Design Of Functionalized Electrospun Nanofibers For Circulating Tumor Cell Capture And Tumor Therapy Applications

In recent years,cancer has become a huge threat to human lives,and over 90%of cancer-related death is caused by tumor metastasis and recurrence.Due to the metastatic and invasive properties,tumor cells are able to shed from primary tumor tissue,enter into blood circulatory system and become circulating tumor cells?CTCs?.Detection of CTCs in peripheral blood is of great significance for the early diagnosis,the prediction of cancer development,and the evaluation of efficacy,prognosis and individualized treatment of tumors.However,it is very difficult to isolate or capture CTCs from blood via conventional means due to the extremely low concentration and heterogeneity of CTCs in the blood.Electrospun nanofibers have the advantages of large surface area to volume ratio,good biocompatibility,ability to mimic native extracellular matrix,and the advantages of easy preparation and surface modifications,thus enabling them to serve as an ideal platform for cancer cell capture applications.In this thesis,a series of functionalized electrospun nanofiber-based platforms were constructed.Through nanofiber surface modification and integration of microfluidic technology,the nanofiber-integrated microfluidic chips were fabricated for efficient capture and release of CTCs.And monodispersed short nanofibers or drug-loaded fibrous rings obtained by homogenization process were designed for CTCs sorting and tumor therapy applications.The main contents of the thesis are as follows:Aiming at the problem of low capture purity and difficult release of CTCs,we constructed a microfluidic nanoplatform integrated with zwitterion-functionalized electrospun nanofibers for specific capture and intact release of CTCs.Firstly,electrospun polyethylenimine/polyvinyl alcohol?PEI/PVA?nanofibers were brominated and modified with zwitterion poly?2-methacryloyloxyethyl phosphorylcholine??PMPC?via an atom transfer radical polymerization?ATRP?.After that,targeting ligand of arginine-glycine-aspartic acid?RGD?peptide linked with polyethylene glycol?PEG?was modified on the surface of nanofibers via a benzoic imine linker through the bromide group.The functionalized nanofibers were then integrated with a herringbone microfluidic channel system for dynamic capture and release of cancer cells.We show that zwitterion functionalization could significantly reduce the nonspecific adhesion of blood cells,and thus improve the capture purity.The functionalized nanofiber-integrated microfluidic chips are able to specifically capture?_v?₃ integrin-overexpressed cancer cells or CTCs with a high capture efficiency?91.8%?and considerable purity?33.1%?and to rapidly and non-destructively release the cells under an acidic pH condition within 30 min.Furthermore,the detection results of clinical blood samples reveal that the developed microfluidic chip integrated with zwitterion-functionalized PEI/PVA nanofibers may be potentially used for CTCs sorting in clinical utility.Based on the above research,we designed a unique microfluidic chip system integrated with zwitterion-functionalized aligned PEI/PVA nanofibers for efficient capture and rapid release of CTCs,through combining the strategies of aligned nanofibers,zwitterionic modification and disulfide bond cleavage.Firstly,aligned PEI/PVA nanofibers were brominated and modified with zwitterions of PMPC via an ATRP reaction.Meanwhile,the targeting ligand containing a disulfide bond was synthesized by thiol oxidation of cysteine and SH-PEG-FA?a PEGylated folic acid with one end of sulfydryl group?,and then was modified on the surface of nanofibers.The functionalized nanofibers were systematically characterized using different techniques and then integrated with a herringbone microfluidic channel system for capture and release of cancer cells.We show that PMPC immobilization renders the nanofibers with excellent antifouling properties against blood cell attachment,and the attached blood cells were further reduced to improve the capture purity of cancer cells by using aligned nanofiber strategy.The dynamic capture assay using the fiber-integrated microfluidic platform demonstrates that FA receptor-expressing cancer cells can be isolated with a high capture efficiency?92.7%?and considerable purity?43.4%?and can be rapidly detached from the nanofibrous substrates within 5 min by the introduction of high-efficient reducing agent tris?2-carboxyethyl?phosphine.These results as well as the isolation and detection of CTCs from blood of cancer patients suggest that the developed microfluidic chip may be potentially used for clinical cancer diagnosis applications.To solve the problem that CTCs isolated by conventional magnetic separation methods are tightly coated by magnetic materials,and difficult to be effectively released for the subsequent analysis,we combined the advantages of electrospun nanofibers and magnetic separation to design a novel magnetic short nanofiber?MSNF?system for efficient capture and release of CTCs.Firstly,PEI-stabilized iron oxide?Fe₃O₄?NPs?Fe₃O₄@PEI NPs?were synthesized through a one-step hydrothermal method.Afterwards,the synthesized Fe₃O₄@PEI NPs were incorporated within PEI/PVA nanofibers through a blended electrospinning process.The hybrid nanofibrous mat was crosslinked with glutaraldehyde vapor and homogenized to obtain MSNFs.Then,DNA aptamer,specifically targeting to epithelial cell adhesion molecule?EpCAM?was immobilized onto the surface of MSNFs via the linkage of 3-?maleimido?propionic acid N-hydroxysuccinimide ester to get the final product of aptamer-MSNFs.The formed aptamer-MSNFs with a mean diameter of 350 nm and an average length of 9.6?m display a quick magnetic response property,are capable to specifically capture cancer cells with an efficiency up to 87%,and enable non-destructive release of cancer cells with a release efficiency of 91% after nuclease treatment.In particular,the prepared aptamer-MSNFs displayed a significantly higher release efficiency than commercial magnetic beads.Thus,the designed aptamer-MSNFs may hold a great promise for cell sorting applications.Inhibition of tumor metastasis and invasion is crucial for effective cancer therapy and prolonged survival time of cancer patients.Herein,we propose a completely new concept to generate a multifunctional drug-loaded poly?lactic-co-glycolic acid??PLGA?fibrous ring system for simultaneous therapy of primary tumors and inhibition of the tumor metastasis via reducing the formation of CTCs from primary tumors.Firstly,we prepared electrospun PLGA fibrous mats loaded with anticancer drug doxorubicin?DOX?,homogenized the fibrous mats in an aqueous solution with a high ionic strength to surprisingly generate fibrous rings,and decorated the surface of fibrous rings with PEI partially modified with gadolinium?Gd?chelates and DNA aptamer.The generated multifunctional fibrous rings?DOX@PLGA-PEI-Gd/aptamer?have an average outer diameter of 11.1?m,and exhibit good biocompatibility,long-term drug release profile,and a high r₁ relaxivity(4.46 mM^-1s^-1).Due to the ring structure and the DNA aptamer functionalization,the multifunctional fibrous rings are able to target and anchor tumor cells for a relatively long retention time,consequently decreasing the tumor cell shedding into the blood to form circulating tumors cells and inhibiting tumor metastasis and invasion.Furthermore,in vitro cell experiments and in vivo 4T1 tumor model reveal that the formed DOX@PLGA-PEI-Gd/aptamer have a long-term therapeutic efficacy and could effectively inhibit the tumor metastasis via reducing the formation of CTCs.The developed multifunctional fibrous rings may hold a great promise to be used as a unique platform for simultaneous inhibition of primary tumors and tumor metastasis.In summary,we constructed a series of functionalized platforms based on electrospun nanofibers and explored their potential for CTCs capture and tumor therapy.The nanofiber-integrated microfluidic chips are expected to be used for capture and release of CTCs in clinic.The prepared monodisperse nanofibers also show great potential in the field of cell sorting applications and tumor therapy.