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Biodegradable pH-sensitive surfactants (BPS) in liposome-mediated nucleic acid cellular uptake and distribution
European Journal of Pharmaceutical Sciences 11 (2000) 199–205 www.elsevier.nl / locate / ejps
Biodegradable pH-sensitive surfactants (BPS) in liposome-mediated nucleic acid cellular uptake and distribution 1
Earvin Liang , Marla N. Rosenblatt, Preeti S. Ajmani, Jeffrey A. Hughes* Department of Pharmaceutics, College of Pharmacy, University of Florida, P.O. Box 100494, Gainesville, FL 32610, USA Received 11 November 1999; received in revised form 8 February 2000; accepted 13 April 2000
Abstract The impact of biodegradable pH-sensitive surfactant (BPS)-liposomes on nucleic acid, i.e., oligonucleotide and plasmid DNA, cellular delivery was examined. Fluorescein-labeled nucleic acids complexed with 1,2-dioleoyl-3-trimethylammonium propane cationic liposomes and BPS at a charge ratio (1 / 2) of 10 were incubated in CV-1 cells and analyzed by flow cytometry. The fluorescence intensity of oligonucleotides but not plasmid DNA complexed with BPS-liposomes was higher than those complexed with BPS-free liposomes at early time points. However, when cells were fixed to equalize the intracellular pH since fluorescein, a pH-sensitive fluorophore, has higher fluorescence intensity in alkaline pH than acidic, no difference in intensity was observed. This indicated the incorporation of BPS in liposomes did not increase oligonucleotide cellular uptake over control liposomes, but redistributed oligonucleotides into a more basic environment, e.g., cytoplasm. An explanation consistent with the presented data is the formation of small transient membrane defects within the endosomal membrane as presented previously [Liang, E., Hughes, J.A., 1998a. Membrane fusion and rupture in liposomes: effect of biodegradable pH-sensitive surfactants. J. Membr. Biol. 166, 37–49.]. The above findings suggested that BPS may be effective agents of disrupting one of the major barriers, endosomal membrane, to enhance nucleic acid cellular transport. 2000 Elsevier Science B.V. All rights reserved. Keywords: Biodegradable pH-sensitive surfactant; Liposomes; Oligonucleotides; Flow cytometry
1. Introduction The theoretical basis of nucleic acid, i.e., oligonucleotide and plasmid DNA (pDNA), utilization is very attractive since it allows for the inhibition or production of specific proteins. Nucleic acids are internalized mostly through endocytosis (Akhtar and Juliano, 1992; Zabner et al., 1995) but the cellular uptake of free nucleic acids is inefficient in tissue culture models. Nucleic acids accumulate in the endosomes, intracellular compartments with an acidic intraluminal pH, and are eliminated in the later lysosomal stage (McGraw and Maxfield, 1991). One strategy to improve the delivery of nucleic acids is by using liposomes to increase intracellular accumulation; however, the obstacle of escaping from endosomes still *Corresponding author. Tel.: 11-352-846-2725; fax: 11-352-392-444. E-mail address: [email protected] (J.A. Hughes). 1 Current address: 900 Ridgebury Rd. / P.O. Box 368, Pharmaceutics Department, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT 06877, USA.
remains (Juliano and Akhtar, 1992; Zabner et al., 1995). Consequently, the development of accessory compounds that enhance endosome to cytoplasmic transfer may be vital to nucleic acid based therapies (Wagner, 1998). Biodegradable pH-sensitive surfactants (BPS) were developed to address this particular pitfall and to enhance nucleic acid cellular delivery. Dodecyl 2-(19-imidazolyl) propionate, a member of the BPS family, has demonstrated that is has the ability to increase the biological effects of oligonucleotides (Hughes et al., 1996) and pDNA (Liang and Hughes, 1998b). It has also been demonstrated that acidic pH can trigger the activation of BPS, inducing membrane fusion and rupture (Liang and Hughes, 1998a). However, the mechanisms and the extent to which BPS enhances the activity of nucleic acids in cells has not been determined. The objective of this paper is to investigate BPSliposome-induced nucleic acid cellular delivery using a flow cytometry-based assay. The assay permits measurements of relatively quantitative differences in treatments and allows examination of a single member in the entire
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cell population. In addition to dodecyl 2-(19-imidazolyl) propionate, two other agents loosely grouped as BPS, methyl 1-imidazolyl laureate and N-dodecyl imidazole, were used to corroborate the findings of the study.
BPS at different molar fractions were added to DOTAP lipid to prepare cationic liposomes as described previously (New, 1990). After the liposomes were rehydrated in sterile water, a Sonic Dismembrator 60 probe (Fisher Scientific, Pittsburgh, PA, USA) was used to form small unilamellar vesicles by applying 5 W of power for 10 s to the liposome suspension and maintaining the samples on ice for 30 s. The cycle was repeated until a clear solution was observed. The size of the liposomes (volume–weight Gaussian distribution) was determined by a dynamic light scattering method using a NICOMP 380 ZLS Zeta Potential / Particle Sizer (Santa Barbara, CA, USA) and varied between 30 and 70 nm.
pyruvate solution, 13 MEM amino acids solution, and 10% heated fetal bovine serum (FBS). DOTAP or dodecyl 2-(19-imidazolyl) propionate containing DOTAP (molar ratio 0.3) liposomes (37.5 nmol) were complexed with oligonucleotides (0.25 nmol) at a charge ratio of 10 (6) for 30 min, the optimal ratio between liposomes and oligonucleotides proposed by Zelphati and Szoka (1996). In the consequent similar study, therefore, the charge ratio between the liposomes (20 mg) and pDNA (1 mg) was also fixed at 10 (1 / 2) to investigate the size effect on nucleic acid transport without the interference of charge. The original growth media were removed and the cells were washed with phosphate-buffered saline (PBS). The liposome–nucleic acids complex was added to 500 ml of fresh serum-free MEM media and exposed to cells for 4 h. After the exposure, either the cells were analyzed by flow cytometry or the serum-free media, including the lipid–nucleic acids, was discarded and replaced with 1 ml of fresh MEM growth media containing 10% FBS. The cells were then collected at additional various time points and analyzed. In the second part of the experiment, all three BPS agents were incorporated into cationic DOTAP liposomes at four molar ratios (0, 0.1, 0.2, and 0.3). The rest of the procedure was identical to the first part of experiment except only after the exposure of cells to the complexes (4 h), samples was collected and analyzed. Thereafter, the cells were washed twice with cold PBS, scraped from the wells, and centrifuged at 1200 rpm for 5 min. The supernatant (800 ml) was decanted and the cell pellet was resuspended in 800 ml of cold PBS. The above procedure was repeated twice and the samples were kept on ice until analysis. To address intracellular differences in nucleic acid cellular uptake, in some studies instead of PBS, 2% formaldehyde was added into the tubes for 30 min to fix the cells in the final step. The samples were analyzed using a Becton-Dickinson FACSort (San Jose, CA, USA). Green fluorescence was monitored with a 530 / 30 nm bandpass filter, and photomultiplier tube pulses were amplified logarithmically. Ten thousand cells were counted at a flow rate between 100 and 200 cells / s. Live cells were gated with their morphological properties, forward scatter and side scatter, set on logarithmic mode. The mean fluorescence intensity of the related populations of cells was calculated using histograms and expressed in arbitrary units corresponding to an intensity channel number ranging from 0 to 1,023 using a LYSYS II software program (Becton-Dickinson).
2.3. Flow cytometry studies
2.4. Statistical analysis
CV-1 cells were plated in 24-well plates (5310 5 cells / well) with 1 ml of MEM growth media per well and incubated at 378C, 5% CO 2 , and a 100% humidity environment for 24 h. The media included 100 U / ml penicillin, 100 mg / ml streptomycin, 1 mM MEM sodium
Statistical differences between the treatments were determined using analysis of variance where appropriate (StatView 4.53; Abacus Concepts, Berkeley, CA, USA) with P,0.05 considered statistically significant. Fisher’s (PLSD) post hoc t-test was applied when necessary.
2. Materials and methods
2.1. Chemicals and cells Formaldehyde solution (37%) was obtained from Fisher Scientific (Pittsburgh, PA, USA). Label IT fluorescein Nucleic Acid Labeling Kit was purchased from Mirus (Madison, WI, USA). 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Minimum Eagle medium (MEM) and Dulbecco’s modified Eagle’s (DMEM) media were obtained from Life Technologies (Grand Island, NY, USA). The three BPS agents were synthesized as previously reported (Liang and Hughes, 1998a). Fifteen bases of poly-A phosphorothioate oligonucleotides labeled with fluorescein isothiocyanate (FITC) at the 59-end were synthesized in the DNA Core Synthesis Lab at the University of Florida. pGL3 pDNA (Promega) was isolated from E. coli (strain JM-109) using a Wizard Plus Megaprep DNA Purification Kit and labeled with fluorescein using a Mirus Label IT Nucleic Acid labeling Kit of which the circular form of pDNA was maintained. The CV-1 (monkey kidney fibroblast) cell line was a generous gift from Dr. M.C. Cho (Chapel Hill, University of North Carolina, NC, USA).
2.2. Liposome preparation
E. Liang et al. / European Journal of Pharmaceutical Sciences 11 (2000) 199 – 205
3. Results
3.1. Oligonucleotide cellular delivery Oligonucleotide intensities of both fixed and live cell types with delivery systems were significantly (P,0.05) higher than the controls (no liposomes) at all observed time points (Fig. 1). The mean cellular fluorescent intensity was highest at 4 h indicating that the oligonucleotides were associated with the cells. As the oligonucleotide-containing media was removed, the mean cellular fluorescent intensity gradually decreased as a function of time (Fig. 1). Oligonucleotide signals acquired from dodecyl 2-(19-imidazolyl) propionate containing DOTAP liposomes (molar ratio 0.3) in live cells at early time points (4 and 5 h) were significantly (P,0.05) higher compared to DOTAP liposomes (Fig. 1A). When the cells were fixed to equalize the intracellular compartments with regards to pH, no difference in the fluorescence intensity was observed between these two groups (Fig. 1B). When the live and fixed cells were compared, the cellular fluorescent intensity from liposomes without dodecyl 2-(19-imidazolyl) propionate were significantly higher in fixed cells than those in live ones between 4 and 5 h but not in dodecyl 2-(19-imidazolyl) propionate containing DOTAP liposomes (molar ratio 0.3). On the contrary, there were no major differences in the fluorescence intensities between the live and fixed cells at later time points (after 5 h). Oligonucleotide cellular uptake of the three BPS agents at different molar ratios was further investigated. After 4 h of incubation in live CV-1 cells, significant differences (P,0.05) in the fluorescence intensity were observed between the dodecyl 2-(19-imidazolyl) propionate treated liposome groups (molar ratios 0.1, 0.2, and 0.3) and blank
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liposomes (Fig. 2A). However, no difference in the fluorescence intensity was observed among the dodecyl 2-(19-imidazolyl) propionate containing DOTAP liposome groups. When CV-1 cells were fixed, there was no difference in fluorescence intensity among all liposome groups (Fig. 2A). Similar fluorescence intensity profiles were also seen with the other BPS (methyl 1-imidazolyl laureate and N-dodecyl imidazole) containing liposomes among all molar ratio groups (0, 0.1, 0.2, and 0.3) in both live and fixed cells (Fig. 2B,C). In addition, there was no difference in fluorescence intensity among the three BPS agents at any molar ratio groups.
3.2. pDNA cellular delivery Like oligonucleotides in the above experiment, pDNA intensities for both cell types with delivery systems were significantly (P,0.05) higher than those without delivery systems at all observed time points. pDNA associated with cells was progressively eliminated with time, after the pDNA-containing media was replaced with fresh media (Fig. 3). Contrary to oligonucleotides, no significant difference in the fluorescence intensity of pDNA between DOTAP liposomes and dodecyl 2-(19-imidazolyl) propionate containing DOTAP liposomes (molar ratio 0.3) was observed throughout the entire time course in both live and fixed CV-1 cells (Fig. 3). In addition, pDNA intensities were similar for live and fixed cells in both DOTAP liposomes and dodecyl 2-(19-imidazolyl) propionate containing DOTAP liposomes (molar ratio 0.3). The cellular uptake of pDNA as influenced by dodecyl 2-(19-imidazolyl) propionate, methyl 1-imidazolyl laureate, and N-dodecyl imidazolyl at different molar ratios was also investigated. After 4 h of incubation in both live and
Fig. 1. Time course study of cationic liposomes on FITC-labeled oligonucleotide cellular uptake in CV-1 cells (n53). Oligonucleotide (0.25 nmol) without (m) and with a delivery system, i.e., DOTAP liposomes plus (j) or minus (♦) dodecyl 2-(19-imidazolyl) propionate (molar ratio 0.3) at a charge ratio of 10 (1 / 2)) were compared. After 4 h of incubation, the complex-containing medium was replaced with fresh media containing 10% FBS. The fluorescence intensities (mean6standard deviation (S.D.)) were subsequently recorded by flow cytometry at various time points in: (A) live CV-1 cells; (B) fixed CV-1 cells.
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Fig. 2. Effect of cationic BPS containing DOTAP liposomes at various molar ratios: 0 (open bars); 0.1 (solid bars); 0.2 (vertical bars); and 0.3 (horizontal bars); on FITC-labeled oligonucleotide (0.25 nmol) cellular uptake in CV-1 cells (n53). The fluorescence signals (mean6S.D.) were recorded by flow cytometry after 4 h of exposure of cells to the complexes. (A) Dodecyl 2-(19-imidazolyl) propionate; (B) methyl 1-imidazolyl laureate; (C) N-dodecyl imidazole.
fixed CV-1 cells, no differences in the fluorescent signal was observed among all dodecyl 2-(19-imidazolyl) propionate-containing liposome groups (molar ratio 0, 0.1, 0.2, and 0.3) (Fig. 4A). Similar fluorescence intensity profiles were also observed in the other BPS containing liposome groups (methyl 1-imidazolyl laureate and N-dodecyl imidazole), in both the live and fixed cells (Fig. 4B,C).
4. Discussion BPS are designed to disrupt the endosomal membrane barrier to nucleic acid delivery in a pH-dependent manner. Before being protonated in a basic environment, BPS stay within the lipid bilayers with minimum surface activities, maintaining the integrity of liposomes. Therefore, by
Fig. 3. Time course study of cationic liposomes on fluorescein-labeled pDNA cellular uptake in CV-1 cells (n53). pDNA (1 mg) without (m) and with a carrier system, i.e., DOTAP liposomes plus (j) or minus (♦) dodecyl 2-(19-imidazolyl) propionate (molar ratio 0.3) at a charge ratio of 10 (1 / 2)) were compared. After 4 h of incubation, the complex-containing medium was replaced with fresh media containing 10% FBS. The fluorescence signals (mean6S.D.) were then recorded by flow cytometry at different time in (A) live CV-1 cells; (B) fixed CV-1 cells.
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Fig. 4. Effect of cationic BPS containing DOTAP liposomes at various molar ratios: 0 (open bars); 0.1 (solid bars); 0.2 (vertical bars); and 0.3 (horizontal bars) on fluorescein-labeled pDNA (1 mg) cellular uptake in CV-1 cells (n53). The fluorescence signals (mean6S.D.) were recorded by flow cytometry after 4 h of incubation of cells to the complexes. (A) Dodecyl 2-(19-imidazolyl) propionate; (B) methyl 1-imidazolyl laureate; (C) N-dodecyl imidazole.
incorporating BPS into the liposome delivery system, like other ordinary liposomes, BPS-liposomes can also protect nucleic acids from degradation and result in an increase of nucleic acid cellular uptake (Akhtar and Juliano, 1992; Hofland et al., 1996). As the pH decreases during endocytosis, the hydrogen ions protonate the pre-surfactants, and induce membrane destabilization and release the membrane contents, including nucleic acids. Flow cytometry studies were conducted to investigate the impact of BPS in cationic liposomes on nucleic acid cellular uptake. When fluorescent signals of oligonucleotides were analyzed in live CV-1 cells, the dodecyl 2-(19imidazolyl) propionate-containing liposomes demonstrated increased fluorescence signals during the early time points (4 and 5 h) as compared to control liposomes. The oligonucleotide labeling material FITC, a pH-sensitive fluorophore, demonstrates much higher fluorescence intensity in basic solutions than in acidic ones. Other fluorescent compounds, e.g., folate-DM-NERF-dextran and Texas Red-dextran have been used to estimate intracellular pH (Lee et al., 1996). The results obtained with live cells indicated that with the addition of dodecyl 2-(19-imidazolyl) propionate, liposomes could induce either higher oligonucleotide cellular association or different oligonucleotide intracellular distribution, e.g., from endosome to cytoplasm. When the cells were fixed with formaldehyde to equalize the pH of the subcellular compartments, there was no difference in the fluorescence signal over the entire time course for both liposomes. It eliminated the possibility that the differences in intensity were a result of the total amount of oligonucleotide interacting with the cells,
including cellular adsorption and uptake. Instead, BPS containing liposomes perhaps are able to redistribute the oligonucleotides after they were brought into cells (Hughes et al., 1999). However, due to the possible excretion of oligonucleotides in BPS-liposomes and re-distribution of oligonucleotides in control liposomes, additional information of oligonucleotide delivery in both types of liposomes at later time points (after 5 h) was inconclusive. It is not known where this event occurs or whether the event at this time is pH sensitive. The implications were further validated when the signals acquired from live and fixed cells in both types of liposomes were compared. There was no difference in the mean cellular associated fluorescence of dodecyl 2-(19-imidazolyl) propionate containing liposomes in live and fixed cells. Non-dodecyl 2-(19-imidazolyl) propionate liposomes had relatively low cellular associated fluorescence signals while the cells were alive. However, the oligonucleotide intensities were elevated after the cells were fixed, indicating the pH shift of the oligonucleotides surrounding environment to a more basic one. The above experiments suggest similar oligonucleotide cellular uptake and possible transport of oligonucleotides to a more acidic environment, e.g., lysosome, with liposomes in the absence of BPS, and to a more basic atmosphere, e.g., endosome or cytoplasm, with liposomes in the presence of BPS. As the molar ratios of BPS was increased, greater signals from the oligonucleotides with BPS containing liposomes were observed after 4 h of incubation in the live cells compared to control liposomes. When the cells were fixed, no difference in the signal was detected. However,
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no correlation between the three BPS agents or different molar ratios of BPS to liposomes and the fluorescence intensity could be established. It is possible that the maximum concentration of BPS had been reached, and additional BPS did not increase membrane defects. Further detailed examinations regarding the selection of BPS, molar ratio of BPS to liposomes, sampling time, cell type, oligonucleotide concentration, and charge ratio are needed to expand and distinguish this relationship. In our subsequent studies, BPS seemed to alter the pH–time profile of labeled pDNA at either the observed time course or at any molar ratio of BPS. A possible explanation may be that the membrane pores promoted by BPS were not big enough to allow the transfer of large molecules like pDNA (5256 bp) into the cytoplasm, but could only allow the seepage of smaller molecules like oligonucleotides (15 b). The double-stranded structure of pDNA may also hinder its movement. The different backbones of pDNA, i.e., phosphorodiester, and the oligonucleotides, i.e., phosphorothioate, may not only affect their stability, but their transfection efficiency as well. However, the use of BPS to increase the pDNA transfer has been demonstrated in a previous study (Liang and Hughes, 1998b). Other mechanisms may be involved in BPS-induced pDNA delivery or it may be possible that our experiments were not sensitive enough to notice small differences in transport, since only a few molecules of pDNA may be able to elicit an effect. Translocation of exogenous DNA through the nuclear membrane is a major concern of gene delivery technologies. For pDNA, increasing endosomal exit may not increase nuclear localization and nuclear gene expression. Nuclear localization signal peptides have been shown to enhance transgene expression (Aronsohn and Hughes, 1998; Zanta et al., 1999). In summary, the mechanism by which BPS increases the cellular-associated fluorescence was probably not through an increase in cellular uptake of liposome-conjugated nucleic acids. Once oligonucleotides were brought into the intracellular compartment, they were redistributed into areas with a higher pH. An explanation consistent with the presented data is the formation of small membrane pores between the endosomal membrane and liposome. BPS were developed to enhance the intracellular delivery of nucleic acids (Hughes et al., 1996; Liang and Hughes, 1998b). There was no statistical difference between the three BPS agents in our study. The three agents have similar critical micelle concentration values (Liang and Hughes, 1998b) in their ionized state and may behave similarly in our model system. Nevertheless, there are several critical factors that could refine the entire BPS delivery system. Nucleic acid cellular delivery by BPSliposomes relies on a number of variables including the liposome composition (Zhou and Huang, 1994; Bennett et al., 1995), charge ratio (Jaaskelainen et al., 1994; Zelphati and Szoka, 1996; Arima et al., 1997), incubation period (Zelphati and Szoka, 1996), and presence of serum (Fel-
gner et al., 1987). By modulating any one of these variables, the cellular delivery of the oligonucleotides by BPS-liposomes may be strongly influenced and eventually improved.
Acknowledgements The authors wish to express their gratitude to National Institutes of Health (P01-AG10485 and R29 HL55770) for funding. Support from the Centers of Gene Therapy and Neurobiology of Aging at the University of Florida is also gratefully acknowledged. We also wish to acknowledge the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida for the use of the flow cytometer.
References Akhtar, S., Juliano, R.L., 1992. Cellular uptake and intracellular fate of antisense oligonucleotides. Trends Cell Biol. 2, 139–144. Arima, H., Aramaki, Y., Tsuchiya, S., 1997. Effects of oligodeoxynucleotides on the physicochemical characteristics and cellular uptake of liposomes. J. Pharm. Sci. 86, 438–442. Aronsohn, A.I., Hughes, J.A., 1998. Nuclear localization signal peptides enhance cationic liposome-mediated gene therapy. J. Drug Target. 5, 163–169. Bennett, M.J., Nantz, M.H., Balasubramaniam, R.P., Gruenert, D.C., Malone, R.W., 1995. Cholesterol enhances cationic liposome-mediated DNA transfection of human respiratory epithelial cells. Biosci. Rep. 15, 47–53. Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M., Danielsen, M., 1987. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. USA 84, 7413–7417. Hofland, H.E., Shephard, L., Sullivan, S.M., 1996. Formation of stable cationic lipid / DNA complexes for gene transfer. Proc. Natl. Acad. Sci. USA 93, 7305–7309. Hughes, J.A., Aronsohn, A.I., Avrutskaya, A.V., Juliano, R.L., 1996. Evaluation of adjuvants that enhance the effectiveness of antisense oligodeoxynucleotides. Pharm. Res. 13, 404–410. Hughes, J.A., Astriab, A., Yoo, H., Alahari, S., Liang, E., Sergueev, D., Shaw, B.R., Juliano, R.L., 1999. In vitro transport and delivery of antisense oligonucleotides. In: Phillips, M.I. (Ed.), Methods in Enzymology. Academic Press, New York, pp. 342–358. Jaaskelainen, I., Monkkonen, J., Urtti, A., 1994. Oligonucleotide-cationic liposome interactions. A physicochemical study. Biochim. Biophys. Acta 1195, 115–123. Juliano, R.L., Akhtar, S., 1992. Liposomes as a drug delivery system for antisense oligonucleotides. Antisense Res. Dev. 2, 165–176. Lee, R.J., Wang, S., Low, P.S., 1996. Measurement of endosome pH following folate receptor-mediated endocytosis. Biochim. Biophys. Acta 1312, 237–242. Liang, E., Hughes, J.A., 1998. Membrane fusion and rupture in liposomes: effect of biodegradable pH-sensitive surfactants. J. Membr. Biol. 166, 37–49. Liang, E., Hughes, J.A., 1998b. Characterization of a pH-sensitive surfactant, dodecyl-2-(19-imidazolyl) propionate (DIP), and preliminary studies in liposome mediated gene transfer. Biochim. Biophys. Acta 1369, 39–50. New, R.R., 1990. Preparation of liposomes. In: New, R.R. (Ed.),
E. Liang et al. / European Journal of Pharmaceutical Sciences 11 (2000) 199 – 205 Liposomes: A Practical Approach. Oxford University Press, New York, pp. 33–104. McGraw, T.E., Maxfield, F.R., 1991. Internalization and sorting of macromolecules: Endocytosis. In: Juliano, R.L. (Ed.), Targeted Drug Delivery. Springer-Verlag, New York, pp. 11–42. Wagner, E., 1998. Effects of membrane-active agents in gene delivery. J. Control. Release 53, 155–158. Zabner, J., Fasbender, A.J., Moninger, T., Foellinger, K.A., Welsh, M.J., 1995. Cellular and molecular barriers to gene transfer by a cationic lipid. J. Biol. Chem. 270, 18997–19007.
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Zanta, M.A., Belguise-Valladier, P., Behr, J.P., 1999. Gene delivery: a single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus. Proc. Natl. Acad. Sci. USA 96, 91–96. Zelphati, O., Szoka, Jr. F.C., 1996. Intracellular distribution and mechanism of delivery of oligonucleotides mediated by cationic lipids. Pharm. Res. 13, 1367–1372. Zhou, X., Huang, L., 1994. DNA transfection mediated by cationic liposomes containing lipopolylysine: characterization and mechanism of action. Biochim. Biophys. Acta 1189, 195–203.
Biodegradable pH-sensitive surfactants (BPS) in liposome-mediated nucleic acid cellular uptake and distribution 1
Earvin Liang , Marla N. Rosenblatt, Preeti S. Ajmani, Jeffrey A. Hughes* Department of Pharmaceutics, College of Pharmacy, University of Florida, P.O. Box 100494, Gainesville, FL 32610, USA Received 11 November 1999; received in revised form 8 February 2000; accepted 13 April 2000
Abstract The impact of biodegradable pH-sensitive surfactant (BPS)-liposomes on nucleic acid, i.e., oligonucleotide and plasmid DNA, cellular delivery was examined. Fluorescein-labeled nucleic acids complexed with 1,2-dioleoyl-3-trimethylammonium propane cationic liposomes and BPS at a charge ratio (1 / 2) of 10 were incubated in CV-1 cells and analyzed by flow cytometry. The fluorescence intensity of oligonucleotides but not plasmid DNA complexed with BPS-liposomes was higher than those complexed with BPS-free liposomes at early time points. However, when cells were fixed to equalize the intracellular pH since fluorescein, a pH-sensitive fluorophore, has higher fluorescence intensity in alkaline pH than acidic, no difference in intensity was observed. This indicated the incorporation of BPS in liposomes did not increase oligonucleotide cellular uptake over control liposomes, but redistributed oligonucleotides into a more basic environment, e.g., cytoplasm. An explanation consistent with the presented data is the formation of small transient membrane defects within the endosomal membrane as presented previously [Liang, E., Hughes, J.A., 1998a. Membrane fusion and rupture in liposomes: effect of biodegradable pH-sensitive surfactants. J. Membr. Biol. 166, 37–49.]. The above findings suggested that BPS may be effective agents of disrupting one of the major barriers, endosomal membrane, to enhance nucleic acid cellular transport. 2000 Elsevier Science B.V. All rights reserved. Keywords: Biodegradable pH-sensitive surfactant; Liposomes; Oligonucleotides; Flow cytometry
1. Introduction The theoretical basis of nucleic acid, i.e., oligonucleotide and plasmid DNA (pDNA), utilization is very attractive since it allows for the inhibition or production of specific proteins. Nucleic acids are internalized mostly through endocytosis (Akhtar and Juliano, 1992; Zabner et al., 1995) but the cellular uptake of free nucleic acids is inefficient in tissue culture models. Nucleic acids accumulate in the endosomes, intracellular compartments with an acidic intraluminal pH, and are eliminated in the later lysosomal stage (McGraw and Maxfield, 1991). One strategy to improve the delivery of nucleic acids is by using liposomes to increase intracellular accumulation; however, the obstacle of escaping from endosomes still *Corresponding author. Tel.: 11-352-846-2725; fax: 11-352-392-444. E-mail address: [email protected] (J.A. Hughes). 1 Current address: 900 Ridgebury Rd. / P.O. Box 368, Pharmaceutics Department, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT 06877, USA.
remains (Juliano and Akhtar, 1992; Zabner et al., 1995). Consequently, the development of accessory compounds that enhance endosome to cytoplasmic transfer may be vital to nucleic acid based therapies (Wagner, 1998). Biodegradable pH-sensitive surfactants (BPS) were developed to address this particular pitfall and to enhance nucleic acid cellular delivery. Dodecyl 2-(19-imidazolyl) propionate, a member of the BPS family, has demonstrated that is has the ability to increase the biological effects of oligonucleotides (Hughes et al., 1996) and pDNA (Liang and Hughes, 1998b). It has also been demonstrated that acidic pH can trigger the activation of BPS, inducing membrane fusion and rupture (Liang and Hughes, 1998a). However, the mechanisms and the extent to which BPS enhances the activity of nucleic acids in cells has not been determined. The objective of this paper is to investigate BPSliposome-induced nucleic acid cellular delivery using a flow cytometry-based assay. The assay permits measurements of relatively quantitative differences in treatments and allows examination of a single member in the entire
0928-0987 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0928-0987( 00 )00101-9
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E. Liang et al. / European Journal of Pharmaceutical Sciences 11 (2000) 199 – 205
cell population. In addition to dodecyl 2-(19-imidazolyl) propionate, two other agents loosely grouped as BPS, methyl 1-imidazolyl laureate and N-dodecyl imidazole, were used to corroborate the findings of the study.
BPS at different molar fractions were added to DOTAP lipid to prepare cationic liposomes as described previously (New, 1990). After the liposomes were rehydrated in sterile water, a Sonic Dismembrator 60 probe (Fisher Scientific, Pittsburgh, PA, USA) was used to form small unilamellar vesicles by applying 5 W of power for 10 s to the liposome suspension and maintaining the samples on ice for 30 s. The cycle was repeated until a clear solution was observed. The size of the liposomes (volume–weight Gaussian distribution) was determined by a dynamic light scattering method using a NICOMP 380 ZLS Zeta Potential / Particle Sizer (Santa Barbara, CA, USA) and varied between 30 and 70 nm.
pyruvate solution, 13 MEM amino acids solution, and 10% heated fetal bovine serum (FBS). DOTAP or dodecyl 2-(19-imidazolyl) propionate containing DOTAP (molar ratio 0.3) liposomes (37.5 nmol) were complexed with oligonucleotides (0.25 nmol) at a charge ratio of 10 (6) for 30 min, the optimal ratio between liposomes and oligonucleotides proposed by Zelphati and Szoka (1996). In the consequent similar study, therefore, the charge ratio between the liposomes (20 mg) and pDNA (1 mg) was also fixed at 10 (1 / 2) to investigate the size effect on nucleic acid transport without the interference of charge. The original growth media were removed and the cells were washed with phosphate-buffered saline (PBS). The liposome–nucleic acids complex was added to 500 ml of fresh serum-free MEM media and exposed to cells for 4 h. After the exposure, either the cells were analyzed by flow cytometry or the serum-free media, including the lipid–nucleic acids, was discarded and replaced with 1 ml of fresh MEM growth media containing 10% FBS. The cells were then collected at additional various time points and analyzed. In the second part of the experiment, all three BPS agents were incorporated into cationic DOTAP liposomes at four molar ratios (0, 0.1, 0.2, and 0.3). The rest of the procedure was identical to the first part of experiment except only after the exposure of cells to the complexes (4 h), samples was collected and analyzed. Thereafter, the cells were washed twice with cold PBS, scraped from the wells, and centrifuged at 1200 rpm for 5 min. The supernatant (800 ml) was decanted and the cell pellet was resuspended in 800 ml of cold PBS. The above procedure was repeated twice and the samples were kept on ice until analysis. To address intracellular differences in nucleic acid cellular uptake, in some studies instead of PBS, 2% formaldehyde was added into the tubes for 30 min to fix the cells in the final step. The samples were analyzed using a Becton-Dickinson FACSort (San Jose, CA, USA). Green fluorescence was monitored with a 530 / 30 nm bandpass filter, and photomultiplier tube pulses were amplified logarithmically. Ten thousand cells were counted at a flow rate between 100 and 200 cells / s. Live cells were gated with their morphological properties, forward scatter and side scatter, set on logarithmic mode. The mean fluorescence intensity of the related populations of cells was calculated using histograms and expressed in arbitrary units corresponding to an intensity channel number ranging from 0 to 1,023 using a LYSYS II software program (Becton-Dickinson).
2.3. Flow cytometry studies
2.4. Statistical analysis
CV-1 cells were plated in 24-well plates (5310 5 cells / well) with 1 ml of MEM growth media per well and incubated at 378C, 5% CO 2 , and a 100% humidity environment for 24 h. The media included 100 U / ml penicillin, 100 mg / ml streptomycin, 1 mM MEM sodium
Statistical differences between the treatments were determined using analysis of variance where appropriate (StatView 4.53; Abacus Concepts, Berkeley, CA, USA) with P,0.05 considered statistically significant. Fisher’s (PLSD) post hoc t-test was applied when necessary.
2. Materials and methods
2.1. Chemicals and cells Formaldehyde solution (37%) was obtained from Fisher Scientific (Pittsburgh, PA, USA). Label IT fluorescein Nucleic Acid Labeling Kit was purchased from Mirus (Madison, WI, USA). 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Minimum Eagle medium (MEM) and Dulbecco’s modified Eagle’s (DMEM) media were obtained from Life Technologies (Grand Island, NY, USA). The three BPS agents were synthesized as previously reported (Liang and Hughes, 1998a). Fifteen bases of poly-A phosphorothioate oligonucleotides labeled with fluorescein isothiocyanate (FITC) at the 59-end were synthesized in the DNA Core Synthesis Lab at the University of Florida. pGL3 pDNA (Promega) was isolated from E. coli (strain JM-109) using a Wizard Plus Megaprep DNA Purification Kit and labeled with fluorescein using a Mirus Label IT Nucleic Acid labeling Kit of which the circular form of pDNA was maintained. The CV-1 (monkey kidney fibroblast) cell line was a generous gift from Dr. M.C. Cho (Chapel Hill, University of North Carolina, NC, USA).
2.2. Liposome preparation
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3. Results
3.1. Oligonucleotide cellular delivery Oligonucleotide intensities of both fixed and live cell types with delivery systems were significantly (P,0.05) higher than the controls (no liposomes) at all observed time points (Fig. 1). The mean cellular fluorescent intensity was highest at 4 h indicating that the oligonucleotides were associated with the cells. As the oligonucleotide-containing media was removed, the mean cellular fluorescent intensity gradually decreased as a function of time (Fig. 1). Oligonucleotide signals acquired from dodecyl 2-(19-imidazolyl) propionate containing DOTAP liposomes (molar ratio 0.3) in live cells at early time points (4 and 5 h) were significantly (P,0.05) higher compared to DOTAP liposomes (Fig. 1A). When the cells were fixed to equalize the intracellular compartments with regards to pH, no difference in the fluorescence intensity was observed between these two groups (Fig. 1B). When the live and fixed cells were compared, the cellular fluorescent intensity from liposomes without dodecyl 2-(19-imidazolyl) propionate were significantly higher in fixed cells than those in live ones between 4 and 5 h but not in dodecyl 2-(19-imidazolyl) propionate containing DOTAP liposomes (molar ratio 0.3). On the contrary, there were no major differences in the fluorescence intensities between the live and fixed cells at later time points (after 5 h). Oligonucleotide cellular uptake of the three BPS agents at different molar ratios was further investigated. After 4 h of incubation in live CV-1 cells, significant differences (P,0.05) in the fluorescence intensity were observed between the dodecyl 2-(19-imidazolyl) propionate treated liposome groups (molar ratios 0.1, 0.2, and 0.3) and blank
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liposomes (Fig. 2A). However, no difference in the fluorescence intensity was observed among the dodecyl 2-(19-imidazolyl) propionate containing DOTAP liposome groups. When CV-1 cells were fixed, there was no difference in fluorescence intensity among all liposome groups (Fig. 2A). Similar fluorescence intensity profiles were also seen with the other BPS (methyl 1-imidazolyl laureate and N-dodecyl imidazole) containing liposomes among all molar ratio groups (0, 0.1, 0.2, and 0.3) in both live and fixed cells (Fig. 2B,C). In addition, there was no difference in fluorescence intensity among the three BPS agents at any molar ratio groups.
3.2. pDNA cellular delivery Like oligonucleotides in the above experiment, pDNA intensities for both cell types with delivery systems were significantly (P,0.05) higher than those without delivery systems at all observed time points. pDNA associated with cells was progressively eliminated with time, after the pDNA-containing media was replaced with fresh media (Fig. 3). Contrary to oligonucleotides, no significant difference in the fluorescence intensity of pDNA between DOTAP liposomes and dodecyl 2-(19-imidazolyl) propionate containing DOTAP liposomes (molar ratio 0.3) was observed throughout the entire time course in both live and fixed CV-1 cells (Fig. 3). In addition, pDNA intensities were similar for live and fixed cells in both DOTAP liposomes and dodecyl 2-(19-imidazolyl) propionate containing DOTAP liposomes (molar ratio 0.3). The cellular uptake of pDNA as influenced by dodecyl 2-(19-imidazolyl) propionate, methyl 1-imidazolyl laureate, and N-dodecyl imidazolyl at different molar ratios was also investigated. After 4 h of incubation in both live and
Fig. 1. Time course study of cationic liposomes on FITC-labeled oligonucleotide cellular uptake in CV-1 cells (n53). Oligonucleotide (0.25 nmol) without (m) and with a delivery system, i.e., DOTAP liposomes plus (j) or minus (♦) dodecyl 2-(19-imidazolyl) propionate (molar ratio 0.3) at a charge ratio of 10 (1 / 2)) were compared. After 4 h of incubation, the complex-containing medium was replaced with fresh media containing 10% FBS. The fluorescence intensities (mean6standard deviation (S.D.)) were subsequently recorded by flow cytometry at various time points in: (A) live CV-1 cells; (B) fixed CV-1 cells.
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Fig. 2. Effect of cationic BPS containing DOTAP liposomes at various molar ratios: 0 (open bars); 0.1 (solid bars); 0.2 (vertical bars); and 0.3 (horizontal bars); on FITC-labeled oligonucleotide (0.25 nmol) cellular uptake in CV-1 cells (n53). The fluorescence signals (mean6S.D.) were recorded by flow cytometry after 4 h of exposure of cells to the complexes. (A) Dodecyl 2-(19-imidazolyl) propionate; (B) methyl 1-imidazolyl laureate; (C) N-dodecyl imidazole.
fixed CV-1 cells, no differences in the fluorescent signal was observed among all dodecyl 2-(19-imidazolyl) propionate-containing liposome groups (molar ratio 0, 0.1, 0.2, and 0.3) (Fig. 4A). Similar fluorescence intensity profiles were also observed in the other BPS containing liposome groups (methyl 1-imidazolyl laureate and N-dodecyl imidazole), in both the live and fixed cells (Fig. 4B,C).
4. Discussion BPS are designed to disrupt the endosomal membrane barrier to nucleic acid delivery in a pH-dependent manner. Before being protonated in a basic environment, BPS stay within the lipid bilayers with minimum surface activities, maintaining the integrity of liposomes. Therefore, by
Fig. 3. Time course study of cationic liposomes on fluorescein-labeled pDNA cellular uptake in CV-1 cells (n53). pDNA (1 mg) without (m) and with a carrier system, i.e., DOTAP liposomes plus (j) or minus (♦) dodecyl 2-(19-imidazolyl) propionate (molar ratio 0.3) at a charge ratio of 10 (1 / 2)) were compared. After 4 h of incubation, the complex-containing medium was replaced with fresh media containing 10% FBS. The fluorescence signals (mean6S.D.) were then recorded by flow cytometry at different time in (A) live CV-1 cells; (B) fixed CV-1 cells.
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Fig. 4. Effect of cationic BPS containing DOTAP liposomes at various molar ratios: 0 (open bars); 0.1 (solid bars); 0.2 (vertical bars); and 0.3 (horizontal bars) on fluorescein-labeled pDNA (1 mg) cellular uptake in CV-1 cells (n53). The fluorescence signals (mean6S.D.) were recorded by flow cytometry after 4 h of incubation of cells to the complexes. (A) Dodecyl 2-(19-imidazolyl) propionate; (B) methyl 1-imidazolyl laureate; (C) N-dodecyl imidazole.
incorporating BPS into the liposome delivery system, like other ordinary liposomes, BPS-liposomes can also protect nucleic acids from degradation and result in an increase of nucleic acid cellular uptake (Akhtar and Juliano, 1992; Hofland et al., 1996). As the pH decreases during endocytosis, the hydrogen ions protonate the pre-surfactants, and induce membrane destabilization and release the membrane contents, including nucleic acids. Flow cytometry studies were conducted to investigate the impact of BPS in cationic liposomes on nucleic acid cellular uptake. When fluorescent signals of oligonucleotides were analyzed in live CV-1 cells, the dodecyl 2-(19imidazolyl) propionate-containing liposomes demonstrated increased fluorescence signals during the early time points (4 and 5 h) as compared to control liposomes. The oligonucleotide labeling material FITC, a pH-sensitive fluorophore, demonstrates much higher fluorescence intensity in basic solutions than in acidic ones. Other fluorescent compounds, e.g., folate-DM-NERF-dextran and Texas Red-dextran have been used to estimate intracellular pH (Lee et al., 1996). The results obtained with live cells indicated that with the addition of dodecyl 2-(19-imidazolyl) propionate, liposomes could induce either higher oligonucleotide cellular association or different oligonucleotide intracellular distribution, e.g., from endosome to cytoplasm. When the cells were fixed with formaldehyde to equalize the pH of the subcellular compartments, there was no difference in the fluorescence signal over the entire time course for both liposomes. It eliminated the possibility that the differences in intensity were a result of the total amount of oligonucleotide interacting with the cells,
including cellular adsorption and uptake. Instead, BPS containing liposomes perhaps are able to redistribute the oligonucleotides after they were brought into cells (Hughes et al., 1999). However, due to the possible excretion of oligonucleotides in BPS-liposomes and re-distribution of oligonucleotides in control liposomes, additional information of oligonucleotide delivery in both types of liposomes at later time points (after 5 h) was inconclusive. It is not known where this event occurs or whether the event at this time is pH sensitive. The implications were further validated when the signals acquired from live and fixed cells in both types of liposomes were compared. There was no difference in the mean cellular associated fluorescence of dodecyl 2-(19-imidazolyl) propionate containing liposomes in live and fixed cells. Non-dodecyl 2-(19-imidazolyl) propionate liposomes had relatively low cellular associated fluorescence signals while the cells were alive. However, the oligonucleotide intensities were elevated after the cells were fixed, indicating the pH shift of the oligonucleotides surrounding environment to a more basic one. The above experiments suggest similar oligonucleotide cellular uptake and possible transport of oligonucleotides to a more acidic environment, e.g., lysosome, with liposomes in the absence of BPS, and to a more basic atmosphere, e.g., endosome or cytoplasm, with liposomes in the presence of BPS. As the molar ratios of BPS was increased, greater signals from the oligonucleotides with BPS containing liposomes were observed after 4 h of incubation in the live cells compared to control liposomes. When the cells were fixed, no difference in the signal was detected. However,
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no correlation between the three BPS agents or different molar ratios of BPS to liposomes and the fluorescence intensity could be established. It is possible that the maximum concentration of BPS had been reached, and additional BPS did not increase membrane defects. Further detailed examinations regarding the selection of BPS, molar ratio of BPS to liposomes, sampling time, cell type, oligonucleotide concentration, and charge ratio are needed to expand and distinguish this relationship. In our subsequent studies, BPS seemed to alter the pH–time profile of labeled pDNA at either the observed time course or at any molar ratio of BPS. A possible explanation may be that the membrane pores promoted by BPS were not big enough to allow the transfer of large molecules like pDNA (5256 bp) into the cytoplasm, but could only allow the seepage of smaller molecules like oligonucleotides (15 b). The double-stranded structure of pDNA may also hinder its movement. The different backbones of pDNA, i.e., phosphorodiester, and the oligonucleotides, i.e., phosphorothioate, may not only affect their stability, but their transfection efficiency as well. However, the use of BPS to increase the pDNA transfer has been demonstrated in a previous study (Liang and Hughes, 1998b). Other mechanisms may be involved in BPS-induced pDNA delivery or it may be possible that our experiments were not sensitive enough to notice small differences in transport, since only a few molecules of pDNA may be able to elicit an effect. Translocation of exogenous DNA through the nuclear membrane is a major concern of gene delivery technologies. For pDNA, increasing endosomal exit may not increase nuclear localization and nuclear gene expression. Nuclear localization signal peptides have been shown to enhance transgene expression (Aronsohn and Hughes, 1998; Zanta et al., 1999). In summary, the mechanism by which BPS increases the cellular-associated fluorescence was probably not through an increase in cellular uptake of liposome-conjugated nucleic acids. Once oligonucleotides were brought into the intracellular compartment, they were redistributed into areas with a higher pH. An explanation consistent with the presented data is the formation of small membrane pores between the endosomal membrane and liposome. BPS were developed to enhance the intracellular delivery of nucleic acids (Hughes et al., 1996; Liang and Hughes, 1998b). There was no statistical difference between the three BPS agents in our study. The three agents have similar critical micelle concentration values (Liang and Hughes, 1998b) in their ionized state and may behave similarly in our model system. Nevertheless, there are several critical factors that could refine the entire BPS delivery system. Nucleic acid cellular delivery by BPSliposomes relies on a number of variables including the liposome composition (Zhou and Huang, 1994; Bennett et al., 1995), charge ratio (Jaaskelainen et al., 1994; Zelphati and Szoka, 1996; Arima et al., 1997), incubation period (Zelphati and Szoka, 1996), and presence of serum (Fel-
gner et al., 1987). By modulating any one of these variables, the cellular delivery of the oligonucleotides by BPS-liposomes may be strongly influenced and eventually improved.
Acknowledgements The authors wish to express their gratitude to National Institutes of Health (P01-AG10485 and R29 HL55770) for funding. Support from the Centers of Gene Therapy and Neurobiology of Aging at the University of Florida is also gratefully acknowledged. We also wish to acknowledge the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida for the use of the flow cytometer.
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