The Effect And Mechanism Of Polyglutamic Acid Grafted CA4 And BLZ945 Nanoparticle On Renal Cell Carcinoma

Background:Renal carcinoma is a malignancy affecting the urinary system.Renal cell carcinoma,primarily originating from renal parenchymal epithelial cells,constitutes over 90% of the cases.The global incidence and mortality rates of renal cell carcinoma are increasing annually.Current research emphasizes the exploration of specific tumor markers and therapeutic strategies.Although surgical intervention remains the primary approach,recurrence or metastasis after surgery remains a concern in some patients.Considering that renal cell carcinoma is characterized by angiogenesis and hypoxia dependency,treatment strategies involve the integration of molecular-targeted therapies and immune checkpoint inhibitors into combination regimens.Resistance to targeted agents and immunotherapy-related adverse effects significantly affect clinical outcomes,underscoring suboptimal prognoses.Ongoing efforts are focused on devising novel treatment modalities to prevent and manage renal cancer.Vascular disrupting agents have garnered considerable attention for their capacity to selectively obstruct abnormal blood vessels within tumors.CA4-NPs,a type of nanoparticle belonging to this category,operate by facilitating the targeted accumulation of CA4 around tumor vasculature,inducing oxygen and nutrient deprivation,ultimately culminating in tumor necrosis.Despite its efficacy,CA4-NPs may contribute to tumor recurrence,attributed to M2 macrophage infiltration.BLZ945,a macrophage colony-stimulating factor-1 receptor inhibitor,demonstrates the ability to induce apoptosis of M2 macrophages within tumors.Leveraging the properties of these compounds,Tang et al.from Changchun Institute of Applied Chemistry conjugated CA4 and BLZ945 to PLG-g-m PEG via esterification,resulting in the development of co-bonded vascular disrupting agents and BLZ945 polymeric nanoparticle,termed CB-NPs.This nanoparticle exhibits promising anti-tumor effects in both the C26 colon cancer and H22 liver cancer models in mice,offering a novel therapeutic avenue for cancer treatment.Nevertheless,the optimal ratio of CA4 to BLZ945 within this nanoparticle remains to be elucidated.Based on these results,we hypothesized that CB-NPs could inhibit tumor growth in renal cancer models.We screened CB-NP formulations and determined their tissue distribution,pharmacokinetics,hematoxylin and eosin staining,and median lethal dose to elucidate the fundamental characteristics of the optimal CB-NP ratio.We then assessed the antitumor effects in a renal cancer model.We established an evaluation framework for proteins associated with hypoxia and angiogenesis to clarify the efficacy of CB-NPs in treating renal cell carcinoma and explore the related mechanisms.We selected the Hsp90 inhibitor BIIB021 for combination therapy to assess its antitumor effects.Methods:First,to ensure the antitumor effect of CB-NPs and reduce drug toxicity,we conducted formula screening of CB-NPs in H22 liver cancer models,which previously validated for their antitumor effects.CB-NPs were prepared at various ratios for tumor inhibition experiments.Optimization was achieved by altering the injection dose of CBNPs,the ratio of BLZ945/CA4,and the loading rates of BLZ945 and CA4 to identify the ratio with optimal tumor inhibition and minimal drug toxicity.Following formula screening,a series of clinical trials were conducted in BALB/c mouse models to elucidate the fundamental characteristics of the selected optimal CB-NPs ratio,providing a basis for subsequent renal cancer model experiments.We employed the CCK-8 detection method and flow cytometry to investigate the inhibitory and apoptotic impacts of CB-NPs on Renca renal cancer cells.After confirming that CB-NPs can inhibit their growth at the cellular level,a Renca renal cell carcinoma subcutaneous tumor model was established.Tumor inhibition experiments were conducted at varying doses(low,medium,and high),with measurements of tumor tissue volume and mouse weight taken every two days.Subsequently,tumor growth trends and changes in mouse weight were graphed based on recorded data,informing the selection of appropriate doses for subsequent experiments.Histological staining with H&E was performed on separate tumor tissue to assess CB-NPs’ effect on renal cancer tissue.Additionally,the influence of CB-NPs on the expression of HIF-1α and VEGFA in tumor tissues was investigated via q PCR,western blotting,ELISA,and immunohistochemical staining techniques.The impact of CB-NPs on the quantity of macrophages in tumor tissue was examined using flow cytometry.Finally,we used the Hsp90 inhibitor BIIB021 in combination to assess the comparative antitumor effects between monotherapy and combination therapy groups.Additionally,we conducted a preliminary evaluation of the combined efficacy of the two drugs.Results:1.Modifying the dosage of CA4,the B/C ratio,and the loading rates of CA4 and BLZ945 in CB-NPs,alongside tumor inhibition experiment outcomes and mouse weight variations,reveals optimal tumor suppression with B/C ratio of 0.45/1,and B+C at 20.7%.The liver and kidneys primarily metabolize CB-NPs,influencing tissue morphology in tumors,the liver,and the spleen.The half-lives of CA4 and BLZ945 in vivo are 5.75±0.26 h and 6.31±0.29 h respectively,with a median lethal dose of 43.12mg/kg.CB-NPs manifest toxic effects in vivo,precipitating significant weight loss in mice at high doses,potentially leading to mortality.2.Experimental findings on antitumor effects in the Renca renal cancer model revealed that CB-NPs effectively suppressed tumor growth when the tumor volume was substantial(V = 220 mm3).Within a specific dosage range,increasing the CB-NP dosage correlated with enhanced antitumor effects,resulting in tumor inhibition rates of 39.7%(20 mg/kg),72.4%(30 mg/kg),and 88.0%(40 mg/kg).3.The CCK-8 assay revealed that CB-NPs exerted potent toxicity on Renca cells within 72 h post-administration,exhibiting prolonged efficacy.Specifically,the 24-hour CCK-8 test demonstrated the concentration-dependent toxicity of CB-NPs on Renca cells.Ki67 staining indicated a significantly reduced area of Ki67-positive expression in the CB-NPs group compared to the PBS group;CB-NPs inhibited tumor cell proliferation in the Renca renal cancer model and consequently inhibited tumor growth.H&E staining further supported these results,showing a significantly larger necrotic area in the tumor tissue of the CB-NP group than the PBS group,indicating that CBNPs can cause massive necrosis of tumor tissue.4.Flow cytometry analysis revealed that CB-NPs significantly augmented the proportion of apoptotic cells across multiple stages of Renca cell lines,encompassing total apoptosis,early apoptosis,and late apoptosis,under both normoxic and hypoxic conditions.This suggests the apoptotic promotion effect of CB-NPs on Renca cells.Comparative analysis of apoptosis ratios between normoxic and hypoxic conditions indicated a more pronounced ability of CB-NPs to induce cell apoptosis under normoxic conditions.Hence,it is hypothesized that mitigating hypoxia levels within tumor cells could enhance the anti-tumor efficacy of CB-NPs.5.The expression of HIF-1α in Renca renal carcinoma tissue,as detected by ELISA,gradually increases within 72 hours.Additionally,the expression of VEGFA gradually increases in the first 48 hours and decreases from 48 to 72 hours.6.In the late stage of treatment,the expression of HIF-1α and VEGFA in tumor tissues of the CB-NPs group of Renca carcinoma was assessed using q PCR,western blot,ELISA,and immunohistochemical staining.The expression of HIF-1α was increased in the CB-NPs group compared with that in the PBS group,whereas the expression of VEGFA was decreased.7.Flow cytometry analysis revealed no significant increase in M2 macrophages following treatment with CB-NPs,suggesting inhibition of M2 macrophage polarization.This inhibition correlates with suppressed tumor invasion,metastasis,and reduced tumor recurrence.8.Comparative analysis between the PBS group and treatment groups(CB-NPs,BIIB021,and combination therapy)demonstrated tumor growth inhibition across all groups.Notably,the combination therapy exhibited the lowest average tumor volume and mass,with respective tumor inhibition rates of 45.6%(20mg/kg CB-NPs),33.9%(60mg/kg BIIB021),and 87.3%(combination).The calculated Q value of 1.36 suggests a synergistic effect between the two drugs.9.Antitumor effects of CB-NPs,BIIB021,and combination therapy were evaluated on Renca renal cell carcinoma.H&E staining indicated greater tumor tissue necrosis under all treatment regimens compared to the PBS group,with the combination therapy group exhibiting the largest necrotic area.Ki67 test results revealed a substantially lower positive expression rate of Ki67 in all three treatment groups compared to the PBS group,with the combination therapy group showing the lowest Ki67 positive rate.TUNEL test results demonstrated significantly increased positive expression rates of TUNEL in all treatment groups compared to the PBS group,with the combination therapy group exhibiting the highest positive rate.Western blot analysis revealed that,unlike CB-NPs,both BIIB021 monotherapy and combination therapy resulted in a simultaneous decrease in the expression of HIF-1α and VEGFA.Conclusion:1.Through screening the formulation of co-bonded vascular-disrupting agents and BLZ945 polymeric nanoparticle(CB-NPs),an optimal and stable ratio was established for tumor inhibition(B/C=0.45/1,B+C=20.7%),laying the groundwork for subsequent experimental efficacy and safety evaluations.2.CB-NPs demonstrate potent antitumor activity in the Renca renal cell carcinoma model,exhibiting a direct correlation with their concentration.They effectively impede tumor cell proliferation,induce apoptosis,induce necrosis in tumor tissue,downregulate VEGFA expression,and hinder tumor growth.Simultaneously,inhibition of M2 macrophages significantly reduces tumor recurrence.3.CB-NPs synergistically enhance the inhibitory effect of BIIB021 on CB-NPsinduced HIF-1α expression,thereby enhancing anti-tumor efficacy.