Preparation Of Smart Drug Delivery System Based On ZIF-8 And Their Effects On Autophagy In HeLa Cells

Metal-organic frameworks(MOFs),as a kind of hybrid materials,composed of metal ions and organic ligands,have received wide attention.Due to their attracting characteristics,such as high stability,large internal surface areas,uniform porosity and a great diversity of architectures,many attempts have been made to employ MOFs in drug delivery system(DDS).Zn-MOFs is regarded as a potential drug carrier owning to the wonderful biodegradability and lowcytotoxicity.As a representative of Zn-MOFs,zeolitic imidazolate framework nanoparticles(ZIF-8 NPs)are the most promising drug delivery hosts because of excellent chemical and thermal stabi lity under neutral conditions.More importantly,the pH-sensitive property allows ZIF-8 NPs to be decomposed and to control drug release in the acid environment of cancer cells.To enhance therapeutic effects of an anticancer drug and to reduce side effects on normal cells,some target ligands can be decorated on the surface of drug carrier.It is well-known that folate receptors(FR)are generally overexpressed on a number of cancer cell surfaces and can identify folic acid(FA),which can promote a selective uptake into cancer cells.However,covalent binding of FA on ZIF-8 requires a series of complicated processes via chemical modification and leads to the aggregation of nanoparticles.Since polyethylene glycol(PEG)is considered as a stability and compatibility material,FA-PEG attached on the surface of ZIF-8NPs is tried to be an ideal targeting molecule.Recently,studies have reported that autophagy is an important mechanism for cancer treatment.The induced autophagy can supply cancer cells with energy source,which allows cancer cells surviving in rugged environments.Furthermore,clinical trials have also exhibited the favorable effects of chloroquine,a classic autophagy inhibitor,as a novel anti-cancer drug.When autophagy is suppressed through autophagy inhibitor,it can block the recycle of nutrients and then kill cancer cells.Although chloroquine is usually studied in autophagy as an autophagy inhibitor,its derivative,chloroquine diphosphate(CQ),is rarely studied.Recent investigations suggest that autophagy deficiency can be induced by CQ as an autophagy inhibitor.The functionalities of CQ are considered more likely to be the coordination sites.Thus,based on ZIF-8 NPs and CQ,a novel smart DDS with specific target and pH-responsive release is reported in this work.The ZIF-8 nanoparticles encapsulating CQ(CQ@ZIF-8 NPs)were fabricated by a simple one-pot method.The coordination polymer was formed by the self-assembly of metal ions and drug molecules in which the drug molecules were then replaced by 2-methylimidazole.With the formation of MOF structure,drug molecules were encapsulated in MOFs NPs.CQ@ZIF-8 were then decorated by methoxy poly(ethylene glycol)-folate(FA-PEG),a special identifier for cancer cells,to form FA-PEG/CQ@ZIF-8.A series of analysis methods including scanning electron microscope(SEM),transmission electron microscope(TEM),powder X-ray diffraction patterns(PXRD),dynamic light scattering(DLS),fourier transform infrared spectroscopy(FTIR)and ultraviolet-visible spectroscopy(UV-Vis)were used to determine the size,crystal structure and drug loading capacity of the NPs.The FA-PEG/CQ@ZIF-8 NPs whose size is 250 nm,is regular octahedron structure and have high drug loading capacity(up to 18 wt%of CQ).The effects of different form of drug(CQ,CQ@ZIF-8 NPs,CQ+ZIF-8 NPs,FA-PEG/CQ@ZIF-8 NPs)on the viability of cells were determined by MTT assay.The experiments proved that CQ was encapsulated in ZIF-8 rather than by physical mixing processes and the existence of ZIF-8 enhanced the cytotoxicity of CQ on HeLa cells.The results of in vitro drug release showed that CQ in ZIF-8 frameworks was released fully only under low pH conditions,demonstrating FA-PEG/CQ@ZIF-8 NPs had good pH-responsive CQ release ability.CQ release from CQ@ZIF-8 was further probed under biomimetic cell membrane like environment of liposomes and SDS micelles.The fluorescence quenching behavior shows more hydrophobic interaction of CQ@ZIF-8 with SDS micelles than CQ with SDS micelles.The bioadhesion of CQ@ZIF-8 with cell membrane like environment offers a strong stand towards its bioavailability.The cellular uptake of nanoparticles of different form drug was qualitatively and quantitatively determined by transmission electron microscope(TEM)and inductively coupled plasma mass spectrometry(ICP-MS),respectively.The target identification of FA-PEG/CQ@ZIF-8 NPs,compared with CQ@ZIF-8 NPs,makes increasing number of the NPs internalized into HeLa cells,which decreases the loss of drug and leads to high cytotoxicity of CQ for cancer cells.The effects of different form of drug on autophagy inhibition were further explored with the combination of Western Blot assay,fluorescence labeling and flow cytometry.The quantitative measurements of autophagy related proteins and the detection of autophagy flux in HeLa cells suggest that the autophagosome formation and autophagy flux are appreciably blocked after the cells treated with FA-PEG/CQ@ZIF-8 NPs and there is a maximum LC3-I/LC3-? ratio in HeLa cells treated with FA-PEG/CQ@ZIF-8 NPs among all the investigated samples.Furthermore,the effect of autophagy inhibition of FA-PEG/CQ@ZIF-8 NPs against HeLa cells is better than that against HEK293 cells.These results reveals that the prepared FA-PEG/CQ@ZIF-8 NPs display targeting delivery in HeLa cells.The pH-responsive and tumor-targeted properties of smart drug delivery system can control the drug release and enhance the efficiency of autophagy inhibition.This work broadens the promising application of MOFs and autophagy inhibitors in drug delivery system of biomedical field.