TMC was synthesized from high viscosity chitosan as described previously [11]. ¹H-NMR was used to characterize the synthesized TMC polymer. The ¹H-NMR spectrum was recorded in D₂O with a Bruker 600 MHz spectrometer (Bruker, Faellanden, Switzerland) at 80°C. Minimum interference was observed from the water peak at this temperature and suppression of the water peak was not necessary.

Several approaches have been employed for the formation large vesicles. In a recent study large vesicles were formed combining an electric field and calcium ions [15]. Large Unilamellar Vesicles (LUV), containing TMC, consisted from L-a-Phosphatidyl Choline (PC) (Avanti Lipids, Alabama US) were prepared as has been previously described [16]. Briefly, 13 μmol of the lipid (PC) was dissolved in chloroform in a spherical flask. The organic solvent was removed in a rotary evaporator and the thin film was rehydrated with 2 mL of PBS pH 7.4 containing different amounts of TMC (0, 0.25, 0.50 and 1% w/v respectively). Highly purified water (18.2 MΩ.cm) was used for the preparation of all the solutions. The dispersion was subjected to temperature controlled (20 ^oC) bath sonication for 20 minutes. The LUV were prepared by extrusion through two stacked polycarbonate filters of 0.4 μm pore size (Nucleopore, Corning Costar Corp., MA, USA) under nitrogen pressure 30 psi.

The size distribution (mean diameter and polydispersity index) and the zeta (ζ) potential of the liposomes were measured by DLS on a ZetasizerNano-ZS (Malvern Instruments Ltd., UK), which enabled to obtain the mass distribution of particle size as well as the electrophoretic mobility. Measurements were made at 25 °C with a fixed angle of 137 °. Samples for three different batches were measured.

Thermal analysis of the formulations was performed using a Perkin Elmer calorimeter (Perkin Elmer Diamond DSC) in a nitrogen atmosphere. Samples consisted of a 30 μL of liposome suspension in PBS buffer were placed in standard aluminium DSC pans of 40 μL with a pierced lid at a heating scan rate of 2 ^oC/min.

Advancing (θA) contact angles of small drops (2 μL) at 20 °C were measured using a goniometer with an enclosed thermostated cell (Kruss G10, Hamburg, Germany). The contact angles of droplets (× 3) placed on the horizontal PMMA (× 2) and were obtained for both ‘left’ and ‘right’ contact after approximately 15 s after the initial drop-surface contact. PMMA was used as a model substrate because its surface free energy is a reasonable estimate for hydrophobic compounds which are known to have problematic wetting.

A yield of 67% was obtained with the synthesis). The polymer synthesised was characterised by ¹H-NMR to determine the degree of quarternization, which indicates the percentage of N-groups methylated to quaternary ammonium groups. The degree of substitution was 21.2% which falls within the range typically observed for a one step synthesis reported previously [17].

The effect of different amounts of TMC on the particle size of PC liposomes is illustrated in Fig. (1A). The particle size of TMC-PC liposomes is decreased with increasing concentration of polymer solution. More specifically, the particle size of plain liposomes decreased from 350 to 150 nm with the addition of 0.25% w/v TMC, followed by a decrease to 115 nm and 150 nm when 0.5% and 1% w/v of TMC were added, respectively. The Polydispersity Index (PDI) values for 0, 0.25%, 0.5% and 1% were respectively 0.157, 0.198, 0.245 and 0.259, respectively. This is suggestive of interaction between the polymer and the lipid bilayer (Fig. 1A). The electrical properties of the liposomes in the presence of TMC were assessed by means of ζ-potential measurements. The ζ-potential values for both formulations are shown in (Fig. 2B). PC is a zwitterionic lipid with no net charge [18] while TMC at pH 7.4 is positively charged due to the quaternary ammonium groups. An increase to the ζ-potential values of the PC formulation from -0,65 mV (plain liposomes) to 8.46 mV (1% w/v TMC) was observed after increasing the amount of polyelectrolyte, indicating the incorporation of the polymer in the vesicles’ structure, possible as a layer on the surface of the vesicles.

The relative low values of ζ-potential measured for the liposomal formulations could be attributed to the high ionic strength of the buffer (PBS, pH 7.4) which suppresses the thickness of the electric double layer of the vesicles [18].

To further investigate the interactions of the produced vesicles DSC and contact angle goniometry were employed. The miscibility of TMC with PC liposomes was assessed by means of DSC. Plain PC liposomes have a clear endotherm transition peak at T_m = -10,6°C (Fig. 2A), which is in line with values reported previously [19]. This suggests that the presence of TMC induced changes to the main Transition Temperature, T_m, of the liposomes. More specifically, when 0.25% w/v of TMC was added to vesicles, the T_m was dramatically decreased from -10.8°C to -18.5°C, and was further decreased to -19.3°C and -18.9°C when 0.5% w/v and 1% w/v of TMC was respectively present in the membrane an indication of the miscibility of polyelectrolyte with the phospholipid (Fig. 2A). The main transition is shifted by the presence of TMC in both formulations due to a change, from a gel-like to a liquid-like packing, of the hydrophobic tails of the lipids [20]. That suggests that the packing of the lipids is disturbed within the bilayer; i.e. their order is reduced. This can be attributed to the penetration of the polyelectrolyte into the lipid bilayers. The changes noticed with PC liposomes advocating that the length of the fatty acyl chain seems to play a role on the localization of the polyelectrolyte into the membrane. In an attempt to investigate the potential changes of the lipophilic character of the liposomes upon the addition of the polyelectrolyte, Contact Angle Goniometry (CAG) studies were performed. Fig. (2B) illustrates the CAG values obtained for TMC-PC and liposomes as a function of the polyelectrolyte concentration. In the PC formulation, the θ° value of plain liposomes is increased significantly from 63.42 to 72 (t-test p<0.05) when 0.25% of the polyelectrolyte was added to the vesicles indicating that TMC is located in the lipid bilayer. However, the contact angle values declined up to 55.75 after the addition of high concentrations of TMC (1% w/v). The significantly lower CAG values, compared with the control (t-test p<0.001), confirm the presence of hydrophilic moieties of the polyelectrolyte (TMC) to the surface of liposomes.

Fig. (1). The effect of different TMC concentration on (A.) the size of TMC-coated liposomes in PBS pH 7.4, (B.) the ζ-potential of TMC-coated liposomes in PBS pH 7.4. The results are the mean of three different experiments.

Fig. (2). (A.) DSC thermogramm of TMC-PC liposomes, (B.) Contact angle goniometry values of TMC-PC liposomes. The results are the mean of three different experiments.