Electrostatics of PEGylated Micelles and Liposomes Containing

The electrostatics of large unilamellar vesicles (LUVs) of various lipid compositions were determined and correlated with steric stabilization. The co...
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Langmuir 2005, 21, 2560-2568

Electrostatics of PEGylated Micelles and Liposomes Containing Charged and Neutral Lipopolymers Olga Garbuzenko,† Samuel Zalipsky,‡ Masoud Qazen,‡ and Yechezkel Barenholz*,† Laboratory of Membrane and Liposome Research, Hebrew UniversitysHadassah Medical School, Jerusalem 91120, Israel, and ALZA Corporation, 1900 Charleston Road, Mountain View, California 94043 Received August 22, 2004. In Final Form: January 5, 2005 The electrostatics of large unilamellar vesicles (LUVs) of various lipid compositions were determined and correlated with steric stabilization. The compositional variables studied include (a) degree of saturation, comparing the unsaturated egg phosphatidylcholine (EPC) and the fully hydrogenated soy phosphatidylcholine (HSPC) as liposome-forming lipids; (b) the effect of 40 mol % cholesterol; (c) the effect of mole % of three methyl poly(ethylene glycol) (mPEG)-lipids (the negatively charged mPEG-distearoyl phosphoethanolamine (DSPE) and two uncharged lipopolymers, mPEG-distearoyl glycerol (DSG) and mPEG-oxycarbonyl-3-amino-1,2-propanediol distearoyl ester (DS)); and (d) the negatively charged phosphatidyl glycerol (PG). The lipid phases were as follows: liquid disordered (LD) for the EPC-containing LUV, solid ordered (SO) for the HSPC-containing LUV, and liquid ordered (LO) for either of those LUV with the addition of 40 mol % cholesterol. The LUV zeta potential and electrical surface potential (ψ0) were determined. It was found that progressive addition of mPEG2k-DSPE to liposomes increases negative surface potential and reduces surface pH to a similar extent as the addition of PG. However, due to the “hidden charge effect”, zeta potential was more negative for liposomes containing PG than for those containing mPEG2k-DSPE. Replacing mPEG-DSPE with mPEG2k-DS or mPEG-DSG had no effect on surface pH and surface potential, and zeta potential was approximately zero. Addition of low concentrations of cationic peptides (protamine sulfate and melittin) to PG- or mPEG-DSPE-containing liposomes neutralized the liposome negative surface potential to a similar extent. However, only in liposomes containing PG, did liposome aggregation occur. Replacing the negatively charged lipopolymer mPEG-DSPE with the neutral lipopolymers mPEG-DS or mPEG-DSG eliminates or reduces such interactions. The relevance of this effect to the liposome performance in vivo is discussed.

1. Introduction One of the major breakthroughs in medical liposome application is the development of sterically stabilized liposomes (SSLs).1-4 The common denominator of all SSLs is the inclusion of a lipopolymer in the liposome lipid bilayer either before the lipid hydration step or by inserting it into the preformed liposomes.2-5 Since 1990, the SSLs were the focus of intense studies on the relationships between their physicochemical properties and their unique pharmacological performance, especially in order to explain benefits of liposome steric stabilization in cancer treatment of animals and humans by DOXIL (100 nm SSLs loaded with doxorubicin) through ammonium sulfate gradient3,6-8 and in arthritis animal models.9,10 Various * To whom correspondence should be addressed: phone 972 2 6758507; fax 972 2 6757499; e-mail [email protected]. † Hebrew UniversitysHadassah Medical School. ‡ ALZA Corporation. (1) Poly (ethylene glycol). Chemistry and Biological Applications; Harris, J. M., Zalipsky, S., Eds.; ACS Symposium Series 680; American Chemical Society: Washington, DC, 1997. (2) Lasic, D. D.; Martin, F. Stealth Liposomes; CRC Press: Boca Raton, FL, 1995. (3) Barenholz, Y. Curr. Opin. Colloid Interface Sci. 2001, 6, 66-77. (4) Long-circulating liposomes; Woodle, M. C., Storm, G., Eds.; Springer: Berlin, 1997. (5) Uster, P. S.; Allen, T. M.; Daniel, B. E.; Mendez, C. J.; Newama, M. S.; Zhu, G. FEBS Lett. 1996, 386, 243-246. (6) Gabizon, A.; Barenholz, Y. Liposomes: Rational Design; Janoff, A. S., Ed.; Marcel Dekker: New York, 1999; pp 343-362. (7) Gabizon, A.; Papahadjopoulos, D. Biochim. Biophys. Acta 1992, 1103, 94-100. (8) Gabizon, A.; Shmeeda, H.; Barenholz, Y. Pharmacokinetics of pegylated liposomal Doxorubicin: Review of animal and human studies. Clin. Pharmacokinet. 2003, 42, 419-436.

physicochemical properties of SSLs were studied: thermotropic behavior,11 level of hydration,12 and electrostatics.13-18 A very important question in order to further improve and extend SSL therapeutic performance is to better understand the effects of the lipopolymers which are relevant to the steric stabilization on SSL side effects. For example, the phenomena of steric stabilization and DOXIL side effects19 are still not fully understood. So far the most common lipopolymer used for this purpose is mPEG2k-distearoyl phosphoethanolamine (DSPE), in which the lipid part is linked to a 2000 Da mPEG (45 oxyethylene units) via a carbamoyl linker that connects the primary amino group of the ethanolamine and the free hydroxyl group of the mPEG. Thus, in contrast to (9) Metselaar, J.; Wauben, M.; Boerman, O.; Van Lent, P.; Storm, G. Cell Mol. Biol. Lett. 2002, 7, 291-292. (10) Metselaar, J.; Wauben, M.; Wagenaar-Hilbers, J.; Boerman, O.; Storm, G. Arthritis Rheum. 2003, 48, 2059-2066. (11) Kenworthy, D. A. K.; Simon, S. A.; McIntosh, J. Biophys. J. 1995, 68, 1903-1920. (12) Tirosh, O.; Barenholz, Y.; Katzhendler, J.; Priev, A. Biophys. J. 1998, 74, 1371-1379. (13) Woodle, M. C.; Collins, L. R.; Sponsler, E.; Kosovsky, N.; Papahadjopoulos, D.; Martin, F. J. Biophys. J. 1992, 61, 902. (14) Jones, M. N. Adv. Colloid Interface Sci. 1995, 54, 93-128. (15) Price, M. E.; Cornelius, R. M.; Brash, J. L. Biochim. Biophys. Acta 2001, 1512, 191-205. (16) Silvander, M.; Hansson, P. H.; Edwards, K. Langmuir 2000, 16, 3696-3702. (17) Woodle, M. C. Chem. Phys. Lipids 1993, 64, 249-262. (18) Woodle, M.; Newman, M.; Cohen, J. J. Drug Targeting 1994, 2, 397-403. (19) Szebeni, J.; Baranyi, L.; Savay, S.; Milosevits, J.; Bunger R.; Laverman, P.; Metsellar, J. M.; Storm, G.; Chanan-Khan, A.; Liebes, L.; Muggia, F. M.; Cohen, R.; Barenholz, Y.; Alving, C. R. J. Liposome Res. 2002, 12 (1 & 2), 165-172.

10.1021/la0479105 CCC: $30.25 © 2005 American Chemical Society Published on Web 02/10/2005

Electrostatics of Large Unilamellar Vesicles

DSPE, the mPEG-DSPE is not zwitterionic, but due to its phosphodiester moiety it is negatively charged. However, the role of this charge and the type of linker used to attach the PEG polymer to the lipid and in SSL side effects has not yet been studied. This study is aimed to fill this gap, focusing mainly on two different parameters that define the liposome electrostatics, zeta potential, and electrical surface potential (ψ0). To achieve this goal, we compared three different lipopolymers: the commonly used negatively charged mPEG-DSPE, and two others that are both uncharged but differ in their linkersthe mPEG-oxycarbonyl-3amino-1,2-propanediol distearoyl ester (mPEG-DS) and mPEG-distearoylglycerol (mPEG-DSG). Previous studies on large unilamellar vesicles (LUVs) containing mPEG-DSPE show that these liposomes, like the phosphatidyl glycerol (PG)-containing liposomes, are indeed negatively charged.13,14,16,18 Our studies, which confirm the above, also demonstrate that when uncharged lipopolymers are used for SSL preparation the SSLs are uncharged. One major implication of the difference between using mPEG-DSPE and the use of mPEG-DS or mPEG-DSG is interactions with positively charged peptides and inorganic ions which occur with the negative charge of the phosphodiester of mPEG-DSPE, but does not occur with the SSLs having uncharged lipopolymers. These comparisons hold for micelles of the lipopolymers and for 100 nm LUVs, which are commonly used for intravenous (iv) drug delivery. Similar effects were obtained for the three different physical states of the lamellar phases: solid ordered (SO), liquid disordered (LD), and liquid ordered (LO). Furthermore, cationic peptides and small inorganic cations neutralize the negative surface potential of PGand PEG-DSPE-containing LUVs to a similar extent. However the PEG moiety of PEG-DSPE-containing liposomes prevents intervesicle aggregation with cationic peptides which occurs in the case of PG-containing liposomes. 2. Experimental Design First we compared the electrostatic properties of mPEG-DSPE, mPEG-DS, and mPEG-DSG lipopolymers. The PEG moiety comprised on average 15, 44, 112, or 271 ethylene glycol units and had an average Mw of 750, 2000, 5000, or 12000 Da, respectively, for mPEGDSPE and mPEG-DS. For mPEG-DSG only the molecules having a PEG moiety of 2000 Da were used. We studied the effect of negatively charged lipopolymer mPEG-DSPE and the neutral lipopolymers mPEG-DS and commercially available mPEG-DSG on the electrostatics of two states of lipid aggregationssmicelles and liposomes. For liposomes we studied three phases: liquid disordered (LO), using EPC as the liposome forming lipid (Tm -5 °C); solid ordered (SO), using HSPC as liposome forming lipid (Tm 52 °C), and liquid ordered (LO), using lipid mixtures of either EPC/Chol or HSPC/Chol in ratio 60:40 mol %. To assess electrical surface potential (ψ0) and surface pH, we used the pH- and electrical field-sensitive fluorophore 7-heptadecyl 4-hydroxycoumarin (C17HC) that was incorporated into all LUVs and micelles studied.16,20-24 (20) Barenholz, Y.; Pal, R.; Wagner, R. R. Methods Enzymol. 1993, 220, 288-312. (21) Pal, R.; Petri, W. A.; Barenholz, Y. Wagner, R. R. Biochim. Biophys. Acta 1983, 729, 185-192. (22) Pal, R.; Petri, W.; Ben-Yashar, V.; Wagner, R.; Barenholz, Y. Biochemistry 1985, 24, 573-581. (23) Fromherz, P. Methods Enzymol. 1989, 171, 376-387.

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For LUVs of most lipid compositions, in addition to ψ0C17HC, we also determined the zeta potential from their electrophoretic mobility and compared it with the electrical surface potential determined by means of C17HC ionization. 3. Material and Methods 3.1. Materials. mPEG-distearoyl phosphatidylethanolamine (mPEG-DSPE) was obtained from ALZA Corporation, Mountain View, CA. mPEG-oxycarbonyl-3-amino-1,2-propanediol distearoyl ester (mPEG-DS) was prepared as described below; mPEG-disteroyl glycerol (mPEG-DSG) was obtained from NOF Corporation, Tokyo, Japan; EPC, HSPC, egg phosphatidyl glycerol (EPG), and hydrogenated soy PG (HSPG) were obtained from Lipoid, Ludwigshafen, Germany. All lipids were g98% pure and were used without further purification. Figure 1A shows molecular structures of most lipids used and C17HC. The structural differences between mPEG-DSPE, mPEG-DS, and mPEG-DSG are illustrated in Figure 1B. The electrical surface potential and surface pH probe 7-heptadecyl-4-hydroxycoumarin (C17HC) was purchased from Molecular Probes, Eugene, OR; 4-methylubelliferone (MU), protamine sulfate (MW ∼2000), melittin (MW ∼2800), spermine (MW ∼200), and sodium bicarbonate and boric acid were obtained from Sigma, St. Louis, MO; L-histidine monohydrochloride was obtained from Merck, Darmstadt, Germany; sodium carbonate was obtained from J.T. Baker, Phillipsburg, NJ; sodium chloride, and calcium chloride, from Frutarom, Haifa, Israel. Only highly pure water purified with WaterPro PS HPLC/UV Ultrafilter Hybrid model (LABCONCO, Kansas City, MO), which delivers 18.2 ΜΩ/cm, sterile (pyrogen-free to 0.06 e.u./mL), was used throughout all these studies. 3.2. Chemical Synthesis of mPEG-DS. mPEG-DS is a novel lipopolymer synthesized according to Zalipsky.26 The procedure described below for mPEG2k-DS was also followed for derivatives containing mPEGs of other molecular weights. Figure 1C presents the main steps of mPEG-DS synthesis. Synthesis of mPEG-Oxycarbonyl-3-amino-1,2-propanediol. Forty milliliters of a solution of mPEG2k (4.0 g, 2.0 mM) in toluene was rotary evaporated to azeotropically remove any water from the polymer. The remaining waxy solid was dissolved in 20 mL of dry chloroform. The solution was stirred in an ice bath for cooling to 0 °C, treated with p-nitrophenylchloroformate (0.604 g, 3.0 mmol) and then triethylamine (0.84 mL, 6.0 mmol), and allowed to react at 25 °C. After 6 h, 3-amino-1,2-propanediol (APD, 0.6 g, 6.0 mmol) in 1 mL of acetonitrile was added to the reaction mixture, which was then stirred overnight at 25 °C. The solution was filtered and evaporated to dryness. The product was recrystallized twice from 2-propanol, collected by filtration, and dried in vacuo over P2O5. The yield was 2.8 g (65%). 1H NMR analysis (360 MHz, DMSO-d6) gave the following ppm values: 2.90 (m, NCH2 of APD, 1 H), 3.05 (m, NCH2 of APD, 1 H), 3.23 (s, OCH3, 3 H), 3.65 (s, PEG, 180 H), 4.05 (t, CO2CH2, 2 H), 4.42 (t, 1 OH, 1H), 4.57 (d, 2 OH, 1 H), 6.98 (t, NH, 1 H). Synthesis of mPEG-Oxycarbonyl-3-amino-1,2-propanediol Distearoyl Ester (mPEG-DS). mPEG-oxycarbonyl-3-amino-1,2propanediol (2.3 g, 1.08 mmol, 2.16 mequiv of OH) was dissolved in 30 mL of toluene and azeotropically dried by partial removal of 10 mL of the solvent. The solution was cooled to room temperature and treated with 4 mL of pyridine and then 1 g of 4.3 M stearoyl chloride, leading to the immediate formation of a white precipitate. The reaction mixture was refluxed overnight, filtered while still warm (40 °C), and evaporated to dryness. The product was purified by two recrystallizations from 2-propanol and dried in vacuo over P2O5. The yield was 2.26 g (80%). Product purity was confirmed by thin-layer chromatography on Silica gel G with chloroform/methanol (9:1) as mobile phase. Treatment with iodine vapor revealed a single spot product with an Rf of 0.53, which was unambiguously different from the Rf of 0.27 of the more polar adduct mPEG-diol. 1H NMR analysis (360 MHz, (24) Viard, M.; Gallay, J.; Vincent, M.; Meyer, O.; Robert, B.; Paternostre, M. Biophys. J. 1997, 73, 2221-2234. (25) Priev, A.; Zalipsky, S.; Cohen, R.; Barenholz, Y. Langmuir 2002, 18, 612-617. (26) Zalipsky, S. Int. Patent Appl. WO 0105873; 2001.

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c

Figure 1. (A) Molecular structure and membrane location of the C17HC fluorophore with respect to phospholipids (EPC), cholesterol, and mPEG-DSPE. (B) Structural formulas of mPEG-DSG, mPEG-DS, and mPEG-DSPE. (C) Synthesis of mPEG-DS. DMSO-d6) resulted in the following ppm values: 0.89 (t, CH3, 6H), 1.26 (s, CH2, 56 H), 1.50 (t, 2CH2, 4 H), 2.24 (t, 2CH2CH2Cd O, 4 H), 3.23 (s, OCH3, 3 H), 3.50 (s, PEG, 180 H), 4.00 (dd, CH2 of APD, 1 H), 4.02 (t, CH2OCdON, 2 H), 4.20 (dd, CH2 of APD, 1 H), 4.98 (m, CHOCO, 1 H), 7.34 (m, NH, 1 H). 1HNMR spectra for all of the mPEG-DS lipopolymers were essentially the same, except that the PEG signal at 3.50 ppm integrated for 68, 180, 450, and 1100 protons for PEG0.75k, PEG2k, PEG5k, and PEG12k, respectively. Molecular weights of the mPEG-DS products were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (performed by Charles Evans & Associates, Sunnyvale, CA), which exhibited in all cases a typical bell-shaped

PC,a mol %

cholesterol, mol %

mPEG-lipidb or PG,c mol %

100 98.0 96.0 95.0 94.0 92.0 88.0 75.0 60.0 58.8 57.6 57.0 56.4 55.2 52.8 45.0 0

0 0 0 0 0 0 0 0 40.0 39.2 38.4 38.0 37.6 36.8 35.2 30.0 0

0 2 4 5 6 8 12 25 0 2 4 5 6 8 12 25 100

a EPC or HSPC. b mPEG-DSPE, mPEG-DS, or mPEG-DSG. EPG or HSPG.

distribution of lines spaced 44 Da apart. This mass difference corresponds to the molecular weight of one ethylene oxide unit. 3.3. Labeling Micelles and Liposomes with C17HC. 3.3.1. C17HC-Labeled Micelles. Micelles of Triton-X-100 (nonionic detergent micelles, size is ∼5 nm), or mPEG-DSPE micelles having either PEG0.75k, or PEG2k, or PEG5k, or PEG12k moieties, or the respective mPEG-DS lipopolymers (size is ∼15-20 nm) containing C17HC in mole ratio 1:400 (C17HC/lipopolymer), were prepared by colyophilization of the lipids and C17HC, followed by hydrating the dried powder in the desired aqueous solutions to a final concentration above the lipopolymer critical micelle concentration. 3.3.2. C17HC-Labeled Liposomes. Liposomes were based either on unsaturated EPC or on saturated HSPC as the liposomeforming lipid, with or without cholesterol, and varied in their mole percent of mPEG-DSPE (PEG2k or PEG5k), mPEG2k-DS, mPEG-DSG, or PG. Table 1 lists the various lipid combinations used for individual liposome preparations. All liposomes contained C17HC. To prepare C17HC-labeled liposomes, the desired lipid components were dissolved in tert-butyl alcohol at a lipid/ C17HC mole ratio of 400:1. The lipid/C17HC solutions were lyophilized and at the desired time hydrated in 10 mM histidine buffer (pH 6.7) in 18.2 MΩ/cm highly pure water. Hydration was performed at room temperature for EPC-based systems and at 65 °C for HSPC-based systems. LUVs were prepared by extrusion through a polycarbonate filter with a pore size of 100 nm27 using the LiposoFast (Avestin, Inc., Ottawa, Canada) system. The phospholipid concentration was quantified as described elsewhere28,29 and adjusted to a final concentration of 15 mM phospholipid in 10 mM histidine buffer, pH 6.7. Liposome size distribution, as determined by dynamic light scattering, was unimodal, having the mean size of 110 ( 10 nm. 3.4. Determination of Surface pH and Electrical Surface Potential (Ψ0). To determine surface pH and electrical surface potential (Ψ0) of liposomes, we used C17HC and therefore we refer to it as ψ0C17HC. For this we measured the degree of the fluorophore C17HC ionization over a broad range of pH values (between 6 and 13) using three different buffers: bicarbonate (pH range 6-8), borate (pH range 8-9), and carbonate/bicarbonate (pH range 9-13). An aliquot of 30 µL of liposomes was diluted in 1.5 mL of each of the following buffers. All samples were sonicated for about 5 s in a water bath to ensure pH equilibrium between the inside and the outside of the LUV. The bulk pH was determined before and immediately after each fluorescence measurement. To measure the C17HC ionization state, C17HC (27) MacDonald, R. C.; MacDonald, R. I.; Menco, B. P.; Takenshita, K.; Subbaro, N. K.; Hu, L. Biochim. Biophys. Acta 1991, 1061, 297-303. (28) Barenholz, Y.; Amselem, S. In Liposome Technology: Liposome Preparation and Related Techniques; Gregoriadis, G., Ed.; CRC Press: Boca Raton, FL, 1993; Vol. 1, p 527. (29) Shmeeda, H.; Even-Chen, S.; Honen, R.; Cohen, R.; Weintraub, C.; Barenholz, Y. Methods Enzymol. 2003, 367, 270-290.

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fluorescence excitation spectra were recorded at room temperature (22 °C) using an LS550B luminescence spectrometer (Perkin-Elmer, Norwalk, CT). Measurements were carried out at two excitation wavelengths: 330 nm, which is pH independent isosbestic point and represents the total amount of C17HC (unionized + ionized) in the lipid environment, and 380 nm, which reflects only the ionized C17HC-. The emission wavelength was 450 nm for both excitation wavelengths. Excitation and emission bandwidths of 2.5 nm were used. For each lipid composition, the apparent pKa of C17HC was calculated from the shift of the ratio of excitation wavelengths 380/330 as a function of bulk pH. A shift in the apparent pKa of C17HC, which represents its apparent proton binding constant, relative to a reference neutral surface, is indicative of the surface pH and the electrical surface potential in the immediate environment of the C17HC fluorophore. The values for electrical surface potential (Ψ0) and surface pH were calculated using the equations30,31

Ψ0 ) -

∆pKelkT e ln 10

pHsurface ) 6.7 +

Ψ0e kT ln 10

where 6.7 is the bulk pH for which surface pH and surface potential were calculated. For more details see Zuidam and Barenholz.32,33 3.5. Determination of Zeta Potential. Zeta potential was measured at 25 °C using a Zetasizer 3000 HAS, Malvern Instruments Ltd., Malvern, U.K. An aliquot of 40 µL of LUV was diluted in 20 mL of 10 mM NaCl (pH 6.7) and the solutions were passed through a 0.2-µm syringe filter (Minisart, Sartorius, Germany). The principle of measurement is the following: when an electrical field is applied to a suspension of charged particles in an electrolyte, the velocity of their movement toward the electrode of opposite polarity depends on the strength of the field, the dielectric constant, the viscosity of the medium, and the zeta potential. The relationship of zeta potential to the particle velocity in a unit electric field (electrophoretic mobility) is described by the Henry equation

UE ) zf(Ka)/6πη where UE ) electrophoretic mobility, z ) zeta potential,  ) dielectric constant, and η ) viscosity. f(Ka) is a function of the electric double layer thickness and particle diameter. In aqueous media or moderate electrolyte concentrations (10 mM NaCl), f(Ka) value is 1.5, which is used in the Smoluchowski approximation

UE ) z/4πη At 25 °C, the zeta potential can be approximated as

Ζ ) 12.85UE mV

4. Results and Discussion 4.1. Electrical Surface Potential (Ψ0C17HC) and Surface pH of Micelles and Liposomes. Figure 2 shows titration curves of the C17HC dissociation degree in micelles and liposomes with various amounts of two different PEG-lipids. The negatively charged mPEG2kDSPE (Figure 2A) and the neutral mPEG2k-DS (Figure 2B) were compared. The ratio of the excitation fluorescence intensities at 380 and 330 nm (emission for both at 450 nm) expressed as a percentage of the maximum value is (30) Cevc, G. Biochim. Biophys. Acta 1990, 1031, 311-382. (31) Toccane, J. F.; Teissie, J. Biochim. Biophys. Acta 1990, 1031, 111-142. (32) Zuidam, N. J.; Barenholz, Y. Biochim. Biophys. Acta 1997, 1329, 211-222. (33) Zuidam, N. J.; Barenholz, Y. Biochim. Biophys. Acta 1998, 1368, 115-128.

Figure 2. Dissociation degree of C17HC in liposomes and micelles as monitored by the ratio of excitation fluorescence intensities at 380 nm/330 nm (expressed as percentage of the maximum value) against bulk pH: mPEG2k-DSPE micelles and EPC:mPEG2k-DSPE LUV (A); mPEG2k-DS micelles and EPC:mPEG2k-DS LUV (B). Table 2. Micelle Apparent pKa and Calculated Values of Surface Potential (ψ0) and Surface pH at Bulk pH 6.7 micelles

apparent pKa

MU Triton X-100 mPEG0.75k-DSPE mPEG2k-DSPE mPEG5k-DSPE mPEG12k-DSPE mPEG0.75k-DS mPEG2k-DS mPEG12k-DS mPEG2k-DSG

7.90 8.83 11.29 11.24 10.74 10.86 9.06 8.99 9.03 9.09

ψ0 (mV)a

surface pH ( 0.02

-144.0 -141.0 -111.7 -118.8 -13.4 -9.4 -11.7 -15.2

(6.70)b 6.70 4.24 4.29 4.79 4.67 6.47 6.54 6.50 6.44

a Standard deviation is (6.5 in the case of mPEG-DSPE and (0.95 in the case of mPEG-DS or mPEG-DSG. b Water-soluble fluorophore (no surface).

plotted against the bulk pH. Values for the apparent pKa, the surface potential, and the surface pH of various compositions of micelles are presented in Table 2 and of various compositions of LUVs in Table 3. 41.1. Micelle Electrostatics. In a previous study25 we found that mPEG2k-DSPE and mPEG2k-DS both have similar critical micelle concentration (cmc) values,25 about 10 µM. The cmc value of mPEG-DSG having an mPEG moiety of 2000 Da determined as described by Priev et al.25 was also ∼10 µM. All of these three lipopolymers can be inserted into liposomes either during their preparation or postpreparation. Micelles composed of Triton X-100

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Table 3. Apparent pKa and Calculated Values of Electrical Surface Potential (ψ0) and Surface pH for PC Liposomes with Different mol % of MPEG2k-DSPE (A), MPEG2k-DS (B), MPEG5k-DSPE (C), and EPG or HSPG (D) lipid compositions (mole ratio)

pKa

EPC EPC:mPEG2k-DSPE 98:2 EPC:mPEG2k-DSPE 96:4 EPC:mPEG2k-DSPE 94:6 EPC:mPEG2k-DSPE 88:12 EPC:mPEG2k-DSPE 75:25 EPC:Chol EPC:Chol:mPEG2k-DSPE 58.8:39:2:2 EPC:Chol:mPEG2k-DSPE 57.6:38.4:4 EPC:Chol:mPEG2k-DSPE 56.4:37.6:6 EPC:Chol:mPEG2k-DSPE 52.8:35.2:12 EPC:Chol:mPEG2k-DSPE 45:30:25

ψ0 (mV)a

surface pH

9.50 9.65 9.79 9.91 10.31 10.65

-8.80 -17.01 -24.02 -47.40 -67.32

6.70 6.55 6.41 6.29 5.89 5.55

9.07 9.05

1.21

6.70 6.72

9.34

-15.80

6.43

9.58

-29.80

6.19

9.83

-44.50

5.94

10.00

-54.41

5.77

lipid compositions (mole ratio)

pKa

ψ0 ( mV)a

surface pH

A HSPC HSPC:mPEG2k-DSPE 98:2 HSPC:mPEG2k-DSPE 96:4 HSPC:mPEG2k-DSPE 94:6 HSPC:mPEG2k-DSPE 92:8 HSPC:mPEG2k-DSPE 88:12 HSPC:mPEG2k-DSPE 75:25 HSPC:Chol HSPC:Chol:mPEG2k-DSPE 58.8:39:2:2 HSPC:Chol:mPEG2k-DSPE 57.6:38.4:4 HSPC:Chol:mPEG2k-DSPE 56.4:37.6:6 HSPC:Chol:mPEG2k-DSPE 55.2:36.8:8 HSPC:Chol:mPEG2k-DSPE 52.8:35.2:12 HSPC:Chol:mPEG2k-DSPE 45:30:25

9.73 10.31 10.35 10.57 10.72 10.78 10.86 9.39 9.44

-2.90

6.70 6.13 6.08 5.86 5.71 5.65 5.57 6.70 6.65

9.66

-15.81

6.43

9.99

-35.12

6.10

10.17

-45.62

5.92

10.18

-46.20

5.91

10.24

-49.71

5.85

HSPC HSPC:mPEG2k-DS 98:2 HSPC:mPEG2k-DS 96:4 HSPC:mPEG2k-DS 94:6 HSPC:mPEG2k-DS 92:8 HSPC:mPEG2k-DS 88:12 HSPC:mPEG2k-DS 75:25 HSPC:Chol HSPC:Chol:mPEG2k-DS 58.8:39:2:2 HSPC:Chol:mPEG2k-DS 57.6:38.4:4 HSPC:Chol:mPEG2k-DS 56.4:37.6:6 HSPC:Chol:mPEG2k-DS 55.2:36.8:8 HSPC:Chol:mPEG2k-DS 52.8:35.2:12 HSPC:Chol:mPEG2k-DS 45:30:25

9.73 10.04 10.06 10.01 9.9 9.87 9.72 9.39 9.41

HSPC HSPC:mPEG5k-DSPE 98:2 HSPC:mPEG5k-DSPE 95:5 HSPC:Chol HSPC:Chol:mPEG5k-DSPE 58.8:39:2:2 HSPC:Chol:mPEG5k-DSPE 57:38:5

9.70 10.22 10.27 9.39 9.77

HSPC HSPC:HSPG 98:2 HSPC:HSPG 96:4 HSPC:HSPG 92:8 HSPC:HSPG 88:12 HSPC:Chol HSPC:Chol:HSPG 58.8:39:2:2 HSPC:Chol:HSPG 57.6:38.4:4 HSPC:Chol:HSPG 55.2:36.8:8 HSPC:Chol:HSPG 52.8:35.2:12

9.73 11.09 11.16 11.19 11.41 9.39 9.75 9.92 10.21 10.22

-33.40 -36.30 -49.22 -57.90 -61.41 -66.14

B EPC EPC:mPEG2k-DS 98:2 EPC:mPEG2k-DS 96:4 EPC:mPEG2k-DS 94:6 EPC:mPEG2k-DS 92:8 EPC:mPEG2k-DS 88:12 EPC:mPEG2k-DS 75:25 EPC:Chol EPC:Chol:mPEG2k-DS 58.8:39:2:2 EPC:Chol:mPEG2k-DS 57.6:38.4:4 EPC:Chol:mPEG2k-DS 56.4:37.6:6 EPC:Chol:mPEG2k-DS 55.2:36.8:8 EPC:Chol:mPEG2k-DS 52.8:35.2:12 EPC:Chol:mPEG2k-DS 45:30:25

9.50 9.56 9.60 9.58 9.56 9.64 9.54 9.07 9.36

-17.00

6.70 6.64 6.77 6.62 6.64 6.56 6.66 6.70 6.41

9.24

-9.90

6.53

9.22

-8.80

6.55

9.12

-2.91

6.65

9.13

-3.54

6.64

9.04

-1.7

6.73

-3.53 -4.12 -4.70 -3.51 -8.22 -2.34

-1.21

6.70 6.39 6.37 6.42 6.53 6.56 6.71 6.70 6.68

9.42

-1.80

6.67

9.44

-2.91

6.65

9.61

-12.32

6.49

9.49

-5.81

6.61

9.53

-8.20

6.56

-18.10 -19.31 -16.22 -9.90 -8.20 0.63

C EPC EPC:mPEG5k-DSPE 98:2 EPC:mPEG5k-DSPE 95:5 EPC:Chol EPC:Chol:mPEG5k-DSPE 58.8:39:2:2 EPC:Chol:mPEG5k-DSPE 57:38:5

9.50 10.06 10.38 9.07 9.52

EPC EPC:EPG 98:2 EPC:EPG 96:4 EPC:EPG 94:6 EPC:EPG 88:12 EPC:Chol EPC:Chol:EPG 58.8:39:2:2 EPC:Chol:EPG 57.6:38.4:4 EPC:Chol:EPG 56.4:37.6:6 EPC:Chol:EPG 52.8:35.2:12

9.50 9.55 9.69 9.73 10.01 9.07 9.41 9.39 9.82 9.94

9.85

-26.31

6.70 6.14 5.82 6.70 6.25

-45.62

5.92

-32.80 -51.50

10.17

-22.21

6.70 6.21 6.16 6.70 6.32

-45.60

5.92

-28.70 -31.61

D

a

-2.90 -11.10 -13.50 -29.80 -19.91 -18.72 -43.91 -50.90

6.70 6.65 6.51 6.47 6.19 6.7 6.36 6.38 5.95 5.83

-79.60 -83.70 -85.40 -97.70 -21.10 -29.80 -47.40 -48.60

6.70 5.34 5.27 5.24 5.03 6.7 6.34 6.19 5.89 5.87

Standard deviation is e5%.

served as neutral reference micelles for studying the electrostatics due to the similarity between the PEG moiety of the three lipopolymers used and Triton X-100. Accuracy of pKa measurement was evaluated for MU, the water-soluble fluorophore of C17HC; a pKa value of 7.9 determined in this study is in a good agreement with previously published data.32 As can be seen from Table 2,

all mPEG-DS and mPEG-DSG micelles tested had surface potentials close to zero, as expected for uncharged amphiphiles, and their surface pH was similar to the bulk pH (6.7) used in the study. In contrast, the micelles of all mPEG-DSPE species used were highly negatively charged, and their surface pH was significantly lower than the bulk pH. Within the tested range of 750-12000 Da,

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Table 4. Electrophoretic Mobility and Zeta Potential of Liposomes and Micelles at pH 6.7 in 10 mM NaCl at Room Temperature

lipid compositions (mole ratio) mPEG2k-DSPE micelles mPEG2k-DS micelles mPEG2k-DSG micelles EPC EPC:mPEG2k-DSPE 98:2 EPC:mPEG2k-DSPE 92:8 EPC:mPEG2k-DS 98:2 EPC:mPEG2k-DS 92:8 EPC:mPEG-DSG 91.5:8.5 EPC:Chol EPC:Chol:mPEG2k-DSPE 58.8:39:2:2 EPC:Chol:mPEG2k-DSPE 55.2:36.8:8 EPC:Chol:mPEG2k-DS 58.8:39:2:2 EPC:Chol: mPEG2k-DS 55.2:36.8:8 EPC:EPG 98:2 EPC:EPG 92:8 HSPC HSPC: mPEG2k-DSPE 98:2 HSPC: mPEG2k-DSPE 92:8 HSPC: mPEG2k-DS 98:2 HSPC: mPEG2k-DS 92:8 HSPC:mPEG-DSG 91.5:8.5 HSPC:Chol HSPC:Chol: mPEG2k-DSPE 58.8:39:2:2 HSPC:Chol: mPEG2k-DSPE 55.2:36.8:8 HSPC:Chol: mPEG2k-DS 58.8:39:2:2 HSPC:Chol: mPEG2k-DS 55.2:36.8:8 HSPC:HSPG 98:2 HSPC:HSPG 92:8 Doxil [HSPC:Chol:mPEG2kDSPE 56.4:38.3:5.3]

electrophoretic mobility (µm‚cm/V s), ζ potential (0.04 (mV), (0.5 -0.708 -0.098 -0.197 -0.624 -0.769 -0.857 -0.028 -0.144 0.131 -0.673 -0.782 -0.869 -0.007 -0.040 -1.740 -3.103 -0.524 -0.667 -0.718 -0.289 -0.211 0.055 -0.360 -0.715 -0.812 -0.045 -0.056 -0.907 -2.105 -1.046

-9.0 -1.3a -2.5a -7.9 -9.8 -10.9 -0.4a -0.8a 1.7 -8.6 -10.0 -11.3 -0.1a -0.7a -22.2 -39.5 -6.7 -7.5 -8.9 -3.7 -2.7 0.7 -4.6 -9.1 -10.3 -0.74a -0.79a -11.5 -26.8 -13.3

a According to the instrument guide, values in the range of 0 ( 2 mV are considered to be uncharged.

the molecular weight of the PEG moiety had only a minimal effect on the surface potential and surface pH. While ψ0 of the mPEG-DSPE micelles is highly negative (Table 2), the zeta potential of these micelles is surprisingly low (only -9.0 mV, Table 4). 4.1.2. Effect of PEG-Lipid or PG on Liposome Electrostatics. Table 3 shows the apparent pKa, surface potential, and surface pH values for liposomes with different lipid compositions. Increasing the mPEG2kDSPE mole concentration in liposomes (EPC or HSPC, with or without cholesterol) makes the surface potential more negative and decreases surface pH (Table 3 A). Addition of the negatively charged phospholipids EPG (for EPC liposomes) or HSPG (for HSPC liposomes) had a similar effect on the surface potential and surface pH of liposomes (Table 3D). In contrast, an increase in the mole percentage of mPEG2k-DS had only a minimal effect on the negative surface charge of PC or PC/Chol liposomes (Table 3 B). Liposomes containing mPEG2k-DS showed a higher apparent pKa than the mPEG2k-DS micelles. This can be explained by the suggestion of Fromherz23 that the HC fluorophore is located closer to the negatively charged phosphodiester than to the positively charged choline moiety of the PC (see Figure 1A). Table 3C shows that the effect of mPEG5k-DSPE on liposome surface potential and surface pH is practically the same as that of mPEG2k-DSPE. 4.1.3. Effect of Matrix-Lipid Composition on Liposome Electrostatics. From the results obtained (Table 3A,B), the surface of HSPC seems to be slightly more negatively charged than that of EPC. This may be related to the fact that HSPC is fully saturated, being very high in distearoyl PC (83%), and also contains

1-palmitoyl, 2-stearoyl PC (15%) (Lipoid, certificate of analysis), which explains its high Tm of 52.5 °C, similar to that of DSPC. In contrast, EPC has one saturated and one unsaturated acyl chain,34 and therefore the EPC is more hydrated and less densely packed than HSPC.35 The presence of cholesterol (40 mol %) in the liposome membrane reduces bilayer compressibility due to reduction in free volume30 and reduces charge density (surface dilution), resulting in a small shift of surface potential to slightly less negative values and, consequently, a small increase in surface pH (Table 3). These results agree well with those of Silvander et al.16 Another effect of cholesterol that should be considered is its ability to “dry” the liposome surface.40 4.2. Zeta Potential of Micelles and Liposomes. Table 4 presents the electrophoretic mobility and zeta potential of most of the liposomes and micelles described in Tables 2 and 3. As expected from the fact that mPEG2kDS and mPEG2k-DSG lack any charged groups, both micelles and liposomes containing these neutral lipopolymers are almost neutral, and the absolute value of their zeta potential is less negative than that of PC or PC/Chol liposomes not containing mPEG-DS or mPEG2k-DSG. Inclusion of PG into the liposome membrane causes a substantially higher negative zeta potential; this effect of PG on the zeta potential is proportional to the mole percentage of PG incorporated in liposomes for all compositions studied (EPC; EPC/Chol; HSPC; HSPC/Chol) and is in agreement with the results of Bu¨rner et al.36 mPEG2k-DSPE micelles and LUV (including DOXIL) containing up to 8 mol % of mPEG2k-DSPE have a larger negative zeta potential than PC or PC/Chol liposomes. However, the effect of mPEG2k-DSPE on the zeta potential of LUV per mole percentage negative charge is smaller than that of PG and increases less with increasing mole percentage of mPEG2k-DSPE, although both mPEG2kDSPE and PG have identical phosphodiester groups that are fully charged over a broad pH range (pH 3-14) and are expressed similarly when ψ0C17HC is determined. The finding that the zeta potential of lipid assemblies (micelles and liposomes) containing mPEG2k-DSPE is lower than expected is in agreement with previously reported data13,14,17,18,36 and fits the concept of the “hidden charge effect” suggested for liposomes sterically stabilized through grafting of mPEG-DSPE having PEG moiety g0.75 kDa.13,37 This “hidden charge effect” of the grafted PEG (also found for DOXIL, see Table 4) may result from the PEG moiety (but not the glycerol in PG) moving the plane of shear further away from the lipid/water interface.18 Therefore, the difference between ψ0 and zeta potential is much larger for mPEG-DSPE-containing LUV compared with PG-containing LUV. This effect of relatively large ψ0 and much smaller zeta potential is even larger for mPEG-DSPE micelles (see 4.1.1). This fits well with the finding that in micelles the PEG moieties are in a brush conformation12 and therefore the plane of shear is much further away from the phospholipid headgroup, which makes the “hidden charge effect” in the micelles more striking than that in the LUV. According to current scaling models,38,39 the extension of PEG2k from the lipid surface is about 3.5 nm for the mushroom regime (0-4 (34) Samuni, A. M.; Barenholz, Y. Free Radical Biol. Med. 1997, 22, 165-174. (35) Marsh, D. Handbook of Lipid Bilayers; CRC Press: Boca Raton, FL, 1990. (36) Bu¨rner, H.; Winterhalter, M.; Benz, R. J. Colloid Interface Sci. 1994, 168, 183-189. (37) Gaspar, M.; Martins, M.; Corvo, M.; Cruz, M. Biochim. Biophys. Acta 2003, 1609, 211-217. (38) DeGennes, P. G. Adv. Colloid Interface Sci. 1987, 27, 189-209.

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Table 5. Comparison between Corrected Zeta Potential and Surface Potential (Ψ0)

lipid compositions (mole ratio) mPEG2k-DSPE mPEG2k-DS EPC EPC:mPEG2k-DSPE 98:2 EPC:mPEG2k-DSPE 92:8 EPC:mPEG2k-DS 98:2 EPC:mPEG2k-DS 92:8 EPC:Chol EPC:Chol:mPEG2k-DSPE 58.8:39:2:2 EPC:Chol:mPEG2k-DSPE 55.2:36.8:8 EPC:Chol:mPEG2k-DS 58.8:39:2:2 EPC:Chol:mPEG2k-DS 55.2:36.8:8 EPC:EPG 98:2 EPC:EPG 92:8 HSPC HSPC:mPEG2k-DSPE 98:2 HSPC:mPEG2k-DSPE 92:8 HSPC:mPEG2k-DS 98:2 HSPC:mPEG2k-DS 92:8 HSPC:Chol HSPC:Chol:mPEG2k-DSPE 58.8:39:2:2 HSPC:Chol:mPEG2k-DSPE 55.2:36.8:8 HSPC:Chol:mPEG2k-DS 58.8:39:2:2 HSPC:Chol:mPEG2k-DS 55.2:36.8:8 HSPC:HSPG 98:2 HSPC:HSPG 92:8 Doxil [HSPC:Chol:mPEG2kDSPE 56.4:38.3:5.3]

ζ potential (mV) corrected value

Ψ0 (C17HC) (mV)

-9.0 -1.3a 0a -2.1a -3.2

-141 -9.4 0 -9.0 -35.0 -3.5 -3.5 0 0 -35.0 -17.0 -2.9 -2.9 -26.0 0 -33.4 -57.9 -18.2 -9.9 0 -2.9 -45.6 -1.2 -12.3 -79.6 -90.0

0a 0a -1.4a -2.7a 0a 0a -14.3 31.6 0a -0.8a -2.2a -0a -0a 0.5a -4.5 -5.7 -0a -0a -4.8 -20.1 -8.7

a According to the instrument guide, values in the range of 0 ( 2 mV are considered to be uncharged.

mol % of grafted PEG2k in LUV) and up to 10 nm for the brush regime (above 4 mol % of grafted PEG).18,40 Therefore, the location of the plane where zeta potential is measured is different for the PG- and mPEG2k-DSPEcontaining liposomes, and even more so for mPEG-DSPE micelles, to the extent that the comparison of ψ0 and zeta potential may be invalid. 4.3. Zeta Potential versus Electrical Surface Potential. The electrical surface potential is the electrical potential determined in the plane of the negatively charged phosphodiester of the phospholipid (Figure 1B).23 The zeta potential is the electrokinetic potential determined further away from the actual phospholipid headgroup plane (where electrostatic potential is equal to zeta potential) and closer to the boundary between the fixed and the mobile electrical parts of the Stern double layer.41 Therefore, the measurements with fluorescent probes, such as C17HC, can give more accurate information about liposome surface charge at the phospholipid headgroup region than measurements of their electrophoretic mobility (zeta potential), although measurement by such fluorescent probes may be biased by the exact location of the fluorophore.23,42 Table 5 presents values of surface potential and zeta potential of liposomes containing mPEG2k-lipids and PG corrected for those of PC and PC/Chol liposomes not containing mPEG2k-lipids or PG. For most LUVs containing increasing amounts of PG or mPEG2k-DS, and for mPEG2k-DS micelles, there is good agreement between (39) Hristova, K.; Needham, D. J. Colloid Interface Sci. 1994, 168, 302-314. (40) Priev, A.; Samuni, A.; Tirosh, O.; Barenholz, Y. In Targeting of Drugs 6: Strategies for Stealth Therapeutic Systems; Gregoriadis, G., McCormack, B., Eds.; Plenum Press: New York, 1998; Vol. 300, p 147. (41) Egorova, E. Electophoresis 1994, 15, 1125-1131. (42) Kalmanzon, E.; Zlotkin, E.; Barenholz, Y. In Handbook of Nonmedical Applications of Liposomes; Barenholz, Y., Lasic, D. D., Eds.; CRC Press: Boca Raton, FL, 1996; Vol. 4, p 183.

the changes in surface potential and zeta potential. In contrast, changes in surface potential and zeta potential differ from one another for micelles or LUVs containing mPEG2k-DSPE, due to the “hidden charge effect” of the PEG moiety (discussed above). 4.4. Effect of Liposome Electrostatics on Interaction with Cationic Peptides and Inorganic Cations. We studied the effect of various cationic molecules on electrical surface potential (Ψ0) and vesicle aggregation of C17HC-labeled liposomes. Changes in Ψ0 are indicative of close contact (