Nov.2024 27
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“All in one” lipid-polymer nanodelivery system for gene therapy of ischemic diseases

Introduction
This study develops lipid-polymer hybrid nanocarriers (FLNPs) to enhance gene delivery for ischemic diseases. FLNPs improve gene transfection, avoid lysosomes, and target the nucleus. Loaded with Hepatocyte Growth Factor (pHGF) and Catalase (pCAT) plasmids, FLNPs boosted cell resistance to oxidative stress and improved recovery in a mouse ischemia model, without causing metabolic disturbances. FLNPs show promise for effective gene therapy in ischemia.
Details


 1. Introduction
This study focuses on improving gene therapy for lower limb ischemia, a condition caused by reduced blood flow that leads to pain, ulcers, and gangrene. The researchers developed a hybrid gene delivery system combining lipid-polymer nanoparticles (FLNPs) to enhance gene transfection efficiency. This system co-encapsulates two therapeutic genes, Hepatocyte Growth Factor (pHGF) and Catalase (pCAT), to promote angiogenesis and reduce oxidative stress. The FLNPs are designed to fuse with cell membranes, deliver genes directly into cells, and target the nucleus, avoiding lysosomal degradation. In vivo, the FLNPs improved gene expression, promoted blood vessel growth, and aided recovery of motor function in mice with ischemic lower limbs. The approach holds promise for more effective gene therapy in ischemic diseases.

 3. Results and disscusion
 3.1. Preparation and characterization of lipid-polymer nanodelivery system
This study developed a hybrid lipid-polymer nanoparticle (FLNPs) system for efficient gene delivery. The nanoparticles were designed with a double-layer structure: a polymer core encapsulating therapeutic genes (pHGF and pCAT) and a cationic liposome membrane for enhanced cellular uptake. The surface of the nanoparticles was further modified with anti-LMNB1 antibodies for nuclear targeting. Additionally, the envelope of Japanese hemagglutinin virus (HVJ-E) was adsorbed onto the liposomes, enabling membrane fusion and avoiding lysosomal damage during gene delivery. The FLNPs showed good stability, effective gene encapsulation, and sustained release. The system demonstrated enhanced gene transfection and is expected to improve the delivery of therapeutic genes for ischemic disease treatment.

 3.2. Detection of drug delivery, gene transfection and cell compatibility of lipid-polymer nanodelivery system
This study evaluated the drug delivery capabilities of FLNPs using Coumarin 6 as a model drug. The results showed that FLNPs enhanced cellular uptake of Coumarin in C2C12 cells, even under oxidative stress (H₂O₂ exposure), compared to other formulations. Additionally, FLNPs demonstrated superior lysosomal escape ability, with most nanoparticles staying on the cell membrane, suggesting membrane fusion facilitated intracellular drug delivery after HVJ-E adsorption.
When co-incubated with Coumarin, FLNPs also exhibited significant nuclear accumulation of the drug, confirming their ability to target the nucleus. The gene transfection efficiency of FLNPs was significantly higher than that of other groups, with GFP expression 14.18 times higher than the GFP group and 4.12 times higher than GFP-NPs.
Furthermore, when loaded with Hepatocyte Growth Factor (pHGF) and Catalase (pCAT), HGF/CAT-FLNPs did not affect C2C12 cell proliferation under normal conditions but significantly promoted cell proliferation under oxidative stress (H₂O₂-induced damage). This suggests that FLNPs can enhance cell resistance to oxidative damage, offering potential for improved therapeutic gene delivery in ischemic conditions.







 3.3. In vivo effects of lipid-polymer nanodelivery system

This study assessed the metabolic effects of FLNPs, which were prepared using the Sendai virus envelope for gene delivery in a hindlimb ischemia model. Metabolomics was used to analyze gene and metabolite expression in the gastrocnemius muscle after treatment with Hepatocyte Growth Factor (HGF) and HGF-FLNPs.

Principal Component Analysis (PCA) revealed metabolic differences between the HGF and HGF-FLNPs groups, with FLNPs causing changes in lipid metabolism, likely due to the liposomes on the surface of the FLNPs. Further analysis using KEGG pathway annotation identified differential metabolites between the groups, with notable changes in lipid metabolism pathways. Hierarchical clustering and volcano plots visualized the differences in metabolite content, showing that 65 genes were up-regulated and 105 down-regulated in the HGF-FLNPs group.

The top 10 metabolites with the largest changes were displayed in a radar chart, including Anabasine, chondroitin D-glucuronate, and 17 alpha,21-dihydroxypregnenolone. KEGG pathway enrichment analysis highlighted significant changes in the regulation of metabolic pathways, particularly the down-regulation of the lipoic acid metabolic pathway and the up-regulation of the longevity-regulating pathway. The findings suggest that HGF-FLNPs may modulate cellular metabolism and promote longevity-related signaling, likely through the up-regulation of oleic acid.

 
3.4. HGF/CAT-FLNPs significantly improved gangrene, motor function and blood perfusion of ischemic hindlimbs

This study evaluated the effectiveness of FLNPs (lipid-polymer hybrid nanoparticles) for gene delivery in treating hindlimb ischemia in mice. The HGF/CAT-FLNPs, containing pHGF (pro-angiogenesis) and pCAT (hydrogen peroxide regulation), significantly improved ischemic hindlimb gangrene, tissue repair, and motor function. Compared to other groups, FLNPs enhanced blood perfusion recovery and inhibited muscle atrophy and collagen fiber production. Blood flow in the ischemic leg reached 86.38% of normal levels in the HGF/CAT-FLNPs group, demonstrating superior therapeutic efficacy. FLNPs effectively delivered genes via membrane fusion, lysosome avoidance, and nuclear targeting, offering a promising approach for ischemia treatment.


Original article URL link:
“All in one” lipid-polymer nanodelivery system for gene therapy of ischemic diseases - ScienceDirect

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