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Ionization Behavior of Amino Lipids for siRNA Delivery: Determination of Ionization Constants, SAR, and the Impact of Lipid pKa on Cationic Lipid-Biomembrane Interactions Jingtao Zhang,* Haihong Fan, Dorothy A. Levorse, and Louis S. Crocker Department of Pharmaceutical Sciences, Merck Research Laboratories, Merck & Co., Inc., West Point, Pennsylvania, United States Received November 18, 2010 Ionizable amino lipids are being pursued as an important class of materials for delivering small interfering RNA (siRNA) therapeutics, and research is being conducted to elucidate the structure-activity relationships (SAR) of these lipids. The pKa of cationic lipid headgroups is one of the critical physiochemical properties of interest due to the strong impact of lipid ionization on the assembly and performance of these lipids. This research focused on developing approaches that permit the rapid determination of the relevant pKa of the ionizable amino lipids. Two distinct approaches were investigated: (1) potentiometric titration of amino lipids dissolved in neutral surfactant micelles; and (2) pH-dependent partitioning of a fluorescent dye to cationic liposomes formulated from amino lipids. Using the approaches developed here, the pKa values of cationic lipids with distinct headgroups were measured and found to be significantly lower than calculated values. It was also found that lipid-lipid interaction has a strong impact on the pKa values of lipids. Lysis of model biomembranes by cationic lipids was used to evaluate the impact of lipid pKa on the interaction between cationic lipids and cell membranes. It was found that cationic lipid-biomembrane interaction depends strongly on lipid pKa and solution pH, and this interaction is much stronger when amino lipids are highly charged. The presence of an optimal pKa range of ionizable amino lipids for siRNA delivery was suggested based on these results. The pKa methods reported here can be used to support the SAR screen of cationic lipids for siRNA delivery, and the information revealed through studying the impact of pKa on the interaction between cationic lipids and cell membranes will contribute significantly to the design of more efficient siRNA delivery vehicles.
Introduction Since the recent discovery of RNA interference as a powerful mechanism for regulating gene expression,1,2 the potential to use siRNA (small interfering RNA) as a novel therapeutic modality has attracted significant attention and has been heavily exploited in academia and the biotech and pharmaceutical industries.3,4 Although the use of naked siRNA for localized treatment in lung and eye has progressed rapidly through preclinical and clinical stages,4-6 significant challenges still remain for the systemic administration of siRNA for broader therapeutic indications. Among them, one of the biggest hurdles is the lack of highly efficient delivery vehicles to enable safe and efficacious delivery of siRNA for systemic administration.7 Lipid-based delivery systems such as liposomes, lipoplexes, and lipid nanoparticles have been broadly utilized to deliver plasmid *Corresponding author. E-mail:
[email protected]. (1) Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.; Mello, C. C. Nature 1998, 391, 806–11. (2) Elbashir, S. M.; Harborth, J.; Lendeckel, W.; Yalcin, A.; Weber, K.; Tuschl, T. Nature 2001, 411, 494–498. (3) Bumcrot, D.; Manoharan, M.; Koteliansky, V.; Sah, D. W. Y. Nat. Chem. Biol. 2006, 2, 711–719. (4) Castanotto, D.; Rossi, J. J. Nature 2009, 457, 426–433. (5) de Fougerolles, A.; Vornlocher, H. P.; Maraganore, J.; Lieberman, J. Nat. Rev. Drug Discovery 2007, 6, 443–453. (6) Sepp-Lorenzino, L.; Ruddy, M. K. Clin. Pharmacol. Ther. 2008, 84, 628–632. (7) Whitehead, K. A.; Langer, R.; Anderson, D. G. Nat. Rev. Drug Discovery 2009, 8, 129–138. (8) Heyes, J.; Palmer, L.; Bremner, K.; MacLachlan, I. J. Controlled Release 2005, 107, 276–287. (9) Santel, A.; Aleku, M.; Keil, O.; Endruschat, J.; Esche, V.; Fisch, G.; Dames, S.; Loffler, K.; Fechtner, M.; Arnold, W.; Giese, K.; Klippel, A.; Kaufmann, J. Gene Ther. 2006, 13, 1222–1234. (10) Akinc, A.; Zumbuehl, A.; Goldberg, M.; Leshchiner, E. S.; Busini, V.; Hossain, N.; et al. Nat. Biotechnol. 2008, 26, 561–569.
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DNA, and more recently to deliver siRNA.8-12 In general, cationic lipids as well as various helper lipids are used to promote the encapsulation of DNA or siRNA and the formation of these lipid delivery systems with desired physicochemical and pharmacokinetic properties.13 Currently, lipid nanoparticles consisting of amino lipids with ionizable amine headgroups are being pursued as a promising approach to siRNA delivery, and research is being conducted to elucidate the structure-activity relationships (SAR) of these amino lipids.10,12,14 Among the physicochemical properties of these ionizable amino lipids, the ionization constant (Ka) of the lipid headgroups is one of the most important attributes, since it directly determines the charge interaction behavior of the lipids, as well as the surface charge properties of the assembled lipid nanoparticles. Because the interaction between cationic lipids and nucleic acids is strongly influenced by charge-charge interaction,15 lipid pKa is expected to play a very important role in determining not only the encapsulation of nucleic acids in the assembled lipid particles but also the release of nucleic acids from the particles during intracellular delivery. In addition to its effect on lipid-nucleic acid interaction, the pKa of ionizable amino lipids also affects the surface charge behavior of assembled lipid nanoparticles, since surface charge densities of lipid nanoparticles (11) Abrams, M. T.; Koser, M. L.; Seitzer, J.; Williams, S. C.; DiPietro, M. A.; Wang, W. M.; Shaw, A. W.; Mao, X. Z.; Jadhav, V.; Davide, J. P.; Burke, P. A.; Sachs, A. B.; Stirdivant, S. M.; Sepp-Lorenzino, L. Mol. Ther. 2010, 18, 171–180. (12) Semple, S. C.; Akinc, A.; Chen, J.; Sandhu, A. P.; Mui, B. L.; Cho, C. K.; et al. Nat. Biotechnol. 2010, 28, 172–6. (13) Li, W. J.; Szoka, F. C. Pharm. Res. 2007, 24, 438–449. (14) Spelios, M.; Nedd, S.; Matsunaga, N.; Savva, M. Biophys. Chem. 2007, 129, 137–147. (15) Kennedy, M. T.; Pozharski, E. V.; Rakhmanova, V. A.; MacDonald, R. C. Biophys. J. 2000, 78, 1620–1633.
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depend not only on the ratio of the amino lipids to neutral lipids, but also on the ionization state of the amine headgroup. It is widely known that physicochemical properties of lipid nanoparticles, such as particle size, morphology, and especially surface charge, have a dramatic impact on the performance of delivery vehicles.13,16-20 For example, it was demonstrated in DNA delivery that surface charges of lipid-DNA nanoparticles under physiological conditions played a critical role in determining the interaction of lipid nanoparticles with cell membranes and ultimately determined the internalization of the nanoparticles by cells and the toxicity induced during delivery.19-23 Later, it was also suggested that the surface charge densities of lipid-DNA nanoparticles in acidic environments such as those found in cellular endosomes determined the rate of membrane fusion between lamellar lipid vehicles and endosomes, which was one of the ratelimiting steps for transfection processes.17,24,25 Therefore, understanding the charge state of ionizable amino lipids by measuring pKa could prove to be crucial to the understanding of the charge behavior of lipids and lipid nanoparticles during assembly, systemic circulation, internalization, and intracellular trafficking, and could contribute significantly to the design and development of lipid nanoparticles for siRNA delivery. Most conventional approaches to determining the pKa of pharmaceutical compounds,26-28 such as potentiometric titration, spectrophotometric titration, capillary electrophoresis, or chromatographic measurements, cannot be directly applied to determine the relevant pKa value of ionizable amino lipids used for siRNA delivery. For example, potentiometric titration, the best option in determining ionization constants, requires the titrated compounds to have sufficient aqueous solubility and be fully dissolved in solution; unprotonated amino lipids generally possess extremely low aqueous solubility, and they form colloidal aggregates after protonation due to their amphiphilic nature. In addition, it is widely known that the value of pKa is strongly affected by environmental conditions such as dielectric constant, ionic strength, and the presence of neighboring charges.29,30 For instance, the pKa of undecyl-hydroxycoumarin shifted significantly higher in a hydrophobic environment compared with the value when it was in aqueous solution; the measured pKa values also changed dramatically depending on whether it was incorporated in a cationic or anionic surfactant micelle.29 Consequently, in order to gain a relevant understanding of the ionization status of amino lipids, the determination of pKa should be conducted in an (16) Barteau, B.; Chevre, R.; Letrou-Bonneval, E.; Labas, R.; Lambert, O.; Pitard, B. Curr. Gene Ther. 2008, 8, 313–323. (17) Ewert, K. K.; Ahmad, A.; Evans, H. M.; Safinya, C. R. Expert Opin. Biol. Ther. 2005, 5, 33–53. (18) Resina, S.; Prevot, P.; Thierry, A. R. PLoS One 2009, 4, 11. (19) Ahmad, A.; Evans, H. M.; Ewert, K.; George, C. X.; Samuel, C. E.; Safinya, C. R. J. Gene Med. 2005, 7, 739–748. (20) Caracciolo, G.; Pozzi, D.; Caminiti, R.; Marchini, C.; Montani, M.; Amici, A.; Amenitsch, H. J. Phys. Chem. B 2008, 112, 11298–11304. (21) Lv, H. T.; Zhang, S. B.; Wang, B.; Cui, S. H.; Yan, J. J. Controlled Release 2006, 114, 100–109. (22) Mislick, K. A.; Baldeschwieler, J. D. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12349–12354. (23) Mounkes, L. C.; Zhong, W.; Cipres-Palacin, G.; Heath, T. D.; Debs, R. J. J. Biol. Chem. 1998, 273, 26164–26170. (24) Budker, V.; Gurevich, V.; Hagstrom, J. E.; Bortzov, F.; Wolff, J. A. Nat. Biotechnol. 1996, 14, 760–764. (25) Lin, A. J.; Slack, N. L.; Ahmad, A.; George, C. X.; Samuel, C. E.; Safinya, C. R. Biophys. J. 2003, 84, 3307–3316. (26) Albert, A.; Serjeant, E. P. Ionization Constants of Acids and Bases: A Laboratory Manual; Methuen: London, 1962. (27) Allen, R. I.; Box, K. J.; Comer, J. E. A.; Peake, C.; Tam, K. Y. J. Pharm. Biomed. Anal. 1998, 17, 699–712. (28) Poole, S. K.; Patel, S.; Dehring, K.; Workman, H.; Poole, C. F. J. Chromatogr., A 2004, 1037, 445–454. (29) Fernandez, M. S.; Fromherz, P. J. Phys. Chem. 1977, 81, 1755–1761. (30) Fromherz, P. Biochim. Biophys. Acta 1973, 323, 326–334.
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environment similar to what lipids will encounter in delivery vehicles. Heyes et al. reported a fluorescence-based approach to assessing the ionization of amino lipids by taking advantage of the pHdependent partitioning of a fluorescent dye to lipid nanoparticles consisting of amino lipids.8 This represented an important advancement toward determining the pKa values of amino lipids in a relevant environment and could serve as an indirect approach to determining the ionization status of lipid nanoparticles. However, the determination of pKa by this method requires the assembly of lipids and nucleic acids into lipid nanoparticles, and the value of pKa could depend on the dye and the particle assembly process employed. Therefore, it is challenging to apply it to a large-scale lipid screening where the pKa values of individual lipids are required prior to the assembly of lipid nanoparticles. To support the structure-activity relationship (SAR) screening of cationic lipids for siRNA delivery, our research sought to develop more straightforward approaches to determine the pKa of amino lipids bearing ionizable amine headgroups. Here, we report our use of two distinct approaches to determining the pKa of amino lipids for siRNA delivery: (1) potentiometric titration of amino lipids dissolved in neutral surfactant micelles; and (2) pH-dependent partitioning of a fluorescent dye into cationic liposomes formulated from amino lipids. Using the approaches described here, the pKa values of amino lipids with distinct headgroups are measured and compared to calculated values. To determine the potential impact of lipid pKa on the interaction between cationic lipids and cell membranes, pH-dependent lysis of model membranes caused by amino lipids is evaluated and found to be highly dependent on the pKa values of amino lipids. The pKa methods reported here can be used to support the SAR screen of cationic lipids for siRNA delivery, and the information revealed through studying the impact of pKa on the interaction between cationic lipids and cell membranes will contribute significantly to the design of more efficient siRNA delivery vehicles.
Materials and Methods Materials. All amino lipids and the PEGylated lipid (PEGDMG) used in the study were synthesized in Merck Research Laboratories and purities by HPLC using a charged aerosol detection (CAD) detector were determined to be greater than 90%.31 n-Octyl-β-D-glucopyranoside and reduced Triton X-100 were obtained from Sigma-Aldrich (Milwaukee, WI). TNS (2-(p-toluidino)6-naphthalene sulfonic acid) and carboxyfluorescein were obtained from Molecular Probes (Eugene, OR). DOPC, DOPE, DOPS, and cholesterol were obtained from Avanti Polar Lipids (Alabaster, AL) and used without further purification. All buffers used in the study were prepared from the acid or sodium salt form of the components. Preparation of Amino Lipids in Surfactant Micelles and Potentiometric Titration. Surfactant micelle solutions used for potentiometric titrations (120 mM) were prepared by dissolving the appropriate amount of n-Octyl-β-D-glucopyranoside in a 0.15 M NaCl solution. To aid the protonation of amino lipids in micelles, solution pH was adjusted to 2.0 by the addition of 1 M hydrochloric acid. Appropriate amounts of amino lipids were then weighed and directly dissolved in the micelle solutions. The ratio of amino lipids to surfactants was generally kept at 1:100 unless otherwise noted to ensure that at most one lipid was incorporated in each micelle. The resulting lipid solutions were then vigorously vortexed to permit the distribution of lipids in surfactant micelles and were equilibrated at 4 °C for one to two days. Potentiometric titration of the micelles was performed on a (31) Chen, T.; Vargeese, C.; Vagle, K.; Wang, W.; Zhang, Y. Lipid Nanoparticle Based Compositions and Methods for the Delivery of Biologically Active Molecules. U.S. Patent 7,641,915 B2, 2010.
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Sirius GLpKa/D-PAS instrument (Sirius Analytical Instruments, East Sussex, UK) using a double junction electrode. The sample was titrated with 0.5 M KOH at 25 °C from pH 2.0 to pH 11.0, and the data were analyzed using Refinement Pro v 2.2 software or processed manually using buffer capacity analysis. Duplicate measurements of pKa using this method generally differed from one another by no more than 0.2.
Formulation of Cationic Lipids into Cationic Liposomes. Cationic lipids were formulated into cationic liposomes as described.11 Briefly, cationic lipids (30 mM) were directly suspended in a pH 4.0 citrate buffer (25 mM citrate and 100 mM NaCl) containing 3 mM PEG-DMG (PEG 2000-dimyristoyl glycerol). The resulting lipid suspension was vigorously vortexed and sonicated for 30 min to form a homogeneous solution.
TNS Fluorescence Assay for the Determination of Lipids pKa. Except as noted otherwise, 30 mM cationic lipid liposome
solution was diluted to 75 μM cationic lipid in a series of buffers with pH ranging between 3 and 12 (buffers are composed of 10 mM citrate, 10 mM phosphate, 10 mM borate, and 150 mM NaCl). A stock solution of TNS dissolved in DMSO was then added to the above buffer solution to make a 6 μM TNS solution. All procedures were performed using a Tecan Evo liquid handling robot. The fluorescence of the resulting solution was read on a SpectraMax M5 fluorescence plate reader (Molecular Devices, Sunnyvale, CA) with the excitation wavelength (λex) set at 325 nm and the emission wavelength (λem) set at 435 nm. pH of the samples was confirmed using an automated Tiamo pH measurement robot. The fluorescence of TNS was plotted against pH and fitted using a three-parameter-sigmoid function (see below). It is assumed that in the presence of amino lipids TNS fluorescence reaches a maximum when 100% of the amino lipids are ionized, while TNS has little fluorescence when the amino lipids are in the un-ionized state.8 The pH values at which half of the maximum fluorescence is reached in the TNS fluorescence assay are reported as the apparent pKa values of amino lipids. Fitting function : FL ¼
a ! pH - pKa 1 þ exp b
where FL is TNS fluorescence, pKa is apparent pKa of amino lipid, a is maximum TNS fluorescence in the presence of amino lipids, b is a measure of the cooperativeness of the protonation process of amino lipids; and pKa-b is the pH at which fluorescence reaches 73% of maximum fluorescence.
Liposome Lysis for Determining the Impact of Lipid pKa on Cationic Lipid-Biomembrane Interactions. Liposomes mimicking cell membranes were prepared from phospholipid mixtures containing DOPC, DOPE, DOPS, and cholesterol at a weight ratio of 45:20:20:15.32 Briefly, 10 mg of lipid mixture dissolved in chloroform was evaporated on a rotary evaporator to remove the bulk solvent and further dried overnight under vacuum. The resulting lipid film was hydrated with 1 mL of carboxyfluorescein solution (100 mM carboxyfluorescein, 20 mM phosphate, 20 mM citrate, pH 8.0) and sonicated for 60 min to form ∼100 nm membrane-mimicking liposomes as determined by dynamic light scattering (Zetasizer Nano, Malvern Instruments). Unencapsulated carboxyfluorescein was separated from liposomes through size exclusion chromatography with a NAP-25 column (GE Healthcare) using a pH 8.0 buffer (20 mM phosphate, 20 mM citrate, and 100 mM NaCl) as eluant. To evaluate the pH-dependent lysis of carboxyfluoresceinliposomes by cationic lipid liposomes, purified carboxyfluorescein-liposomes were further diluted 200-fold in pH 7.5, 6.0, and 5.4 buffers (20 mM phosphate, 20 mM citrate, and 100 mM NaCl) and the final lipid concentration was ∼25 μM; varying amounts of (32) Koynova, R.; Wang, L.; MacDonald, R. C. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 14373–14378.
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cationic lipid liposomes were then added to the above liposome solution to reach final cationic lipid concentrations of 50 μM. All procedures were performed using a Tecan Evo liquid handling robot. Fluorescence intensities of carboxyfluorescein (λex = 480 nm, λem = 520 nm) were measured after 30 min on a fluorescence plate reader. Fluorescence intensities at maximum (I100) or minimum (I0) lysis were determined in the presence of 0.5% reduced Triton X-100 or a blank phosphate-citrate buffer at the same pH. Lysis activities caused by the cationic lipids were then calculated as % liposome lysis = (I - I0)/(I100 - I0) 100, and data were reported as the average of triplicate measurements.
Results and Discussion Potentiometric Titration of Amino Lipids Prepared in Surfactant Micelles. Although the pKa of an acid or base in aqueous solution can be conveniently determined through potentiometric titration by strong base or acid, the low solubility of amino lipids in aqueous solutions prohibits the direct measurement of pKa using this approach. In addition, the pKa values of amino lipids determined in an aqueous environment generally do not represent the “true” protonation and deprotonation behavior when amino lipids are incorporated in lipid nanoparticles due to the change in the chemical environment that these lipids will encounter. For example, amino lipids in lipid nanoparticles will be surrounded predominately by other lipids with low dielectric constants, while molecules in aqueous solutions encounter a high dielectric constant solvent-water. This large difference in dielectric constants will shift the acid-base equilibrium toward noncharged species and effectively decrease the pKa of an amine.29 Dissolving amino lipids in neutral surfactant micelles is an effective approach to solubilize them and could be used to determine the apparent pKa of amino lipids in a hydrophobic environment similar to the one in lipid nanoparticles.24 Due to the amphiphilic nature of neutral surfactants, they self-assemble in aqueous solutions to form micelles with hydrophobic tails facing in and hydrophilic headgroup facing out (i.e., water) when the concentration is above the critical micelle concentration (CMC). Because the amino lipid is also amphiphilic when protonated, it can be easily solubilized in surfactant micelles and will orient itself in a micelle with the lipid tail buried inside the micelle. This configuration closely resembles the conditions that the lipid will experience in a lipid nanoparticle and also permits potentiometric titrations in a conventional manner. To facilitate the titration of amino lipids in neutral surfactant micelles, 1.2 mM amino lipid in 120 mM n-octyl-β-D-glucopyranoside solution was prepared. The ratio of surfactants to amino lipids was chosen to be greater than the micelle aggregate number to minimize potential charge interactions between multiple amino lipids (see section II for more discussion). Figure 1 shows a representative titration curve and corresponding buffer capacity analysis obtained from the titration of lipid 1 in 0.15 M NaCl solution. Table 1 lists the pKa value obtained from the titration curve of lipid 1 as well as the measured and calculated pKa values of other amino lipids and model compounds with diverse chemical structures (the structure of lipids is shown below and in Supporting Information Figure S1).
Lipid drug delivery formulations routinely utilize sterols as functional excipients to adjust biomembrane fluidity to achieve DOI: 10.1021/la104590k
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Figure 1. (a) Potentiometric titration of lipid 1 (1.2 mM) prepared in neutral surfactant micelles (120 mM); surfactant micelles and titration
media were in 0.15 M ionic strength adjusted water. (b) Buffer capacity analysis (d(OH-)/d pH) of the potentiometric titration in part (a); the squares (0) correspond to experimental data and the line came from the fitting of the data. The peak in maximum buffer capacity corresponded to the pKa of the titrated lipid. Table 1. Measured (Micelle Method and TNS Fluorescence Method) and Calculated pKa Values of Amino Lipids and Model Compounds at 25 °Ca ID
lipid headgroup
lipid tail L1
lipid tail L2
measured pKa (micelle)
measured pKa (TNS)
calculated pKab
1 dimethyl amine linoleyl octyl cholesteryl ether 7.9 7.7 8.6 2 dimethyl amine linoleyl butyl cholesteryl ether 8.2 8.1 8.6 3 morpholine linoleyl butyl cholesteryl ether 5.7 6.1 6.8 4 pyrrolidine linoleyl butyl cholesteryl ether 8.4 8.0 9.6 5 dimethyl amine oleyl hexyl cholesteryl ether 8.1 8.2 8.6 6 diethyl amine oleyl hexyl cholesteryl ether 7.9 7.3 9.6 7 1-methyl pyrrolidine oleyl hexyl cholesteryl ether 7.6 7.3 9.7 8 3-fluoropiperidine oleyl hexyl cholesteryl ether 6.6 6.1 6.6 9 pyrrolidine oleyl hexyl cholesteryl ether 8.1 7.8 9.6 10 piperidine oleyl hexyl cholesteryl ether 7.4 7.1 8.5 11 morpholine oleyl hexyl cholesteryl ether 5.3 4.8 6.8 12 imidazole oleyl hexyl cholesteryl ether 5.6 5.6 6.9 13 benzyl amine oleyl hexyl cholesteryl ether 5.2 4.1 7.7 14 methyl piperazine oleyl hexyl cholesteryl ether 2.8/7.9 7.9 3.3/7.6 9.1 8.8 3-(dimethylamino)-1,2-propanediolc c 9.7 8.8 3-piperidino-1,2-propanediol a Measured pKa values for amino lipids are determined either by potentiometric titration in the presence of neutral surfactants (micelle method) or by a TNS fluorescence approach. b Calculated pKa values are obtained from ACD Laboratories v 11.0 software. c pKa values of model compound are determined directly in the absence of micelles.
desired pharmacokinetic properties. Recently, strategies to incorporate sterols as part of lipid structures were shown to further improve biomembrane properties, and formulations based on these sterol-modified lipids were successfully applied in drug delivery and siRNA delivery.11,31,33 For instance, a new class of asymmetric cationic lipids consisting of a cholesteryl ether tail and an aliphatic tail (see the above generic lipid structure) was found effective in mediating the delivery of siRNA in vitro and in vivo.11,31 To understand the relevant ionization behavior of these lipids, we decided to develop appropriate methods to evaluate the pKa of a series of these ionizable amino lipids. Headgroups of these ionizable amino lipids (Table 1) were selected to span across a broad range of calculated pKa values to evaluate the SAR of lipids. A subset of the lipids was chosen to have identical tails to allow for a simple assessment of the impact of headgroup structures on pKa. From Figure 1a, it is clear that potentiometric titration of lipids dissolved in micelles resembled those expected from the titration of fully soluble molecules. This shows that the micelle titration method can be used to understand the ionization behavior of cationic lipids. Buffer capacity analysis in Figure 1b demonstrates that the pKa of cationic lipid can be easily obtained from the potentiometric titration data. Comparison between the pKa values of amino lipids prepared in micelles and the pKa values of (33) Huang, Z.; Szoka, F. C., Jr. J. Am. Chem. Soc. 2008, 130, 15702–12.
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model compounds consisting of lipid headgroups alone (i.e., lipid 1, 2, 5, and 10 vs model compounds) reveals that the pKa values of these ionizable amine headgroups are lower by more than 1 after lipids are incorporated in micelles. Similar decreases are observed between the measured pKa values and those calculated through modeling. This decrease in the pKa values of lipids assembled in micelles is consistent with the increase in the hydrophobicity around lipid headgroups after they are incorporated in micelles and suggests that the pKa of amino lipids in lipid nanoparticles could also be lower than those calculated and those of the model compounds. It is noted that, although majority of lipid headgroups appear to have decreased measured pKa values compared with calculated values, the extent of this decrease appears to depend on headgroup structures. For example, certain lipid headgroups (e.g., benzyl amine: lipid 13) can have a greater difference between measured and calculated pKa values compared with other lipid headgroups (e.g., 3-fluoropiperidine: lipid 8). This might be due to the differences in the hydrophobicities of lipid headgroups and suggests that it will be difficult to find a universal correction factor between measured and calculated pKa values. Additionally, these results also show that the use of only calculated pKa values to guide the SAR evaluation of cationic lipid headgroups might be insufficient to obtain the correct understanding on the impact of lipid pKa on siRNA delivery. However, it is further noted that three subsets of lipids with the same headgroups and different tail structures (i.e., dimethyl amine: lipids 1, Langmuir 2011, 27(5), 1907–1914
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Table 2. Impact of Multiple Amino Lipids on the Apparent pKa of Amino Lipidsa amino lipid in surfactant (mol %)
estimated average numbers of measured apparent amino lipid molecules pKa of amino lipid per surfactant micelleb
0.5 0.4 8.2 1 0.8 8.2 1.25 1 8.2 1.5 1.2 8.1 2 1.6 8.0 2.5 2 7.9 5 4 7.7 10 8 7.4 a pKa values for amino lipids are determined by potentiometric titration in the presence of neutral surfactants (micelle method). b Micelle aggregate number of 80 is used in the calculation.34
2, and 5; morpholine: lipids 3 and 11; pyrrolidine: lipids 4 and 9) show similar but measurable difference in measured pKa values. This suggests that measured pKa values are mostly determined by the chemical structures of headgroups while tail structures will also contribute to the results, likely by influencing the conformation of cationic lipids within micelles. Therefore, it might be possible to estimate the pKa range of other lipids with similar headgroups using the data reported here. Finally, although only lipids with asymmetric tail and glycerol backbone structures were tested here, it was observed that this micelle titration method could also be applied to lipids with symmetric tails or different backbone structures (data not shown). This suggests that the micelle preparation method to determine lipid pKa is a convenient approach that could be broadly applicable to other classes of ionizable amino lipids. Potentiometric Titration of Multiple Amino Lipids Incorporated in a Micelle. The use of surfactant micelles to dissolve amino lipids allows for the assessment of the impact of charge interactions between lipid headgroups on the value of pKa. Most pKa values reported previously in the literature are determined under conditions where amines or acids are soluble in solution and will not interact with each other during ionization. However, in a self-assembled lipid delivery vehicle, cationic lipids are packed within a ∼100 nm particle and headgroups of lipids are in close contact with each other. Therefore, due to charge repulsion, the charge on a protonated amine headgroup will inhibit the acceptance of another proton by the neighboring amine headgroup. To assess the impact of charge interaction and lipid packing on the value of pKa, surfactant micelles were prepared with varying ratios of surfactants to lipid (lipid 2). Table 2 lists the measured pKa values of lipid 2 in surfactant micelles as a function of the number of amino lipid molecules encapsulated per micelle. When cationic lipid concentration is less than 1.25%, the ratio of surfactant to amino lipid is greater than the micelle aggregate number (∼80)34 and there is less than one cationic lipid per micelle. The pKa at these concentrations corresponds to lipid pKa without any contribution from the charge interaction between cationic lipids, and the measured values are independent of the lipid concentration in the solution. Although measurement of pKa at a low lipid concentration is desirable for obtaining the intrinsic value of lipid pKa, ionization of the dissolved carbonate in solution will interfere with the measurement at low lipid concentrations. Therefore, intrinsic pKa values of amino lipids are generally measured at a lipid concentration of 1% (∼1 mM amino lipid). At cationic lipid concentrations between 1.5% and 10%, there will be more than one lipid within each micelle. It is obvious that the (34) Lorber, B.; Bishop, J. B.; Delucas, L. J. Biochim. Biophys. Acta 1990, 1023, 254–265.
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presence of multiple lipids within micelles significantly lowers the pKa values of lipids and the pKa values decreased systematically with the increasing amount of amino lipids within micelles. The decrease in pKa values is likely due to the increasing charge repulsion between protonated amine headgroups. It is noted that the highest level of tested cationic lipid concentration in a micelle is 10% due to the decreased solubility of amino lipids in micelles. However, cationic lipid concentration in lipid nanoparticles could be as high as 50%. This suggests that the pKa values of cationic lipids in lipid nanoparticles where lipids are in close proximity with each other will likely be even lower than the pKa values determined here. TNS Fluorescence Approach to Determining the Apparent pKa of Amino Lipids. An alternative nonpotentiometric approach for determining the pKa values of amino lipids is to assemble amino lipids into liposomes and utilize the chargedependent binding property of TNS (2-(p-toluidino)-6-naphthalene sulfonic acid) to indirectly measure the apparent pKa.35 TNS is a negatively charged fluorescent dye, and the partition of TNS is stronger into positively charged lipid membrane due to electrostatic interactions. After sequestration of TNS in lipid membranes, the fluorescence of TNS is significantly increased due to the removal of water, which acts as a fluorescence quencher, from the environment of the TNS molecule. Therefore, the increase of TNS fluorescence in the presence of ionized amino lipids could be used to estimate the surface charges of lipid membranes and provides an approach to measure the apparent pKa of amino lipids. To determine the pKa of amino lipids using TNS fluorescence, cationic lipids were formulated into cationic lipid liposomes by suspending cationic lipids (30 mM) with a PEG-DMG lipid solution (3 mM) and further homogenized using sonication. The resulting liposomes resembled unilamellar vesicles by CryoTEM (data not shown) and were generally in the range 100500 nm. To determine the optimum lipid concentration for TNS fluorescence, varying amounts of lipid 2 were combined with a fixed concentration of TNS (6 μM) in buffers at three different levels of pH. The fluorescence results are shown in Figure 2. As shown, TNS fluorescence shows a strong dependence on both buffer pH and lipid concentration. For example, TNS fluorescence at pH 5.0 increased linearly as a function of lipid concentration until the fluorescence plateaus when lipid concentration is above 75 μM. The increase in TNS fluorescence in the presence of lipid suggests that TNS partitions into the hydrophobic lipid environment. However, the concentration dependence behavior of TNS fluorescence suggests that at low lipid concentrations the binding of TNS to cationic lipids is limited by the amount of binding sites in lipids, while at high lipid concentrations, all TNS molecules bind to cationic lipids and fluorescence values correspond to the maximum TNS fluorescence when they are all in a hydrophobic environment. It is noted that the TNS/lipid ratio at the transition point should correspond to the maximum TNS concentration in cationic lipids (i.e., solubility of TNS in lipid membranes). This value (1/12.5) is significantly lower than 1, which suggests that the binding of TNS to lipid is significantly hindered by the accessibility of lipids to TNS. To maximize fluorescence signal and minimize lipid usage, 75 μM lipid concentration was used in all later experiments. Binding of TNS to amino lipids at higher pH (8 and 11) showed similar concentration-dependent profiles although with significantly reduced fluorescence values. The decrease of TNS fluorescence as the pH increases is consistent with the hypothesis that less TNS binds to surfaces as the charges of amino lipids are neutralized at high pH. (35) Bailey, A. L.; Cullis, P. R. Biochemistry 1994, 33, 12573–12580.
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Figure 2. TNS fluorescence as a function of lipid 2 concentration at three levels of pH; all solutions contain the same concentration of TNS (6 μM). Fluorescence values and error bars are the results from triplicate measurements.
This suggests that TNS can be used as a reporter molecule for the charge state of amino lipids. To further determine the usefulness of TNS to measure pKa of amino lipids, TNS fluorescence was evaluated at a broader pH range for multiple lipids. Figure 3 shows the representative plot of TNS fluorescence as a function of pH in the presence of lipid 2 and five other cationic lipids. It is clear that TNS fluorescence is significantly affected by the charge states of the lipids, and the pHdependent fluorescence data follow a simple sigmoid function. The profiles shown here are consistent with the TNS binding data on other lipids previously reported in the literature.8,14 The resemblance of these curves to the sigmoid function of an ionization event suggests that TNS can be used to measure the pKa of amino lipids. A fit of the data using a sigmoid function reveals the pH at which fluorescence values are half of the maximum fluorescence values. These pH values correspond to apparent pKa values of the amino lipids. Apparent pKa values of amino lipids from TNS fluorescence assays are reported in Table 1 and compared with the pKa values measured from micelle titration method and calculation. It was shown that the apparent pKa values determined from TNS method are generally in good agreement with the values determined from the micelle titration method, while both values are significantly lower than those estimated from modeling. This shows again that the determination of pKa of amino lipids in a relevant hydrophobic environment is critical in understanding the ionization behavior of amino lipids. It is noted that the TNS fluorescence assay for pKa determination relies on the binding of TNS to the positive charges of the lipid headgroups, which have a relatively smaller size compared with TNS molecule. As mentioned above, binding of TNS to lipids is limited by the accessibility of lipids to TNS. Therefore, if amino lipids bearing multiple amines are used, it might be challenging for multiple TNS molecules to bind around one lipid headgroup due to even greater steric hindrance, and therefore, no increases in TNS fluorescence will be observed even if more than one amine on a single lipid headgroup are ionized. In fact, evaluation of the TNS fluorescence of lipid 14 (dibasic amine with measured pKa around 2.8 and 7.9 (based on the micelle approach)) at pH 2 showed the same fluorescence value as pH 5, while the cationic lipid liposome showed much greater surface charge at pH 2 compared with pH 5. A further evaluation of the lipid 14 concentration-dependent fluorescence profiles at pH 1 and 5 showed overlapping fluorescence values at all concentration 1912 DOI: 10.1021/la104590k
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Figure 3. Representative plots showing TNS fluorescence as a function of pH in the presence of cationic lipids (75 μM); all solutions contain the same concentration of TNS (6 μM). Fluorescence values shown here are normalized by the maximum TNS fluorescence values of each lipid. The apparent pKa values of the amino lipids are the pH values at which TNS fluorescence is half of the maximum fluorescence and are obtained after fitting the data using a sigmoid function (discussed in detail in the Methods and Materials section).
ranges (data not shown). This suggests that TNS does not respond to two charges on a single lipid molecule, and it is unlikely that the TNS fluorescence approach will be able to differentiate among the individual pKa values of the multiple amines on the same lipid headgroup. Impact of Lipid pKa on the Interaction between Cationic Lipids and Model Cell Membranes. The charge state of the amine headgroups on cationic lipids directly determines surface charge properties of lipid nanoparticles, which are mostly composed of cationic lipids and neutral lipids and can have a dramatic impact on the efficacy and toxicity of these lipid nanoparticles as siRNA delivery vehicles. For example, the delivery of siRNA using lipid nanoparticles generally needs to overcome several critical extracellular and intracellular barriers such as blood protein adsorption, biodistribution, cell uptake, endosomal escape, dissociation of siRNA from delivery vehicles, and binding of siRNA with RNA interference silencing complex (RISC).36 A common feature in many of these steps is the involvement of negatively charged biological components including peptides, proteins, and most importantly the external and internal cell membranes. It was hypothesized that the charge state of cationic lipids mostly affects its biological performance by influencing the charge-charge interaction between cationic lipids and negatively charged cell membranes and blood proteins.21-23,37-39 In addition to affecting the interaction with external cell membranes which happens at physiological pH (∼7), the charge state of amino lipids at acidic pH significantly affects the ability of cationic lipids to interact with internal cell membranes in an acidified environment such as those in the endosomes (pH 5-6) and therefore plays an important role in determining the efficiency of endosomal escape of lipid nanoparticles, a key ratelimiting step in siRNA delivery.24,25 (36) Juliano, R.; Bauman, J.; Kang, H.; Ming, X. Mol. Pharmaceutics 2009, 6, 686–695. (37) Xu, Y.; Szoka, F. C., Jr. Biochemistry 1996, 35, 5616–23. (38) Zelphati, O.; Szoka, F. C., Jr. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 11493–8. (39) Caracciolo, G.; Pozzi, D.; Amenitsch, H.; Caminiti, R. Langmuir 2007, 23, 8713–8717.
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Figure 4. Lysis of membrane-mimicking liposomes by ionizable amino lipids with diverse amine headgroups. Amino lipids tested here all have the same tail structure of one oleyl and one hexyl cholesteryl ether tail. The pKa values of amino lipids are determined using the micelle method (see Table 1 for detail). Liposome lysis is conducted in buffer solutions at pH 5.4, 6.0, and 7.5 using carboxyfluorescein as a reporter for lysis. Extent of liposome lysis is reported as an average of triplicate measurements of lysis activities at 50 μM cationic lipids concentration.
To evaluate the impact of the ionization of lipid headgroups on the interaction between cationic lipids and biomembranes, cationic lipids with pKa values ranging between 5 and 8 are formulated into cationic lipid liposomes, and their ability to lyse a model cell membrane over a range of physiologically relevant pH conditions (pH 7.5, 6.0, and 5.4) was used to assess their tendency to interact with biomembranes. A phospholipid mixture of DOPC/DOPE/ DOPS/cholesterol (45:20:20:15, w/w) was used to construct liposomes to mimic cellular membranes,32 with carboxyfluorescein encapsulated as a reporter for membrane lysis.40 Due to the selfquenching of carboxyfluorescein at high concentrations when encapsulated inside liposomes mimicking biomembranes, little or no fluorescence should be detected. Upon incubation with cationic lipid liposomes, interactions between cationic lipids and model biomembranes will cause fusion and breakup of liposome membranes and the subsequent release of carboxyfluorescein into solution. An increased fluorescence signal can be detected due to the dilution of carboxyfluorescein in solution and the reduced quenching effect at low carboxyfluorescein concentrations. The extent of liposome lysis is quantified based on the relative amount of fluorescence signal increase, normalized to the fluorescence increase from complete lysis caused by the addition of surfactants. The liposome lysis activities of five ionizable amino lipids with different headgroups (with pKa spanning across the physiologically relevant pH range), and otherwise, the same tail structures are compared in Figure 4. Tail structures of lipids were kept the same to ensure that lysis differences observed only resulted from the differences in the lipid headgroups (i.e., ionization states of amine headgroups). As shown, lysis activities of cationic lipids depended strongly on the pKa values of the amino lipids. For example, amino lipids with high pKa headgroups (pyrrolidine, dimethyl amine, and piperidine) had strong lysis activities at pH 5.5, while cationic lipids with much lower pKa (e.g., morpholine headgroup) demonstrated little or no lysis activities at all three pH conditions. The lack of lysis activity for the amino lipid with morpholine headgroup is probably due to the lack of ionization in the lipid headgroup as its pKa is lower than the lowest pH conditions tested (5.4). This suggests that cationic charges in the lipid headgroup play an important role in determining the interaction between amino lipid and cell membranes. Consistent (40) Ralston, E.; Hjelmeland, L. M.; Klausner, R. D.; Weinstein, J. N.; Blumenthal, R. Biochim. Biophys. Acta 1981, 649, 133–137.
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with this result, closer inspection of the lysis activities of cationic lipids with high pKa headgroups (pyrrolidine, dimethyl amine, piperidine, and fluoropiperidine) shows that their lysis activities have a strong pH dependency, with a much higher lysis activity under acidic pH conditions and almost no lysis activity at pH 7.5. This is related to the increased ionization of lipids at low pH, as amino lipids will be more positively charged at acidic conditions and are expected to interact more strongly with the negatively charged biomembranes. In contrast, amino lipids with pKa close to or below 7.5 will have little tendency to interact with membranes at physiological pH since most of these amino lipids will remain neutral at this pH. Further inspection of the data shows that the greatest differences in pH-dependent lysis activities were achieved with the cationic lipids with pKa values close to physiological pH (pH 7.5). For example, the cationic lipid bearing a piperidine headgroup showed a difference of 37% between the lysis activities at pH 7.5 and 5.4, while the cationic lipid with a 3-fluoropiperidine group (pKa of 6.6) showed only a difference of 12%. Results in Figure 4 clearly show that the interaction between amino lipid and cell membranes depends heavily on the ionization state of the amino lipids and can be tuned systematically by adjusting the pKa of the lipid headgroup. Importantly, these data suggest that the use of a lipid with an ionizable lipid headgroup could be beneficial for siRNA delivery, since they can mediate a pH-dependent membrane interaction. A pH-dependent ability of cationic lipids to interact with cell membranes is desirable because a lower level of interaction with biomembranes at physiological pH can help to avoid toxicity during systemic circulation, while stronger interactions with endosomal membranes under acidic environments can cause higher membrane fusion rates and could potentially lead to improved endosomal escape.13,41 From a pure charge-charge membrane interaction perspective, there will be an optimal pKa value for amino lipids to achieve the optimal pHdependent membrane interaction behavior. For siRNA intracellular delivery, we speculate that the optimal pKa value of the amino lipids will be in the range 5-8, because the headgroup of amino lipids needs to be protonated within the acidified endosomes (pH 5-6.5) to interact with the membranes while the cationic (41) Semple, S. C.; Klimuk, S. K.; Harasym, T. O.; Dos Santos, N.; Ansell, S. M.; Wong, K. F.; Maurer, N.; Stark, H.; Cullis, P. R.; Hope, M. J.; Scherrer, P. Biochim. Biophys. Acta 2001, 1510, 152–166.
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charge of the headgroup need to be masked during systemic circulation (pH 7.4). It will be interesting to evaluate this hypothesis in vitro or in vivo and identify the optimal lipid pKa value for siRNA delivery. However, it is noted that the optimal pKa value could depend heavily on the extent of acidification in endosomes and thus could be different when delivering to different cell types or going through different intracellular pathways. It is further noted that successful intracellular siRNA delivery depends on not only the endosomal escape of delivery vehicle, but also the final release of siRNA from the delivery vehicle, a step which could also be affected by the pKa of amino lipids and the interaction strength between amino lipid and siRNA.19,20,39,42 Therefore, it might be difficult to find a universal optimal lipid pKa value for siRNA delivery. Finally, it is expected that coupling of the lipid pKa determination methods described here with methods that can permit quantitative understanding of endosomal escape efficiency or membrane interaction mechanism of amino lipids could greatly help to advance the rational design and use of amino lipid for siRNA delivery.
Summary and Perspectives Ionizable amino lipids represent an important class of materials for siRNA therapeutics delivery. Rapid synthesis and structural variation on ionizable amino lipids are being carried out to optimize the lipids for delivery, and demand approaches for the physicochemical properties of amino lipids to inform structureactivity relationships (SAR). pKa of lipids, an important parameter in SAR study, is critical for understanding the phase and self-assembly property of the ionizable amino lipids. It also plays a very important role in determining the interaction between amino lipids and cell membranes as well as blood proteins, which ultimately determines the delivery efficacy and toxicity of the assembled lipid carrier. We described two distinct approaches that permit the rapid determination of pKa of ionizable amino (42) Tarahovsky, Y. S.; Koynova, R.; MacDonald, R. C. Biophys. J. 2004, 87, 1054–1064.
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lipids. The first method determines the pKa of lipids using potentiometric titration by suspending amino lipids in neutral surfactant micelles, while the second method takes advantage of the charge-dependent partition of TNS to cationic lipids. Both methods are straightforward, can be easily automated, and are well-suited for supporting the pharmaceutical development of cationic lipids for siRNA delivery. However, due to the ease of sample preparation and the ability to measure headgroups with multiple charges, the micelle method appears to be more broadly applicable to determine the pKa of ionizable amino lipids. Using the methods developed here, pKa values of a set of different amino lipids are measured and observed to be significantly lower than calculated values. These values are also found to be highly dependent on the amount of cationic lipids incorporated per micelle. The impact of pKa of lipid headgroups on cationic lipidbiomembrane interaction is assessed using lipids with pKa values ranging between 5 and 8. It is found that cationic lipidbiomembrane interaction is strongly affected by the ionization state of amino lipid headgroup and is strongest when amino lipids are highly charged. The use of lipid headgroups with pKa in the range 6-8 provides pH-dependent membrane interaction within physiological pH range, which suggests the presence of an optimal amino lipid pKa for siRNA delivery. Approaches developed here can be used to support the SAR evaluation of cationic lipids for siRNA delivery and help to advance the development of siRNA as a novel therapeutic modality. Acknowledgment. We would like to thank Ye Zhang for the discussions on liposome lysis and Andrew Shaw for the methods of preparing cationic lipid liposomes. Review and comments on the manuscript by Merck colleagues are gratefully acknowledged. Provision of cationic lipids and PEGylated lipids by Merck medicinal chemists is gratefully acknowledged. Supporting Information Available: Additional figure as described in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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