| Glioma is one of the most common intracranial tumors,and is characterized by high malignancy and recurrence rate.At present,glioma is mainly treated by surgery,radiotherapy,chemotherapy and immunotherapy,as well as the combination of chemotherapy and immunotherapy.However,the blood-brain barrier(BBB)and blood-brain tumor barrier(BBTB)significantly limit drug access to the glioma tissues.Therefore,in addition to effective chemotherapeutic and immunotherapeutic drugs against glioma,it is also critical to develop novel drug delivery platforms that combine the advantages of both.Biomimetic nano-delivery systems(BDDS)use biological components to achieve prolonged retention,targeted accumulation and immune escape of the drug cargo.The cell membrane-encapsulated carriers for instance are increasingly being used in anticancer therapy,and have been shown to improve targeted delivery of the drugs and enhance immunogenicity of cancer vaccines.However,the scaffolds of biomimetic nanoparticles(NPs)are made of synthetic materials,which often leads to low drug loading.In addition,functionalization of the NPs with ligands for targeted drug delivery easily destroys the membrane proteins and affects the stability of the targeting system.In order to achieve targeted delivery of glioma drugs,we fused glioma cell membranes with that of dendritic cells(DCs)without any exogenous immune adjuvants or modified ligands.This type of a double-membrane fusion vector can target the homologous tumor cells and deliver high concentrations of chemotherapeutic drugs to the lesions across the BBB.In addition,the chimeric membrane carries abundant and complete tumor antigens,as well as DC-derived components that promote the uptake of these antigens and trigger immune responses either directly or indirectly.The synergistic effect of chemotherapy and immunotherapy can effectively ablate the glioma cells.In the following sections,we have summarized the construction and characterization of biomimetic nanosuspensions,preliminary safety evaluation in vitro and in vivo,tumor targeting,immune activation,and glioma inhibition.(1)Synthesis and characterization of(DNS-[C6&DC]m)We constructed a biomimetic nano-drug delivery system for DTX through the following three steps:preparation of nanosuspensions,membrane fusion,and the preparation of biomimetic nanosuspensions.The nanosuspension was prepared by ultrasonic precipitation using particle size,potential and stability as the indicators,and optimized by single factor experiment and response surface method.Transmission electron microscopy indicated that the particles in the nanosuspension were uniformly sized and relatively round.The successful preparation of DTX nanosuspension was also verified by X-ray diffraction,differential scanning calorimetry and infrared spectroscopy.The tumor cell and DC membranes were respectively isolated by hypotonic extraction and freeze-thaw cycles.To verify absence of tumor cell nucleus or any genetic material in the membrane fraction,the separation of C6 cell membrane and nucleus were dectected by laser confocal microscopy;ultrasonic fusion method was used for membrane fusion,and fluorescence resonance energy transfer was used to verify the fusion.Confocal microscopy preliminarily verified the fusion of tumor cell and DC membranes on the basis of co-localization signals.The biomimetic membrane nanosuspension was prepared by the extrusion method,and characterized in terms of particle size,electric potential,drug loading capacity and encapsulation rate.The ratio of cell membrane to the NPs was determined by measuring the protein concentration,and the number of extrusions was calculated to be 20.The membrane to drug ratio was3:1;and three batches of biomimetic nanosuspensions were prepared and physico-chemically characterized.Transmission electron microscopy revealed that the double-membrane fusion biomimetic nanosuspension had a core-shell structure.Particle size analysis further showed that the increase in the thickness of the NPs following cell membrane encapsulation was equivalent to the thickness of a membrane bilayer.Consistent with this,the chimeric membrane shell also increased the potential of the NPs,and the enhanced stability of DNS as a result of the shielding effect of the membrane was verified through suitable experiments.The protein content of the samples was measured by the BCA method,which indicated low protein loss during the preparation process.In addition,the protein content of the fusion membrane was the sum of the proteins from the two distinct cell membranes.Almost all membrane proteins of DCs and tumor cells were detected in the chimeric membrane through western blotting,including specific homologous binding adhesion molecules and immune-related antigens.Circular dichroism chromatography further showed that the secondary structure of the proteins was not affected by membrane fusion,which laid an effective structural foundation for the functioning of these biomimetic nanocarriers.The in vitro drug release experiments indicated that membrane encapsulation improved controlled drug release from the NPs,which suggested that the nanosuspension can prolong drug retention and the time window of its action in vivo.(2)DNS-[C6&DC]m showed good biosafety at the cellular and animal levelsThe toxicity of the biomimetic nanocarriers was preliminarily evaluated in cellular and animal models.There was no obvious toxicity in the mice in terms of histological findings or serum biochemical indices at the administered dose,indicating that the biomimetic nanocarriers are safe.(3)DNS-[C6&DC]m can promote DC maturation and T cell activationThe immunological effect of the biomimetic nano-delivery system was measured in vitro through flow cytometry.The percentage of CD80~+and CD86~+DCs increased significantly following exposure to DNS-[C6&DC]m compared to that in the other groups.The abundant tumor antigens on the chimeric membrane were internalized by the DCs,which then underwent maturation.The latter is essential to the antigen presentation function of the DCs in vivo.The cytotoxic CD8~+T cells are the primary effectors against cancer cells,whereas the CD4~+T cells promote the proliferation and activity of the CD8~+T cells.We found that the chimeric membrane-encapsulated DNS had a stronger effect on the activation level and proliferation of the T lymphocytes compared to either of the single membrane-encapsulated particles.Consistent with the in vitro results,tumor-bearing mice injected with DNS-[C6&DC]m markedly increased the proportion of CD8 and CD4 T cells in the spleen,lymph nodes and tumor sites,and further enhanced the immune response via the antigen presenting function of DC-derived membrane.(4)DNS-[C6&DC]m can cross the blood-brain barrier and target glioma sitesTo assess the targeting ability of the biomimetic nanocarrier system,we analyzed its permeability through the BBB and BBTB in cellular and animal models.The biomimetic nanosuspension was able to cross the BBB and BBTB in vitro,and resulted in the highest inhibition rate of glioma cell growth.We also established a glioma-bearing mouse model,and in vivo imaging revealed that the biomimetic particles were evenly distributed in the brain tissue.Taken together,these findings indicate that DNS-[C6&DC]m can effectively target the brain tumor lesions and remain in circulation for a prolonged duration.(5)DNS-[C6&DC]m can effectively ablate glioma through targeted drug delivery and immune system activationThe anti-tumor effect of DNS-[C6&DC]m was also evaluated in cellular and animal models.The biomimetic nanocarrier significantly increased the apoptosis rates of C6 glioma cells and decreased their viability.In vivo pharmacodynamic studies showed that the nanosuspension significantly extended the survival of glioma-bearing mice.Glioma growth was monitored through MRI,which indicated a significant inhibition in growth.Furthermore,histopathological examination of the tumors showed larger necrotic regions and fewer malignant cells in the tissues sections obtained from mice treated with DNS-[C6&DC]m.CD31 and TUNEL staining of these tissue sections showed high numbers of apoptotic tumor cells.In addition,the mice treated with the nanosuspension did not exhibit any significant weight loss or other adverse effects,which is consistent with the enhanced targeting and therapeutic effect of DTX in intracranial gliomas by the biomimetic nanocarrier.In conclusion,we constructed a multifunctional biomimetic nano-delivery system without exogenous immune adjuvants and targeting ligands,which was able to achieve glioma ablation through synergistic chemotherapy and immunotherapy.Given the therapeutic efficacy,high drug loading and easy preparation,the nanocarrier has the potential of clinical transformation. |
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