In Vitro Study Of DOX-loaded Nanoparticles Delivered By Macrophages To Kill Tumor Cells

Macrophages are expected to be served as cellular carriers for tumor-targeted therapy due to their intrinsic tumor-homing properties and phagocytosis abilities.How to reduce cytotoxicity and increase the drug loading in living cells has become a hot and difficult point in current research.In this study,we have intended to construct a biomimetic delivery system(BDS)by loading doxorubicin(DOX)-loaded mesoporous silica nanoparticles(DOX/MSNs)into a mouse macrophage-like cell line(RAW264.7).DOX/MSNs exhibit high drug loading efficiency without affecting the activity of the cell RAW264.7,hoping that the newly BDS can achieve efficient drug delivery,kill tumor cells and reduce the toxic and side effects of DOX.This study includes the following two parts:Part 1 Preparation and characterization of drug-loaded MSNsObjective To prepare MSNs and DOX/MSNs and evaluate their characterization.Methods Hydrothermal synthesis method was conducted with Benzylcetyldimethylammonium chloride(BCDAC)as a porogen for the formation of mesostructured and a phase transfer agent,TEOS as the silica precursor and alkali as the catalyst.The pH,temperature,and reaction time of the system were precisely controlled,MSNs were fabricated by removing the template several times from a 2%concentrated hydrochloric acid absolute ethanol mixed solution.Doxorubicin(DOX)was encapsulated in mesoporous silica nanoparticles(MSNs)to prepare DOX/MSNs by heating solvent evaporation.The morphology,pore size,particle size,and surface potential of the MSNs nanoparticles were cell-defined by transmission electron microscopy(TEM),scanning electron microscopy(SEM),fully automatic specific surface measuring instrument,particle size potential analyzer in vitro.Changes in particle size were measured for 72 h and then evaluate the stability of MSNs.The drug encapsulation rate,drug loading rate and drug release of DOX/MSNs were observed by multifunctional microplate reader.Results(1)MSNs prepared by hydrothermal synthesis showed a uniform white powder with an average hydrated particle size of 307.4±16.0 nm and a surface potential of-18.30±4.16 mV The morphology was identified by transmission electron microscopy(TEM)as a monodispersed sphere with a grid structure.Scanning electron microscopy(SEM)has further confirmed that MSNs has a monodisperse sphere structure with uniform particle size and pores on the surface.The pore size of the MSNs examined by the TriStar Ⅱ full-automatic specific surface analyzer is about 3 nm. (2)The changes of hydrated particle size of MSNs were measured continuously for 72 h,the particle size of MSNs did not change significantly over time,and it remained at about 310 nm. (3)As the dosage of DOX increases,the drug encapsulation rate and drug loading rate of DOX/MSNs continue to increase.When the ratio of MSNs to DOX is 1:2,the drug encapsulation rate of DOX/MSNs is 99.96±0.2%,drug loading rate was 65.9 ±0.04%. (4)The microplate reader detects the drug release rate,the drug cumulative release rate in the carrier reaches 54.94%at 48 hours,.After that,the drug is hardly released further and is in a platform stage.Conclusion In this study,MSNs with good stability were successfully prepared,and a nano drugloading system DOX/MSNs with high encapsulation efficiency was constructed,which can slowly release drugs in vitro.Part 2 Construction of macrophage drug delivery system and evaluation of its killing effect on tumor cellsObjective To explore the chemotaxis and tumor cell killing effect of RAW264.7 that phagocyted DOX/MSNs on mouse prostate cancer cell line RM-1.Methods(1)The fluorescently labeled MSNs were co-incubated with macrophages RAW 264.7 at different times,the fluorescence intensity in the cells was observed by confocal laser microscope to determine the optimal incubation time.(2)CCK8 test was used to determine the toxic effect of MSNs on cell RAW264.7 under different incubation times(3)DOX and DOX/MSNs were co-incubated with RAW264.7 at the same drug concentration.The intracellular DOX fluorescence was observed by confocal laser microscope,and the phagocytosis rate was well-quantified by FACS(Fluorescence Activating Cell Sorter).(4)CCK8 test was used to determine the toxicity effect of DOX and DOX/MSNs on RAW264.7 under different concentration gradients.(5)Transwell migration experiment was conducted to evaluate the chemotaxis properties of MΦ-DOX/MSNs group,MΦ-DOX group and MΦ.6 fields were selected randomly under the fluorescence microscope to count the number of cells that migrated to the lower chamber.(6)In order to evaluate the killing effect on RM-1 tumor cells of MΦ-DOX/MSNs,MΦ-DOX in vitro,the CCK8 test was carried out and the relative cell viability was calculated.Results(1)The CLSM results demonstrated that the fluorescence intensity in the cells increased with the incubation time and almost saturated at 6 h.(2)CCK8 test proves that under the same conditions,MSNs have no obvious toxic effect on RAW264.7 when the incubation time is less than 12 h.(3)The CLSM results showed that compared with the intensity of red fluorescence in cells,DOX/MSNs group is much larger than DOX group.Flow cytometry results indicated that 99.4 ± 0.61% of RAW264.7 cells showed significant uptake of MSNs comparing with the blank group(21.8 ± 1.35%).There are significant statistical differences between the two groups,P <0.01.(4)The CCK8 test evaluated the toxic effects on macrophages between DOX/MSNs group and DOX group.At a drug concentration of 5μg /m L,the survival rate of the RAW264.7 cells treated with DOX/MSNs was 86.9 ± 0.05%,the survival rate of cells in the DOX group was 63.6 ± 0.01%,which had a significant statistical difference,P <0.01.With the increase of drug concentration,significant statistical differences remained between the two groups,P<0.01.(5)Transwell migration experiment show that the number of migrating cells per well of MΦ-DOX/MSNs group is significantly greater than that of MΦ-DOX group(P<0.01),but there is no significant difference with MΦ group,P>0.05.(6)The results of the CCK8 test showed that MΦ-DOX/MSNs group showed strong ability to kill tumor cells.In contrast,the killing ability of MΦ-DOX group was weak,there are significant statistical differences between the two groups,P <0.01.Conclusion Macrophages can engulf a large number of DOX/MSNs and their activity is not affected.The constructed macrophage drug-loading system(MΦ-DOX/MSNs)can chemoattract mouse prostate cancer cells and produce a killing effect.