Targeted Anti-tumoral Mechanism Of C-PC/CMC-CD55sp Nanospheres On Cervical Cancer HeLa Cells

Objective:Early experiments in vitro have confirmed that phycocyanin（C-PC）has an inhibitory effect on the growth of cervical cancer HeLa cells.In this experiment,the phycocyanin/carboxymethyl chitosan（CMC）-CD55 ligand peptide（CD55sp）nanospheres were used to achieve targeted drug delivery,and the nanospheres targeting anti-tumor effects for cervical cancer HeLa cells were further evaluated in vitro and in vivo.Methods:C-PC/CMC nanospheres were constructed by CMC embedded C-PC by ion-crosslinking method（CaCl₂ as cross-linking agent）,and then C-PC/CMC-CD55sp nanospheres were synthesized by modification of CD55sp.The chemical bond changes of CMC nanospheres,C-PC/CMC nanospheres and C-PC/CMC-CD55sp nanospheres were detected by infrared spectrometer.The particle size and potential of the three nanospheres were detected by laser particle size analyzer.The morphology of the nanospheres was observed by electron microscopy.The experiment was divided into four groups:control group,C-PC group,C-PC/CMC group and C-PC/CMC-CD55sp group.Flow cytometry and imaging system in vivo were used to detect the targeting of drugs in the C-PC group,C-PC/CMC group and C-PC/CMC-CD55sp group.In vitro,the inhibitory effects of three groups of drugs on cervical cancer HeLa cells were verified by CCK8,flow cytometry,and Tunel.The apoptotic morphology of cells was observed by electron microscopy.The levels of apoptosis-related proteins and autophagy-related proteins were detected by western blot.In vivo,the inhibitory effects of three groups of drugs on the growth of tumor tissue were proved by constructing tumor-bearing nude mice and the levels of related immune cytokines in the blood were examined by ELISA.Results:All CMC nanospheres,C-PC/CMC nanospheres and C-PC/CMC-CD55sp nanospheres showed regular spherical shapes,with the particle sizes of 160.5±48.06 nm,146.6±53.3 nm,and 258.9±40.505 nm,and the zeta potentials of-7.66±3.83 mV,-19.7±2.53 mV and-13.1±3.28 mV,respectively.Compared with CMC nanospheres,C-PC/CMC nanospheres had changed two chemical bonds,and C-PC/CMC-CD55sp nanospheres had five chemical bonds changed.In vitro,compared with C-PC group and C-PC/CMC group,HeLa cells in C-PC/CMC-CD55sp group had stronger fluorescence reaction,indicating that C-PC/CMC-CD55sp nanospheres could target HeLa cells;in vivo,compared with the C-PC group and the C-PC/CMC group,the tumor tissues in the C-PC/CMC-CD55sp group had stronger fluorescence reaction and normal tissue and organ（heart,liver,spleen,kidney）showed no obvious fluorescence reaction,indicating that C-PC/CMC-CD55sp nanospheres could target tumor tissues in nude mice.In vitro,the growth state of HeLa cells depended on the drug concentration in the C-PC group,the C-PC/CMC group and the C-PC/CMC-CD55sp group,and the proliferation-inhibiting effect of C-PC/CMC-CD55sp on HeLa cells was more stronger than that of C-PC and the C-PC/CMC group.The apoptosis rate of HeLa cells induced by C-PC/CMC-CD55sp group was significantly higher than that of C-PC group and C-PC/CMC group.Compared with C-PC group and C-PC/CMC group,C-PC/CMC-CD55sp could significantly inhibit the expression of anti-apoptotic protein Bcl-2 and promote Caspase 3 to cleave into apoptotic protein Cleaved Caspase 3.Compared with the control group,the levels of autophagy-associated proteins mTOR in the C-PC group,C-PC/CMC group and C-PC/CMC-CD55sp group detected were increased,and the level of Beclin1 and LC3was decreased,and the effect of C-PC/CMC-CD55sp group was the most obvious,indicating that C-PC/CMC-CD55sp nanospheres might inhibit the autophagy of HeLa cells.In vivo,it was also found that the C-PC/CMC-CD55sp group had a stronger inhibitory effect on tumor tissue growth than the C-PC group and the C-PC/CMC group,and made the relevant immune cytokine TGF-βlevel lower,IL-6 and TNF-αlevels higher in the blood,suggesting that immunomodulation triggered by C-PC/CMC-CD55sp nanospheres in nude mice played an important role in tumor suppression.Conclusion:C-PC/CMC-CD55sp nanospheres were successfully constructed and proved to be targeting to cancer tissues.Cell experiments confirmed that the nanospheres could inhibit proliferation,promote apoptosis and inhibit autophagy.Animal experiments further confirmed that the nanospheres could inhibit the growth of tumor tissue,and also played an anti-tumor role by regulating the immune response in nude mice.