| Ganoderma lucidum spore powder(GLSP)has attracted increasing attention due to its abundant bioactive substances and multiple functions.Here,the bioactive substances(chitosan,proteoglycan)was obtained from GLSP using deacetylation and maceration extraction,respectively.Then,the applications of chitosan and proteoglycan in drug delivery with core-shell structure have been completed via electrospinning technology and electrohydrodynamic 3D printing technology.And obtained the drug delivery possessed multiple functions of regulating drug release rate,combination drug therapy,and synergistic therapy.And thereby,the therapy that can fully exert the pharmaceutical value of natural bioactive component and other commercial drugs with minimizing adverse effects together with optimizing efficacy was fulfilled in theory.Firstly,chitosan was prepared from GLSP.Among them,the effects of thermochemical deacetylation(TCD)and ultrasound assisted deacetylation(USAD)on the physicochemical characteristics(degree of deacetylation-DD,dynamic viscosity-[η],and viscosity average molecular weight-Mv)were compared;then,the effects of dual-frequency ultrasound on these physicochemical characteristics were studied further.Besides,a novel natural proteoglycan from GLSP was obtained using maceration(deionized water)extraction;and then,the differences among the characteristics,such as amino acids content,monosaccharides content,hypoglycemic effects,and inhibition effects on HeLa cells between proteoglycan-C(from cracked well GLSP)and proteoglycan-UC(from uncracked well GLSP)were explored.As the results shown,chitosan obtained via both processes displayed good thermostability,excellent biocompatibility;although the chitosan prepared from USAD exhibited much significantly improved fibroblast(L929 cell)viability and enhanced antibacterial zones for both Escherichia coli(E.coli)and Staphylococcus aureus(S.aureus).The morphology of chitosan from TCD was smooth,comparing with the tiny embossments covered the surface of chitosan from USAD.And the USAD possessed the advantages of less time consumption,higher efficiency,lower concentration of required alkali solution,and lower temperatureBesides,the enhanced effects can be obtained from dual frequency ultrasound,which improved the deacetylation process and increased the DD value,although the chitosan obtained from parallel orientation possessed lower[η]and Mv compared with orthogonal orientation.The generated proteoglycan showed effective inhibition on the proliferation of HeLa cells and antibacterial effects on E.coli and S.aureus.While,the molecular weight of protein contained in proteoglycan-UC(55,72,95 kDa)was different to that of proteoglycan-C(43,95 kDa).These amino acids(Glu,Arg,Leu and Lys)content and monosaccharides(Fuc,Gal,Glc,Man)content between proteoglycan-C(7.38%,0%,6.35%,6.32%;3.31‰,13.32‰,247.96‰,2.6‰)and proteoglycan-UC(9.06%,10.74%,3.87%,0%;1.19‰,7.42‰,524.28‰,6.39‰)were differed obviously.The 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)(ABTS)and 1,1-Diphenyl-2-picrylhydrazyl(DPPH)radical scavenging activities of proteoglycan-C were higher than that of proteoglycan-UC.The proteoglycan-UC exhibited stronger hypoglycemic and antibacterial effects against E.coli and S.aureus.Secondly,the randomly arranged core-shell structure fiber membrane was fabricated using electrospinning technology,the 5-FU and chitosan was encapsulated into core and shell level,respectively.The water-soluble polyvinyl pyrrolidone(PVP)and water-insoluble polycaprolactone(PCL)was used as core and shell matrix respectively,which for the achievement of therapy on cutaneous melanoma(B16F10 cells)with minimizing adverse effects.As the results indicated,the yielded nanofibers exhibited an average diameter of 503 nm with high drug-encapsulating efficiency and good mechanical properties.Moreover,the rapid release of 5-FU significantly inhibited B16F10 cells,and the sustained release of chitosan exhibited "remedying effects" on L929 cells after suffering adverse effects from 5-FU treatment.For the B16F10 cells,the early apoptosis cells increased from 0.8%to 62.2%after being treated for 24 hours;for the L929 cells,the vital cells increased from 68.9%to 77.0%,and the early apoptosis of stage cells decreased from 12.3%to 10.9%.The results here indicate this synergistic treatment for cutaneous melanoma can effectively inhibit the proliferation of B16F10 cells,but also reduce the side effects of 5-FU on normal cells,providing a reference for antitumor with minimal adverse effects.Finally,a patterned core-shell structure fiber membrane was fabricated using EHD 3D printing technology.Cellulose acetate and polyacrylic resin Ⅱ was used as outer and inner layer matrix,respectively,and the stachyose and proteoglycan were loaded using the membranes.The impact of membrane geometry and drug-loaded site on drug release behavior and physicochemical properties of the resulting fibers were studied As the results shown,the membrane with the three geometries can be stacked precisely to 10 levels,the distribution of fiber mean diameter was 46-88 μm.The printed fibrous membranes possess a biphasic drug release profile with a burst release within the first 12 hours and a slower sustained release up to 72 hours,and the release rate of stachyose was higher in outer layer.The obvious differences of mechanical properties showed among these geometries’ membrane,the maximum tensile strengths are 3.3’MPa(full circle),2.3 MPa(semi-circular),2.9 MPa(grid).The fiber membrane can increase the proliferation of Bifidobacterium bifidum(B.bifidum)to 294.2%and at the same time reduce the proliferation of E.coli to 37.0%.These results here indicated the composite fiber membranes can significantly promote the proliferation of B.bifidum,and significantly inhibit the proliferation of E.coli at the same time.Thus,a method can significantly improve the intestinal microbial ecosystem was found,providing a theoretical reference for the improvement of the human intestinal microbial system. |
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