Mechanical and transport properties of chitosan-zwitterionic phospholipid vesicles

Highlights

• Mechanical properties and water permeability of chitosan: SOPC vesicles were measured using micropipette aspiration.

• Increase in bending modulus with increasing chitosan: SOPC ratio was more pronounced at pH 7 compared to that at pH 4.5.

• Water permeability of chitosan: SOPC vesicles decreased with increasing chitosan: SOPC molar ratio.

• Phospholipid diffusivity in supported lipid bilayer decreased in presence of chitosan.

Abstract

Chitosan is a polysaccharide that has shown promise in liposomal drug delivery because of certain desirable properties such as muco-adhesivity, biodegradability and low toxicity. In this study, chitosan-bearing 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles were prepared using inverse phase precursor method to measure their mechanical and transport properties. We show that while an increase in chitosan: lipid molar ratio in the vesicle bilayer at pH 7 led to a substantial increase in its bending modulus, chitosan-mediated change in bending modulus was diminished at pH 4.5. Water permeability across the vesicle bilayer, as well as phospholipid diffusivity within supported lipid bilayers, were also found to decrease with increasing chitosan: lipid molar ratio. Together, these findings demonstrate that incorporation of chitosan in phospholipid bilayers modulates the mechanical and transport properties of liposomes which may affect their in vivo circulation time and drug release rate.

Introduction

Liposomes are vesicles formed by the self-assembly of phospholipid bilayers in an aqueous phase. Even as liposomes are increasingly used for controlled and targeted drug delivery, their detection and rapid clearance by the reticuloendothelial system is a major challenge [[1], [2], [3]]. To overcome this hurdle, several efforts have been reported where surface properties of liposomes were modified with both, natural and synthetic polymers [4].

Chitosan, a cationic linear polysaccharide composed of N-acetyl-d-glucosamine and β-(1,4)-linked d-glucosamine units has been widely used in drug delivery because of its biodegradability, biocompatibility, non-toxicity and mucoadhesive properties [[5], [6], [7]]. For instance, when compared to non-coated liposomes, oral administration of liposomes coated with chitosan (MW:150kDa) showed a 30 % increase in adhesion to the intestinal lining of male wistar rats [8]. Additionally, it was shown that chitosan-coated liposomes, which are negatively-charged at physiological pH, become positively-charged at acidic pH [9,10]. This pH sensitive property of chitosan was used to selectively improve cellular uptake efficiency of doxorubicin-loaded liposomes in tumors which have an acidic environment [11].

Interactions between chitosan and the liposomal phospholipid bilayer have been shown to change the thickness and charge of the bilayer as well as the size and polydispersity of the liposomes [12]. Electron paramagnetic resonance spectroscopic studies have shown that, while at a lower chitosan concentration the polymer chains are well-extended and their adsorption to the lipid bilayer leads to a reduction in membrane fluidity, at a higher concentration, the chitosan chains self-aggregate resulting in an increase in membrane fluidity [13,14]. Moreover, Quemeneur et.al demonstrated that the stability of pure and chitosan-decorated 1,2-dioleoy-sn-glycero-phosphocholine (DOPC) liposomes was adversely affected on reduction of pH or by increasing salt concentration of the suspending medium [10]. At an acidic pH of 4.5 and in the presence of chitosan, Henriksen and coworkers observed significantly higher aggregation of negatively-charged liposomes comprising of phosphatidylcholine (PC) and phosphatidylglycerol (PG) (PC:PG ratio 90:10), when compared to neutral liposomes made of pure PC [15]. The acidic pH below 4.5 is critical to chitosan solubility and its incorporation into liposomes. Mertins and Dimova concluded that at acidic pH, the chitosan chains become positively-charged and cause bridging of liposomes made of negatively-charged phospholipids thereby resulting in liposome aggregation [16]. In contrast, in the case of liposomes comprising of chitosan and neutral phospholipids, the net charge on the bilayer becomes positive resulting in repulsion between vesicles and thereby preventing their aggregation [16]. Another study by Mertins and Dimova conducted at pH 4.5 showed that strong electrostatic interaction between chitosan and negatively-charged phospholipids resulted in pore formation and leakage in the liposomes [17]. Other studies showed that an increase in degree of unsaturation and/or reduction in the length of the hydrocarbon chain of the phospholipid itself results in increased water permeability of the vesicle membrane [18] as well as faster leakage of water soluble drugs [19].

Differences in chitosan organization on the bilayer was observed to depend on the preparation method of chitosan-lipid vesicles [12,20]. Previous studies reported two different approaches for the incorporation of chitosan into the lipid bilayer: (i) incubation method and (ii) inverse phase precursor method [17,21,22]. In the first approach, pre-formed vesicles were incubated in a chitosan solution leading to chitosan adsorption only to the outer leaflet of vesicle bilayer. In the second method, chitosan is incorporated into the lipid bilayer during its self-assembly prior to vesicle formation, resulting in chitosan presence on both the inner and outer leaflets of the vesicles [22]. Using small angle x-ray scattering, Mertins et.al have shown that for dried chitosan-phospholipid films prepared by inverse phase precursor method (also known as reverse phase emulsion method), presence of chitosan reduces the thickness of the phospholipid bilayer in the organogel [22]. This suggests that the bilayer is sandwiched between polymer chains, and vesicles formed from the organogel contain chitosan in inner and outer leaflet of the membrane [22].

Mertins and coworkers measured chitosan content in chitosan-DOPC vesicles and showed that chitosan incorporation in the bilayer by the inverse phase precursor method remains stable over time and that the thermodynamic stability bilayer is also enhanced due to effective shielding of phospholipid head group by chitosan [20,23]. Using fluctation analysis, Mertins and Dimova showed that at low concentrations chitosan, the bending rigidity of chitosan-DOPC giant unilamellar vesicles (GUVs) was found to decrease [17]. However, measurements could not be made for higher chitosan: DOPC molar ratios (≥2 × 10⁻⁴) as membrane undulations reduce to unobservable levels thereby rendering fluctation analysis inapplicable [17].

In the present work, chitosan-bearing 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) GUVs prepared by inverse phase precursor method [23]. We employed micropipette aspiration assay which enabled us to measure the mechanical and transport properties of the GUVs at high chitosan : SOPC molar ratios (upto 3 × 10⁻³). We confirmed the presence of chitosan in the bilayer by laser scanning confocal microscopy (LSCM) and also quantified the amount of chitosan associated with vesicles. Further, we estimated the bending and area compressibility moduli, water permeability and lysis tension of these chitosan-SOPC GUVs. In addition, the impact of solution pH on surface charge as well as bending and area compressibility moduli of chitosan-SOPC vesicles was measured. Phospholipid diffusivity in chitosan-SOPC supported lipid bilayers (SLBs) was also estimated.