2460
Biomacromolecules 2010, 11, 2460–2464
Cationic Liposome Colloidal Stability in the Presence of Guar Derivatives Suggests Depletion Interactions May be Operative in Artificial Tears Anil Khanal,† Yuguo Cui,† Liang Zhang,† Robert Pelton,*,† Yuanyuan Ren,† Howard Ketelson,‡ and James Davis‡ Department of Chemical Engineering, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4L7, and Alcon Research, Ltd., 6201 South Freeway, Fort Worth, Texas 76134-2099 Received June 12, 2010; Revised Manuscript Received July 20, 2010
The colloidal stability of cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) liposomes was measured in the presence of guar, carboxymethyl guar, hydroxypropyl guar (HPG), and in mixtures of HPG with boric acid. Carboxymethyl guar induced DOTAP aggregation, presumably by bridging flocculation. The interaction with HPG-borate, an anionic polyelectrolyte with labile charge groups, depended on ionic strength. Without added salt, HPG-borate (pH 9.2) adsorbed on the liposomes and destabilized them. In contrast, in 0.1 M NaCl, HPG-borate did not adsorb onto the liposomes. HPG, HPG-borate, and guar induced depletion flocculation of the liposomes at high polymer concentration. Depletion flocculation may be an important mechanism when HPG is employed in artificial tear formulations. Scheme 1. Structure of Hydroxypropyl Guar with a Bound Borate (HPG-Borate)a
Introduction This work describes the interactions of hydroxypropyl guar (HPG) with cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) liposomes. The structure of DOTAP is given in the Supporting Information. Our interest in this system arises because HPG in conjunction with boric acid has been shown to be particularly effective in artificial tears used to treat dry eye syndrome.1-3 Our approach has been to characterize the interactions of HPG-borate with a series of model solutions and suspensions mimicking various ophthalmic components in the tear film. Before explaining the relevance of liposomes, some background is required. HPG is a synthetic derivative of guar, which, in turn, is a linear polymannose chain with single galactose rings on every other mannose unit. Hydroxypropylation is employed to increase the ease of dissolution and is typically at a level of 0.4 DS (i.e., degree of substitution, the number of substituents per sugar ring) and is approximately random.4 Although HPG is a nonionic water-soluble polymer, in the presence of borate ions it becomes a labile anionic polyelectrolyte.5 Borate ions condense onto the galactose units of guar and HPG, introducing negative charge groups. However, the borate binding energy is low (K binding is typically 100 L/mol), meaning the charge density on HPG is sensitive to the local environment, hence the name “labile anionic polyelectrolyte”. Herein we use the term HPG-borate to describe this polyelectrolyte form in situ. (See Scheme 1.) However, it is critical to recognize that HPG-borate is not a unique composition; the charge density varies with pH and concentrations of borate and borate binding sites. For example, at pH 9.2 in 50 mmol boric acid, the charge density of HPG-borate, expressed as a degree of substitution (i.e., the number of bound borates per sugar ring) is 0.25, whereas in the same mixture at pH 7.4, the DS of HPG is only 0.05. * To whom correspondence should be addressed. Fax: +1-905-528-5114. Tel: +1-905-525-9140 ext. 27045. E-mail:
[email protected]. † McMaster University. ‡ Alcon Research, Ltd.
a In this case, the degree of substitution of borate and of hydroxypropyl groups is 0.333.
The external surface of the human tear film is protected by a complex lipid layer that lowers the rate of tear evaporation.6 In an effort to understand the mechanisms by which HPG-borate might interact with the tear film lipid layer, we have employed two model interfaces, polystyrene latex7 and surfactant micelles, as models for lipids.8 The surface of anionic surfactant-free polystyrene latex is hydrophobic polystyrene decorated with a low density of negatively charged sulfate groups (∼3 nm2 per charged group9) or, in the case of cationic latex, positively charged amidine groups. In other work, we showed that anionic HPG-borate flocculated cationic polystyrene latex7 and formed adsorbed monolayers on anionic latex. Therefore, irrespective of the latex charge, HPG-borate adsorbed on polystyrene, perhaps driving hydrophobic effects, such as those proposed to explain guar adsorption on talc10 and other hydrophobic surfaces.11
10.1021/bm100655j 2010 American Chemical Society Published on Web 08/06/2010
Cationic Liposome Colloidal Stability
Biomacromolecules, Vol. 11, No. 9, 2010
2461
We have also investigated the interactions of HPG-borate with cationic micelles formed from dodecyltrimethyl ammonium bromide (DTAB). The surfactant micelles are much smaller than the latex particles and have a 10 times higher density of quaternary ammonium groups (∼0.4 nm2 per ammonium group12) on the micelle surface. We found that the cationic micelles were aggregated by HPG-borate and not by HPG without borate.8 Therefore, unlike the latex, the DTAB micelles interactions with HPG-borate were dominated by electrostatic interactions. In an effort to explore in more detail the interplay of hydrophobic and electrostatic interactions between HPG-borate and interfaces, we employed liposomes because they offer a great range of surface chemistry. In this work, we report our initial results with cationic DOTAP micelles. Herein we show two effects that are relevant to the ophthalmic applications of HPG. First, we show that compared with carboxymethyl guar (CMG), the interactions of HPG-borate with DOTAP are highly sensitive to background electrolyte. Second, under conditions of ophthalmic relevance, HPG induces depletion flocculation of DOTAP liposomes with or without the presence of borate. On the basis of these results, we propose that depletion effects may contribute to lipid layer stability in the eye.
Experimental Section Materials. HPG (ALCON HPGG-87) with MW ) 1.5 MDa and hydroxypropyl DS ) 0.36, native guar (guar), and partially hydrolyzed native guar (PH guar) were provided by Alcon Laboratories (Fort Worth). The CMG was prepared by derivatizing the native guar to give a carboxyl content DS ) 0.39.8 The properties of the four guar derivatives are summarized in the Supporting Information. Boric acid, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), and NaCl were purchased from Sigma-Aldrich, whereas DOTAP was purchased from Avanti Polar Lipid. All chemicals were used without further purification. Preparation of DOTAP Liposomes. DOTAP liposome suspension was prepared as described by Bordi et al.13 In a typical experiment, 220 mg (31.5 mM) of DOTAP lipid was dissolved in 10 mL of chloroform and methanol (1:1). Then, the solution was evaporated in a round-bottomed flask using a rotary evaporator at 45 °C. Afterward, the lipid film was dried with nitrogen for an additional 0.5 h. We then added 50 mL of milli-Q water, and the mixture was placed in an ultrasonic bath (Branson-3510) for 5 h. The final dispersions were highly transparent. The resulting liposome stock solution was stored at 5 °C and showed good colloidal stability. The diameter and polydispersity index of 1 mM DOTAP were around 95 nm and 0.1, respectively, as measured by dynamic light scattering (DLS). Liposome Colloidal Stability. Suspensions of DOTAP liposomes in a range of buffers and HPG or guar concentrations were prepared in 5 mL volume metric flasks and were stored in a water bath at 25 or 35 °C. The lower temperature results facilitated comparisons with the literature, whereas the higher temperature results simulated tear film temperature. After 24 h, the liposome stability was assessed by UV-vis at 500 nm and by DLS. Only the DLS results are shown herein because the UV-vis results were the same. For all experiments, the DOTAP concentration was 1 mM, which approximately corresponds to a liposome concentration of 6.69 × 1012 particles/mL.13 Measurements were made in the following electrolyte environments: 0.1 M NaCl, pH 7.4; 50 mM boric acid, pH 9.2, 50 mM boric acid plus 100 mM NaCl, pH 9.2; 50 mM boric acid at pH 7.4; and 50 mM HEPES plus 100 mM NaCl at pH 7. The normal order of addition was DOTAP, guar or HPG, and finally buffer plus salt. However, no sensitivity to the order of addition was observed. Dynamic Light Scattering. DLS measurements were carried out with a BI-9000 AT digital correlator (Brookhaven) apparatus at a fixed
Figure 1. Influence of carboxymethyl guar (CMG) on (A) the electrophoretic mobility and (B) the average diameter of DOTAP liposomes. Adsorption of anionic CMG changes the net charge (A) on the liposomes and induces aggregation (B).
90° scattering angle. Correlation functions were analyzed by the software 9kdlsw32 version 3.34 using the cumulants method. Electrophoresis. Electrophoretic mobilities were measured at 25 °C with a Brookhaven Zetapals Microelectrophoresis apparatus in the phase analysis light scattering mode using BIC Pals Zeta Potential Analyzer software version 2.5. Measurements were repeated three times, and the standard errors of the mean were
