Construction And Application Of Gene/Drug Carriers Based On Lentinan/Polydeoxynucleotide Composite

Natural polysaccharides have been widely applied in gene/drug delivery and other biomedical fields due to the specific characteristics of nontoxicity,renewability,biodegradability and easily modified property.Lentinan(LNT)is a kind ofβ-(1→3)-D-glucan with triple helix chain conformation,which possesses anti-inflammation,antitumor and immunomodulatory activities.More interestingly,the chain conformation of LNT can be transformed from the single random coils(s-LNT)to the triple helix chain conformation(t-LNT)by changing the solvent environment.And two s-LNT chains can selectively combine one single chain of polydeoxyadenylic acid(poly(dA))to form a hetero-triple helical composite(s-LNT/poly(dA)composite)via hydrogen bonding under the re-assembling condition of s-LNT.The s-LNT/poly(dA)composite owning good stability and inheriting the biosecurity,biocompatibility and biodegradability of LNT has been successfully used to fabricate intelligent and efficient gene carriers,evidenced by in vitro cell experiments.However,the study of the application in vivo of the carrier based on s-LNT/poly(dA)composite has not been reported.Therefore,this paper aims to optimize the s-LNT/poly(dA)system for constructing more novel drug/gene vectors,and evaluate their effects through their animal experiments,so as to further extend the application of LNT and s-LNT/poly(dA)composite in the biomedical field.The innovative points of this work are as follows.(1)For the first time,the antisense oligodeoxyribonucleotide of TNF-αwith the function of down-regulation of TNF-αlevel was successfully delivered to the inflamed colon by the s-LNT/poly(dA)composite,which was encapsulated into the chitosan-alginate(CA)hydrogel,through intragastric gavage.(2)The acid-sensitive LNT-doxorubicin(LNT-Dox)conjugate was synthesized through the sodium periodate oxidation,and two LNT-Dox single chains(s-LNT-Dox)combined with one poly(dA)chain to form s-LNT-Dox/poly(dA)composite with an extended chain morphology.(3)Dox and the dye could be simultaneously delivered into HeLa cells or the tumors with a prolonged duration time by the s-LNT-Dox/poly(dA)-fluorophore composite,for the first time.LNT-Dox conjugates showed enhanced cytotoxicity against HeLa cancer cells with time,and effectively inhibited tumor growth and decreased adverse effects of the free Dox in vivo.(4)The aptamer and polyethyleneimine(PEI)were introduced into the s-LNT/poly(dA)system for improving the cellular uptake of s-LNT/poly(dA)composite,for the first time.The main contents and conclusions in this project are divided into the following parts.s-LNT/poly(dA)composite carrying antisense oligodeoxyribonucleotide of TNF-αwas encapsulated in the CA hydrogel for the targeted treatment of colitis.The experimental results from an inverted fluorescence microscope,scanning electronic microscopy,circular dichroism spectra and fluorescence spectra demonstrated that the s-LNT/poly(dA)composite was uniformly dispersed in the CA hydrogel without apparent leakage in the neutral buffer solution and the simulated gastric acid fluid.The in vitro release experiments demonstrated that the CA hydrogel could protect the encapsulated agents against the external extreme conditions(pH 1.2 and pH 7.4),and specially release the loaded cargoes under the simulated inflammatory colonic fluid(pH 6.8).The in vivo bio-distribution and ex vivo imaging studies indicated that the CA hydrogel could prevent the leakage of the loaded cargoes,leading to a tiny amount of loaded drugs penetrating the intestinal wall into the blood circulation causing side effects.The CA hydrogel encapsulated the s-LNT/poly(dA)composite could be quickly transferred to the intestinal canal after intragastric gavage,avoiding the adverse impact of the acid condition in stomach on the CA hydrogel.And the CA hydrogel loaded with s-LNT/poly(dA)composites was cleared along with excrement within 24 h.The images of cryosection of the colon tissue indicated that the loaded s-LNT/poly(dA)composites could be specifically released from the CA hydrogel and were internalized into colon cells.The treatment of colitis in vivo demonstrated that the CA hydrogel loaded with s-LNT/poly(dA)composites could alleviate the weight loss of the mice and the lesion of colon and spleen in mice,and down-regulate pro-inflammatory factors including TNF-α,myeloperoxidase(MPO)and malondialdehyde(MDA),thereby relieving of the mice colitis.This work proposes a novel strategy for alleviating inflammation through oral antisense oligonucleotide,which has a broad application prospect in the targeted treatment of intestinal inflammation.The t-LNT-Dox conjugate was prepared based on the special linkage of the?-(1?3)-D-glucan and?-(1?6)-D-glucose side group of t-LNT through Schiff-base reaction,and the interaction between the single chain of t-LNT-Dox(s-LNT-Dox)and poly(dA)was studied.The experimental results from ~1H NMR spectra,FT-IR spectrum,UV-vis spectrophotometry,and atomic force microscopy(AFM)demonstrated that the t-LNT-Dox conjugate was successfully prepared,and the Dox content in t-LNT-Dox was measured to be 6.2%.t-LNT-Dox exhibited the extended chain morphology in accordance with the isolated chains of t-LNT,however t-LNT-Dox showed the stronger aggregation tendency than t-LNT due to the hydrophobic Dox.In addition,Dox could be released from t-LNT-Dox in a controlled manner under the acidic condition due to the acid-responsive oxime bond(-CH=N)in t-LNT-Dox.The experimental results from AFM images,transmission electron microscope,CD spectra and agarose gel electrophoresis retardation assay demonstrated that s-LNT-Dox possessed the same ability as s-LNT of binding with poly(dA)to form the s-LNT-Dox/poly(dA)composite.Moreover,it was proved that Dox and the dye could be simultaneously delivered into HeLa cells by the s-LNT-Dox/poly(dA)-fluorophore composite,which exhibited efficient tumor accumulation and bioimaging in vivo.The studies of cellular uptake and cytotoxicity of t-LNT-Dox in vitro revealed the effective cellular internalization and antiproliferative activity of t-LNT-Dox against HeLa cells.The t-LNT-Dox conjugates also could effectively inhibited tumor growth and significantly reduced the adverse effects of Dox in vivo.This work provides a safe and efficacious strategy to combine cancer chemotherapy and imaging into one platform,showing great potential in the construction of theranostic nanomaterials.The cell uptake of s-LNT/poly(dA)composite was enhanced through introduction of a nucleic acid aptamer and PEI.The results of CD spectra demonstrated that the AS1411 aptamer linked at the tail of poly(dA)hardly affected the interaction between s-LNT and poly(dA).Meanwhile,the fluorescin isothiocyanate(FITC)was grafted to t-LNT to prepare t-LNT-FITC,the single chain(s-LNT-FITC)of which could interact with poly(dA)-AS1411 to form s-LNT-FITC/poly(dA)-AS1411 composites.The results of confocal microscopy and flow cytometry experiments showed that the AS1411 aptamer could effectively enhance the cellular uptake of s-LNT/poly(dA)and s-LNT-FITC/poly(dA)composites.In addition to AS1411,the aptamer MUC1 also used to improve the cellular uptake of s-LNT-FITC/poly(dA)-MUC1,and the cellular uptake increased with the concentration of poly(dA)-MUC1.The results of CD spectra demonstrated that the intruction of PEI hardly affected the stability of the s-LNT/poly(dA)composite.The cell experiments proved that the cellular uptake of s-LNT/poly(dA)composites was significantly enhanced after intructing PEI,and the interaction of s-LNT and poly(dA)to form the s-LNT/poly(dA)composite was necessary for improving the cell uptake.Meanwhile,the addition of PEI could significantly promote the cellular uptake of s-LNT-FITC/poly(dA)-AS1411 composite.This work proposes an effectively strategy for improving the cellular uptake of the s-LNT/poly(dA)system,which will greatly promote the application of the s-LNT/poly(dA)system in the biomedical field.In summary,a gene vector for the treatment of colitis and a theranostic platform for simultaneous delivery of Dox and fluorophore were fabricated based on the s-LNT/poly(dA)composite in this thesis,and a promising strategy was proffered to improving the cellular uptake of s-LNT/poly(dA)system by introducing of the aptamer and PEI.This thesis dramatically extends the application range of the s-LNT/poly(dA)composite,and provides scientific data for the biomedical application of lentinan,showing important academic values and potential applications.