| Glioblastoma,the most invasive and primary central nervous system(CNS)tumor,is notorious for its high morbidity and mortality with a high tendency of recurrence.In the treatment of gliomas,blood brain barrier(BBB)has proved to be a serious one of the key factors that restrict therapeutic effects due to BBB maintains brain homeostasis by shielding the brain from potential neurotoxins and impedes the delivery of chemotherapeutics simultaneously,so the prognosis is far below satisfactory.Therefore,how to improve drugs to cross the BBB,achieving targeting and integration has become an urgent problem to be solved.In recent years,a large number of studies have shown that ultrasound-targeted microbubble destruction(UTMD)can reversibly and instantaneously open BBB with a high degree of spatial and temporal specificity.And now,it has become the unique method that can open the BBB safely and reversibly which alleviates the bottleneck on therapeutic interventions for brain drug delivery.Sonodynamic Therapy(SDT)relies on the deep penetration of ultrasound and the preferential tumor accumulation of sonosensitizer,which has been explored for various tumor treatment with effectiveness,low-cost and high-safety.Many studies have shown that ultrasound can pass through the skull for deep imaging and disease diagnosis,so the application of SDT in the treatment of glioma has a splendid advantage.So how to efficiently transport the sonosensitizer to the tumor area is the most important part in the sonodynamic glioma therapy.Nano-Drug delivery system(Nano-DDS)is a research hotspot of novel drug delivery systems.It has good targeting and safety,and can improve the bioavailability of drugs while reducing side effects.In many nanocarriers,such as liposomes,polymer micelles,dendrimers,and other inorganic nanoparticles etc.,they all can be used as carriers to carry anticancer drugs or small molecules into the tumor target,in order to significantly improve the disease therapeutic effect and reduce the toxicity of the drug.In addition,we found that SDT initiates autophagic responses during the induction of tumor cell apoptosis.Autophagy is an intracellular homeostatic mechanism,in which cellular components including damaged organelles and some dysfunctional proteins or other macromolecules can be degraded in the lysosome for the recycle of energy.In other therapeutic modalities,such as radiation and chemotherapy,the outcomes of autophagy response differ among different models,different triggers,and different stimuli intensities or mechanisms.Autophagy enables to protect cancer cells from extraneous stresses,allows cells to survive particularly during the pathogenesis of cancer,so as to compromises the therapeutic outcome.Therefore,autophagy inhibitors can be as potential anti-tumor drugs,cutting off the self-protection pathway of tumors in various treatments,providing a novel modality to enhance the anti-glioma efficiency.Based on the above research background,a smart "all-in-one" nanosensitizer platform by incorporating Ce6 and HCQ into angiopep-2 peptide-modified liposomes was evaluated as potential DDS for glioma theranostics.We utilized UTMD to reversibly open BBB for the delivery of liposomes to assess glioma targeting capabilities of peptide modified liposomes.At the optimal time-window,the sequential ultrasound stimulus was employed to generate SDT effects.In this study,we mainly investigated the synthesis and targeted modification of co-loaded liposomes,and the killing effect of SDT on glioma in both cell-level and animal experiments in vivo.The purpose is to improve the anti-tumor efficacy of SDT and provide a valuable theoretical and experimental basis,our study will be discussed from the following three aspects:Part 1 The preparation and characterization of liposomesThe standard preparation of PEGylated liposomes was studied as a drug loading platform for sonosensitizer Ce6 and autophagy inhibitor HCQ.The lipophilic Ce6 was located into the phospholipid bilayer of the liposome,and the water-soluble HCQ was encapsulated in the hydrophilic core of liposomes.The chlorin e6 liposome(CL),the targeting peptide Angiopep-2 modified chlorin e6 liposome(ACL),the chlorin e6 and the hydroxychloroquine co-loaded liposomes(CHL),and the targeting peptide Angiopep-2 modified chlorin e6 and hydroxychloroquine co-loaded liposomes(ACHL)were prepared by membrane hydration method.The morphology of the prepared liposomes was first observed by transmission electron microscopy;The particle size distribution,dispersion coefficient of different liposomes and the stability of serum were determined by dynamic light scattering particle size analyzer.UV spectrophotometry was established to determine the entrapment efficiency of HCQ and Ce6 and the linear range was investigated.And the in vitro drug release under ultrasound conditions was evaluated.The main conclusions are as follows:?The prepared liposome has uniform particle size,regular morphology and stable properties,which lays a good foundation for subsequent experiments.?The encapsulation efficiency of Ce6 in CL is 82.46±4.26%,Ce6 in ACL The encapsulation efficiency was 77.68 ± 3.15%.The entrapment efficiency of Ce6 and HCQ in CHL co-loaded liposomes were 66.74±3.58%and 70.42±2.88%.The encapsulation efficiency of Ce6 and HCQ in ACHL co-loaded liposomes were 54.16±2.19%and 63.21±3.01%.?In the 0?60 min,the release rate of Ce6 or HCQ in the ACHL under ultrasonic treatment was greatly increased and showed a burst release tendency,and the drug accumulation and release amount reached 70%at 60 min.In contrast,the drug was slowly released in the control group,and there was no significant change in drug release within 6 h..Part 2 The anti-tumor effect of ultrasound combined with ACHL liposome on glioma in vitroWe selected the mouse glioma cell line GL261,mouse brain endothelial microvascular cell line bEND.3 and mouse embryonic fibroblast cell line NIH3T3 with different LRP expression levels to evaluate the in vitro targeting of ACL.The enrichment of free Ce6,CL and ACL in GL261 cells were analyzed.The distribution of Ce6 was also observed by laser confocal microscopy.The MTT method and Calcein probe were used to investigate the cytotoxic effects of CL,ACL,CHL and ACHL combined with ultrasonic irradiation.Fluorescence microscopy,flow cytometry combined with DCFH-DA study the generation of reactive oxygen species in GL261 cells under different treatment modes;Confocal microscopy and flow cytometry were utilized identify mitophagy in glioma cells.Western blot,TEM,and immunocytochemistry were used to detect mitophagy in the presence of HCQ.The main conclusions arc as follows:?The expression of low-density lipoprotein receptor on the cell surface significantly affects the uptake of liposomes by cells,and the number of LRP receptors on glioma cells and brain endothelial microvascular cells is notably higher than that of normal cells.?The sonosensitizer Cc6 is localized in the mitochondria and Lysosom,the cells are time-dependent on the uptake of Ce6.?Drug-loaded liposomes combined with ultrasound irradiation show a strong synergistic killing effect,and the target peptide-modified liposome has a higher cell killing effect than normal liposomes.?Glioma cells can produce ROS after SDT treatment,and the presence of HCQ significantly increases the intracellular reactive oxygen species level for which enhances the endogenous ROS from the damaged mitochondria,resulting in regenerative feedback of oxidative damage to improve the ROS production.After SDT treatment,laser confocal microscopy showed that autophagosomes and mitochondria co-localization produced by glioma cells(mitophagy)and flow cytometry showed that the number of mitochondria in the treated group was significantly reduced.?The presence of HCQ prevented the degradation of autophagosomes thus caused a large accumulation of mitochondria autophagosomes.Autophagy inhibition enhances autophagosome production and produced a positive feedback loop for ROS,further promoting glioma cell death or apoptosis.Part 3 The anti-tumor effect of ultrasound combined with ACHL liposome on glioma in vivoThe BBB model in vivo and in vitro were established to detect the opening of the blood-brain barrier under UTMD.Then the living imager was utilized to assess the drug enrichment tendency in the tumor and major organs.To investigate the inhibitory effect of ACHL combined with ultrasound and UTMD on the growth of orthotopic glioma in GL261 mice,we evaluated the glioma size,weight change in mice,mouse survival monitoring,HE observation of major organs,the morphological examination,immunohistochemical analysis and TEM observation in vivo respectively.The experimental results showed that ultrasound combined with microbubbles can be reversibly open BBB,and after sonication for 2 h BBB open extremely in the in vivo model.Under UTMD,mice injected ACL liposomes will reach the maximum amount at 36 h.ACHL combined with ultrasound irradiation significantly inhibited the growth of brain tumors,improved the quality of life,prolonged the survival time of mice and even produced more autophagy and apoptosis.At the same time,under the selected drug dose and ultrasound parameters,different treatments had no significant side effects on the main organs of the mice,and the safety of treatment was good.ConclusionsIn summary,this project successfully designed and developed a liposome that co-loads a sonosensitizer and an autophagy inhibitor for sonodynamic glioma therapy.The therapy can significantly inhibit the growth of glioma cells in both in vitro and in vivo experiments,resulting in a strong SDT killing effects.Therefore,the potential therapeutic effects of this platform will facilitate its future clinical applications. |
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