Aluminum Nanoparticles Deliver Apatinib And PD-1 Antibodies For Enhancing Anti-tumor Therapy In Non-small Cell Lung Cancer

Non-small cell lung cancer(NSCLC)is the primary histological type of lung cancer,accounting for approximately 85% of all lung cancer cases,with the highest incidence and mortality in China and the world.Roughly one-half of NSCLC is considered advanced(stage 4)when it has spread outside where the tumor began growing.Thus,over 40% of the patients diagnosed with NSCLC die within one year of diagnosis,and the 5-year survival is around 20%.In recent years,with the development of drugs against immune checkpoint inhibitors(ICIs),tumor vaccines,vascular endothelial growth factor,and vascular endothelial growth factor receptors,a new approach with the combination of anti-angiogenic drugs and ICIs to tumor treatment has emerged.The potential synergistic efficacy of VEGF/VEGFR targeting compounds and ICIs in treating NSCLC has been demonstrated.As one kind of nanosized metal particle,aluminum nanoparticles(Al NPs)are nanoparticles with acceptable biocompatibility,operable size and shape,and accessible surface modifiers,resulting in easier targeted penetration into tumor cells and easy uptake by cells.Moreover,Al NPs can encapsulate various active molecules such as drugs,peptides,and oligonucleotides to achieve the goal of multi-target synergistic therapy.Therefore,we use Al NPs as a drug carrier to synergistically deliver Apatinib and PD-1 antibodies for treating Lewis lung cancer to play a more potent anti-tumor effect.In this study,the toxicity effects of Al NPs on cells were investigated.Our result showed that Al NPs had no harmful impact on BEAS-2b cells and LLC cells in normal human lung epithelial cells.The Lewis lung cancer(LLC)cells from NSCLC were used in this study to establish a mouse model of the Lewis lung cancer subcutaneous transplanted tumor.Next,the anti-tumor effect of the aluminum nanoparticle-based drug delivery system with the delivery of Apatinib and PD-1 antibody was explored in vivo.Thirty-two mice were randomly divided into four groups(n=8).The groups and treatment were as follows: 1)PBS group: PBS buffer containing 1% DMSO 200 μL each mouse;2)Al NPs group: 1mg Al NPs each mouse;3)Apa+a PD1 group: 200 μg Apatinib + 50 μg PD-1 antibody each mouse;4)Al NP@Apa/a PD-1 group: 1 mg Al NPs+200 μg Apatinib +50 μg PD-1 antibody each mouse.The tumor volume in each group was recorded during the treatment period.The mice were sacrificed after 20 days of the treatment,and the tumor tissues of each group were isolated,photographed,and weighed.Meantime,the proportion of NK cells and PD-1 expression in lymphocytes of tumor tissues.Similarly,the proportion of Treg cells in lymph nodes and the expression of IFN-γ were detected by flow cytometry in mice from each group.Then,the expression of CD31 and VEGFR-2 was detected by immunohistochemistry.In addition,the apoptosis of tumor cells in tumor tissues was detected by TUNEL immunofluorescence and Western blot.Furthermore,the anti-tumor effects of Al NPs delivered with Apatinib(Al NPs@Apa)and Al NPs combined with Apatinib(Al NPs+Apa)were further investigated in vivo.The main experimental contents and results of this study are as follows:1.LLC cell was used to construct a subcutaneous tumor graft model in C57BL/6J mice,and the anti-tumor effect of the aluminum nanoparticle drug delivery system was evaluated in mice.The results showed that Apatinib combined with PD-1 antibody inhibited tumor growth,and the inhibitory effect was more robust after treatment with Al NP@Apa/a PD-1 compared to the PBS group.2.Relevant immune cell indexes in tumor tissue and lymph nodes of each group were detected by Flow cytometry.The results showed that compared with the other groups,Al NPs deliver Apatinib and PD-1 antibodies could increase the proportion of effector NK cells in tumor cells and inhibit the expression of PD-1 in infiltrating lymphocytes of tumor tissue.Meantime,the proportion of Treg cells in lymph node cells was reduced,and higher levels of IFN-γ were produced.3.The relative quantitative analysis of CD31 and VEGFR-2 in tumor tissue slices of each group was performed by immunohistochemistry to evaluate the effect of the aluminum nanoparticle delivery system on tumor angiogenesis.The results showed that Al NPs deliver Apatinib and PD-1 antibodies can reduce the expression of CD31 and VEGFR-2 and inhibit tumor angiogenesis,compared with other groups.4.The apoptosis of tumor cells of each group was detected by TUNEL immunofluorescence.The results showed that the degree of apoptosis of tumor cells was significantly enhanced after treatment by the aluminum nanoparticle delivery system compared with the other groups,resulting in promoting the apoptosis of tumor cells more efficiently.5.The expression of cleaved Caspase-3 protein was detected by Western blot to confirm further the promoting effect of the aluminum nanoparticle delivery system on tumor cell apoptosis.The results showed that Al NP delivery of Apatinib and PD-1antibody could significantly promote the expression of cleaved Caspase-3 and promote the apoptosis of tumor cells,compared with other groups.6.Based on the anti-angiogenesis inhibitor Apatinib,the difference of tumor inhibition between Al NPs@Apa and Al NPs+Apa was explored.The results showed that both of Al NPs@Apa and Al NPs+Apa could significantly inhibit tumor growth compared with the PBS group,but there was no significant difference in tumor inhibitory effect between the two groups.This study confirmed that the combination of Al NPs with Apatinib and PD-1antibody showed synergistic anti-angiogenesis and anti-tumor effects,which has potential clinical application value and provides a new idea for the multi-target combination drug strategy of NSCLC treatment in the future...