| Bladder cancer(BCa)has become the most prevalent urological malignancy worldwide,in which around 75% are initially diagnosed as non-muscle invasive bladder cancer(NMIBC)in clinical.Clinical treatment of NMIBC often begins with transurethral resection of bladder tumor(TURBT)followed by intravesical instillation of chemotherapeutic agents to prevent recurrence.However,nearly 75% of patients still suffer from cancer recurrence or malignant progression within 5 years,while chemotherapeutic agents are limited by poor targeting,rapid clearance rate,and side effects.Outer membrane vesicles(OMVs)have received increasing attention for their recognized advantages in vaccine development and therapeutic drug delivery.Bacteria derived OMVs have been confirmed as an emerging immune adjuvant benefiting from their abundant pathogen-associated molecular patterns(PAMPs),which enable the activation of anticancer immune response.Comparing to attenuated bacteria vaccines,the acellular OMVs represent better effectiveness and safety when applied in the interior of organism.Manipulating genetically engineered bacteria to produce OMVs encapsulated therapeutic protein agents has been confirmed with cogent feasibility.Therefore,development of bioengineered OMVs containing anticancer peptide provide a novel exploring area for cancer treatment.In this work,we successfully fabricated a high-performance multifunctional antitumor agent using bacterial outer membrane vesicles as nanocarriers to encapsulate MPI fusion peptides generated by genetic engineering technology.Its efficacy and biocompatibility in treating tumors were evaluated at the cellular level and in a solid tumor model,respectively.Our work describes a novel general-purpose nanoplatform that provides a new avenue for clinical bladder cancer treatment.The subject is divided into 3 chapters.In the first chapter,a plasmid expressing Polybia-mastoparan I(MPI)was constructed by genetic engineering,and OMVs containing MPI fusion peptides(OMV-MPI)were obtained from E.coli transformed with the recombinant plasmid.The protein fractions were analyzed by SDS-PAGE,Western Blot,transmission electron microscope(TEM)to observe the morphology of OMVs,and nanoparticle size and zeta potential analyzer to measure the particle size and potential.In the second chapter,the inhibition of proliferation of engineered OMV-MPI on bladder cancer MB49 and UMUC3 cells in vitro was demonstrated and the half-inhibitory concentrations of various drugs were analyzed comparatively.The ability of OMV-MPI to inhibit cell migration and promote apoptosis was examined using cell scratch assay and flow cytometry.In addition,the stimulatory effect of OMVs on bone marrow-derived dendritic cells(BMDCs)was explored,and the results showed that it could stimulate immature dendritic cells(DCs)to mature DCs and stimulate the role in tumor-specific immune response.In the third chapter,a mouse subcutaneous tumor model of bladder cancer was established with intratumoral injection of OMVs,and the tumor suppressive effect of OMV-MPI was evaluated by tumor volume changes,pathological analysis,etc.The type and status of immune cells in the tumor were also analyzed,and the content of pro-inflammatory cytokines in tumor tissues was measured to further confirm the antitumor immune effect of the organism.The results showed that the growth of subcutaneous MB49 tumors was significantly restricted after the injection of genetically engineered OMVs into the tumors.the intrinsic immune stimulation of OMVs triggered the maturation of DCs,the recruitment of macrophages,and the infiltration of cytotoxic T lymphocytes(CTLs)and helper T lymphocytes(Ths),causing the secretion of proinflammatory cytokines(IL-6,TNF-α and IFN-γ)increased.At the same time,some evidence also suggests that bioengineered OMVs have a satisfactory biosafety profile.Taken together,this study provides an important strategy for expanding the application of MPI peptides and OMVs in cancer therapy.Figure 24 Table 21 Reference 101... |
