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Chitosan microspheres as an alveolar macrophage delivery system of ofloxacin via pulmonary inhalation
International Journal of Pharmaceutics 441 (2013) 562–569
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Chitosan microspheres as an alveolar macrophage delivery system of ofloxacin via pulmonary inhalation Ju-Hwan Park a , Hyo-Eon Jin a , Dae-Duk Kim a , Suk-Jae Chung a , Won-Sik Shim b,∗∗ , Chang-Koo Shim a,∗ a b
National Research Laboratory of Transporters Targeted Drug Design, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea College of Pharmacy, Gachon University, Hambakmoeiro 191, Yeonsu-gu, Incheon 406-799, Republic of Korea
a r t i c l e
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Article history: Received 1 September 2012 Received in revised form 29 October 2012 Accepted 30 October 2012 Available online 8 November 2012 Keywords: Ofloxacin Chitosan Microspheres Delivery Alveolar macrophage Inhalation
a b s t r a c t Because Mycobacterium tuberculosis, which causes tuberculosis, survives mainly in the alveolar macrophages, the remedial efficiency of anti-tuberculosis drugs such as ofloxacin may be improved by their direct delivery to the lungs via pulmonary inhalation. For this purpose, ofloxacin-loaded, glutaraldehyde-crosslinked chitosan microspheres (OCMs) were prepared using a water-in-oil emulsification method. The particle size of the OCMs was around 1–6 m, and the content of ofloxacin was 27% (w/w). A twin-stage impinger (TSI) study revealed that the device-removal efficiency of the drug from the capsule and the arrival rate of the drug to stage II of the apparatus were substantially improved for OCMs compared to ofloxacin itself (i.e., 81 vs. 98% and 13 vs. 45%, respectively). Also, the in vitro uptake of ofloxacin from the OCMs to alveolar macrophages (NR8383) was substantially accelerated: the cellular ofloxacin concentrations at 4 and 24 h after the application were >3.5-fold greater than those for free ofloxacin. The above results indicate that pulmonary inhalation of OCMs might improve the delivery efficiency of ofloxacin to the alveolar macrophages, thereby shortening the length of time that is required to cure tuberculosis with the drug—usually at least 6 months when administered orally. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Tuberculosis (TB) is a serious infectious disease worldwide that is caused by Mycobacterium tuberculosis. The important thing is that M. tuberculosis can survive in alveolar macrophages for extended periods of time by preventing the fusion of phagosomes and lysosomes—a bactericidal mechanism of macrophages (Bermudez, 1994). M. tuberculosis causes granuloma and pneumorrhagia in the lung, and necrosis of other organs by the infection through lymph or plasma. To treat TB, several anti-TB drugs such as ethambutol, isoniazid and rifampinusually are administered orally. Although these drugs demonstrate an adequate antibiotic effect against M. tuberculosis, they cannot sufficiently reach the lung and alveolar macrophages via oral administration (Vyas et al., 2004). For that reason, the oral administration of the drugs should be continued for at least 6 months to 2 years to cure TB (Vilarica et al., 2010). In addition to inconvenience and irritability, the administration of drugs for such long periods of time can provoke adverse effects. Also, problems with tolerance or resistance by M. tuberculosis to the administered drugs may be induced. Indeed, multidrug
∗ Corresponding author. Tel.: +82 2 880 7873; fax: +82 2 888 5969. ∗∗ Corresponding author. Tel.: +82 32 899 6060; fax: +82 32 899 6061. E-mail addresses: [email protected] (W.-S. Shim), [email protected] (C.-K. Shim). 0378-5173/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2012.10.044
resistant tuberculosis (MDR-TB) strains of bacteria that are resistant to at least two anti-TB drugs have caused serious problems in efforts to control TB (Tomioka, 2000). Therefore, it seems desirable to shorten the period of drug administration to cure TB by elevating the delivery efficiency of relevant drugs by any means. Coincidentally, inhalation of drugs via the respiratory route has been a frequent approach for delivery. Therefore, pulmonary inhalation is expected to shorten the drug treatment period for a regimen that can cure TB, if an efficient delivery of the drugs to the alveolar macrophages can be achieved (Vyas et al., 2004). The pulmonary delivery of drugs would be achievable by the inhalation of appropriately sized microspheres that contain relevant drugs, because the microspheres are likely to be actively phagocytosed by the alveolar macrophages, enhancing the influx of the drugs into the macrophages (Champion et al., 2007). In the present study, the development of an alveolar macrophage delivery system for ofloxacin was the aim. In order to develop the system, ofloxacin-loaded, glutaraldehyde-crosslinked chitosan microspheres (OCMs) were prepared using a water-inoil emulsification method. There have been various attempts to encapsulate anti-tuberculosis drugs to micro-sized formulation for pulmonary delivery, and poly-lactic-co-glycolic acid (PLGA) represents the polymers that have been used to encapsulate the drugs (Muttil et al., 2009). For example, rifampicin-loaded PLGA microspheres enhanced the delivery of rifampicin to alveolar macrophages compared to free rifampicin (Hirota et al., 2010).
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PLGA is a biocompatible polymer that is useful in sustaining the release of entrapped drugs in vivo (Gupta et al., 2010). However, the problem with this polymer is that because it is hydrophobic, it is difficult to load a sufficient amount of hydrophilic drugs, such as fluoroquinolones, into PLGA particles. Furthermore, the degradation rate of the polymer—in other words, the drug release rate—is difficult to control, which can frequently provoke drug release problems for PLGA particles (Meenach et al., 2012). However, chitosan, another biocompatible polymer, is hydrophilic and soluble in acidic solvents, and thus it is easy to encapsulate hydrophilic drugs. In addition, due to its mucoadhesive properties, chitosan formulations are likely to adhere to mucous membranes (El-Shabouri, 2002). In fact, the clearance of inhaled chitosan-coated PLGA nanospheres in the lung tissue was retarded because of the enhanced mucoadhesion of the nanospheres by the chitosan coating (Yamamoto et al., 2005). Also, chitosan particles interact with the mannose receptors of macrophages, which results in the phagocytosis of the particles in macrophages followed by the degradation of lysozymes and N-acetyl--d-glucosaminidase in phagosomes (Bianco et al., 2000; Shibata et al., 1997). Because of these strong points, chitosan has attracted the attention of many researchers as a polymer that can encapsulate hydrophilic anti-tuberculosis drugs in the development of alveolar drug delivery systems. Thus, in the present study, ofloxacin-loaded, glutaraldehydecrosslinked chitosan microspheres (OCMs) were prepared, and their potential to deliver ofloxacin directly to alveolar macrophages via the respiratory route was examined. Ofloxacin, afluoroquinolone antibiotic, was selected because it demonstrates superior antibiotic activity against various M. tuberculosis strains, including MDR-TB (Berning et al., 1995; Yew et al., 2000). Indeed, ofloxacin has been used to treat tuberculosis patients who have a resistance to other anti-tuberculosis drugs (Ziganshina and Squire, 2008). Also, the drug is effective against other respiratory pathogens, such as Haemophilus influenzae and Streptococcus pneumoniae (Plouffe et al., 1996), and has shown an effective response to acute exacerbations of chronic bronchitis (T’Jonck and Willems, 1993). 2. Materials and methods 2.1. Materials Ofloxacin, medium molecular weight chitosan (deacetylation ratio of 75–85%), glutaraldehyde, antipyrine, phosphate buffered saline (PBS) and Percoll solution were purchased from Sigma–Aldrich (St. Louis, MO, USA) and Span 80, acetic acid, hydrochloric acid, sodium hydroxide and dichloromethane were obtained from Daejung Chemical (Gyonggi-do, Korea). Paraffin liquid and diethyl ether were purchased from Samchun Chemical (Gyonggi-do, Korea). Fetal bovine serum (FBS), penicillin (100 units/mL) and streptomycin (100 g/mL) were from Welgene Inc., Korea. 2.2. Preparation of ofloxacin-loaded, glutaraldehyde-crosslinked chitosan microspheres (OCMs) OCMs were prepared according to a modified water-in-oil emulsification method (Menon et al., 2010), as shown in Fig. 1. First, 240 mg of chitosan was dissolved in 24 mL of 1% (w/v) acetic acid (pH 2.72), and 240 mg of ofloxacin was dissolved in the chitosan solution with shaking and sonication. The aqueous phasewas centrifuged at 1000 rpm for 10 min to remove undissolved chitosan debris. The supernatant phase was emulsified as follows in a mixed oil phase, which was composed of 20 mL of dichloromethane and
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Fig. 1. Preparation process forofloxacin-loaded, glutaraldehyde-crosslinked chitosan microspheres (OCMs).
20 mL of liquid paraffin, containing Span 80 (1%, v/v, in the mixture, as an emulsifier) and lecithin (0.1%, v/v, in the mixture, as a deaggregation agent) (Menon et al., 2010): 20 mL of the aqueous phase (i.e., ofloxacin–chitosan solution) was added dropwise to 40 mL of the mixed-oil phase at the rate of 2 mL/min under continuous homogenization (IKA-Ultra-Turrax T25 basic, IKA-Labortechnik, Germany) and at a speed of 13,500 rpm. The water-in-oil emulsion thus formed was further homogenized for 10 min under the dropwise addition of 2 mL of glutaraldehyde solution (5%, v/v). The emulsion was then homogenized for another 10 min, transferred to 20 mL of pre-heated liquid paraffin, and heated at 170 ◦ C with stirring for 1 h. The heating was to remove dichloromethane and aqueous solvent from the emulsion by evaporation. The remaining oil phase was cooled to room temperature, and centrifuged at 2000 rpm for 10 min. That speed (2000 rpm) was found to be appropriate to separate ofloxacin debris and nano-sized particles from the paraffin-suspended OCMs. The resultant microsphere pellet was dispersed in fresh liquid paraffin and centrifuged again at 2000 rpm for 10 min to remove non-encapsulated ofloxacin debris. The obtained microsphere pellets were washed with diethyl ether three times to remove residual paraffin, and dried overnight in an oven at 50 ◦ C. Most of the added ofloxacin (85%) was recovered from the final product, suggesting insignificant degradation of the drug during the preparation of OCMs, if any. Besides, chitosan seemed to be stable during the preparation of OCMs because it was reported to be degraded at temperatures over 250 ◦ C (Sakurai et al., 2000). 2.3. Particle size analysis The OCMs were suspended in DDW and sonicated for 30 s to uniformly disperse the particles, and their particle size was measured
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five times using a particle size analyzer (ELS-Z, Otsuka Electronics, Hirakata, Japan). The size of the particles was analyzed at 90◦ light scattering angle and with the intensity around 15,000 (Doh et al., 2012). 2.4. Determination of ofloxacin content in OCMs An HPLC method (Hwang et al., 2008) was used for the analysis of ofloxacin. The OCMs (5 mg) were dispersed in 1 mL of 0.1N HCl. The dispersion was sonicated for 30 s, vortexed for 4 h, and centrifuged (13,200 rpm, 5 min). The upper layer solution was filtered with a syringe filter (pore size, 0.2 m), and 50 L aliquots of the filtrate were injected to an HPLC system composed of a 717 autosampler, a 515 pump, a HPLC 2487 dual absorbance detector (all from Waters, Milford, MA, USA), and a Capcell Pak C18 (5 m, 4.6 mm × 50 mm) column (Shiseido Fine Chemicals, Tokyo, Japan). In this system, a mixture of 20 mM potassium phosphate buffer (pH 2.4) and acetonitrile (20:80, v/v) was used as a mobile phase, the flow rate of the mobile phase was adjusted to 0.8 mL/min, and the absorbance wavelength was set to 294 nm. 2.5. Scanning electron microscopy (SEM) The OCMs were platinum-coated with sputter coater (BalTec/SCD005, Switzerland), and the morphology was examined by SEM (SUPRA 55VP, Carl Zeiss, Germany). 2.6. X-ray diffraction (XRD) study An XRD analysis was conducted for OCMs, free ofloxacin, chitosan, and a 1:1 physical mixture of these two substances in order to compare their crystal structures. 2.7. Morphology of OCMs in the cell culture medium In order to examine whether the morphology of OCMs is maintained in the lung following pulmonary inhalation, OCMs were appropriately dispersed in a cell culture medium, i.e., Ham’s F12K medium containing 15% fetal bovine serum (FBS), penicillin (100 units/mL) and streptomycin (100 g/mL), and the morphology was observed as a function of time using an inverted microscope (CKX31, Olympus, Japan) at 200× magnification. 2.8. In vitro uptake of ofloxacin from OCMs by alveolar macrophages NR8383 cells (rat alveolar macrophage cells, ATCC, Manassas, VA, USA) were suspended at a concentration of 1 × 106 cell/mL in the cell culture medium. The concentration of FBS in the medium was lowered to 4% in order to keep the number of cells constant during the uptake test (Hirota et al., 2010). We transferred 1 mL each of the cell suspensions to a 12-well plate (Costar, Corning, NY), which was incubated at 37 ◦ C and 5% CO2 in a CO2 incubator. After the cells had sunk to the bottom of the plate (approximately 12 h after the incubation start), either a suspension of OCMs or free ofloxacin powder (all 1 mg/mL as ofloxacin, 100 L each) was applied onto the wells of the plate (n = 6 for each suspension at each sampling time point). The suspensions were prepared by dispersing the OCMs and free ofloxacin powder, respectively, in the incubation medium under sonication for 10 s. Then the cells were incubated at 37 ◦ C and 5% CO2 in the CO2 incubator for 0, 2, 4, 24, and 48 h. At given time points, 500 L of trypsin EDTA (0.25%, w/v) was applied to the well sat room temperature, and the cells were detached from the plate at 5 min
after the application via careful pipetting. The collected cell suspensions were then centrifuged at 9000 × g for 5 min, and the upper solution layers were removed via careful pipetting. The resultant cell pellets were re-dispersed in 500 L of the fresh culture medium. In order to separate macrophages and macrophage-adsorbed microspheres, a 500 L aliquot of the re-dispersed suspension was slowly layered onto 500 L of 70% Percoll solution and centrifuged at 9000 × g for 5 min. The macrophage cells, which existed as a layer in the interface between the culture medium and the Percoll solution, were collected by pipetting and washed twice with phosphate buffered saline (PBS, pH 7.4). The PBS was then removed by centrifugation, and the remaining cell pellets were dissolved in 250 L of 0.2N NaOH. The concentration of ofloxacin in the NaOH was quantified as follows: 150 L aliquot of the NaOH solution was mixed with 37.5 L of 0.8 N HCl and 375 L of acetonitrile that contained antipyrin (an internal standard, 3 g/mL) for 3 min, and the mixed solution was centrifuged at 13,200 rpm for 3 min. A 500 L aliquot of the supernatant was evaporated at 40 ◦ C under nitrogen gas, and the remaining residue was reconstituted with 200 L of the mobile phase and a mixture of 20 mM potassium phosphate buffer (pH 2.4) and acetonitrile (20:80, v/v), followed by analysis using the above-mentioned HPLC method (Hwang et al., 2008). The amount of ofloxacinin the macrophages was normalized by the protein content of macrophages, which was quantified using the BCA method (Stoscheck, 1990). 2.9. Mechanism of ofloxacin uptake by the macrophages from OCMs The uptake of OCMs by macrophages is often mediated via the phagocytosis mechanism. In the present study, therefore, the involvement of phagocytosis in the macrophage uptake of ofloxacin from OCMs was tested. Because phagocytosis is accelerated in the presence of ATP (Leeper-Woodford and Mills, 1992), and inhibited in the absence of ATP (Holtzman, 1989), the effect of ATP on the uptake of ofloxacin in NR8383 cells was examined. Basically, uptake experiments were conducted in accordance with the above-mentioned process, if not specifically described. The ATP of NR8383 cells was depleted by exposing the cells to 50 mM 2-deoxyglucose and 10 mM sodium azide solution (37 ◦ C) for 1 h (Bhattacharjee et al., 2011). 2-Deoxyglucose and sodium azide were used as a glycolysis inhibiting agent and a mitochondrial electron transfer-blocking agent, respectively (Dawson et al., 1993). Then, either a suspension of OCMs or free ofloxacin powder was applied to the cells, which were incubated for 2 h, collected, and assayed for the entrapped amount of ofloxacin. 2.10. Twin stage impinger (TSI) study The delivery of OCM microspheres or free ofloxacin powder to the lung was evaluated by measuring the device-removal efficiency (DRE) and aerosolization efficiency (AE) using the twin stage impinge (TSI) apparatus (Copley Scientific, Nottingham, UK): the amount of ofloxacin emitted from the device (E)/ofloxacin dose (D) for DRE, and the amount of ofloxacin reaching Phase II as fine particles (F)/ofloxacin dose (D) for AE, respectively. The OCMs and ofloxacin powder were respectively blended with varied amounts of lactose (i.e., 1:0, 1:1, 1:2, 1:3, and 1:5) using a vortex mixer, because lactose is often used to control the flow ability of the particles in commercial inhalants. For that purpose, ␣-lactose was sieved using standard sieves (KP VIII, Chunggae Industrial Co., Seoul, Korea), and particle sizes that ranged between #170 and #230 (63–90 m) were used for the blending (Hwang et al., 2008; Zeng et al., 1998). The blended mixtures (10 mg) were
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encapsulated in gelatin capsules (size no. 3), and the capsule was put in the dosage chamber of a Handihaler® . The Handihaler® was inserted into a mouthpiece that had been connected to a TSI apparatus. When assembling the TSI apparatus, two collecting chambers were filled with PBS (7 mL in stage I and 30 mL in stage II). Before the aspiration of the blend, an HCP5 pump (Copley Scientific, Nottingham, UK) was connected to the TSI apparatus and turned on. After stabilization of the vacuum conditions for 10 s, the flow rate was set to 60 mL/min by using a calibrated flowmeter (DFM 2, Copley Scientific, Nottingham, UK). Then the encapsulated blend was aspirated by breaking the capsule with the Handihaler® . After the aspiration, the TSI apparatus was disassembled, and each chamber and the remaining capsule were washed with PBS (5 mL for the capsule, 10 mL for the throat, 10 mL for the stage I, and 40 mL for the stage II). The PBS washes were collected and the concentration of ofloxacin in the collected PBS was analyzed by the above-mentioned HPLC method. The amount of ofloxacin that was recovered from all parts of the TSI apparatus and the capsules was regarded as the loaded dose (D), and the ofloxacin amount recovered from all parts of the TSI apparatus, with the exception of the capsules, was regarded as the emitted dose (E) in the calculation of DRE. The amount of ofloxacin recovered from stage II was regarded as the amount of ofloxacin that had reached Phase II as fine particles (F) in the calculation of AE (Snell and Ganderton, 1999). 2.11. Statistical data analysis All of the data were collected from at least three independent experiments, and the data were expressed as the mean ± standard deviation (SD). Comparisons of two different groups were performed using an unpaired Student’s t-test.
Fig. 2. Scanning electron microscope (SEM) images (5000×) of OCMs (A) and ofloxacin powder (B).
3. Results 3.1. Physicochemical properties of OCMs Brown-colored ofloxacin-loaded, glutaraldehyde-crosslinked chitosan microspheres (OCMs) with a particle size of 4.44 ± 0.65 m (n = 5) and anofloxacin content of 27.41 ± 0.74% (w/w, n = 3) were prepared according to the above-described water-in-oil emulsification method (Fig. 1). Scanning electron microscopy (SEM) revealed that the OCM particles were spherical and distributed in sizes that ranged from 1 to 6 m (Fig. 2A). However, the free ofloxacin powder was pointed and rod-shaped in sizes that ranged from 2 to 12 m (Fig. 2B). X-ray diffraction (XRD) analysis demonstrated that the crystal structure of the OCMs was distinct from that of the free ofloxacin powder and the ofloxacin–chitosan physical mixture (Fig. 3): the ofloxacin-specific peaks remained apparent for the physical mixture, but disappeared for the OCMs, which was consistent with the formation of a eutectic mixture of ofloxacin and chitosan in an OCM (Menon et al., 2010). The spherical shape and uniform scattering of OCM particles was maintained for a substantial period of time when the particles were dispersed in the cell culture medium (Fig. 4). No significant degradation or size change could be detected even at 6 h after the dispersion of OCMs. However, when the free ofloxacin powder was dispersed in the medium, the particles disappeared immediately due to the fast dissolution of ofloxacin powder in the medium. That result suggests that the particulate characteristics of OCMs can be maintained in the lung for a fairly long period of time following pulmonary inhalation of the microspheres, thereby enabling the phagocytosis of the microspheres by alveolar macrophages.
3.2. In vitro uptake of ofloxacin by alveolar macrophages from OCMs In vitro uptake of ofloxacin by alveolar macrophages was measured for OCMs and free ofloxacin powders. The viability of the macrophages, as checked by MTT assay, could be maintained at more than 85% for 5 days under the conditions of the uptake study. This result (Fig. 5) shows that the uptake of ofloxacin from the OCMs was substantially greater compared to that from the ofloxacin
Fig. 3. X-ray diffractograms of ofloxacin (A), chitosan (B), physical mixture of ofloxacin and chitosan (C), and OCMs (D).
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Fig. 4. Microscopicmorphologies (200×) of OCMs at 0.5, 2, 4 and 6 h after dispersion in Ham’s F-12K medium containing 15% fetal bovine serum.
powder. For example, the amount of ofloxacin entrapped in the cell at 4 h was only 0.247 ± 0.100 g/mg of cell protein for the free powder, but the amount increased to 0.950 ± 0.129 g/mg for the OCMs, which was a 3.8-fold increase. This tendency was maintained for 24 h. As a result, a 3.6-fold greater area under the drug amount per cell-time curve (AUA) was obtained for the OCMs, compared with that of the ofloxacin powder, suggesting superiority of OCMs in terms of the in vitro delivery of ofloxacin to the macrophages.
Fig. 5. In vitro accumulation of ofloxacin by rat alveolar macrophage (NR8383) cells from ofloxacin powder (control, ) and OCMs () for 48 h. The cell density was 1 × 106 cells/mL, and the applied drug amount was 100 g. The data are expressed as the mean ± SD for 6 experiments. *Significantly (p < 0.05) increased compared to the control.
The cellular accumulation of ofloxacin from the OCMs was dramatically decreased in the absence of ATP in the cell culture medium (Fig. 6), which was induced by the addition of 2-deoxyglucose and sodium azide to the medium, indicating that the uptake process is likely to be ATP-dependent. An active transport mechanism, probably phagocytosis, appears to be operative in the uptake of ofloxacin from the OCMs. The ATP-dependent
Fig. 6. In vitro uptake of ofloxacin from the ofloxacin powder and OCMs in NR8383 alveolar macrophage cells in the presence (white column) and absence (black column) of ATP. The depletion of ATP was achieved by the treatment of the macrophages with 50 mM 2-deoxyglucose and 10 mM sodium azide. Each column represents the mean ± SD of four experiments. *Significantly (p < 0.01) decreased by the ATPdepletion.
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Fig. 7. Effect of ATP-depletion on the microscopic photographs (200×) of macrophages at 2 h following the application of OCMs and ofloxacin.
decrease was not observed for free ofloxacin powder. Immediate dissolution of ofloxacin from the powder appears to be responsible for the ATP-independent uptake of free ofloxacin. The inverted microscopy demonstrated that the morphology of alveolar macrophages was not changed by the addition of ofloxacin powder, regardless of the presence or absence of ATP in the medium (Fig. 7). On the other hand, the morphology following the application of OCMs differed depending on the presence of ATP: in the presence of ATP (control group), most of the OCMs were found to exist inside the macrophages, while most of the OCMs were found outside the macrophages in the absence of ATP. Therefore, phagocytosis, an ATP-dependent active transport mechanism, appeared to be involved in the accelerated uptake of ofloxacin from the OCMs. 3.3. Delivery efficiency to deep sites in the lung: a TSI study The effect of lactose blending on the device-removal efficiency (DRE) and aerosolization efficiency (AE) of OCMs and ofloxacin powder was measured using a twin stage impinger (TSI). As shown in Fig. 8A, the DRE of ofloxacin powder (81%) was increased to more than 90% by lactose blending, regardless of the blending ratio (Fig. 8A). The DRE of OCMs was more than 90% regardless of the lactose-blending with a maximum value of 98% without lactose blending (Fig. 8A). That result indicates that OCMs themselves are superior to the free ofloxacin powder in terms of DRE. On the other hand, the two formulations differed in terms of AE (Fig. 8B)— i.e., the AE of the powder was around 13%, regardless of the lactose blending, while the AE of OCMs was 45%. It is worth noting that the value of 45% for lactose-free OCMs decreased gradually as the lactose ratio in the blend was increased. This indicates that a much higher AE is expected from the OCMs compared with the ofloxacin powder. Because the AE of preparations indicates the efficiency of the drug to reach stage II of the TSI apparatus, a more efficient delivery of ofloxacin to deep sites of the lung, where alveolar macrophages are located, might be plausible by applying lactose-free OCMs instead of the lactose-blended ofloxacin powder.
Fig. 8. Effect of lactose blending on the device removal efficiency (DRE, A) and aerosolization efficiency (AE, B) of OCMs (gray column) and ofloxacin powder (white column) in a twin stage impinger (TSI). Each column indicates the mean ± SD of three experiments.
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4. Discussion Several inhalation formulations including a sugar microparticle formulation of isoniazid (Sawatdee et al., 2006), burst-releasing proliposome formulation of isoniazid (Rojanarat et al., 2011), and spray-dried poly lactic acid microparticles of ofloxacin (Palazzo et al., in press) have been reported to possess potential as pulmonary delivery systems of hydrophilic antituberculosis drugs. The precise evaluation of antituberculosis formulations should be performed based on the quantitative estimation of macrophageuptake of loaded drugs, at least in vitro. However, no such information was available from those studies for hydrophilic antituberculosis drugs. Microspheres are often prepared using PLGA as a polymer. In the present study, however, chitosan was selected as the polymer that was used to prepare OCMs. This was because the ofloxacin content in the PLGA microspheres was very low (<5%, w/w) in our preliminary studies, when they were prepared according to the water-in-oil-in-water emulsion method (Yang et al., 2000). The content could be increased to as high as 27.41% in the present study, far exceeding the content of ofloxacin in PLGA microspheres (<15%; Abazinge et al., 2000). The hydrophilic characteristics of both chitosan and ofloxacin might be associated with the increase. Besides, electrostatic interaction between protonated chitosan and deprotonated ofloxacin (Singh and Dutta, 2010) might have potentiated the increase. The OCMs prepared in the present study were dispersed uniformly in the cell culture medium, and their spherical shape was well maintained for a fairly long period of time (Fig. 4). However, when the glutaraldehyde-crosslinking was not conducted, such physicochemical properties could not be achieved. Therefore, the crosslinking seemed essential to prepare sufficiently rigid spherical microspheres. Microsphere particles would be taken up by the alveolar macrophages following pulmonary inhalation, probably via phagocytosis, which was followed by hydrolytic degradation promoted by the enzymes in the phagosomes (Bianco et al., 2000). Owing to the characteristics of the OCMs, the uptake of ofloxacin from the OCMs to alveolar macrophages (NR8383 cells) was substantially (3.6-fold) increased for the OCMs, compared with that of the free ofloxacin powder (Fig. 5). The uptake from the OCMs was ATP-dependent (Figs. 6 and 7), which was consistent with the active phagocytosis of solid particles for the OCMs. The substantial increase in the macrophage uptake may be explainable in association with the immunological characteristics of chitosan. N-acetyl glucosamine residues in chitosan are known to cause immune stimulation of macrophages (Peluso et al., 1994), accelerating the phagocytosis of chitosan microspheres (Ueno et al., 2001). The electrostatic interaction between positive charge of chitosan and negative charge of the cell membrane might have further enhanced the phagocytosis, as was previously reported for positively charged particles (Mutsaers and Papadimitriou, 1988). The degree of drug delivery to the deep portions of the lung could be estimated by measuring the device-removal efficiency (DRE) and aeorosolization efficiency (AE) of the particles using the twin stage impinger (TSI) (Fig. 8A). In the case of ofloxacin powder, most of the drug was emitted well from the capsule (i.e., DRE > 80%), and the DRE value was increased slightly by the addition of a small amount of lactose to the blend, indicating that lactose is useful to discharge ofloxacin powder from the capsule in the TSI apparatus. The DRE of lactose-free OCMs was higher than that of ofloxacin powder, and was decreased slightly by the lactose blending. However, the AE of OCMs (45%) was much higher than that of ofloxacin powder (13%) (Fig. 8B). The AE of ofloxacin powder was not improved by lactose blending. Considering that the particle sizes of OCMs and ofloxacin powder are similar, only differing in morphology (Fig. 2), the spherical shape of OCMs appears to be
advantageous compared with the rod shape of ofloxacin powder in terms of aeorosolization. Both the AE and DRE values were highest for lactose-free OCMs, which suggested that these particles can be inhaled more efficiently in order to reach deep inside the lung. More information regarding the tendency to deposit in deep lungs (alveoli) would be obtainable from the aerodynamic diameter (particle effective density) of the particles, which could not be measured in the present study. Despite the superiority of OCMs over ofloxacin powder in terms of in vitro macrophage uptake of ofloxacin and in vitro degree of drug delivery to the deep lung (i.e., phase II in TSI study), the conclusion on the potential of OCMs as an alveolar macrophage-targeted delivery system should await in vivo experimental verification, which will require the collection of substantial amounts of alveolar macrophages from rats. 5. Conclusion Ofloxacin-loaded, glutaraldehyde-crosslinked chitosan microspheres (OCMs) were prepared using a water-in-oil emulsification method, and their potential to deliver ofloxacin directly to alveolar macrophages via the respiratory route was examined. In comparisons with ofloxacin powder, the OCM particles demonstrated better aerosolization efficiency (AE) and enhanced ofloxacin uptake by the alveolar macrophages in vitro. These results suggest the potential of OCMs as an efficient delivery system of ofloxacin to alveolar macrophages where tuberculosis-causing M. tuberculosis mainly survives. However, the extrapolation of this conclusion to other antituberculosis drugs, or conclusions concerning the possible advantages of microsphere inhalants over conventional forms of oral dosage in the treatment of tuberculosis, should await careful in vivo verification. Acknowledgements This study was supported by the Korea Science and Engineering Foundation (KOSEF) through the National Research Laboratory program, which was funded by the Ministry of Science and Technology (no. ROA-2006-000-10190-0). References Abazinge, M., Jackson, T., Yang, Q., Owusu-Ababio, G., 2000. Comparison of in vitro and in vivo release characteristics of sustained release ofloxacin microspheres. Drug Deliv. 7, 77–81. Bermudez, L.E., 1994. Use of liposome preparation to treat mycobacterial infections. Immunobiology 191, 578–583. Berning, S.E., Madsen, L., Iseman, M.D., Peloquin, C.A., 1995. Long-term safety of ofloxacin and ciprofloxacin in the treatment of mycobacterial infections. Am. J. Respir. Crit. Care Med. 151, 2006–2009. Bhattacharjee, S., Ershov, D., Gucht, J.V., Alink, G.M., Rietjens, I.M., Zuilhof, H., Marcelis, A.T., 2011. Surface charge-specific cytotoxicity and cellular uptake of tri-block copolymer nanoparticles. Nanotoxicology, http://dx.doi.org/10.3109/17435390.2011.633714. Bianco, I.D., Balsinde, J., Beltramo, D.M., Castagna, L.F., Landa, C.A., Dennis, E.A., 2000. Chitosan-induced phospholipase A2 activation and arachidonic acid mobilization in P388D1 macrophages. FEBS Lett. 466, 292–294. Champion, J.A., Katare, Y.K., Mitragotri, S., 2007. Particle shape: a new design parameter for micro-and nanoscale drug delivery carriers. J. Control. Release 121, 3–9. Dawson, T.L., Gores, G.J., Nieminen, A.L., Herman, B., Lemasters, J.J., 1993. Mitochondria as a source of reactive oxygen species during reductive stress in rat hepatocytes. Am. J. Physiol. Cell Physiol. 264, C961–C967. Doh, H.J., Jung, Y., Balakrishnan, P., Cho, H.J., Kim, D.D., 2012. A novel lipid nanoemulsion system for improved permeation of granisetron. Colloids Surf. B Biointerfaces 101C, 475–480. El-Shabouri, M.H., 2002. Positively charged nanoparticles for improving the oral bioavailability of cyclosporin-A. Int. J. Pharm. 249, 101–108. Gupta, H., Aqil, M., Khar, R.K., Ali, A., Bhatnagar, A., Mittal, G., 2010. Sparfloxacinloaded PLGA nanoparticles for sustained ocular drug delivery. Nanomedicine 6, 324–333. Hirota, K., Hasegawa, T., Nakajima, T., Inagawa, H., Kohchi, C., Soma, G., Makino, K., Terada, H., 2010. Delivery of rifampicin-PLGA microspheres into alveolar
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Chitosan microspheres as an alveolar macrophage delivery system of ofloxacin via pulmonary inhalation Ju-Hwan Park a , Hyo-Eon Jin a , Dae-Duk Kim a , Suk-Jae Chung a , Won-Sik Shim b,∗∗ , Chang-Koo Shim a,∗ a b
National Research Laboratory of Transporters Targeted Drug Design, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea College of Pharmacy, Gachon University, Hambakmoeiro 191, Yeonsu-gu, Incheon 406-799, Republic of Korea
a r t i c l e
i n f o
Article history: Received 1 September 2012 Received in revised form 29 October 2012 Accepted 30 October 2012 Available online 8 November 2012 Keywords: Ofloxacin Chitosan Microspheres Delivery Alveolar macrophage Inhalation
a b s t r a c t Because Mycobacterium tuberculosis, which causes tuberculosis, survives mainly in the alveolar macrophages, the remedial efficiency of anti-tuberculosis drugs such as ofloxacin may be improved by their direct delivery to the lungs via pulmonary inhalation. For this purpose, ofloxacin-loaded, glutaraldehyde-crosslinked chitosan microspheres (OCMs) were prepared using a water-in-oil emulsification method. The particle size of the OCMs was around 1–6 m, and the content of ofloxacin was 27% (w/w). A twin-stage impinger (TSI) study revealed that the device-removal efficiency of the drug from the capsule and the arrival rate of the drug to stage II of the apparatus were substantially improved for OCMs compared to ofloxacin itself (i.e., 81 vs. 98% and 13 vs. 45%, respectively). Also, the in vitro uptake of ofloxacin from the OCMs to alveolar macrophages (NR8383) was substantially accelerated: the cellular ofloxacin concentrations at 4 and 24 h after the application were >3.5-fold greater than those for free ofloxacin. The above results indicate that pulmonary inhalation of OCMs might improve the delivery efficiency of ofloxacin to the alveolar macrophages, thereby shortening the length of time that is required to cure tuberculosis with the drug—usually at least 6 months when administered orally. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Tuberculosis (TB) is a serious infectious disease worldwide that is caused by Mycobacterium tuberculosis. The important thing is that M. tuberculosis can survive in alveolar macrophages for extended periods of time by preventing the fusion of phagosomes and lysosomes—a bactericidal mechanism of macrophages (Bermudez, 1994). M. tuberculosis causes granuloma and pneumorrhagia in the lung, and necrosis of other organs by the infection through lymph or plasma. To treat TB, several anti-TB drugs such as ethambutol, isoniazid and rifampinusually are administered orally. Although these drugs demonstrate an adequate antibiotic effect against M. tuberculosis, they cannot sufficiently reach the lung and alveolar macrophages via oral administration (Vyas et al., 2004). For that reason, the oral administration of the drugs should be continued for at least 6 months to 2 years to cure TB (Vilarica et al., 2010). In addition to inconvenience and irritability, the administration of drugs for such long periods of time can provoke adverse effects. Also, problems with tolerance or resistance by M. tuberculosis to the administered drugs may be induced. Indeed, multidrug
∗ Corresponding author. Tel.: +82 2 880 7873; fax: +82 2 888 5969. ∗∗ Corresponding author. Tel.: +82 32 899 6060; fax: +82 32 899 6061. E-mail addresses: [email protected] (W.-S. Shim), [email protected] (C.-K. Shim). 0378-5173/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2012.10.044
resistant tuberculosis (MDR-TB) strains of bacteria that are resistant to at least two anti-TB drugs have caused serious problems in efforts to control TB (Tomioka, 2000). Therefore, it seems desirable to shorten the period of drug administration to cure TB by elevating the delivery efficiency of relevant drugs by any means. Coincidentally, inhalation of drugs via the respiratory route has been a frequent approach for delivery. Therefore, pulmonary inhalation is expected to shorten the drug treatment period for a regimen that can cure TB, if an efficient delivery of the drugs to the alveolar macrophages can be achieved (Vyas et al., 2004). The pulmonary delivery of drugs would be achievable by the inhalation of appropriately sized microspheres that contain relevant drugs, because the microspheres are likely to be actively phagocytosed by the alveolar macrophages, enhancing the influx of the drugs into the macrophages (Champion et al., 2007). In the present study, the development of an alveolar macrophage delivery system for ofloxacin was the aim. In order to develop the system, ofloxacin-loaded, glutaraldehyde-crosslinked chitosan microspheres (OCMs) were prepared using a water-inoil emulsification method. There have been various attempts to encapsulate anti-tuberculosis drugs to micro-sized formulation for pulmonary delivery, and poly-lactic-co-glycolic acid (PLGA) represents the polymers that have been used to encapsulate the drugs (Muttil et al., 2009). For example, rifampicin-loaded PLGA microspheres enhanced the delivery of rifampicin to alveolar macrophages compared to free rifampicin (Hirota et al., 2010).
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PLGA is a biocompatible polymer that is useful in sustaining the release of entrapped drugs in vivo (Gupta et al., 2010). However, the problem with this polymer is that because it is hydrophobic, it is difficult to load a sufficient amount of hydrophilic drugs, such as fluoroquinolones, into PLGA particles. Furthermore, the degradation rate of the polymer—in other words, the drug release rate—is difficult to control, which can frequently provoke drug release problems for PLGA particles (Meenach et al., 2012). However, chitosan, another biocompatible polymer, is hydrophilic and soluble in acidic solvents, and thus it is easy to encapsulate hydrophilic drugs. In addition, due to its mucoadhesive properties, chitosan formulations are likely to adhere to mucous membranes (El-Shabouri, 2002). In fact, the clearance of inhaled chitosan-coated PLGA nanospheres in the lung tissue was retarded because of the enhanced mucoadhesion of the nanospheres by the chitosan coating (Yamamoto et al., 2005). Also, chitosan particles interact with the mannose receptors of macrophages, which results in the phagocytosis of the particles in macrophages followed by the degradation of lysozymes and N-acetyl--d-glucosaminidase in phagosomes (Bianco et al., 2000; Shibata et al., 1997). Because of these strong points, chitosan has attracted the attention of many researchers as a polymer that can encapsulate hydrophilic anti-tuberculosis drugs in the development of alveolar drug delivery systems. Thus, in the present study, ofloxacin-loaded, glutaraldehydecrosslinked chitosan microspheres (OCMs) were prepared, and their potential to deliver ofloxacin directly to alveolar macrophages via the respiratory route was examined. Ofloxacin, afluoroquinolone antibiotic, was selected because it demonstrates superior antibiotic activity against various M. tuberculosis strains, including MDR-TB (Berning et al., 1995; Yew et al., 2000). Indeed, ofloxacin has been used to treat tuberculosis patients who have a resistance to other anti-tuberculosis drugs (Ziganshina and Squire, 2008). Also, the drug is effective against other respiratory pathogens, such as Haemophilus influenzae and Streptococcus pneumoniae (Plouffe et al., 1996), and has shown an effective response to acute exacerbations of chronic bronchitis (T’Jonck and Willems, 1993). 2. Materials and methods 2.1. Materials Ofloxacin, medium molecular weight chitosan (deacetylation ratio of 75–85%), glutaraldehyde, antipyrine, phosphate buffered saline (PBS) and Percoll solution were purchased from Sigma–Aldrich (St. Louis, MO, USA) and Span 80, acetic acid, hydrochloric acid, sodium hydroxide and dichloromethane were obtained from Daejung Chemical (Gyonggi-do, Korea). Paraffin liquid and diethyl ether were purchased from Samchun Chemical (Gyonggi-do, Korea). Fetal bovine serum (FBS), penicillin (100 units/mL) and streptomycin (100 g/mL) were from Welgene Inc., Korea. 2.2. Preparation of ofloxacin-loaded, glutaraldehyde-crosslinked chitosan microspheres (OCMs) OCMs were prepared according to a modified water-in-oil emulsification method (Menon et al., 2010), as shown in Fig. 1. First, 240 mg of chitosan was dissolved in 24 mL of 1% (w/v) acetic acid (pH 2.72), and 240 mg of ofloxacin was dissolved in the chitosan solution with shaking and sonication. The aqueous phasewas centrifuged at 1000 rpm for 10 min to remove undissolved chitosan debris. The supernatant phase was emulsified as follows in a mixed oil phase, which was composed of 20 mL of dichloromethane and
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Fig. 1. Preparation process forofloxacin-loaded, glutaraldehyde-crosslinked chitosan microspheres (OCMs).
20 mL of liquid paraffin, containing Span 80 (1%, v/v, in the mixture, as an emulsifier) and lecithin (0.1%, v/v, in the mixture, as a deaggregation agent) (Menon et al., 2010): 20 mL of the aqueous phase (i.e., ofloxacin–chitosan solution) was added dropwise to 40 mL of the mixed-oil phase at the rate of 2 mL/min under continuous homogenization (IKA-Ultra-Turrax T25 basic, IKA-Labortechnik, Germany) and at a speed of 13,500 rpm. The water-in-oil emulsion thus formed was further homogenized for 10 min under the dropwise addition of 2 mL of glutaraldehyde solution (5%, v/v). The emulsion was then homogenized for another 10 min, transferred to 20 mL of pre-heated liquid paraffin, and heated at 170 ◦ C with stirring for 1 h. The heating was to remove dichloromethane and aqueous solvent from the emulsion by evaporation. The remaining oil phase was cooled to room temperature, and centrifuged at 2000 rpm for 10 min. That speed (2000 rpm) was found to be appropriate to separate ofloxacin debris and nano-sized particles from the paraffin-suspended OCMs. The resultant microsphere pellet was dispersed in fresh liquid paraffin and centrifuged again at 2000 rpm for 10 min to remove non-encapsulated ofloxacin debris. The obtained microsphere pellets were washed with diethyl ether three times to remove residual paraffin, and dried overnight in an oven at 50 ◦ C. Most of the added ofloxacin (85%) was recovered from the final product, suggesting insignificant degradation of the drug during the preparation of OCMs, if any. Besides, chitosan seemed to be stable during the preparation of OCMs because it was reported to be degraded at temperatures over 250 ◦ C (Sakurai et al., 2000). 2.3. Particle size analysis The OCMs were suspended in DDW and sonicated for 30 s to uniformly disperse the particles, and their particle size was measured
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five times using a particle size analyzer (ELS-Z, Otsuka Electronics, Hirakata, Japan). The size of the particles was analyzed at 90◦ light scattering angle and with the intensity around 15,000 (Doh et al., 2012). 2.4. Determination of ofloxacin content in OCMs An HPLC method (Hwang et al., 2008) was used for the analysis of ofloxacin. The OCMs (5 mg) were dispersed in 1 mL of 0.1N HCl. The dispersion was sonicated for 30 s, vortexed for 4 h, and centrifuged (13,200 rpm, 5 min). The upper layer solution was filtered with a syringe filter (pore size, 0.2 m), and 50 L aliquots of the filtrate were injected to an HPLC system composed of a 717 autosampler, a 515 pump, a HPLC 2487 dual absorbance detector (all from Waters, Milford, MA, USA), and a Capcell Pak C18 (5 m, 4.6 mm × 50 mm) column (Shiseido Fine Chemicals, Tokyo, Japan). In this system, a mixture of 20 mM potassium phosphate buffer (pH 2.4) and acetonitrile (20:80, v/v) was used as a mobile phase, the flow rate of the mobile phase was adjusted to 0.8 mL/min, and the absorbance wavelength was set to 294 nm. 2.5. Scanning electron microscopy (SEM) The OCMs were platinum-coated with sputter coater (BalTec/SCD005, Switzerland), and the morphology was examined by SEM (SUPRA 55VP, Carl Zeiss, Germany). 2.6. X-ray diffraction (XRD) study An XRD analysis was conducted for OCMs, free ofloxacin, chitosan, and a 1:1 physical mixture of these two substances in order to compare their crystal structures. 2.7. Morphology of OCMs in the cell culture medium In order to examine whether the morphology of OCMs is maintained in the lung following pulmonary inhalation, OCMs were appropriately dispersed in a cell culture medium, i.e., Ham’s F12K medium containing 15% fetal bovine serum (FBS), penicillin (100 units/mL) and streptomycin (100 g/mL), and the morphology was observed as a function of time using an inverted microscope (CKX31, Olympus, Japan) at 200× magnification. 2.8. In vitro uptake of ofloxacin from OCMs by alveolar macrophages NR8383 cells (rat alveolar macrophage cells, ATCC, Manassas, VA, USA) were suspended at a concentration of 1 × 106 cell/mL in the cell culture medium. The concentration of FBS in the medium was lowered to 4% in order to keep the number of cells constant during the uptake test (Hirota et al., 2010). We transferred 1 mL each of the cell suspensions to a 12-well plate (Costar, Corning, NY), which was incubated at 37 ◦ C and 5% CO2 in a CO2 incubator. After the cells had sunk to the bottom of the plate (approximately 12 h after the incubation start), either a suspension of OCMs or free ofloxacin powder (all 1 mg/mL as ofloxacin, 100 L each) was applied onto the wells of the plate (n = 6 for each suspension at each sampling time point). The suspensions were prepared by dispersing the OCMs and free ofloxacin powder, respectively, in the incubation medium under sonication for 10 s. Then the cells were incubated at 37 ◦ C and 5% CO2 in the CO2 incubator for 0, 2, 4, 24, and 48 h. At given time points, 500 L of trypsin EDTA (0.25%, w/v) was applied to the well sat room temperature, and the cells were detached from the plate at 5 min
after the application via careful pipetting. The collected cell suspensions were then centrifuged at 9000 × g for 5 min, and the upper solution layers were removed via careful pipetting. The resultant cell pellets were re-dispersed in 500 L of the fresh culture medium. In order to separate macrophages and macrophage-adsorbed microspheres, a 500 L aliquot of the re-dispersed suspension was slowly layered onto 500 L of 70% Percoll solution and centrifuged at 9000 × g for 5 min. The macrophage cells, which existed as a layer in the interface between the culture medium and the Percoll solution, were collected by pipetting and washed twice with phosphate buffered saline (PBS, pH 7.4). The PBS was then removed by centrifugation, and the remaining cell pellets were dissolved in 250 L of 0.2N NaOH. The concentration of ofloxacin in the NaOH was quantified as follows: 150 L aliquot of the NaOH solution was mixed with 37.5 L of 0.8 N HCl and 375 L of acetonitrile that contained antipyrin (an internal standard, 3 g/mL) for 3 min, and the mixed solution was centrifuged at 13,200 rpm for 3 min. A 500 L aliquot of the supernatant was evaporated at 40 ◦ C under nitrogen gas, and the remaining residue was reconstituted with 200 L of the mobile phase and a mixture of 20 mM potassium phosphate buffer (pH 2.4) and acetonitrile (20:80, v/v), followed by analysis using the above-mentioned HPLC method (Hwang et al., 2008). The amount of ofloxacinin the macrophages was normalized by the protein content of macrophages, which was quantified using the BCA method (Stoscheck, 1990). 2.9. Mechanism of ofloxacin uptake by the macrophages from OCMs The uptake of OCMs by macrophages is often mediated via the phagocytosis mechanism. In the present study, therefore, the involvement of phagocytosis in the macrophage uptake of ofloxacin from OCMs was tested. Because phagocytosis is accelerated in the presence of ATP (Leeper-Woodford and Mills, 1992), and inhibited in the absence of ATP (Holtzman, 1989), the effect of ATP on the uptake of ofloxacin in NR8383 cells was examined. Basically, uptake experiments were conducted in accordance with the above-mentioned process, if not specifically described. The ATP of NR8383 cells was depleted by exposing the cells to 50 mM 2-deoxyglucose and 10 mM sodium azide solution (37 ◦ C) for 1 h (Bhattacharjee et al., 2011). 2-Deoxyglucose and sodium azide were used as a glycolysis inhibiting agent and a mitochondrial electron transfer-blocking agent, respectively (Dawson et al., 1993). Then, either a suspension of OCMs or free ofloxacin powder was applied to the cells, which were incubated for 2 h, collected, and assayed for the entrapped amount of ofloxacin. 2.10. Twin stage impinger (TSI) study The delivery of OCM microspheres or free ofloxacin powder to the lung was evaluated by measuring the device-removal efficiency (DRE) and aerosolization efficiency (AE) using the twin stage impinge (TSI) apparatus (Copley Scientific, Nottingham, UK): the amount of ofloxacin emitted from the device (E)/ofloxacin dose (D) for DRE, and the amount of ofloxacin reaching Phase II as fine particles (F)/ofloxacin dose (D) for AE, respectively. The OCMs and ofloxacin powder were respectively blended with varied amounts of lactose (i.e., 1:0, 1:1, 1:2, 1:3, and 1:5) using a vortex mixer, because lactose is often used to control the flow ability of the particles in commercial inhalants. For that purpose, ␣-lactose was sieved using standard sieves (KP VIII, Chunggae Industrial Co., Seoul, Korea), and particle sizes that ranged between #170 and #230 (63–90 m) were used for the blending (Hwang et al., 2008; Zeng et al., 1998). The blended mixtures (10 mg) were
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encapsulated in gelatin capsules (size no. 3), and the capsule was put in the dosage chamber of a Handihaler® . The Handihaler® was inserted into a mouthpiece that had been connected to a TSI apparatus. When assembling the TSI apparatus, two collecting chambers were filled with PBS (7 mL in stage I and 30 mL in stage II). Before the aspiration of the blend, an HCP5 pump (Copley Scientific, Nottingham, UK) was connected to the TSI apparatus and turned on. After stabilization of the vacuum conditions for 10 s, the flow rate was set to 60 mL/min by using a calibrated flowmeter (DFM 2, Copley Scientific, Nottingham, UK). Then the encapsulated blend was aspirated by breaking the capsule with the Handihaler® . After the aspiration, the TSI apparatus was disassembled, and each chamber and the remaining capsule were washed with PBS (5 mL for the capsule, 10 mL for the throat, 10 mL for the stage I, and 40 mL for the stage II). The PBS washes were collected and the concentration of ofloxacin in the collected PBS was analyzed by the above-mentioned HPLC method. The amount of ofloxacin that was recovered from all parts of the TSI apparatus and the capsules was regarded as the loaded dose (D), and the ofloxacin amount recovered from all parts of the TSI apparatus, with the exception of the capsules, was regarded as the emitted dose (E) in the calculation of DRE. The amount of ofloxacin recovered from stage II was regarded as the amount of ofloxacin that had reached Phase II as fine particles (F) in the calculation of AE (Snell and Ganderton, 1999). 2.11. Statistical data analysis All of the data were collected from at least three independent experiments, and the data were expressed as the mean ± standard deviation (SD). Comparisons of two different groups were performed using an unpaired Student’s t-test.
Fig. 2. Scanning electron microscope (SEM) images (5000×) of OCMs (A) and ofloxacin powder (B).
3. Results 3.1. Physicochemical properties of OCMs Brown-colored ofloxacin-loaded, glutaraldehyde-crosslinked chitosan microspheres (OCMs) with a particle size of 4.44 ± 0.65 m (n = 5) and anofloxacin content of 27.41 ± 0.74% (w/w, n = 3) were prepared according to the above-described water-in-oil emulsification method (Fig. 1). Scanning electron microscopy (SEM) revealed that the OCM particles were spherical and distributed in sizes that ranged from 1 to 6 m (Fig. 2A). However, the free ofloxacin powder was pointed and rod-shaped in sizes that ranged from 2 to 12 m (Fig. 2B). X-ray diffraction (XRD) analysis demonstrated that the crystal structure of the OCMs was distinct from that of the free ofloxacin powder and the ofloxacin–chitosan physical mixture (Fig. 3): the ofloxacin-specific peaks remained apparent for the physical mixture, but disappeared for the OCMs, which was consistent with the formation of a eutectic mixture of ofloxacin and chitosan in an OCM (Menon et al., 2010). The spherical shape and uniform scattering of OCM particles was maintained for a substantial period of time when the particles were dispersed in the cell culture medium (Fig. 4). No significant degradation or size change could be detected even at 6 h after the dispersion of OCMs. However, when the free ofloxacin powder was dispersed in the medium, the particles disappeared immediately due to the fast dissolution of ofloxacin powder in the medium. That result suggests that the particulate characteristics of OCMs can be maintained in the lung for a fairly long period of time following pulmonary inhalation of the microspheres, thereby enabling the phagocytosis of the microspheres by alveolar macrophages.
3.2. In vitro uptake of ofloxacin by alveolar macrophages from OCMs In vitro uptake of ofloxacin by alveolar macrophages was measured for OCMs and free ofloxacin powders. The viability of the macrophages, as checked by MTT assay, could be maintained at more than 85% for 5 days under the conditions of the uptake study. This result (Fig. 5) shows that the uptake of ofloxacin from the OCMs was substantially greater compared to that from the ofloxacin
Fig. 3. X-ray diffractograms of ofloxacin (A), chitosan (B), physical mixture of ofloxacin and chitosan (C), and OCMs (D).
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Fig. 4. Microscopicmorphologies (200×) of OCMs at 0.5, 2, 4 and 6 h after dispersion in Ham’s F-12K medium containing 15% fetal bovine serum.
powder. For example, the amount of ofloxacin entrapped in the cell at 4 h was only 0.247 ± 0.100 g/mg of cell protein for the free powder, but the amount increased to 0.950 ± 0.129 g/mg for the OCMs, which was a 3.8-fold increase. This tendency was maintained for 24 h. As a result, a 3.6-fold greater area under the drug amount per cell-time curve (AUA) was obtained for the OCMs, compared with that of the ofloxacin powder, suggesting superiority of OCMs in terms of the in vitro delivery of ofloxacin to the macrophages.
Fig. 5. In vitro accumulation of ofloxacin by rat alveolar macrophage (NR8383) cells from ofloxacin powder (control, ) and OCMs () for 48 h. The cell density was 1 × 106 cells/mL, and the applied drug amount was 100 g. The data are expressed as the mean ± SD for 6 experiments. *Significantly (p < 0.05) increased compared to the control.
The cellular accumulation of ofloxacin from the OCMs was dramatically decreased in the absence of ATP in the cell culture medium (Fig. 6), which was induced by the addition of 2-deoxyglucose and sodium azide to the medium, indicating that the uptake process is likely to be ATP-dependent. An active transport mechanism, probably phagocytosis, appears to be operative in the uptake of ofloxacin from the OCMs. The ATP-dependent
Fig. 6. In vitro uptake of ofloxacin from the ofloxacin powder and OCMs in NR8383 alveolar macrophage cells in the presence (white column) and absence (black column) of ATP. The depletion of ATP was achieved by the treatment of the macrophages with 50 mM 2-deoxyglucose and 10 mM sodium azide. Each column represents the mean ± SD of four experiments. *Significantly (p < 0.01) decreased by the ATPdepletion.
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Fig. 7. Effect of ATP-depletion on the microscopic photographs (200×) of macrophages at 2 h following the application of OCMs and ofloxacin.
decrease was not observed for free ofloxacin powder. Immediate dissolution of ofloxacin from the powder appears to be responsible for the ATP-independent uptake of free ofloxacin. The inverted microscopy demonstrated that the morphology of alveolar macrophages was not changed by the addition of ofloxacin powder, regardless of the presence or absence of ATP in the medium (Fig. 7). On the other hand, the morphology following the application of OCMs differed depending on the presence of ATP: in the presence of ATP (control group), most of the OCMs were found to exist inside the macrophages, while most of the OCMs were found outside the macrophages in the absence of ATP. Therefore, phagocytosis, an ATP-dependent active transport mechanism, appeared to be involved in the accelerated uptake of ofloxacin from the OCMs. 3.3. Delivery efficiency to deep sites in the lung: a TSI study The effect of lactose blending on the device-removal efficiency (DRE) and aerosolization efficiency (AE) of OCMs and ofloxacin powder was measured using a twin stage impinger (TSI). As shown in Fig. 8A, the DRE of ofloxacin powder (81%) was increased to more than 90% by lactose blending, regardless of the blending ratio (Fig. 8A). The DRE of OCMs was more than 90% regardless of the lactose-blending with a maximum value of 98% without lactose blending (Fig. 8A). That result indicates that OCMs themselves are superior to the free ofloxacin powder in terms of DRE. On the other hand, the two formulations differed in terms of AE (Fig. 8B)— i.e., the AE of the powder was around 13%, regardless of the lactose blending, while the AE of OCMs was 45%. It is worth noting that the value of 45% for lactose-free OCMs decreased gradually as the lactose ratio in the blend was increased. This indicates that a much higher AE is expected from the OCMs compared with the ofloxacin powder. Because the AE of preparations indicates the efficiency of the drug to reach stage II of the TSI apparatus, a more efficient delivery of ofloxacin to deep sites of the lung, where alveolar macrophages are located, might be plausible by applying lactose-free OCMs instead of the lactose-blended ofloxacin powder.
Fig. 8. Effect of lactose blending on the device removal efficiency (DRE, A) and aerosolization efficiency (AE, B) of OCMs (gray column) and ofloxacin powder (white column) in a twin stage impinger (TSI). Each column indicates the mean ± SD of three experiments.
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4. Discussion Several inhalation formulations including a sugar microparticle formulation of isoniazid (Sawatdee et al., 2006), burst-releasing proliposome formulation of isoniazid (Rojanarat et al., 2011), and spray-dried poly lactic acid microparticles of ofloxacin (Palazzo et al., in press) have been reported to possess potential as pulmonary delivery systems of hydrophilic antituberculosis drugs. The precise evaluation of antituberculosis formulations should be performed based on the quantitative estimation of macrophageuptake of loaded drugs, at least in vitro. However, no such information was available from those studies for hydrophilic antituberculosis drugs. Microspheres are often prepared using PLGA as a polymer. In the present study, however, chitosan was selected as the polymer that was used to prepare OCMs. This was because the ofloxacin content in the PLGA microspheres was very low (<5%, w/w) in our preliminary studies, when they were prepared according to the water-in-oil-in-water emulsion method (Yang et al., 2000). The content could be increased to as high as 27.41% in the present study, far exceeding the content of ofloxacin in PLGA microspheres (<15%; Abazinge et al., 2000). The hydrophilic characteristics of both chitosan and ofloxacin might be associated with the increase. Besides, electrostatic interaction between protonated chitosan and deprotonated ofloxacin (Singh and Dutta, 2010) might have potentiated the increase. The OCMs prepared in the present study were dispersed uniformly in the cell culture medium, and their spherical shape was well maintained for a fairly long period of time (Fig. 4). However, when the glutaraldehyde-crosslinking was not conducted, such physicochemical properties could not be achieved. Therefore, the crosslinking seemed essential to prepare sufficiently rigid spherical microspheres. Microsphere particles would be taken up by the alveolar macrophages following pulmonary inhalation, probably via phagocytosis, which was followed by hydrolytic degradation promoted by the enzymes in the phagosomes (Bianco et al., 2000). Owing to the characteristics of the OCMs, the uptake of ofloxacin from the OCMs to alveolar macrophages (NR8383 cells) was substantially (3.6-fold) increased for the OCMs, compared with that of the free ofloxacin powder (Fig. 5). The uptake from the OCMs was ATP-dependent (Figs. 6 and 7), which was consistent with the active phagocytosis of solid particles for the OCMs. The substantial increase in the macrophage uptake may be explainable in association with the immunological characteristics of chitosan. N-acetyl glucosamine residues in chitosan are known to cause immune stimulation of macrophages (Peluso et al., 1994), accelerating the phagocytosis of chitosan microspheres (Ueno et al., 2001). The electrostatic interaction between positive charge of chitosan and negative charge of the cell membrane might have further enhanced the phagocytosis, as was previously reported for positively charged particles (Mutsaers and Papadimitriou, 1988). The degree of drug delivery to the deep portions of the lung could be estimated by measuring the device-removal efficiency (DRE) and aeorosolization efficiency (AE) of the particles using the twin stage impinger (TSI) (Fig. 8A). In the case of ofloxacin powder, most of the drug was emitted well from the capsule (i.e., DRE > 80%), and the DRE value was increased slightly by the addition of a small amount of lactose to the blend, indicating that lactose is useful to discharge ofloxacin powder from the capsule in the TSI apparatus. The DRE of lactose-free OCMs was higher than that of ofloxacin powder, and was decreased slightly by the lactose blending. However, the AE of OCMs (45%) was much higher than that of ofloxacin powder (13%) (Fig. 8B). The AE of ofloxacin powder was not improved by lactose blending. Considering that the particle sizes of OCMs and ofloxacin powder are similar, only differing in morphology (Fig. 2), the spherical shape of OCMs appears to be
advantageous compared with the rod shape of ofloxacin powder in terms of aeorosolization. Both the AE and DRE values were highest for lactose-free OCMs, which suggested that these particles can be inhaled more efficiently in order to reach deep inside the lung. More information regarding the tendency to deposit in deep lungs (alveoli) would be obtainable from the aerodynamic diameter (particle effective density) of the particles, which could not be measured in the present study. Despite the superiority of OCMs over ofloxacin powder in terms of in vitro macrophage uptake of ofloxacin and in vitro degree of drug delivery to the deep lung (i.e., phase II in TSI study), the conclusion on the potential of OCMs as an alveolar macrophage-targeted delivery system should await in vivo experimental verification, which will require the collection of substantial amounts of alveolar macrophages from rats. 5. Conclusion Ofloxacin-loaded, glutaraldehyde-crosslinked chitosan microspheres (OCMs) were prepared using a water-in-oil emulsification method, and their potential to deliver ofloxacin directly to alveolar macrophages via the respiratory route was examined. In comparisons with ofloxacin powder, the OCM particles demonstrated better aerosolization efficiency (AE) and enhanced ofloxacin uptake by the alveolar macrophages in vitro. These results suggest the potential of OCMs as an efficient delivery system of ofloxacin to alveolar macrophages where tuberculosis-causing M. tuberculosis mainly survives. However, the extrapolation of this conclusion to other antituberculosis drugs, or conclusions concerning the possible advantages of microsphere inhalants over conventional forms of oral dosage in the treatment of tuberculosis, should await careful in vivo verification. Acknowledgements This study was supported by the Korea Science and Engineering Foundation (KOSEF) through the National Research Laboratory program, which was funded by the Ministry of Science and Technology (no. ROA-2006-000-10190-0). References Abazinge, M., Jackson, T., Yang, Q., Owusu-Ababio, G., 2000. Comparison of in vitro and in vivo release characteristics of sustained release ofloxacin microspheres. Drug Deliv. 7, 77–81. Bermudez, L.E., 1994. Use of liposome preparation to treat mycobacterial infections. Immunobiology 191, 578–583. Berning, S.E., Madsen, L., Iseman, M.D., Peloquin, C.A., 1995. Long-term safety of ofloxacin and ciprofloxacin in the treatment of mycobacterial infections. Am. J. Respir. Crit. Care Med. 151, 2006–2009. Bhattacharjee, S., Ershov, D., Gucht, J.V., Alink, G.M., Rietjens, I.M., Zuilhof, H., Marcelis, A.T., 2011. Surface charge-specific cytotoxicity and cellular uptake of tri-block copolymer nanoparticles. Nanotoxicology, http://dx.doi.org/10.3109/17435390.2011.633714. Bianco, I.D., Balsinde, J., Beltramo, D.M., Castagna, L.F., Landa, C.A., Dennis, E.A., 2000. Chitosan-induced phospholipase A2 activation and arachidonic acid mobilization in P388D1 macrophages. FEBS Lett. 466, 292–294. Champion, J.A., Katare, Y.K., Mitragotri, S., 2007. Particle shape: a new design parameter for micro-and nanoscale drug delivery carriers. J. Control. Release 121, 3–9. Dawson, T.L., Gores, G.J., Nieminen, A.L., Herman, B., Lemasters, J.J., 1993. Mitochondria as a source of reactive oxygen species during reductive stress in rat hepatocytes. Am. J. Physiol. Cell Physiol. 264, C961–C967. Doh, H.J., Jung, Y., Balakrishnan, P., Cho, H.J., Kim, D.D., 2012. A novel lipid nanoemulsion system for improved permeation of granisetron. Colloids Surf. B Biointerfaces 101C, 475–480. El-Shabouri, M.H., 2002. Positively charged nanoparticles for improving the oral bioavailability of cyclosporin-A. Int. J. Pharm. 249, 101–108. Gupta, H., Aqil, M., Khar, R.K., Ali, A., Bhatnagar, A., Mittal, G., 2010. Sparfloxacinloaded PLGA nanoparticles for sustained ocular drug delivery. Nanomedicine 6, 324–333. Hirota, K., Hasegawa, T., Nakajima, T., Inagawa, H., Kohchi, C., Soma, G., Makino, K., Terada, H., 2010. Delivery of rifampicin-PLGA microspheres into alveolar
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