Preparation of pH-Sensitive Poly(glycidol) Derivatives with Varying

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Bioconjugate Chem. 2008, 19, 1040–1048

Preparation of pH-Sensitive Poly(glycidol) Derivatives with Varying Hydrophobicities: Their Ability to Sensitize Stable Liposomes to pH Naoki Sakaguchi, Chie Kojima, Atsushi Harada, and Kenji Kono* Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan. Received December 19, 2007; Revised Manuscript Received February 16, 2008

We have previously shown that modification with succinylated poly(glycidol) (SucPG) provides stable egg yolk phosphatidylcholine (EYPC) liposomes with pH-sensitive fusogenic property. Toward production of efficient pHsensitive liposomes, in this study, we newly prepared three carboxylated poly(glycidol) derivatives with varying hydrophobicities by reacting poly(glycidol) with glutaric anhydride, 3-methylglutaric anhydride, and 1,2cyclohexanedicarboxylic anhydride, respectively, designated as GluPG, MGluPG, and CHexPG. Correlation between side-chain structures of these polymers and their respective abilities to sensitize stable liposomes to pH was investigated. These polymers are soluble in water at neutral pH but became water-insoluble in weakly acidic conditions. The pH at which the polymer precipitated was higher in the order SucPG < GluPG < MGluPG < CHexPG, which is consistent with the number of carbon atoms of these polymers’ side chains. Although CHexPG destabilized EYPC liposomes even at neutral pH, attachment of other polymers provided pH-sensitive properties to the liposomes. The liposomes bearing polymers with higher hydrophobicity exhibited more intense responses, such as content release and membrane fusion, at mildly acidic pH and achieved more efficient cytoplasmic delivery of membrane-impermeable dye molecules. As a result, modification with appropriate hydrophobicity, MGluPG, produced highly potent pH-sensitive liposomes, which might be useful for efficient cytoplasmic delivery of bioactive molecules, such as proteins and genes.

INTRODUCTION Recent advances in protein and nucleic acid based therapies, such as immunotherapy and gene therapy, require efficient delivery systems of such labile bioactive molecules into the cytoplasm of target cells. In general, these molecules are not able to permeate a cellular membrane, although their site for action exists inside a cell, such as the nucleus. Therefore, it is necessary to use carrier systems that deliver these molecules into the cytoplasmic space of the target cell. Although various systems have been attempted for application to cytoplasmic delivery, pH-sensitive liposomes are a promising system. pHsensitive liposomes are stable at physiological pH, but become destabilized and/or fusogenic under mildly acidic conditions. In general, a main pathway for cellular internalization of liposomes is shown to be endocytosis. Therefore, after being taken up by a cell, liposomes are trapped in endosomes and are eventually degraded in lysosome. Because the endosome has a weakly acidic environment, pH-sensitive liposomes induce destabilization of endosomes and/or fusion with endosomes, resulting in the release of encapsulated materials into the cytosol. To date, various approaches to the production of pH-sensitive liposomes have been attempted. For example, pH-sensitive amphiphiles, such as oleic acid (1) and cholesteryl hemisuccinate (2), have been mixed with non-bilayer-forming phospholipid dioleoylphosphatidylethanolamine to yield pH-sensitive liposomes. In this case, pH-sensitive amphiphiles act as a pHsensitive stabilizer, which stabilizes unstable liposomes only when they are negatively charged. However, the lipid molecules used for this type of pH-sensitive liposome have a strong * Corresponding author. Kenji Kono; Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan; Tel: +81-72254-9330; Fax: +81-722-54-9330; E-mail [email protected].

tendency to form a hexagonal II structure; consequently, these liposomes are readily destabilized (3). Another efficient method for pH-sensitization of liposomes is modification of stable liposomes with pH-sensitive membraneactive molecules. Various kinds of fusion peptides derived from viral fusogenic proteins and their analogues have been used as liposomal membranes. Synthetic polymers with carboxyl groups have also been used to sensitize liposomes to pH (4, 5). In comparison to peptide-based pH-sensitizers, synthetic polymers might be advantageous because of their simplicity of preparation and low immunogenicity. Tirrell and co-workers have demonstrated that some poly(alkylacrylic acid)s interact with phospholipid membranes in a pHdependent manner (4, 6). In particular, poly(2-ethylacrylic acid) exhibits a strong interaction with phospholipid membranes at mildly acidic pH: it permeabilizes and, at sufficiently high concentration, solubilizes them by micelle formation (7-9). In addition, they showed that attachment of this polymer onto the liposome surface gave pH-sensitive content release properties (10). Hoffman et al. further derivatized poly(2-ethacrylic acid) and obtained poly(2propylacrylic acid), which exhibited even higher membranelytic activity than poly(2-ethylacrylic acid) (11-13). In a previous study, we developed succinylated poly(glycidol), named SucPG, for sensitization of liposomes (Figure 1) (13, 14). This polymer has a backbone that resembles that of poly(ethylene glycol) (PEG) and carboxyl groups on the side chains, which control interaction of the polymer backbone with lipid membranes in a pH-dependent manner. We have shown that modification with SucPG gave stable phosphatidylcholine liposome pH-sensitive properties. The SucPG-modified liposomes are stable at neutral pH but exhibit considerable destabilization under mildly acidic conditions. In contrast to poly(2-ethacrylic acid), which induced destabilization by solubilizing liposomal lipid molecules upon protonation of its pendant carboxyl groups, SucPG caused fusion of liposomes

10.1021/bc7004736 CCC: $40.75  2008 American Chemical Society Published on Web 04/18/2008

pH-Sensitive Poly(glycidol) Derivatives

Figure 1. Optical densities at 500 nm for solutions of SucPG (2), GluPG ([), MGluPG (b), and CHexPG (9) dissolved in 30 mM sodium acetate and 120 mM NaCl at various pHs (0.25 mg/mL) at 25 °C. Each point is the mean ( SD (n ) 3).

in the same situation (8, 13). Because the fusion ability of liposomes is useful for cytoplasmic delivery of membraneimpermeable molecules, such as proteins and oligonucleotides, the fusogenic properties of the SucPG are of great importance. In fact, SucPG-modified liposomes were shown to deliver calcein into the cytosol through fusion with endosome/lysosome membranes (14). Fusion capabilities of SucPG-modified liposomes were used to increase the transfection activities of various lipoplexes: complexation of the SucPG-modified liposomes with lipoplexes generates efficient gene vectors (15, 16). Although the mechanism of destabilization of liposome membranes mediated by SucPG has not been clarified, it might be assumed that hydrophobic interaction of its backbone with the lipid membrane and hydrogen bond formation between the polymer carboxyl groups and phosphate groups of the phospholipid membrane are responsible for inducing fusion (13). Therefore, one strategy for production of polymers with high fusogenic activity might be synthesis of poly(glycidol) with carboxyl groups and enhanced hydrophobic characteristics. For this study, we prepared poly(glycidol) derivatives with various side chains having carboxyl groups with different chemical structures (Scheme 1). Correlation between hydrophobicity of the side chains of poly(glycidol) derivatives and their membrane active properties was investigated. The ability of these poly(glycidol) derivatives to provide pH-sensitive properties to stable liposomes has been described.

MATERIALS AND METHODS Materials. Egg yolk phosphatidylcholine (EYPC) were kindly donated by Nippon Oil and Fats Co. (Tokyo, Japan). Pyrene, pyranine, and Triton X-100 was obtained from Tokyo Kasei Kogyo (Tokyo, Japan). p-Xylene-bis-pyridinium bromide (DPX) was from Molecular Probes (Oregon, USA). N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)dioleoylphosphatidylethanolamine (NBD-PE) and Lissamine rhodamine B-sulfonyl phosphatidylethanolamine (Rh-PE) were purchased from Avanti Polar Lipids (Birmingham, AL, USA). 1-Aminodecane was purchased from Nakalai Tesque (Kyoto, Japan). Glutaric anhydride and 1,2-cyclohexanedicarboxylic anhydride were from Tokyo Kasei Kogyo (Tokyo, Japan). 3-Methylglutaric anhydride was from Aldrich (Milwaukee, WI). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was from Dojindo (Kumamoto, Japan).Triton X-100 was obtained from Tokyo Kasei Kogyo (Tokyo, Japan). Two kinds of poly(glycidol) [PG-1: number average molecular weight (Mn) 82 000, weight average molecular weight (Mw) 13 000; PG-2: Mn 8700, Mw 16 100] were

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prepared as previously reported (13, 14). Succinylated poly(glycidol) (SucPG) was prepared using PG-1 as previously reported (13). Synthesis of Poly(glycidol) Derivatives. Glutarylated poly(glycidol) (GluPG) was prepared by reaction of poly(glycidol) with glutaryl anhydride. Poly(glycidol) (PG-1) (2.0 g) was dissolved in N,N-dimethylformamide (19 mL) and 3.0 equiv of glutaryl anhydride (9.2 g) was added to the solution. The mixed solution was kept at 80 °C for 6 h with stirring. Then, the reaction mixture was evaporated and dialyzed against water for 7 days. The product was recovered by freeze-drying. 3-Methylglutarylated poly(glycidol) (MGluPG) and 2-carboxy-cyclohexanoylated poly(glycidol) (CHexPG) were prepared by the same procedure by the reaction of poly(glycidol) (PG-2) with 3-methylglutaric anhydride and 1,2-cyclohexanedicarboxylic anhydride, respectively. As anchor moieties for fixation of poly(glycidol) derivatives onto liposome membranes, 1-aminodecane was combined with carboxyl groups of these poly(glycidol) derivatives. Each polymer was dissolved in water around pH 5, and 1-aminodecane (0.15 equiv to carboxyl group of polymer) was reacted to carboxyl groups of the polymer using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.2 equiv to carboxyl group of polymers) at 4 °C for two days with stirring. The resultant polymers were purified by washing with chloroform and subsequent dialysis in water. Precipitation pH. Precipitation pHs of polymers were determined by measuring the optical density of aqueous polymer solutions (0.25 mg/mL) at various pH. Polymers were dissolved in 2.0 mL of acetate buffer (30 mM sodium acetate, 120 mM NaCl) adjusted to various pH. After 5 min incubation at 25 °C, optical densities (OD) of the polymer solutions at 500 nm were measured by using a spectrophotometer (Jasco V-520). Precipitation pH were determined by optical density-pH profile as the pH at which OD drastically rose. Preparation of Pyranine-Loaded Liposomes. To a dry, thin membrane of EYPC (7 mg) was added 500 µL of pyranine solution containing 35 mM pyranine, 50 mM DPX, and 25 mM MES of pH 7.4, and the mixture was sonicated for 2 min using a bath-type sonicator. The liposome suspension was further hydrated by freeze and thaw, and extruded through a polycarbonate membrane with a pore size of 50 nm. The liposome suspension was applied to a G75 column to remove free pyranine from the pyranine-loaded liposomes. Modification of Liposomes with Polymers. Liposome suspensions were obtained by the above procedure. To a liposome suspension (lipid concentration 17.5 mM) in the 25 mM MES and 125 mM NaCl buffer was added polymer solution (final concentration 10 mg/mL), and the mixture was incubated 60 min at room temperature. Liposomes were purified by gel filtering with a G75 column to separate excess polymer and nonencapsulated pyranine in the case of pyranine containing polymer-modified liposome. Release of Pyranine from Liposome. Release of pyranine from liposome was measured as previously reported (17). Pyranine fluorescence was quenched by DPX inside of the liposomes, but this molecule exhibits intense fluorescence when released from the liposome (17). To a suspension of pyranineloaded liposomes (lipid concentration 2.0 × 10-5 M) in the 25 mM MES and 125 mM NaCl buffer of varying pHs was added varying amounts of the polymer dissolved in the same buffer. Pyranine fluorescence at 512 nm with excitation at 416 nm was monitored by using a spectrofluorometer (Jasco FP-6500). 100% release was achieved by adding Triton X-100 (final concentration 0.1%) to the liposome suspension. Fusion Assay. Fusion between bare EYPC liposomes and polymer-modified liposomes was detected by measuring reso-

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Scheme 1. Synthetic Route for Various Poly(glycidol) Derivativesa

a

EDC represents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.

nance energy transfer between NBD-PE and Rh-PE (15, 19). Polymer-modified liposomes containing NBD-PE and Rh-PE were prepared via the above method using the membrane composed of EYPC, NBD-PE, Rh-PE (98.8:0.6:0.6, mol/mol/ mol), and varying amounts of the polymers. The labeled liposomes (final concentration of lipid 0.125 M) were mixed with fluorescent probe-free EYPC liposomes (final concentration of lipid 0.25 M) in 25 mM MES, and 125 mM NaCl solution of varying pHs and fluorescence intensities NBD-PE and RhPE followed. Their fusion was followed by monitoring the fluorescence intensity ratio of NBD-PE to Rh-PE (R). The excitation wavelength of NBD-PE was 450 nm, and monitoring wavelengths for NBD-PE and Rh-PE were 520 and 580 nm, respectively. Percentage increase in R was defined as Percentage increase in R ) (Rt - R0) ⁄ (R100 - R0) × 100

(1)

where R0 and Rt represent the initial and intermediary R values. R100 is the R of the labeled liposomes when the liposomes fused completely. The fluorescent lipid-labeled liposomes and the unlabeled liposomes were dissolved in methanol, dried by evaporation, and resuspended in MES buffer. The R value of the suspension was taken as R100 (15). Pyrene Fluorescence. A known amount of pyrene in acetone solution was added to an empty flask, and acetone was removed under vacuum. Polymer (0.2 mg/mL) in the 25 mM MES and 125 mM NaCl buffer solutions of varying pHs were added the flask, yielding 1 µM concentration of pyrene. The sample solution was stirred overnight at room temperature, and emission spectra with excitation at 337 nm were recorded. The fluorescence intensity ratio of the first band at 373 nm to the third band at 384 nm (I1/I3) was analyzed as a function of pH of the solution. Interaction of Liposomes with Cells. Polymer-modified liposomes containing calcein were prepared via the above procedure using an aqueous calcein solution (63 mM, pH 7.4) instead of an aqueous pyranine solution. HeLa cells (1.0 × 105) were seeded on glass-bottomed culture dishes and incubated in 10% FCS/DMEM overnight. The cells were washed with PBS and reincubated with fresh medium with 10% FCS (0.5 mL). Then, liposomes in the 20 mM Hepes and 130 mM NaCl suspension (0.5 mL) were added to the dishes (final concentration of liposomal lipid: 0.5 mM), and the cells were incubated for 4 h. For the incubation in the presence of NH4Cl, cells were preincubated in 10% FCS/DMEM containing NH4Cl (20 mM) for 1 h before the addition of liposome suspension. After the incubation with the liposomes, the cells were washed with PBS and were observed using a fluorescence microscope (BX51WI Olympus). In addition, cells were incubated for another 2 h in

10% FCS/DMEM with or without NH4Cl (20 mM) and were observed using a fluorescence microscope.

RESULT AND DISCUSSION Properties of Poly(glycidol) Derivatives. For a previous study, we developed SucPG, which generates fusogenic activity under weakly acidic conditions and is useful to produce pHsensitive liposomes that destabilize at mildly acidic pH (13). In fact, we observed that SucPG-modified liposomes delivered membrane-impermeable fluorescent dye calcein efficiently into the cytosol through fusion with endosomal/lysosomal membranes (14). For the present study, we attempted to develop new polymers with even higher pH-responsive properties than SucPG. We postulated that both a PEG-like structure of the backbone and carboxyl groups on the side chains are important for their pH-dependent fusogenic activity (13). Therefore, an increase in the side chains hydrophobicity is expected to strengthen their interaction with the lipid membranes, thereby producing polymers with a high ability to destabilize lipid membranes in a pH-dependent manner. Three kinds of polyglycidol derivatives with more hydrophobic side chains than those of SucPG were designed (Scheme 1). We synthesized them using the reaction of poly(glycidol) with various acid anhydrides according to Scheme 1. Compositions of these polymers were estimated using 1H NMR and are listed in Table 1. For all polymers, only a small percentage of unreacted glycidol units around 2.0-3.8% remained on the polymer backbone after the reaction of poly(glycidol) with acid anhydrides, indicating that these reactions were very efficient; fundamentally, every repeating unit possesses a carboxyl group in the resultant polymers. We observed that ester bonds of these polymers were essentially not hydrolyzed during dialysis against distilled water for several days, indicating their high stability in water. Many studies have shown that hydrophobic moieties, such as stearyl groups and cholesteryl groups, were incorporated into polymer chains to act as an anchor for their fixation to liposome membranes (22-24). We showed that attachment of 1-aminodecane to about 8 unit % of a SucPG chain is sufficient to fix the polymer chain onto an EYPC liposome membrane (13). On the basis of a previous study (13), we combined decylamine to about 10 unit % of the polymer chains in the present study using a condensing agent, EDC (Scheme 1). As shown in Table 1, decyl chains were attached to around 10 unit % of the polymer chains for all poly(glycidol) derivatives. We first investigated the pH sensitivity of the poly(glycidol) derivatives because these polymers contain many carboxyl groups. Acid-base titration of these polymers was performed to estimate the pKa values. As presented in Table 2, SucPG, GluPG, and MGluPG had almost equal pKa values around 6.2-6.4, although CHexPG showed a somewhat higher pKa



Table 1. Compositions of Various Poly(glycidol) Derivativesa

polymer

l (mol %)

m (mol %)

n (mol %)

SucPG GluPG MGluPG CHexPG SucPG-C10 GluPG-C10 MGluPG-C10 CHexPG-C10

2.4 2.0 3.8 2.3 3.0 3.7 5.6 3.2

97.6 98.0 96.2 97.7 85.7 83.8 86.6 85.7

11.3 12.5 7.8 11.1

a

Estimated by 1H NMR. SucPG-C10, GluPG-C10, MGluPG-C10, and CHexPG-C10 are anchor-carrying types of corresponding poly(glycidol) derivatives. Table 2. pKa and Precipitation pH of Various Poly(glycidol) Derivatives polymer

pKa

precipitation pH

R at precipitation pH

SucPG GluPG MGluPG CHexPG

6.2 6.4 6.3 7.0

4.0 4.7 5.0 5.4

0.93 0.95 0.93 0.99

value of 7.0. Hydrophobicity of side groups and conformation of polyelectrolytes are known to affect the dielectric constant around charged groups, thereby imparting different ionization characteristics (25-27). Because CHexPG has highly hydrophobic side groups, hydrophobic interactions among these side groups might engender a compact conformation, which enhances charge-charge interactions, resulting in suppression of dissociation of carboxyl groups (27). Hydrophobicity of carboxylated polymers affects their precipitation behaviors (28). For that reason, we estimated the pH at which the polymers precipitate using the following optical densities (ODs) of these polymer solutions as the pH of the solution was decreased (Figure 1). These polymers were soluble in water at neutral pH; their solutions were transparent. However, the polymer solutions suddenly became turbid at a specific pH, which was defined as the precipitation pH. The precipitation pH thresholds for these poly(glycidol) derivatives were estimated as listed in Table 2. Apparently, the precipitation pH shifted to higher pH values as the number of carbon atoms in the side chains of the polymers increased. This result indicates that the precipitation pH increases with increasing hydrophobicity of the polymer side groups and is consistent with previous observation for methacrylic acid copolymers with varying hydrophobicities (28). Acid-base titration for these polymers indicated that the degree of protonation for their carboxyl groups was at pH 0.93-0.99 of the precipitation pH: most carboxyl groups must be protonated to elicit precipitation of these polymers. Hydrophobicity of the polymers was further investigated using fluorescence of pyrene. An emission intensity ratio of the first (373 nm) to the third (384 nm) peaks of pyrene, I1/I3, is known to be sensitive to the microenvironmental polarity surrounding the pyrene molecule (19). Consequently, this ratio has been widely used to estimate the hydrophobic nature of polymers (20, 21). Figure 2 depicts the I1/I3 ratio of pyrene fluorescence in the buffer dissolving various polymers as a function of pH. In buffers dissolving SucPG and GluPG, the I1/I3 ratios of pyrene were appropriately 1.8 at any pH and were the same as the ratio in the buffer without polymers. This result suggests that these polymers formed no domains with a hydrophobic nature, even

Figure 2. I1/I3 of pyrene fluorescence in MES 25 mM and NaCl 125 mM solution with SucPG (2), GluPG ([), MGluPG (b), and CHexPG (9), and without polymer (O). Concentration of polymers and pyrene were 0.2 mg/mL and 1 µM, respectively. I1/I3 was defined as the fluorescence intensity ratio of the first band at 373 nm to the third band at 384 nm.

after protonation of most carboxyl groups of the polymer chain. The presence of MGluPG affected the I1/I3 ratio, which tends to decrease below pH 5.5, indicating that this polymer formed domains with a hydrophobic nature. A more significant decrease in the I1/I3 ratio was shown by the presence of CHexPG, which caused a marked decrease in the ratio below pH 8.0. This polymer formed hydrophobic domains, even at physiological pH. Interaction of Poly(glycidol) Derivatives with Lipid Membrane. We investigated the ability of these polymers to interact with liposomal membranes. The poly(glycidol) derivatives were added to liposomes encapsulating both pyranine and its fluorescence quencher DPX suspended in the buffer of varying pHs; leakage of pyranine from the liposomes was monitored by following pyranine fluorescence. Figure 3A,B depicts the time course and pH dependence of the pyranine release from the liposomes induced by the polymers. Although CHexPG induced a marked release at pH 6.5, other polymers exhibited no ability to induce a release of pyranine at any pH, indicating that SucPG, GluPG, and MGluPG have very weak or no ability to destabilize liposomal membrane. In contrast, when their counterparts with anchor moieties, namely, SucPG-C10, GluPG-C10, MGluPG-C10, and CHexPGC10, were added to the pyranine-loaded liposomes, an intensive release of the contents was observed for all polymers (Figure 3C,D). Considering that most of these polymers without anchors failed to induce content release, even at acidic pH, these polymers are necessarily associated with the liposome membranes before their protonation to induce content release from the liposomes. Because their backbone with PEG-like structure can form hydrogen bonding with carboxyl groups, both the polymer backbone and side-chain carboxyl groups might not efficiently interact with liposomal membranes when the polymer chains are dissolved in solution. However, when bound onto the liposome membrane, these polymers might strongly interact with the liposome membrane through hydrogen bonding between the side-chain carboxyl groups and the phosphate groups of the liposomal lipid and hydrophobic interaction between the polymer backbone and the lipid membrane, generating structural defects in the liposome membrane (13). As shown in Figure 2D, the pH region in which the content release was triggered increased in the order of SucPG-C10, GluPG-C10, MGluPG-C10, and CHexPG-C10. Apparently, the polymers with higher hydrophobicity trigger the release at higher pH. Actually, CHexPG caused the release, even at neutral pH, whereas other polymers induced the release below pH 6.5 or 6,


Sakaguchi et al.

Figure 3. Pyranine release from egg yolk phosphatidylcholine liposomes induced by various poly(glycidol) derivatives. Time course at pH 6.5 (A) and pH dependence (B) of pyranine release induced by SucPG (4), GluPG (]), MGluPG (O), and CHexPG (0). Time course at pH 6.5 (C) and pH dependence (D) of pyranine release induced by SucPG-C10 (2), GluPG-C10 ([), MGluPG-C10 (b ), and CHexPG-C10 (9), and without polymer (1). Measurements were performed in MES 25 mM and NaCl 125 mM solution at 25 °C. Percent release of pyranine after 10 min incubation was shown (B,D). Polymer and lipid concentrations were 0.1 mg/mL and 2.0 × 10-5 M, respectively. Each point is the mean ( SD (n ) 3).

Figure 4. Correlation between dissociation degree (R) of polymers and their induced content release of liposomes. Percent release of pyranine after 10 min incubation with SucPG-C10 (2), GluPG-C10 ([), MGluPGC10 (b), and CHexPG-C10 (9) was plotted against R of polymer.

which corresponds to the pH of endosome. In addition, their ability to destabilize the liposome membrane in the neutral and weakly acidic region increased with increasing hydrophobicity of the polymer side chains. Because these polymers acquire the ability to destabilize the liposome membrane upon protonation of carboxyl groups on the side chains, we examined the correlation between the protonation degree and their membrane-destabilizing ability. Figure 4 depicts the percent release induced by the polymer as a function of dissociation degree R of the polymers evaluated using acid-base titration. Highly hydrophobic CHexPG can acquire the ability to induce the content release when only about 25% of carboxylate anions are protonated. In contrast, hydrophilic SucPG cannot induce the release until about 70-80% of carboxylate anions lose their negative charges by protonation. Indeed, dissociation of carboxyl groups might be affected in

Figure 5. Mean diameters of egg yolk phosphatidylcholine liposomes after overnight incubation with SucPG-C10 (closed bars), MGluPGC10 (open bars), and CHexPG-C10 (gray bars) at varying pHs. Mean diameters of the same liposomes after overnight incubation without polymer were also shown (diagonal bars). Measurements were performed in MES 25 mM and NaCl 125 mM solution at 25 °C. Polymer and lipid concentrations were 0.83 mg/mL and 1.7 × 10-4 M, respectively. Each point is the mean ( SD (n ) 3).

the peripheral region of the liposome. Therefore, the result indicated that polymers with higher hydrophobicity acquire membrane-destabilizing activity on a state with a lesser degree of protonation. Interaction of the polymers with the liposomes was also investigated through inspection of the liposome size change. The EYPC liposomes with 60 nm diameter were incubated with SucPG-C10, MGluPG-C10, or CHexPG-C10 at varying pH overnight; their diameters were evaluated using DLS (Figure 5). The liposome size only slightly changed after incubation with SucPG-C10, even at pH 5.5. Similarly, the liposome size was not affected when incubated with MGluPG-C10 at pH 7.4 and 6.0, but incubation at pH 5.5 caused an increase in diameter. Interaction with this polymer might induce destabilization of the liposome membrane, engendering their aggregation. Ag-



Figure 6. Pyranine release behaviors of egg yolk phosphatidylcholine liposomes modified with SucPG-C10 (triangles), GluPG-C10 (diamonds), and MGluPG-C10 (squares). (A) Time course at pHs 7.4 (open symbols), 6.0 (gray symbols), and 5.5 (closed symbols). (B) Percent release of pyranine from the polymer-modified liposomes after 10 min incubation as a function of pH. Lipid concentrations were 2.0 × 10-5 M. Each point is the mean ( SD (n ) 3).

Figure 7. pH-sensitive fusogenic properties of polymer-modified liposomes. Time course at pH 5.0 (A) and pH dependence of percent increase in R values after mixing of fluorescent lipid-labeled liposomes modified with SucPG ([), GluPG (9), and MGluPG (2) after 3 h incubation at varying pHs. Measurements were performed in 25 mM MES and 125 mM NaCl at 37 °C. Each point is the mean ( SD (n ) 3).

gregation of liposomes became more intensive when incubated with CHexPG-C10. Indeed, polymer-induced aggregation was enhanced with decreasing pH, but this polymer exhibited the ability to induce liposome aggregation, even at neutral pH. Preparation of pH-Sensitive Liposomes with Poly(glycidol) Derivatives. Results of a previous study showed that modification of liposomes with pH-sensitive polymers is an efficient means to prepare pH-sensitive liposomes (10, 13, 24, 29). Especially, modification with SucPG provided pH-sensitive fusion ability, which is useful for cytoplasmic delivery of membrane-impermeable molecules (14). Therefore, we next examined the ability of poly(glycidol) derivatives to render stable EYPC liposomes pH-sensitive. For this purpose, polymers are necessary to exhibit activity only under mildly acidic conditions. However, CHexPG exhibits membrane-destabilizing activity even at neutral pH; we examined three poly(glycidol) derivatives aside from CHexPG. The liposomes were incubated with varying amounts of the polymers for varying periods and the liposomes were separated from the free polymers using gel filtration to achieve modification of EYPC liposomes with the polymers. Comparison of their content release performance revealed that incubation of liposomes (17.5 mM) in more than 2 mg/mL of the polymer solutions (pH 7.4) for more than 60 min yielded the highest performance (Supporting Information Figure S1). In addition, the zeta potential of these polymer-modified liposomes with the

highest performance showed a constant value of about -31 mV (Supporting Information Figure S1). These results indicate that the polymer chains were fully incorporated into the liposome surface by incubation under these conditions. On the basis of these results, we prepared EYPC liposomes modified with the poly(glycidol) derivatives by incubating the liposomes in 10 mg/mL of the polymer solutions for 1 h, with subsequent gel filtration. Figure 6 depicts pH-sensitive content release behaviors of liposomes modified with the SucPG-C10, GluPG-C10, and MGluPG-C10. All liposomes retained pyranine at pH 7.4, indicating that they are stable at physiological pH. However, these liposomes enhanced their content release below pH 6.0; almost complete release was achieved at pH 5.5. This result demonstrates that these liposomes were able to destabilize the inside of early and late endosomes, which have respective environments with pH 6.5-6.0 and pH 6.0-5.0 (30). As portrayed in Figure 6, all liposomes exhibited a similar pH response. This observation differs greatly from the case of content release induced by addition of these polymers into the liposome suspensions, in which MGluPG-C10, GluPG-C10, and SucPG-C10, respectively, exhibited content release enhancement around 6.5, 6.0, and 5.5 (Figure 3D). Apparently, the content release was enhanced in a higher pH region for the polymers attached to the liposomes than for the free polymers. Protonation of carboxylate anions of these polymers might be enhanced in


Sakaguchi et al.

Figure 8. Fluorescence micrographs of HeLa cells treated with calcein-loaded liposomes modified with varying polymers or without polymer modification. The cells were incubated with liposomes for 4 h at 37 C° and were washed with PBS. Then, the cells were observed just after washing (left column) or after additional incubation in DMEM with 10% FCS at 37 C° for 2 h (right column) using a fluorescence microscope. Cells treated with liposomes in the presence of NH4Cl were also shown.

the peripheral region of the liposome membrane, which is an environment with lower polarity than the aqueous medium. Therefore, the difference in hydrophobicity of these polymers’ side chains might not reflect the pH region, where their protonation took place. We also examined the pH response of these polymer-modified liposomes with respect to fusion ability. For this purpose, we prepared polymer-modified EYPC liposomes containing NBDPE and Rh-PE, which have been used widely to detect fusion of liposomes (18). Fusion of the labeled liposomes with

unlabeled liposomes causes dilution of these fluorescent lipids in the membrane. Therefore, their fusion results in decreased resonance energy transfer efficiency between these fluorescent lipids. In addition, previous results have shown that the fluorescence intensity ratio of NBD-PE to Rh-PE (R) is useful to monitor liposome fusion (14, 15). Polymer-modified liposomes with the fluorescent lipids were mixed with the unlabeled EYPC liposomes and the fluorescence intensity ratio of NBD-PE to Rh-PE (R) for the mixed liposome suspension was calculated (Figure 7). Figure 7A depicts the


percentage increase in the R-value (see the Materials and Methods section) for the liposome suspensions at pH 5.0. The liposomes modified with SucPG exhibited a slight increase in the R-value, suggesting that this polymer increases the fusion ability of the liposome only slightly at pH 5.0. However, the liposomes modified with GluPG showed a marked increase in the R-value with time; the MGluPG-modified liposomes exhibited an even more significant increase in R, indicating that these polymers have a higher ability to render the stable liposomes fusogenic than SucPG. Their ability is influenced by differences of side-chain structures. Figure 7B portrays the percent increase in the R-value after 3 h incubation for polymer-modified liposomes and plain liposomes. Irrespective of the polymer, these liposomes displayed no change in R-value at pH 7.4-6.0, but the R-values increased below pH 6.0, which indicates that these liposomes became fusogenic in the mildly acidic environment. Their fusion ability varies depending on the polymer attached to liposomes around pH 5.0. However, a further decrease in the ambient pH strengthened their fusion ability, and these polymer-modified liposomes acquired similar and strong fusion ability around pH 4.5-4.0. Actually, SucPG has fewer hydrophobic side chains than other polymers. For that reason, this polymer, with many charged groups, might not strongly destabilize the lipid membrane to cause liposome fusion. In contrast, GluPG and MGluPG, which possess hydrophobic side chains, might destabilize the liposome membrane even through their side chains are partly charged. Therefore, MGluPG, of which side chains are most hydrophobic among these polymers, produced liposomes with the highest fusion capability. Interaction of Polymer-Modified Liposomes with Cells. Finally, we estimated their potential as a cytplasmic delivery vehicle for membrane-impermeabile molecules. For this purpose, we loaded calcein, which is a water-soluble fluorescent dye, in the polymer-modified EYPC liposomes. It is well-known that fluorescence of calcein is strongly quenched when encapsulated in the liposome at a high concentration, but becomes highly fluorescent once released from the liposome (31). Therefore, intracellular localization of calcein molecules released from the liposomes can be estimated from their fluorescence. We incubated HeLa cells with the calcein-loaded liposomes for 4 h and washed them with PBS. Then, the treated cells were observed using a fluorescence microscope just after washing or after additional incubation in the medium for 2 h. As is seen in Figure 8, the cells treated with the calcein-loaded bare liposome displayed punctuate fluorescence. Considering that the liposomes were generally taken up by a cell via endocytosis, it is highly likely that calcein molecules were still trapped in endosome and/or lysosome (14, 32). In contrast, the cells treated with the calcein-loaded, polymer-modified liposomes showed diffused fluorescence, indicating that calcein molecules existed in cytoplasm. Because these liposomes have the ability to destabilize and fuse with lipid membranes under a weakly acidic environment, it is likely that calcein molecules were released from endosome or lysosome into cytoplasm. On the basis of the comparison of calcein fluorescence observed in the liposome-treated cells, it seems that the ability of cytoplamic calcein delivery may differ among these polymermodified liposomes. Punctuate fluorescence was mainly observed in the cells just after the incubation with SucPG-C10modified liposome, though additional 2 h incubation caused diffused fluorescence in the cell. In contrast, intensive diffused fluorescence was observed in the cells treated with MGluPGC10-modified liposomes, indicating that these polymers might possess a stronger ability to destabilize endosomal and lysosomal membranes than SucPG-C10. In addition, when the treatment with the MGluPG-C10-modified liposomes was performed in


the presence of ammonium chloride, which is known to inhibit acidification of endosome and lysosome, punctuate fluorescence appeared in the cells, suggesting that acidification of these intracellular organelles is necessary for the transfer of calcein molecules into cytosol. These results demonstrate that modification of stable liposomes with fusogenic polymers, which strongly destabilize lipid membranes at weakly acidic pH, can produce pH-sensitive liposomes that achieve efficient cytoplasmic delivery of membrane-impermeable molecules.

CONCLUSION We have previously used SucPG for pH sensitization of stable liposomes. In this study, we newly synthesized three pHsensitive poly(glycidol) derivatives with side chains of varying hydrophobicities, GluPG, MGluPG, and CHexPG, and investigated a correlation between their side-chain structure and their ability to render stable liposomes pH-sensitive. Their ability for pH sensitization of liposomes was enhanced with increasing side-chain hydrophobicity. In particular, MGluPG, which possesses side chains with appropriate hydrophobicity, produced highly pH-sensitive liposomes that undergo content release and membrane fusion at mildly acidic pH, which corresponds to endosomal and lysosomal pH. Such properties of the liposomes are important for efficient cytoplasmic delivery of membraneimpermeable molecules because destabilization and fusion with endosome and/or lysosome membranes improves the transfer of liposomal contents to cytosol. In fact, MGluPG-modified liposomes encapsulating calcein efficiently delivered their contents into the cytosol of a cell. Considering their application into body, the increased hydrophobicity of the polymer may affect biodistribution of the liposomes. However, further incorporation of poly(ethylene glycol) grafts to the liposomes might cover the liposome surface and produce liposomes with high pH sensitivity and long circulating properties. Information obtained from this study is expected to be useful for development of highly efficient pH-sensitive liposomes and related vehicles for efficient cytoplasmic delivery of bioactive molecules, such as proteins and genes.

ACKNOWLEDGMENT This work was supported in part by a Grant-in-Aid for Research on Nanotechnical Medicine from the Ministry of Health, Labor and Welfare of Japan and by a Grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture in Japan. Supporting Information Available: Release of pyranine from SucPG-modified liposomes prepared by incubation of the liposomes with SucPG for varying periods. Release of pyranine from SucPG-modified liposomes prepared by incubation of liposomes with SucPG at varying concentrations. Zeta potentials of SucPG-modified liposomes prepared by incubation of liposomes with SucPG at varying concentrations for 1 h. This material is available free of charge via the Internet at http:// pubs.acs.org.

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Preparation of pH-Sensitive Poly(glycidol) Derivatives with Varying

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