Zeta Potential of Liposome Production
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Empty and drug loaded liposomes were prepared with different lipid compositions and by different methods. These liposomes were analysed as this types of liposome have particle size in the range needed for aerosol delivery and delivery of drug to the deep lung deposition. In order to interpret which parameters are significant for the preparation of liposomes that can be used for such applications, eight different lipid membrane compositions were studied. The drug molecule used was tobramycin, as there is therapeutic rationale for its use in cystic fibrosis, but also because of its complex structure, hydrophilic nature and it is also believed that it is difficult drug to encapsulate into liposome vesicles. Thus results and findings of this study can be used as a basis for the formulation of liposomes intended for delivery to the lungs by nebulisation.
Liposome Surface Charge
The zeta-potential of different liposome formulation without tobramycin were obtained, values (Table 6) indicates difference in charges based on different lipids used in the formulation.
As DPPC is a neutral lipid, it produces liposomes with no surface charge on the liposomes as seen in formulation T1 and T2. The addition of cholesterol to the liposome formulation caused no change in surface charge. Values with DSPE-PEG lipids were found close to zero, indicating an overall neutral charge for these liposomes (zeta potential for T3 and T4 liposomes formulation was measured to be -6.46±0.42 mV and -7.24±0.14 mV respectively). As expected for negatively charged (DPPG-Na) liposomes, the zeta potential was more negative (-66.43± 2.95 mV), which indicates lipid membranes are negatively charged. It is readily observed that the zeta-potential values of the negatively charged liposome, is decreased by presence of DSPE-PEG and cholesterol such as in formulation T6, T7 and T8. DPPG represents the main component of bacterial membranes and is a minor component of lung surfactant. Unlike DPPC, head groups of DPPG lipid molecules have a net negative charge (anionic) (Ianoul, et al., 2007) and higher membrane rigidity (Kinman, et al., 2006). It is therefore expected that since the tobramycin is a positively charged, their interaction with DPPG-Na phospholipid will be stronger.
Size of Liposomes
The particle size distributions of the different formulation with and without tobramycin obtained from Mastersizer, presented in Tables 7 and 8; are found to be unimodal and range from 1.0 to 4.0 µm.
Liposome formulations with or without cholesterol did not show any great differences in the size characteristics. With the addition of tobramycin, it is observed that size of the liposome decreases this is due to the fact that tobramycin has hydrophilic nature and positive charge. It can be seen that after including DPPG-Na, size of the liposomes decreases as it is negatively charged and tobramycin is positively charged so due to electrostatic interaction, its size is reduced or decreased aggregation. It can be seen from the results, liposome of tobramycin are within the range which are required for an optimal deep lung deposition.
Size distributions of the various types of liposome after sonication for 10 min are presented in tables 9 and 10. Effect of various lipids and cholesterol is seen on the size distribution of different liposome formulation with or without tobramycin.
All liposomes formulations are within range from 90.0 to 250 nm (i.e. small unilamellar liposomes or small multilamellar liposomes). In most liposome formulation, the polydispersity indexes measured are low indicating that the vesicles are monodisperse. Addition of cholesterol in the lipid membranes of liposomes resulted in increased vesicle size as previously reported (Zaru, et al., 2007). Incorporation of tobramycin to the liposome formulation (Table 10) increased the size of liposome in comparison to empty liposomes prepared under identical conditions. These liposomes incorporate tobramycin in an aqueous core as it is hydrophilic drug (Ramana, et al., 2010). The bulky size of tobramycin may responsible for the increased size of the tobramycin loaded liposomes compared to empty liposomes.
Selected formulations were directed into the Sympatec particle sizer in order to determine the size of the aerolised particles as presented in Table 11 and 12. It can be observed from both tables that droplet size is in range 3-5 µm as required for the pulmonary delivery.
From table 11, it can be interpreted that addition of cholesterol to the empty liposomes had very little effect on droplet size. DSPE-PEG increases the size of the droplet size in both empty as well as drug loaded liposome while DPPG-Na decreases the size of the droplet size. Droplet sizes are in the respirable range. There is no major difference in droplet size, in comparison to with or without tobramycin. Droplet size is a function of the nebuliser rather than the liposomes formulations.
Liposome Encapsulation Efficiency
The liposome encapsulation efficiency % measured for encapsulation of tobramycin liposomes with different lipid composition and different methods used, is presented in Figure 7. Formulations (T1 - T9) are prepared by the proliposome method and without sonicated samples were used. Formulations TFM1 and TFM2 are prepared by the thin film method. As seen, tobramycin liposome encapsulation is within range from 5 to 20% for proliposome method. Lipid composition is a very important determinant of Tobramycin encapsulation. The amount of tobramycin encapsulated in liposomes increased significantly when liposomes are formed from DSPE-PEG and DPPG-Na lipids compared to DPPC based liposomes. Inclusion of cholesterol in the lipid bilayers, results in a decrease of tobramycin encapsulation efficiency % in formulations T2, T4and T6 (due to displacement of drug from the bilayers by cholesterol). Usually, the charged liposomes containing DPPG-Na lipid express considerably higher encapsulation ability (Liposome Encapsulation Efficiency % is 15 and 10% for T5 and T7 formulations, respectively) compared to the equivalent uncharged liposomes.
In the formulation T9, the effect of chitosan coating on tobramycin loaded liposomes was observed as liposome encapsulation efficiency. As previously reported, when liposomes are prepared with chitosan solution, it forms chitosan coated liposomes as polymer adheres to the liposomal surface (Takeuchi, et al, 1996 and Takeuchi, et al., 2003). From the results obtained from this study, it is evident that electrostatic interactions are involved in the liposome coating with chitosan solution. This can be proven by the fact that negative charge (DPPG-Na) of liposome is modified as they are coated with polymer; while non-charged liposome, were not significantly modified, therefore they have very low coating efficiency. In case of neutral liposomes, they can be coated with chitosan solution but they will have lower efficiency in comparison to negatively charged ones (Mobed, et al., 1992). It has been previously noted that chitosan coating on neutral liposome involves hydrogen bonding between the phospholipid head groups and the polysaccharides (Perugini, et al., 2000). In respect to tobramycin encapsulation efficiency, an increase in encapsulation efficiency was observed in chitosan coated liposomes (Figure 7) in comparison to non-coated liposome formulations.
Thin film hydration is a simple technique for the preparation of liposomes, but its major disadvantage is poor encapsulation efficiency of hydrophilic drugs. Furthermore, encapsulation of the drug decreases with the reduction in size of liposome (Sharma and Sharma, 1997). Therefore, it indicates from the result that encapsulation efficiency (Figure 7) of liposomes prepared by thin film hydration method (TFM 1 AND TFM 2) is low compared to those formulations prepared by the proliposome method.
Figure 8, represents surface association of tobramycin solution on to the empty liposomes. Drug loading technique like adsorption on to preformed empty liposome is widely described. It is difficult to encapsulate the drug with small size particles; therefore, the drug is adsorbed onto the surface of the empty liposomes rather than encapsulation (Almeida and Souto, 2007). Adsorption % from the graph (1 - 1.5 %) is negligible in comparison to encapsulation of the drug. Therefore, encapsulation is a better method for drug entrapment onto the liposomes.
Twin Stage Impinger (TSI) deposition of nebulised formulations
Figure 9, represents drug deposition after the sonicated liposome formulations were subjected to nebulisation. Tobramycin liposomal formulation is more efficiently delivered to stage 2 (cut-off diameter of particles delivered to stage 2 is less than 6.4 µm) of TSI than the tobramycin solution. In all the liposome formulations, nebulisation efficiency was higher than 65 percent of initial materials contained in the nebuliser and this is not practically affected by lipid composition present in different liposome formulation. It is interested to note that the presence of lipids distinctly enhances the deposition in stage 2 of the twin stage impinger (TSI). As shown in Figure 9, the presence of lipid coat around tobramycin particles allowed decrease in deposition in the nebuliser device, while there is increase in deposition in stage 2. The stage 2 deposition of uncoated tobramycin is around 49%, which is increased by up to 66-71% for the lipid coated formulations. Therefore it is extremely helpful for the patients in terms of deep lung penetration and drug targeting efficiency. As expected, there is negligible amount of tobramycin observed in stage 1 of TSI. There is no effect after addition of cholesterol to the lipid membrane, on nebulisation efficiency. This may be due to the hydrophilic nature of the drug and because cholesterol is forming leaky lipid membranes.
The deposition of the non-sonicated liposome formulation after nebulisation is shown in Figure 10. The aerosolisation and deposition performance of the tobramycin liposome were analysed at two stages of twin stage impinger. This finding has resulted in reduction of deposition in nebuliser device, whereas it increased deposition in the lower stage of twin stage impinger. From the Figure 10, it can be said that after inclusion of DSPE-PEG to the formulation, deposition in lower stage is increased from 63 - 80% (liposome formulation T5 and T6) to 96% (liposome formulation T7 and T8). There was no tobramycin detected in upper stage of TSI. After comparison of the nebulisation efficiency of the sonicated and non-sonicated, it can be observed that deposition in lower stage is more with non-sonicated samples but this experiment was performed only once.
The size of the nebulised liposomes was measured before and after nebulisation at different stages of TSI as shown in Table 13 and 14. As seen in Table 13, size of the sonicated liposomes is not changed on passage through nebuliser for the formulation T5 and T6. But there is reduction in size after nebulisation for the formulation T7 and T8, this may be due to the fact that liposomes were disrupted and further loss of the entrapped drug, which is hydrophilic in nature (Elhissi, et al., 2007).
From Table 14, it can be observed that size is decreased in nebuliser compared to before nebulisation for non-sonicated liposomes but there is very little difference in particle size present in nebuliser and stage 2 for the formulations T5 and T6. For the formulation T7 and T8, it can be seen that size of the particle is increased in stage 2 as compared to nebuliser and stage 1. It is to be expected that liposomes have aggregated in the lower stage impinger after they were delivered from the air jet nebulizer and this may be due to close proximity of the liposomes or disruption of the liposomes which led to aggregation of liposome fragments (Elhissi, et al., 2010).
From this study it can be concluded that depending on the composition of lipid and method of preparation of lipid, liposomes have advantage of acting as carrier for delivering tobramycin to the lung. Also based on coating of liposome with chitosan, various mixtures of phospholipid and cholesterol used can improve delivery of tobramycin to deep lung deposition. The size of all the liposome formulation was within the range which is suitable for lung deposition. Drug loading may be increased with the use of chitosan solution. From this study, it demonstrated the effect of negatively charged lipid (DPPG-Na) with the positively charged tobramycin that encapsulation efficiency and nebulisation efficiency can be increased in comparison to non-charged ones. Therefore, liposomal tobramycin is good formulation in comparison to tobramycin solution.
From the results of this study, it has been demonstrated that chitosan coated liposomes offers advantage over non-coated ones. Liposome prepared by chitosan solution has more encapsulation efficiency. But limitation from this study is that it was performed with only one lipid compoistion. So liposome of different lipid compositions can be formulated with chitosan solution and characterised for its liposome size, drug loading and twin stage impinger for further studies. Even negatively charged lipid is a promising lipid for further studies with positively charged tobramycin. The twin stage impinger studies can be done with the use of a different nebuliser, particularly a vibrating-mesh nebuliser would be useful.
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