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Optimization of Statin-Loaded Delivery Nanoparticles for Treating Chronic Liver Diseases by Targeting Liver Sinusoidal Endothelial Cells.

ABSTRACT: In this study, we developed functionalized polymeric micelles (FPMs) loaded with simvastatin (FPM-Sim) as a drug delivery system to target liver sinusoidal endothelial cells (LSECs) for preserving liver function in chronic liver disease (CLD). Polymeric micelles (PMs) were functionalized by coupling peptide ligands of LSEC membrane receptors CD32b, CD36 and ITGB3. Functionalization was confirmed via spectroscopy and electron microscopy. In vitro and in vivo FPM-Sim internalization was assessed by means of flow cytometry in LSECs, hepatocytes, Kupffer and hepatic stellate cells from healthy rats. Maximum tolerated dose assays were performed in healthy mice and efficacy studies of FPM-Sim were carried out in bile duct ligation (BDL) and thioacetamide (TAA) induction rat models of cirrhosis. Functionalization with the three peptide ligands resulted in stable formulations with a greater degree of in vivo internalization in LSECs than non-functionalized PMs. Administration of FPM-Sim in BDL rats reduced toxicity relative to free simvastatin, albeit with a moderate portal-pressure-lowering effect. In a less severe model of TAA-induced cirrhosis, treatment with FPM-CD32b-Sim nanoparticles for two weeks significantly decreased portal pressure, which was associated with a reduction in liver fibrosis, lower collagen expression as well as the stimulation of nitric oxide synthesis. In conclusion, CD32b-FPM stands out as a good nanotransporter for drug delivery, targeting LSECs, key inducers of liver injury.

SUBMITTER: Gil M

PROVIDER: S-EPMC11340786 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Optimization of Statin-Loaded Delivery Nanoparticles for Treating Chronic Liver Diseases by Targeting Liver Sinusoidal Endothelial Cells.

Gil Mar M Khouri Lareen L Raurell Imma I Rafael Diana D Andrade Fernanda F Abasolo Ibane I Schwartz Simo S Martínez-Gómez María M Salcedo María Teresa MT Pericàs Juan Manuel JM Hide Diana D Wei Mingxing M Metanis Norman N Genescà Joan J Martell María M

Pharmaceutics 20231014 10

In this study, we developed functionalized polymeric micelles (FPMs) loaded with simvastatin (FPM-Sim) as a drug delivery system to target liver sinusoidal endothelial cells (LSECs) for preserving liver function in chronic liver disease (CLD). Polymeric micelles (PMs) were functionalized by coupling peptide ligands of LSEC membrane receptors CD32b, CD36 and ITGB3. Functionalization was confirmed via spectroscopy and electron microscopy. In vitro and in vivo FPM-Sim internalization was assessed b ...[more]

PMID: 37896223

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Project description:Phenotypic transformation of liver sinusoidal endothelial cells is one of the most important stages of liver metastasis progression. The miRNA effects on liver sinusoidal endothelial cells during liver metastasis have not yet been analysed. Herein, analysis of miRNA expression in these cells during colorectal liver metastasis revealed repressed expression of microRNA-20a. Importantly, downregulation of miR-20a occurs in parallel with upregulation of its known protein targets. To restore normal miR-20a levels in liver sinusoidal endothelial cells, we developed chondroitin sulfate-sorbitan ester nanoparticles conjugated with miR-20a in a delivery system that specifically targets liver sinusoidal endothelial cells. The restoration of normal mir-20a levels in these cells induced downregulation of the expression of its protein targets, and this also resulted in a reduction of in vitro LSEC migration and a reduction of in vivo activation and tumor-infiltrating capacity and ability of the tumor decreased by approximately 80% in a murine liver metastasis model. Animals: Balb/c mice (6- to 8-week-old males) were obtained from Charles River Laboratories Spain SA (Barcelona, Spain). All the procedures were approved by the Ethical Committee for Animal Experimentation (CEEA) of the University of the Basque Country (EHU/UPV) in accordance with institutional, national and international guidelines regarding the protection and care of animal use for scientific purposes. Mice were kept in the animal facility of EHU/UPV and had access to standard chow and water ad libitum. Colorectal cancer cells: Murine colorectal cancer C26 cells (ATCC, Manassas, VA) syngeneic with Balb/c mice were used. The cells were grown under standard conditions in RPMI medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 microg/ml) and amphotericin B (0.25 microg/ml), all purchased from Life technologies, Carlsbad, CA. Control and tumor-activated LSECs culture: Control and tumour activated primary cultures of LSECs were isolated form livers with colorectal cancer metastasis or from healthy ones. To do so, Balb/c mice were anesthetized with isofluorano and small cut done in their left broadside. Then, 50 microl for each mice of C26 colon carcinoma cells were inoculated in the spleen at 2 x106 cells/ml concentration and the peritoneum and skin were sutured. The control mice were inoculated with PBS 1x buffer. Fourteen days later, all mice were sacrificed and the tumour activated LSECs and control LSECs were isolated by differential centrifugation. To do so, mice were perfused with collagenase P from Clostridium histolyticum (Sigma-Aldrich, St.Louis, MO, USA) through cava vein. The obtained cell suspension was twice centrifuged resulting in a parenchymal (PC)-enriched pellet and a non PC-enriched supernatant. Then, the non PC-enriched supernatant was layered on Percoll gradients (25% on top of 50%) to obtain LSEC. After a centrifugation the interphase between the two density cushions was recollected with purified non-PC enriched LSECs and Kupffer cells (KC). Then, the KCs were separated by adherence and isolated LSECs were washed and cultured with RPMI 1640 medium (Sigma-Aldrich, St.Louis, MO, USA) supplemented with 10% FBS and used in different experiments 24 hours after the isolation. (Life technologies, Carlsbad, CA). miRNA microarray analysis : The purified total RNA was analysed using Agilent mouse miRNA microarrays. Release 18.0, 8x60K (v18) microarray slides (Agilent Technologies, USA), with 1,200 mouse miRNAs represented, were used. Briefly, 100 ng of total RNA was labelled and hybridized following the standard Agilent Protocol for the miRNA Microarray System with a miRNA Complete Labelling and Hybridization Kit, including Agilent miRNA Spike-ins, and the results were scanned using an Agilent Microarray Scanner G2565CA (Agilent Technologies, USA). The scanned TIFF image files were processed using Agilent Feature Extraction Software vs 10.7.3.1 to extract raw data and obtain QC reports. The microarray raw data were normalized using the quantile method. To find the statistically significant differentially expressed miRNAs (DEMs) between two groups of samples, we calculated the mean of each probe across all the samples of the group. Next, we filtered out all the probes whose absolute value of the difference of mean values between the two groups was less than a selection threshold thetaDEM = 4 (that corresponds to a fold change of 2 in log2 scale), and we applied a Student's t-test with a significance threshold alphaDEM = 0.05. The PCA and the hierarchical clustering of genes and samples were performed with one minus the correlation metric and the unweighted average distance (UPGMA) linkage method. All the analysis algorithms and graphics were implemented functions in Matlab.

Project description:Diabetes mellitus (DM) is the most prevalent cause of diabetic retinopathy (DRP). DRP has been recognized for a long time as a microvascular disease. Many drugs were used to treat DRP, including vildagliptin (VLD). In addition to its hypoglycemic effect, VLD minimizes ocular inflammation and improves retinal blood flow for individuals with type 2 diabetes mellitus. Nevertheless, VLD can cause upper respiratory tract infections, diarrhea, nausea, hypoglycemia, and poor tolerability when taken orally regularly due to its high water solubility and permeability. Effective ocular administration of VLD is achieved using solid lipid nanoparticles (SLNPs), which improve corneal absorption, prolonged retention, and extended drug release. Ocuserts (OCUs) are sterile, long-acting ocular dosage forms that diminish the need for frequent dosing while improving residence time and stability. Therefore, this study intends to develop VLD solid lipid nanoparticle OCUs (VLD-SLNPs-OCUs) to circumvent the issues commonly associated with VLD. SLNPs were prepared using the double-emulsion/melt dispersion technique. The optimal formula has been implemented in OCUs. Optimization and development of VLD-SLNPs-OCUs were performed using a Box-Behnken Design (BBD). VLD-SLNPs-OCUs loading efficiency was 95.28 ± 2.87%, and differential scanning calorimetry data (DSC) showed the full transformation of VLD to an amorphous state and the excellent distribution in the prepared OCUs matrices. The in vivo release of VLD from the optimized OCUs after 24 h was 35.12 ± 2.47%, consistent with in vitro drug release data of 36.89 ± 3.11. The optimized OCUs are safe to use in the eye, as shown by the ocular irritation test. VLD-SLNPs-OCUs provide extended VLD release, an advantageous alternative to conventional oral dose forms, resulting in fewer systemic adverse effects and less variation in plasma drug levels. VLD-SLNPs-OCUs might benefit retinal microvascular blood flow beyond blood glucose control and may be considered a promising approach to treating diabetic retinopathy.

Project description:BackgroundHepatocellular carcinoma (HCC) developed in fibrotic liver does not respond well to immunotherapy, mainly due to the stromal microenvironment and the fibrosis-related immunosuppressive factors. The characteristic of liver sinusoidal endothelial cells (LSECs) in contributing to fibrosis and orchestrating immune response is responsible for the refractory to targeted therapy or immunotherapy of HCC. We aim to seek a new strategy for HCC treatment based on an old drug simvastatin which shows protecting effect on LSEC.MethodThe features of LSECs in mouse fibrotic HCC model and human HCC patients were identified by immunofluorescence and scanning electron microscopy. The effect of simvastatin on LSECs and hepatic stellate cells (HSCs) was examined by immunoblotting, quantitative RT-PCR and RNA-seq. LSEC-targeted delivery of simvastatin was designed using nanotechnology. The anti-HCC effect and toxicity of the nano-drug was evaluated in both intra-hepatic and hemi-splenic inoculated mouse fibrotic HCC model.ResultsLSEC capillarization is associated with fibrotic HCC progression and poor survival in both murine HCC model and HCC patients. We further found simvastatin restores the quiescence of activated hepatic stellate cells (aHSCs) via stimulation of KLF2-NO signaling in LSECs, and up-regulates the expression of CXCL16 in LSECs. In intrahepatic inoculated fibrotic HCC mouse model, LSEC-targeted nano-delivery of simvastatin not only alleviates LSEC capillarization to regress the stromal microenvironment, but also recruits natural killer T (NKT) cells through CXCL16 to suppress tumor progression. Together with anti-programmed death-1-ligand-1 (anti-PD-L1) antibody, targeted-delivery of simvastatin achieves an improved therapeutic effect in hemi-splenic inoculated advanced-stage HCC model.ConclusionsThese findings reveal an immune-based therapeutic mechanism of simvastatin for remodeling immunosuppressive tumor microenvironment, therefore providing a novel strategy in treating HCC.

Dataset Information

Optimization of Statin-Loaded Delivery Nanoparticles for Treating Chronic Liver Diseases by Targeting Liver Sinusoidal Endothelial Cells.

Publications

Optimization of Statin-Loaded Delivery Nanoparticles for Treating Chronic Liver Diseases by Targeting Liver Sinusoidal Endothelial Cells.

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