| The glioma is one of the most lethal brain tumors in the world,which is called the"disaster star" of life.The glioma is characterized by the poor prognosis,high mortality and less than 5%five-year survival rate.Due to the complex structure of the nervous system,invasive growth of gliomas and unclear boundary of the tumor,the complete surgical removal of gliomas is almost impossible,making it extremely easy to relapse.Chemotherapy plays a crucial part in the treatments of gliomas.However,the blood brain barrier(BBB)prevents more than 98%of small molecule drugs and almost 100%of macromolecular drugs from reaching the brain tumor tissue,severely hindering the therapeutics of glioma.Therefore,overcoming the BBB and promoting the enrichment of drugs in tumor tissues is a prerequisite for the treatment of glioma.Exosomes are endogenous nanoscale vesicles derived from cells or body fluids.exosomes as new endogenous nanoscale drug delivery systems have a widespread use in the treatment of neurological diseases such as glioma and Parkinson due to their low immunogenicity,excellent biocompatibility and can across the physiological and pathological barriers(especially BBB).Moreover,although some exosomes possess an intrinsic ability to cross biological barriers,the delivery of therapeutic drugs to entire tumors at concentrations that permit effective treatments,remain largely undefined.In order to improve the targeting delivery of exosomes and enrichment of drugs in tumors,studies have focused on chemical modifications and genetic engineering,but these studies are time-consuming,labor-intensive,and off-target cannot be avoided.From the physical factors of opening BBB,Focused ultrasound(FUS)has been approved by the FDA for the diagnosis and treatment of brain disease due to its non-invasive nature,reversibility,and instantaneous local-opening of the BBB.There are limitation of treatment of gliomas in current situation and opportunities and challenges when exosomes are used as drug delivery systems for treatment of brain diseases.Based on that,the thesis develops a natural and safe drug delivery system(DDS)which can combine FUS and then prompt exosomes target to deliver doxorubicin to gliomas.The study compares and analyzes the advantages of macrophage-derived exosomes(R-Exos)and blood-derived exosomes(B-Exos)in this system,aiming to screen a better and larger clinical transformation platform.In vitro,both R-Exos and B-Exos can cross the BBB and accumulate in gliomas with the aid of ultrasound.There are no significant differences in physical features,drug release,specific glioma targeting and cell toxicity.Vivo imaging results show that FUS can promote the enrichment of exosomes(B-Exos)in the tumor site and inhibit tumor growth significantly and without obvious side effects.Therefore,Exos combined with FUS is expected to be a promising method for the treatment of brain tumor diseases.In this paper,the study is conducted mainly from the the extraction and representation of exosomes,the drug release promoted by ultrasound,the cytotoxic effect in vitro,exosomes across the BBB and the antitumor effects in vivo and so on which are summarized from the following aspects:Part 1 Extraction and characterization of R-Exos and B-ExosWe obtained exosomes derived from macrophages and blood and used them as drug carriers for the chemotherapeutic drug doxorubicin(Dox).The high drug-loading Exos-Dox was prepared and characterized by optimizing the drug loading method.The size and potential of exosomes were tested by NTA.The morphology of exosomes was observed by TEM.The marker proteins of exosomes were confirmed by Western Blot.The results indicated that R-Exos and B-Exos were prepared with good particle size,zeta potential and stability.The marker proteins of exosomes expressions were consistent with relevant reports,suggesting exosomes could be used as an excellent drug carrier for following research.Part2 Drug loading and FUS boosted drug release from Exos.The efficiency of exosome-loaded doxorubicin was determined according to a standardcurve by establishing a standard curve of doxorubicin concentration and fluorescence value.Vitro drug release study showed that the rate of drug release was low at 4? without shaking,indicating the good drug carrier potential of exosomes.At 37?,simulating an in vivo environment,the rate of drug release was relatively slow.But with the help of FUS and with the increase of the intensity of FUS,the rate of drug accelerated.The drug release of R-Exos-Dox is similar to B-Exos-Dox.When the drugs reach the lesion site and accumulate to high levels,the applied FUS promotes a burst of drug-release,leading to more efficient tumor killing.Part 3 Cellular uptake and toxicity of exosomes combined with ultrasoundThe cellular uptake of R-Exos-Dox and B-Exos-Dox by GL261 cells was detected by laser confocal and flow cytometry,and the cell viability was determined by MTT assay.The results showed that the cell uptake of R-Exos-Dox and B-Exos-Dox in a time-dependent manner.Uptake tendencies of exosomes vary from cell to cell and the uptake efficiency of tumor cells is higher than that of normal cells under identical conditions.Moreover,US treatment enhances drug uptake efficiency.Both R-Exos and B-Exos had no effect on survival rate of cells.The cytotoxicity of Dox,R-Exos-Dox and B-Exos-Dox were concentration-dependent,and the combination with US can greatly reduce cell survival efficiency.Part 4 FUS facilitated exosomal drug delivery in orthotopic glioma.A cell-based in vitro trans-well model of the BBB was established to investigate whether ultrasound promoted exosomes to across the BBB.The results showed that the exosomes could cross the BBB,and the combination with ultrasound greatly promoted the efficiency of exosomes crossing the BBB.To evaluate the capacity of exosomes to cross intact BBB,normal mice models with intact BBB were used.The results clearly demonstrated that exosomes can transfer drugs and then cross the BBB into the brain and FUS further greatly promoted this process.By establishing an orthotopic glioma model to explore that ultrasound promote exosomes to enrich at the tumor site.The results showed that ultrasound greatly promoted drug accumulation in the tumor site,and the amount of enrichment reached the maximum after the 12-hour injection of exosomes.Therefore,12h after injection of exosomes will be used as the time point when the second ultrasound promotes drug release.Part 5 FUS enhanced exosomal chemotherapy in orthotopic gliomasOrthotopic gliomas models were established,the effect of tumor inhibition was evaluated by using Xenogen IVIS Lumina ? system to monitor the change of tumor size.The safety of treatment model can be discussed from different aspects such as mice's body weight,mice's survival period,and change of mice's organs.The results showed that on the 15th day after treatment,B-Exos-Dox with twice FUS treatment resulted in the most obvious effect of tumor inhibition,an extended survival time than other treatment groups,and the insignificant change of mice's body weight.Moreover,H&E staining indicates that exosome-encapsulated Dox greatly reduced cardiotoxicity,indicating that our platform is relatively safe.ConclusionIn this study,we designed a new strategy of exosome-loaded doxorubicin combined with FUS in the treatment of glioma,compared and analyzed the production and therapeutic effect of exosomes derived from different sources that are loaded with Dox.It was found that both R-Exos and B-Exos could effectively deliver Dox into the glioma tumors with the help of FUS,while with no significant difference in physical features,ultrasound-responsiveness,and specific glioma targeting.Due to the abundant source,high-yield,and easy access of blood serum derived exosomes,it would have more clinical transformation potentials compared to R-Exos.Therefore,the platform using a combination of FUS and natural B-Exos provides a valuable future strategy for glioma and other brain diseases. |
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