Study Of Silk Fibroin-based Drug Loaded Intestinal Stents

As the increasing incidence of intestinal obstruction,especially the patients with acute obstruction caused by colorectal cancer(CRC)and in late phase,who could not tolerate surgery risk.If they failed to get effective treatment in time,it would endanger their safety.In clinical,stents combined radio-chemotherapy is an effective palliative treatment for CRC patients with advanced disease,high emergent surgical risk,multiple transfer rate and in poor general health.In the present project,a noval intestinal stent with good compatibility,degradability and local delivery ability was developed.The inner layer of the degradable stent was knitted from polydioxanone(PDO)monofilament and SF yarns;5-fluorouracil(5-FU)and curcumin(CUR)were dissolved in SF solution,and the solutions were eletrospun onto the weft-knitted stent.Firstly,PDO monofilament with a linear density of 105±5 tex and SF multifilament with a linear density of 5±1 tex were knitted into a tubular stent,the mechanical properties of prepared intestinal stent was higher than commercial enteral stent,meet the mechanical performance requirements of expand patients’ intestinal,realizing defecation and relieve obstruction.SF solution(23 wt%)and Poly(ethyleneglycol)(PEG)with a molecular weight of 400(PEG-400)(60 wt%)are mixed uniformly according to the volume ratio of 10:1(v/v).The given voltage was 24±0.5 k V,the injection speed of spinning solution was set at 0.6±0.05 m L/h and the distance between the needle tip and the roller was 20±0.5 cm.Three different drug loaded membranes were prepared by mixing CUR/5-FU at the concentration ratios of 0.15/0.25 wt%,0.3/0.5 wt% and 0.45/0.75 wt% by weight to SF solution of 23 wt%.The diameter of fibers increased,when the drug loadings of CUR and 5-FU increased to 0.45 wt% and 0.75 wt%,respectively.In the first two groups,spinning solution is stable,flow velocity is normal,and diameter of the fiber is uniform.When the drug loadings increased to 0.45 wt% and 0.75 wt%,the fiber beads,irregular shape,uneven diameter distribution of fibers were observed,even some fibers with the phenomenon of concave,flat and so on.The conductivity and viscosity of silk fibroin solution were tested respectively.Pore distribution,hydrophobicity,and secondary structure(FT-IR),crystal structure(XRD)and mechanical properties of silk fibroin membrane were tested.Meanwhile,the release of two drugs was detected.The drug-loaded membranes showed a burst-release behavior within 72 hours,the phenomenon of “sudden release” appears and then remained stable during 210-400 hours.Particularly,the release percentage of 5-FU reached 95.63±0.05 wt% at 96-hour,while the CUR reached this percentage until 210-hour.This is because the water solubility of 5-FU is much higher than that of CUR.For all three membranes with different drug loadings,CUR and 5-FU released completely within 400 hours,the highest release rate was 96.74±0.05 wt % and 99.97±0.05 wt% respectively.Human colorectal cancer cells(HCT-116)and normal colonic epithelial cells(NCM-460)were cultured in vitro.The effects of drug loaded fibrous membrane on cell proliferation,differentiation,cycle and apoptosis were studied.Meanwhile,thymus immune deficiency BALB/c nude mice were used to construct a subcutaneous colorectal tumor model in nude mice.The safety and anti-tumor effect of silk fibroin membrane coated with 5-FU and curcumin were tested.The combination of curcumin and 5-FU on cell growth inhibition,S arrest effect and apoptosis rate were significantly higher than use alone respectively,which could enhance the anti-tumor effect and inhibite the growth of BALB/c nude mice tumor volume.The SF/PEG fiber membrane has excellent biocompatibility as a carrier material for antitumor drugs.Meanwhile,SF/PEG fiber membrane could make use of its own material advantages to play the role of drug release,and realize the steady release of CUR/5-FU.Tumor cells absorbed completely,achieved the effect of cumulative therapy,improved the effect of cell S phase block,and increased the rate of cell apoptosis,so as to inhibit the growth of tumor volume at the highest degree,and no other side effects of BALB/c nude mice.Immunohistochemical staining and Western blot results showed that the CUR and 5-FU loaded fibrous membrane(0.3/0.5 wt%)significantly increased the inhibition of Stat-3 and NF-κB(P-65)protein expression,especially for the former.The antitumor effect was significantly higher than intraperitoneal injection of CUR(LD50)and 5-FU(LD50).In conclusion,this thesis developed the silk fibroin drug loaded fibrous membrane intestinal stent can play the role in expanding the narrow intestine,local sustainning release antitumor drugs,inducing apoptosis of tumor cell and inhibiting the tumor tissue growth.Therefore,the drug loaded stents will have great potentials for the use in the treatment of intestinal cancers in the future.