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Rational design of a drug delivery system with enhanced therapeutic potency is critical for efficient tumor chemotherapy. Many protein-based drug delivery platforms have been designed to deliver drugs to target sites and improve the therapeutic efficacy. In this study, paclitaxel (PTX) molecules were encapsulated within an apoferritin nanocage-based drug delivery system with the modification of an extracellular-signal-regulated kinase (ERK) peptide inhibitor at the C-terminus of ferritin (HERK). Apoferritin is an endogenous nano-sized spherical protein which has the ability to specially bind to a majority of tumor cells via interacting with transferrin receptor 1. The ERK peptide inhibitor is a peptide which can disrupt the interaction of MEK with ERK in the mitogen-activated protein kinase/ERK pathway. By combining the targeted delivery effect of ferritin and the inhibitory effect of the ERK peptide inhibitor, the newly fabricated ferritin carrier nanoparticle HERK could still be taken up by tumor cells, and it displayed higher cell cytotoxicity than the parent ferritin. After loading with PTX, HERK-PTX displayed a favorable anticancer effect in human breast cancer cells MDA-MB-231 and lung carcinoma cells A549. The remarkable inhibitory effect on MDA-MB-231 tumor spheroids was also identified. These results indicated that the constructed HERK nanocarrier is a promising multi-functional drug delivery vehicle to enhance the therapeutic effect of drugs in cancer therapy.

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PURPOSE: The aims of this study were to test the feasibility, targeting specificity and anticancer therapeutic efficacy of CendR motif tLyP-1 functionalized at the N-terminal of ferritin for paclitaxel (PTX) delivery. METHODS: A tumor homing and penetrating peptide tLyP-1 was fused to the N-terminal of human H chain ferritin (HFtn) to generate a dual-targeting nanoparticle delivery system. PTX molecules were encapsulated into the HFtn nanocage using the disassembly/assembly method by adjusting pHs. Cellular uptake was examined by confocal laser scanning microscopy (CLSM) and flow cytometry. The MTT assay was used to test the cytotoxicity of various PTX-loaded NPs against MDA-MB-231 and SMMC-7721 tumor cells. The wound healing and cell migration assays were conducted to assess the inhibitory effect on cell motility and metastasis. The inhibition effect on the SMMC-7721 tumor spheroids was studied and penetration ability was evaluated by CLSM. The antitumor efficacy of PTX-loaded NPs was assessed in MDA-MB-231 breast cancer xenografted in female BALB/c nude mice. RESULTS: Compared with HFtn-PTX, in vitro studies demonstrated that the tLyP-1-HFtn-PTX displayed enhanced intracellular delivery and better cytotoxicity and anti-invasion ability against both SMMC-7721 and MDA-MB-231 cells. The better penetrability and growth inhibitory effect on SMMC-7721 tumor spheroids were also testified. In vivo distribution and imaging demonstrated that the tLyP-1-HFtn-PTX NPs were selectively accumulated and penetrated at the tumor regions. Verified by the breast cancer cells model in BABL/c nude mice, tLyP-1-HFtn-PTX displayed higher in vivo therapeutic efficacy with lower systemic toxicity. CONCLUSION: Ferritin decorated with tumor-homing penetration peptide tLyP-1 at the N terminal could deliver PTX specifically inside the cell via receptor-mediated endocytosis with better efficacy. The peptide tLyP-1 which is supposed to work only at the C terminus showed enhanced tumor tissue penetration and antitumor efficacy, demonstrating that it also worked at the N-terminal of HFtn.

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We describe the development of a neuropilin-1 binding peptide (RGERPPR)-ferritin nanocage that specifically targets tumor cells. Herein, the tumor-penetrating peptide RGERPPR motif was modified at the C-terminal of human H chain ferritin (HFtn) using flexible linker moieties. Since the C-terminal of HFtn is positioned toward the inner cavity, relatively long linkers (GGGGS)4 were used, in which the MMP-2 cleavage site was inserted in the linker. The RGERPPR motif was proved to be exposed outside the protein shell by the effective cleavage at the linker region by MMP-2. The loading of paclitaxel (PTX) and HFtn-mMMP2-RGE was prepared by using the low concentration of urea. In vitro studies demonstrated that HFtn-mMMP2-RGE-PTX nanoparticles exhibited better cytotoxicity and could specifically bind to and be taken up by human lung cancer cells A549 that highly express NRP-1 receptor. Better penetrability and growth inhibitory effect were also verified by the 3D tumor spheroid experiment. The results confirmed that the tumor-targeting and penetration peptide RGERPPR-modified ferritin had great potential in enhancing tumor therapy and could be a promising therapeutic agent.

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Paclitaxel (PTX), an excellent chemotherapeutic antitumor drug, is widely used to treat patients with various cancers. However, its clinical applications are greatly restricted by poor solubility and lack of targeting. Herein, we applied natural human H chain ferritin (HFtn) nanocages that can bind to tumor cells via interacting with the human transferritin receptor 1 (TfR1) leading to its endocytosis as the PTX carrier for the targeted delivery. PTX molecules were encapsulated into HFtn cavity using disassembly/reassembly method through adjusting pH. According to the requirements of drugs suitable for clinical trials, HFtn can be easily purified in high yields with no ligand modification or property modulation. We demonstrated that PTX molecules were successfully encapsulated in the protein nanocages. The HFtn-PTX nanoparticles exhibited similar morphology and structural characteristics to the hollow cage and showed significant cytotoxicity in vitro than the naked PTX. Flow cytometry, confocal laser scanning microscopy, and in vivo imaging of MDA-MB-231 tumor demonstrated the HFtn-PTX nanoparticles targeting ability to tumor cells. Cell apoptosis assay showed that HFtn-PTX had similar apoptotic characteristics on MDA-MB-231 cells as that of the free PTX. HFtn-PTX nanoparticles have higher in vivo therapeutic efficacy and lower systemic toxicity. The BALB/c mice model also confirmed the effectiveness of the nanoparticles. Specifically targeting to tumors and solving the solubility issue of water-insoluble drugs thus alleviating the side effects, HFtn can be an efficient hydrophobic drug delivery nanocarrier for further applications in cancer therapy.