A Study On Vaccine Delivering System Of Active Percutaneous Targeting APCs By Microneedles

Because of poor adaptability, low immune efficiency and easily-generatingdrug-resistance of present approachs for vaccination, it has always been the hot spot tosearch for good adaptative, more convenient novel vaccination and deliver system inimmunology and pharmaceutics. Transcutaneous immunization (TCI) is a novel strategyfor immune vaccination in recent years, which applies antigen and adjuvant locally to skinto reduce systemic immune response. This new method could overcome the drawbacks ofpresent injection vaccination, therefore becomes a promising application. The currentobstacle is the skin barrier function of cuticle, which makes it difficult for macro-molecularantigen to penetrate skin surface and enter the active epidermis layer which is rich inAntigen-presenting cells，APCs. Microneedles are complicated needlelike structures whichare machined by Micro-electro mechanical Systems(MEMS), size in micro-metre andmake from silicon, polymers or metals. As drug delivery system, microneedles couldeffectively overcome the obstacle of the barrier function of cuticle, at the same time,because of its accuracy, painless, high efficiency and convenience, microneedles becomesthe most efficient method in transdermal delivery.There are numerous Antigen-presenting cells locating in the active epidermis layer ofskin. These cells mainly include Dendritic cells(DCs) and immature Langerhans cells(LCs). Both cells can effectively uptake, deal with and present foreign antigens,furthermore, they effectively trigger the antigen-specific cells and humoral immuneresponse after moving into draining lymph node, as a consequence, active epidermis layeris taken as the best position for vaccination. Microneedles can be initially used in activetargeting to active epidermis layer and deliver antigen to active epidermis layer whereAPCs capture antigen and trigger efficient systemic immune response.In this study, immunoliposome delivery system active-targeting to DCs is successfullyconstructed with Ovalbumin (OVA) as the antigen model. Combined with the mechanismof microneedles transdermal drug delivering and the study on enhancing macromoleculetransdermal efficiency, experiments are carried out on its targeting capacity and mechanism, immunoliposome’s effect on DCs’ function and mechanism, the effect and mechanism ofthe immunoliposome to DCs. Based on all above, the characteristics of the immuneresponse is analyzed to search out the mechanism and route to enhance the immuneresponse and practical prospect of this project.Part I:we apply film dispersion method and extrusion method to prepare OVA loadedimmunoliposome, then thiolate the immunoliposome to make it react with the maleimideof the lipospme material to form covalent bond which links CD11c monoclone antibody tothe surface of the liposome, successfully construct and purify OVA loadedimmunoliposome with active targeting to DCs. Furthermore, the immunoliposome ischaracterized with particle size, morphology, encapsulation and zeta potential. The resultsindicate, the liposome has a smooth surface, homogeneous in particle size, with the rangefrom145-160nm. Compared with OVA-liposomes, CD11c-OVA-imliposomes haveincreased particle size, with the zeta potencial (-31.2±1.7)mv. Flow cytometry data indicatethe specific targeting to CD11c is preserved after CD11s monoclonal antibody conjugatingto the surface of the immunoliposomes.Part II: we probe into the effects and mechanism of microneedles’ improvement onmacromolecular antigen’s transdermal behavior. We find out the amount ofimmunoliposomes and the macromolecule and antigen loaded permeating and detainedgreatly increased. That means a good enhancement on trandemal permeation. Marked by2fluorescence and under laser confocal microscopy, observations indicate,immunoliposomes can get into skin through the microtunnels made by microneedle matrix.At a depth of200μm, strong fluorescence is observed, which implies, immunoliposomesand the macromolecule antigen loaded in it are effectively delivered to the skin epidermislayer and the top of the dermis layer. Also in the experiment it is found that pores of skincan be the tunnel to deliver the immunoliposomes.We observe the formation of the drugdelivery channel and the changes of skin microstructure with SEM and dyeing the skinwith trypan blue and HE. TEWL(Transepidermal water loss) data show treating withmicodeedles can increase the TEWL value; compared with the untreated group, treatingwith microneedle matrix greatly increases the transdermal and retention amount of OVA loaded antigen in immunoliposome and shows outstanding transdermal permeability.Meanwhile, results of the evaluation of the irratation response indicatemicroneedletransdermal has good safety in practice.Part III:CD11c-OVA-immunoliposomes’ specific targeting to DCs is proved by flowcytometer and confocal microscopy. We discover the active targeting immunoliposomescan effectively enhance the expression of cell maturity-related molecule such as Iab、CD40、CD80、CD86, therefore promote the mature of DCs to benefit the immunepresenting function.Meanwhile, immunoliposomes can effectively enhance DCs’ uptakeand endocytosis of antigen, also stimulate DCs to secrets functional cell factors TNF-α,which imply immunoliposomes effectively improve the function maturity of the DCs, andbenefit for exerting the immune presenting function. We also find out, the mechanism ofDCs targeting to immunoliposomes and enhancement of antigen uptake is the specificconjugation of monoclonal antibody of immunoliposomes and its ligand. Additionally, wehave observed that DCs can effectively deliver the uptake immunoliposomes and theantigen loaded to draining lymph node, spleen and other secondary lymphoid organ.Part IV: we take advantage of the EG7-OVA cell model whose surface specificallyexpresses OVA，to measure the tumor growth and mice survival rates after applyingmicroneedles loading CD11c-OVA-imliposomes to tumor-bearing mice, herein vivoeffect and immune response character are evaluated. Compared with negative groups,tumors of those mice treated with microneedles loading CD11c-OVA-imliposomes growmuch slower, survival time is prolonged.Determination of specific anti-OVA antibody inmice indicates transdermal vaccination of CD11c-OVA-imliposomes effectively trigger themice’s antigen specific immune response in vivo. More significantly, in the experimentswe discover a higher antibody’s titer ratio of IgG1/IgG2a, which indicates the immuneresponse triggered in this way is Th1polarized, which is beneficial to generating cellularimmune.Further observation on the immunoliposomes targeting to DCs stimulating cellularimmunity of mice in vivo shows, after microneedle transcutaneous immunization, the immunoliposomes antigen system greatly activates the T cell with antigenic specificity,induces Th1cell factor to secrete IFN-γ and generate CTL with OVA antigenic specificityto fully stimulate cellular immune response. Correspondently，although cholera toxin virusas transdermal adjuvant or directly subcutaneous injection can trigger Th1type response,but its mechanism may be differs from immunization of immunoliposomes targeting toDCs transcutaneous immunization alone.Based on microneedle drug delivery system, making use of monoclonal antibody’sactive targeting and adopting chemical crosslinking technique, monoclonal antibodies fromthe surface of DCs are linked to the surface of liposome to form immune liposome whichcan active target to antigen presenting cells. In this way, macromolecules loaded inimmune liposome can be effectively delivered to DCs, then enhance the capacity ofendocytosis, uptake and presenting the antigen molecules to trigger more effective immuneresponse. In this study, microneedles and immunoliposome are combined to exert theaccurate position and immunoliposome’s targeting to specific cells. Two fold localizations(from microneedles to active epidermis layer and from monoclonal antibody to antigenpresenting cells) are achieved, therefore it is a totally new promising and effective immunemethod and route.