Hyaluronan Modified Drug Delivery System Of Milk Exosomes

Exosomes are essentially membrane-derived nano-vesicles?30-150 nm?that can transfer functional cargoes including proteins,nucleic acid as well as lipids into recipient cells,which have been recognized as a vital means for the intercellular communication.Moreover,the lipid bilayer structure of exosome exhibits high biocompatibility,stability in the circulation and low immunogenicity,rendering them as viable drug and gene delivery vectors for cancer therapy.However,exosomes are mainly extracted from cell culture fluid nowadays,and the preparation process is cumbersome and expensive.In addition,exosomes lack targeting ability when used as nanocarriers and cannot deliver drugs to tumor sites specifically.In order to overcome the above defects,in this study,we selected economical available milk as the source of exosomes,and milk exosome?mExo?was isolated from milk by simple means such as isoelectric precipitation.At the same time,the hyaluronan?HA?conjugate DSPE-PEG₂₀₀₀-HA that can modify mExo was synthesized by chemical methods.Doxorubicin?Dox?and the tumor suppressor gene miRNA204 were selected to construct two types of mExo drug delivery systems for CD44 specifically targeting delivery,and in the subsequent experiments,we confirmed the targeted delivery ability and antitumor activity of the nano drug delivery system HA-mExo-Dox and the gene delivery system HA-mExo-miRNA204.The main conclusions are as follows:?1?Exosomes with complete membrane structure were isolated from milk by isoelectric point precipitation and ultrahigh speed centrifugation.The hydrated particle size of mExo was mainly distributed around 123.9 nm and the Zeta potential was-4.24 mV.?2?A nano-drug delivery system HA-mExo-Dox was constructed,and its good nanocarrier characteristics were verified by morphological observation,stability and in vitro sustained release;the drug delivery capability of HA-mExo-Dox was evaluated by fluorescence microscopy observation and flow cytometry analysis,the results showed that HA-mExo-Dox possessed the ability to efficiently deliver drugs to tumor cells;finally,the tumor cell killing ability was evaluated,and the results showed the killing capacity of HA-mExo-Dox against CD44 overexpressing MDA-MB-231,MCF-7 and A549 cells were 1.7,1.3 and 3.7 times than that of free Dox,respectively.?3?A gene transport system HA-mExo-miRNA204 was constructed,and its good nanocarrier characteristics were verified by morphological observation,particle size distribution and storage stability;fluorescence observation analysis showed that HA-mExo-mi RNA204 could deliver miRNA204 to CD44 over expressing tumor cells specifically;intracellular fluorescence quantitative analysis showed that HA-mExo-miRNA204 could increase the content of miRNA204 in MDA-MB-231,MCF-7 and A549 cells by 20.3,10.7 and9.3 times,respectively;in vitro cell proliferation experiments showed that HA-mExo-mi RNA204 could induce proliferation inhibition by 42.9%,35.0%and 21.1%of MDA-MB-231,MCF-7 and A549 cells,respectively.