Targeted inhibition of autophagy in hepatic stellate cells by hydroxychloroquine: An effective therapeutic approach for the treatment of liver fibrosis.

BACKGROUND AND PURPOSE: Liver fibrosis is a wound-healing reaction which is the main cause of chronic liver diseases worldwide. The activated hepatic stellate cell (aHSC) is the main driving factor in the development of liver fibrosis. Inhibiting autophagy of aHSC can prevent the progression of liver fibrosis, but inhibiting autophagy of other liver cells has opposite effects. Hence, targeted inhibition of autophagy in aHSC is quite necessary for the treatment of liver fibrosis, which prompts us to explore the targeted delivery system of small molecule autophagy inhibitor hydroxychloroquine (HCQ) that can target aHSC and alleviate the liver fibrosis. METHODS: The delivery system of HCQ@retinol-liposome nanoparticles (HCQ@ROL-LNPs) targeting aHSC was constructed by the film dispersion and pH-gradient method. TGF-ß-induced HSC activation and thioacetamide (TAA)-induced liver fibrosis mice model were established, and the targeting ability and therapeutic effect of HCQ@ROL-LNPs in liver fibrosis were studied subsequently in vitro and in vivo. RESULTS: HCQ@ROL-LNPs have good homogeneity and stability. They inhibited the autophagy of aHSC selectively by HCQ and reduced the deposition of extracellular matrix (ECM) and the damage to other liver cells. Compared with the free HCQ and HCQ@LNPs, HCQ@ROL-LNPs had good targeting ability, showing enhanced therapeutic effect and low toxicity to other organs. CONCLUSION: Construction of HCQ@ROL-LNPs delivery system lays a theoretical and experimental foundation for the treatment of liver fibrosis and promotes the development of clinical therapeutic drugs for liver diseases.

Construction of GSH-responsive polyethyleneimine-based delivery vector for effective gene transfection.

The rise of gene therapy has solved many diseases that cannot be effectively treated by conventional methods. Gene vectors is very important to protect and deliver the therapeutic genes to the target site. Polyethyleneimine (PEI) modified with mannitol could enhance the gene transfection efficiency reported by our group previously. In order to further control and improve the effective gene release to action site, disulfide bonds were introduced into mannitol-modified PEI to construct new non-viral gene vectors PeiSM. The degrees of mannitol linking with disulfide bonds were screened. Among them, moderate mannitol-modified PEI with disulfide bonds showed the best transfection efficiency, and significantly enhanced long-term systemic transgene expression for 72 hin vivoeven at a single dose administration, and could promote caveolae-mediated uptake through up-regulating the phosphorylation of caveolin-1 and increase the loaded gene release from the nanocomplexes in high glutathione intracellular environment. This functionalized gene delivery system can be used as an potential and safe non-viral nanovector for further gene therapy.

Injectable hybrid hydrogels enable enhanced combination chemotherapy and roused anti-tumor immunity in the synergistic treatment of pancreatic ductal adenocarcinoma.

Chemotherapy and immunotherapy have shown no significant outcome for unresectable pancreatic ductal adenocarcinoma (PDAC). Multi-drug combination therapy has become a consensus in clinical trials to explore how to arouse anti-tumor immunity and meanwhile overcome the poorly tumoricidal effect and the stroma barrier that greatly hinders drug penetration. To address this challenge, a comprehensive strategy is proposed to fully utilize both the ferroptotic vulnerability of PDAC to potently irritate anti-tumor immunity and the desmoplasia-associated focal adhesion kinase (FAK) to wholly improve the immunosuppressive microenvironment via sustained release of drugs in an injectable hydrogel for increasing drug penetration in tumor location and averting systematic toxicity. The injectable hydrogel ED-M@CS/MC is hybridized with micelles loaded with erastin that exclusively induces ferroptosis and a FAK inhibitor defactinib for inhibiting stroma formation, and achieves sustained release of the drugs for up to 12 days. With only a single intratumoral injection, the combination treatment with erastin and defactinib produces further anti-tumor performance both in xenograft and KrasG12D-engineered primary PDAC mice and synergistically promotes the infiltration of CD8+ cytotoxic T cells and the reduction of type II macrophages. The findings may provide a novel promising strategy for the clinical treatment of PDAC.

Construction of mPEI/pGPX4 gene therapeutic system for the effective treatment of acute lung injury.

Acute lung injury (ALI) can be induced by various injury factors, which is closely related to the inflammatory reaction and cellular ferroptosis reported recently. Glutathione peroxidase (GPX4) palys an important role in the inflammatory reaction, which also is the core regulatory protein of ferroptosis. Up-regulation of GPX4 can be helpful to inhibit the cellular ferroptosis and inflammatory reaction to treat ALI. mPEI/pGPX4 gene therapeutic system based on mannitol-modified polyethyleneimine (mPEI) was constructed. Compared with PEI/pGPX4 nanoparticles using commoditized gene vector PEI 25k, mPEI/pGPX4 nanoparticles achieved caveolae-mediated endocytosis and improved the gene therapeutic effect. mPEI/pGPX4 nanoparticles could up-regulate the gene expression of GPX4, inhibit inflammatory reaction and the cellular ferroptosis, thereby alleviating the ALIin vitroandin vivo. The finding indicated that gene therapy with pGPX4 is a potential therapeutic system for the effective treatment of ALI.

Tumor microenvironment and redox dual stimuli-responsive polymeric nanoparticles for the effective cisplatin-based cancer chemotherapy.

The serious side effects of cisplatin hindered its clinical application and the nanotechnology might be the potential strategy to address the limitation. However, rapid clearance in the blood circulation and ineffective controlled drug release from nanocarriers hamper the therapeutic efficacy of the nano-delivery system. We constructed a tumor microenvironment and redox dual stimuli-responsive nano-delivery system PEG-c-(BPEI-SS-Pt) by cross-linking the disulfide-containing polymeric conjugate BPEI-SS-Pt with the dialdehyde group-modified PEG2000via Schiff base. After optimized the cross-linking time, 72 h was selected to get the nano-delivery system.1H NMR and drug release assays showed that under the acidic tumor microenvironment (pH 6.5-6.8), the Schiff base can be broken and detached the PEG cross-linked outer shells, displaying the capability to release the drugs with a sequential pH- and redox-responsive manner. Moreover, PEG-c-(BPEI-SS-Pt) showed more effective anti-tumor therapeutic efficacyin vivowith no significant side effects when compared with the drug of cisplatin used in the clinic. This strategy highlights a promising platform with the dual stimuli-responsive profile to achieve better therapeutic efficacy and minor side effects for platinum-based chemotherapy.

New Steroidal Saponins Isolated from the Rhizomes of Paris mairei.

The genus Paris is an excellent source of steroidal saponins that exhibit various bioactivities. Paris mairei is a unique species and has been widely used as folk medicine in Southwest China for a long time. With the help of chemical methods and modern spectra analysis, five new steroidal saponins, pamaiosides A-E (1-5), along with five known steroidal saponins 6-10, were isolated from the rhizomes of Paris mairei. The cytotoxicity of all the new saponins was evaluated against human pancreatic adenocarcinoma PANC-1 and BxPC3 cell lines.

Polyethylenimine-based nanovector grafted with mannitol moieties to achieve effective gene delivery and transfection.

Polyethylenimine (PEI), a kind of cationic non-viral gene delivery vector, is capable of stable and efficient transgene expression for gene delivery. However, low transfection efficiency in vivo along with high toxicity limited the further application of gene therapy in the clinic. To enhance gene transfection performance and reduce cytotoxicity of polyethylenimine, branched polyethylenimine-derived cationic polymers BPEI25 k-man-S/L/M/H with different grafting degree with mannitol moieties were prepared and the transfection efficiency was evaluated. Among them, BPEI25 k-man-L showed the best transfection efficiency, lower toxicity, and significantly enhanced long-term systemic transgene expression for 96 h in vivo even at a single-dose administration. The results of cellular uptake mechanism and western-blot experiments revealed that the mannitol modification of BPEI25 k induced and up-regulated the phosphorylation of caveolin-1 and thus enhanced the caveolae-mediated cellular uptake. This class of gene delivery system highlights a paradigmatic approach for the development of novel and safe non-viral vectors for gene therapy.

Enhance transgene responses through improving cellular uptake and intracellular trafficking by bio-inspired non-viral vectors.

BACKGROUND: Gene therapy remains a significant challenge due to lots of barriers limiting the genetic manipulation technologies. As for non-viral delivery vectors, they often suffer insufficient performance due to inadequate cellular uptake and gene degradation in endosome or lysosome. The importance of overcoming these conserved intracellular barriers is increasing as the delivery of genetic cargo. RESULTS: A surface-functionalized non-viral vector involving the biomimetic mannitol moiety is initiated, which can control the cellular uptake and promote the caveolae-mediated pathway and intracellular trafficking, thus avoiding acidic and enzymatic lysosomal degradation of loaded gene internalized by clathrin-mediated pathway. Different degrees of mannitol moiety are anchored onto the surface of the nanoparticles to form bio-inspired non-viral vectors and CaP-MA-40 exhibits remarkably high stability, negligible toxicity, and significantly enhanced transgene expression both in vitro and in vivo. CONCLUSIONS: This strategy highlights a paradigmatic approach to construct vectors that need precise intracellular delivery for innovative applications.

Mitochondria-targeted cyclosporin A delivery system to treat myocardial ischemia reperfusion injury of rats.

BACKGROUND: Cyclosporin A (CsA) is a promising therapeutic drug for myocardial ischemia reperfusion injury (MI/RI) because of its definite inhibition to the opening of mitochondrial permeability transition pore (mPTP). However, the application of cyclosporin A to treat MI/RI is limited due to its immunosuppressive effect to other normal organ and tissues. SS31 represents a novel mitochondria-targeted peptide which can guide drug to accumulate into mitochondria. In this paper, mitochondria-targeted nanoparticles (CsA@PLGA-PEG-SS31) were prepared to precisely deliver cyclosporin A into mitochondria of ischemic cardiomyocytes to treat MI/RI. RESULTS: CsA@PLGA-PEG-SS31 was prepared by nanoprecipitation. CsA@PLGA-PEG-SS31 showed small particle size (~ 50 nm) and positive charge due to the modification of SS31 on the surface of nanoparticles. CsA@PLGA-PEG-SS31 was stable for more than 30 days and displayed a biphasic drug release pattern. The in vitro results showed that the intracellular uptake of CsA@PLGA-PEG-SS31 was significantly enhanced in hypoxia reoxygenation (H/R) injured H9c2 cells. CsA@PLGA-PEG-SS31 delivered CsA into mitochondria of H/R injured H9c2 cells and subsequently increased the viability of H/R injured H9c2 cell through inhibiting the opening of mPTP and production of reactive oxygen species. In vivo results showed that CsA@PLGA-PEG-SS31 accumulated in ischemic myocardium of MI/RI rat heart. Apoptosis of cardiomyocyte was alleviated in MI/RI rats treated with CsA@PLGA-PEG-SS31, which resulted in the myocardial salvage and improvement of cardiac function. Besides, CsA@PLGA-PEG-SS31 protected myocardium from damage by reducing the recruitment of inflammatory cells and maintaining the integrity of mitochondrial function in MI/RI rats. CONCLUSION: CsA@PLGA-PEG-SS31 exhibited significant cardioprotective effects against MI/RI in rats hearts through protecting mitochondrial integrity, decreasing apoptosis of cardiomyocytes and myocardial infract area. Thus, CsA@PLGA-PEG-SS31 offered a promising therapeutic method for patients with acute myocardial infarction.

Osteoclasts and tumor cells dual targeting nanoparticle to treat bone metastases of lung cancer.

Bone is one of the prone metastatic sites of lung cancer. Osteoclast plays an important role in bone resorption and the growth of bone metastases of lung cancer. In order to treat bone metastases of lung cancer, we reported a docetaxel (DTX)-loaded nanoparticle, DTX@AHP, which could target dually at osteoclasts and bone metastatic tumor cells. The in vitro drug release from DTX@AHP exhibited pH and redox responsive characteristics. DTX@AHP displayed high binding affinity with bone matrix. In addition, DTX@AHP significantly inhibited the differentiation of RAW264.7 into osteoclast and effectively inhibited the proliferation of osteoclasts and tumor cells in in-vitro 3D bone metastases model of lung cancer. DTX@AHP could accumulate in bone metastases sites in vivo. Consequently, DTX@AHP not only markedly inhibited the growth of bone metastases of lung cancer but also reduced osteolysis in tumor-bearing mice. DTX@AHP exhibited great potential in the treatment of bone metastases of lung cancer.

Mitochondria-targeted antioxidant delivery for precise treatment of myocardial ischemia-reperfusion injury through a multistage continuous targeted strategy.

Efficient delivery of antioxidant drugs into mitochondria of ischemic cardiomyocytes where reactive oxygen species largely induced is a major challenge for precise treatment of myocardial ischemia-reperfusion injury. Herein, we report a smart dual-shell polymeric nanoparticle, MCTD-NPs, which utilizes multistage continuous targeted strategy to deliver reactive oxygen species scavenger specifically to mitochondria of ischemic cardiomyocytes upon systemic administration. In vitro experiments indicated that the intracellular uptake of MCTD-NPs was specifically enhanced in hypoxia reoxygenation injured H9c2 cells. MCTD-NPs selectively delivered resveratrol to mitochondria of hypoxia reoxygenation injured H9c2 cells. In addition, MCTD-NPs increased the viability of H/R injured H9c2 cell through eliminating mitochondrial ROS, decreasing mPTP opening and blocking mitochondria-dependent apoptotic pathway. In vivo experiments revealed that MCTD-NPs increased the distribution of resveratrol in the ischemic myocardium and subsequently reduced infarct size in MI/RI rats. These results demonstrated a novel platform for specific delivery of antioxidant to mitochondria to treat MI/RI.

Charge Reversible and Mitochondria/Nucleus Dual Target Lipid Hybrid Nanoparticles To Enhance Antitumor Activity of Doxorubicin.

The experiment aims to increase antitumor activity while decreasing the systemic toxicity of doxorubicin (DOX). Charge reversible and mitochondria/nucleus dual target lipid hybrid nanoparticles (LNPs) was prepared. The in vitro experimental results indicated that LNPs released more amount of DOX in acidic environment and delivered more amount of DOX to the mitochondria and nucleus of tumor cells than did free DOX, which resulted in the reduction of mitochondrial membrane potential and the enhancement of cytotoxicity of LNPs on tumor cells. Furthermore, the in vivo experimental results indicated that LNPs delivered more DOX to tumor tissue and significantly prolonged the retention time of DOX in tumor tissue as compared with free DOX, which consequently resulted in the high antitumor activity and low systemic toxicity of LNPs on tumor-bearing nude mice. The above results indicated that charge reversible mitochondria/nucleus dual targeted lipid hybrid nanoparticles greatly enhanced therapeutic efficacy of DOX for treating lung cancer.

Construction and evaluation of BSA-CaP nanomaterials with enhanced transgene performance via biocorona-inspired caveolae-mediated endocytosis.

Non-viral nanovectors have attracted much attention owing to their ability to condense genetic materials and their ease of modification. However, their poor stability, low biocompatibility and gene degradation in endosomes or lysosomes has significantly hampered their application in vivo and in the clinic. In an attempt to overcome these difficulties a series of bovine serum albumin (BSA)-calcium phosphate (CaP) nanoparticles were constructed. The CaP condenses with DNA to form nanocomplexes coated with a biomimetic corona of BSA. Such complexes may retain the inherent endocytosis profile of BSA, with improved biocompatibility. In particular the transgene performance may be enhanced by stimulating the cellular uptake pathway via caveolae-mediated endocytosis. Two methods were employed to construct and optimize the formulation of BSA-CaP nanomaterials. The optimized BSA-CaP-50-M2 nanoparticles prepared by our second method exhibited good stability, negligible cytotoxicity and enhanced transgene performance with long-term expression for 72 h in vivo even with a single dose. Determination of the cellular uptake pathway and Western blot revealed that cellular uptake of the designed BSA-CaP-50-M2 nanoparticles was mainly via caveolae-mediated endocytosis in a non-degradative pathway in which the biomimetic uptake profile of BSA was retained.

The enhancement of siPLK1 penetration across BBB and its anti glioblastoma activity in vivo by magnet and transferrin co-modified nanoparticle.

In order to enhance the penetration of small interference RNA against the polo-like kinase I (siPLK1) across BBB to treat glioblastoma (GBM), transferrin (Tf) modified magnetic nanoparticle (Tf-PEG-PLL/MNP@siPLK1) was prepared. The in vitro experiments indicated that Tf-PEG-PLL/MNP@siPLK1 enhanced the cellular uptake of siPLK1, which resulted in an increase of gene silencing effect and cytotoxicity of Tf-PEG-PLL/MNP@siPLK1 on U87 cells. Besides, Tf-PEG-PLL/MNP@siPLK1 significantly inhibited the growth of U87 glioblastoma spheroids and markedly increased the BBB penetration efficiency of siPLK1 with the application of external magnetic field in in-vitro BBB model. The in vivo experiments indicated that siPLK1 selectively accumulated in the brain tissue, and markedly reduced tumor volume and prolonged the survival time of GBM-bearing mice after Tf-PEG-PLL/MNP@siPLK1 was injected to GBM-bearing mice via tail vein. The above data indicated that magnet and transferrin co-modified nanoparticle enhanced siPLK1 penetration across BBB and increased its anti GBM activity in vivo.

The toxicological mechanisms and detoxification of depleted uranium exposure.

Depleted uranium (DU) has been widely applied in industrial and military activities, and is often obtained from producing fuel for nuclear reactors. DU may be released into the environment, polluting air, soil, and water, and is considered to exert both radiological and chemical toxicity. In humans and animals, DU can induce multiple health effects, such as renal tubular necrosis and bone malignancies. This review summarizes the known information on DU's routes of entry, mechanisms of toxicity, and health effects. In addition, we survey the chelating agents used in ameliorating DU toxicity.

Chitosan cross-linked with poly(ethylene glycol)dialdehyde via reductive amination as effective controlled release carriers for oral protein drug delivery.

The covalently cross-linked chitosan-poly(ethylene glycol)1540 derivatives have been developed as a controlled release system with potential for the delivery of protein drug. The swelling characteristics of the hydrogels based on these derivatives as the function of different PEG content and the release profiles of a model protein (bovine serum albumin, BSA) from the hydrogels were evaluated in simulated gastric fluid with or without enzyme in order to simulate the gastrointestinal tract conditions. The derivatives cross-linked with difunctional PEG1540-dialdehyde via reductive amination can swell in alkaline pH and remain insoluble in acidic medium. The cumulative release amount of BSA was relatively low in the initial 2h and increased significantly at pH 7.4 with intestinal lysozyme for additional 12h. The results proved that the release-and-hold behavior of the cross-linked CS-PEG1540H-CS hydrogel provided a swell and intestinal enzyme controlled release carrier system, which is suitable for oral protein drug delivery.

Design and application of cationic amphiphilic ß-cyclodextrin derivatives as gene delivery vectors.

The nano self-assembly profiles of amphiphilic gene delivery vectors could improve the density of local cationic head groups to promote their DNA condensation capability and enhance the interaction between cell membrane and hydrophobic tails, thus increasing cellular uptake and gene transfection. In this paper, two series of cationic amphiphilic ß-cyclodextrin (ß-CD) derivatives were designed and synthesized by using 6-mono-OTs-ß-CD (1) as the precursor to construct amphiphilic gene vectors with different building blocks in a selective and controlled manner. The effect of different type and degree of cationic head groups on transfection and the endocytic mechanism of ß-CD derivatives/DNA nanocomplexes were also investigated. The results demonstrated that the designed ß-cyclodextrin derivatives were able to compact DNA to form stable nanocomplexes and exhibited low cytotoxicity. Among them, PEI-1 with PEI head group showed enhanced transfection activity, significantly higher than commercially available agent PEI25000 especially in the presence of serum, showing potential application prospects in clinical trials. Moreover, the endocytic uptake mechanism involved in the gene transfection of PEI-1 was mainly through caveolae-mediated endocytosis, which could avoid the lysosomal degradation of loaded gene, and had great importance for improving gene transfection activity.

Enhanced gene transfection performance and biocompatibility of polyethylenimine through pseudopolyrotaxane formation with α-cyclodextrin.

Polyethylenimine (PEI), a commercially available gene transfection reagent, is a promising nonviral vector due to its inherent ability to efficiently condense genetic materials and its successful transfection performance in vitro. However, its low transfection efficiency in vivo, along with its high cytotoxicity, limit any further applications in gene therapy. To enhance the gene transfection performance and reduce the cytotoxicity of linear polyethylenimine, pseudopolyrotaxane PEI25k/CD and the polyrotaxanes PEI25k/CD-PA and PEI25k/CD-PB were prepared and their transfection efficiencies were then evaluated. The pseudopolyrotaxane PEI25k/CD exhibited better transfection efficiency and lower cytotoxicity than the transfection reagent linear PEI25k, even in the presence of serum. It also showed a remarkably higher cell viability, similar DNA protecting capability, and better DNA decondensation and release ability, and could be useful for the development of novel and safe nonviral gene delivery vectors for gene therapy.

pH-Triggered Surface Charge Reversed Nanoparticle with Active Targeting To Enhance the Antitumor Activity of Doxorubicin.

PLGA nanoparticles are widely used in tumor targeting drug delivery systems. However, the naked PLGA nanoparticles (NNPs) not only have low drug loading but also can be rapidly removed from blood circulation by the immune system. The aim of this study was to prepare pH-triggered surface charge reversed lipid hybrid PLGA nanoparticles (LNPs) to enhance drug loading and drug delivery efficiency. CHO-Arg-His-OMe and FA-PEG-DSPE were synthesized to modify PLGA nanoparticles to prepare LNPs. The drug loading and encapsulation rate of LNPs were greatly improved as compared with NNPs. In pH 7.4 medium, doxorubicin (DOX)-loaded LNPs showed negative charge and released DOX slowly. In pH 5.0 medium, DOX-loaded LNPs exhibited positive charge and released DOX quickly. DOX-loaded LNPs delivered more DOX to the nucleus of KB cells and MBA-MD-231/ADR cells than did free DOX. In addition, DOX-loaded LNPs significantly inhibited the proliferation of KB cells and MBA-MD-231/ADR cells. Compared with free DOX, the same dose of the DOX-loaded LNPs delivered more DOX to tumor tissue. Thus, DOX-loaded LNPs significantly inhibited the growth of tumor in tumor-bearing nude mice and obviously reduced the systemic toxicity of DOX. In conclusion, pH-triggered surface charge reversed DOX-loaded LNPs significantly enhanced the antitumor activity of DOX in vitro and in vivo. DOX-loaded LNPs had great potential in tumor targeted chemotherapy.

Cholesterol derived cationic lipids as potential non-viral gene delivery vectors and their serum compatibility.

Cholesterol derivatives M1-M6 as synthetic cationic lipids were designed and the biological evaluation of the cationic liposomes based on them as non-viral gene delivery vectors were described. Plasmid pEGFP-N1, used as model gene, was transferred into 293T cells by cationic liposomes formed with M1-M6 and transfection efficiency and GFP expression were tested. Cationic liposomes prepared with cationic lipids M1-M6 exhibited good transfection activity, and the transfection activity was parallel (M2 and M4) or superior (M1 and M6) to that of DC-Chol derived from the same backbone. Among them, the transfection efficiency of cationic lipid M6 was parallel to that of the commercially available Lipofectamine2000. The optimal formulation of M1 and M6 were found to be at a mol ratio of 1:0.5 for cationic lipid/DOPE, and at a N/P charge mol ratio of 3:1 for liposome/DNA. Under optimized conditions, the efficiency of M1 and M6 is greater than that of all the tested commercial liposomes DC-Chol and Lipofectamine2000, even in the presence of serum. The results indicated that M1 and M6 exhibited low cytotoxicity, good serum compatibility and efficient transfection performance, having the potential of being excellent non-viral vectors for gene delivery.