Preparation,Characterization And Performance Of Folate-targeted Polysaccharides Composite Hydrogel

Folate（FA）is high affinity between folate receptors（FR）,which are overexpressed in cancer cells.Folic acid targeting delivery system is designed by coupling folic acid to the surface of the drug delivery system and loads antitumor drug to improve drug bioavailability and reduce side effects.Plant polysaccharide and its drug carrier are a kind of natural polymer materials with high biocompatibility and environment-friendly.Currently,folate-targeted polysaccharides and their drug delivery carrier are mainly focus on the research of cellulose and starch.The folate-targeted polysaccharides carrier systems are beneficial to antitumor drug targeting release,and become research hotspots in the field of polysaccharides.In this study,FA conjugated sodium alginate（FA-ASA）were synthesized by applying1-（3-Dimethylaminopropyl）-3-ethylcarbodiimidehydrochloride/N-hydroxysuccinimide（EDC/NHS）activation method of SA and FA.Primary amine（NH₂）of ethylenediamine couples with activated-folate and activated-SA..Folate-targeted composite polysaccharide hydrogels were prepared by FA-ASA and Fenugreek gum（FG）which has high apparent viscosity characteristics.The targeting effect of FA composite polysaccharide hydrogels was evaluated by cell experiments in vitro.The research contents of this thesis as follows:1.Synthesis and characterization of aminated sodium alginateAminated sodium alginate（ASA）were synthesized by using ethylenediamine.HCl was added to SA to promote protonation and form alginate.Alginate can bind with N of formamide that destroy intramolecular hydrogen bonding of SA,and increase solubility of SA.The order of dropping material liquid directly have relate to type of the ASA,adding SA into ethylenediamine is conducive to form monoamide which are propitious to subsequently couple with FA.In the choice of activators,EDC firstly coupled with SA forming reactive intermediates,the adding of NHS made the intermediates convert into the active esters which the intermediates and active esters can react with ethylenediamine.The elemental analysis were investigated that reaction activities was not significantly increased for amidation reaction when NHS was added into EDC.ASA was synthesized as EDC catalysis has the highest N content（11.747%）with substituted degree 0.796.The synthesized ASA are characterized by FT-IR,X-ray photoelectron spectroscopy（XPS）and ¹H NMR and ¹³C NMR.FT-IR spectrum indicated that the peak at 1615.72 cm^-1 and 1545.81 cm^-1 stand for the amide of ASA.XPS analysis results showed that the N signal intensity of the amide was slightly higher than the ethylenediamine,and revealed a small amount of bisamide has been produced.It was concluded that the amide reaction has been occured between the-COO^-of SA and the-NH₂ of ethylenediamine.New hydrogen were occured inδ6.379 andδ7.9 in ¹H NMR,Furthermore,in ¹³C NMR analysis,the signals of new carbon were occured inδ164.76 andδ164.23.It were demonstrated that ethylenediamine and SA were successfully coupled.2.Synthesis and characterization of folate modified ASAFA modified sodium alginate（FA-ASA）were synthesized using ASA as raw material applying three catalysts ethylamine（TEA）,pyridine（Py）and 4-two methyl pyridine（DMAP）,respectively.The elemental analysis showed that the content of N（%）of FA-ASA systhesized by TEA、Py and DMAP（with highest DS of 0.304）were16.882%、13.86%and 18.348%,respectively.DMAP enhanced the nucleophilic attack ability of N atoms because of the methyl push effect on the pyridine ring.FT-IR results showed that the characteristic peaks of benzene ring has been occured;XPS analysis results incidated the Na signal of the FA-ASA has been disappeared and N signal was enhanced compared with ASA.The change of binding energy for carbon inner electron demonstrated that FA and ASA were reacted.The new hydrogen signals atδ8.816、δ3.892、δ2.785、δ3.892、δ0.767 in solid ¹H NMR,and new carbon signals atδ155.7、δ149.2、δ129.3、δ113.95、δ45.19、δ31.75 in solid ¹³C NMR indicated FA and ASA was successfully coupled.3.Preparation and performances of composite drug-loaded hydrogel based FA-ASAFA-ASA_composite/MTX drug-loaded hydrogels were prepared using FA-ASA,SA and FG as raw materials,methotrexate（MTX）as model drug,through calcium carbonate/D-gluconate lactone（CaCO₃-GDL）in situ.The drug-loaded capacity of FA-ASA_composite/MTXhydrogelswas9595.80μg/gdetectedbyUV spectrophotometry.Swelling behavior showed that the swelling degree of FA-ASA_composite hydrogels was higher than SA hydrogel.The rheological results showed that the storage modulus（G’）of FA-ASA_composite hydrogels was slightly lower than that of SA hydrogel.As a result,the cross-linking sites of the carboxyl group and Ca²⁺were reduced by the effective cross-linking strength of composite gel.In vitro release results showed that the swelling ability and the effective cross-linking strength of composite gels affect the release rate of drugs.At low pH environment,MTX release rate of FA-ASAcomposite/MTX hydrogel（600min release completely）was slightly faster than that of SA/MTX drug-loaded gels.Human hepatoma HepG-2 cells and human lung cancer A549 cell models in vitro were established to verify the targeting ability of FA-ASA_composite/MTX drug-loaded gels.The IC₅₀ of FA-ASA_composite/MTX was reduced to 180.5 and 81.3μg/mL for A549 and HepG-2,respectively.It has been indicated that folate modified composites could mediate more MTX enriched in tumor cells.After adding free folate occupied surface folate receptor of tumor cells,the IC₅₀ of FA-ASA_composite/MTX increased to 97572.38 and 497.9μg/mL（6 h）for A549 and HepG-2,respectively.It was confirmed that the free FA and folate modified composite compete for FR on the tumor cell surface reducing MTX entry into the tumor cells via the folate receptor pathway.Further,A549 cells nucleus fluorescence were stained by 4’,6-two-2-guanyl phenylindole（DAPI）.The change of nucleus shape showed that FA-ASA_composite/MTX could accelerate the cracking of nucleus of A549 cells and produce large numbers of apoptotic bodies.But,the cracking of A549 cell nucleus was weakened when the free FA was increased.The results that demonstrate that FA of FA-ASA_composite/MTX gel can identify with folate receptors,thereby mediating the MTX entry into tumor cells to achieve drug delivery.In summary,folate-targeted of polysaccharides hydrogel were prepared to improve the targeting effect of vitro tumor cells in our studied.However,the different agent of folate targeted polysaccharide drug delivery is still at the primary stage.More subjects should be designed for microspheres,nanometergels and micelles of polysaccharide-based.Our research provided guiding significance for the preparation of folate targeting polysaccharide-based drug delivery carriers.