Browsing by Author "Chan, Chia-Ling"

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Endosomal escape and transfection efficiency of PEGylated cationic liposome-DNA complexes prepared with an acid-labile PEG-lipid
(2012-06-01)
Chan, Chia-Ling
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Majzoub, Ramsey N.
;
Shirazi, Rahau S.
;
Ewert, Kai K.
;
Chen, Yen-Ju
Cationic liposome-DNA (CL-DNA) complexes are being pursued as nonviral gene delivery systems for use in applications that include clinic trials. However, to compete with viral vectors for systemic delivery in vivo, their efficiencies and pharmacokinetics need to be improved. The addition of poly (ethylene glycol)-lipids (PEGylation) prolongs circulation lifetimes of liposomes, but inhibits cellular uptake and endosomal escape of CL-DNA complexes. We show that this limits their transfection efficiency (TE) in a manner dependent on the amount of PEG-lipid, the lipid/DNA charge ratio, and the lipid membrane charge density. To improve endosomal escape of PEGylated CL-DNA complexes, we prepared an acid-labile PEG-lipid (HPEG2K-lipid, PEG MW 2000) which is designed to lose its PEG chains at the pH of late endosomes. The HPEG2K-lipid and a similar but acid-stable PEG-lipid were used to prepare PEGylated CL-DNA complexes. TLC and dynamic light scattering showed that HPEG2K-CL-DNA complexes are stable at pH 7.4 for more than 24 h, but the PEG chains are cleaved at pH 5 within 1 h, leading to complex aggregation. The acid-labile HPEG2K-CL-DNA complexes showed enhanced TE over complexes stabilized with the acid-stable PEG-lipid. Live-cell imaging showed that both types of complexes were internalized to quantitatively similar particle distributions within the first 2 h of incubation with cells. Thus, we attribute the increased TE of the HPEG2K-CL-DNA complexes to efficient endosomal escape, enabled by the acid-labile HPEG2K-lipid which sheds its PEG chains in the low pH environment of late endosomes, effectively switching on the electrostatic interactions that promote fusion of the membranes of complex and endosome. (c) 2012 Elsevier Ltd. All rights reserved.
Fluorescence microscopy colocalization of lipid-nucleic acid nanoparticles with wildtype and mutant Rab5-GFP: A platform for investigating early endosomal events
(2015-06-01)
Majzoub, Ramsey N.
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Chan, Chia-Ling
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Ewert, Kai K.
;
Silva, Bruno F. B.
;
Liang, Keng S.
Endosomal entrapment is known to be a major bottleneck to successful cytoplasmic delivery of nucleic acids (NAs) using cationic liposome-NA nanopartides (NPs). Quantitative measurements of distributions of NPs within early endosomes (EEs) have proven difficult due to the sub-resolution size and short lifetime of wildtype EEs. In this study we used Rab5-GFP, a member of the large family of GTPases which cycles between the plasma membrane and early endosomes, to fluorescently label early endosomes. Using fluorescence microscopy and quantitative image analysis of cells expressing Rab5-GFP, we found that at early time points (t < 1 h), only a fraction (approximate to 35%) of RGD-tagged NPs (which target cell surface integrins) colocalize with wildtype EEs, independent of the NP\'s membrane charge density. In comparison, a GTP-hydrolysis deficient mutant, Rab5-Q79L, which extends the size and lifetime of EEs yielding giant early endosomes (GEEs), enabled us to resolve and localize individual NPs found within the GEE lumen. Remarkably, nearly all intracellular NPs are found to be trapped within GEEs implying little or no escape at early time points. The observed small degree of colocalization of NPs and wildtype Rab5 is consistent with recycling of Rab5-GDP to the plasma membrane and not indicative of NP escape from EEs.Taken together, our results show that endosomal escape of PEGylated nanoparticles occurs downstream of EEs i.e., from late endosomes/lysosomes. Our studies also suggest that Rab5-Q79L could be used in a robust imaging assay which allows for direct visualization of NP interactions with the luminal membrane of early endosomes. (C) 2015 Elsevier B.V. All rights reserved.
Live Cell Imaging of GFP-Rab Gtpases allows for Intracellular, Spatiotemporal Tracking of Surface Functionalized Lipid-DNA Complexes
(2013-01-29)
Majzoub, Ramsey N.
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Chan, Chia-Ling
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Ewert, Kai K.
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Liang, Keng S.
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Safinya, Cyrus R.
Live Cell Imaging with Rab-Gtpases Elucidates Intracellular Pathways of RGD and iRGD Tagged Cationic Lipid-DNA Nanoparticles
(2014-01-28)
Majzoub, Ramsey N.
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Ewert, Kai K.
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Kotamraju, Venkata R.
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Chan, Chia-Ling
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Liang, Keng S.
Optimizing cationic and neutral lipids for efficient gene delivery at high serum content
(2014-03-01)
Chan, Chia-Ling
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Ewert, Kai K.
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Majzoub, Ramsey N.
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Hwu, Yeu-Kuang
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Liang, Keng S.
BackgroundCationic liposome (CL)-DNA complexes are promising gene delivery vectors with potential application in gene therapy. A key challenge in creating CL-DNA complexes for application is that their transfection efficiency (TE) is adversely affected by serum. In particular, little is known about the effects of a high serum content on TE, even though this may provide design guidelines for application in vivo. MethodsWe prepared CL-DNA complexes in which we varied the neutral lipid [1,2-dioleoyl-sn-glycerophosphatidylcholine, glycerol-monooleate (GMO), cholesterol], the headgroup charge and chemical structure of the cationic lipid, and the ratio of neutral to cationic lipid; we then measured the TE of these complexes as a function of serum content and assessed their cytotoxicity. We tested selected formulations in two human cancer cell lines (M21/melanoma and PC-3/prostate cancer). ResultsIn the absence of serum, all CL-DNA complexes of custom-synthesized multivalent lipids show high TE. Certain combinations of multivalent lipids and neutral lipids, such as MVL5(5+)/GMO-DNA complexes or complexes based on the dendritic-headgroup lipid TMVLG3(8+) exhibited high TE both in the absence and presence of serum. Although their TE still dropped to a small extent in the presence of serum, it reached or surpassed that of benchmark commercial transfection reagents, particularly at a high serum content. ConclusionsTwo-component vectors (one multivalent cationic lipid and one neutral lipid) can rival or surpass benchmark reagents at low and high serum contents (up to 50%, v/v). We propose guidelines for optimizing the serum resistance of CL-DNA complexes based on a given cationic lipid. Copyright (c) 2014 John Wiley & Sons, Ltd.
pH-Sensitive Pegylated Cationic Lipid-DNA Complexes for Gene Delivery: Transfection Efficiency and Live Cell Imaging Studies
(2012-01-31)
Chan, Chia-Ling
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Majzoub, Ramsey
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Shirazi, Rahau S.
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Liang, Keng-San
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Ewert, Kai K.
pH-Sensitive Pegylated Cationic Lipid-DNA Complexes for Gene Delivery: Transfection Efficiency and Live Cell Imaging Studies
(2012-01-31)
Chan, Chia-Ling
;
Majzoub, Ramsey
;
Shirazi, Rahau S.
;
Liang, Keng-San
;
Ewert, Kai K.
Uptake and transfection efficiency of PEGylated cationic liposome-DNA complexes with and without RGD-tagging
(2014-06-01)
Majzoub, Ramsey N.
;
Chan, Chia-Ling
;
Ewert, Kai K.
;
Silva, Bruno F. B.
;
Liang, Keng S.
Steric stabilization of cationic liposome DNA (CL DNA) complexes is required for in vivo applications such as gene therapy. PEGylation (PEG: poly(ethylene glycol)) of CL DNA complexes by addition of PEG2000-lipids yields sterically stabilized nanoparticles but strongly reduces their gene delivery efficacy. PEGylation-induced weakening of the electrostatic binding of CL DNA nanoparticles to cells (leading to reduced uptake) has been considered as a possible cause, but experimental results have been ambiguous. Using quantitative live-cell imaging in vitro, we have investigated cell attachment and uptake of PEGylated CL DNA nanopartides with and without a custom synthesized RGD-peptide grafted to the distal ends of PEG2000-lipids. The RGD-tagged nanoparticles exhibit strongly increased cellular attachment as well as uptake compared to nanoparticles without grafted peptide. Transfection efficiency of RGD-tagged PEGylated CL DNA NPs increases by about an order of magnitude between NPs with low and high membrane charge density (sigma(M); the average charge per unit area of the membrane; controlled by the molar ratio of cationic to neutral lipid), even though imaging data show that uptake of RGD-tagged particles is only slightly enhanced by high am. This suggests that endosomal escape and, as a result, transfection efficiency of RGD-tagged NPs is facilitated by high sigma(M). We present a model describing the interactions between PEGylated CL DNA nanoparticles and the anionic cell membrane which shows how the PEG grafting density and membrane charge density affect adhesion of nanoparticles to the cell surface. (C) 2014 Elsevier Ltd. All rights reserved.