A Detachable And Dissolvable Bilayer Microneedle Patch For Combined Chemo-immunotherapy

Breast cancer,as the most common malignant tumor in women,is highly invasive and has a high mortality rate.Traditional treatment methods may eliminate or control the primary tumor but cannot prevent its metastasis and recurrence.As an emerging tumor treatment method,immunotherapy is expected to permanently cure cancer by activating the autoimmune system against cancer cells throughout the body.However,the efficacy of immunotherapy is limited by the low antigen presentation efficiency of immune cells at the tumor site and the protection of tumor cells in the surrounding immunosuppressive microenvironment.Therefore,it is expected to promote the efficacy of cancer immunotherapy by combining with other treatment methods.At present,immunotherapy combined with chemotherapy is a common clinical treatment strategy among various combination therapies.It has great toxicity and side effects during systemic administration by directly delivery of free nonspecific chemotherapeutic drugs and immunotherapeutic agents,and on the other hand,they are easily degraded after local administration.The delivery systems designed by nanotechnology and some receptors on tumor cells can make chemotherapeutic drugs and other therapeutic agents passively or actively target tumor sites.However,this kind of systemic drug delivery still has many problems,such as high administered doses,low bioavailability,as well as early drug leakage during transportation in the body.Moreover,the preparation method of "all-in-one" nano-carrier is too cumbersome to control the quality between batches,which makes it nearly impossible to achieve industrial production.Exactly,microneedle patches can puncture the stratum corneum of skin for drug delivery,which breaks the limitation of drug types for local transdermal drug delivery.In addition,multiple therapeutic agents can be delivered simultaneously by microneedle,which can more easily achieve sequential release of drugs by layered design than hydrogels.This new drug delivery system with simple preparation process and convenient application has recently shown certain application potential in synergistic anti-tumor therapy.In view of this,this paper designed and carried out the following research works:Firstly,carrier-free nanoparticles FPC-NPs with suitable size and potential were formed by the electrostatic adsorption between CpG and fluorine polyethyleneimine(F-PEI)with opposite charges.The properties of FPC-NPs were characterized in vitro,such as the adsorption of specific tumor antigens,the activation of dendritic cells(DCs),and the polarization of macrophages.On this basis,a separable bilayer soluble microneedle patch was designed for programmed delivery of chemotherapeutic drug and the formulated FPC-NPs.The tip and outer hyaluronic acid(HA)layer of MNs were loaded with doxorubicin(DOX),while the inner methylated hyaluronic acid(MeHA)layer was loaded with FPC-NPs.In addition,some low molecular weight HA solutions were added between the tape and the needles as the separating layer.When the microneedle patch was applied to the tumor site,DOX was rapidly released from the tip and the outer layer during the dissolution of the matrix material,which induced immunogenic cell death of the orthotopic tumor to release tumor-associated antigens(TAAs).The network structure of the inner cross-linked material gradually loosens instead of dissolving directly after absorbing tissue fluid,leading to the slow release of the nanoparticles.Then,the nanoparticles adsorped TAAs via electrostatic interaction and in vivo self-assembled personalized tumor vaccines,which could activate a strong immune response.The experimental results showed that the bilayer MNs structure designed in this paper can realize the graded sequential release of DOX and FPC-NPs by using polymers with different dissolution and swelling properties,then obtain personalized tumor nanovaccines through simple in vivo self-assembly.The combined chemotherapy and immunotherapy significantly increased the maturation of DCs,activation and proliferation of antigen-specific cytotoxic T lymphocytes(CTLs)and helper T lymphocytes(Ths)in tumor-draining lymph nodes(TDLNs)and spleen.In addition,the proportion of regulatory T lymphocytes(Tregs)and M2 macrophages were reduced,while the natural killer(NK)cells were increased in the spleen cells.Meanwhile,the combination therapy can increase the infiltration of immune effector cells(CTLs,Ths,NK cells)and reduce the proportion of immunosuppressive cells(Tregs,M2 TAMs)in both primary and distant tumors,which effectively improve the immunosuppressive tumor microenvironment.Moreover,the combination therapy with aPD-1 effectively inhibited the growth of both primary and distant tumors in 4T1 breast cancer models,successfully prevented lung metastasis of the tumor,thus achieving enhanced synergistic chemotherapy-immunotherapy against tumors.