| Gastric carcinoma,which is a malignant tumor of gastric mucosa epithelium.Due to the lack of obvious clinical symptoms in the early stage,many patients with gastric cancer have reached terminal stage or have metastatic lesion at the time of diagnosis,and cannot be cured by surgery.5-Fluorouracil（5-FU）is an effective chemotherapeutic agent for the treatment of gastric cancer and is widely used as a nucleoside analogue in clinical treatment.However,there are two main problems associated with the clinical use of.One is the lack of targeting,which is easy to produce side effects,such as neurological disorders,abnormal renal and myocardial functions.The otheris the long-term use of 5-FU can lead to drug resistance.To solve the problem of non-targeting,targeted nano-drug delivery systems are usually constructed.Biomaterial-based carriers such as cationic peptides（RALA）and polyhydroxyalkanoates（PHA）have been used in the delivery of 5-FU due to their good biocompatibility,biodegradability,and non-cytotoxicity.Cur,as a polyphenolic compound extracted from Curcuma longa Linn.,has almost no toxicity and can sensitize cancer cells to 5-FU by blocking the drug efflux.However,in view of the different solubility of the two drugs,it brings great challenges in the process of drug delivery.Therefore,it is necessary to develop a drug delivery system compatible with the co-delivery of drugs with different solubility and realize the synergistic treatment of multiple drugs.In this study,two drug delivery systems were designed for cancer treatment based on the similar electronegativity characteristics of the 5-FU metabolite deoxyfluuridine（FUdR）to DNA.One is a targeted delivery system,which applies a targeted nanocarrier of cationic peptide carrier RALA fused with target molecule Affibody to adsorb electronegative FUdR chains to achieve targeted delivery of FUdR for antitumor therapy.The other is a hydrophobic microsphere formed by self-assembly of PHA andRALA to co-deliver the hydrophobic drug Cur and hydrophilic FUdR chains to achieve synergistic therapy of two drugs with different solubilities.The main studies are as follows:（1）Design and successful construction of affibody-RALA mediated 15 FUdR chains(FUdR15S)nanomicrosphere drug delivery system(FUdR15S@affi-RA).targeting molecule Affibody bound to RALA（affi-RA）,using the positive charge of affi-RA with negatively charged FUdR15S chains（15 consecutive FUdR）self-assembled by electrostatic interactions.The final microspheres with a particle size of 50 nm and rough surface were formed with a Zeta potential of+16.24 m V.Among them,the loading efficiency of FUdR15S@affi-RA is 1 mol RALA can load 105 mol FUdR,at which the molar ratio of RALA to DNA is 10（N/P=10）.Under simulated physiological conditions,FUdR15S@affi-RA showed excellent stability,and FUdR15S@affi-RA was able to selectively target human epidermal growth factor receptor 2（HER2）overexpressing N87 cells.Under simulated physiological conditions,FUdR15S@affi-RA exhibited excellent stability,and the former showed superior targeting and higher cytotoxicity against human epidermal growth factor receptor 2（HER2）overexpressing gastric cancer N87 cells compared with free FUdR.Furthermore,anticancer mechanism studies showed that FUdR15S@affi-RA significantly enhanced FUdR-induced Bcl-2/Bax expression and caspase 3,8,and 9 activity.This study suggests that the fusion vector affi-RA has broad potential as a nucleoside analogue drug delivery platform for cancer therapy.（2）A cationic RALA-coated PHA granule co-loading nucleic acid drugs FUdR and hydrophobic drugs Cur for the therapy of cancer cells was designed and successfully constructed.Poly（3-hydroxybutyrate-co-3-hydroxyhexanoate）（PHBHHx）,a member of PHAs family,served as the polyester material due to its efficient entrapment and sustained release.Firstly,Cur is wrapped in PHA particles by the emulsification/solvent evaporation method to form the hydrophobic core of Cur@PHA.Secondly,a Pha P-RALA fusion protein（RP）was constructed.Under the hydrophobic force,RP and Cur@PHA self-assemble into an amphiphilic cationic particle Cur@PHA-RP.Then,FUdR15S is adsorbed on the surface of Cur@PHA through electrostatic interaction with RALA,forming a new structure of Cur@PHA-RP/FUdR15S.The encapsulation rate of Cur in Cur@PHA-RP/FUdR15Swas 88.3%,and the drug load was 7.79%.The moprhology of Cur@PHA-RP/FUdR15Swas characterized by DLS,SEM and TEM in vitro.The results showed that Cur@PHA-RP/FUdR15S was a rough microsphere with a particle size of 100nm and a Zeta potential of+23.17 m A.In vitro experiments showed that Cur@PHA-RP/FUdR15S maintained a stable structure in serum and neutral environment without being destroyed.The results of MTT and flow cytometry suggested that the combined drug group produced more cytotoxicity than the single drug,which was due to Cur increase the sensitivity of FUdR to cells.Compared with the FUdR/Cur physical mixed group,Cur@PHA-RP/FUdR15Sshowed greater cytotoxicity and stronger anti-tumor effects.It indicated that PHA-RP protected FUdR and Cur from being degraded during the transportation process,and made the drug release continuously invivo,so as to playthe greatest effect in the body.In addition,the anticancer mechanism research concluded that,Cur@PHA-RP/FUdR15S could cause a decrease in Bcl-2expression in cells,an increase in Bax expression and an increase in caspase activity,which lead to cell apoptosis.As a biocompatible andnon-cytotoxic drug delivery platform,PHA-RP opens up the possibility of co-delivery of nucleic acid drugs and hydrophobic drugs. |
