Near Infrared Light Mediated Nanomicelles Enhanced Efficacy Of Paclitaxel Against Small Cell Lung Cancer

BackgroundSmall cell lung cancer?SCLC?is the most aggressive form of lung cancer.It is characterized by rapid growth,early and widespread metastasis,and poor prognosis.As a bedrock treatment for SCLC,systemic chemotherapy has reached a therapeutic efficacy plateau.Hence,there is an urgent need for more effective SCLC treatment.Paclitaxel?PCL?is widely used in various types of cancer treatment.Nevertheless,its poor aqueous solubility has limited its clinical applications.The currently available commercial formulations may cause serious adverse reactions.Therefore,it is essential to develop a novel delivery system which can overcome the above drawbacks,hence enhancing anti-cancer activity of PCL.Among the emerging nanoscopic carrier systems,polymeric micelles are one of the most promising and well-investigated approaches that have improved the solubility and stability of hydrophobic drugs.In recent years,photodynamic therapy?PDT?is becoming an emerging research hotspot under active clinical trials owing to its outstanding advantages,including enhanced therapeutic efficiency,reduced side effect and improved spatiotemporal management.Uncoordinated mutant number-45 myosin chaperone A?UNC-45A?plays an important role in regulating cytoskeletal-associated functions including cytokinesis,exocytosis,cell motility,and neuronal development.Cumulative evidence suggests that UNC-45A contributes to tumorigenesis and its overexpression correlates with poor clinical outcome in human cancers.However,the molecular mechanism by which UNC-45A regulates cancer cell remains largely unknown.Moreover,there is no report on the role of UNC-45A in the development of SCLC.AimsThe objective of the present study was to investigate the in vitro and in vivo efficacy as well as safety profiles of paclitaxel loaded functionalized fullerene-?polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer?-?polyethylene glycol succinate?micelles?PCL-FSTM?combined with PDT against SCLC,and to explore the role of UNC-45A in the treatment.MethodsThe first part focused on the characterization of PCL-FSTM and the investigation of the in vitro effcicay of PCL-FSTM combined with PDT against SCLC.First,the particle size and zeta potential of PCL-FSTM were investigated by dynamic light scattering.Then,the effect of PCL-FSTM on cytotoxicity in the SCLC cell lines NCI-H446 and NCI-H1688 was investigated.Next,flow cytometry was applied to detect the effects of PCL-FSTM on the cell cycle and apoptosis of NCI-H446 cells.Finally,cell counting kit-8?CCK8?assay was employed to quantitatively analyze the PDT toxicity of PCL-FSTM in NCI-H446 cells upon near-infrared?NIR?irradition.In the second part,we explored the mechanism of PCL-FSTM against SCLC.Firstly,reactive oxygen species?ROS?and mitochondrial membrane potential?MMP?levels of NCI-H446 cells in different treatment groups were determined using 2',7'-dichlorodihydrofluorescein diacetate?DCFH-DA?and JC-1,respectively.Next,quantitative reverse transcription-polymerase chain reaction?qRT-PCR?and Western blot assay were respectively applied to analyze the expression levels of mRNAs and proteins related to cell cycle or apoptosis in NCI-H446 cells.Finally,the effect of UNC-45A overexpression on the proteins related to cell cycle in NCI-H446 was further studied by lentivirus transfection technique.In the third part,we investigated the in vivo pharmacodynamics as well as the systemic toxicity of PCL-FSTM combined with PDT against SCLC.NCI-H446 tumor bearing mice were chosen for systematically investigating antitumor efficacy of PCL-FSTM combined with PDT in vivo.The tumor volume and tumor weight were used as evaluation indexes.Moreover,the main organs of the NCI-H446 tumor bearing mice were obtained,weighted and fixed for Hematoxylin-eosin?HE?staining.At the same time,the serum of the blood samples was collected.Then the biological markers of liver and kidney function were measured by enzyme-linked immunosorbent assay?ELISA?.In the final part,we further explored the mechanism of PCL-FSTM combined with PDT against SCLC with the xenograft tumor specimens.The expression level of UNC-45A protein was detected by immunofluorescence assay.The expression levels of other proteins involved in the UNC-45A/CDK1/NEK7/Eg5 pathway were examined with Western blot assay.ROS level was determined using dihydroethidium?DHE?.The effect of PCL-FSTM combined with PDT on tumor apoptosis was further analyzed with terminal deoxynucleotidyl transferase dUTP Nick end labeling?TUNEL?staining.ResultsIn the first part,PCL-FSTM exhibited a hydrodynamic diameter of?67.23±1.35?nm and a zeta potential of?-1.88±0.23?mV.It could be inferred from CCK8 assay that PCL as well as PCL-FSTM inhibited both NCI-H446 and NCI-H1688 cells in a dose-and time-dependent manner.Compared with PCL,PCL-FSTM exhibited better antitumor activity against the two SCLC cell lines.After treatment for 48 h,the IC₅₀ value of PCL-FSTM on NCI-H446 cells?0.094?0.008?g/mL?was about 12.2-fold less than that of PCL?1.146?0.195?g/mL?.In addition,the IC₅₀ value of PCL-FSTM on NCI-H1688 cells?0.094?0.014?g/mL?was about 2.3-fold less than that of PCL?0.220?0.056?g/mL?.The results of flow cytometry revealed that PCL and PCL-FSTM both selectively arrested the cell cycle of NCI-H446 cells at G2/M phase.Moreover,the effect of PCL-FSTM was significantly stronger than that of PCL?P<0.001?.While,both of them had no effect on the apoptosis of NCI-H446 cells?P>0.05?.Furthermore,PCL-FSTM showed PDT cytotoxicity against NCI-H446 cells upon NIR irradiation.In the second part,the flow cytometry analysis revealed that the ROS level of PCL-FSTM treated NCI-H446 cells increased as compared to that of the control?P<0.05?.However,there was no significant difference in the ROS level between the PCL-FSTM group and the PCL group.The results of fluorescence microscopy showed that there was no significant difference in MMP among the groups.After treatment with PCL and PCL-FSTM for 48 h,qRT-PCR and Western blot results showed that PCL and PCL-FSTM significantly decreased the mRNA and protein expression levels of UNC-45A,CDK1,NEK7 and Eg5 in NCI-H446,and PCL-FSTM had more significant effect?P<0.05?.In the third part,the in vivo therapeutic efficacy in nude mice bearing NCI-H446 xenograft tumors confirmed that PCL-FSTM combined with PDT showed significant inhibitory potency on tumor growth.What's more,no pathological changes were found in the HE staining of the main organs in each group.Besides,the biological markers of liver and kidney function were all in the normal range,demonstrating the good biocompatibility of PCL-FSTM combined with PDT.In the final part,immunofluorescence experiments and Western blot assays were carried out to investigate the expression levels of UNC-45A and its related proteins in the xenograft tumors.And the decreased protein expression levels of UNC-45A,CDK1,NEK7 and Eg5 were detected in the group with the treatment of PCL-FSTM combined with PDT?P<0.05?.Besides,the increased fluorescence in DHE and TUNEL staining indicated that PCL-FSTM combined with PDT also promoted cell apopotosis.In summary,PCL-FSTM combined with PDT exerted significant suppressive effects on the growth of the xenografted SCLC by inducing cell cycle arrest at G2/M phase and cell apoptosis at the same time.ConculsionsThis nanoplatform for combining chemotherapy and PDT were more effective to inhibit the growth of SCLC than the parent drug both in vitro and in vivo without eliciting systemic toxicity,indicating its great potential in treating SCLC.PCL-FSTM combined with PDT could not only enhance G2/M arrest via the UNC-45A/CDK1/NEK7/Eg5 pathway,but also promote apoptosis of tumor cells.Moreover,UNC-45A played an important role in the regulation of tumor progression,and could be used as a new target in the treatment of SCLC and other malignant tumors.