leading to the gradual loss of dopaminergic (DA) neurons in the substantia nigra. Although α-syn expression can be attenuated by antisense oligonucleotides (ASOs) and heteroduplex oligonucleotide (HDO) by intracerebroventricular (ICV) injection, the challenge to peripheral targeted delivery of oligonucleotide safely and effectively into DA neurons remains unresolved. Here, we designed a new DNA/DNA double-stranded (complementary DNA, coDNA) molecule with cholesterol conjugation (Chol-HDO (coDNA)) based on an α-syn-ASO sequence and evaluated its silence efficiency. Further, Chol-HDO@LMNPs, Chol-HDO-loaded, cerebrovascular endothelial cell membrane with DSPE-PEG2000-levodopa modification (L-DOPA-CECm)-coated nanoparticles (NPs), were developed for the targeted treatment of PD by tail intravenous injection. CECm facilitated the blood-brain barrier (BBB) penetration of NPs, together with cholesterol escaped from reticuloendothelial system uptake, as well as L-DOPA was decarboxylated into dopamine which promoted the NPs toward the PD site for DA neuron regeneration. The behavioral tests demonstrated that the nanodecoys improved the efficacy of HDO on PD mice. These findings provide insights into the development of biomimetic nanodecoys loading HDO for precise therapy of PD. STATEMENT OF SIGNIFICANCE: The accumulation of α-synuclein (α-syn) aggregates is a hallmark of PD. Our previous study designed a specific antisense oligonucleotide (ASO) targeting human SNCA, but the traumatic intracerebroventricular (ICV) is not conducive to clinical application. Here, we further optimize the ASO by creating a DNA/DNA double-stranded molecule with cholesterol-conjugated, named Chol-HDO (coDNA), and develop a DA-targeted biomimetic nanodecoy Chol-HDO@LMNPs by engineering cerebrovascular endothelial cells membranes (CECm) with DSPE-PEG2000 and L-DOPA. The in vivo results demonstrated that tail vein injection of Chol-HDO@LMNPs could target DA neurons in the brain and ameliorate motor deficits in a PD mouse model. This investigation provides a promising peripheral delivery platform of L-DOPA-CECm nanodecoy loaded with a new Chol-HDO (coDNA) targeting DA neurons in PD therapy.

Bacterial outer membrane vesicles-based therapeutic platform eradicates triple-negative breast tumor by combinational photodynamic/chemo-/immunotherapy.

Bacterial outer membrane vesicles (OMVs) are potent immuno-stimulating agents and have the potentials to be bioengineered as platforms for antitumor nanomedicine. In this study, OMVs are demonstrated as promising antitumor therapeutics. OMVs can lead to beneficial M2-to-M1 polarization of macrophages and induce pyroptosis to enhance antitumor immunity, but the therapeutic window of OMVs is narrow for its toxicity. We propose a bioengineering strategy to enhance the tumor-targeting ability of OMVs by macrophage-mediated delivery and improve the antitumor efficacy by co-loading of photosensitizer chlorin e6 (Ce6) and chemotherapeutic drug doxorubicin (DOX) into OMVs as a therapeutic platform. We demonstrate that systemic injection of the DOX/Ce6-OMVs@M therapeutic platform, providing combinational photodynamic/chemo-/immunotherapy, eradicates triple-negative breast tumors in mice without side effects. Importantly, this strategy also effectively prevents tumor metastasis to the lung. This OMVs-based strategy with bioengineering may serve as a powerful therapeutic platform for a synergic antitumor therapy.

Nonviral delivery systems for antisense oligonucleotide therapeutics.

Antisense oligonucleotides (ASOs) are an important tool for the treatment of many genetic disorders. However, similar to other gene drugs, vectors are often required to protect them from degradation and clearance, and to accomplish their transport in vivo. Compared with viral vectors, artificial nonviral nanoparticles have a variety of design, synthesis, and formulation possibilities that can be selected to accomplish protection and delivery for specific applications, and they have served critical therapeutic purposes in animal model research and clinical applications, allowing safe and efficient gene delivery processes into the target cells. We believe that as new ASO drugs develop, the exploration for corresponding nonviral vectors is inevitable. Intensive development of nonviral vectors with improved delivery strategies based on specific targets can continue to expand the value of ASO therapeutic approaches. Here, we provide an overview of current nonviral delivery strategies, including ASOs modifications, action mechanisms, and multi-carrier methods, which aim to address the irreplaceable role of nonviral vectors in the progressive development of ASOs delivery.

Apoptotic bodies for advanced drug delivery and therapy.

Extracellular vesicles (EVs) have emerged as promising candidates for multiple biomedical applications. Major types of EVs include exosomes, microvesicles, and apoptotic bodies (ABs). ABs are conferred most properties from parent cells in the final stages of apoptosis. A wide variety of sources and stable morphological features are endowed to ABs by the rigorous apoptotic program. ABs accommodate more functional biomolecules by relying on the larger volume and maintaining their naturalness in circulation. The predominant body surface ratio of ABs facilitates their recognition by recipient cells and is advantageous for interactions with microenvironments. ABs can modulate and alleviate symptoms of numerous diseases for their origins, circulation, and high biocompatibility. In addition, ABs have been emerging in disease diagnosis, immunotherapy, regenerative therapy, and drug delivery. Here, we aim to present a thorough discussion on current knowledge about ABs. Of particular interest, we will summarize the application of AB-based strategies for diagnosis and disease therapy. Perspectives for the development of ABs in biomedical applications are highlighted.

Establishing and evaluating physician-pharmacist collaborative clinics to manage patients with type 2 diabetes in primary hospitals in Hunan province: study protocol of a multi-site randomized controlled trial in the era of COVID-19 pandemic.

BACKGROUND: The COVID-19 pandemic has exerted an unprecedented and universal impact on global health system, resulting in noticeable challenges in traditional chronic disease care, of which diabetes was reported to be most influenced by the reduction in healthcare resources in the pandemic. China has the world's largest diabetes population, and current diabetes management in China is unsatisfactory, particularly in rural areas. Studies in developed countries have demonstrated that physician-pharmacist collaborative clinics are efficient and cost-effective for diabetes management, but little is known if this mode could be adapted in primary hospitals in China. The aim of this proposed study is to develop and evaluate physician-pharmacist collaborative clinics to manage type 2 diabetes mellitus (T2DM) in primary hospitals in Hunan province. METHODS: A multi-site randomized controlled trial will be conducted to evaluate the effectiveness and cost-effectiveness of the physician-pharmacist collaborative clinics compared with usual care for Chinese patients with T2DM. Six primary hospitals will participate in the study, which will recruit 600 eligible patients. Patients in the intervention group will receive services from both physicians and pharmacists in the collaborative clinics, while the control group will receive usual care from physicians. Patients will be followed up at the 3rd, 6th, 9th and 12th month. Comparison between the two groups will be conducted by assessing the clinical parameters, process indicators and costs on diabetes. A satisfaction survey will also be carried out at the end of the study. DISCUSSION: If effective, the physician-pharmacist collaborative clinics can be adapted and used in primary hospitals of China to improve glycemic control, enhance medication adherence, decrease incidence of complications and reduce patients' dependence on physicians. Findings from the present study are meaningful for developing evidence-based diabetes care policy in rural China, especially in the COVID-19 pandemic era. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR2000031839 , Registered 12 April 2020.

Hybrid Cell Membrane-Functionalized Biomimetic Nanoparticles for Targeted Therapy of Osteosarcoma.

PURPOSE: In order to prepare a biomimetic nano-carrier which has inflammatory chemotaxis, homologous targeting and reduce immune clearance, for targeted chemotherapy of osteosarcoma, we fabricated the paclitaxel-loaded poly(lactic-co-glycolic) acid (PLGA) nanoparticles coated with 143B-RAW hybrid membrane (PTX-PLGA@[143B-RAW] NPs) and evaluate its anti-cancer efficacy in vitro and vivo. METHODS: PTX-PLGA@[143B-RAW] NPs were prepared by the ultrasonic method and were characterized by size, zeta potential, polymer dispersion index (PDI), Coomassie bright blue staining, transmission electron microscopy (TEM) and high performance liquid chromatography (HPLC). Cellular uptake, cell viability assay, flow cytometry and chemotactic effect of PTX-PLGA@[143B-RAW] NPs were evaluated in vitro. Biodistribution, anti-cancer therapeutic efficacy and safety of PTX-PLGA@[143B-RAW] NPs were evaluated in 143B osteosarcoma xenograft mice. RESULTS: The hybrid membrane successfully coated onto the surface of PLGA nanoparticles. PTX-PLGA@[143B-RAW] NPs had a drug loading capacity of 4.24 ± 0.02% and showed targeting ability to osteosarcoma. PTX-PLGA@[143B-RAW] NPs showed high cellular uptake and improved anti-cancer efficacy against 143B cells. More importantly, PTX-PLGA@[143B-RAW] NPs treatment suppressed tumor growth in tumor-bearing mice with minimal damage to normal tissues. CONCLUSION: PTX-PLGA@[143B-RAW] NPs could be used for targeted drug delivery and osteosarcoma therapy.

Does Prolonged Infusion Time Really Improve the Efficacy of Meropenem Therapy? A Prospective Study in Critically Ill Patients.

INTRODUCTION: Meropenem is a carbapenem antibiotic, which has demonstrated excellent antimicrobial activity against gram-negative clinical isolates. It is also commonly used in critically ill patients. This study aimed to determine the pharmacokinetics/pharmacodynamics of meropenem in critically ill patients and whether prolonged injection duration is really beneficial to meropenem therapy. METHODS: We included 209 samples in 64 patients in this prospective study. PPK analysis and Monte Carlo dosing simulations were developed using Phoenix. RESULTS: A two-compartment model described the data adequately. Clearance (CL), volume (V), clearance of peripheral compartment (CL2), and volume of peripheral compartment (V2) were 6.15 l/h, 2.83 l/h, 17.40 l, and 17.48 l, respectively. Creatinine clearance and uric acid were significant covariates. Patients with creatinine clearance ≤ 60 ml/min and uric acid > 400 µmol/l could achieve the target > 90% under the minimum inhibitory concentration (MIC) of 8 mg/l, even with the administration dose of 500 mg/8 h with a 2-h infusion. Prolonging the infusion time significantly improved the therapeutic effect when MIC < 4. However, for the pharmacodynamic (PD) effects of 100% fT > MIC and 100% fT > 4 MIC, no significant statistical difference was observed in critically ill patients. CONCLUSIONS: Critically ill patients with lower creatinine clearance and higher uric acid levels tended to need a lower dosage of meropenem. Prolonged infusion time was not always beneficial for those who needed a higher therapeutic target (100% fT > MIC, 100% fT > 4 MIC) or with MIC > 4 mg/l. Increasing dose or alternative therapeutic strategies may be required for critically ill patients with drug-resistant or severe infections. The study is of great significance to guide the rational use of meropenem in critically ill patients. TRIAL REGISTRATION: The trial was registered in the China Clinical Trial (ChiCTR1900020672). Registered on 12 January 2019.

Meropenem is commonly used empirically or targeted in critically ill patients for bacterial infection. Many studies have reported that prolonged infusion time can improve the efficacy of meropenem therapy. However, we are skeptical about that. Meanwhile, prolonged injections can sometimes cause mobility problems for patients. A quantitative method is used to evaluate meropenem use. It is called the population pharmacokinetic model or pharmacodynamic study. Using this method, we found two significant influencing factors of meropenem metabolism: creatinine clearance and uric acid level. It is likely that patients with a lower level of creatinine clearance and a high uric acid level tend to require lower dosages of meropenem. As for the effect of infusion time, Monte Carlo simulation was used, which can do 3000 simulations on an individual. The result was complex. We found infusion time was beneficial only when bacteria were sensitive to meropenem. The evidence suggests that prolonged injection duration sometimes does not significantly improve the outcome of antimicrobial therapy.

Factors Affecting Voriconazole Trough Concentration and Optimal Maintenance Voriconazole Dose in Chinese Children.

Voriconazole is a triazole antifungal agent commonly used for the treatment and prevention of invasive aspergillosis (IA). However, the study of voriconazole's use in children is limited. The present study was performed to explore maintenance dose to optimize voriconazole dosage in children and the factors affecting voriconazole trough concentration. This is a non-interventional retrospective clinical study conducted from 1 January 2016 to 31 December 2020. The study finally included 94 children with 145 voriconazole trough concentrations. The probability of achieving a targeted concentration of 1.0-5.5 µg/mL with empiric dosing increased from 43 (45.3%) to 78 (53.8%) after the TDM-guided adjustment. To achieve targeted concentration, the overall target maintenance dose for the age group of less than 2, 2 to 6, 6 to 12, and 12 to 18 years old was approximately 5.71, 6.67, 5.08 and 3.31 mg·kg-1/12 h, respectively (p < 0.001). Final multivariate analysis found that weight (p = 0.019), dose before sampling (p < 0.001), direct bilirubin (p < 0.001), urea nitrogen (p = 0.038) and phenotypes of CYP2C19 were influencing factors of voriconazole trough concentration. These factors can explain 36.2% of the variability in voriconazole trough concentration. Conclusion: In pediatric patients, voriconazole maintenance doses under the target concentration tend to be lower than the drug label recommended, but this still needs to be further studied. Age, body weight, dose, direct bilirubin, urea nitrogen and phenotypes of CYP2C19 were found to be influencing factors of voriconazole concentration in Chinese children. The influence of these factors should be taken into consideration during voriconazole use.

Multifunctional exosome-mimetics for targeted anti-glioblastoma therapy by manipulating protein corona.

Targeted drug delivery to the glioblastoma (GBM) overcoming blood-brain barrier (BBB) has been challenging. Exosomes are promising vehicles for brain tumor drug delivery, but the production and purification hinder its application for nanomedicine. Besides, the formation of protein corona (PC) may affect the behaviour of nanocarriers. Here, multifunctional exosomes-mimetics (EM) are developed and decorated with angiopep-2 (Ang) for enhancing GBM drug delivery by manipulating PC. Docetaxel (DTX)-loaded EM with Ang modification (DTX@Ang-EM) show less absorption of serum proteins and phagocytosis by macrophages. Ang-EM show enhanced BBB penetration ability and targeting ability to the GBM. Ang-EM-mediated delivery increase the concentration of DTX in the tumor area. The multifunctional DTX@Ang-EM exhibits significant inhibition effects on orthotopic GBM growth with reduced side effects of the chemotherapeutic. Findings from this study indicate that the developed DTX@Ang-EM provide a new strategy for targeted brain drug delivery and GBM therapy.

Piperazine ferulate prevents high-glucose-induced filtration barrier injury of glomerular endothelial cells.

Filtration barrier injury induced by high glucose (HG) levels leads to the development of diabetic nephropathy. The endothelial glycocalyx plays a critical role in glomerular barrier function. In the present study, the effects of piperazine ferulate (PF) on HG-induced filtration barrier injury of glomerular endothelial cells (GEnCs) were investigated and the underlying mechanism was assessed. Immunofluorescence was used to observe the distribution of the glycocalyx as well as the expression levels of syndecan-1 and Zonula occludens-1 (ZO-1). Endothelial permeability assays were performed to assess the effects of PF on the integrity of the filtration barrier. Protein and mRNA expression levels were measured by western blotting and reverse transcription-quantitative PCR analyses, respectively. In vitro experiments revealed that adenosine monophosphate-activated protein kinase (AMPK) mediated HG-induced glycocalyx degradation and endothelial barrier injury. PF inhibited the HG-induced endothelial barrier injury and restored the expression levels of heparanase-1 (Hpa-1), ZO-1 and occludin-1 by AMPK. In vivo assays demonstrated that PF reduced the expression levels of Hpa-1, increased the expression levels of ZO-1 and attenuated glycocalyx degradation in the glomerulus. These data suggested that PF attenuated HG-induced filtration barrier injury of GEnC by regulating AMPK expression.

Artificial exosomes for translational nanomedicine.

Exosomes are lipid bilayer membrane vesicles and are emerging as competent nanocarriers for drug delivery. The clinical translation of exosomes faces many challenges such as massive production, standard isolation, drug loading, stability and quality control. In recent years, artificial exosomes are emerging based on nanobiotechnology to overcome the limitations of natural exosomes. Major types of artificial exosomes include 'nanovesicles (NVs)', 'exosome-mimetic (EM)' and 'hybrid exosomes (HEs)', which are obtained by top-down, bottom-up and biohybrid strategies, respectively. Artificial exosomes are powerful alternatives to natural exosomes for drug delivery. Here, we outline recent advances in artificial exosomes through nanobiotechnology and discuss their strengths, limitations and future perspectives. The development of artificial exosomes holds great values for translational nanomedicine.

Exosomes and biomimetic nanovesicles-mediated anti-glioblastoma therapy: A head-to-head comparison.

Exosomes (Exos) are promising vehicles for brain drug delivery due to nanosize and the ability to breach the blood-brain barrier (BBB). But the low yield of natural exosomes limits its application for nanomedicine. The generation of bioinspired nanovesicles (BNVs) that mimicking Exos is attractive, but there is a lack of comparative evaluation of Exos and BNVs. Here, we perform the first head-to-head comparison study of Exos and BNVs for brain tumor drug delivery. We show that BNVs derived from brain-derived endothelial cells are competent alternative nanocarrier to natural exosomes. The drug-loading capacity of Exos and BNVs are similar, but the yield of BNVs is substantially higher (500-fold) than Exos. Doxorubicin (DOX)-loaded BNVs (BNV/DOX) and DOX-loaded Exos (Exo/DOX) showed similar pharmacokinetic profiles and prolonged circulation od DOX. Despite inconsistent mechanisms, BNV/DOX can across the BBB, and exhibit suppression effects similar to Exo/DOX on the progress of glioblastoma (GBM) in zebrafish and in vivo subcutaneous and orthotopic xenografts mice models, with minimal systemic toxicity. Findings from this head-to-head comparison study indicate that autologous BNVs is a effective alternative of Exos for brain tumor nanomedicine.

From blood to brain: blood cell-based biomimetic drug delivery systems.

Brain drug delivery remains a major difficulty for several challenges including the blood-brain barrier, lesion spot targeting, and stability during circulation. Blood cells including erythrocytes, platelets, and various subpopulations of leukocytes have distinct features such as long-circulation, natural targeting, and chemotaxis. The development of biomimetic drug delivery systems based on blood cells for brain drug delivery is growing fast by using living cells, membrane coating nanotechnology, or cell membrane-derived nanovesicles. Blood cell-based vehicles are superior delivery systems for their engineering feasibility and versatile delivery ability of chemicals, proteins, and all kinds of nanoparticles. Here, we focus on advances of blood cell-based biomimetic carriers for from blood to brain drug delivery and discuss their translational challenges in the future.

Biomimetic Liposome with Surface-Bound Elastase for Enhanced Tumor Penetration and Chemo-Immumotherapy.

Dense extracellular matrix (ECM) in the tumor stroma has been a challenge for drug penetration and cytotoxic T lymphocyte (CTL) infiltration. Neutrophil elastase (NE), in surface-bound form, can destruct ECM rapidly, may be used for remodeling tumor ECM, and overcoming tumor stromal barrier. Focusing on elastosis in triple-negative breast tumor, biomimetic liposomes with chimeric cell membrane proteins (LMP) are developed and for the first time, it is demonstrated that LMP with surface-bound elastase (NE-LMP) can target and degrade ECM effectively in tumor stroma, with minimal toxicity to normal tissues. The pretreatment of NE-LMP increases the accumulation of chemotherapeutics at the tumor site and enhances antitumor effects. Also, NE-LMP facilitates CTL infiltration in tumors and exhibits enhanced chemo-immunotherapy in combination of PD-1 immune checkpoint blockade treatment in orthotopic 4T1 tumor-bearing mice, with significantly prolonged survival. Moreover, the remodeling of the tumor ECM by NE-LMP shows inhibiting effects on metastasis in the lung. Findings from this study suggest that NE-LMP holds promise for enhancing deep penetration of drug and infiltration of CTL in desmoplastic tumor by effective degrading ECM in the tumor stroma.

Piperazine ferulate attenuates high glucose­induced mesangial cell injury via the regulation of p66Shc.

Diabetic nephropathy (DN) is a severe microvascular complication of diabetes. Hyperglycemia­induced glomerular mesangial cells injury is associated with microvascular damage, which is an important step in the development of DN. Piperazine ferulate (PF) has been reported to exert protective effects against the progression of DN. However, whether PF prevents high glucose (HG)­induced mesangial cell injury remains unknown. The aim of the present study was to investigate the effects of PF on HG­induced mesangial cell injury and to elucidate the underlying mechanisms. Protein and mRNA expression levels were determined via western blot analysis and reverse transcription­quantitative PCR, respectively. IL­6 and TNF­α levels were measured using ELISA. Reactive oxygen species levels and NF­κB p65 nuclear translation were determined via immunofluorescence analysis. Apoptosis was assessed by measuring lactate dehydrogenase (LDH) release, as well as using MTT and flow cytometric assays. The mitochondrial membrane potential of mesangial cells was determined using the JC­1 kit. The results revealed that LDH release were increased; however, cell viability and mitochondrial membrane potential were decreased in the HG group compared with the control group. These changes were inhibited after the mesangial cells were treated with PF. Moreover, PF significantly inhibited the HG­induced production of inflammatory cytokines and the activation of NF­κB in mesangial cells. PF also attenuated the HG­induced upregulation of the expression levels of fibronectin and collagen 4A1. Furthermore, the overexpression of p66Src homology/collagen (Shc) abolished the protective effect of PF on HG­induced mesangial cell injury. In vivo experiments revealed that PF inhibited the activation of inflammatory signaling pathways, glomerular cell apoptosis and mesangial matrix expansion in diabetic mice. Collectively, the present findings demonstrated that PF attenuated HG­induced mesangial cells injury by inhibiting p66Shc.

Comparative evaluation of methods for isolating small extracellular vesicles derived from pancreatic cancer cells.

BACKGROUND: Small extracellular vesicles (sEVs) are nanosized vesicles involved in cell-to-cell communication. sEVs have been widely studied for clinical applications such as early detection of diseases and as therapeutics. Various methods for sEVs isolation are been using, but different methods may result in different qualities of sEVs and impact downstream analysis and applications. Here, we compared current isolation methods and performed a comparative analysis of sEVs from supernatant of cultured pancreatic cancer cells. METHODS: Ultracentrifugation, ultrafiltration and co-precipitation as concentration methods were firstly evaluated for yield, size, morphology and protein level of pellets. Then, isolate sEVs obtained by four different purification methods: size exclusion chromatography, density gradient ultracentrifugation, ultracentrifugation, and immunoaffinity capturing, were analysed and compared. RESULTS: For the concentration process, ultracentrifugation method obtained high quality and high concentration of pellets. For the purification process, immunoaffinity capturing method obtained the purest sEVs with less contaminants, while density gradient ultracentrifugation-based method obtained sEVs with the smallest size. Proteomic analysis revealed distinct protein contents of purified sEVs from different methods. CONCLUSIONS: For isolating sEVs derived from supernatant of cultured pancreatic cancer cell line, ultracentrifugation-based method is recommended for concentration of sEVs, density gradient ultracentrifugation-based method may be applied for obtaining purified sEVs with controlled size, immunoaffinity capturing may be suitable for studies requiring sEVs with high purity but may loss subtypes of sEVs without specific protein marker.

Preservation of small extracellular vesicles for functional analysis and therapeutic applications: a comparative evaluation of storage conditions.

Extracellular vesicles (EVs) are nanovesicles involved in multiple biological functions. Small EVs (sEVs) are emerging as therapeutics and drug delivery systems for their contents, natural carrier properties, and nanoscale size. Despite various clinical application potentials, little is known about the effects of storage conditions on sEVs for functional analysis and therapeutic use. In this study, we evaluated the stability of sEVs stored at 4 °C, -20 °C, and -80 °C up to 28 days and compared them to fresh sEVs. Also, the effect of freeze-thawing circles on the quantity of sEVs was assessed. We found that different storage temperatures, along with shelf life, impact the stability of sEVs when compared to freshly isolated sEVs. Storage changes the size distribution, decreases quantity and contents, and impacts cellular uptake and biodistribution of sEVs. For functional studies, isolated sEVs are suggested to be analyzed freshly or stored at 4 °C or -20 °C for short-term preservation depending on study design; but -80 °C condition would be more preferable for long-term preservation of sEVs for therapeutic application.

3,5,4'-Trimethoxy-trans-stilbene loaded microemulsion for cutaneous melanoma therapy by transdermal drug delivery.

For therapy of skin cancer, transdermal administration has been a potential way to enhance chemotherapy. However, the drug delivery efficacy remained unsatisfactory because of the physiological barriers from the skin to the tumor, which hindered the effect of 3,5,4'-trimethoxy-trans-stilbene (BTM), a drug that has toxicity to cancer. Herein, we prepared an oil-in-water (O/W) microemulsion to load BTM (BTM-ME) for transdermal therapy of melanoma. BTM-ME was characterized by size, zeta potential, and polymer disperse index (PDI). B16F10 melanoma cell line was used for cell experiments and animal models. And cell uptake, viability assay, and flow cytometry were to test the cell internalization and the ability of BTM-ME to induce cancer cell apoptosis. Skin penetration testing was to detect its penetration efficiency to the skin. And tumor-bearing mice were used to prove the improvement of anti-cancer efficacy of BTM-ME with the combination of Taxol. BTM was successfully loaded in O/W microemulsion, with a drug loading capacity of 24.82 mg/mL. BTM-ME can penetrate the skin and increase the retention of BTM in the epidermis. And the combination of Taxol and BTM-ME effectively suppressed tumor growth and has lower toxicity to normal organs. BTM-ME provides adjuvant therapy to cutaneous melanoma and the combination of Taxol and BTM-ME has the clinical potential for skin cancer therapy. Graphical abstract.

Population pharmacokinetics, safety and dosing optimization of voriconazole in patients with liver dysfunction: A prospective observational study.

AIMS: Voriconazole is a broad-spectrum antifungal agent for the treatment of invasive fungal infections. There is limited information about the pharmacokinetics and appropriate dosage of voriconazole in patients with liver dysfunction. This study aimed to explore the relationship between voriconazole trough concentration (Ctrough ) and toxicity, identify the factors significantly associated with voriconazole pharmacokinetic parameters and propose an optimised voriconazole dosing regimen for patients with liver dysfunction. METHODS: The study prospectively enrolled 51 patients with 272 voriconazole concentrations. Receiver operating characteristic curves were used to explore the relationship between voriconazole Ctrough and toxicity. The pharmacokinetic data was analysed with nonlinear mixed-effects method. Dosing simulations stratified by total bilirubin (TBIL, TBIL-1: TBIL < 51 µmol/L; TBIL-2: 51 µmol/L ≤ TBIL < 171 µmol/L; TBIL-3: TBIL ≥ 171 µmol/L) were performed. RESULTS: Receiver operating characteristic curve analysis revealed that voriconazole Ctrough of ≤ 5.1 mg/L were associated with significantly lower the incidence of adverse events. A 1-compartment pharmacokinetic model with first-order absorption and elimination was used to describe the data. Population pharmacokinetic parameters of clearance, volume of distribution and oral bioavailability were 0.88 L/h, 148.8 L and 88.4%, respectively. Voriconazole clearance was significantly associated with TBIL and platelet count. The volume of distribution increased with body weight. Patients with TBIL-1 could be treated with a loading dose of 400 mg every 12 hours (q12h) for first day, followed by a maintenance dose of 100 mg q12h administered orally or intravenously. TBIL-2 and TBIL-3 patients could be treated with a loading dose of 200 mg q12h and maintenance doses of 50 mg q12h or 100 mg once daily and 50 mg once daily orally or intravenously, respectively. CONCLUSIONS: Lower doses and longer dosing intervals should be considered for patients with liver dysfunction. TBIL-based dosing regimens provide a practical strategy for achieving voriconazole therapeutic range and therefore maximizing treatment outcomes.