Chitosan-coated liposomes for delivery to lungs by nebulisation

Colloids and Surfaces B: Biointerfaces 71 (2009) 88–95

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Chitosan-coated liposomes for delivery to lungs by nebulisation Marco Zaru a,b , Maria-Letizia Manca a,b , Anna Maria Fadda b , Sophia G. Antimisiaris a,c,∗ a

Laboratory of Pharmaceutical Technology, Department of Pharmacy, University of Patras, Rio 26510, Greece Department Farmaco Chimico Tecnologico, University of Cagliari, via Ospedale 72, 09124 Cagliari, Italy c Institute of Chemical Engineering and High-Temperature Processes-FORTH, GR-26500 Patras, Greece b

a r t i c l e

i n f o

Article history: Received 20 October 2008 Received in revised form 5 January 2009 Accepted 10 January 2009 Available online 20 January 2009 Keywords: Liposome Chitosan Lipid Alveolar delivery Rifampicin Nebulisation Aerosol Particles Mucoadhesive Cytotoxicity

a b s t r a c t The preparation of Chitosan (CHT)-coated liposomes and their applicability as a carrier for delivery of drugs to the lungs by nebulisation was investigated. Empty SUV (small unilamellar) liposomes were initially prepared (with different lipid compositions) and coated with CHT by dropwise addition of CHT solution in the liposome dispersion. CHT-coating efficiency was calculated after separation of coated/noncoated liposomes by centrifugation, and measurement of lipid in each fraction. After establishing the best conditions for CHT-coating (concentration of CHT in the solution), RIF-loaded CHT-coated liposomes, with different lipid compositions (negatively charged and non-charged) were constructed, and their encapsulation efficiency (EE) and nebulisation efficiency (NE%)/stability (NER%) were evaluated. Charged liposomes (containing phosphatidylglycerol [PG]) can be coated with CHT better compared to non-charged ones. The EE of CHT-coated liposomes (that contain PG) is slightly increased while their stability after nebulisation is significantly increased (NER%). Mucoadhesive properties of CHT-coated liposomes were substantially better (compared to non-coated ones) while the toxicity of liposomal RIF towards A549 epithelial cells was lower compared to free drug for all the types of vesicles evaluated, and especially the CHT-coated ones. Thereby, it is concluded that CHT-coated liposomes have advantages (compared to non-coated) when the delivery of drugs to the lungs by nebulisation is considered. © 2009 Elsevier B.V. All rights reserved.

1. Introduction In general, it is well accepted that increased residence time of particulate delivery systems at mucosal surfaces may facilitate (and increase) the uptake of such agents. In accordance to this, mucoadhesive agents provide a strategy that may increase the residence time and consequently the uptake of biodegradable particulate drug carriers when administered by the pulmonary route. Many agents as carbopol [1,2], hyaluronic acid [3,4] and chitosan (CHT) [5,6], are promising mucoadhesive agents with potential for use in pulmonary delivery. For this reason we selected to study the performance of CHT-coated liposomes during nebulisation. Although mucoadhesive properties may not be beneficial in the case of RIF delivery to the lungs; since macrophage targeting is the objective in this specific case, we chose to use RIF as a model drug for this study, since it was previously studied in our laboratory and thus results about nebulisation efficiency and stability of RIF-loaded MLV and DRV liposomes, are available for comparison.

∗ Corresponding author at: Laboratory of Pharmaceutical Technology, Department of Pharmacy, University of Patras, Rio 26510, Greece. Tel.: +30 2610 969332; fax: +30 2610 996302. E-mail address: [email protected] (S.G. Antimisiaris). 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.01.010

In a recent study [7], it was found that: (i) Liposome membrane composition is an important determinant of the stability of vesicles (retention of encapsulated drug) during the nebulisation process ([NER%]). More rigid liposomal membranes as those composed of distearoyl-glycero phosphocholine (DSPC) demonstrated increased NER% compared to liposomes composed of phosphatidylcholine (PC), while inclusion of Chol in the latter vesicles significantly increased their stability. (ii) The percent of liposomal lipid being nebulised (NE%) is influenced by the lipid concentration of the dispersions used for nebulisation, due to the fact that more concentrated dispersions have higher viscosity [8–10]. For the same reason, when the rigidity of liposomal membranes increased the NE% decreased (i.e. DSPC liposomes with 50 mol% of Chol had significantly lower NE% compared to the same liposomes with 33 mol% Chol). Since both factors NE% and NER% are important for the final outcome of liposomal drug nebulisation, a fine tuning of the liposomal dispersion properties is required. Indeed, it was observed that multilamellar (MLV) or dried rehydrated (DRV) liposomes consisting of DSPC/Chol 2:1 mol/mol (lipid composition) were preferable for the delivery of Rifampicin (RIF), compared to liposomes with different lipid compositions. Herein, various types of RIF-encapsulating vesicles were coated with CHT. In addition to their nebulisation efficiency and stability, the RIF encapsulation efficiency (EE%) of the different types of

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RIF-loaded CHT-coated liposomes was evaluated as well as their mucoadhesive properties and their cytotoxicity towards alveolar epithelial cells. 2. Materials and methods Egg phosphatidylocholine (PC), Phosphatidylglycerol (PG) and Distearoylglycero-phosphatidyl-choline (DSPC) were purchased from Lipoid, Gmbh (Ludwigshafen, Germany) and were demonstrated to give single spots on TLC [11]. Cholesterol (Chol), rifampicin, mucin, bradford reagent (Coomasie® dye binding protein assay), diphenylhexatriene (DPH), medium molecular weight chitosan (CHT) [with a deacetylation grade of 87%], and all other reagents used were of analytical grade and were purchased from Sigma–Aldrich OM, Athens, Greece. The A549 epithelial alveolar cells were a kindly provided from Dr. Ben Forbes (School of Pharmacy, Kings College, London). All media used for cell growth and handling were purchased from Biochrom (Berlin, Germany), and were of cell culture grade. 2.1. Preparation of CHT-coated liposomes 2.1.1. Empty liposomes—investigation of liposome coating procedure For liposome coating with CHT, a previously described technique [12–15], was investigated, in order to establish the requirements (vesicle lipid composition and CHT/Lipid ratio) for optimum vesicle coating. For this, empty small unilamellar (SUV) liposomes were prepared by probe sonication of empty multilamellar vesicle (MLV) dispersions. The preparation of MLV liposomes was done by the lipid thin film hydration technique, as reported before [7,16]. PBS buffer pH 7.40, (containing 5 mM Sodium Phosphate and 20 mM NaCl), was used as hydrating solution. Four different lipid compositions were evaluated for CHT-coating ability: PC/Chol 2:1 (mol/mol); PC/PG/Chol 9:1:5 (mol/mol/mol); DSPC/Chol 2:1 (mol/mol) and DSPC/PG/Chol 9:1:5 (mol/mol/mol). The SUVs were prepared with a Vibra-cell sonicator (Sonics and Materials, U.K.) equipped with a tapered micro-tip by applying at least two 10 min cycles of sonication - or more if needed- until the initially turbid liposomal dispersion became transparent. The Ti-fragments and any multilamellar vesicles or liposomal aggregates were removed by centrifugation at 10,000 × g for 15 min. Following their formation, the SUV liposomes were left to stand for 2 h at a temperature above the transition temperature of the lipids used in each case, in order to correct any structural defects. For the coating with CHT, an appropriate amount of the polymer was dissolved in isotonic acetate buffer (pH 4.4) in order to prepare various CHT solutions that would results in 0.001 up to 0.66, CHT/lipid (w/w) ratios, when mixed with an equal volume of the SUV liposome dispersions. In each case, an aliquot of the SUV liposome dispersion was mixed with an equal volume of polymer solution, which was added dropwise to the liposomes, under continuous stirring. After this, the mixture was incubated at 20 ◦ C for 1 h. After preparation, the different types of CHT-coated empty liposomes were evaluated for their CHT-coating efficiency, as described below (Section 2.2). 2.1.2. RIF-loaded liposomes In order to evaluate if the CHT-coating influences drug entrapment and vesicle performance during nebulization, CHT-coated and RIF-loaded MLV liposomes were prepared, using a 0.1 (w/w) CHT/lipid ratio (which was found to be sufficient for maximum coating of the liposomes (see below)). The same lipid compositions, as used for empty liposomes, were prepared in the case of RIF-loaded liposomes. For the preparation of these liposomes,

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RIF-loaded MLV liposomes were initially prepared as previously described [7], and after that, the liposomes were coated with CHT by the method mentioned above. Non-coated liposomes were also prepared and evaluated, for comparison. In all cases, for measurement of drug entrapment in liposomes, liposomal RIF was separated from non-entrapped drug by gel filtration on a Sephadex G-50 (1 cm × 30 cm) column eluted with PBS buffer. 2.2. Determination of the coating efficiency of CHT-coated liposomes The coating efficiency (CE%) of the empty liposomes was evaluated in order to establish the optimum conditions for liposome coating with CHT. The coating efficiency was determined as the phospholipid content within the CHT-coated liposomal fraction over the total phospholipid content of liposomes, as previously described [12] (Eq. (1)). For this, the CHT-coated liposomes were subjected to centrifugation at 14,000 rpm for 15 min until complete sedimentation of the coated liposomes. The obtained precipitate was re-suspended in the same initial volume of PBS, stirred until complete homogenization and used in order to measure the phospholipid content by the Stewart assay (as described in the following paragraph). The total content of phospholipid was determined by the same method in samples drawn from the liposome dispersion prior to centrifugation. CE% =

LPRE (lipid-in-precipitate) × 100 LTOT (Total Lipid)

(1)

2.3. RIF encapsulation measurement RIF encapsulation in liposomes is determined as the molar ratio of drug over lipid [D/L (mmol/mol)] in the RIF encapsulating liposomes. For the calculation of RIF encapsulation efficiency of the RIF-loaded CHT-coated (and non-coated) liposomes, the drug as well as the lipid content of each liposome preparation was measured, as described below. For the measurement of the RIF amount, a 200 ␮L sample of each liposome dispersion was completely dissolved in 5 mL of methanol and the RIF concentration was calculated by the optical density of the methanol solution at 485 nm, according to a calibration curve constructed by standard solutions of rifampicin in methanol (linear in the 2–50 ppm range). For the measurement of phospholipid concentration of RIFloaded liposomes, the DPH fluorescence method was initially used [17], as also described before [7] due to the fact that RIF absorption maxima is exactly in the same wavelength area (485 nm) used for measurement of lipid concentration in most routine analytical methods, as the Stewart assay [18] or the enzymatic assay used for phospholipids measurement [19]. After establishing that for CHT-coated liposomes (as also seen previously for non-coated vesicles [7]), the results obtained by the DPH fluorescence assay and those obtained by Stewart assay are statistically equal in the 0–200 ␮g/mL phospholipid concentration range (due to the fact that the amount of RIF in the diluted liposomes, is very low, and thus it practically does not interfere with the phospholipid concentration measurement), the colorimetric assay was used for routine measurement of the lipid concentration of liposome dispersions (in RIF-encapsulating as well as empty liposomes). 2.4. Liposome nebulization ability (NE%) In order to evaluate the effect of CHT-coating on the liposome nebulization ability, the RIF-loaded and CHT-coated MLV liposomes were nebulised (the same procedure was followed in the case of non-coated liposomes which were used as controls). A volume of

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3 mL of each liposome dispersion was used and liposome aerosols were generated as previously [7] using an efficient high-output continuous-flow Medeljet nebuliser (0.38 L/min), driven by a Medel Z17 compressor operated at 11 L/min. The RIF-loaded liposome containing aerosols were collected in a buffer solution using a homemade 2-stage glass impinger. The impinger device was utilized after placing 3 mL of buffer in the collecting flask. The aerosol was introduced into the collecting flask through a calibrated glass tube delivering the jet of aerosol 5 mm above the bottom of the flask. After nebulisation (which had an approximate duration of 10 min) both, RIF and lipid content of the impinger were assayed, before and after vesicle separation from any released drug, in order to evaluate the total output or nebulisation efficiency (NE%) and the effect of nebulisation on liposome stability (drug leakage NER%). Sample dilution was accounted for, after measuring the exact volume of dispersion collected. The nebulisation efficiency (NE%) was calculated by Eq. (2), after considering the drug/lipid (D/L) collected on the impinger and was expressed as a percentage of the initial D/L submitted to nebulisation: NE% =

Aerolised(D/L)(collected-in-flask) × 100 Initial(D/L)(placed-in-nebuliser)

(2)

2.5. Evaluation of RIF ampicin retention in liposome after nebulisation (NER%) The membrane integrity of the RIF-loaded and CHT-coated (or non-coated, control) MLV liposomes after nebulisation, was evaluated. For this, the percentage of drug entrapped in the nebulised vesicles was calculated, after separating the nebulised vesicles from released drug and measuring the drug and lipid content of the samples, as described in detail above. Finally the retention of RIF in the nebulised vesicles (NER%) was calculated by Eq. (3): NER% =

Aerolised and Purified (D/L) (collected-in-flask) × 100 (3) Aerolised(D/L)(collected-in-flask)

2.6. Measurement of liposome size and surface charge The size distribution (Mean diameter and Polydispersity Index) and -potential of the liposome dispersions were measured by dynamic light scattering (DLS) and laser Doppler electrophoresis (LDE), respectively, on a Nano-ZS (Nanoseries, Malvern Instruments), which measures the mass distribution of particle size as well as the dispersed particles electrophoretic mobility. Measurements were made at 25 ◦ C with a fixed angle of 137◦ . Sizes quoted are the z-average mean (dz) for the liposomal hydrodynamic diameter (nm). Calculation of -potential (mV) was done by the instrument (from electrophoretic mobility).

the amount of mucin adsorbed on the liposomes (from the difference between total and free mucin). A mucin calibration curve was prepared by measuring mucin standard solutions (0.25, 0.5, 0.75, 1 and 1.25 mg/mL). All samples (of known and unknown mucin concentration) were incubated at 37 ◦ C for 20 min after addition of the Bradford reagent, and then their absorbance at 595 nm was measured (Shimatzu UV-1205 spectrophotometer). The mucin content of each liposome type was calculated from the standard calibration curve. 2.8. Cell culture studies Human A549 alveolar cells (at passage 35) were grown as monolayers in 35 mm tissue culture dishes incubated in 100% humidity and 5% CO2 at 37 ◦ C. HAM’s medium containing 365 mg/L l-glutamine, supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin, and 100 ␮g/mL streptomycin was used as growth media. The cells, that form monolayers, were harvested with trypsin (0.25%), centrifuged at low speed (1600 × g, 4 min), re suspended in fresh medium and plated at a concentration of 2 × 105 cells/dish. The cells were grown to confluence on tissue culture dishes after 3–4 days of incubation. 2.8.1. RIF and liposomal RIF cytotoxicity assessment—MTT assay The effect of empty and RIF-loaded (CHT-coated or noncoated) MLV liposomes on the viability of cells was determined by the [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] MTT assay [21]. For this, A549 cells (105 cells/well) were cultured on 24-well plates with 500 mL of medium. The cells were incubated for 24 h with and without the tested compounds. Then 50 ␮L of MTT (5 mg/mL in PBS) was added to each well and after 2 h of incubation, the formazan crystals formed (the MTT dye is reduced in mitochondria by succinic dehydrogenase to an insoluble violet formazan product) were dissolved in DMSO. Absorbance at 580 nm was measured with a plate reader—spectrophotometer. Identical cell viability experiments with free RIF at the same concentrations with the RIF loaded in the vesicles were also carried out for comparison. On the basis of this assay %cell viability values were obtained in three independent experiments for each formulation. 2.9. Statistical treatment of experimental results For the statistical evaluation of differences between results, the one-way Anova, to check the significance between RIF encapsulation (D/L), NE%, NER%, mucoadhesive properties and cell viability values between the different liposome types and compositions, was used. In all cases, a probability value of less than 0.05 was considered to be significant.

2.7. Mucoadhesive studies—adsorption of mucin on liposomes 3. Results The adsorption of mucin on the vesicle surface was used as a method to assess the mucoadhesive properties of the RIFloaded liposomes [14]. For this, 1 mL of mucin aqueous solution (0.5 mg/mL) was mixed (vortexed) with 1 mL of each liposome dispersion (the concentration of lipid in these dispersions was fixed at 2 mg/mL) at room temperature. Then, the dispersions were centrifuged at 15,000 rpm for 30 min, and the supernatant was used for the measurement of free mucin. PC/Chol and DSPC/Chol liposomes were studied. Additionally we evaluated the effect of: (i) adding negative charge on liposomes – by including 10 mol% of PG (a negative charge lipid) in their membranes – and (ii) coating the liposomes with CHT (as described above) on the vesicle mucoadhesive properties. The Bradford colorimetric method [20] was used to determine free mucin concentration in order to assess

3.1. Coating efficiency of empty SUV liposomes with CHT The CE% values measured for coating of SUV liposomes with different lipid compositions, by CHT, when using increasing CHT/Lipid ratios, is presented in Fig. 1. As seen, CE% increases as the CHT/lipid w/w ratio increases from 0 to 0.1, and then after reaches, a saturated state. In the case of DSPC/PG/Chol liposomes, saturation is achieved at a 10 times lower concentration of CHT. In general, the charged liposomes (containing PG lipid) demonstrate a substantially higher coating ability (CE% is 88 and 76% for DSPC/PGChol and PC/PG/Chol liposomes, respectively) compared to the equivalent uncharged vesicles (CE% of uncharged vesicles was lower than 20% for both DSPC/Chol and PC/Chol liposomes). This observation

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Table 1 Mean hydrodynamic diameter (nm) of empty SUV liposomes with various lipid compositions before and after coating with CHT. CHT/Lipid (w/w) ratios of 0.005, 0.1 and 0.3 were evaluated. Each measurement is the mean of at least 5 measurements taken from each of 3 different formulations, and the standard deviation of the mean is reported. PI is the Polydispersity Index of the liposomal dispersions. Lipid Composition

No CHT

PC/Chol PC/PG/Chol DSPC/Chol DSPC/PG/Chol

97.2 78.3 98.8 93.4

± ± ± ±

2.5 3.2 3.5 8.1

PI

CHT/Lipid 0.005

PI

CHT/Lipid 0.1

PI

CHT/Lipid 0.3

0.28 0.21 0.25 0.40

NM 651 ± 33 NM 820 ± 41

NM 0.28 NM 0.38

NM 2491 ± 77 NM 2520 ± 25

NM 0.44 NM 0.41

114 4381 162 5030

± ± ± ±

5 73 31 30

PI 0.27 0.43 0.40 0.86

NM = not measured.

Fig. 1. Coating efficiency (%) of liposomes as a function of CHT/Lipid (w/w) ratio applying during the coating procedure. Empty SUVs composed of PC, DSPC, PC/PG (9:1, mol/mol) and DSPC/PG (9:1, mol/mol) and always containing Chol at a lipid/chol 2:1, mol/mol ratio, were prepared and coated with CHT, using different CHT/Lipid w/w ratios, as described in detail in Section 2. Each value is the mean from at least 3 liposome preparations. Bars represent standard deviations of the mean. Figure Key is included in figure insert.

proves that electrostatic interactions are very important for the coating of the vesicles with CHT, as suggested before. In Fig. 2 the -potential values measured for the different types of CHT-coated SUV liposomes, are presented as a function of the CHT/Lipid ratio used in each case. It is readily observed that the

-potential values of the charged vesicle types, is substantially modified by CHT, whereas the uncharged vesicles are only slightly (if at all) affected. As seen (by comparing Figs. 1 and 2), the -potential values of the vesicles increase as the CHT/Lipid ratio increases, in a similar mode with the increase of CE% The effect of CHT coating on the size distribution (mean hydrodynamic diameter and PI) of the various types of SUV liposomes tested is seen in Table 1. As seen, the size distribution of the uncharged liposome types tested (PC/Chol and DSPC/Chol) is also minimally affected by CHT, compared to the charged vesicles (PC/PG/Chol and DSPC/PG/Chol) which become substantially larger as the CHT/Lipid ratio increases and at the CHT/Lipid ratio of 0.3 both types of vesicles have more than 54 times larger hydrodynamic diameters, compared to the diameters of the same vesicles without CHT-coating. An interesting observation made during the vesicle coating procedure is that when using CHT solutions at concentrations at which CHT/Lipid ratio were below or equal to 0.1 (w/w), all liposomes (charged and uncharged) were individually and uniformly dispersed in the suspension after coating. However, when CHT solutions with higher concentrations were used for coating, liposomal clusters formed (especially in the case of charged vesicles). For this reason, and since the CHT/Lipid ratio of 0.1 (w/w) was able to achieve the maximum CE%, in all the cases evaluated (as seen in Fig. 1) this CHT/Lipid ratio was used for the coating of RIF-loaded liposomes (as described in Section 2.1.2). 3.2. RIF encapsulation and nebulization of CHT-coated RIF-loaded liposomes Fig. 3 shows the RIF entrapment efficiencies of all the RIF-loaded liposome types studied, calculated as mmoles of RIF encapsu-

Fig. 2. Zeta potential (mV) of liposomes as a function of CHT/Lipid (w/w) ratio applying during the vesicle coating procedure. Empty SUVs composed of PC, DSPC, PC/PG (9:1, mol/mol) and DSPC/PG (9:1, mol/mol) and always containing Chol at a lipid/chol 2:1, mol/mol ratio, were prepared and coated with CHT, using different CHT/Lipid w/w ratios, as described in detail in Section 2. Each value is the mean from at least 3 liposome preparations and each measurement is the mean of 10. Bars represent standard deviations of the mean. Figure Key is included in figure insert.

Fig. 3. Drug to Lipid ratio (D/L) of RIF-loaded liposomes, composed of different phospholipids and coated or not with CHT. Each value is the mean calculated from at least 3 separate preparations and standard deviation of mean is presented as bars. Differences between CHT-coated and equivalent (with the same lipid composition) non-coated vesicles were evaluated by One-Way-Anova (** significant at p = 0.01; * significant at p = 0.05).

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Table 2 Nebulisation efficiency [NE%] (percent of material that was nebulised) and stability during nebulisation (percent of RIF retained in vesicles in the nebulised fraction) [NER%], for CHT-coated and non-coated RIF-loaded liposomes with different lipid compositions. In all cases the vesicles were nebulised immediately after preparation (and separation from non-incorporated drug). Each value is the mean calculated from at least 3 separate preparations and standard deviation of mean is presented in parenthesis. Differences between CHT-coated and non-coated vesicles (with the same lipid composition) were evaluated by One-Way-Anova. Lipid Composition

NE%

NER%

PC/Chol [PC/Chol]CHT PC/PG/Chol [PC/PG/Chol]CHT DSPC/Chol [DSPC/Chol]CHT DSPC/PG/Chol [DSPC/PG/Chol]CHT

71.2 (6.1) 53.5 (8.3)* 61.4 (7.1) 59.4 (2.5) 58.3 (8.1) 55.5 (3.3) 77.5 (8.2) 45.4 (2.4)**

32.5 (4.3) 25.0 (5.1) 24.8 (6.3) 48.2 (1.9)** 70.8 (6.9) 59.3 (9.4) 31.2 (4.2) 85.3 (7.6)**

* **

Significant at p = 0.05. Significant at p = 0.01.

lated per mole of lipid. As seen, in the non-coated liposomes the D/L ratios range between 25 and 36, and DSPC/Chol liposomes incorporate significantly larger amounts of RIF (per mole of lipid) compared to the PC/Chol ones. The presence of CHT has a positive effect (increase) on RIF entrapment for all the liposomal formulations evaluated (D/L ratios are between 32 and 44). Indeed the D/L ratios increased from 13 to 23% when liposomes were coated with CHT. The difference between coated and non-coated liposome RIF EE, was statistically significant in all cases. In order to evaluate liposome stability during nebulisation both CHT-coated and non-coated RIF-encapsulating liposomal preparations were evaluated for their nebulisation efficiency (NE%) and drug leakage during nebulisation (NER%). It has been already demonstrated [7] that both NE% and NER% are affected by the vesicle lipid composition. In the present cases of CHT-coated vesicles, as seen in Table 2, CHT-coating results in a decrease of the NE% for two of the liposome types studied; PC/Chol liposomes for which a 24% reduction of NE% occurred and DSPC/PG/Chol liposomes that were affected even more (41% reduction of NE%). Oppositely NE% was not modified by CHT-coating in the cases of PC/PG/Chol and DSPC/Chol liposomes. The effect of CHT-coating of liposomes on their stability during the nebulisation process (NER%) was seen to be clearly influenced by the vesicle charge. Indeed from the results presented in Table 2 it is seen that although CHT-coating did not have a (statistically) significant effect on NER% values of the non-charged liposome types studied (PC/Chol and DSPC/Chol), the charged liposome types (PC/PG/Chol and DSPC/PG/Chol) were found to exhibit significantly higher NER% values after they were coated with CHT (NER% increased by 94% and 173% for the two types of liposomes, respectively). From the NER% values obtained for the various types of RIFloaded liposomes studied (Table 2) two other very interesting observations can be made: First, the addition of a negatively charged lipid (PG) in the vesicle membrane and thus the generation of a negative surface charge to the vesicles, results in a significant reduction of the vesicle NER, which is more pronounced in the case of DSPC/Chol liposomes. In other words negatively charged vesicles are less stable during nebulisation compared to their uncharged equivalents. Second, for the uncharged liposomes studied, CHTcoating results in a slight reduction of NER%, which although not statistically significant, corresponds to loss of the additional amount of drug encapsulated in the coated vesicles (compared to the non-coated ones with equivalent lipid composition).

Table 3 Percent mucin associated with RIF-loaded liposomes with various lipid compositions before and after they were coated with CHT. All the liposomes contain Chol at a 2:1 Lipid/Chol mol/mol ratio. Mucin association was measured after incubating the liposomes with mucin and separation of liposomes from the non-adsorbed (on vesicles) mucin by centrifugation. Liposome-mucin incubation conditions as well as mucin measurement details are described under Section 2.6). Triplicate samples were run in each case. Liposome composition

Mucin adsorbed on liposomes (%) Mean value (SD)

PC/Chol PC/PG/Chol [PC/Chol]CHT [PC/PG/Chol]CHT DSPC/Chol DSPC/PG/Chol (9:1:5) [DSPC/Chol]CHT [DSPC/PG/Chol]CHT

17.0 7.4 47.1 90.9 46.2 25.1 66.6 93.1

± ± ± ± ± ± ± ±

8.3a 4.4a 1.2 7.6 4.1a 8.1a 2.2 4.1

Zeta-Potential (mV)

+0.09 −22.9 +4.4 +24.98 +0.93 −19.9 +5.4 +24.43

± ± ± ± ± ± ± ±

0.54a 2.1a 1.9 0.91 0.77a 2.3a 2.7 0.62

a These values were taken from our previous publication [7] and are added here for direct comparison of the results.

3.3. Mucoadhesive properties of CHT-coated RIF-loaded liposomes Uncoated and CHT-coated RIF-loaded liposomes were tested for their mucoadhesive properties. As seen in Table 3, the non-coated negatively charged liposomes showed the lowest mucoadhesion from all the liposome types tested, as anticipated because of their negative charge, followed by the non-coated uncharged liposomes, and finally the CHT-coated ones. However, within the last set of liposomes (CHT-coated) mucin absorption was significantly higher for the PG-containing liposomes (charged vesicles) compared to the liposomes that did not contain PG. This is in line with the higher CHT-coating efficiency demonstrated for liposomes that are initially negatively charged (Fig. 1). As seen in Fig. 4 there is a linear correlation between the mucin percent absorbed on the vesicles and their corresponding zeta potential values (R2 = 0.8873) which becomes better (R2 = 0.9767) when the point corresponding to the PC/Chol liposomes (circled) is excluded. 3.4. Cell toxicity The viability of A549 cells was estimated after exposure of the cells for 24 h to empty (Fig. 5A) or RIF-loaded liposomes (that contain 0.25 mg/mL of RIF) (Fig. 5B), which were coated with CHT or non-coated. Empty and RIF-loaded CHT-coated liposomes and non-

Fig. 4. Effect of vesicle zeta potential on their mucoadhesive properties. Both, vesicle zeta potential and mucoadhesive properties (expressed as percent mucin absorbed on the vesicles), were measured as described in detail in Section 2.

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Fig. 5. Viability (% of control) of A549 alveolar cells after they have been incubated for 24 h with empty (A) or RIF-loaded (0.25 mg/mL) (B), non-coated or CHT-coated liposomes (Key is presented in figure insert). Viability was measured by the MTT assay, as explained in detail in Section 2.7. In all cases, liposomes contain Chol in their membranes (at a 2/1 lipid/Chol mol/mol) and the result presented is the mean value calculated (SD of the mean is presented as bar) from at least two different formulations (and 3 different wells/formulation). The grid line in graph B denotes the viability in presence of 0.25 mg/mL free RIF. Differences between CHT-coated and equivalent (with the same lipid composition) non-coated vesicles were evaluated by One-Way-Anova (** significant at p = 0.01; * significant at p = 0.05).

coated charged vesicles (containing PG) were evaluated at the same lipid composition incubated with cells, which ranged between 5.6 and 7.5 mg/mL. Results for PC/Chol and DSPC/Chol liposomes from our previous study [7] were included in the graphs for comparison. Nevertheless, comparison between these non-coated and the equivalent CHT-coated liposomes, which were constructed and studied herein, should not be considered, because different lipid concentrations were used in the previous study. Only comparison between the empty and the RIF-loaded PC/Chol and DSPC/Chol liposomes is safe. It is obvious from Fig. 5B that for all the liposome types studied, the cytotoxicity of liposomal-RIF is lower than that of the free drug (the horizontal line included in the figure denotes viability of cells after incubation with 0.25 mg/mL of free RIF), as demonstrated also previously for the PC/Chol and DSPC/Chol liposomes. Furthermore, in almost all of the cases evaluated, CHT-coating results in increased cell viability, and only in the case of DSPC/Chol liposomes a slight reduction of viability was caused by coating the vesicles with CHT, however, as stated above this comparison may not be safe since different lipid concentrations were used in the two studies. By comparing the different liposome types studied, it is interesting that PG containing liposomes are always significantly more toxic compared to those containing the same phospholipids but no PG. This result is related with the higher cytotoxicity of the empty carriers that contain PG, as seen in Fig. 5A. When comparing the results of empty (Fig. 5A) and equivalent RIF-loaded liposomes (Fig. 5B) for the different cases studied, we see that in all cases the %viability values towards A549 cells were reduced by 3 up to 22% for the RIF-loaded vesicles (compared to the empty ones). It is evident that the presence of RIF affects the cell viability, most possibly due to the cytotoxic effect of RIF that is endocytosed after release from the liposomes, or by uptake of the RIF-loaded liposomes by the cells. 4. Discussion In this study the effect of coating RIF-loaded liposomes with CHT on the vesicle behaviour during nebulisation as well as on

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their physicochemical characteristics, mucoadhesive properties and cytotoxicity, was evaluated. Initially, a previously reported technique for coating vesicles with CHT (12–15), was evaluated on empty SUV liposomes in order to select the optimum conditions for coating the RIF-loaded vesicles. In accordance with previous reports when liposomes are mixed with CHT solutions, the polymer adheres to the liposomal surface and CHT-coated vesicles are formed [13,22]. This coated layer does not desorb during washing. From the experimental results obtained in this study it is evident that electrostatic interactions are implicated in the vesicle coating procedure. Proof for this is provided by the fact that the zeta-potential of the vesicles that initially have negative charge (PC/PG/Chol and DSPC/PG/Chol) is drastically modified (as observed by comparing Fig. 2 with Fig. 1) as the vesicles become coated with polymer; whilst the zeta-potential of the non-charged vesicles, for which very low coating efficiencies were measured, were not significantly modified. Nevertheless, in addition to the electrostatic attraction between positively charged CHT and negatively charged liposomes, other mechanisms seem to be also implicated in the process of vesicle coating with CHT. Indeed, the fact that neutral liposomes can also be coated with CHT (at a lower efficiency compared to negatively charged ones) as seen before [13,22,23] and observed by us as well (Fig. 1), provides further proof of the hypothesis mentioned above. In fact, it has been previously suggested that coating neutral PC liposomes with chitosan involves hydrogen bonding between the polysaccharide and the phospholipid head groups [24]. The CHT-coated vesicles that demonstrated high coating efficiencies (Fig. 1) and also large modifications in their surface charge (Fig. 2) were also found to be much larger in size; as seen in Table 1, since their mean diameters were higher than 2 and 4 ␮m when they were coated with 0.1 and 0.3 CHT/Lipid (w/w), respectively. Oppositely, the vesicles with low CHT coating efficiency demonstrated a comparably very small size increase, even when coated with 0.3 CHT/Lipid (vesicle mean diameter increased from 97 to 114 and 98 to 162 nm for PC/Chol and DSPC/Chol liposomes, respectively). The size modifications of the vesicles due to CHT-coating are in line with the measured coating efficiencies of the two types of vesicles studied (charged and uncharged). In respect to RIF EE, an increase of RIF encapsulation was observed in CHT-coated vesicles (Fig. 3). This positive effect of CHT on RIF encapsulation in liposomes can be explained by considering the physicochemical characteristics of the drug. As reported earlier [25] and proved also recently [7] at physiologic conditions RIF interacts with bilayer lipids, due to its lipophilic nature. Thereby, in addition to being encapsulated in the aqueous entrapped volume of vesicles, a portion of RIF is incorporated in the vesicle lipid membrane. It is thus highly likely, that CHT-coating can increase the retention of the portion of the drug that is incorporated in the lipid bilayer that would elsewise tend to be released from the lipid phase upon dilution of the liposome dispersion (dilution takes place during the separation of free from liposomal drug, which is the first step of the procedure followed for measurement of drug encapsulation as described above). According to relevant studies by our group [7,26] and others [10] liposome and in general particle NE% is affected by the viscosity of the particular liposome/particle dispersion that is subjected to nebulisation. In the case of liposomes, the dispersion viscosity is related to vesicle size and membrane rigidity. The fact that more CHT is associated with the charged DSPC/PG/Chol liposomes compared to the uncharged DSPC/Chol ones, and thereby the first case of coated liposomes will be larger and consequently their dispersions will exhibit higher viscosity values (compared to the second liposome composition), explains the difference between the two DSPC-containing liposome types, in terms of NE% (Table 2).

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However, it is difficult to explain why the PC/Chol liposomes are negatively affected by CHT-coating (in terms of NE%) and not the PC/PG/Chol ones. A possible explanation is that in the case of vesicles with less rigid liposome membranes (compared to those formed by DSPC) other factors, as the surface charge of the coated particles produced (after CHT-coating) should also be considered, as the implication of surface charge of coated vesicles on their aggregation. Since charged liposomes have higher CHT-coating efficiency and finally result in the formation of particles with higher positive surface charge (compared to the uncharged liposomes) aggregation between particles is probably reduced (compared to particles with lower surface charge). This reduction of vesicle aggregation would result in dispersions with lower viscosity, compared with dispersions of aggregated vesicles (irrespective of the initial particle size). 4.1. NER% In regards to the effect of CHT-coating on NER% of liposomes, it was demonstrated herein that the extra amount of RIF encapsulated due to the CHT-coating (Fig. 3) is not retained in the vesicles during nebulisation (Table 2), if they are not initially bearing negative surface charge in order to allow electrostatic interaction between the vesicles and CHT to occur. On the other hand, the highly increased NER% of the initially charged (PG-containing) CHT-coated liposomes (Table 2) is not only due to the retention of the extra RIF encapsulated as a result of CHT-coating (Fig. 3). Thereby, it seems that either the positive charge of vesicles or the stable adhesion of CHT with the vesicles due to electrostatic interactions, or perhaps both, are important factors for formation of vesicles that are highly stable during nebulisation. Liposome surface charge has been observed to influence the stability of liposomes during nebulisation also by others [8]. Indeed, positively charged liposomes (by the addition of stearylamine in the membranes) composed of hydrogenated soy PC were found to retain higher amount of vesicle encapsulated dye compared to uncharged ones. This effect was attributed to the better molecular packing of the liposome lipid bilayer. Nevertheless, we could not claim that the vesicle surface charge is the sole parameter that affects their stability during nebulisation, since many other factors, between which the interactions of the loaded drug with the components of the liposomes, are also very important. The most important aspect when nebulising liposomal drugs (when liposomal retention of the drug is required for therapeutic purposes) is the final amount of drug retained in the vesicles after nebulisation. When the total output, during nebulisation of RIF-encapsulated vesicles is considered (the percent of initially encapsulated drug that is nebulised and remains associated with the vesicles in the nebulised dispersion, which is the product of NE% multiplied by NER%), it is calculated that the best formulation between the CHT-coated RIF-loaded liposomes studied herein, is that of (CHT-coated-) DSPC/PG/Chol liposomes. In fact these liposomes have similar outcome (38.7 ± 5.3%) with that of DSPC/Chol liposomes (41.3 ± 3.2%), which were found before [7] to be the best type of non-coated liposomes. Furthermore, when the higher EE of the CHT-coated liposomes (compared to the non-coated ones) is taken into account, the CHT-coated DSPC/PG/Chol liposomes are proven to be superior. Good mucoadhesive properties of particles are generally the result of interactions which take place between mucus and the particle surface. It has been stated before [27] and it is proven in this study (Table 3 and Fig. 4), that electrostatic attraction plays an important role in interactions between mucin (negatively charged) and the vesicle surface (in the present case positively charged due to the presence of CHT). Indeed, stearylamine-containing cyclosporine-A loaded liposomes with positive surface charge

were found to have higher mucoadhesion properties in intestine mucoadhesion test studies, compared to non-charged or negatively charged vesicles [28]. Nevertheless, in other studies, particle mucoadhesion was not found to be directly connected with a more positive surface charge as in the case of self-assembling pectinliposome nanocomplexes which were studied previously for their mucoadhesive ability by in vivo confocal scanning microscopy [29]. Thereby, it should be kept in mind, that, in addition to electrostatic interactions other mechanisms may be also implicated in mucoadhesion. We suspect that the observed influence of RIF-loaded liposome type on their cytotoxicity towards A549 cells (Fig. 5) are linked to the fact that particle zeta-potential is an important factor for their cytotoxicity. In the results of this study presented in Fig. 5A for empty liposomes and Fig. 5B for RIF-loaded ones, it is seen that in the charged liposome types studied (the ones containing PG); CHT coating results in an increased cell viability (compared to that of the non-coated liposomes). This effect of the CHT-coating may be linked with its effect on the vesicle surface charge (substantial change of surface charge from highly negative to highly positive). Indeed, it has been reported in several cases that the surface charge of liposomes or nanoparticles influences their interaction with cells, and depending on the cell types and the specific particles studied, in some cases cationic charge is claimed to enhance cytotoxicity, as in one case of positively charged nanoparticles [30] or positively charged liposomes [containing stearylamine or cationic lipids] [31,32]; or enhanced uptake of positively charged liposomes by macrophages [33]; while in other cases negatively charged liposomes are found to be more cytotoxic [34]. Whether the lower cytotoxicity of liposomal RIF compared to free RIF demonstrated for all types of vesicles evaluated is due to lower uptake of liposomal RIF by the cells, or slow release of RIF from liposomes, or other factors, we cannot be sure. Nevertheless, reduced cytotoxicity (compared to that of free drug) towards lung epithelial cells is considered as an advantage for any formulation designed for pulmonary delivery of drugs. 5. Conclusions From the results of this study it is concluded that when liposomes are coated with CHT, depending on their lipid composition, they may have several advantages when considering their performance as carriers to deliver drugs to alveolar sites after nebulisation of vesicle dispersions. Indeed, drug loading may be increased, especially in the case of drugs that may interact with lipid membranes, as RIF or perhaps also other drugs that may interact with CHT, as well. Furthermore, the vesicle stability during nebulisation (NER%) may be increased, depending on the liposome formulation construction. The results of the current study show that in order to achieve high liposome stability during nebulisation it is important to have vesicles that contain a negatively charged lipid (as PG that was used in our case) that can lead to efficient liposome-coating with the polymer due to electrostatic interactions between the positively charged polymer and the negatively charged liposome surface. Furthermore, a very important advantage of CHT-coated liposomes, compared to non-coated ones, is that they posses highly improved mucoadhesive properties which are required when drug delivery to the lungs by particulate drug carriers is considered [33,35]. Acknowledgements EU funded this work under the Marie Curie Early Stage Scholarship Program, Project name: Towards a Euro-PhD in advances drug delivery, Contract No.: MEST-CT-2004 - 504992.

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