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Stable vesicle assemblies on surfaces of hydrogel nanoparticles formed from a polysaccharide modified with lipid moieties
Chemical Engineering Journal 263 (2015) 38–44
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Stable vesicle assemblies on surfaces of hydrogel nanoparticles formed from a polysaccharide modified with lipid moieties Insung Kang a, Jungju Yoon a, Yebin Lee a, Jaehyuk Choi a, Jinho Yang a, Suk Tai Chang a, Jonghwi Lee a, Jaehwi Lee b, Pil J. Yoo c, Juhyun Park a,⇑ a b c
School of Chemical Engineering and Materials Science, Chung-Ang University, Seoul 156-756, Republic of Korea College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea School of Chemical Engineering and SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon 440-746, Republic of Korea
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
We synthesize hyaluronic acids
attached with methacrylates and phospholipids. Modified hyaluronic acids form nanohydrogels via a surfactant free route. Lipids attached to hyaluronic acids anchor foreign lipids for assembling peripheral vesicles. Nanohydrogels swell with pH variations for disassembling peripheral vesicles.
a r t i c l e
i n f o
Article history: Received 29 May 2014 Received in revised form 23 October 2014 Accepted 29 October 2014 Available online 11 November 2014 Keywords: Hyaluronic acid Hydrogel Inverse microemulsion Nanoparticle Lipid assembly
a b s t r a c t Micro- and nanoparticle-supported lipid assemblies have significant potential for being used in biology and medicine for sensing, mimicking cellular membranes, and delivering drugs or cosmetic agents. Here, we introduce a new type of nanohydrogels based on the modification of a polysaccharide with lipid moieties, followed by the formation of nanoparticles and assembling lipid bilayers on the particle surfaces. The lipophilic compound 1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine (DMPE) and the UVcrosslinkable methacrylic anhydride were covalently attached to hyaluronic acid (HA) and the formation of hydrogel nanoparticles via a surfactant-free inverse emulsion mechanism was demonstrated. As an anchoring group, the lipophilic DMPE moiety enables the formation of hydrogel nanoparticles with a spherical morphology in nonpolar media and allows for the stable assembly of lipid bilayers bearing amphiphiles on HA nanohydrogel surfaces. The conjugated oligoelectrolyte, 4,40 -bis[40 -(N,N-bis(600 (N,N,N-trimethylammonium)hexyl)amino)styryl] stilbene tetraiodide (DSSN+), was incorporated into the nanohydrogel-supported lipid bilayers, resulting in the formation of stable multilamellar peripheral vesicular structures due to the similarity in chemical structure of DMPE and the assembled lipid molecules. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Lipid bilayers self-assembled on microspheres of silica [1], polymers such as polystyrene [2] and polyvinyl alcohols [3], and ⇑ Corresponding author. Tel.: +82 2 820 5735; fax: +82 2 824 3495. E-mail address: [email protected] (J. Park). http://dx.doi.org/10.1016/j.cej.2014.10.096 1385-8947/Ó 2014 Elsevier B.V. All rights reserved.
hydrogels such as agarose–gelatin [4] and poly(N-isopropylacrylamide) [5] have received considerable attention for over the last two decades due to their potential in biomedical applications. Pharmaceutical agents or biofunctional proteins could be embedded inside such microsphere-liposome assemblies within the microsphere or supported lipid membranes on the microsphere while maintaining mechanical stability. The so-called lipobeads
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or lipogels [6,7] have been used as artificial biological cells to study membrane biophysics [8], as biosensors coupled with microfluidics [9], in drug delivery [10,11], and in dermatologic applications [12–14]. Hydrogel-liposome assemblies have been fabricated using electrostatic attraction between lipids and hydrogel surfaces [15–17], by transferring aqueous gel beads in an oil medium onto a saturated lipid monolayer at a planar oil–water interface [18], and by forming liposomes followed by gelation of their interior [19]. Other methods of preparation of hydrogel–liposome assemblies include coating microgels with layer-by-layer self-assembled polyelectrolyte multilayers and subsequent formation of lipid shells by vesicle adsorption and disruption [20], and modification of microgel surfaces with hydrophobic fatty acids [3,8,21] after gel formation to anchor lipid molecules and to induce their selfassembly. However, further decreases in bead or gel size to the nanometer scale are required for future pharmaceutical and cosmetic applications [10,15,17]. When the size of a colloidal system decreases, it is expected that the circulation time in the blood would increase or that the response time for swelling or shrinking under stimulus would decrease, which could improve targeting, transmembrane delivery, and controlled release of drugs [13–17,12,22]. Nanohydrogels are also useful for transdermal delivery and cosmetics. The stratum corneum, the outermost layer of skin, has a morphology that can be represented by a brick (corneocyte) and mortar (intercellular lipid layer) model [14]. Smaller-sized colloids show enhanced permeation into these microstructures and allow for transport of hydrophobic drugs or cosmetic components through the intercellular lipid layers [12,23]. Herein, we demonstrate a new type of hydrogel–liposome assemblies with a nanogel core formed by the hydrophobic anchoring group of a phospholipid. The novel aspects of our work include the formation of nanogels via a surfactant-free inverse emulsion route and the demonstration of stable peripheral lipid vesicular structures bearing an amphiphilic agent. We modified hyaluronic acid (HA) with a phospholipid moiety, 1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine (DMPE), and with UV-crosslinkable methacrylic anhydride (MA). It is anticipated that the modified HA (HA–MA–DMPE) can form nanospheres in an inverse emulsion media without the use of surfactants, generating nanogel particles upon UV irradiation with phospholipid moieties displayed on the nanogel surfaces. Thus, the phospholipid bilayers, which have a similar chemical structure to that of the surfaceanchoring moieties and can accommodate hydrophobic or amphiphilic agents, can self-assemble on the surface of HA nanogels, as
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illustrated in Scheme 1. As a proof of concept, we demonstrate the assembly of the DMPC lipid bilayers bearing the amphiphilic conjugated oligoelectrolyte 4,40 -bis[40 -(N,N-bis(600 -(N,N,N-trimethylammonium)hexyl)amino)styryl] stilbene tetraiodide (DSSN+) on a nanogel surface [24,25]. Using cryogenic transmission electron microscopy (cryo-TEM) and the polarity-dependent photoluminescence of the conjugated oligoelectrolyte, we demonstrate the successful assembly of the lipid bilayers on the HA nanogel surface. 2. Experimental 2.1. Materials Sodium hyaluronate (MW = 10,000–20,000 g/mol, Cat. No. HA10K) was obtained from Lifecore Biomedical, Inc. (Minnesota, USA). Methacrylic anhydride (MA), 2-morpholinoethanesulfonic acid (MES), N-hydroxy succinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), and a photo initiator (Irgacure D-2959) were purchased from Sigma–Aldrich Co. (Montana, USA). A cellulose membrane (MWCO = 3500 g/mol) from Spectrum Laboratories Inc. (California, USA) was used for dialysis. 1,2-Ditetradecanoyl-sn-glycero-3-phosphoethanolamine (DMPE) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were purchased from Avanti Polar Lipids, Inc. (Alabama, USA). A MES buffer solution (pH 6.4) was prepared from 0.5 M sodium chloride and 50 mM MES. 2.2. Synthesis of methacrylated hyaluronic acid (HA–MA) To prepare methacrylated HA (HA–MA), a synthetic route similar to that described in the literature [26] was used. In brief, HA (0.5 g, 1.25 mmol on a repeat unit basis, [(C14H20NO11Na)n] = 401 g/mol) was dissolved in deionized water (25 mL), followed by the addition of a 20-fold excess of methacrylic anhydride (3.7 mL, 25 mmol) relative to the primary hydroxyl groups in HA. The reaction mixture was then stirred for 24 h in an ice bath after adjusting the solution pH to 8.0 using a 5 M NaOH solution. The solution of HA–MA was purified by dialysis for 3 days against distilled water, filtered using 0.2 lm Whatman filter paper, and freeze-dried. Degree of substitution: 15.0%. 1H NMR (300 MHz, D2O): d 6.06– 5.61 (d, J = 135 Hz, 2H, C = C-H, Haa1), 2.57 (s, 2H; Hc), 1.89 (s, 3H; Hb1), 1.82 (s, 3H; Hb); IR (KBr): m = 3427 (s), 2922 (w), 1739 (m), 1652 cm1 (s).
Scheme 1. Assembly of the DMPC lipid bilayers bearing DSSN+ conjugated oligoelectrolytes on the surface of a hyaluronic acid hydrogel nanoparticle formed via the inverse microemulsion mechanism under UV irradiation after modification with methacrylates and DMPE.
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2.3. Synthesis of HA–MA modified with DMPE (HA–MA–DMPE) To covalently attach DMPE to HA–MA via amide bonding, a DMPE solution was first prepared by dissolving DMPE (0.1 g, 0.157 mmol) in 10 mL of a solvent mixture (THF:methanol:water = 60:30:10) using sonication for 10 min at 30 °C. HA– MA (0.5 g, 1.24 mmol on a repeat unit basis) was dissolved in 100 mL of a solvent mixture (MES buffer solution:THF:methanol = 60:30:10), followed by the addition of NHS (18 mg, 0.157 mmol) and EDC (24.4 mg, 0.157 mmol) to activate the carboxylic acids in HA–MA at the amounts corresponding to DMPE. After stirring the HA–MA solution for 1 h, the DMPE solution was added, and the resulting reaction mixture was stirred for 24 h at room temperature. HA–MA attached to DMPE (HA–MA–DMPE) was purified by dialysis for 36 h against the first solvent mixture (H2O:THF:methanol = 60:30:10), and then against the second solvent mixture (H2O:methanol = 70:30) for another 36 h. The resulting solutions were filtered and freeze-dried. Degree of substitution: 10.2%. 1H NMR (300 MHz, D2O + acetone-d6): d 5.37–5.32 (1H, N-H, Hq), 4.34–4.29 (m, 1H, Hn), 4.21–4.04 (m, 4H, Hl, Hm), 3.22–3.17 (m, 2H, Hp), 2.48–2.38 (m, 4H, Hk), 1.71–1.62 (4H, Hj), 1.43–1.27 (s, 40H, He, Hf, Hg, Hh, Hi), 0.960.91 (t, J = 3.3 Hz, 6H, Hd). 2.4. Preparation of hydrogel nanoparticles of HA–MA–DMPE HA–MA–DMPE (0.1 g) was dissolved in 2 mL of a solvent mixture (methanol:H2O = 70:30), followed by the addition of Irgacure D-2959 (0.5 mg). The polar solution of HA–MA–DMPE was added to 10 mL of dodecane and the resulting emulsion was sonicated for 2 h and cured under irradiation at 365 nm for 2 h. For purification, 10 ml of ethanol was added to the reaction mixture and the solution was centrifuged to remove the nonpolar medium and unreacted curing agent. The ethanol rinsing process was repeated 5 times. 2.5. Vesicle assembly on nanohydrogel surfaces DMPC (58 mg, 0.086 mmol) and DSSN+ (2.6 mg, 1.72 lmol, 2 mol% to DMPC, see reference [24] for the synthetic procedure) were dissolved in 2 mL of ethanol and the resulting solution was mixed with a nanohydrogel dispersion in ethanol (0.5 mL, 1 wt% of solid content). We used an excess amount of DMPC to observe the formation of multilamellar structure on nanohydrogel surfaces. The calculated molar mixing ratio of HA–MA–DMPE on a repeat unit basis to DMPC was 1:20. In addition, samples at mixing ratios of 1:1 and 1:7 were prepared. The mixture was placed in a vial and was dried under a nitrogen flow at 30 °C to remove the solvent and to form a thin film, followed by vacuum drying for 12 h at room temperature. Deionized water (2 mL) was added to the dried film in the vial and the solution was ultrasonicated for 2 h. To isolate nanohydrogels assembled with DMPC and DSSN+, the solution was centrifuged at 4000 rpm for 10 min, and the nanohydrogels were re-dispersed in deionized water at pH 6.5. To investigate the stability of lipid structures assembled on nanohydrogels upon swelling, the nanohydrogels were dispersed in deionized water at pH 10.5, and used for measuring particle size, zeta potential and photoluminescence. 2.6. Characterization of the compounds 1 H NMR spectra were recorded at room temperature on a Gemini 2000 300 MHz NMR spectrometer (Varian). FT-IR spectra were measured using a Nicolet 6700 spectrophotometer (Thermo Scientific). Hydrogel nanoparticles were characterized using a Mastersizer 3000 particle size analyzer (Malvern Instruments
Ltd.), a ELS-8000 zeta potential analyzer (Otsuka Electronics Co. Ltd.), a QM-3/2004SE photoluminescence spectrometer (PTI), a SIGMA scanning electron microscope (Carl Zeiss), and a Tecnai F20 cryogenic transmission electron microscope (FEI Com.). 3. Results and discussion 3.1. Hyaluronic acid modified with methacrylate and the phospholipid HA–MA–DMPE was prepared by chemical conjugation of MA with the hydroxyl groups of HA, followed by the formation of amide bonds between the amine-terminated phospholipid (DMPE) and the carboxylic acid groups of the HA backbone. The synthesis of HA–MA was carried out in an aqueous environment at pH 8.0 with an excess of MA with respect to the hydroxyl groups in HA, and the resulting compound was characterized by means of 1H NMR spectroscopy. In comparison with the 1H NMR spectrum of HA (Fig. 1a), the 1H NMR spectrum of HA–MA (Fig. 1b) shows new peaks at 1.82 (3H, Hb), 5.62, and 6.05 ppm (2H, Haa1), which were assigned to the protons of the methacrylate moieties. The degree of substitution of the methacrylate groups was determined from the integration ratio of the two vinyl proton peaks at 5.62 and 6.05 ppm to the methyl peak of pure HA at 1.89 ppm (3H, b1) and was found to be 15.0%, which indicates an adequate amount of photocrosslinkable units for the formation of a stable hydrogel upon UV irradiation. The covalent attachment of DMPE to HAMA was carried out by activating the carboxylic acid groups in the HA backbone using NHS and EDC at pH 6.4. Because the solubility of the reaction product, HAMADMPE, was poor in aqueous medium, we used a solvent mixture of MES buffer, THF, and methanol, which is key to the synthesis of HAMADMPE. The 1H NMR spectrum of HA–MA–DMPE in a mixture of deuterated acetone and water (Fig. 1c) shows the characteristic methyl and methylene peaks of DMPE. The assignment of these peaks to the DMPE hydrogens was made based on solid and solution state 1H NMR spectra of DMPC [27] and DMPE (cf. Supplementary information Figs. S1 and S2). The degree of substitution of DMPE was estimated to be 10.2% from the integration ratio of the vinyl proton peaks of MA at 5.85 and 5.54 ppm to the methyl peak of DMPE at 0.94 ppm. We attempted to use higher amounts of DMPE to increase the DMPE substitution, but it was limited to 10.2% due to precipitation during the reaction, as the lipophilicity increased with the degree of DMPE substitution. 3.2. Hydrogel nanoparticles formed via a surfactant-free inverse emulsion route The amphiphilicity of HA–MA–DMPE allows the polymer to be dissolved in polar media. Due to the increase in hydrophobicity with DMPE substitution, HA–MA–DMPE could not be dissolved in water, in contrast to the salt form of pure HA that is completely water-soluble. In addition, the solubility of HA–MA–DMPE was poor in low molecular weight alcohols such as methanol and ethanol [28–30]. The preparation of a HA–MA–DMPE solution was possible in a solvent mixture of water and methanol at a mixing ratio of 70:30, at which the hydrophilicity and hydrophobicity were balanced. The dropwise addition of the HA–MA–DMPE solution into a nonpolar medium with vigorous stirring followed by 2 h sonication resulted in a surfactant-free inverse emulsion due to the fact that the hydrophobic alkyl chains in DMPE were located at the interface and partitioned between the polar droplets and the nonpolar medium. Subsequent UV-crosslinking using methacrylate substituents and water-soluble photoinitiators resulted in the formation of an
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should be mentioned that the formation of hydrogel nanoparticles was not successful with toluene, which is a typical nonpolar medium for the formation of inverse emulsions. Thus, an alkane solvent with a chemical structure compatible with the alkyl chains of DMPE is seemingly a better nonpolar medium than toluene for preparing well-dispersed nanoparticles of HA–MA–DMPE. 3.3. Lipid bilayer assembly on the hydrogel nanoparticle surface
Fig. 1. 1H NMR spectra of (a) hyaluronic acid (HA), (b) HA after conjugation with methacrylate (HA–MA), and (c) HA–MA after conjugation with DMPE.
HA hydrogel. As demonstrated in Fig. 2a and b, hydrogel nanoparticles with a mean diameter 302 ± 20 nm were successfully synthesized when dodecane was employed as the nonpolar phase. It
Assembly of lipid bilayers on the surfaces of the hydrogel nanoparticles was carried out using a conventional unilamellar vesicle preparation procedure. The phospholipid DMPC containing 14 carbons in its chain was used in this reaction. The only difference in chemical structure between DMPC and DMPE is the functional group at the polar head, which is a quaternary amine in DMPC and a primary amine in DMPE, as shown in Scheme 1. Hydrogel nanoparticles, DMPC, and DSSN+ as a model amphiphilic substance were dissolved in ethanol, a co-solvent in which all three chemicals were soluble. After evaporating ethanol, deionized water was added. A subsequent long (>4 h) sonication at about 30 °C a temperature above the melting point of DMPC induced the disruption of the mixture and self-assembly of the lipid bilayers bearing DSSN+ on the surface of the hydrogel nanoparticles. The cryo-TEM images in Fig. 3a and b shows the hydrogel nanoparticles before and after the assembly of the lipid bilayers on their surfaces, respectively. The dark circular lines surrounding the hydrogel nanoparticles in Fig. 3b indicate a material with high electron density, demonstrating the incorporation of DSSN+ in the lipid bilayers. It can be seen that the vesicle structure surrounding nanoparticles is multilamellar. In contrast, when only DSSN+ and DMPC were used in the vesicle formation procedure, only unilamellar vesicles with a diameter less than 100 nm bearing DSSN+ in lipid bilayers were formed, as we previously reported [24,25]. In Fig. 3b, small unilamellar vesicles are found around HA hydrogel nanoparticles with the multilamellar structure, which can be explained by the use of an excess amount of DMPC to ensure the formation of a multilamellar structure on nanohydrogel surfaces. The average percentage of nanohydrogels with the peripheral multilayer structure was 5.9% at a 1:20 M mixing ratio of nanohydrogels to DMPC, which was estimated by analyzing the number of nanohydrogels and unilamellar vesicles observed in three cryo-TEM images over an area of 1 1 lm2. In addition, no multilamellar structure was formed on nanohydrogels when a 1:1 M mixing ratio of nanohydrogels to DMPC was used, indicating that an excess of the lipid molecules was necessary. Thus, the formation of a multilamellar structure indicates that the anchoring power of the alkyl chains on the nanoparticle surface is significantly enhanced due to the association of alkyl tails in DMPC and
Fig. 2. (a) SEM image of hyaluronic acid hydrogel nanoparticles prepared in dodecane and (b) hydrogel nanoparticle size analysis data.
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Fig. 3. Cryogenic TEM images of (a) the pristine nanohydrogels, (b) nanohydrogels after assembling DMPC lipid layers bearing DSSN+ on their surfaces, (c) TEM image of nanohydrogels assembled with DMPC without DSSN+, and (d) fluorescence spectra of DSSN+ in water (dashed line), in the multilamellar structure on nanohydrogel surfaces (solid line) and in unilamellar vesicles (dotted line).
DMPE, despite a long sonication time. We suggest that the alkyl tails in DMPC and DMPE facilitate the self-assembly of DMPC on the nanoparticle surface, providing strong anchoring power due to the enhanced hydrophobic attraction, as the short-range hydrophobic attraction force increases with increasing contact between hydrophobic groups. Such an effect was also demonstrated in the thin film deposition of a polyelectrolyte onto the surface of Teflon [31]. It is notable that nanohydrogels with the multilamellar structure could not be successfully prepared without DSSN+. As shown in Fig. 3c, agglomerates of nanohydrogels were formed when we used only pristine nanohydrogels and DMPC. We presume that the incorporation of DSSN+ in the DMPC multilayer structure induces cationic charges on nanohydrogel surfaces by charge compensation with polar lipid heads in DMPC and that the resulting electrostatic repulsion between the nanoparticles hinders the formation of agglomerates. The zeta potential value of 2.56 mV measured for nanohydrogels with the multilamellar structure isolated by centrifuging supports this presumption (Table 1). At the same time, it seems that such electrostatic repulsion due to cationic charges in the polar head region does not seriously affect the formation of the multilamellar structure on the nanohydrogel surface. As we previously reported, the zeta potential value of unilamellar vesicles bearing DSSN+ was approximately 40 mV [25]. Thus, the zeta potential value of 2.56 mV, measured for nanohydrogels with the multilamellar structure, indicates that a significant fraction of
Table 1 pH dependent variations of zeta potential values, particle diameters, and the wavelength of the maximum photoluminescence for nanohydrogels before and after assembling multilamellar structure on their surfaces. Samples Lipid-assembled nanohydrogel Lipid-assembled nanohydrogel
Solution pH
Diameter (nm)
Zeta potential (mV)
kmax (nm)
6.5
357
2.56
540
10.5
747
9.27
523
DSSN+ is embedded in the multilamellar structure and that a small fraction of DSSN+ is exposed at the outermost surface of the nanohydrogels. The incorporation of DSSN+ into DMPC layers was further confirmed using photoluminescence, as shown in Fig. 3d. The photoluminescence of DSSN+ is highly dependent on the polarity of the environment, showing solvatochromism [24,25,32]. When DSSN+ is dissolved in a polar medium such as water, water dipoles align with the DSSN+ dipoles, stabilizing the excited state of DSSN+. Consequently, the photoluminescence spectrum of DSSN+ is significantly red-shifted and shows a broad featureless peak. In contrast, when DSSN+ is located in a nonpolar medium such as the interior of lipid bilayers, there is a hypsochromic shift and the photoluminescence peak appears at a shorter wavelength. Fig. 3d shows that
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the peak corresponding to DSSN+ in unilamellar vesicles (dotted line) has a maximum at 516 nm, while the peak of water-dissolved DSSN+ has a maximum at 571 nm (dashed line), which confirms the incorporation of DSSN+ in lipid bilayers. The peak of DSSN+ assembled with DMPC on the nanoparticle surfaces has a maximum at 540 nm (solid line), which shows a moderate blue shift in comparison with the spectrum of water-dissolved DSSN+. We speculate that the stabilization of the energy levels takes place as a result of the association among DSSN+ molecules in the multilamellar structure. We further investigated the stability of the multilamellar structure assembled on the nanohydrogel surfaces by varying the solution pH and measuring the changes in particle sizes, zeta potential values, and photoluminescence spectra (Table 1 and Figs. S3 and S4). The stability of the multilamellar structure relying on the variation of solution pH and resulting swelling and deswelling of nanohydrogels is a significant feature of the nanohydrogels. At pH 6.5, the diameter of the nanohydrogels with the multilamellar structure was 357 nm, which is 55 nm larger than that of the pristine nanohydrogels, reflecting the existence of the peripheral multilamellar structure; the zeta potential value was 2.56 mV. Interestingly, the average diameter of the nanoparticles drastically increased to 747 nm when the solution pH was adjusted to 10.5. At the same time, the zeta potential value of the solution increased to 9.27 mV. The increase in the diameter of the nanoparticles with the increase of pH indicates that carboxylic acid groups in the HA nanoparticles are ionized at high pH, inducing the swelling of the nanoparticles. Furthermore, the increase in the zeta potential values indicates that the cation groups of DSSN+ embedded in the multilamellar structure are more exposed to water due to possible structural rearrangement taking place during the swelling. To elucidate the increase in zeta potential value and particle size at high pH, we measured photoluminescence spectra. Notably, the maximum photoluminescence was at 523 nm, which is similar to the maximum value of 516 nm in the spectrum of DSSN+ in unilamellar vesicles (Fig. 3d). A significant blue shift indicates that the multilamellar structure assembled on the nanohydrogel surface is destabilized with the pH increase and swelling. It is possible that the multilamellar structure is disrupted and shattered into unilamellar vesicles. The high zeta potential value of 9.27 mV also supports this explanation because similar high values were observed for the DMPC/DSSN+ unilamellar vesicles dispersed in deionized water in our previous report [25].
4. Conclusions HA substituted with 15.0 mol% methacrylate and 10.2 mol% phospholipid at the primary hydroxyl and carboxyl groups in the repeat units of HA has been used for preparing a surfactant-free inverse emulsion. The assembly of lipid bilayers of DMPC lipids and amphiphilic DSSN+ on hydrogel nanoparticle surfaces showed that the phospholipid hydrophobic anchoring group plays a significant role in the self-assembly of phospholipid bilayers. Multiple analyses demonstrated the formation of stable multilamellar peripheral vesicular structures on the nanoparticle surfaces, suggesting potential applications in the biomedical field for delivery of cosmetic or drug agents.
Acknowledgements This work was supported by the Ministry of Health and Welfare (Grant No. HN10C0032020013), Republic of Korea and by the Chung-Ang University Excellent Student Scholarship.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cej.2014.10.096.
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Short communication
Stable vesicle assemblies on surfaces of hydrogel nanoparticles formed from a polysaccharide modified with lipid moieties Insung Kang a, Jungju Yoon a, Yebin Lee a, Jaehyuk Choi a, Jinho Yang a, Suk Tai Chang a, Jonghwi Lee a, Jaehwi Lee b, Pil J. Yoo c, Juhyun Park a,⇑ a b c
School of Chemical Engineering and Materials Science, Chung-Ang University, Seoul 156-756, Republic of Korea College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea School of Chemical Engineering and SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon 440-746, Republic of Korea
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
We synthesize hyaluronic acids
attached with methacrylates and phospholipids. Modified hyaluronic acids form nanohydrogels via a surfactant free route. Lipids attached to hyaluronic acids anchor foreign lipids for assembling peripheral vesicles. Nanohydrogels swell with pH variations for disassembling peripheral vesicles.
a r t i c l e
i n f o
Article history: Received 29 May 2014 Received in revised form 23 October 2014 Accepted 29 October 2014 Available online 11 November 2014 Keywords: Hyaluronic acid Hydrogel Inverse microemulsion Nanoparticle Lipid assembly
a b s t r a c t Micro- and nanoparticle-supported lipid assemblies have significant potential for being used in biology and medicine for sensing, mimicking cellular membranes, and delivering drugs or cosmetic agents. Here, we introduce a new type of nanohydrogels based on the modification of a polysaccharide with lipid moieties, followed by the formation of nanoparticles and assembling lipid bilayers on the particle surfaces. The lipophilic compound 1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine (DMPE) and the UVcrosslinkable methacrylic anhydride were covalently attached to hyaluronic acid (HA) and the formation of hydrogel nanoparticles via a surfactant-free inverse emulsion mechanism was demonstrated. As an anchoring group, the lipophilic DMPE moiety enables the formation of hydrogel nanoparticles with a spherical morphology in nonpolar media and allows for the stable assembly of lipid bilayers bearing amphiphiles on HA nanohydrogel surfaces. The conjugated oligoelectrolyte, 4,40 -bis[40 -(N,N-bis(600 (N,N,N-trimethylammonium)hexyl)amino)styryl] stilbene tetraiodide (DSSN+), was incorporated into the nanohydrogel-supported lipid bilayers, resulting in the formation of stable multilamellar peripheral vesicular structures due to the similarity in chemical structure of DMPE and the assembled lipid molecules. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Lipid bilayers self-assembled on microspheres of silica [1], polymers such as polystyrene [2] and polyvinyl alcohols [3], and ⇑ Corresponding author. Tel.: +82 2 820 5735; fax: +82 2 824 3495. E-mail address: [email protected] (J. Park). http://dx.doi.org/10.1016/j.cej.2014.10.096 1385-8947/Ó 2014 Elsevier B.V. All rights reserved.
hydrogels such as agarose–gelatin [4] and poly(N-isopropylacrylamide) [5] have received considerable attention for over the last two decades due to their potential in biomedical applications. Pharmaceutical agents or biofunctional proteins could be embedded inside such microsphere-liposome assemblies within the microsphere or supported lipid membranes on the microsphere while maintaining mechanical stability. The so-called lipobeads
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or lipogels [6,7] have been used as artificial biological cells to study membrane biophysics [8], as biosensors coupled with microfluidics [9], in drug delivery [10,11], and in dermatologic applications [12–14]. Hydrogel-liposome assemblies have been fabricated using electrostatic attraction between lipids and hydrogel surfaces [15–17], by transferring aqueous gel beads in an oil medium onto a saturated lipid monolayer at a planar oil–water interface [18], and by forming liposomes followed by gelation of their interior [19]. Other methods of preparation of hydrogel–liposome assemblies include coating microgels with layer-by-layer self-assembled polyelectrolyte multilayers and subsequent formation of lipid shells by vesicle adsorption and disruption [20], and modification of microgel surfaces with hydrophobic fatty acids [3,8,21] after gel formation to anchor lipid molecules and to induce their selfassembly. However, further decreases in bead or gel size to the nanometer scale are required for future pharmaceutical and cosmetic applications [10,15,17]. When the size of a colloidal system decreases, it is expected that the circulation time in the blood would increase or that the response time for swelling or shrinking under stimulus would decrease, which could improve targeting, transmembrane delivery, and controlled release of drugs [13–17,12,22]. Nanohydrogels are also useful for transdermal delivery and cosmetics. The stratum corneum, the outermost layer of skin, has a morphology that can be represented by a brick (corneocyte) and mortar (intercellular lipid layer) model [14]. Smaller-sized colloids show enhanced permeation into these microstructures and allow for transport of hydrophobic drugs or cosmetic components through the intercellular lipid layers [12,23]. Herein, we demonstrate a new type of hydrogel–liposome assemblies with a nanogel core formed by the hydrophobic anchoring group of a phospholipid. The novel aspects of our work include the formation of nanogels via a surfactant-free inverse emulsion route and the demonstration of stable peripheral lipid vesicular structures bearing an amphiphilic agent. We modified hyaluronic acid (HA) with a phospholipid moiety, 1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine (DMPE), and with UV-crosslinkable methacrylic anhydride (MA). It is anticipated that the modified HA (HA–MA–DMPE) can form nanospheres in an inverse emulsion media without the use of surfactants, generating nanogel particles upon UV irradiation with phospholipid moieties displayed on the nanogel surfaces. Thus, the phospholipid bilayers, which have a similar chemical structure to that of the surfaceanchoring moieties and can accommodate hydrophobic or amphiphilic agents, can self-assemble on the surface of HA nanogels, as
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illustrated in Scheme 1. As a proof of concept, we demonstrate the assembly of the DMPC lipid bilayers bearing the amphiphilic conjugated oligoelectrolyte 4,40 -bis[40 -(N,N-bis(600 -(N,N,N-trimethylammonium)hexyl)amino)styryl] stilbene tetraiodide (DSSN+) on a nanogel surface [24,25]. Using cryogenic transmission electron microscopy (cryo-TEM) and the polarity-dependent photoluminescence of the conjugated oligoelectrolyte, we demonstrate the successful assembly of the lipid bilayers on the HA nanogel surface. 2. Experimental 2.1. Materials Sodium hyaluronate (MW = 10,000–20,000 g/mol, Cat. No. HA10K) was obtained from Lifecore Biomedical, Inc. (Minnesota, USA). Methacrylic anhydride (MA), 2-morpholinoethanesulfonic acid (MES), N-hydroxy succinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), and a photo initiator (Irgacure D-2959) were purchased from Sigma–Aldrich Co. (Montana, USA). A cellulose membrane (MWCO = 3500 g/mol) from Spectrum Laboratories Inc. (California, USA) was used for dialysis. 1,2-Ditetradecanoyl-sn-glycero-3-phosphoethanolamine (DMPE) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were purchased from Avanti Polar Lipids, Inc. (Alabama, USA). A MES buffer solution (pH 6.4) was prepared from 0.5 M sodium chloride and 50 mM MES. 2.2. Synthesis of methacrylated hyaluronic acid (HA–MA) To prepare methacrylated HA (HA–MA), a synthetic route similar to that described in the literature [26] was used. In brief, HA (0.5 g, 1.25 mmol on a repeat unit basis, [(C14H20NO11Na)n] = 401 g/mol) was dissolved in deionized water (25 mL), followed by the addition of a 20-fold excess of methacrylic anhydride (3.7 mL, 25 mmol) relative to the primary hydroxyl groups in HA. The reaction mixture was then stirred for 24 h in an ice bath after adjusting the solution pH to 8.0 using a 5 M NaOH solution. The solution of HA–MA was purified by dialysis for 3 days against distilled water, filtered using 0.2 lm Whatman filter paper, and freeze-dried. Degree of substitution: 15.0%. 1H NMR (300 MHz, D2O): d 6.06– 5.61 (d, J = 135 Hz, 2H, C = C-H, Haa1), 2.57 (s, 2H; Hc), 1.89 (s, 3H; Hb1), 1.82 (s, 3H; Hb); IR (KBr): m = 3427 (s), 2922 (w), 1739 (m), 1652 cm1 (s).
Scheme 1. Assembly of the DMPC lipid bilayers bearing DSSN+ conjugated oligoelectrolytes on the surface of a hyaluronic acid hydrogel nanoparticle formed via the inverse microemulsion mechanism under UV irradiation after modification with methacrylates and DMPE.
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2.3. Synthesis of HA–MA modified with DMPE (HA–MA–DMPE) To covalently attach DMPE to HA–MA via amide bonding, a DMPE solution was first prepared by dissolving DMPE (0.1 g, 0.157 mmol) in 10 mL of a solvent mixture (THF:methanol:water = 60:30:10) using sonication for 10 min at 30 °C. HA– MA (0.5 g, 1.24 mmol on a repeat unit basis) was dissolved in 100 mL of a solvent mixture (MES buffer solution:THF:methanol = 60:30:10), followed by the addition of NHS (18 mg, 0.157 mmol) and EDC (24.4 mg, 0.157 mmol) to activate the carboxylic acids in HA–MA at the amounts corresponding to DMPE. After stirring the HA–MA solution for 1 h, the DMPE solution was added, and the resulting reaction mixture was stirred for 24 h at room temperature. HA–MA attached to DMPE (HA–MA–DMPE) was purified by dialysis for 36 h against the first solvent mixture (H2O:THF:methanol = 60:30:10), and then against the second solvent mixture (H2O:methanol = 70:30) for another 36 h. The resulting solutions were filtered and freeze-dried. Degree of substitution: 10.2%. 1H NMR (300 MHz, D2O + acetone-d6): d 5.37–5.32 (1H, N-H, Hq), 4.34–4.29 (m, 1H, Hn), 4.21–4.04 (m, 4H, Hl, Hm), 3.22–3.17 (m, 2H, Hp), 2.48–2.38 (m, 4H, Hk), 1.71–1.62 (4H, Hj), 1.43–1.27 (s, 40H, He, Hf, Hg, Hh, Hi), 0.960.91 (t, J = 3.3 Hz, 6H, Hd). 2.4. Preparation of hydrogel nanoparticles of HA–MA–DMPE HA–MA–DMPE (0.1 g) was dissolved in 2 mL of a solvent mixture (methanol:H2O = 70:30), followed by the addition of Irgacure D-2959 (0.5 mg). The polar solution of HA–MA–DMPE was added to 10 mL of dodecane and the resulting emulsion was sonicated for 2 h and cured under irradiation at 365 nm for 2 h. For purification, 10 ml of ethanol was added to the reaction mixture and the solution was centrifuged to remove the nonpolar medium and unreacted curing agent. The ethanol rinsing process was repeated 5 times. 2.5. Vesicle assembly on nanohydrogel surfaces DMPC (58 mg, 0.086 mmol) and DSSN+ (2.6 mg, 1.72 lmol, 2 mol% to DMPC, see reference [24] for the synthetic procedure) were dissolved in 2 mL of ethanol and the resulting solution was mixed with a nanohydrogel dispersion in ethanol (0.5 mL, 1 wt% of solid content). We used an excess amount of DMPC to observe the formation of multilamellar structure on nanohydrogel surfaces. The calculated molar mixing ratio of HA–MA–DMPE on a repeat unit basis to DMPC was 1:20. In addition, samples at mixing ratios of 1:1 and 1:7 were prepared. The mixture was placed in a vial and was dried under a nitrogen flow at 30 °C to remove the solvent and to form a thin film, followed by vacuum drying for 12 h at room temperature. Deionized water (2 mL) was added to the dried film in the vial and the solution was ultrasonicated for 2 h. To isolate nanohydrogels assembled with DMPC and DSSN+, the solution was centrifuged at 4000 rpm for 10 min, and the nanohydrogels were re-dispersed in deionized water at pH 6.5. To investigate the stability of lipid structures assembled on nanohydrogels upon swelling, the nanohydrogels were dispersed in deionized water at pH 10.5, and used for measuring particle size, zeta potential and photoluminescence. 2.6. Characterization of the compounds 1 H NMR spectra were recorded at room temperature on a Gemini 2000 300 MHz NMR spectrometer (Varian). FT-IR spectra were measured using a Nicolet 6700 spectrophotometer (Thermo Scientific). Hydrogel nanoparticles were characterized using a Mastersizer 3000 particle size analyzer (Malvern Instruments
Ltd.), a ELS-8000 zeta potential analyzer (Otsuka Electronics Co. Ltd.), a QM-3/2004SE photoluminescence spectrometer (PTI), a SIGMA scanning electron microscope (Carl Zeiss), and a Tecnai F20 cryogenic transmission electron microscope (FEI Com.). 3. Results and discussion 3.1. Hyaluronic acid modified with methacrylate and the phospholipid HA–MA–DMPE was prepared by chemical conjugation of MA with the hydroxyl groups of HA, followed by the formation of amide bonds between the amine-terminated phospholipid (DMPE) and the carboxylic acid groups of the HA backbone. The synthesis of HA–MA was carried out in an aqueous environment at pH 8.0 with an excess of MA with respect to the hydroxyl groups in HA, and the resulting compound was characterized by means of 1H NMR spectroscopy. In comparison with the 1H NMR spectrum of HA (Fig. 1a), the 1H NMR spectrum of HA–MA (Fig. 1b) shows new peaks at 1.82 (3H, Hb), 5.62, and 6.05 ppm (2H, Haa1), which were assigned to the protons of the methacrylate moieties. The degree of substitution of the methacrylate groups was determined from the integration ratio of the two vinyl proton peaks at 5.62 and 6.05 ppm to the methyl peak of pure HA at 1.89 ppm (3H, b1) and was found to be 15.0%, which indicates an adequate amount of photocrosslinkable units for the formation of a stable hydrogel upon UV irradiation. The covalent attachment of DMPE to HAMA was carried out by activating the carboxylic acid groups in the HA backbone using NHS and EDC at pH 6.4. Because the solubility of the reaction product, HAMADMPE, was poor in aqueous medium, we used a solvent mixture of MES buffer, THF, and methanol, which is key to the synthesis of HAMADMPE. The 1H NMR spectrum of HA–MA–DMPE in a mixture of deuterated acetone and water (Fig. 1c) shows the characteristic methyl and methylene peaks of DMPE. The assignment of these peaks to the DMPE hydrogens was made based on solid and solution state 1H NMR spectra of DMPC [27] and DMPE (cf. Supplementary information Figs. S1 and S2). The degree of substitution of DMPE was estimated to be 10.2% from the integration ratio of the vinyl proton peaks of MA at 5.85 and 5.54 ppm to the methyl peak of DMPE at 0.94 ppm. We attempted to use higher amounts of DMPE to increase the DMPE substitution, but it was limited to 10.2% due to precipitation during the reaction, as the lipophilicity increased with the degree of DMPE substitution. 3.2. Hydrogel nanoparticles formed via a surfactant-free inverse emulsion route The amphiphilicity of HA–MA–DMPE allows the polymer to be dissolved in polar media. Due to the increase in hydrophobicity with DMPE substitution, HA–MA–DMPE could not be dissolved in water, in contrast to the salt form of pure HA that is completely water-soluble. In addition, the solubility of HA–MA–DMPE was poor in low molecular weight alcohols such as methanol and ethanol [28–30]. The preparation of a HA–MA–DMPE solution was possible in a solvent mixture of water and methanol at a mixing ratio of 70:30, at which the hydrophilicity and hydrophobicity were balanced. The dropwise addition of the HA–MA–DMPE solution into a nonpolar medium with vigorous stirring followed by 2 h sonication resulted in a surfactant-free inverse emulsion due to the fact that the hydrophobic alkyl chains in DMPE were located at the interface and partitioned between the polar droplets and the nonpolar medium. Subsequent UV-crosslinking using methacrylate substituents and water-soluble photoinitiators resulted in the formation of an
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should be mentioned that the formation of hydrogel nanoparticles was not successful with toluene, which is a typical nonpolar medium for the formation of inverse emulsions. Thus, an alkane solvent with a chemical structure compatible with the alkyl chains of DMPE is seemingly a better nonpolar medium than toluene for preparing well-dispersed nanoparticles of HA–MA–DMPE. 3.3. Lipid bilayer assembly on the hydrogel nanoparticle surface
Fig. 1. 1H NMR spectra of (a) hyaluronic acid (HA), (b) HA after conjugation with methacrylate (HA–MA), and (c) HA–MA after conjugation with DMPE.
HA hydrogel. As demonstrated in Fig. 2a and b, hydrogel nanoparticles with a mean diameter 302 ± 20 nm were successfully synthesized when dodecane was employed as the nonpolar phase. It
Assembly of lipid bilayers on the surfaces of the hydrogel nanoparticles was carried out using a conventional unilamellar vesicle preparation procedure. The phospholipid DMPC containing 14 carbons in its chain was used in this reaction. The only difference in chemical structure between DMPC and DMPE is the functional group at the polar head, which is a quaternary amine in DMPC and a primary amine in DMPE, as shown in Scheme 1. Hydrogel nanoparticles, DMPC, and DSSN+ as a model amphiphilic substance were dissolved in ethanol, a co-solvent in which all three chemicals were soluble. After evaporating ethanol, deionized water was added. A subsequent long (>4 h) sonication at about 30 °C a temperature above the melting point of DMPC induced the disruption of the mixture and self-assembly of the lipid bilayers bearing DSSN+ on the surface of the hydrogel nanoparticles. The cryo-TEM images in Fig. 3a and b shows the hydrogel nanoparticles before and after the assembly of the lipid bilayers on their surfaces, respectively. The dark circular lines surrounding the hydrogel nanoparticles in Fig. 3b indicate a material with high electron density, demonstrating the incorporation of DSSN+ in the lipid bilayers. It can be seen that the vesicle structure surrounding nanoparticles is multilamellar. In contrast, when only DSSN+ and DMPC were used in the vesicle formation procedure, only unilamellar vesicles with a diameter less than 100 nm bearing DSSN+ in lipid bilayers were formed, as we previously reported [24,25]. In Fig. 3b, small unilamellar vesicles are found around HA hydrogel nanoparticles with the multilamellar structure, which can be explained by the use of an excess amount of DMPC to ensure the formation of a multilamellar structure on nanohydrogel surfaces. The average percentage of nanohydrogels with the peripheral multilayer structure was 5.9% at a 1:20 M mixing ratio of nanohydrogels to DMPC, which was estimated by analyzing the number of nanohydrogels and unilamellar vesicles observed in three cryo-TEM images over an area of 1 1 lm2. In addition, no multilamellar structure was formed on nanohydrogels when a 1:1 M mixing ratio of nanohydrogels to DMPC was used, indicating that an excess of the lipid molecules was necessary. Thus, the formation of a multilamellar structure indicates that the anchoring power of the alkyl chains on the nanoparticle surface is significantly enhanced due to the association of alkyl tails in DMPC and
Fig. 2. (a) SEM image of hyaluronic acid hydrogel nanoparticles prepared in dodecane and (b) hydrogel nanoparticle size analysis data.
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Fig. 3. Cryogenic TEM images of (a) the pristine nanohydrogels, (b) nanohydrogels after assembling DMPC lipid layers bearing DSSN+ on their surfaces, (c) TEM image of nanohydrogels assembled with DMPC without DSSN+, and (d) fluorescence spectra of DSSN+ in water (dashed line), in the multilamellar structure on nanohydrogel surfaces (solid line) and in unilamellar vesicles (dotted line).
DMPE, despite a long sonication time. We suggest that the alkyl tails in DMPC and DMPE facilitate the self-assembly of DMPC on the nanoparticle surface, providing strong anchoring power due to the enhanced hydrophobic attraction, as the short-range hydrophobic attraction force increases with increasing contact between hydrophobic groups. Such an effect was also demonstrated in the thin film deposition of a polyelectrolyte onto the surface of Teflon [31]. It is notable that nanohydrogels with the multilamellar structure could not be successfully prepared without DSSN+. As shown in Fig. 3c, agglomerates of nanohydrogels were formed when we used only pristine nanohydrogels and DMPC. We presume that the incorporation of DSSN+ in the DMPC multilayer structure induces cationic charges on nanohydrogel surfaces by charge compensation with polar lipid heads in DMPC and that the resulting electrostatic repulsion between the nanoparticles hinders the formation of agglomerates. The zeta potential value of 2.56 mV measured for nanohydrogels with the multilamellar structure isolated by centrifuging supports this presumption (Table 1). At the same time, it seems that such electrostatic repulsion due to cationic charges in the polar head region does not seriously affect the formation of the multilamellar structure on the nanohydrogel surface. As we previously reported, the zeta potential value of unilamellar vesicles bearing DSSN+ was approximately 40 mV [25]. Thus, the zeta potential value of 2.56 mV, measured for nanohydrogels with the multilamellar structure, indicates that a significant fraction of
Table 1 pH dependent variations of zeta potential values, particle diameters, and the wavelength of the maximum photoluminescence for nanohydrogels before and after assembling multilamellar structure on their surfaces. Samples Lipid-assembled nanohydrogel Lipid-assembled nanohydrogel
Solution pH
Diameter (nm)
Zeta potential (mV)
kmax (nm)
6.5
357
2.56
540
10.5
747
9.27
523
DSSN+ is embedded in the multilamellar structure and that a small fraction of DSSN+ is exposed at the outermost surface of the nanohydrogels. The incorporation of DSSN+ into DMPC layers was further confirmed using photoluminescence, as shown in Fig. 3d. The photoluminescence of DSSN+ is highly dependent on the polarity of the environment, showing solvatochromism [24,25,32]. When DSSN+ is dissolved in a polar medium such as water, water dipoles align with the DSSN+ dipoles, stabilizing the excited state of DSSN+. Consequently, the photoluminescence spectrum of DSSN+ is significantly red-shifted and shows a broad featureless peak. In contrast, when DSSN+ is located in a nonpolar medium such as the interior of lipid bilayers, there is a hypsochromic shift and the photoluminescence peak appears at a shorter wavelength. Fig. 3d shows that
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the peak corresponding to DSSN+ in unilamellar vesicles (dotted line) has a maximum at 516 nm, while the peak of water-dissolved DSSN+ has a maximum at 571 nm (dashed line), which confirms the incorporation of DSSN+ in lipid bilayers. The peak of DSSN+ assembled with DMPC on the nanoparticle surfaces has a maximum at 540 nm (solid line), which shows a moderate blue shift in comparison with the spectrum of water-dissolved DSSN+. We speculate that the stabilization of the energy levels takes place as a result of the association among DSSN+ molecules in the multilamellar structure. We further investigated the stability of the multilamellar structure assembled on the nanohydrogel surfaces by varying the solution pH and measuring the changes in particle sizes, zeta potential values, and photoluminescence spectra (Table 1 and Figs. S3 and S4). The stability of the multilamellar structure relying on the variation of solution pH and resulting swelling and deswelling of nanohydrogels is a significant feature of the nanohydrogels. At pH 6.5, the diameter of the nanohydrogels with the multilamellar structure was 357 nm, which is 55 nm larger than that of the pristine nanohydrogels, reflecting the existence of the peripheral multilamellar structure; the zeta potential value was 2.56 mV. Interestingly, the average diameter of the nanoparticles drastically increased to 747 nm when the solution pH was adjusted to 10.5. At the same time, the zeta potential value of the solution increased to 9.27 mV. The increase in the diameter of the nanoparticles with the increase of pH indicates that carboxylic acid groups in the HA nanoparticles are ionized at high pH, inducing the swelling of the nanoparticles. Furthermore, the increase in the zeta potential values indicates that the cation groups of DSSN+ embedded in the multilamellar structure are more exposed to water due to possible structural rearrangement taking place during the swelling. To elucidate the increase in zeta potential value and particle size at high pH, we measured photoluminescence spectra. Notably, the maximum photoluminescence was at 523 nm, which is similar to the maximum value of 516 nm in the spectrum of DSSN+ in unilamellar vesicles (Fig. 3d). A significant blue shift indicates that the multilamellar structure assembled on the nanohydrogel surface is destabilized with the pH increase and swelling. It is possible that the multilamellar structure is disrupted and shattered into unilamellar vesicles. The high zeta potential value of 9.27 mV also supports this explanation because similar high values were observed for the DMPC/DSSN+ unilamellar vesicles dispersed in deionized water in our previous report [25].
4. Conclusions HA substituted with 15.0 mol% methacrylate and 10.2 mol% phospholipid at the primary hydroxyl and carboxyl groups in the repeat units of HA has been used for preparing a surfactant-free inverse emulsion. The assembly of lipid bilayers of DMPC lipids and amphiphilic DSSN+ on hydrogel nanoparticle surfaces showed that the phospholipid hydrophobic anchoring group plays a significant role in the self-assembly of phospholipid bilayers. Multiple analyses demonstrated the formation of stable multilamellar peripheral vesicular structures on the nanoparticle surfaces, suggesting potential applications in the biomedical field for delivery of cosmetic or drug agents.
Acknowledgements This work was supported by the Ministry of Health and Welfare (Grant No. HN10C0032020013), Republic of Korea and by the Chung-Ang University Excellent Student Scholarship.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cej.2014.10.096.
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