Experimental Study Of Anti-G250 Nanobody-targeted Drug-loaded Nanobubbles In The Treatment Of Renal Cell Carcinoma

Renal cell carcinoma?RCC?has the high morbidity and poor prognosis.It is the most common malignant tumor of the kidney and is not sensitive to traditional chemotherapy and radiotherapy.Early detection can improve the prognosis of patients by partial or radical resection,but due to the insidious disease,early diagnosis is difficult,and there has the high incidence of postoperative recurrence and distant metastasis,those factors may reduce the survival expectations of patients with RCC.Molecular targeted therapies via the VEGF/PDGFR/mTOR pathway which have replaced immunotherapy as the first-line treatment for patients with advanced kidney cancer,but few patients have achieved complete remission,due to the delivery disorders,and more systemic side effects.Therefore,the treatment of RCC still needs to develop a more effective targeted drug or drug delivery system to achieve higher response rate,lower side effects and durable remission.Nano-scale ultrasound microbubbles?NBs?can be used not only for the diagnosis and treatment of tumors,but also as a safe and effective carrier for delivering drugs or genes.Studies have shown that targeted drug-loaded ultrasound nanobubbles combined with ultrasound targeted nanobubble destruction?UTND?can deliver drugs/therapeutic genes to specific tumors,enhance antitumor efficacy,and reduce side effects.In the previous study,we prepared nano-microvesicles targeted by G250 monoclonal antibody,which confirmed that the targeted microvesicles can bind to renal cancer cells expressing kidney cancer-associated antigen G250.The nanobody which could specifically bind to the extracellular domain of RCC-associated antigen G250 was synthetized by phage display technology.The aim of this study is to prepare the targeted drug-loaded nano-ultrasound microbubbles for local release,by dissolving the hydrophobic molecular targeted drug temsirolimus?TEM?in the microbubble lipid shell,and then connect the G250 nanobody to the microbubble surface by biotin-avidin bridge method.Furthermore,we will study its ability to specifically target RCC,and combined with UTND for precise treatment of RCC.Part I Preparation and characterization of targeted temsirolimus lipid nanobubblesObjective:To prepare the temsirolimus lipid nanobubbles?TNBs?by dissolving temsirolimus?TEM?,a molecular targeted drug,in the nanobubble lipid shell,and then attached the G250 nano-antibody to the nanobubble surface to prepare the targeted drug-loaded nanobubble G250-TNBs.Metnods:TNBs were prepared by membrane hydrationcombined with mechanical shock and electrostatic interaction.G250-TNBs were obtained by attaching G250 to the surface of TNBs by the method of biotin-streptavidin bridge,and then NbG250 coupled with micro-vesicle were verified by immunofluorescence.The distribution of G250-TNBs was observed by optical microscope,their morphological characteristics was observed by transmission electron microscope,and the average particle size and zeta potential were measured by Malvern particle size potential analyzer.The encapsulation efficiency?EE?and drug loading efficiency?LE?were determined by high performance liquid chromatography?HPLC?.Under the effects of ultrasound,the drug release efficiency of G250-TNBs was evaluated by the dialysis bag method.Results:The prepared G250-TNBs were dispersed in dots observed by the ordinary light microscope,and the concentration was about?4.50±1.18?×10⁹/mL.Under the transmission electron microscope,which were as the uniform spherical shape structure and the average diameter was about 400 nm.Measured by the Malvern particle size potential analysis,the average particle size of G250-TNBs was?399.67±99.67?nm,and the average zeta potential was?-23.33±2.63?mV.Immunofluorescence confirmed that anti-G250Nanobody was evenly distributed on the surface of drug-loaded nanobubbles.Analyzed by HPLC,the drug encapsulation efficiency of G250-TNBs was?87.73±5.44?%,and the drug-loading efficiency was?7.99±0.49?%.The drug carried by G250-TNBs was slowly released within 72 hours,but released quickly after the ultrasonic irradiation.Conclusion:The Nb^G250-targeted lipid nanobubbles coupled with temsirolimus were successfully prepared with high drug encapsulation efficiency.The drug-loaded nano-microbubbles tend to be stable within 72 hours,and the drug can be rapidly released under ultrasonic irradiation to achieve targeted drug delivery.Part?Study on the binding ability of G250-TNBs to human renal cell carcinoma 786-O cells and the efficiency to inhibit cells proliferation and induce apoptosis of G250-TNBs combined with ultrasound irradiationObjective:To investigate the ability of G250-TNBs binding to human renal cell carcinoma 786-O cells,and the efficiency of G250-TNBs combined with ultrasound irradiation to inhibit the growth of 786-O cells.Methods:The ability of G250-TNBs binding to786-O cells was observed by rosette formation assay and rosette blockade assay.The inhibitory effects of G250-TNBs combined with UTND on 786-O cell proliferation were detected by CCK-8.One-step TUNEL Staining and flow cytometry were used to detect the effect of G250-TNBs combined with UTND on apoptosis of 786-O cells.Result:In vitro cell experiments revealed that G250-TNBs have the ability of specific targeted to 786-O cells.The inhibition of 786-O cells'proliferation in the free TEM group was significantly higher than that in the TNBs group and the G250-TNBs group?P<0.05?.Combined with UTND,effects of inhibiting the proliferation and inducing apoptosis were significantly enhanced in both of the TNBs group and the G250-TNBs group?P<0.05?.The most important was that the ability of inhibiting 786-O cells proliferation and inducing cells apoptosis in G250-TNBs+US group were significantly higher than those in other groups?P<0.05?.Conclusion:In vitro,G250-TNBs can targeted binding to human renal cell carcinoma786-O cells,and combined with UTND could significantly inhibit the cells proliferation.Part III Study on the binding ability of G250-TNBs to renal carcinoma cells in vivo and their ability to inhibit the renal carcinoma progress combined with UTNDObjective:To explore the ability of G250-TNBs binding to targeted 786-O cells via enhanced ultrasound imaging in renal carcinoma-bearing nude mice.To observe the efficiency of G250-TNBs combined with ultrasound-targeted nano-microbubble to induce renal carcinoma cells apoptosis,and then to inhibit the progress of RCC.Methods:Same concentrations and volumes of G250-TNBs or TNBs were randomly injected into tumor-bearing nude mice,and continuous dynamic ultrasound images were collected by ultrasound imaging system for small animal imaging.The peak intensity?dB value?and the duration of the collected images were analyzed using special software,then to plot the time-intensity curves and calculate the areas under the curve.To evaluate the anti-tumor effect of G250-TNBs combined with UTND on renal carcinoma-bearing mice,30 tumor-bearing mice were randomly assigned to 6 groups?n=5?:the control group,the TEM group,the TNBs group,the G250-TNBs group the TNBs+US group and the G250-TNBs+US group.After the treatments,tumor weights and sizes were measured to calculate the volumes and the inhibitive efficiency.With hematoxylin and eosin?H&E?staining,observing the histopathological changes of the RCC tissues,and TUNEL assay was used to evaluate tumor cells apoptosis.The apoptotic index?AI?was calculated by the positive tumor cells stained divided by the total tumor cells counted of 5 randomly selected high power fields.Result:Through the enhanced imaging experiments in nude mice,we found that enhanced imaging effects of G250-TNBs were statistically significant better compared with TNBs?P<0.05?,and this pointed out that G250-TNBs had the ability binding to the targeted RCC cells We also found in all the treatment groups,tumor volumes decreased more significant in TNBs or G250-TNBs combined with UTND?P<0.05?.More importantly,G250-TNBs combined with UTND group had higher tumor inhibition rates and apoptotic indexes than TNBs combined with UTND group?P<0.05?.Conclusion:G250-TNBs have the ability of binding to targeted renal carcinoma cells in tumor-bearing mice.G250-TNBs combined with UTND can effectively inhibit the proliferation and induce apoptosis of renal cancer cells in xenografted tumors.It can be used as a potential targeted therapy in the treatment of RCC.