Polymeric Short Fibers As Injectable Drug Carriers And Cell Substrates For Spheroid Culture

Drug screening and tissue repairs are the important research areas of biomedical materials.Determining the efficacy and toxicity of drugs is an important part of drug screening before clinical research.Two-dimensional(2D)culture is a commonly used in in vitro drug screening,however the results are always far from the actual clinical observations.Organ transplantation is the appropriate therapeutic strategy for tissue repairing.Inevitably it always suffers from the limited source of organ donors,immunological rejection after transplantation,and secondary damage to the body after the use of self-tissues.Therefore,the construction of tissues-like with similar functional and structures could provide an ideal in vitro model for drug screening and alternative organ transplantation for tissue repair.So it designed that short fibers as a scaffold cultured with cells on on agarose-coated plates for hepatocyte spheroids,tumor cell spheroids and mesenchymal stem cell spheroids,which was used for drug screening,tissue mimicry,cartilage repair,and treatment of arthritis.On the other hand,with injectable short fiber as a drug delivery carrier to treatment tumor by intratumoral injection to realize local targeted treatment of tumor.The establishment of a reliable in vitro liver model for drug screening remains challenging with respect to tethering the growth of hepatocyte spheroids and adapting to the current high-throughput system.In the current study,short fibers are utilized as scaffolds for the generation of size-controlled hepatocyte spheroids that recapitulate in vivo hepatic phenotypes and functions.The spheroid formation is modulated by the length and galactose/RGD grafts of short fibers,and short fiber with 50 ?m,95%PSMA blending ratio,grafting density of galactose and RGD were135.7 ± 4.5 nmol/mg and 14.8 ± 0.5 pmol/mg motivate the spheroid formation with optimal hepatic function.Short fibers distribute throughout the entire spheroid for tethering hepatocyte growth to form compact spheroids.When compared with scaffold-free spheroid culture on agarose-coated plates and examined the metabolic clearance rates of tolbutamide,s-warfarin,midazolam,testosterone,and acetaminophen,the spheroid culture with short fibers achieves higher clearance rates of model drugs and provides a better prediction of the in vivo drug clearance rate with a correlation value of 0.886.In addition,the drug metabolism capability is highly sensitive to the inducers and inhibitors of metabolizing enzymes,and the responsiveness is maintained during 20 days of culture,exhibiting an efficient in vitro model for determining drug-drug interactions.The liver regulates glucose and lipid metabolism.Spheroid culture of rat primary hepatocytes(rPH)and hepatoma cell lines(HepG2)is established using injectable short fibers with galactose grafts(gSF)as the substrate to imitate liver for research of glucose and lipid metabolism.Compared with HepG2 spheroids,rPH spheroids after coculture with gSF show strong glucose metabolism abilities in terms of glucose consumption,intracellular glycogen content,gluconeogenesis rate and sensitivity to glucose modulator hormones like insulin and glucagon.On the other hand,HepG2 spheroids after coculture with gSF display strong lipid metabolism abilities from high levels of total cholesterol and triglyceride.The above experimental results were verified by characterization of the activity of phosphoenolpyruvate carboxykinase(PEPCK)and adenosine 5'-monophosphate-activated protein kinase(AMPK).Compared with those spheroids without short fibers,the fiber-supported spheroid culture models show significantly higher sensitivity and clinically related response to hypoglycemic(dexamethasone,metformin and pioglitazone)and lipid-lowering drugs(metformin and lovastatin).Thus,the culture configuration suggests a predictable in vitro platform for defining glucose and lipid metabolism profiles and screening therapeutic agents for metabolism disorders like diabetes and obesity.There was only a small percentage of drug delivered to tumors after systemic administration,and solid tumors also have many barriers to prevent drug penetration within tumors.In the current study,intratumoral injection of drug-loaded fiber fragments was proposed to overcome these barriers,allowing drug accumulation at the target site to realize the therapeutic efficacy.Fragmented fibers with hydroxycamptothecin(HCPT)loaded were constructed by cryocutting of aligned electrospun fibers,and the fiber lengths of 5(FF-5),20(FF-20),and 50 ?m(FF-50)could be easily controlled by adjusting the slice thickness.Fragmented fibers were homogeneously dispersed into 2%sodium alginate solution,and could be smoothly injected syringe needles.FF-5,FF-20 and FF-50 fiber fragments indicated similar release profiles except a lower burst release from FF-50.In vitro viability tests showed that FF-5 and FF-20 fiber fragments caused higher cytotoxicity and apoptosis rates than FF-50.After intratumoral injection into murine H22 subcutaneous tumors,fragmented fibers with longer lengths indicated a higher accumulation into tumors and a better retention at the injection site,but showed less apparent diffusion within tumor tissues.In addition to the elimination of invasive surgery,HCPT-loaded fiber fragments showed superior in vivo antitumor activities and fewer side effects than intratumoral implantation of drug-loaded fiber mats.Compared with FF-5 and FF-50,FF-20 fiber fragments indicated optimal spatial distribution of HCPT within tumors and achieved the most significant effects on the animal survival,tumor growth inhibition and tumor cell apoptosis induction.It is suggested that the intratumoral injection of drug-loaded fiber fragments provided an efficient strategy to improve patient compliance,allow the retention of fragmented fibers and spatial distribution of drugs within tumor tissues to achieve a low systemic toxicity and an optimal therapeutic efficacy.In order to establish a tumor-like,it designed short fiber as scaffold based on poly(lactic acid)-polyethylene glycol/polyethylene oxide(PELA/PEO),and incorporated with 17?-estradiol(E2)which could promote breast cancer cell line(MCF-7)proliferation spheres.E2 loaded short fibers were used as scaffold culturing with MCF-7 into spheroid.The content of PEO and the loading amount of E2 had effect on drug release and the viability,proliferation and adhesion of MCF-7.When the content of PEO was 3%and the loading amount of E2 was 0.01%in E2 loaded short fibers,the mature malignant tumor MCF-7 spheroids with 250 ?m could be obtained after culturing with this scaffold for 6 days.The MCF-7 spheroids were similar to malignant tumors in structure and expression of factors,that is,cells in central of spheroids grew densely and suffered from internal hypoxia,which always found in malignant tumors.MCF-7 spheroids were characterized by,the secretion of E-cadherin and malignant factors,and the expression of genes related to epithelial-mesenchymal transformation(EMT)compared to malignant tumors,and it showed that MCF-7 spheroids had a tendency of malignant invasion and metastasis.The gene expression of cancer stem cell(CSC)phenotype of MCF-7 spheroids and the construction of tumor in vivo indicated that these spheroid had strong tumorigenicity.All results indicate that MCF-7 spheroids were similar to malignant solid tumors,therefore can be used as an tumor-like model in vitro for new drugs development and tumor diseases researchCartilage has no self-repairing function,so cartilage tissue engineering was used to repair cartilage defects.Kartogenin(KGN),a chondrocyte inducer small molecule drug,incorporated into PELA short fibers cultured with bone marrow mesenchymal stem cells(BMSC)for BMSC spheroids,which have a potential to differentiate into cartilage.BMSC spheroids were encapsulated with an injectable hydrogel to prevent cell loss from defects.The results showed that the loading amount of KGN,the mechanical properties of hydrogel and the injection process would affect the vitality and chondrocyte differentiation of the BMSC spheroids.And the mechanical of hydrogel was strongly correlated with hydrogel concentration,reaction ratio and the amount of short fibers incorporated into hydrogel.Finally,the desired parameter of scaffold was that the hydrogel concentration was 3%,the proportion of sulfhydryl/acryloyl(HS/Acr)was 1/1.2,the short fiber doping concentration was 1%,and loading amount of KGN was 0.1%,which was most effective in promoting BMSC differentiation into chondrocytes.In vivo cartilage repair,the defect was filled with newborn mature cartilage after treatment with BMSC spheroids composite scaffold for 6 weeks and completely repaired after 12 weeks.Subsequently,this cartilage tissue engineering was combined with the anti-arthritis drug celecoxib(CLX)to treat osteoarthritis.The results showed that the synergistic effect of two component could inhibit the development of arthritis.After 12 weeks,the symptoms of arthritis were relieved and the lesions cartilage defects were repaired.In conclusion,short fibers are used as injectable drug carrier for subcutaneous tumors treatment by intratumoral injection,and can be used as a scaffold for culturing cell spheroids to simulate liver-like,tumor-like and cartilage-like tissue for liver drug-metabolizing screening,glucose and lipid metabolism drug screening,tumor-like construction in vitro,cartilage repair and arthritis treatment in vivo respectively,which offer new ideas and means for drug screening,organ repair and disease treatment developments.