Construction Of Stem Cell-based Novel Multi-functional Targeting Vehicle And Its Application In Ischemic Stroke Therapy

Objective:Based on the idea of Targeting Drug Delivery System (TDDS), the present study aimed at constructing a novel multi-functional gene delivery vehicle for targeting therapy of brain diseases. Starting from screening of targeting peptide for ischemic lesion, mesenchymal stem cells (MSCs) were modified with this targeting peptide in order to enhance homing capability. Meanwhile, non-viral vector that had high transfection efficiency with low cytotoxicity was used to delivery therapeutic RNAi/gene into MSCs. This combined method could make MSCs as a promising targeting therapeutic agent with its targeting delivery ability of RNAi/genes, as well as its own therapeutic effect. The study evaluated potential of MSCs as a targeting vehicle for brain diseases and prospect of MSC-based targeting therapy for ischemic stroke.Methods:1. Synthesis of palmitated (PA)-CLEVSRKNC, modification of PA-peptide onto cell membrane via lipid raft and screening of optimum modification condition.2. Characterization of PA-peptide modified MSCs (Pep-MSCs) through flow cytometry and fluorescence images, evaluation of cytotoxicity of PA-peptide by MTT assay, evaluation of influence of PA-peptide on the differentiation of MSCs into neural cells, thus giving a clear clue of modification efficiency and bio-safety of PA-peptide modification.3. Establishment of oxygen-glucose deprivation (OGD) model using Neuro-2A and astrocytes to assess migration ability of Pep-MSCs and protection effect of MSCs for injured neural cells.4. Establishment of middle cerebral artery occlusion (MCAO) rat model, assessment of the targeting ability of Pep-MSCs for ischemic lesion and distribution of Pep-MSCs in MCAO rat using in vivo imaging system and frozen section analysis.5. Non-viral vector spermine-pullulan (SP) which was synthesized in our lab was used for MSCs transfection, transfection efficiency was evaluated.6. Evaluation of therapeutic benefits of MSCs with combination of miR-133b transfection and PA-peptide modification (Pep-133b-MSCs), neurological severity score (NSS) and histologic sections were evaluated.Results:The multi-functional stem cell-based targeting vehicle was successfully constructed (Pep-MSCs). It was evidenced that PA-peptide successfully coat onto surface of MSCs with high modification efficiency and low cytotoxicity. In the OGD model, Pep-MSCs showed enhanced migration compared to naive MSCs. More importantly, PA-peptide could enhance homing of MSCs to ischemic lesion, as well as reducing accumulation of MSCs in other tissues, after transplantation of Pep-MSCs into MCAO rat. Furthermore, non-viral vector SP could delivery miR-133b into MSCs while miR-133b transfected MSCs showed better protection effect for injured neural cells. With combination of miR-133b transfection and PA-peptide modification, Pep-133b-MSCs exhibited better therapeutic effect with better neural protection benefits in MCAO rats. Either the miR-133b transfection or PA-peptide modification influenced neural differentiation of MSCs in MCAO rats.Conclusion:Pep-MSCs, successfully constructed in the present study, showed enhance targeting ability for brain ischemic lesion. Meanwhile, Pep-MSCs that could deliver miR-133b to target tissue and exhibited own therapeutic effects of MSCs served as a multi-functional targeting vehicle, and showed potential in stem cell-based therapy for ischemic stroke.