Polyamidoamine dendrimers, which can deliver drugs and genetic materials to resistant cells, are attracting increased research attention, but their transportation behavior in resistant cells remains unclear. In this paper, we performed a systematic analysis of the cellular uptake, intracellular transportation, and efflux of PAMAM-NH
2
dendrimers in multidrug-resistant breast cancer cells (MCF-7/ADR cells) using sensitive breast cancer cells (MCF-7 cells) as the control. We found that the uptake rate of PAMAM-NH
2
was much lower and exocytosis of PAMAM-NH
2
was much greater in MCF-7/ADR cells than in MCF-7 cells due to the elimination of PAMAM-NH
2
from P-glycoprotein and the multidrug resistance-associated protein in MCF-7/ADR cells. Macropinocytosis played a more important role in its uptake in MCF-7/ADR cells than in MCF-7 cells. PAMAM-NH
2
aggregated and became more degraded in the lysosomal vesicles of the MCF-7/ADR cells than in those of the MCF-7 cells. The endoplasmic reticulum and Golgi complex were found to participate in the exocytosis rather than endocytosis process of PAMAM-NH
2
in both types of cells. Our findings clearly showed the intracellular transportation process of PAMAM-NH
2
in MCF-7/ADR cells and provided a guide of using PAMAM-NH
2
as a drug and gene vector in resistant cells.
Considerable effort has been devoted to the development of gene carriers over the years. However, toxicity, immunogenicity, and low transfection efficiency are still major barriers. How to overcome these obstacles has become a burning question in gene delivery. In the present study, a simple cationic human serum albumin (CHSA)-based gene-delivery system containing nuclear localization signals (NLSs) was constructed to conquer the limitations. CHSA/NLS/plasmid DNA (pDNA) complexes were prepared and characterized by Hoechst 33258 intercalation, gel retardation assay, morphological analysis, circular dichroism (CD) spectroscopy, particle size, and zeta potential measurements. Results showed that CHSA/NLS/pDNA complexes were able to condense and protect pDNA with high encapsulation efficiency. The complexes displayed a nutritional effect on cells at a low concentration and there was no significant cytotoxicity or immunogenicity. In addition, CHSA/NLS/pDNA complexes exhibited excellent cellular uptake rates and the mechanism was mainly the clathrin or macropinocytosis-dependent endocytosis pathway. Furthermore, CHSA/NLS/pDNA significantly enhanced gene expression efficiency in vitro. More importantly, CHSA/NLS/pDNA complexes showed a desired antitumor effect in vivo, exhibiting the highest inhibition rate (57.3%) and significant upregulation in p53 protein. All these results confirm that CHSA/NLS/pDNA complexes have a bright future as a safe and effective delivery system for gene therapy.
Vaccination is a widely-accepted resort against the invasion or proliferation of bacteria, parasites, viruses, and even cancer, which accounts heavily on an active involvement of CD8 + T cells. As one of the pivotal strategies taken by dendritic cells (DCs) to promote the responsiveness of CD8 + T cells to exogenous antigens, cross presentation culminates in an elevated overall host defense against cancer or infection. However, the precise mechanisms regulating such a process remains elusive, and current attempts to fuel cross presentation usually fail to exert efficiency. Here, model antigen OVA-loaded, endoplasmic reticulum (ER)-targeting cationic liposome (OVA@lipoT) is developed and characterized with a booster effect on the activation and maturation of DCs. Moreover, OVA@lipoT pulsed DCs exhibit overwhelming superiority in triggering cytotoxic T lymphocyte response both in vivo and in vitro. Data reveal that lipoT alters the intracellular trafficking and presenting pathway of antigen, which promotes cross presentation and bears close relationship to the ER-associated degradation (ERAD). These results may drop a hint about the interconnectivity between cross presentation and ER-targeted antigen delivery, provide extra information to the understanding of ERAD-mediated cross priming, and even shed new light on the design and optimization of vaccines against currently intractable cancers or virus-infection.
Currently, mRNA-based tumor therapies are in full flow because in vitro-transcribed (IVT) mRNA has the potential to express tumor antigens to initiate the adaptive immune responses. However, the efficacy of such therapy relies heavily on the delivery system. Here, a pardaxin-modified liposome loaded with tumor antigen-encoding mRNA and adjuvant (2ʹ,3ʹ-cGAMP, (cyclic [G(2ʹ,5ʹ)pA(3ʹ,5ʹ)p])), termed P-Lipoplex-CDN is reported. Due to an nonlysosomal delivery route, the transfection efficiency on dendritic cells (DCs) is improved by reducing the lysosome disruption of cargos. The mRNA modified DCs efficiently induce tumor antigen-specific immune responses both in vitro and in vivo. As prophylactic vaccines, mRNA transfected DCs significantly delay the occurrence and development of tumors, and several immunized mice are even completely resistant to tumors. Interestingly, the efficacy depends on the major histocompatibility complex class I (MHC-I) expression level on tumor cells. Furthermore, epigenetic modification (decitabine, DAC) is applied as a combination strategy to deal with malignant tumor progression caused by deficient tumor MHC-I expression. This study highlights the close relationship between mRNA-DCs vaccine efficacy and the expression level of tumor cell MHC-I molecules. Moreover, a feasible strategy for tumor MHC-I expression deficiency is proposed, which may provide clinical guidance for the design and application of mRNA-based tumor therapies.
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