Exploratory Study Of Stem Cells And Engineered Exosomes In The Treatment Of Diabetes And Malignant Tumors

Background:For the past five years,cell treatment has become more prevalent in medical and healthcare applications.Cell therapy describes medical procedures that use autologous or donor living cells to repair injured tissue,stimulate the immune system,or promote tissue regeneration.Autologous cell therapy includes chimeric antigen receptor T-cell immunotherapy(CAR-T)therapy and allogeneic cell therapy includes mesenchymal stem cell(MSC)-related therapy.The main applications are in the treatment of immune-related diseases,such as autoimmune diseases where immunomodulatory ability is used to improve the immune system’s attack on autologous tissues and inflammatory environment,such as the treatment of diabetes and complications with MSC;and malignant tumors where CAR-T technology is used to activate the immune system to attack tumor cells in order to exert tumor-killing ability.In addition,based on the differentiation ability of stem cells,MSC,induced pluripotent stem cell(i PSC)and hematopoietic stem cell(HSC)are also used in tissue regeneration research.The "cell-free" cell therapy technology,known as exosome technology,has just emerged recently from the cell therapy industry as a result of innovations in the biomedical industry.Exosomes are vesicle-like particles with a lipid bilayer structure which are secreted by cells.They carry a significant amount of biological information,particularly nucleic acids and proteins from the original cell,as well as membrane protein species which are close to the parent cell.Exosomes are supposed to be promising to approach and replace the therapeutic ability of parental cells since they can pass through a variety of physiological barriers and have good biocompatibility,non-immunogenicity,oral accessibility,and ease of preservation compared to cells.In addition to this,exosomes can also be used as drug delivery carriers for in vivo delivery of nucleic acids,peptides,and small molecule drugs.Exosomes’ limited ability to carry drugs,short half-life after systemic administration,sensitivity to hepatic clearance,and ineffective targeting,however,restrict their use in clinical settings.Therefore,it is still necessary for innovations to enhance the drug-carrying and drug-forming capabilities of exosomes.Exosome technology will advance concurrently with the forefront of the(stem)cell therapy industry.Objectives:Thus,this work conducted an exploratory study in three parts:1)To explore the preparation of MSC-derived Exo and its immunomodulatory capacity,the feasibility and key mechanisms of MSC differentiation to islet-like β cells,the feasibility of milk-derived Exo(Milk-Exo)loaded with liraglutide(LRT),MSC-Exo combined with MSC-induced islet-like β cells in the treatment of diabetes.2)To investigate the key factors affecting the pharmacological behavior of cell-derived exosome drugs in vivo,and to improve the plasma half-life of engineered exosome by reducing the binding ability of exosome to recipient cells through engineering knockdown of exosome ITGB1 expression and den-glycoylation modification.3)After extending the plasma half-life of exosome systemic administration,we further explored the feasibility of in vivo targeting enrichment after exosome systemic administration by loading targeted antibodies,and developed a cell-engineered exosome-based "cell-free" cell therapy drug for liver cancer using interleukin 12(IL12)as the pharmacodynamic molecule to mimic CAR-T cell therapy.Part I:Methods: The MSCs were characterized by observing the cell morphology,flow cytometry,and cell ability.UC-MSCs were differentiated into islet β-like cells through the induction method reported in literatures.Insulin expression in islet β-like cells after induction was detected by dithizone staining,q PCR and immunofluorescence.T1 DM mouse model was established by streptozotocin(STZ),and the islet β-like cells were transplanted into the T1 DM model mice to test their ability to regulate blood sugar in vivo.MSC-Exo were obtained by cassette membrane filtration-tangential flow filtration-size exclusion chromatography column purification.Characterization of the purified products obtained by nano-flow,electron microscopy and Western Blot.Detection of immunomodulatory capacity by assaying its ability to inhibit IFNγsecretion by CD3/CD28-activated human peripheral blood lymphocytes(h PBMC).Optimization of milk exosome loading liraglutide efficiency by changing the p H of the system at loading.Loading efficiency was tested by ELISA.Validation of the effect of oral administration of Milk-Exo-LRT by its ability to regulate blood glucose in a mouse model of T2 DM.Results: Experiments such as q PCR and immunofluorescence demonstrated that MSCs have a certain ability to induced differentiation to insulin producing cells,but have limited ability to regulate blood sugar in STZ-induced diabetes mouse models.The MSC-derived exosomes could effectively inhibit CD3/CD28 activation of PBMC IFNγsecretion.Milk-Exo-LRT can reach 31.87±4.75 ng/1×108 particles after optimization of loading conditions,and can reduce blood sugar in db/db mice after sublingual administrationPart II:Methods: Exosomes were prepared by cassette membrane filtration-TFF-gradient density centrifugation.Characterization of the purified products obtained by nano-flow,electron microscopy and Western Blot.HEK293F-Exo’s cell adhesion molecule species and expression abundance was detected by quantitative proteomics.The candidate molecule ITGB1 was knocked out at the cellular level by the CRISPR/Cas9 gene editing system,and the knockout effect at the cellular and exosome levels was verified by flow cytometry and Western Blot.The changes of integrin family molecule expression on exosomes after ITGB1 knockout was detected by quantitative protein profiling and differential analysis.The surface N-glycosylation of exosomes was removed by PNGase F enzyme,and the effect of de-N-glycosylation was verified by Western Blot.The integrity of exosomes after digestion was investigated by nanoflow and electron microscopy.Established an exosome detection method by loading luciferase Nano Luc on exosomes.The binding ability of ITGB1 knockdown combined with deglycosylated exosomes to cells of different tissue origins,including Hep G2,RAW264.7,RAW264.7-LPS,AML12 and Hepa1-6,was detected by cell binding assay.The effects of engineering modification on the plasma half-life and tissue distribution of exosomes in vivo were investigated by detecting the distribution of exosomes in BALB/C mice at different times after tail vein injection.Results: Purified product consistent with exosome profile as demonstrated by nano-flow,electron microscopy and Western Blot assay.Through quantitative proteomic assay and analysis of HEK293 F cell-derived exosomes,the cell adhesion molecule ITGB1 was screened as a candidate molecule.Cells and exosomes do not express ITGB1 after knockdown with the CRISPR/Cas9 gene editing system as detected by flow cytometry or nanoflow and Western Blot.ITGB1 knockout led to a broad suppression of integrins on the exosome surface by quantitative protein profiling assay and differential analysis.Western Blot verified that exosomes achieved de-N-glycosylation to a certain extent.Exosomes remain structurally intact after deglycosylation.Nano Luc was loaded intracellularly into exosomes,and its activity showed a linear relationship with the number of exosomal particles,with a lower limit of quantification at 1×106 particles/ml and a minimum limit of detection at 1×105particles/ml.Knockout of ITGB1 inhibited the binding of exosomes to liver tissue-derived cells,but promoted the binding ability to monocyte-macrophage RAW264.7.On the basis of ITGB1 knockout,deglycosylation promoted the binding of exosomes to liver tissue-derived cells but inhibited the binding of activated RAW264.7.And knocking out ITGB1 alone did not significantly change the plasma half-life and tissue distribution of exosomes.Combined with deglycosylation,the plasma half-life was prolonged from 13 min to 30 min,Cmax and AUC were both increased by an order of magnitude,MRT was increased from 10 min to 14 min,Vd was reduced to 1/3 of the original,and reduce the enrichment in liver.Part III:Methods: On the basis of the ITGB1 knockout cell line constructed in part II,the anti-human GPC3 nano-m Ab HN3 and single-chain antibody GC33 sc Fv were loaded on the surface of exosome membrane by constructing a transmembrane expression module.The loading was detected by nanoflow and Western Blot.The binding of HN3+ITGB1-Exo,GC33 sc Fv+ITGB1-Exo and h GPC3 was detected by nanoflow,cytometry,cell binding and laser confocal.Targeted enrichment of HN3+ITGB1-Deg Exo in tumors was examined by establishing mouse Hepa1-6-h GPC3 subcutaneous transplantation tumor and lung metastasis models.The hsc IL12 or msc IL12 molecular were loaded on exosomes via the transmembrane expression module of ITGB1.The function of hsc IL12+Exo,msc IL12+Exo stimulated cells to secrete IFNγ was verified by h PBMC or mouse splenocyte,respectively.The abundance of msc IL12 in HN3+msc IL12+ITGB1-Exo was detected and estimated by quantitative proteomics.The ability of HN3+msc IL12+ITGB1-Deg Exo to inhibit tumor growth in vivo was verified by mouse Hepa1-6-h GPC3 subcutaneous xenograft tumor model.Results:HN3 and GC33 sc Fv molecular were shown to be loaded on exosomal membranes by nanoflow and Western Blot.The specific binding ability between HN3+ITGB1 Exo or GC33 sc Fv+ITGB1 Exo to h GPC3 were detected by nanoflow,flow cytometry,cell binding,and laser confocal.In C57BL/6 mice bearing subcutaneous or pulmonary tumor of Hepa1-6-h GPC3 cells,the HN3+msc IL12+ITGB1-Deg Exo were significant enrichment in the tumor compared to control groups.In a mouse total splenocyte culture,the msc IL12-loaded Exo successfully elicited a significant amount of IFNγ secretion with an EC50 of 5.3×106 particles/m L.The hsc IL12-loaded Exo successfully elicited a significant amount of IFNγ secretion in CD3/CD28 antibody stimulate human PBMCs.Quantitative proteomic analysis indicated that the amount of msc IL12 was approximately 40 ng per 4.7 ×1010 particles.HN3+msc IL12+ITGB1-Deg Exo can suppressed the tumor growth in the murine model of subcutaneous tumor of Hepa1-6-h GPC3 cells without visible toxicity.Conclusion:In summary,in this research we did the following aspects:1)Preliminary demonstration of the feasibility of MSC differentiation into islet-like β-cells.MSC-derived exosomes with strong inflammatory inhibitory activity were obtained.However,due to the low efficiency of MSC differentiation into islet-likeβ cells,they cannot be directly applied to diabetes treatment at present.Meanwhile,a breakthrough has been made in the technology of oral GLP1 formulation with milk-derived exosomes as the carrier,and the initial oral delivery of GLP1 peptide drug has been achieved.The therapeutic effect of this drug in T1 DM will be evaluated in the future.2)The tissue distribution and pharmacokinetic behavior of cellular exosomes in vivo are influenced by cell adhesion-like molecules on their surface and surface glycosylation.By knocking down integrin family molecules on the surface of exosomes and specifically removing N-glycosylated molecules,the non-specific adhesion of cellular exosomes in vivo can be significantly reduced,and the in vivo circulating half-life can be prolonged to improve the drug-forming properties.3)Based on the improved druggability,the simultaneous loading of GPC3 targeting antibody,a marker of hepatocellular carcinoma,and IL12,an immune activating molecule,can achieve the effect of simulated immune cell therapy.This multi-module loading of exosomal drugs extends the therapeutic window of IL12 itself and provides a new idea and technical basis for the development of engineered exosomal drugs,i.e."cell-free" cell therapy technology.