Deoxycholic acid-chitosan coated liposomes combined with in situ colonic gel enhances renal fibrosis therapy of emodin.

BACKGROUND: Renal fibrosis is the final common pathological feature of various chronic kidney diseases (CKD). Despite recent advances, development of new treatments strategy is needed. Emodin (EMO), an important ingredient of Chinese medicine, rhubarb (Polygonaceae Rheum palmatum l.), has been reported to inhibit the development of renal fibrosis effectively. However, the poor oral bioavailability of EMO and the insufficient monotherapy therapy compromise its efficacy. PURPOSE: In order to enhance renal fibrosis therapy of emodin, an innovative combination therapy based on deoxycholic acid-chitosan coated liposomes (DCS-Lips) and in situ colonic gel (IGE) was developed. METHODS: For one, the DCS-Lips were prepared via electrostatic interaction by mixing anionic conventional Lips with cationic DCS, deoxycholic acid conjugated on the backbone of chitosan. The cellular uptake of FITC-labeled DCS-Lips in Caco-2 cell monolayer was evaluated by CLSM and flow cytometry, respectively. Permeability study was carried out using Caco-2 cell monolayer. For another, EMO-loaded in situ colonic gel (EMO-IGE) was prepared by mixing EMO nanosuspensions and plain in situ gel, which was obtained by the cold method. The EMO-IGE was assessed for morphology, gelation temperature, viscosity and in vitro drug release. Finally, the therapeutic efficacy of the combination strategy, oral DCS-Lips formulations and in situ colonic gel, was evaluated in unilateral ureteral obstruction (UUO) rat model. Additionally, 16S rDNA sequencing was performed on rats faces to investigate whether the combination strategy improves the microbial dysbiosis in UUO rats. RESULTS: The prepared DCS-Lips produced small, uniformly sized nanoparticles, and significantly enhanced the cellular uptake and in vitro permeability of EMO compared to non-coated liposomes. Moreover, the EMO-IGE was characterized by short gelation time, optimal gelling temperature, and excellent viscosity. In UUO model, the combination of DCS-Lips (gavage) and IGE (enema) attenuated renal fibrosis effectively. The results of 16S rDNA sequencing illustrated that IGE could restore the gut microbial dysbiosis of UUO rats. CONCLUSION: Overall, the combination of DCS-Lips and EMO-IGE alleviated renal fibrosis effectively, resulting from the improved oral bioavailability of EMO by DCS-Lips and the restoration of gut microbiota by EMO-IGE, thus, presenting an innovative and promising potential for renal fibrosis treatment.

MMP2-responsive dual-targeting drug delivery system for valence-controlled arsenic trioxide prodrug delivery against hepatic carcinoma.

Arsenic trioxide (ATO) is the active ingredient in traditional Chinese medicine, i.e., Arsenic, which has shown excellent therapeutic effects on hepatocellular carcinoma. However, due to its poor tumor distribution and high toxicity, the mass adoption of ATO in clinical applications has been severely impeded. In this study, matrix metalloproteinase 2 (MMP2)-responsive cleaved cell-penetrating peptide (PF) and folate (FA) co-modified liposome coated calcium arsenate nanoparticles (FA/PF-LP-CaAs) were fabricated based on these two considerations: (1) The tumor microenvironment characterized by overexpressed MMP2 in extracellular matrix and folate receptor on the cell membrane can enhance drug accumulation and accelerate endocytosis; (2) leveraging different toxicity of arsenic in different valence states, i.e., AsV can be reduced to more toxic AsIII by glutathione in tumor cells. Furthermore, FA/PF-LP-CaAs could be responsively degraded by the mild acidic tumor environment, and the degraded product could escape from lysosomes after endocytosis. More importantly, in light of the in vivo biodistribution and pharmacodynamic studies, the vehicle was able to accumulate in the tumor efficiently. Also, it was able to exhibit excellent anti-tumor efficacy with minimized side effects when compared to single-modified counterparts. Thus, the novel strategy based on the tumor microenvironment proposed in this work can enhance the tumor-targeting efficiency and intratumor toxicity.

Microwave-triggered/HSP-targeted gold nano-system for triple-negative breast cancer photothermal therapy.

Due to the lack of effective therapeutic targets and the passive delivery of a limited quantity of nanoparticles to the tumors, the photothermal conversion agents used in photothermal therapy (PTT) have not been effective in treating triple-negative breast cancer (TNBC). As a result, there is a need to improve the tumor-targeting ability of these photothermal conversion agents. To address this, a microwave-triggered heat shock protein (HSP)-targeted gold nano-system (cmHSP-AuNC), with a gold nanocage (AuNC) as a photothermal conversion agent and anti-HSP monoclonal antibody (cmHSP) as a targeting ligand, was fabricated. cmHSP-AuNC was characterized based on morphology, particle size, zeta potentials, absorption spectrum, and photothermal conversion ability. The expression of HSP70 in 4T1 cells after microwave irradiation was verified by western blotting, and the optimal treatment conditions to achieve the highest expression were determined. Both in vitro and in vivo results indicated that the induction through microwave irradiation could effectively activate the HSP70 overexpression in TNBC, thereby significantly improving the targeting ability, tumor accumulation and anti-tumor efficacy of cmHSP-AuNC. This study proposes a promising strategy for improving the targeting ability and therapeutic efficacy of PTT.

[Effect of emodin on gut microbiota of rats with acute kidney failure].

The aim of this paper was to investigate the effect of emodin on gut microbiota in acute kidney injury rats( AKI). Rats were randomly divided into several groups: normal group,model group,low-dose of emodin group( 10 mg·kg~(-1)),medium-dose of emodin group( 25 mg·kg~(-1)),high-dose of emodin group( 50 mg·kg~(-1)) and control group( 5 mg·kg~(-1) of benazepril hydrochloride).The AKI model rats were established by intraperitoneal injection of small dose of gentamicin sulfate for 7 days. Two hours after intraperitoneal injection,except for the normal group and the model group,the other groups were given corresponding doses of drugs for 15 days. The serum levels of serum creatinine( SCr),urea nitrogen( BUN),plasma endotoxin level,24 h urinary protein and D-lactate in the plasma were determined by sarcosine oxidase,urease method,tal reagent method,bromo cresol chloroform method and double antibody sandwich enzyme-linked immunoadsorbent assay,respectively. Gut microbial communities were assayed by fluorescent quantitative PCR methods. HE staining was used to detect the pathological changes of the kidneys. Compared with the normal group,there were significant differences in body weight,urinary protein( UTP),bacterial endotoxin,urea nitrogen,creatinine,D-lactate in the plasma and four bacterial contents in the model group( P<0. 05). The urinary protein,urea nitrogen,D-lactate,creatinine and plasma bacterial endotoxin in control group and each emodin group were lower than those in model group,especially for high-dose of emodin( P<0. 01). Moreover,pathology resolution in high-dose emodin was better than other groups. Except for low-dose of emodin group,qRT-PCR data suggested that the amounts of Escherichia coli and Enterococcus in medication administration group were increased,while the amounts of Lactobacilli and Bifidobacterium were reduced compared with model group( P<0. 05),especially for high-dose of emodin( P<0. 01). There is a clear imbalance of gut microbiota in rats with AKI. Emodin could regulate the imbalance of gut microbiota,which might be one of the mechanisms of its effects on AKI rats.

[Preparation and in vitro evaluation of rhein-loaded PEG-PCL-PEI nanoparticles].

This study was aimed to synthesize the polyethyleneglycol-polycaprolactone-polyethyleneimine (PEG-PCL-PEI) three block polymer material, prepareRhein (RH)-loaded PEG-PCL-PEI nanoparticles(PPP-RH-NPS), and then evaluate their physical and chemical properties and biological characteristics in vitro. PEG-PCL-PEI polymer was obtained by adopting thering-opening polymerization and Michael addition reaction, and their physical and chemical properties were analyzed by using NMR and gel permeation chromatography. PEG-PCL-PEI was then used as the carriers to prepare PPP-RH-NPS by applying spontaneous emulsification solvent diffusion method. The results showed that molecular weight of PEG-PCL-PEI polymer was 9.5×103, and critical micelle concentration was 0.723 mmol•L⁻¹. PPP-RH-NPS had pale yellow, opalescence faade, round and smooth without aggregation, formed of (118.3±3.6) nm in particle size with PDI of (0.19±0.08), Zeta potential of (6.3±1.5) mV, entrapment efficiency of (93.64±5.28)%, and drug loading of (8.57±0.53)%. The accumulative release percentage of PPP-RH-NPS was 75.92% in 48h, and the release profiles in PBS conformed to the Higuchi equation： Q=0.121 6t1/2+0.069 5 (R²=0.887 4), presenting slow release characteristics. Within the scope of the 0-0.05 mmol•L⁻¹, the nanoparticles had no obvious hemolysis on rabbit red blood cells and toxicity on HK-2 cells. In the investigation of uptake efficiency by flow cytometry, nanoparticles can be absorbed into cells quickly and internalized within 30 minutes fully, with a high uptake efficiency. In confocal laser scanning microscope observation, the nanoparticles can escape from lysosome into cytoplasm. Herein, this study synthesized the PEG-PCL-PEI polymer and prepared PPP-RH-NPS successfully; the nanoparticles showed uniform particle size, higher encapsulation efficiency and drug-loading rate, slow release characteristics, quick uptake and internalization, lysosome escape property and good biocompatibility. PPP-RH-NPS will be a promising pharmaceutical formulation for further development.

[Preparation of curcumin-loaded long-circulating liposomes and its pharmacokinetics in rats].

Curcumin has a wide spectrum of pharmaceutical properties such as antitumor, antioxidant, antiamyloid, and anti-inflammatory activity. However, poor aqueous solubility and low bioavailability of curcumin are major challenge in its development as a useful drug. To overcome many of these problems, curcumin-loaded long-circulating liposomes (Cur-LCL) were prepared by the ethanol injection method. Morphology of Cur-LCL was observed by transmission electron microscope, mean particle size and Zeta potential were detected by laser particle size analyzer, entrapment efficiency and drug loading were evaluated by ultracentrifugation. The drug release behavior in vitro and pharmacokinetic behavior in rats of Cur-LCL were investigated with curcumin (Cur) and curcumin liposomes (Cur-Lips) as control. The results showed that the mean diameter of Cur-LCL was 110 nm, the Zeta potential was -5.8 mV. The entrapment efficiency and drug loading of Cur-LCL was 80.25%, 2.06%, respectively. The release behavior in vitro studied by dialysis in PBS buffer showed significant sustained release profile that 48.95% Cur were released from Cur-LCL in 7 h, 88.92% in 24 h. The pharmacokinetic parameters showed that compared with Cur and Cur-Lips, the t(1/2beta) of Cur-LCL was extended to 13 and 1.8-fold, respectively. Besides, the AUC values was significantly increased (P < 0.01), and the clearance was evidently decreased (P < 0.01). These results from in vitro and in vivo indicated that Cur-LCL were able to realize controlled drug release and increase circulation time.

[Studies on absorption kinetics characteristics of diterpenoid alkaloids in rats after oral administration of Sini Tang powder].

The purpose of this study was to investigate the absorption kinetics of aconitine, mesaconitine and hypaconitine in rats after oral administration of Sini Tang powder. With cannulate portal and jugular veins cannulated (double-cannulate), conscious moving rats were orally administered Sini Tang. Then samples of portal and systemic blood were collected at the designated periods of time and analyzed for aconitine, mesaconitine and hypaconitine by HPLC. Apparent absorption coefficient of aconitine, mesaconitine and hypaconitine was caculated respectively. The results indicated that the apparent absorption coefficient of aconitine, mesaconitine and hypaconitine come from Sini Tang were 0. 336, 0. 090, 0. 176, respectively, which had some differences among them. It was also suggested that double-cannulated rat was useful for estimating the absorption kinetics of aconitine, mesaconitine and hypaconitine after orally administered Sini Tang by determining the AUC values for drugs in portal and systemic blood samples. The three alkaloids could all be detected in blood, but the absorption differences were existed among the three alkaloids.

[Study on chitosan-modified tripterygium glycoside nanoparticles and its renal targeting property].

OBJECTIVE: To prepare chitosan-modified tripterygium glycoside nanoparticles (LMWC-TG-PLA-NPs), and assess its renal targeting property in rats. METHOD: Chitosan-modified tripterygium glycoside nanoparticles (LMWC-TG-PLA-NPs) were prepared by modified spontaneous emulsification solvent evaporation method, and modified with 50% deacetylated low molecular weight chitosan (LMWC). The shape of nanoparticles was observed under a transmission electron microscope. The mean diameter of nanoparticles was measured by particle size analyzer. The drug encapsulation efficiency and drug loading were measured by centrifuge method. The in vitro release behavior was studied with dialysis bags. Renal microdialysis technique and renal artery administration technique were combined to study the renal targeting property of nanopartcles. LMWC-TG-PLA-NPs were administrated in rats by tail vein injection (TVI) and renal artery administration (RAA), respectively, with TG-PLA-NPs as the control group. Renal dialysis fluid was regularly collected to determine the drug concentration in the dialysis fluid, map drug concentration-time curves, and calculate AUC ratio in kidneys through the two injection approaches as the renal targeting parameter (RTP), in order to assess the renal targeting property of LMWC-TG-PLA-NPs. RESULTS: The prepared LMWC-TG-PLA-NPs looked smooth and round. Their average diameter, polydispersity index, encapsulation efficiency and drug loading were (207.6 +/- 3.4) nm, (0.078 +/- 0.009)%, (61.83 +/- 2.43)%, and (10.70 +/- 0.37)%, respectively. The pH 7.4 PBS buffer solution containing 20% ethanol showed obvious sustained release behavior. LMWC-TG-PLA-NPs showed a RTP of 71.97%, which was 3.6 times of TG-PLA-NPs of the control group. CONCLUSION: The prepared LMWC-TG-PLA-NPs showed high drug encapsulation efficiency and drug loading, with obvious sustained release characteristics and renal targeting property. LMWC-TG-PLA-NPs are expected to become a new type vector for reducing toxic and side effects of tripterygium glycoside. Meanwhile, a new method is established for assessing renal targeting property with AUC ratio in kidneys after administrated through caudal veins and renal arteries as the renal targeting parameter.

[Analysis on the metabolites of mesaconitine in the rat urine by liquid chromatography and electrospray ionization mass spectrometry].

The mesaconitine and its major metabolites in the rat urine were identified by liquid chromatography and electrospray ionization tandem mass spectrometry. The rat urine was collected for consecutive 24 hours from the rat following intragastric infusion of mesaconitine, subsequently which were enriched and purified using solid phase extraction. The metabolites of mesaconitine in the rat urine were analyzed by the liquid chromatography and electrospray ionization tandem mass spectrometry. It is shown that the parent drug mesaconitine and its metabolites were found in the rat urine, such as hypo-mesaconitine glucuronic acid conjugate, 10-hydroxy-mesaconitine, 1-O-demethyl mesaconitine, deoxy-mesaconitine and hypo-mesaconitine. Among the five of metabolites, the hypo-mesaconitine glucuronic acid conjugate (m/z 766) was first discovered as the aconitine in rats phase II metabolites, which revealed a new way of mesaconitine metabolism in rats.