Circulating Tumor Cells-targeted Nanoparticles With Prolonged Blood Circulation

Objectives:The aim of this study was to prepare docetaxel(DTX)-loaded,anti-phagocytosis peptide(PepCD47)and circulating tumor cells(CTCs)recognition peptide(Pep10)-modified solid lipid nanoparticles(SLNs)with prolonged blood circulation,and to characterize the physicochemical properties,safety and effectiveness of SLNs in vitro and in vivo.Methods:The stearic acid(SA)and PEGconjugation(SA-PEG2000 or SA-PEG6000)was synthesized by an amide bond between SA and PEG to improve the lipophilicity of PEG.For the synthesis of SA and peptide conjugation(SA-PepCD47 or SA-Pep10),PepCD47 or Pep10 were firstly prepared by a solid phase peptide synthesis(SPPS)and followed by the connection with SA via an amide bond.DTX-SLNs,DTX-SLNs-PepCD47,DTX-SLNs-Pep10 and DTX-SLNs-PepCD47-Pep10 were prepared by an emulsion-solvent evaporation method.The in vitro release study was carried out in phosphate-buffered saline(pH 7.4),using a dialysis bag method.X-Ray Diffraction(XRD),differential scanning calorimetry(DSC)and fourier transform infrared resonance spectroscopy(FT-IR)were used to observe the status of the drug and its physical changes in nanoparticles.The cell viability of various SLNs in different cell lines(MCF-7 cells,A549 cells,RAW264.7 cells and 293T cells)were determined using the MTT assay.The epithelial cell adhesion molecule(EpCAM)expressionlevel of MCF-7 cells,A549 cellsand293T cells was measured using anti-EpCAM.The anti-phagocytosiscapacity and enhanced specificity to EpCAM positive cancer cell lines of various SLNs were studied by flow cytometry and confocal microscope.The uptake selectivity of SLNs-PepCD47-Pep10 was examined by mixing a series of MCF-7 with RAW264.7 cells at a different mixture ratio MCF-7 cells were intravenously injected into Balb/c nude mice to establish CTCs model.The targeting capacity of SLNs to CTCs in the blood was evaluated by confocal microscope,the tissue distribution and anti-phagocytosiscapacity were analyzed by a small animal imaging system and HPLC-MS.The in vivo efficacy of SLNs on CTCs was investigated by flow cytometry.Moreover,the ABC phenomenon was evaluated in C57BL/6 black mouse after a repeated dose of SLNs.Result:~1H-nuclear magnetic resonance(~1H-NMR)and Fourier transform infrared spectroscopy(FT-IR)results showed that SA-PEG2000 and SA-PEG6000 were successfully synthesized.The purity of SA-PepCD47 and SA-Pep10 were more than 98%checked by high performance liquid chromatography(HPLC)and mass spectrometry(MS).Various peptide-modified SLNs showed a round and regular mophology with a mean particle size less than 200 nm.The DTX release from SLNs at pH7.4 was below 50%after48 h.The results of XRD,DSC and FT-IR showed that there was some interactions between DTX and excipients during the preparation process of DTX-SLNs.The results of MTT assay showed that various SLNs have little toxicity to different cells lines(MCF-7cells,A549 cells,RAW264.7 cells and 293T cells).The cellular uptake of SLNs-PepCD47in RAW264.7 cells was lower than that of SA-PEG2000 and higher than that of SA-PEG6000.After detemined by anti-EpCAM,MCF-7 cells showed high expression of EpCAM,A549 cells showed low expression of EpCAM,and 293T cells were barely expressed with EpCAM.The results of cellular uptake indicated that SLNs-PepCD47-Pep10 could greatly improve the uptake in MCF-7 cells and significantly suppressed the phagocytosis in RAW264.7 cells.In addition,SLNs-PepCD47-Pep10 was selectively captured by MCF-7 cells in MCF-7/RAW264.7 co-culture system.The confocal microscope results showed that SLNs-PepCD47-Pep10 could specifically target the MCF-7cell in the blood and the cellular uptake of SLNs significantly increased.The results of tissue distribution,fluorecence intensity in blood and plasma concentration-time profiles indicated that DTX-SLNs-PepCD47-Pep10 could inhibit the phagocytosis of mononuclear phagocyte system and prolong the circulation time of carriers when compared to DTX-SLNs.A 2.3-fold increase of dead cells was found in DTX-SLNs-PepCD47-Pep10group compared with DTX-SLNs group.ABC phenomenon results showed that the circulation time of SLNs-PepCD47 and SLNs-PEG6000 was similar at 4 h after the first injection,and the circulation time of SLNs-PepCD47 was longer than that of SLNs-PEG6000 at 4 h after the second injection.Conclusion:The CTC-targeted nanoparticles with prolonged blood circulation were successfully prepared.The DTX-SLNs-PepCD47-Pep10 displayed a higher anti-phagocytosis capacity and improved specificity for recognizing EpCAM positive cancer cell lines in vitro and vivo.This study may provide new ideas for cancer diagnosis,postoperative evaluation and cancer metastasis research.