PLGA, chitosan or chitosan-coated PLGA microparticles for alveolar delivery?

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Colloids and Surfaces B: Biointerfaces 62 (2008) 220–231

PLGA, chitosan or chitosan-coated PLGA microparticles for alveolar delivery? A comparative study of particle stability during nebulization Maria-Letizia Manca a,b , Spyridon Mourtas a , Vassileios Dracopoulos c , Anna Maria Fadda b , Sophia G. Antimisiaris a,c,∗ a

Laboratory of Pharmaceutical Technology, Department of Pharmacy, University of Patras, Rio 26510, Greece b 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 Received 31 July 2007; received in revised form 1 October 2007; accepted 6 October 2007 Available online 11 October 2007

Abstract Various types of rifampicin (RIF)-loaded microparticles were compared for their stability during nebulization. Poly(lactide-co-glycolide) (PLGA), chitosan (CHT) and PLGA/CHT microparticles (MPs) were prepared by emulsion or precipitation techniques. MPs ability to be nebulized (NE%) as well as stability during freeze-drying or/and nebulization (NEED%), were evaluated after RIF extraction from MPs and determination by light spectroscopy. MP mean diameters and ␨-potential values were measured by dynamic light scattering, morphology was assessed by SEM, cytotoxicity by MTT method and mucoadhesive properties by mucin association. In all cases, freeze-drying prior to nebulization did not affect EE%, NE or NEED%. In CHT, MPs RIF encapsulation efficiency (EE%) decreased with increasing CHT concentration (viscosity) and CHT-MP NEED% was higher when the polymer was crosslinked by glutaraldehyde. PLGA MPs, exhibited both higher RIF EE% and also higher nebulization ability and NEED%, compared to CHT ones, but also higher cytotoxicity. However, when the two polymers were combined in the PLGA/CHT MPs, EE%, NE% and NEED% increased with increasing MP CHT-content. PLGA/CHT MPs with 0.50% or 0.75% CHT exhibited highest EE% for RIF and also best nebulization ability and stability, compared to all other MP formulations studied. Additionally they had good mucoadhesive properties and comparably low cytotoxicity. © 2007 Elsevier B.V. All rights reserved. Keywords: Microparticle; PLGA; Chitosan; Rifampicin; Nebulisation; Encapsulation; Stability; Mucoadhesion; Cytotoxicity

1. Introduction For drugs that are intended to act topically in the lungs, direct deposition to the lungs by aerosol delivery technologies (as nebulization of liquid solutions) [1] offers many advantages when compared to systemic administration, such as increased drug localization at the site of action and decreased possibility of side effects. When this route of drug administration is combined with the sustained release and/or targeting potential of novel drug delivery systems, therapeutic advantages are even more

∗ 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 © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2007.10.005

pronounced. Nevertheless, for such applications, it is important to have background knowledge about the parameters that would influence the stability of the drug–carrier system during the aerolization process, as well as the ability of the various types of drug delivery systems to be aerolized. From the various available aerosol delivery technologies, nebulizers offer the advantage of delivering colloidal drugs without further processing [2–4]. This of course applies, if the colloidal drug (in aqueous dispersion form) has the appropriate stability (especially towards drug hydrolysis [which depends on the properties of the formulated drug]) in order to provide an adequate shelf life for the formulation. Nevertheless, if formulation stability is a problem and cannot be solved by addition of stabilizer, it may be required to add additional steps in the whole process, in order to lyophilize the formulation (for adequate product shelf life) and re-hydrate the powder before

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nebulization [5]. In such cases, in addition to all other parameters the easiness to rehydrate the powder-lyophilizate produced should also be considered, although another possibility – which will not be considered in this investigation – would be the usage of a dry powder nebulizer. Between the various types of drug delivery systems proposed, microparticles or microspheres composed of biodegradable polymers have been extensively studied. Biodegradable microparticles composed of poly (lactide-co-glycolide) (PLGA) are well established drug delivery systems, having high potential to serve as carriers for drugs or vaccines [6]. However, although experience with synthetic polymers is extensive and encouraging, recently the trend has been to shift towards natural polymers as alginate and chitosan. The major advantages of these polymers are their low cost and compatibility with a wide range of drugs, while they can be formed with minimal use of organic solvents. Furthermore, bio adhesion, stability, safety and approval for human use by the US FDA are additional advantages. Two polymers that are extensively used for drug delivery applications (as discussed above), one synthetic (PLGA) and one natural (Chitosan [CHT]), were selected for evaluation in this study. Additionally, rifampicin (RIF), an amphiphilic drug, was chosen as a model drug because it is a first choice drug for tuberculosis treatment [7,8] and resistance to RIF can develop rapidly [8]. Thereby, there is a therapeutic rational for delivering RIF within microparticles, in order to passively target alveolar macrophages where a large number of tubercule bacilli harbour [9]. For these reasons, several types of novel drug delivery devices have been proposed in the past, for RIF administration, in order to maximize the therapeutic and minimize the toxic and/or side effects [10–14]. From those previous studies, information about incorporation of RIF in various types of colloidal systems is available and can be used for comparison with the results obtained herein. In fact PLGA has been used before for the preparation of RIFloaded microspheres for alveolar delivery of RIF, and it has been established that these particles can reach alveolar macrophages after aerosol delivery and enhance the therapeutic effect of RIF in vivo [13]. Furthermore, several studies have been carried out mainly for evaluation of formulation parameters that influence the release kinetics of RIF from PLGA particles. Recently, the encapsulation of three frontline antituberculosis drugs (ATDs) (rifampicin, isoniazid and pyrazinamide) in alginate microspheres demonstrated promising chemotherapeutic potential [15], while it was shown that a few critical adjustments in the formulation process [16], especially the incorporation of chitosan, could be used for improving the drug(s) encapsulation efficiency and bioavailability and reducing the dose and dosing frequency. Nevertheless, RIF-loaded CHT microparticles have never been –to our knowledge – prepared before. In addition, while RIF-loaded PLGA microspheres have been prepared and evaluated, as mentioned above, no relevant studies involving their behaviour during nebulization have been conducted. In the present study we compared PLGA, CHT or mixtures of the two polymers (CHT-coated PLGA particles) for the prepa-

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ration of drug-loaded microspheres that may be administered to the lungs by nebulization. For this, the three types of particles were prepared and their ability to encapsulate RIF was evaluated. Subsequently, particle ability to be nebulized (the same nebulization device was used in all cases) and their stability during this process were evaluated and compared. The later mentioned characteristic is a very important particle characteristic for alveolar delivery, since association of the drug with the particles is a prerequisite for achieving high drug concentrations in the alveolar macrophages. Morphological assessment of the particles was carried out by environmental scanning electron microscopy. As explained above, depending on the drug used, it may be needed to freeze-dry the colloidal formulation for obtaining adequate shelf life, and rehydrate before nebulization. Thereby, the stability (retention of encapsulated drug) of the MPs after freeze-drying was also evaluated, as well as the stability during nebulization of the resulting – after rehydration – RIF-loaded MPs. Although for the specific application of targeting colloidal RIF to alveolar macrophages the MP formulation may not benefit from increased mucoadhesive ability (a point that is under debate), in other applications mucoadhesive properties of colloidal formulations intended for delivery to the lungs are very important for prolonged retention and slow release of drugs at the site [17,18]. This is why we evaluated the mucoadhesive properties of (some of) the MPs prepared, in order to provide some knowledge about if and how the MP composition influences these properties. Additionally, the toxicity of the MPs (empty and drug-loaded) towards an alveolar epithelial cell line was evaluated, and also compared with that induced by equivalent concentrations of the free drug (in solution) measured under identical experimental conditions. 2. Materials and methods 2.1. Materials Medium molecular weight chitosan (CHT) [with a deacetylation grade of 87%], poly (lactide-co-glycolide) (PLGA) having a monomer ratio [lactic acid/glycolic acid] of 75/25, and MW 85.2 kDa, rifampicin (RIF), sodium sulphate, glutaraldehyde, acetic acid and polyvinyl alcohol (PVA) – MW 30 – 70 kDa, were purchased by Sigma–Aldrich (Athens, Greece), as well as all other reagents used which were always of analytical grade. Water was always demineralised and distilled. 2.2. Preparation of CHT microspheres CHT particles were formulated by the precipitation method [19]. In brief, chitosan at different concentrations was dissolved in an aqueous solution of acetic acid (2%, v/v). A solution of sodium sulphate (20%, w/v) was subsequently added dropwise during vigorous stirring with Ultraturrax® T8, IKA (Germany) at 500 rpm and concurrent bath sonication (Branson 1200). After the addition of the full amount of sodium sulphate, stirring

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Table 1 Preparative parameters – composition of microparticles tested (A) Chitosan MP Formulation

Chitosan (mg/ml)

Acetic acid (␮l)/Na2 SO4 (ml)

Glut 25%

CHT 2.0− CHT 2.5− CHT 5.0− CHT 7.5− CHT 2.0+ CHT 2.5+ CHT 5.0+ CHT 7.5+

2.0 2.5 5.0 7.5 2.0 2.5 5.0 7.5

200/2 200/2 200/4 200/6 200/2 200/2 200/4 200/6

− − − − + + + +

(B) PLGA MP Formulation

PLGA (mg/ml)

PVA (%w/v)

RIF addition

PLGA-1 PLGA-2

2 2

4 4

In organic phase In aqueous phase

(C) PLGA/CHT MP Formulation

PLGA (mg/ml)

PVA (%w/v)

CHT (%w/v)

PLGA/CHT-0% PLGA/CHT-0.1% PLGA/CHT-0.25% PLGA/CHT-0.5% PLGA/CHT-0.75%

2 2 2 2 2

1 1 1 1 1

0 0.10 0.25 0.50 0.75

In all cases RIF was added in the formulations (during their preparation) at a concentration of 2 mg/ml.

and sonication continued for 30 min. In some formulations a solution of glutaraldehyde (25%, w/w) was also added, at this point, in order to evaluate the influence of cross-linking agent on the physicochemical properties of the particles prepared. In all cases, stirring and sonication continued for an additional 30 min period. The different particles formed and their compositions are presented in Table 1. Microspheres were purified by centrifugation for 15 min at 6000 rpm in a Biofuge 28RS, (Hereaus). The sediment was resuspended in water and washed again twice. In some cases the purified microspheres were lyophilized and rehydrated by one-step addition of the appropriate volume of d.d. water before further evaluation. 2.2.1. Preparation of RIF-loaded CHT microparticles For the preparation of RIF-loaded CHT microparticles, RIF and CHT, at different concentrations (presented in Table 1), were initially dissolved in acetic acid (2%, v/v). After this, the method described above was followed for preparation and purification of the microparticles.

taining PVA (4%, w/v). The emulsion formed was homogenised for 10 min with an Ultraturax® T8 IKA homogeniser at 800 rpm. Subsequent evaporation of dichloromethane was carried out with mechanical stirring for 6 h at room temperature (24 ± 3 ◦ C). The microspheres formed, were collected by centrifugation (as above) and washed by dispersion in water and subsequent centrifugation. This final step was repeated three times, while in some cases the MPs prepared were subsequently freeze-dried and rehydrated (as also mentioned above, for CHT microparticles). 2.3.1. Preparation of RIF-loaded PLGA microspheres For the preparation of RIF-loaded PLGA microspheres by the o/w solvent evaporation method described above, two different cases were studied. In the first, PLGA and RIF (20 mg/ml) were dissolved in the organic phase, which was subsequently dispersed in the aqueous phase, as mentioned above. In the second case, PLGA was dissolved in the organic phase while RIF (2 mg/ml) was included in the aqueous (PVA-containing) phase. After initial preparation of the o/w emulsion, the method described above was followed, for both cases. 2.4. Preparation of PLGA/CHT microspheres by emulsion solvent diffusion For the preparation of CHT-coated PLGA microspheres the emulsion solvent diffusion method in water [21] was used. Chitosan was dissolved (in different concentrations [0.1, 0.25, 0.5 and 0.75%, w/v]) in 50 ml of acetic acid buffer, pH 4.4, which also contained PVA (1%, w/v). The PLGA (100 mg) was dissolved in 5 ml DCM which was poured into the coating polymer (CHT) aqueous solution (prepared beforehand) at 2 ml/min under Ultraturrax® stirring (400 rpm), at room temperature. The PVA presence in the aqueous solution of coating polymer prevented aggregation of the emulsion droplets and sticking of the polymers to the propeller shaft during agitation. After particle formation the entire dispersed system was centrifuged (10,000 rpm 15 min) and the sediment was resuspended in distilled water. This process was repeated and the resultant dispersion was subjected to freeze-drying overnight. Control PLGA particles of this type (with no chitosan) were also prepared for comparison with the other PLGA particles. The different types of formulations prepared are presented in Table 1. 2.4.1. Preparation of RIF-loaded PLGA/CHT microspheres For the preparation of RIF-loaded PLGA/CHT microspheres by the method described above, RIF (20 mg/ml) was dissolved in the organic phase together with PLGA, and particles were formed and purified as mentioned above.

2.3. Preparation of PLGA microspheres by emulsion solvent evaporation

2.5. Characterization of RIF-loaded microspheres

PLGA microspheres (MP) were prepared by an O/W solvent evaporation method adapted from Prieto et al. [20]. Twenty milligrams of PLGA were dissolved in 1 ml dichloromethane (DCM). This was dispersed in 10 ml of an aqueous phase con-

2.5.1. Measurement of encapsulation efficiency of RIF in microparticles The RIF content of each lot of microspheres was determined by first extracting RIF from the particles with acetonitrile and

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then quantifying the amount of drug spectrophotometrically. A series of RIF solutions of known concentrations in acetonitrile were prepared and optical densities at 485 nm were measured in order to generate a standard curve. The amount of drug entrapped (encapsulation efficiency, EE%) in CHT, PLGA and PLGA/CHT microparticles was calculated by extracting and quantifying the amount of RIF in a known amount of particles both prior to and after separation of non-entrapped RIF (as mentioned above). Finally, the drug EE% was calculated as the percentage of drug entrapped in microspheres compared to the initial amount of drug recovered in unpurified samples. 2.5.2. Particle size distribution and surface charge (zeta-potential) measurements The size distribution (mean diameter and polydispersity index) and ␨-potential of some of the MP dispersions were measured by dynamic light scattering (DLS) and laser Doppler electrophoresis (LDE), respectively, on a Nano-ZS instrument [22], which enabled the mass distribution of particle size (size range measured by Nano ZS 0.6 nm to 6 ␮m) as well as the electrophoretic mobility to be obtained. The Zetasizer Nano Z, S and ZS measure the scattering information close to 180◦ (173◦ ). This is known as backscatter detection. This technique enables a precise and reliable measurement of the distribution of the hydrodynamic diameters of particles that range between 0.6 nm and 6 ␮m, with minimal background noise due to the application of the Backscatter detection that is done by a patented technology called Non-Invasive Back-Scatter (NIBS) (Zetasizer Nano Series, User Manual, 2005). The particle size measured in a DLS instrument is the diameter of the sphere that diffuses at the same speed as the particle being measured. The Zetasizer system determines the size by first measuring the Brownian motion of the particles in a sample using Dynamic Light Scattering (DLS) and then interpreting a size from this using established theories. Sizes quoted are the z-average means (dz) for the particle hydrodynamic diameters, and the values measured were expressed as % of volume. The ␨-potential (mV) values of the particles were calculated by the instrument (Smoluchowski Equation) from their electrophoretic mobility, which was measured.

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tosan or PLGA microspheres were collected in a buffer solution using a modified 3 stage glass impinger, as described previously [23]. The impinger device was utilized with the collecting flask containing 3 ml of buffer to which the aerosol was introduced through a calibrated glass tube and critical orifice delivering the jet of aerosol 5 mm above the bottom of the flask. Ten minutes after aerosolization, the impinger contents were collected and after washing the inpinger with 2 ml of buffer, samples from the 5 ml dispersion were assayed in order to evaluate the effect of nebulization on microsphere drug content. The secondary aim of this experiment was also to determine the total amount of formulation nebulized (collected into the apparatus). The nebulization efficiency (NE%) of microsphere formulations is defined as the total output of drug collected on the impinger as a percentage of the total amount of drug submitted to nebulization. Aerolized drug (collected in flasks) NE% = × 100 Initial drug (placed in nebulizer) Because nebulization can lead to drug leakage, it is also important to determine the nebulization efficiency of the encapsulated drug (NEED%). This parameter is defined as the percentage of aerosolized drug that remains encapsulated after nebulization. A portion of nebulized sample was purified by centrifugation and the amount of drug in the sample after and before centrifugation was assayed. NEED% =

Aerolized drug (collected in flasks) × 100 Initial drug (placed in nebulizer)

2.7. Mucoadhesive studies – adsorption of mucin on microparticles

2.5.3. Particle morphology evaluation by SEM SEM measurements were performed with a Zeiss SUPRA 35VP-FE working in the high vacuum mode (accelerating voltage 10 kV, WD 4 mm). In order to prevent charging, the samples were coated with gold in a BAL-TEC SCD 004 sputter. For each sample, a drop of the microsphere suspension was placed on an aluminium stub. The samples were evaporated at ambient temperature until completely dried, leaving only a thin layer of particles on the stub.

The adsorption-association of mucin on/with the particles was used as a method to assess mucoadhesive properties of the particles prepared. For this, 1 ml of mucin aqueous solution (0.5 mg/ml) was mixed (vortexed) with 1 ml of each MP dispersion (the concentration of MP’s in these dispersions was fixed at 2 mg/ml) at room temperature. Then, the dispersions were centrifuged at 10,000 rpm for 30 min, and the supernatant was used for the measurement of free mucin. The Bradford colorimetric method [24] was used to determine free mucin concentration in order to assess the amount of mucin adsorbed on the MPs. A mucin calibration curve was also prepared by measuring mucin standard solutions (0.25, 0.5, 0.75, 1 and 1.25 mg/ml). For all samples (of known and unknown mucin concentration) after adding Bradford reagent, the samples were incubated at 37 ◦ C for 20 min and then the absorbance at 595 nm was measured (Shimatzu UV-1205 spectrophotometer). The mucin content of the samples was calculated from a standard calibration curve.

2.6. Nebulization of microspheres

2.8. Cell culture studies

A compressor nebulizer system (Medel Aerofamily, Italy) was used in the study. A volume of 3 ml of sample was used for the nebulization. The aerosols containing RIF-loaded chi-

Human A549 alveolar cells (at passage 31), a kind gift from Dr. Ben Forbes, London, UK, were grown as monolayers in 35 mm tissue culture dishes incubated in 100% humidity and

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5% CO2 at 37 ◦ C. HAM’S medium containing 365 mg/l lglutamine, supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 ug/ml streptomycin was used as growth media. The cells that form monolayers were harvested with trypsin (0.25%) centrifuged at low speed (1600 × g for 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 to 4 days. 2.8.1. RIF and MP RIF cyctotoxicity assessment – MTT assay For dose-dependent studies, cells were treated with both empty and RIF-loaded CHT and PLGA MPs, with varying RIF concentrations. The effect of empty and RIF-loaded MPs on the viability of cells was determined by the MTT [3(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay [25]. The dye is reduced in mitochondria by succinic dehydrogenase to an insoluble violet formazan product. A549 cells (105 cells/well) were cultured on 24-well plates with 500 uL 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 the formazan crystals formed were dissolved in DMSO. Absorbance at 580 nm was measured with a plate reader spectrophotometer. On the basis of this assay %cell viability values were obtained in three independent experiments for each MP type. 2.9. Statistical analysis All experiments were repeated at least three times. Results are expressed as means ± standard deviation. One-way ANOVA and independent t-test were used to check the significance between differences measured (for EE%, NE%, NEED%, etc.) for different particle types, under the same conditions. A difference between means was considered significant if the p-value was less than, or equal to, 0.05.

3. Results and discussion 3.1. Physicochemical properties of RIF-loaded microparticles The physicochemical properties measured for all the RIFloaded microparticles prepared are presented in Table 2. As seen, RIF encapsulation efficiency ranges between 15% and 86% of the amount of drug initially added (2 mg/ml). By comparing the EE% values measured (Table 2) for the different CHT MPs formulated (Table 1), it is obvious that as the concentration of CHT used during MP formation increases, the trapping efficiency of the particles decreases. This may be related to the increased viscosity of the more concentrated CHT solutions, which results in decreased trapping of RIF during the microparticle preparation procedure, as observed also by others [19]. However, even when the lowest polymer concentration is used for particle preparation (2 mg/ml) the CHT particle RIF trapping efficiency, although higher than all other cases of CHT MPs studied, is still significantly lower than that measured for PLGA particles and some of the PLGA/CHT particles. For the CHT particles, it is also obvious (Table 2) that crosslinking of CHT by glutaraldehyde did not provide any advantage in terms of RIF trapping. For the PLGA particles (formulations PLGA1 and PLGA2), a slightly higher RIF EE% was achieved when the drug was initially dissolved in the organic phase. The highest EE% for RIF in this group of particles, 62.9 ± 2.5% (calculated for formulation PLGA 1) was higher than that reported recently for RIF-PLGA nanoparticles (56.97 ± 2.7%), proposed as a delivery system against murine tuberculosis [26], however within the range calculated for RIF-PLGA microspheres (20–30% or 50–67.4%) prepared by solvent evaporation or spray drying techniques [27,28]. For the PLGA/CHT formulations, the EE% values start out from a very low value (37.3%) compared to the other type of PLGA MPs formulated, in which no CHT was added in the for-

Table 2 Physicochemical characteristics of the microparticles prepared, immediately after preparation and cleaning from non-encapsulated drug Polymer/formulation

Encapsulation efficiency (% RIF trapped)

Encapsulation efficiency after FD (% RIF trapped)

Zeta potential (mV)

Mean diameter (␮m)

Yield (%)

CHT 2.0− CHT 2.5− CHT 5.0− CHT 7.5− CHT 2.0+ CHT 2.5+ CHT 5.0+ CHT 7.5+ PLGA 1 PLGA 2 PLGA/CHT-0% PLGA/CHT-0.1% PLGA/CHT-0.25% PLGA/CHT-0.5% PLGA/CHT-0.75%

48.1(1.3) 43.5 (2.6) 35.1 (8.8) 14.9 (3.8) 45.0 (1.6) 39.7 (5.6) 29.4 (4.2) 18.7 (1.8) 62.9 (2.5) 54.8 (2.8) 37.3 (3.8) 44.8 (3.2) 52.5 (9.8) 77.9 (6.6) 86.2 (1.4)

NM 40.9 (6.9) 28.4 (1.7) 17.8 (1.6) NM 48.7 (4.5) 33.6 (5.4) 23.5 (3.7) 62.8 (1.6) 55.6 (2.3) 34.4 (1.2) 42.1 (3.0) 49.1 (4.0) 62.7 (8.4) 81.1 (2.9)

NM +32.5 ± 0.4 +34.7 ± 0.1 +37.0 ± 0.2 NM +23.7 ± 0.6 +21.9 ± 0.2 +15.6 ± 0.2 −4.6 ± 0.2 NM −4.8 ± .10 +14.9 ± 1.0 +19.1 ± .40 +31.1 ± .40 +37.0 ± .40

NM 2.192 ± 0.048, P: 0.243 ± 0.093 1.470 ± 0.051, P: 0.23 ± 0.12 1.710 ± 0.078, P: 0.25 ± 0.13 NM 2.310 ± 0.106, P: 0.160 ± 0.127 NM NM 2.583 ± 0.063, P: 0.209 ± 0.042 NM 2.535 ± 0.058, P: 0.506 ± 0.018 2.900 ± 0.059, P: 0.571 ± 0.018 2.807 ± 0.086, P: 0.459 ± 0.053 2.567 ± 0.019, P: 0.494 ± 0.059 1.407 ± 0.051, P: 0.392 ± 0.020

NM 81 88 86 NM 80 78 84 80 85 82 83 82 86 80

RIF encapsulation efficiency was also calculated after freeze-drying (FD) and re-hydration. Each value is the mean of the corresponding values from at least three different samples and numbers in parenthesis are standard deviation. P is the polydispersity index, a measure of vesicle size distribution.

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Fig. 1. Correlation between RIF EE% (percent of RIF-loaded in the particles) – black squares– and ␨-potential values –blue triangles – of PLGA/CHT MPs with the CHT content (expressed in %w/v) of particles.

mulations. However, EE% increases linearly (Fig. 1, R = 0.9972) with the amount of CHT added, reaching the value of 86% in the particles that contain 0.75% (w/v) CHT. In fact, this is the highest EE% achieved for RIF, between all the MP types prepared. An increase in RIF loading in PLGA microspheres when CHT was used as a stabilizer for their formation was also reported before by others [29]. In all cases (types of MPs prepared), it was observed that FD does not result in significant (p = 0.05) leakage of RIF from the particles. Additionally it should be stated at this point that the powder produced by freeze-drying was very easily re-hydrated by one-step addition of the appropriate volume of H2 O, in all cases studied, a fact that is important for the intended use of the RIF-loaded microparticles. Particle zeta-potential and size distribution of the MP formulations was measured (Table 2). As previously demonstrated [19], drug-loaded CHT-particles have positive surface charge. On the other hand, PLGA MPs are negatively charged, as also demonstrated by others in several cases. For the CHT-coated PLGA particles, the surface charge starts from a negative value for the MPs with no CHT (similar to PLGA 1) and as the CHT content of the particles increases, so does their ␨-potential (Fig. 1). The positive charge of the CHT particles indicates that only a part of the amino groups are neutralized during microsphere formation by the sulfate ions that were used as precipitant. Thus, the residual amino groups are most possibly responsible for the positive ␨-potential. However, these groups are freely accessible for interaction with drugs or other components of the MPs. Glutaraldehyde addition in the CHT particles results in a considerable reduction of ␨-potential, and this is explained by the reaction of glutaraldehyde with amino groups which results in reduction of residual amino groups in the CHT microspheres and consequently similar reduction of their ␨-potential value. Additionally, in the case of the glutaraldehyde-containing MPs, the ␨-potential values were linearly correlated (R = 0.9959) with the amount of RIF encapsulated in the particles (as RIF encapsula-

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tion decreases so does the particle zeta-potential). Perhaps this implies that a fraction of particle-associated RIF is adsorbed on the particle surface and influences the surface charge of the particles (since amino groups are also present on the RIF molecule). Concerning particle mean diameter and size distribution, all MPs prepared had mean diameters ranging between 1.47 ␮m and 2.9 ␮m and are therefore in the size range needed for delivery to the lungs, with the exception of some of the CHT coated PLGA particles. The CHT MPs with highest CHT concentration (5.0 and 7.5%) and the CHT-coated PLGA MPs with the highest CHT content (0.75%) had significantly smaller mean diameters (ranging around 1.5 ␮m) compared to all MPs prepared that had mean diameters >2 ␮m. For all particle types measured, the size distribution was narrow (monodispersed) as judged by the corresponding polydispersity index values (0.16–0.57). The effect of nebulization on particle size (mean diameter and polydispersity values) will be discussed below. 3.2. Particle nebulization efficiency and stability during nebulization In Table 3, the NE% and NEED% values calculated for each one of the microparticle samples studied are presented, for nebulizations performed immediately after the particle preparation (A) or after the particles were FD and re-hydrated (B). As easily observed by comparing the equivalent cases of microparticles between the two sets of results (A and B), freeze-drying and rehydration does not affect neither the amount of RIF nebulized, nor the particle stability during the nebulization process (no significant differences found), for all types of particles formulated (CHT, PLGA and PLGA/CHT). The percentage of RIF collected in nebulized microparticle samples (NE%) ranges from 34.4 to 86.7, while different microparticle formulations offer different nebulization ability for RIF-loaded particles. First of all, it seems that PLGA MPs (PLGA 1 or PLGA 2) are more efficiently nebulized, compared to the CHT ones. However, significant differences were measured for %NE between the different cases of nebulized CHT MPs. Indeed, as the CHT concentration in the particles decreased (which results in a decrease of the MP dispersion viscosity), their NE% increased. A negative effect of viscosity on %NE was also demonstrated before for the nebulization of liposomes [23,30]. When CHT 2.0− and CHT 2.0+ MPs were further diluted prior to nebulization (2×), the NE% for RIF (presented as a second value in Table 3A) increased in the first case, but not in the case of the crosslinked MPs. However, the crosslinked (with glutaraldehyde) CHT particles always demonstrated slightly higher NE% compared to the non-crosslinked ones. Nevertheless, even in these cases (of diluted samples) which resulted in the highest NE% for CHT particles, the NE% values measured were lower compared to those obtained by PLGA particles. PLGA/CHT MPs exhibit a different behaviour depending on their CHT content. From this set of MPs, the plain PLGA particles (with no CHT) demonstrate the lowest NE% (40.9%) a lot lower compared with the other PLGA MPs (PLGA 1 and 2), and as the CHT content of the particles increases so does their neb-

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Table 3 Nebulization efficiency (NE%) (percent of material that was nebulized) and percent of RIF retained in particles in the nebulized fraction (NEED%), for MPs with different compositions Polymer–formulations

Nebulization efficiency%)

Stability after nebulization

% of RIF nebulized (%NE)

% NEED

Final outcome (amount RIF encapsulated after NEB (␮g/ml))

(A) Non freeze dried microparticles CHT 2.0− 56.73 (.38) 67.46 (.54) CHT 2.5− 38.8 (4.0) CHT 5.0− 37.8 (4.0) CHT 7.5− 34.4 (1.5) CHT 2.0+ 59.03 (2.8) 62.1 (2.5) CHT 2.5+ 50.5 (4.1) CHT 5.0+ 44.5 (3.7) CHT 7.5+ 42.9 (3.0) PLGA 1 75.1 (3.9) PLGA 2 70.1 (2.4) PLGA/CHT-0% 40.9 (9.7) PLGA/CHT-0.1% 55.9 (3.0) PLGA/CHT-0.25% 69.5 (4.6) PLGA/CHT-0.5% 77.8 (3.4) PLGA/CHT-0.75% 86.7 (3.7)

15.1 (1.5) 10.8 (2.8) 16.4 (1.1) 14.0 (1.9) 15.8 (1.6) 41.8 (3.2) 15.6 (5.9) 30.4 (2.3) 26.4 (1.5) 26.8 (3.2) 44.5 (2.3) 40.5 (2.0) 30.6 (2.3) 42.2 (4.3) 53.8 (5.6) 58.7 (1.1) 63.5 (2.8)

62.6 (2.3) 49.1 (4.9) 34.1 (5.9) 14.6 (1.5) 165.5 (1.3) 130.6 (4.8) 74.6 (6.5) 41.2 (1.2) 420.9 (1.3) 311.5 (1.6) 94 (12) 211 (22) 383 (11) 724 (35) 961 (57)

(B) Freeze dried microparticles CHT 2.5− CHT 5.0− CHT 7.5− CHT 2.5+ CHT 5.0+ CHT 7.5+ PLGA 1 PLGA 2 PLGA/CHT-0% PLGA/CHT-0.1% PLGA/CHT-0.25% PLGA/CHT-0.5% PLGA/CHT-0.75%

16.9 (1.9) 13.2 (1.5) 13.9 (1.9) 38.2 (4.9) 28.3 (2.7) 26.3 (3.5) 44.1 (2.3) 43.4 (1.9) 31.2 (1.7) 42.49 (.86) 45.5 (1.4) 57.7 (1.7) 67.4 (1.9)

57.7 (6.9) 29.4 (2.1) 16.0 (1.3) 166.9 (4.5) 80.3 (4.4) 40.5 (5.6) 411.8 (3.9) 339.1 (1.5) 86.5 (6.5) 193 (35) 291 (14) 632 (12) 919 (39)

40. 5 (2.5) 35.4 (3.0) 33.3 (2.7) 54.2 (3.8) 44.9 (5.8) 40.4 (4.0) 74.4 (1.8) 70.1 (3.6) 41.28 (.54) 55.0 (2.2) 65.6 (1.2) 77.01 (.71) 83.1 (2.0)

In all cases the MPs were nebulized immediately after their preparation (and separation from non-encapsulated drug). Each value is the mean calculated from at least three separate preparations and standard deviation of mean is presented in parenthesis. Non-freeze dried and freeze dried particles (after one-step rehydration) were evaluated.

ulization efficiency. In fact the particles with the highest CHT content from this group (PLGA/CHT-0.75%), demonstrate the highest NE% between all the particle-types prepared. In addition to the nebulization ability, the retention of the drug in the nebulized particles (NEED%) is also a very important parameter, if not the most important. A lower NE% that would produce stable particles (that retains the drug in high amounts) is preferable to a higher NE% that would result in particles from which most of the drug has been released. In this context, it is obvious that RIF-loaded PLGA particles and especially the ones coated with CHT are superior, compared to all of the CHT particles tested (Table 3). Nevertheless, crosslinking CHT particles with glutaraldehyde definitely results in increased stability during nebulization as concluded by comparing the NEED% values calculated for equivalent formulations of chitosan MPs, with or without glutaraldehyde (CHT 2.5+ and CHT 2.5−, CHT 5.0+ and CHT 5.0− or CHT 7.5+ and CHT 7.5−). In fact, in the case of CHT 2.0+, the stability of the nebulized particles is practically equal with that of the PLGA 1 and PLGA 2 particles, but due to the lower EE% of the CHT MPs the final amount of RIF encapsulated in the nebulized particles is significantly lower compared to PLGA-MPs. When these, sta-

ble, CHT MPs are diluted before nebulization, although the NE% is not modified, their stability is highly and negatively influenced (NEED% is reduced from 42% to 16%). It is most likely that when a lower number of particles are subjected to a specific magnitude of forces and pressure (during nebulization), the impact of these forces on each individual particle is higher. For the CHT-coated PLGA particles, as the content of CHT in the MPs increases, so does their NEED%. This increase follows the same pattern as the increase in NE%, when both values are plotted against CHT content (not shown). In order to have a measure of comparison between the different cases of particles prepared and studied, we calculated and present (last column of Table 3) the final outcome of the RIFloaded particle nebulization procedure. This is the amount of RIF present in microparticle-entrapped form in the nebulized MP samples. PLGA (PLGA 1 and 2) particles exhibited twice the final quantity of encapsulated RIF compared to the best (in terms of final outcome) CHT particles, while from PLGA/CHT particles the ones with 0.50% and 0.75% CHT exhibited two and three times more encapsulated RIF, compared to PLGA 1 and 2, respectively.

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indicating a narrow size distribution for these particles, for the PLGA and PLGA/CHT particles the PIs were 0.778 ± 0.053 and 0.646 ± 0.108, respectively, suggesting a broader size distribution for these MP types. An indication that this is an effect of nebulization is the fact that the PIs measured for the same particles before nebulization were lower (Table 2). 3.3. Particle morphology

Fig. 2. Mean hydrodynamic diameters measured (expressed as % of volume) for different types of RIF-loaded MPs, before and after nebulization. The symbol key is presented in the graph insert.

Additionally, it should be mentioned that after nebilizing the RIF-loaded particles, in all cases of CHT particles tested the nebulizate was present in compartments 1 and 2 of the nebulizer, while in the case of PLGA and most of the PLGA/CHT MPs, a considerable amount of nebulizate was collected from compartment 3, indicating that nebulized PLGA and PLGA/CHT microparticles have a smaller size or broader size distribution compared to those of CHT MPs. This last suggestion is in line with the effect of nebulization on the mean diameters of the particles, as presented in Fig. 2. Indeed, nebulization does not seem to have neither a profound nor a constant effect on the CHT samples measured – particle mean diameter decreases by 22–24% for the low concentration CHT MPs (CHT 2.5− and CHT 2.5+) while it increases (∼30% increase) for the higher concentration CHT MPs (CHT 5.0− and CHT 7.5−). This last observation may be connected with the fact that for the concentrated CHT formulations (CHT 5.0− and CHT 7.5−) the cumulative amount of drug nebulized is very low so consequently also the concentration of the particles in the nebulizate is considerably lower compared to that of the initial sample subjected to nebulization, in each case. Thereby, the explanation for increased particle size may be that when the particles are dispersed in more dilute solutions, perhaps their degree of swelling increases. Oppositely, for PLGA particles, nebulization results in a substantial decrease (59%) of their mean diameter. The same is true in the case of PLGA/CHT MPs for CHT content up to 0.25%. At 0.50% CHT, the difference between mean diameters before and after nebulization is smaller while it is diminished (mean diameter before and after nebulization are statistically equal) for the 0.75% CHT containing PLGA-MPs, implying that the CHTcoating of PLGA/CHT MPs provides substantial stabilization to the particles when CHT concentration is higher than 0.50%. The polydispersity index values measured after particle nebulization also show that for PLGA and most PLGA/CHT MPs the particle size distribution is influenced by the nebulization process. Indeed, while PIs measured for the chitosan particles after nebulization, range between 0.213 and 0.291,

The morphology of CHT (CHT 2.5− and CHT 2.5+), PLGA (1 and 2) and PLGA/CHT (0.10–0.75%) were evaluated by SEM (Figs. 3 and 4). For this, the particles were dehydrated prior to observation. As observed in Fig. 3A and B, the CHT particles seem to have a crumbled structure in comparison to the very smooth surface observed for both PLGA 1 and PLGA 2 MPs (Fig. 3C and D, respectively). This was observed for both types of the CHT particles evaluated, glutaraldehyde containing (Fig. 3A) and not (Fig. 3B), proving that crosslinking CHT does not change the particle behaviour. At this point it should be mentioned that the aggregated structure observed in micrographs of CHT MPs (Fig. 3A and B) is probably due to the SEM sample preparation method, and does not correspond to presence of aggregates in the dispersions, since the PI values measured for these particles range between 0.23 and 0.160, indicating a narrow size distribution that does not correspond to presence of aggregates. Additionally, it is interesting that although the size distribution of the particles present in the micrographs of PLGA MPs (Fig. 3C and D) are in the area of mean diameters measured by DLS for these MPs (Table 2), the CHT particles were mostly smaller (compared with the mean diameter measured). This observation indicates that most possibly the CHT particles shrink during dehydration (applied for sample preparation) and this explains their crumbled morphology. The morphology of two of the PLGA/CHT MPs prepared is presented in Fig. 4. The PLGA/CHT 0.10% particles are presented in micrographs A and B and the PLGA/CHT 0.75% in micrographs C and D. At this point it should be commented that the aggregated structure appearing in Fig. 4 is an artifact caused by the sample preparation technique and the fact that the PLGA/CHT 0.75% dispersion used for sample preparation was less concentrated than the one of PLGA/CHT 0.10% particles. As seen by comparing the two sets of micrographs (better in micrographs B and D that are at higher magnification), the PLGA/CHT MPs with low CHT content (Fig. 4B) have a dual morphology and seem to be constructed by two different parts with completely different morphology (one smooth, as PLGA and one crumbled as CHT). In fact, in some of the larger particles present in the micrograph it is obvious that the crumbled (CHT?) sides are shrinking in the shell of the other side of the particles. Such morphologies, were also observed on the other samples evaluated (with CHT content 0.25% and 0.50%) (not shown). Oppositely, in the case of the PLGA/CHT with 0.75% CHT (Fig. 4D), the particle morphology is more homogenous for all particles observed. In comparison with the plain PLGA particle morphology (Fig. 3C and D), these CHT-coated ones exhibit a slightly modified, less smooth surface morphology, suggesting that their surface is coated with CHT. However, their

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Fig. 3. Surface morphology of CHT (A and B) and PLGA (C and D) RIF-loaded microparticles. CHT MPs without (A) or with (B) glutaraldehyde, and PLGA 1 (C) and 2 (D) MPs were observed. Dimension bar is shown under each micrograph. These PLGA microspheres contain 4% (w/v) PVA. Studies by scanning electron microscopy (SEM) were performed as described in detail under Section 2.

Fig. 4. Surface morphology of different preparations of PLGA/CHT, RIF-loaded microparticles. (A and B) PLGA/CHT 0.1%, at different magnifications, and (C and D) PLGA/CHT 0.75% microparticles. Dimension bar is shown under each micrograph. These PLGA-based microspheres contain 1% (w/v) PVA. Studies by scanning electron microscopy (SEM) were performed as described in detail under Section 2.

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size distribution is absolutely comparable with the mean diameter measured for these MPs by DLS (Table 2). Thereby, it seems that 0.75% (w/v) (or else >0.50%) CHT concentration is needed for complete coating of the PLGA particles, with the preparation technique used in this study. 3.4. Cell toxicity studies The viability of A549 cells was estimated after exposure of the cells for 24 h to different concentrations of empty or RIF-loaded MPs or the same concentration of free RIF (in solution), for comparison. As observed in Fig. 5, when the cells were exposed to empty particles (the highest concentrations used in the case of RIF-loaded MP incubations were selected in each case), cell viability was affected by the empty particles,

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at different degrees. The CHT particles tested affected the cells less compared to the PLGA particles, and resulted in practically no toxicity, in the case of CHT 2.5− MPs, or a very slight (12%) reduction of cell viability, in the case of CHT 2.5+ MPs. Oppositely, both types of (plain) PLGA particles evaluated, PLGA 2 (Fig. 5A) and PLGA/CHT 0% (Fig. 5B), significantly (p > 0.05) reduced A549 cell viability (by 28% and 38%, respectively), under the conditions applying in the cytotoxicity evaluation experiment. These results can be explained by an increase of the local acidity caused by the PLGA particle degradation, which can lead to cell damage, as proposed in previous studies [31,32]. In fact, it is logical to assume that MP-induced cytotoxicity is determined by the particle stability, since, when lower amount of stabilizer (PVA) was used (1% of PVA was in PLGA/Cht 0% MPs) MP-induced cytotoxicity was higher compared to that induced by the same MPs with more stabilizer (4% PVA was used in PLGA 2 MPs). From Fig. 5B, it is obvious that as the amount of CHT used for fabrication of the PLGA/CHT MPs increases (from 0% to 0.75%), there is a gradual decrease in the MP-induced cytotoxicity, from 38% (when no CHT is used) to 18% (for 0.75% CHT content). This phenomenon indicates that the PLGA particles are stabilized by CHT, and thus are degraded slowly and show less cytotoxicity during the 24 h incubation period. The cytotoxicity induced by free RIF was always substantially higher compared to that induced by MP-loaded RIF (same amount of RIF). Whether this is because of a lower uptake of microparticulate-RIF by the cells (compared to free RIF) or due to a different type of interaction of the cells with free RIF and MP-associated RIF (that results in different intracellular distribution of RIF rendering the drug less toxic) we do not know. Nevertheless, the lower cytotoxicity of RIF-loaded MPs (compared to that of free drug) is a considerable advantage for the colloidal drug delivery systems. 3.5. Mucoadhesive properties of RIF-loaded MPs

Fig. 5. Viability (% of control) of A549 alveolar cells after they have been incubated for 24 h with various concentrations (0, 0.10, 0.25 and 0.50 mg/ml) of either free RIF, RIF-incorporated in MPs, or empty MPs. (A) Viability in presence of some of the CHT and PLGA particles and (B) viability in presence of CHT/PLGA particles. Viability was measured by the MTT assay, as explained in detail in Section 2.7. In all cases, result presented is the mean value calculated (S.D. of the mean is presented as bar) from at least two different formulations (and three different wells/formulation). The figure key is presented in the insert of each graph.

Mucoadhesive properties of RIF-loaded MPs were evaluated by measuring mucin adsorption on particles. From the results presented in Table 4, it is obvious that mucoadhesive properties of the MPs tested are determined by their composition. Indeed, MPs composed of CHT have good mucoadhesive properties as demonstrated by the high amounts of particle associated mucin; while on the contrary, PLGA particles associate always dramatically lower amounts of mucin. However, when the PLGA particles are coated with CHT, even the lowest percent of CHT tested herein (0.1%), is enough to provide PLGA MPs with equivalent mucoadhesive properties with those of the CHT MPs. From a methodological point of view the results of Table 4 demonstrate that the mucin-association test gives similar results regardless of the concentration of mucin used (0.025, 0.1 or 0.5 mg/ml). 4. Discussion In this paper the preparation of drug-loaded MPs, that could be used as freeze dried formulations for rapid rehydrationnebulization and delivery of the drug to lung macrophages was investigated. We describe the preparation of RIF-loaded

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Table 4 Percent mucin adsorption/association on RIF-loaded MPs after incubation of the particles with mucin solutions of different concentration (A = 0.025 mg/ml, B = 0.1 mg/ml and C = 0.5 mg/ml) MP-type

CHT 2.5− CHT 5.0− CHT 7.5− CHT 2.5+ CHT 5.0+ CHT 7.5+ PLGA 2 PLGA/CHT-0% PLGA/CHT-0.1% PLGA/CHT-0.25% PLGA/CHT-0.5% PLGA/CHT-0.75%

Mucin adsorbed on cells (%) Mucin Mucin solution A solution B

Mucin solution C

78.7 (4.1) 81.9 (3.4) 85.10 (.79) 76.0 (2.4) 78.9 (1.0) 77.8 (2.1) 8.8 (1.6) 11.05 (.79) 85 (10) 87 (12) 86.9 (1.4) 89.0 (3.1)

98.57 (. 78) 98.48 (.16) 98.63 (.17) 95.28 (.17) 96.21 (.20) 95.53 (.68) 16.9 (1.6) 37.15 (.63) 98.46 (.54) 98.47 (.91) 97.4 (1.3) 97.71 (.71)

94.00 (.68) 94.12 (.86) 96.05 (.34) 91.62 (.68) 92.75 (.20) 94.51 (.51) 14.1 (3.1) 25.6 (1.7) 96.73 (.68) 97.8 (1.2) 94.91 (.54) 96.2 (1.5)

After co-incubation samples were centrifuged for separation of particles from the non-adsorbed/associated mucin fraction. The particle-mucin incubation as well as mucin measurement details are described under materials and methods (§ 2.7). Each value is the mean calculated from at least three separate preparations and standard deviation of mean is presented in parenthesis.

microparticles and chose to evaluate particles consisting of synthetic or natural polymers, (PLGA and CHT) as well as mixtures of the two polymers (PLGA/CHT), under identical conditions in order to compare the different cases. Particles were evaluated in terms of RIF loading, stability during freeze-drying and/or nebulization, cytotoxicity and mucoadhesive properties. When comparing the results for the three different particle types, it is concluded that RIF loading (Table 2) is generally higher in the order PLGA/CHT > PLGA > CHT (although this does not apply for all the different samples evaluated in each group). Within the CHT-group, RIF EE% is affected by the CHT concentration used during CHT particle formation, but in all cases RIF-loading is considerably lower in CHT microspheres, compared to the PLGA ones. For the PLGA MPs, it seems that RIF loading is higher if RIF is initially added in the system through the organic phase (during MP preparation), a fact that is in line with the lipophilic nature of this drug. Comparison between formulations PLGA 1 and PLGA/CHT-0% (with no CHT) reveals that the amount of PVA (which is used as a particle stabilizer) in the particles affects RIF EE%, since this is the only difference between the two compositions (4%, w/v in PLGA 1 and 1%, w/v in PLGA/CHT-0%), most possibly due to the formation of more stable particles when more PVA is used. Finally, within the PLGA/CHT group the CHT content was found to be linearly related with RIF EE% (Fig. 1). Considering the important characteristics of nebulization ability (NE%) and stability (NEED%), the order for particles is again (as for EE%) PLGA/CHT > PLGA > CHT, while significant differences between the different particle compositions within the groups were observed. For plain CHT microparticles, NE% increases significantly with decreasing CHT concentration, due to the easier nebulization of dispersion with lower viscosity [30]. However, RIF is released from the particles at higher amount when more dilute dispersions are subjected to

the forces of the nebulizing apparatus. From the other particle types studied, PLGA with 4% PVA were more stable compared to the ones with lower PVA content, and within the group of CHT coated PLGA particles, both NE% and NEED% or stability increased with increasing CHT-content. The particle types that exhibited higher stability during nebulization (PLGA 1 and 2 and PLGA/CHT 0.75%) are the ones that are denser and have a more stable structure (compared to CHT particles), as proven by their morphological assessment (Figs. 3 and 4). Between the PLGA-based particles the ones that exhibited the highest stability during nebulization were also found to have constant mean diameter before and after the process (Fig. 2). The higher stability demonstrated for PLGA-based particles, during nebulization, may be related to the possibility of reaction between the amino groups of RIF and the terminal carboxyl groups of PLGA [14]. This also explains the higher EE% found for these MPs, compared to plain CHT particles. If this is the case, it would be interesting to evaluate the stability of RIFloaded particles composed of lower molecular weight PLGA that should result in higher and perhaps more stable loading of RIF, due to the presence of more carboxyl groups per unit weight of polymer. Nevertheless, the fact that CHT-coated PLGA particles are even better in terms of RIF EE%, NE% and NEED% compared to plain PLGA MPs (even the ones that contain 4% PVA stabilizer) suggests that CHT acts as a stabilizer for the particles, possibly by interacting with PLGA (the amino groups of CHT with the terminal carboxyl groups of PLGA). This explains the almost linear dependency of particle stability by their CHT content. Recently it was proposed that for improved product shelflife, particles intended for alveolar delivery of drugs could be prepared as freeze dried formulations that will be rehydrated only prior to nebulization [5]. In line with this, all of the particles prepared herein were found to be very stable after freeze-drying and the freeze-drying/rehydration cycle did not affect their NE% nor their NEED% (Tables 2 and 3). Furthermore, the quality of the powder produced was always good (i.e. could be easily rehydrated). In cell culture experiments, RIF-incorporating particles were found to be considerably less cytotoxic towards alveolar epithelial cells compared to free RIF (same RIF concentration in solution), in all cases studied. This may serve as an early indication that microparticulate RIF will be less toxic compared to free RIF, however further studies are needed in order to clarify the mechanism of this observation and the release kinetics of RIF from the different MP types should be evaluated. As concluded by mucin-association experiments, while all types of CHT and also CHT-coated MPs exhibited very good mucoadhesion properties, this was not the case for the noncoated PLGA MPs. When comparing the zeta-potential values of the various types of PLGA/CHT-coated MPs constructed (Table 2) with the mucin association values (Table 4), it is seen that all the MPs with positive zeta potential values had equivalent ability to bind or associate mucin (under the experimental conditions used) although there was a significant difference in the zeta potential value between the particles coated with 0.10 or 0.25% CHT and those coated with more than 0.50% CHT.

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Summarizing the results of this study, we may conclude that the PLGA polymer is better than CHT, for the formation of particles that could deliver RIF to alveolar macrophages after nebulization. However, combination of the two polymers at proper quantities results in the formation of very stable MPs with high loading capacity for RIF, lower cytotoxicity towards alveolar epithelial cells (compared to plain PLGA MPs) and equivalent (with CHT MPs) mucoadhesive properties. In addition to the delivery of rifampicin to the lungs by nebulization, the results and conclusions generated by the present study should be considered during the design of colloidal formulations of other drugs, especially if increased mucoadhesion is considered as an advantage. Indeed, although it may not apply, in particular for RIF-loaded microspheres (which are aimed for macrophage uptake) improvement of the mucoadhesive properties of particles that are intended for delivery to the lungs through surface modification may be required in several different cases [17,18]. In line with this, the fact that PLGA/CHT microspheres, which were demonstrated to be the best in terms of stability during nebulization, have positive surface charge and very good mucoadhesive properties, is indeed a considerable advantage for many therapeutic applications. 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. References [1] S. Suarez, A.J. Hickey, Nebulizors-Aerosol delivery: drug properties affecting aerosol behavior, Respir. Care 45 (2000) 652–666. [2] B.E. Gilbert, P.R. Wyde, S.Z. Wilson, R.K. Robins, Aerosol and intraperitoneal administration of ribavarin and ribavarin triacetate: pharmacokinetics and protection of mice against intracerebral infection with influenza A/WSN virus, Antimicrob. Agents Chemother. 35 (1991) 1448–1453. [3] R. Parthasarathy, B. Gilbert, K. Mehta, Aerosol delivery of liposomal alltrans retinoic acid to the lungs, Cancer Chemother. Pharmacol. 43 (1999) 277–283. [4] C.F. Lange, R.E.W. Hancock, J. Samuel, W.H. Finlay, In Vitro aerosol delivery and regional airway surface liquid concentration of a liposomal cationic peptide, J. Pharm. Sci. 90 (2001) 1647–1657. [5] Y. Darwis, I.W. Kellaway, Nebulization of rehydrated freeze-dried beclomethasone dipropionate liposomes, Int. J. Pharm. 215 (2001) 113–121. [6] V.W. Bramwell, Y. Perrie, Particulate delivery systems for vaccines, Crit. Rev. Ther. Drug Carrier Syst. 22 (2005) 151–214. [7] G.L. Mandell, H.A. Sande, Antimicrobial agents, in: G.A. Gilman, A. Goodman (Eds.), The Pharmacological Basis of Therapeutics, seventh ed., Macmillan, New York, 1985, pp. 1202–1205. [8] H.P. Rang, M.M. Dale, J.M. Rither, Drugs used to treat tuberculosis, in: Pharmacology, fourth ed., Churchill Livingstone, London, 1999, p. 703–707. [9] L.E. Bermudez, Use of liposome preparation to treat mycobacterial infections, Immunobiology 191 (1994) 578–583. [10] C.P. Jain, S.P. Vyas, Preparation and characterizationof niosomes containing rifampicin for lung targeting, J. Microencapsul. 12 (1995) 401–407. [11] S. Nakhare, S.P. Vyas, Multiple emulsion based systems for prolonged delivery of rifampicin: in vitro and in vivo characterization, Die Pharmazie 52 (1997) 224–226.

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