We report a parylene-based flexible electroporation chip for in vivo nucleic acid delivery. Benefited from the flexibility of parylene film, our chip fits the natural shape of the electroporated objects, thereby provides a uniform electric field over a large area. In addition, the subject is less likely to be harmed owing to the chip features, including good biocompatibility, less invasive electroporation and lower applied voltage because of smaller space between electrodes. These characteristics offer vast potential for applications of implantable electroporation devices. Using the proposed chip, we achieved successful plasmid DNA expression and siRNA transfection in both healthy mouse tissue and living tumor with excellent efficacy (90%).
The pharmacokinetics of small interfering RNAs (siRNAs) is a pivotal issue for siRNA-based drug development.In this study, we comprehensively investigated the behavior of siRNAs in vivo in various tissues and demonstrated that intravenously-injected naked siRNA accumulated remarkably in the submandibular gland, bulbourethral gland, and pancreas, with a respective half-life of ~22.7, ~45.6, and ~30.3 h.This was further confirmed by gel separation of tissue homogenates and/or supernatants.In vivo imaging and cryosectioning suggested that delivery carriers significantly influence the distribution and elimination profiles of siRNA.Gene-silencing assays revealed that neither naked nor liposome-formulated siRNA resulted in gene knockdown in the submandibular and bulbourethral glands after systemic administration, suggesting that these glands function as drug reservoirs that enable slow siRNA release into the circulation.But robust gene-silencing was achieved by local injection of liposome-encapsulated siRNA into the submandibular gland.Our results enhance understanding of the pharmacokinetic properties of siRNAs and we believe that they will facilitate the development of siRNA therapy, especially for the submandibular gland.
mRNA is a novel class of therapeutic modality that holds great promise in vaccination, protein replacement therapy, cancer immunotherapy, immune cell engineering etc. However, optimization of mRNA molecules and efficient in vivo delivery are quite important but challenging for its broad application. Here we present an ionizable lipid nanoparticle (iLNP) based on iBL0713 lipid for in vitro and in vivo expression of desired proteins using codon-optimized mRNAs. mRNAs encoding luciferase or erythropoietin (EPO) were prepared by in vitro transcription and formulated with proposed iLNP, to form iLP171/mRNA formulations. It was revealed that both luciferase and EPO proteins were successfully expressed by human hepatocellular carcinoma cells and hepatocytes. The maximum amount of protein expression was found at 6 h post-administration. The expression efficiency of EPO with codon-optimized mRNA was significantly higher than that of unoptimized mRNA. Moreover, no toxicity or immunogenicity was observed for these mRNA formulations. Therefore, our study provides a useful and promising platform for mRNA therapeutic development.
Hydrophobization of cationic polymers, as an efficient strategy, had been widely developed in the structure of cationic polymer micelles to improve the delivery efficiency of nucleic acids. However, the distribution of hydrophobic segments in the polymer chains is rarely considered. Here, we have elaborated three types of hydrophobized polyethylene glycol (PEG)-blocked cationic polymers with different distributions of the hydrophobic segments in the polymer chains PEG–PAM–PDP (E–A–D), PEG–PDP–PAM (E–D–A), and PEG–P(AM/DP) (E–(A/D)), which were synthesized by reversible addition–fragmentation chain transfer polymerization of methoxy PEG, cationic monomer aminoethyl methacrylate, and pH-sensitive hydrophobic monomer 2-diisopropylaminoethyl methacrylate, respectively. In aqueous solution, all of the three copolymers, E–A–D, E–D–A, and E–(A/D), were able to spontaneously form nanosized micelles (100–150 nm) (ME–A–D, ME–D–A, and ME–(A/D)) and well-incorporated small interfering RNA (siRNA) into complex micelles (CMs). The effect of distributions of the hydrophobic segments on siRNA delivery had been evaluated in vitro and in vivo. Compared with ME–D–A and ME–(A/D), ME–A–D showed the best siRNA binding capacity to form stable ME–A–D/siRNA CMs less than 100 nm, mediated the best gene-silencing efficiency and inhibition effect of tumor cell growth in vitro, and showed better liver gene-silencing effect in vivo. In the case of ME–(A/D) with a random distribution of cationic and hydrophobic segments, a gene-silencing efficiency higher than Lipo2000 but lesser than ME–A–D and ME–D–A was obtained. As the mole ratio of positive and negative charges increased, ME–D–A/siRNA and ME–A–D/siRNA showed similar performances in size, zeta potential, cell uptake, and gene silencing, but ME–(A/D)/siRNA showed reversed performances. In addition, ME–A–D as the best siRNA carrier was evaluated in the tumor tissue in the xenograft murine model and showed good anticancer capacity. Obviously, the distribution of the hydrophobic segments in the amphiphilic cationic polymer chains should be seriously considered in the design of siRNA vectors.
AIFM2 is a crucial NADH oxidase involved in the regulation of cytosolic NAD+. However, the role of AIFM2 in the progression of human cancers remains largely unexplored. Here, we elucidated the clinical implications, biological functions, and molecular mechanisms of AIFM2 in hepatocellular carcinoma (HCC). We found that AIFM2 is significantly upregulated in HCC, which is most probably caused by DNA hypomethylation and downregulation of miR-150-5p. High expression of AIFM2 is markedly associated with poor survival in patients with HCC. Knockdown of AIFM2 significantly impaired, while forced expression of AIFM2 enhanced the metastasis of HCC both in vitro and in vivo. Mechanistically, increased mitochondrial biogenesis and oxidative phosphorylation by activation of SIRT1/PGC-1α signaling contributed to the promotion of metastasis by AIFM2 in HCC. In conclusion, AIFM2 upregulation plays a crucial role in the promotion of HCC metastasis by activating SIRT1/PGC-1α signaling, which strongly suggests that AIFM2 could be targeted for the treatment of HCC.
Circular RNAs (circRNAs) are implicated in the progression and radiosensitivity of human cancers, including esophageal carcinoma (ESCA). In this study, we aimed to explore the functions of circRNA 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (circATIC) in ESCA progression.CircATIC expression, miR-10b-3p and Rh family C glycoprotein (RHCG) were examined via quantitative real-time polymerase chain reaction (qRT-PCR), western blot assay or immunohistochemistry (IHC) assay. 5'-ethynyl-2'-deoxyuridine (EdU), wound-healing, transwell, and cell colony formation assays and flow cytometry analysis were conducted to evaluate cell proliferation, migration, invasion, radiosensitivity and apoptosis, respectively. Dual-luciferase reporter assay and RNA pulldown assay were conducted to analyze the relationships among circATIC, miR-10b-3p and RHCG. A murine xenograft model assay was performed to explore the functions of circATIC in tumor formation and radiosensitivity in vivo.CircATIC was decreased in ESCA. CircATIC overexpression suppressed cell proliferation, migration and invasion and promoted radiosensitivity and apoptosis in ESCA cells in vitro and repressed tumor formation and radioresistance in vivo. Functionally, circATIC served as the sponge for miR-10b-3p, which directly targeted RHCG. MiR-10b-3p elevation reversed circATIC-mediated effect on ESCA cell progression. Moreover, miR-10b-3p inhibition suppressed cell growth and metastasis and enhanced radiosensitivity in ESCA cells by targeting RHCG.Overexpression of circATIC hampered ESCA progression and promoted radiosensitivity depending on the regulation of miR-10b-3p and RHCG.
This paper presents a microchip that is used to determine the optimal parameters of cancer electrochemotherapy in vitro rapidly. With a constant concentration of bleomycin, anti-cancer agent, the viability of tumor cells as a function of applied electric field is evaluated. The electric field is localized and linearly varied owing to the design of four-leaf hyperbolic electrodes. Under our experimental condition, MCF-7 (human breast adenocarcinoma) cells are destructively damaged by the synergy of electroper-meabilization and bleomycin when the applied electric field exceeds 4×10 4 V/m. The optimization in in vitro approaches is of guidance to in vivo applications for electrochemotherapy.
Abstract Continuous cell electroporation is an appealing non-viral approach for genetically transfecting a large number of cells. Yet the traditional macro-scale devices suffer from the unsatisfactory transfection efficiency and/or cell viability due to their high voltage, while the emerging microfluidic electroporation devices is still limited by their low cell processing speed. Here we present a flow-through cell electroporation device integrating large-sized flow tube and small-spaced distributed needle electrode array. Relatively large flow tube enables high flow rate, simple flow characterization and low shear force, while well-organized needle array electrodes produce an even-distributed electric field with low voltage. Thus the difficulties for seeking the fine balance between high flow rate and low electroporation voltage were steered clear. Efficient in vitro electrotransfection of plasmid DNA was demonstrated in several hard-to-transfect cell lines. Furthermore, we also explored ex vivo electroporated mouse erythrocyte as the carrier of RNA. The strong ability of RNA loading and short exposure time of freshly isolated cells jointly ensured a high yield of valid carrier erythrocytes, which further successfully delivered RNA into targeted tissue. Both in vitro and ex vivo electrotransfection could be accomplished at high cell processing speed (20 million cells per minute) which remarkably outperforms previous devices.
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