To optimize a receptor-mediated and cell-selective gene transfer with polyethyleneimine (PEI)-based vector, we synthesized three galactosylated PEIs (Gal-PEI) with different molecular weights (PEI(1800), PEI(10,000), and PEI(70,000)) and investigated their potential as a targetable vector to asialoglycoprotein receptor-positive cells. All PEI derivatives formed complexes with plasmid DNA (pDNA), whereas the particle size of the complex became smaller on increasing the molecular weight of PEI. Transfection efficiency in HepG2 cells with PEI was highest with PEI(1800); efficiency was next highest with PEI(10,000), although the cellular association was similar. After galactosylation, Gal(19)-PEI(10,000)/pDNA and Gal(120)-PEI(70,000)/pDNA showed considerable agglutination with a galactose-recognizing lectin, but Gal(9)-PEI(1800) did not, suggesting that galactose units on the Gal(9)-PEI(1800)-pDNA complex are not sufficiently available for recognition. Gal(19)-PEI(10,000)-pDNA and Gal(120)-PEI(70,000)-pDNA complexes showed galactose-inhibitable transgene expression in HepG2 cells. Transfection efficiency was greatest with Gal(19)-PEI(10,000)/pDNA, a result that highlights the importance of obtaining a balance between the cytotoxicity and the transfection activity, both of which are found to be a function of the molecular weight of PEI. After intraportal injection, however, Gal(153)-PEI(70,000)/pDNA having a low N/P ratio was most effective, suggesting that additional variables, such as the size of the complex, are important for in vivo gene transfer to hepatocytes.
By optimizing lipid composition and charge ratio, galactosylated liposome/DNA complexes allow superior in vivo gene transfection in the liver via asialoglycoprotein receptor-mediated endocytosis.
Synchronized bio-distribution of combination therapies has several merits such as synergistic effects and reduced side-effects. Co-delivery of a protein and small molecule drug using a single nanocarrier is challenging because they possess totally different characteristics. Herein, we report the development of sophisticated nanoparticles composed of lipids, calcium carbonate and RGD peptide ligands for the co-delivery of a protein and small molecule drug combination via a simple preparation method. A 'one-step' ethanol injection method was employed to prepare the highly organized nanoparticles. The nanoparticles exhibited a spherical shape with ca. 130 nm diameter, and clearly had an integrated lipid layer covering the periphery. As a ligand, an RGDmodified lipid was post-inserted into the nanoparticles, which was important to overcome the 'PEG dilemma'. The pH-sensitivity of the targeted nanoparticles contributed to the efficient intracellular co-delivery of a protein and drug combination in Colon26 tumor cells, and noticeably improved their accumulation in the tumor region of xenograft mice. Synchronized bio-distribution of the protein and drug was achieved, which was the foundation for the synergistic effects of the combination. The targeting capability of the nanoparticles along with their pH-sensitive drug release and the synchronized bio-distribution of their cargos led to the significant antitumor activity of the SOD and paclitaxel combination in mice. This study provides novel information for the design and preparation of functionalized nanoparticles for the delivery of a protein/drug combination in vivo.
In this study, we demonstrated that the presence of an essential amount of sodium chloride (NaCl) during the formation of cationic liposome/plasmid DNA complexes (lipoplexes) stabilizes the lipoplexes according to the surface charge regulation (SCR) theory. Fluorescence resonance energy transfer analysis revealed that cationic liposomes in an SCR lipoplex (5 and 10 mM NaCl solution in lipoplex) increased fusion. Also, aggregation of SCR lipoplexes was significantly delayed after exposure to saline (150 mM NaCl) as a model of physiological conditions. After intraportal administration, the hepatic transfection activity of galactosylated SCR lipoplexes (5 and 10 mM NaCl solution in lipoplex) was approximately 10- to 20-fold higher than that of galactosylated conventional lipoplexes in mice. The transfection activity in hepatocytes of galactosylated SCR lipoplexes was significantly higher than that of conventional lipoplexes, and preexposure to competitive asialoglycoprotein-receptor blocker significantly reduced the hepatic gene expression, suggesting that hepatocytes are responsible for high hepatic transgene expression of the galactosylated SCR lipoplexes. Pharmacokinetic studies both in situ and in vivo demonstrated a higher tissue binding affinity and a greater expanse of intrahepatic distribution by galactosylated SCR lipoplexes. Moreover, enhanced transfection activity of galactosylated SCR lipoplexes was observed in HepG2 cells, and investigation of confocal microscopic images showed that the release of plasmid DNA in the cell was markedly accelerated. These characteristics partly explain the mechanism of enhanced in vivo transfection efficacy by galactosylated SCR lipoplexes. Hence, information in this study will be valuable for the future use, design, and development of ligand-modified lipoplexes for in vivo applications.
Evaluation methods for determining the distribution of transgene expression in the body and the in vivo fate of viral and non-viral vectors are necessary for successful development of in vivo gene delivery systems. Here, we evaluated the spatial distribution of transgene expression using tissue clearing methods. After hydrodynamic injection of plasmid DNA into mice, whole tissues were subjected to tissue clearing. Tissue clearing followed by confocal laser scanning microscopy enabled evaluation of the three-dimensional distribution of transgene expression without preparation of tissue sections. Among the tested clearing methods (ClearT2, SeeDB, and CUBIC), CUBIC was the most suitable method for determining the spatial distribution of transgene expression in not only the liver but also other tissues such as the kidney and lung. In terms of the type of fluorescent protein, the observable depth for green fluorescent protein ZsGreen1 was slightly greater than that for red fluorescent protein tdTomato. We observed a depth of ~1.5 mm for the liver and 500 μm for other tissues without preparation of tissue sections. Furthermore, we succeeded in multicolor deep imaging of the intracellular fate of plasmid DNA in the murine liver. Thus, tissue clearing would be a powerful approach for determining the spatial distribution of plasmid DNA and transgene expression in various murine tissues.
In this study, we evaluated the effect of blood components (whole blood and serum) on asialoglycoprotein receptor-mediated in vivo gene transfer. The hepatic transfection activity of galactosylated lipoplex preincubated with serum was approximately 10 times higher than that without incubation after intraportal injection in mice. However, preincubation with whole blood significantly reduced hepatic transfection activity. Fluorescent resonance energy transfer analysis and agarose gel electrophoresis revealed that preincubation with serum reduced the degree of destabilization of the galactosylated lipoplex in blood, partially supporting enhanced hepatic transfection activity by preincubation with serum. Inhibition of hepatic transfection activity by predosing galactosylated bovine serum albumin indicated that the galactosylated lipoplex exposed to serum is recognized by asialoglycoprotein-receptors on hepatocytes. Inactivation of serum prior to mixing with galactosylated lipoplex reduced liver accumulation and completely abolished enhancement of hepatic transfection activity by preincubation with active serum, suggesting that not only the stability of the lipoplex in blood but also the serum opsonin activity plays important roles. Alternatively, preincubation with inactivated serum reduced the lung accumulation and inflammatory cytokine production of galactosylated lipoplex. The information provided by this study will be valuable for the future use, design, and development of galactosylated lipoplex for in vivo asialoglycoprotein receptor-mediated gene transfer.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.