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Genipin crosslinked ethyl cellulose–chitosan complex microspheres for anti-tuberculosis delivery
Colloids and Surfaces B: Biointerfaces 103 (2013) 530–537
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Genipin crosslinked ethyl cellulose–chitosan complex microspheres for anti-tuberculosis delivery Hanzhou Feng, Limei Zhang, Chunyan Zhu ∗ Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Beijing 100193, PR China
a r t i c l e
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Article history: Received 12 August 2012 Received in revised form 6 November 2012 Accepted 10 November 2012 Available online 22 November 2012 Keywords: Rifabutin Chitosan Genipin Ethyl cellulose Complex microspheres Intra-tracheal intubation
a b s t r a c t Genipin-crosslinked complex microspheres made of the combination of two polymers, ethyl cellulose and chitosan, were prepared by spray drying method. Rifabutin, an anti-tuberculosis agent was used as a model drug. Effects of various specifications of ethyl cellulose and chitosan, different drug/polymers ratios and crosslinking effect of genipin on complex microspheres and drug release characteristics were compared to obtain optimized manufacturing parameters. The complex microspheres showed a significant different shape as compared to chitosan microspheres. Biphasic release features were observed in the in vitro and in vivo release study, consisting of an initial quick release phase and an extended sustained release phase. Furthermore, pulmonary drug concentrations of rats after administering the complex microspheres were maintained on a therapeutic level for at least 24 days. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Tuberculosis remains a globally serious infectious disease despite the development of various effective anti-tuberculosis drugs and programmatic therapy protocol, 8.8 million people were infected by tuberculosis and 1.4 million died in 2010 by the report of World Health Organization [1]. A standard therapy regime involves chemotherapy using rifampicin (RIP) plus isoniazid (INZ), supplemented with streptomycin or ethambutol for 6–9 months [2], but the therapeutic results were far less satisfactory especially for fibro-cavernous tuberculosis [3,4]. High incidences of relapse and side effects have been reported [5–9]. Extended duration of the therapeutic regime leads to low patient compliance and reproduction of multi-drug resistant tuber bacillus (TB) [1,10,11]. Since TB mainly attacks lungs, enhancing pulmonary anti-tuberculosis drug concentration has been one of the principal goals for improving therapeutic effect. Therefore, growing attention has been given to pulmonary route due to the fact that it allows high drug concentration to be achieved in the lesions and lung tissues and
Abbreviations: RBT, rifabutin; CTS, chitosan; CPM, complex microspheres; EC, ethyl cellulose; GNP, genipin; GTU, glutaraldehyde; TPP, sodium tripolyphosphate; HPLC, high-performance liquid chromatography; BAL, bronchoalvelar lavage; PLGA, poly (lactic-co-glycolic acid); IC50, half maximal (50%) inhibitory concentration. ∗ Corresponding author at: No. 151 Malianwa North Road, Haidian District, Beijing 100193, PR China. Tel.: +86 10 57833276; fax: +86 10 57833276. E-mail address: [email protected] (C. Zhu). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.11.007
minimizes systemic side effects [12–14]. Instead of traditional oral administration route, intra-tracheal instillation has been increasingly used in clinical treatment of pulmonary tuberculosis [15–19]. However, one of the main problems met in current intra-tracheal treatment of tuberculosis includes lack of drug retention in lungs, which leads to the difficulty of maintaining therapeutic drug concentration for a required duration. Microspheres and microparticles have been reported applying in intra-tracheal instillation for their lung-retention property and significant sustained release effect [17,20–22]. In previous study, we designed a drug delivery system using poly (lactic-co-glycolic acid) (PLGA) microspheres loading RIP in combination with sodium alginate gel [23]. The gel provided microspheres with bio-adhesion and our experimental result demonstrated that this delivery system can adhere to lungs for 21 days and has a satisfying restricted release effect. Unfortunately, weighty burden toward lungs could be caused by gelatinization of the sodium alginate carrier when it meets calcium ions. The solid-state gel could be coughed out by patients, limiting its practical utilization. For considerations of avoiding the use of gel carrier while preserving the adhesion of the delivery system, adhesive complex microspheres (CPM) have been prepared by spray drying in this undergoing study. Hydrophobic matrix carrier ethyl cellulose (EC) was used as the main sustained release carrier [24,25]; been commonly used in pharmaceutical and biomedical industries for its high biocompatibility and biodegradability, positive charged natural polysaccharide chitosan (CTS) was used to provide bio-adhesion to CPM [26–30]. In virtue of their good
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dispersion of suitable particle size for pulmonary delivery, CPM were expected to stay in lungs for an extended period. Rifabutin (RBT), an anti-tuberculosis drug with greater lipophilicity and stronger potency toward TB, has been loaded in CPM as the model drug [31–33]. A natural crosslinking agent genipin (GNP) was applied to control the release rate of CPM. In vitro/in vivo release behaviors were studied to clarify sustained release effects and retention properties of CPM. 2. Materials and methods 2.1. Materials EC with different viscosity (10, 20 and 45 cp) was kindly provided by Colorcon, China. CTS (Mw 190,000–310,000) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were purchased from Sigma–Aldrich, USA. GNP was supplied by Linchuan Zhixin Bio-Technology Co., China. RBT was purchased from Hubei Kangbaotai Fine-Chemical Co., Ltd., China. 96 wells plate was purchased from Corning, USA. All the reagents used here were analytical grade. 2.2. Animals Sprague–Dawley (SD) rats were obtained from Vital River Laboratories, China. All experiments were performed in accordance with international accepted guidelines on laboratory animal use, the Guide for the care and use of laboratory animals [34], the protocols were approved by the Beijing Animal Care Committee. 2.3. Cell lines Human lung A549 and Calu-3 airway epithelial cells were purchased from Institute of Basic Medical Sciences, Peking Union Medical College, China. A549 and Calu-3 cells were maintained in F12 and DMEM medium, respectively, both supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) non essential amino acid, 2 mM glutamine, 100 IU/mL penicillin and streptomycin. The medium was changed every 2 days and subcultured every 4 days. 2.4. Cytotoxicity study To compare cytotoxicity of three crosslinking agents, the A549 and Calu-3 cells were plated at a cell density of 3.5 × 105 cells per well in 96-well plates and incubated at 37 ± 1 ◦ C in an atmosphere of 5% CO2 . After 24 h of culture for A549 cells, the medium was replaced with fresh F12 medium containing glutaraldehyde (GTU), sodium tripolyphosphate (TPP) and GNP at the concentrations equivalent to 1 mg mL−1 . After 8 h and 24 h, the medium was discarded and the wells were washed twice with hanks balanced salt solutions (HBSS). 200 L MTT solution (0.5 mg mL−1 in PBS) was added to each well, and incubated 4 h at 37 ± 1 ◦ C for MTT formazan formation. Subsequently, the supernatant was carefully removed and the wells were washed twice with HBSS. 200 L DMSO was added to each well and the plates were then mildly shaken for 15 min to ensure the dissolution of formazan crystals. The optical density values were measured by using MQX200 microplate reader (Bio-Tek, USA) at wavelength 540 nm. Three replicates were read for each sample and mean value was used as the final result. The spectrophotometer was calibrated to zero absorbance using culture medium without cells. The relative cell inhibition ratio (%) related to control wells containing cell culture medium was calculated as:
Inhibition ratio (%) =
1 − [A]test [A]control
× 100%
531
After 24 h of culture for Calu-3 cells, the medium was replaced with fresh DMEM medium containing three crosslinking agents at 5 gradient concentrations, following with the same subsequent process for A549 cells.
2.5. Preparation of microspheres GNP-crosslinked CPM loading RBT were prepared by spraydrying of oil-in-water (O/W) emulsion, using a B-290 mini spray drier (Buchi, Switzerland) with a standard 0.5 mm nozzle. Spraydrying conditions were as follows: the peristaltic pump rate of 3.43 mL min−1 , compressed air flow rate of 536 L h−1 , inlet and outlet air temperature of 135 ◦ C and 85 ◦ C, respectively [35]. CTS was dissolved in 1% (v/v) acetic acid solution at 1% (w/v) concentration, then it was crosslinked by GNP for 2 h at 40 ◦ C. RBT was dissolved in ethyle acetate contained EC with different concentrations. Emulsions were prepared by stirring of the oil phase and the water phase. O/W emulsions were prepared differed in viscosity (10 cp, 20 cp and 45 cp) and concentrations (4% and 1%, w/v) of EC, RBT/EC ratios (1:5, 1:10 and 1:20) and concentrations (1% and 0.5%, w/v) of CTS. The emulsions were mixed for 10 min, in an organic/water phase ratios of 1:5 (v/v) and subjected to spray-drying under process conditions described above. Non-crosslinked CPM were prepared by directly using non-crosslinked CTS solution as water phase, following with the same subsequent preparation process. CTS microspheres were spray dried with the same parameters used in preparation of CPM, but only non-crosslinked and GNP-crosslinked CTS solution loading RBT (RBT/CTS = 1/5, w/w), without EC, were fed to the instrument.
2.6. Determination of RBT encapsulation efficiency and loading capacity RBT was extracted from the microspheres with mixture solution consisted of 0.1 M HCl and ethanol (3:2, v/v) under ultrasonic process for 1 h. The measurements were repeated in triplicate, with analysis by high-performance liquid chromatography (HPLC). Encapsulation efficiency (EE) and loading capacity (LC) were calculated as RBT detected by HPLC of the corresponding theoretical loading quantity and CPM weight, respectively. 0.1 M hydrochloric acid was used to extract RBT from CTS layer in 1 h ultrasonic process to compare the drug loading percentage between layers of CTS and EC.
2.7. Morphology and particle size distribution of microspheres The morphology of CPM and CTS microspheres were characterized by scanning electron microscopy (SEM). Samples were mounted on carbon taped aluminum stubs and gold coated by a sputter coater for 240 s, and they were observed using a JSM-6510 SEM (JEOL, Japan) at an accelerating voltage of 15 kV. The particle size distributions were characterized in a light scattering particle size analyzer (Mastersizer, England) using ethanol as dispersion medium. Three replicates of mean particle diameter and span were determined for each sample and mean value was used as the final result.
2.8. Zeta potential of microspheres Zeta-potential of the CPM and CTS microspheres was determined by photon-correlation spectroscopy (Zeta-sizer 2000, Malvern Instrument, England) in deionized water at 25 ◦ C. Three batches of each formulation were analyzed in triplicate (n = 3).
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2.9. Differential scanning calorimetry (DSC) Differential scanning calorimetry measurements were performed in a Q200 instrument (TA, USA). Accurately weighed samples (5 ± 0.1 mg) of CTS, EC, RBT and their physical mixture and CPM loading RBT were placed into a covered aluminum sample pans. An empty sample pan was used as reference and the runs were performed by heating samples from 20 to 450 ◦ C at a speed of 10 ◦ C min−1 . 2.10. In vitro release In vitro release test was carried out in pH7.4 phosphate-buffered saline (PBS) solution at 37 ± 0.5 ◦ C. 10 mg CPM or CTS microspheres were suspended in 5 mL release medium contained in the dialysis tubes, then they were put in erlenmeyer flask containing 45 mL mediums and incubated in a water bath instrument (SHAB(A), Jintankexi apparatus Co. Ltd., Jiangsu, China) shaken at 100 strokes min−1 for 8 days. At pre-determined time intervals, aliquots of 0.5 mL solutions were withdrawn and filtered through 0.45 m filters. The sample volumes were replaced with equal volume of the fresh medium. RBT released from CPM were quantified by HPLC, three tests were taken for each sample and mean value was used as the final result. 2.11. In vivo release SD rats were divided into two groups, after anesthetizing by pentobarbital sodium (60 mg kg−1 ), control group were administered by intra-tracheal intubation operation with 0.2 mL non-crosslinked CPM suspension and experimental group with the same volume suspension contained crosslinked CPM. Rats were sacrificed at predetermined time intervals, and then the tracheae and lungs were carefully separated and observed. Released drug was extracted by bronchoalveloar lavage (BAL). 2.5 mL PBS solution was injected into the lungs and then it was withdrawn immediately, this process was repeated four times. The lavage fluid was collected and vortex mixed 1 min with methylene chloride (2:1, v/v). The organic layer was separated by centrifuge at 3000 rpm for 5 min and then blown dry with N2 . Finally all samples were re-dissolved by adding 0.2 mL acetonitrile before being analyzed by HPLC. 2.12. HPLC condition RBT was determined using a pump L7100 and a Shimadzu SPD10AVP UV-detector (Shimadzu, Japan). A SLC-10AVP (Shimadzu, Japan) equipped with a Diamonsil ODS (4.6 mm × 150 mm, 5 m) analytical column was used. The column was kept at 40 ◦ C throughout the elution process, which used a mobile phase consisting of acetonitrile: 0.1 M KH2 PO4 (pH 6.45) = 65:35 (v/v) at a flow rate of 1.0 mL min−1 , and the detection wavelength was set to 278 nm. 2.13. Statistical analysis All data are presented as the mean ± standard deviation (SD). Statistical analysis was performed with the SPSS 17.0 software. Values of *P < 0.05 were considered statistically significant. 3. Results and discussion Currently, drugs can be administered by pulmonary route utilizing two techniques: inhalation and intra-tracheal instillation [36,37]. For inhalations, pharyngeal deposition of the drug has been the main problem, which leads to difficulty in measuring the dose inside the lungs and side effects caused by systemic circulation
Fig. 1. Cytotoxicity of three crosslinking agents.
when swallowed [38,39]. Moreover, the presence of partially suppressive drug concentrations promotes the growth of resistant TB [40]. In contrary, intra-tracheal instillation provides a simple, quantifiable way for pulmonary drug delivery and has been increasingly applied in clinical treatment of pulmonary tuberculosis. Microspheres have been widely reported that being applied as a proper formulation in pulmonary instillation administration. According to the simplification principle of formulation design, microspheres using CTS as the only carrier were prepared and evaluated in aspects of release rate and encapsulation efficiency. The investigations of CTS microspheres were regarded as basis for the study of CPM. 3.1. Cytotoxicity study of crosslinking agents (MTT assay) Serious burst effects were observed in previous study of CTS microspheres, therefore, a natural water-soluble crosslinking agent genipin (GNP) was applied in this study to control the release rate. Being extracted from the fruit of Gardenia Jasminoides Ellis, GNP was reported as a fully biocompatible reagent with lower cytotoxicity than traditional chemical crosslinking agent, such as GTU [41–43]. To verify the good biocompatibility of GNP, MTT assay on A549 and Calu-3 cells was carried out by comparing cytotoxicity of GNP to that of other commonly used crosslinking agents, TPP and GTU. When time extended from 8 h to 24 h (Fig. 1), the cytotoxicity of all three crosslinking agents appeared as time-dependent since the inhibition ratio raised (Fig. 1A), while IC50 value decreased (Fig. 1B). Lower cytotoxicity of GNP was shown by SPSS result as compared to GTU and TPP under 1% significance level (P = 0.01). Our experimental result was similar to those reported literatures [44,45]. Owing to its better safety and higher biocompatibility, GNP was used as the crosslinking agent to prepare CPM in the next step.
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Fig. 2. SEM observations of chitosan microspheres.
3.2. Preparation of complex microspheres The cumulative release percentage of RBT released from noncrosslinked CTS microspheres was 68.46 ± 2.87% in 24 h, which decreased to 58.25 ± 3.45% after crosslinking by GNP. Although the significant sustained release effect that GNP exerted on CTS microspheres, it was insufficient to attain our expectation: cumulative drug release percentage lower than 50% in 168 h (7 days). Similar results were observed even by alternating the weight ratio of GNP/CTS from 1/20 to 1/5. Therefore, developing a drug delivery system that can be used in intra-tracheal treatment, meanwhile carrying the functions of restricting drug release and bio-adhesion was the challenge of this study. For these considerations, CPM were prepared in the way that EC functioned like a “core”, encapsulating the hydrophobic model drug RBT and controlling the drug release rate; CTS functioned as the bio-adhesive “Shell”, promoting pulmonary retention property of the delivery system.
3.3. Encapsulation of rifabutin The permeation of RBT from organic phase to water phase during the emulsion process caused part of the drug to be encapsulated in CTS layer. Due to the hydrophilic swelling of CTS, drug would be quickly released from CTS layer as compared to that from EC layer. Theoretically, biphasic drug release would be expected to observe. The quick release phase increased pulmonary drug concentration to therapeutic level in a short time, while sustained release phase helped to maintain the concentration level for an extended period. Factors include different viscosities of EC, EC and CTS at different concentrations and different RBT/EC weight ratios were used to optimize the preparation process of CPM by studying their possible influences on the encapsulation efficiency (EE) and drug loading capacity (LC), as reported in Table 1. Higher EE was observed at 20 cp viscosity and 1% (w/v) concentration of EC, with the EC/drug weight ratio of 5/1. Drug permeation from EC layer to CTS solution was impeded as the viscosity of EC increased from 10 cp to 45 cp, theoretically increasing EE and LC of CPM. Nevertheless, EC with a higher viscosity (45 cp) was difficult to be completely dissolved in ethyl acetate, causing inaccuracy to the preparation process. The inaccuracy possibly accounts for the results that CPM prepared with 20 cp EC had higher EE and LC than that of CPM prepared with 45 cp EC. The results of higher LC were observed in groups of CPM prepared with EC at 4% (w/v) concentration, as compared to that of CPM prepared with EC at 1% (w/v). The different drug inputting amounts, 0.16 g RBT for 4% (w/v) EC and 0.04 g RBT for 1% (w/v) EC, accounted for the results. The optimized parameters for the preparation of CPM were as followed: 20 cp and 4% (w/v) EC, RBT/EC ratios of 1:5 and 1% (w/v) CTS.
The crosslinking effect of GNP significantly improved EE and LC of CPM, which can be also reflected in Table 1. Maintaining all the other parameters constant, EE and LC were increased for CPM crosslinked by GNP. It is thought that the viscosity of CTS solution increased after crosslinking, preventing RBT permeation from EC into CTS. To demonstrate this supposition, the percentages of RBT encapsulated in CTS layers were compared between non-crosslinked and crosslinked CPM, which decreased from 27.43 ± 2.47% to 17.62 ± 2.05% after crosslinking. GNP crosslinking effect on CTS was investigated in pilot test and it was observed that the viscosity of CTS solution gradually increased as the GNP crosslinking reaction continued and the solution finally transformed to a semi-solid gel. This phenomenon was similar to the report of Cao et al. [46]. Therefore, 40 ◦ C was set as the crosslinking temperature to prepare CTS microspheres and CPM. Furthermore, EE and LC of CTS microspheres were higher as GNPcrosslinking time prolonged. The crosslinking degree was inferred to increase from 0.5 h to 4 h, promoting the amount of drug encapsulated in CTS. The maximum crosslinking time for CPM was 2 h, in order to maintain the proper viscosity of the solution for the emulsion process. 3.4. Characterization of complex microspheres 3.4.1. SEM observation CTS microspheres were observed under SEM as a contrast. It can be seen in Fig. 2, both groups of non-crosslinked (Fig. 2A) and GNP crosslinked (Fig. 2B) CTS microspheres had uniform and spherical shape. Shown in Fig. 3, all CPM were spherical, but instead of plicated features of CTS microspheres, only compact layers were observed on surfaces of non-crosslinked (Fig. 3A) CPM, while the surfaces of GNP crosslinked CPM (Fig. 3B) were relatively smoother. This can be attributed to the theoretical “shell” structure of CTS formed on the surfaces of EC “core”. 3.4.2. Particle size distribution As shown by Fig. 4A, the decreased mean particle diameter of CPM was inferred as the result of crosslinking effect, tightening intermolecular distance of CTS. The increased span of particle distribution was possibly as a result of higher viscosity of CTS solution after crosslinking, lowering the uniformity of emulsion droplets when sprayed from the nozzle. Similar results were observed in characterization of CTS microspheres, which can be seen in Fig. 4B. 3.4.3. Zeta potentials Due to the electrostatic interaction between its positive amino groups and anionic substructures in mucous layer or epithelial cells, CTS has been extensively studied as a bio-adhesive material. In this study, zeta potentials of EC microspheres were measured as contrast, which was −19.73 ± 0.64 mV. The presence of CTS at the
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Table 1 Effects of polymers viscosity and concentration (w/v), polymers/drug ratios (w/w) and crosslinking agent GNP on encapsulation efficiency (EE) and loading capacity (LC) of complex microspheres prepared by spray drying. EC viscosity (cp)
EC concentration (%, w/v)
10 10 10 20 20 20 20 20 20 45 45 45
4 1 4 4 4 1 4 4 4 4 1 4
EC/drug ratios (w/w) 5:1 5:1 5:1 5:1 5:1 5:1 10:1 20:1 5:1 5:1 5:1 5:1
CTS concentration (%, w/v)
GNP (g)
EE (%, w/w)
1 1 1 0.5 1 1 1 1 1 1 1 1
– – 0.1 – – – – – 0.1 – – 0.1
45.10 47.06 59.40 52.45 72.63 75.53 54.72 30.42 78.65 61.21 64.68 70.08
± ± ± ± ± ± ± ± ± ± ± ±
3.71 3.95 2.59 2.77 3.22 2.55 2.85 0.96 2.69 2.86 3.85 3.14
LC (%, w/w) 3.66 1.53 4.61 5.75 5.95 2.44 2.33 0.66 6.11 5.00 2.07 5.44
± ± ± ± ± ± ± ± ± ± ± ±
0.46 0.32 0.62 1.06 0.84 0.42 0.37 0.03 0.76 0.63 0.28 0.87
Fig. 3. SEM observations of complex microspheres.
surface of the CPM could be indicated by the positively charged CPM. The crosslinking effect markedly decreased the zeta potential of CPM from 33.60 ± 3.32 mV to 28.97 ± 1.37 mV. Similar results were observed in the study of non-crosslinked and GNP crosslinked CTS microspheres, demonstrating that free amine groups of CTS being bound by GNP. Moreover, zeta potential of CPM was lower than that of CTS microspheres, indicating the possible presence of negative charged EC on the surface. 3.4.4. DSC measurement DSC curve of the CPM loading RBT was drawn in comparison to that of simple EC, CTS, RBT and their physical mixture (Fig. 5). Exothermic peaks were observed around 241DSC curve of the CPM loading RBT was drawn in comparison to for RBT, 313 ◦ C for CTS, 199 ◦ C and 389 ◦ C for EC, attributing to their chemical degradation, respectively. The two exothermic peaks of EC could be explained by its stepwise degradation. All typical exothermic peaks of three pure samples corresponded well with physical mixture. However, only typical exothermic peaks of CTS and EC samples were observed in the curve of CPM loading RBT, indicating the formation of microspheres and its encapsulation effect on RBT. 3.5. In vitro release of complex microspheres
Fig. 4. Particle size distribution.
RBT was demonstrated to be stable in PBS for 14 days at 37.5 ◦ C under static condition, however, its degradation was observed after 48 h in vitro release test. Situations such as different release medium (PBS or de-ionized water), different temperature (25 ◦ C or 37.5 ◦ C) and adding different antioxidants (sodium citrate or sodium sulfite) were examined. No significant improvement to RBT stability was observed. According to the study of Sangshetti et al. [47], RBT was unstable under oxidation condition. Shaking action
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produced by the bath instrument was assumed to cause a more oxidized environment by increasing the contact area between air and release medium. This caused more oxygen to dissolve in the medium than a static system. Similar result was also observed in release study using paddle method with similar action mechanism of stirring. As a result, a modified in vitro release method was performed. This method involved the release medium being replaced with fresh PBS every 24 h, within which RBT was proved to be stable even under shaking action. At the end of the test, microspheres in the dialysis tubes were collected and the encapsulating RBT was extracted for quantification. The sum of drug amount detected in release medium and extracted from microspheres corresponded well with theoretical drug loading capacity. In vitro drug release of RBT-loaded CPM was studied for 192 h at pH 7.4 PBS. Viscosity was chosen as 10 cp, 20 cp and 45 cp while the concentration was 4% and 1% (w/v) respectively. In this study, we compared the release rate of CPM made of EC with each combination (Fig. 6A). In the first four hours, 3.38% of RBT was released for CPM made of 10 cp EC. The percentage of RBT released increased to 15.57% in 24 h followed by a gradual and sustained release up to 20.27% in 192 h. The drug release behavior showed a biphasic pattern indicated by its initial quick release, followed by a very slow release. The initial quick release could be explained by the hydrophilic swelling of CTS, facilitating the drug diffusion from CTS layer to the medium. In 24 h to 192 h, release may have occurred due to the diffusion of drug through CTS corrosion and EC matrix of CPM. This biphasic release profile was also observed in CPM made of 20 cp and 45 cp EC. The release behavior was similar to the biphasic profile discussed above from the curves. The drug release rate of CPM made of 4% EC concentration was significantly faster, due to its higher drug loading rate. To meet the potential clinical requirement, CPM made of 10 cp EC was selected as the subject of in vivo release study. It was demonstrated that in vitro release rate of CPM crosslinked by GNP was significantly slower than non-crosslinked CPM. As shown in Fig. 6B, cumulative release percentages of GNP crosslinked and non-crosslinked CPM were compared, with a result of 1.16% to 3.38% in the first 4 h, 7.89% to 15.57% in 24 h and 10.72% to 20.27% in 192 h, respectively. It is reasonable to infer that the crosslinking effect restricted the drug release by
Fig. 5. DSC study.
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Fig. 6. In vitro release study.
tightening the CTS molecular chain segments, forming more stable heterocyclic-intra-/intermolecular structures. This results in a limitation of hydrophilic pathways in CTS layers thus obstructing the penetration of the medium to trigger swelling action. Also the drug release is decelerated. 3.6. In vivo release Bronchoalveolar lavage (BAL) method was used to extract free RBT from the lungs of administered rats [48,49]. After administration of CPM, lungs were separated to be injected with PBS. The lavage fluid was collected and free drug was extracted by dichloromethane. It is interesting to note that in vivo release curves of noncrosslinked and GNP crosslinked CPM both had double peaks characteristics (Fig. 7). In first 4 h, GNP crosslinking effect significantly slowed the in vivo drug release rate, which corresponded well with in vitro release study result. The initial burst occurred as expected and the RBT load in CTS external layer released
Fig. 7. In vivo release study.
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quickly. In 4–24 h, there was no significant difference between noncrosslinked and GNP crosslinked CPM. The crosslinking effect of GNP on CTS could be weakened by taking the more complicated in vivo environment into account such as enzymes in lungs [50]. It has been reported that CTS mainly be degraded by lysozyme, an enzyme which is synthesized and secreted by surface epithelial cells and macrophages in human airways [51–55]. In 24–192 h, the drug was released faster from GNP crosslinked CPM. The mechanism of diffusion through EC layer following concentration gradient was considered to be the leading role in this stage of in vivo release, therefore GNP crosslinked CPM had a faster release rate for its higher LC. In 596 h (24th day), the local concentration of RBT in lungs was 0.68 ± 0.15 g mL−1 and 1.54 ± 0.23 g mL−1 for noncrosslinked and GNP crosslinked CPM, respectively. They were both higher than the minimal inhibitory concentration (MIC) of RBT, which is 0.015–0.06 g mL−1 [56]. CPM were observed adhering to lungs in bronchoalveolar lavage process through the whole in vivo release time line, demonstrating its good pulmonary retention property. Two peaks – in vivo release curves of CPM indicated that the microspheres exerted such a long pulmonary retention. This was because free drug would quickly be absorbed by pulmonary alveolar and capillaries and then transferred to systemic circulation. The time range of increased drug concentration, 0–1 h and 72–360 h, indicated the drug continuously released from microspheres. As shown by the time range 4–72 h and 360–576 h, decreased drug concentration manifested that the drug absorption rate was faster than drug release rate. The twopeaks in vivo release profile strongly demonstrated that microspheres led to lung pulmonary retention. This data permitted us to conclude that the CPM delivery system could effectively sustain the RBT release and help maintain the local drug concentration at a therapeutic level at length. 4. Conclusion This study has demonstrated that it was feasible to prepare the RBT-loaded CPM made of ethyl cellulose and CTS with spray drying method. The influences of various specifications of ethyl cellulose, CTS and different amount of model drug on encapsulation efficiency and loading capacity were studied. The CPM exhibited sustained release behaviors over an extended period and its initial burst release was restricted by the crosslinking effect of genipin. The result of in vivo release study further demonstrated that the CPM possessed biphasic release and long time pulmonary retention features. We hope this study will be fruitful for the clinical treatment for pulmonary tuberculosis. Acknowledgements This work was supported by the Foundation of 12th Five-Year Important National Science & Technology Specific Projects. Financial support from National Science &Technology Major Special Project on Prevention and Cure of Acquired Immune Deficiency Syndrome and Virus Hepatitis. (Item No. 2012ZX10003009-001002) is gratefully acknowledged. Special thanks to Chunhui Hu whose work paved the way for our research. References [1] P. Nahid, D. Menzies, Update in tuberculosis and nontuberculous mycobacterial disease 2011, Am. J. Respir. Crit. Care Med. 185 (2012) 1266–1270. [2] Short-course chemotherapy in pulmonary tuberculosis, A controlled trial by the British Thoracic and Tuberculosis Association, Lancet 1 (1975) 119–124. [3] V.I. Chukanov, O.G. Komissarova, V. Maishin, R. Abdullaev, A.S. Kononets, Efficiency of a new standard chemotherapy regimen in the treatment of patients with recurrent pulmonary tuberculosis, Probl. Tuberk. Bolezn. Legk. 8 (2006) 9–13.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Genipin crosslinked ethyl cellulose–chitosan complex microspheres for anti-tuberculosis delivery Hanzhou Feng, Limei Zhang, Chunyan Zhu ∗ Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Beijing 100193, PR China
a r t i c l e
i n f o
Article history: Received 12 August 2012 Received in revised form 6 November 2012 Accepted 10 November 2012 Available online 22 November 2012 Keywords: Rifabutin Chitosan Genipin Ethyl cellulose Complex microspheres Intra-tracheal intubation
a b s t r a c t Genipin-crosslinked complex microspheres made of the combination of two polymers, ethyl cellulose and chitosan, were prepared by spray drying method. Rifabutin, an anti-tuberculosis agent was used as a model drug. Effects of various specifications of ethyl cellulose and chitosan, different drug/polymers ratios and crosslinking effect of genipin on complex microspheres and drug release characteristics were compared to obtain optimized manufacturing parameters. The complex microspheres showed a significant different shape as compared to chitosan microspheres. Biphasic release features were observed in the in vitro and in vivo release study, consisting of an initial quick release phase and an extended sustained release phase. Furthermore, pulmonary drug concentrations of rats after administering the complex microspheres were maintained on a therapeutic level for at least 24 days. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Tuberculosis remains a globally serious infectious disease despite the development of various effective anti-tuberculosis drugs and programmatic therapy protocol, 8.8 million people were infected by tuberculosis and 1.4 million died in 2010 by the report of World Health Organization [1]. A standard therapy regime involves chemotherapy using rifampicin (RIP) plus isoniazid (INZ), supplemented with streptomycin or ethambutol for 6–9 months [2], but the therapeutic results were far less satisfactory especially for fibro-cavernous tuberculosis [3,4]. High incidences of relapse and side effects have been reported [5–9]. Extended duration of the therapeutic regime leads to low patient compliance and reproduction of multi-drug resistant tuber bacillus (TB) [1,10,11]. Since TB mainly attacks lungs, enhancing pulmonary anti-tuberculosis drug concentration has been one of the principal goals for improving therapeutic effect. Therefore, growing attention has been given to pulmonary route due to the fact that it allows high drug concentration to be achieved in the lesions and lung tissues and
Abbreviations: RBT, rifabutin; CTS, chitosan; CPM, complex microspheres; EC, ethyl cellulose; GNP, genipin; GTU, glutaraldehyde; TPP, sodium tripolyphosphate; HPLC, high-performance liquid chromatography; BAL, bronchoalvelar lavage; PLGA, poly (lactic-co-glycolic acid); IC50, half maximal (50%) inhibitory concentration. ∗ Corresponding author at: No. 151 Malianwa North Road, Haidian District, Beijing 100193, PR China. Tel.: +86 10 57833276; fax: +86 10 57833276. E-mail address: [email protected] (C. Zhu). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.11.007
minimizes systemic side effects [12–14]. Instead of traditional oral administration route, intra-tracheal instillation has been increasingly used in clinical treatment of pulmonary tuberculosis [15–19]. However, one of the main problems met in current intra-tracheal treatment of tuberculosis includes lack of drug retention in lungs, which leads to the difficulty of maintaining therapeutic drug concentration for a required duration. Microspheres and microparticles have been reported applying in intra-tracheal instillation for their lung-retention property and significant sustained release effect [17,20–22]. In previous study, we designed a drug delivery system using poly (lactic-co-glycolic acid) (PLGA) microspheres loading RIP in combination with sodium alginate gel [23]. The gel provided microspheres with bio-adhesion and our experimental result demonstrated that this delivery system can adhere to lungs for 21 days and has a satisfying restricted release effect. Unfortunately, weighty burden toward lungs could be caused by gelatinization of the sodium alginate carrier when it meets calcium ions. The solid-state gel could be coughed out by patients, limiting its practical utilization. For considerations of avoiding the use of gel carrier while preserving the adhesion of the delivery system, adhesive complex microspheres (CPM) have been prepared by spray drying in this undergoing study. Hydrophobic matrix carrier ethyl cellulose (EC) was used as the main sustained release carrier [24,25]; been commonly used in pharmaceutical and biomedical industries for its high biocompatibility and biodegradability, positive charged natural polysaccharide chitosan (CTS) was used to provide bio-adhesion to CPM [26–30]. In virtue of their good
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dispersion of suitable particle size for pulmonary delivery, CPM were expected to stay in lungs for an extended period. Rifabutin (RBT), an anti-tuberculosis drug with greater lipophilicity and stronger potency toward TB, has been loaded in CPM as the model drug [31–33]. A natural crosslinking agent genipin (GNP) was applied to control the release rate of CPM. In vitro/in vivo release behaviors were studied to clarify sustained release effects and retention properties of CPM. 2. Materials and methods 2.1. Materials EC with different viscosity (10, 20 and 45 cp) was kindly provided by Colorcon, China. CTS (Mw 190,000–310,000) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were purchased from Sigma–Aldrich, USA. GNP was supplied by Linchuan Zhixin Bio-Technology Co., China. RBT was purchased from Hubei Kangbaotai Fine-Chemical Co., Ltd., China. 96 wells plate was purchased from Corning, USA. All the reagents used here were analytical grade. 2.2. Animals Sprague–Dawley (SD) rats were obtained from Vital River Laboratories, China. All experiments were performed in accordance with international accepted guidelines on laboratory animal use, the Guide for the care and use of laboratory animals [34], the protocols were approved by the Beijing Animal Care Committee. 2.3. Cell lines Human lung A549 and Calu-3 airway epithelial cells were purchased from Institute of Basic Medical Sciences, Peking Union Medical College, China. A549 and Calu-3 cells were maintained in F12 and DMEM medium, respectively, both supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) non essential amino acid, 2 mM glutamine, 100 IU/mL penicillin and streptomycin. The medium was changed every 2 days and subcultured every 4 days. 2.4. Cytotoxicity study To compare cytotoxicity of three crosslinking agents, the A549 and Calu-3 cells were plated at a cell density of 3.5 × 105 cells per well in 96-well plates and incubated at 37 ± 1 ◦ C in an atmosphere of 5% CO2 . After 24 h of culture for A549 cells, the medium was replaced with fresh F12 medium containing glutaraldehyde (GTU), sodium tripolyphosphate (TPP) and GNP at the concentrations equivalent to 1 mg mL−1 . After 8 h and 24 h, the medium was discarded and the wells were washed twice with hanks balanced salt solutions (HBSS). 200 L MTT solution (0.5 mg mL−1 in PBS) was added to each well, and incubated 4 h at 37 ± 1 ◦ C for MTT formazan formation. Subsequently, the supernatant was carefully removed and the wells were washed twice with HBSS. 200 L DMSO was added to each well and the plates were then mildly shaken for 15 min to ensure the dissolution of formazan crystals. The optical density values were measured by using MQX200 microplate reader (Bio-Tek, USA) at wavelength 540 nm. Three replicates were read for each sample and mean value was used as the final result. The spectrophotometer was calibrated to zero absorbance using culture medium without cells. The relative cell inhibition ratio (%) related to control wells containing cell culture medium was calculated as:
Inhibition ratio (%) =
1 − [A]test [A]control
× 100%
531
After 24 h of culture for Calu-3 cells, the medium was replaced with fresh DMEM medium containing three crosslinking agents at 5 gradient concentrations, following with the same subsequent process for A549 cells.
2.5. Preparation of microspheres GNP-crosslinked CPM loading RBT were prepared by spraydrying of oil-in-water (O/W) emulsion, using a B-290 mini spray drier (Buchi, Switzerland) with a standard 0.5 mm nozzle. Spraydrying conditions were as follows: the peristaltic pump rate of 3.43 mL min−1 , compressed air flow rate of 536 L h−1 , inlet and outlet air temperature of 135 ◦ C and 85 ◦ C, respectively [35]. CTS was dissolved in 1% (v/v) acetic acid solution at 1% (w/v) concentration, then it was crosslinked by GNP for 2 h at 40 ◦ C. RBT was dissolved in ethyle acetate contained EC with different concentrations. Emulsions were prepared by stirring of the oil phase and the water phase. O/W emulsions were prepared differed in viscosity (10 cp, 20 cp and 45 cp) and concentrations (4% and 1%, w/v) of EC, RBT/EC ratios (1:5, 1:10 and 1:20) and concentrations (1% and 0.5%, w/v) of CTS. The emulsions were mixed for 10 min, in an organic/water phase ratios of 1:5 (v/v) and subjected to spray-drying under process conditions described above. Non-crosslinked CPM were prepared by directly using non-crosslinked CTS solution as water phase, following with the same subsequent preparation process. CTS microspheres were spray dried with the same parameters used in preparation of CPM, but only non-crosslinked and GNP-crosslinked CTS solution loading RBT (RBT/CTS = 1/5, w/w), without EC, were fed to the instrument.
2.6. Determination of RBT encapsulation efficiency and loading capacity RBT was extracted from the microspheres with mixture solution consisted of 0.1 M HCl and ethanol (3:2, v/v) under ultrasonic process for 1 h. The measurements were repeated in triplicate, with analysis by high-performance liquid chromatography (HPLC). Encapsulation efficiency (EE) and loading capacity (LC) were calculated as RBT detected by HPLC of the corresponding theoretical loading quantity and CPM weight, respectively. 0.1 M hydrochloric acid was used to extract RBT from CTS layer in 1 h ultrasonic process to compare the drug loading percentage between layers of CTS and EC.
2.7. Morphology and particle size distribution of microspheres The morphology of CPM and CTS microspheres were characterized by scanning electron microscopy (SEM). Samples were mounted on carbon taped aluminum stubs and gold coated by a sputter coater for 240 s, and they were observed using a JSM-6510 SEM (JEOL, Japan) at an accelerating voltage of 15 kV. The particle size distributions were characterized in a light scattering particle size analyzer (Mastersizer, England) using ethanol as dispersion medium. Three replicates of mean particle diameter and span were determined for each sample and mean value was used as the final result.
2.8. Zeta potential of microspheres Zeta-potential of the CPM and CTS microspheres was determined by photon-correlation spectroscopy (Zeta-sizer 2000, Malvern Instrument, England) in deionized water at 25 ◦ C. Three batches of each formulation were analyzed in triplicate (n = 3).
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2.9. Differential scanning calorimetry (DSC) Differential scanning calorimetry measurements were performed in a Q200 instrument (TA, USA). Accurately weighed samples (5 ± 0.1 mg) of CTS, EC, RBT and their physical mixture and CPM loading RBT were placed into a covered aluminum sample pans. An empty sample pan was used as reference and the runs were performed by heating samples from 20 to 450 ◦ C at a speed of 10 ◦ C min−1 . 2.10. In vitro release In vitro release test was carried out in pH7.4 phosphate-buffered saline (PBS) solution at 37 ± 0.5 ◦ C. 10 mg CPM or CTS microspheres were suspended in 5 mL release medium contained in the dialysis tubes, then they were put in erlenmeyer flask containing 45 mL mediums and incubated in a water bath instrument (SHAB(A), Jintankexi apparatus Co. Ltd., Jiangsu, China) shaken at 100 strokes min−1 for 8 days. At pre-determined time intervals, aliquots of 0.5 mL solutions were withdrawn and filtered through 0.45 m filters. The sample volumes were replaced with equal volume of the fresh medium. RBT released from CPM were quantified by HPLC, three tests were taken for each sample and mean value was used as the final result. 2.11. In vivo release SD rats were divided into two groups, after anesthetizing by pentobarbital sodium (60 mg kg−1 ), control group were administered by intra-tracheal intubation operation with 0.2 mL non-crosslinked CPM suspension and experimental group with the same volume suspension contained crosslinked CPM. Rats were sacrificed at predetermined time intervals, and then the tracheae and lungs were carefully separated and observed. Released drug was extracted by bronchoalveloar lavage (BAL). 2.5 mL PBS solution was injected into the lungs and then it was withdrawn immediately, this process was repeated four times. The lavage fluid was collected and vortex mixed 1 min with methylene chloride (2:1, v/v). The organic layer was separated by centrifuge at 3000 rpm for 5 min and then blown dry with N2 . Finally all samples were re-dissolved by adding 0.2 mL acetonitrile before being analyzed by HPLC. 2.12. HPLC condition RBT was determined using a pump L7100 and a Shimadzu SPD10AVP UV-detector (Shimadzu, Japan). A SLC-10AVP (Shimadzu, Japan) equipped with a Diamonsil ODS (4.6 mm × 150 mm, 5 m) analytical column was used. The column was kept at 40 ◦ C throughout the elution process, which used a mobile phase consisting of acetonitrile: 0.1 M KH2 PO4 (pH 6.45) = 65:35 (v/v) at a flow rate of 1.0 mL min−1 , and the detection wavelength was set to 278 nm. 2.13. Statistical analysis All data are presented as the mean ± standard deviation (SD). Statistical analysis was performed with the SPSS 17.0 software. Values of *P < 0.05 were considered statistically significant. 3. Results and discussion Currently, drugs can be administered by pulmonary route utilizing two techniques: inhalation and intra-tracheal instillation [36,37]. For inhalations, pharyngeal deposition of the drug has been the main problem, which leads to difficulty in measuring the dose inside the lungs and side effects caused by systemic circulation
Fig. 1. Cytotoxicity of three crosslinking agents.
when swallowed [38,39]. Moreover, the presence of partially suppressive drug concentrations promotes the growth of resistant TB [40]. In contrary, intra-tracheal instillation provides a simple, quantifiable way for pulmonary drug delivery and has been increasingly applied in clinical treatment of pulmonary tuberculosis. Microspheres have been widely reported that being applied as a proper formulation in pulmonary instillation administration. According to the simplification principle of formulation design, microspheres using CTS as the only carrier were prepared and evaluated in aspects of release rate and encapsulation efficiency. The investigations of CTS microspheres were regarded as basis for the study of CPM. 3.1. Cytotoxicity study of crosslinking agents (MTT assay) Serious burst effects were observed in previous study of CTS microspheres, therefore, a natural water-soluble crosslinking agent genipin (GNP) was applied in this study to control the release rate. Being extracted from the fruit of Gardenia Jasminoides Ellis, GNP was reported as a fully biocompatible reagent with lower cytotoxicity than traditional chemical crosslinking agent, such as GTU [41–43]. To verify the good biocompatibility of GNP, MTT assay on A549 and Calu-3 cells was carried out by comparing cytotoxicity of GNP to that of other commonly used crosslinking agents, TPP and GTU. When time extended from 8 h to 24 h (Fig. 1), the cytotoxicity of all three crosslinking agents appeared as time-dependent since the inhibition ratio raised (Fig. 1A), while IC50 value decreased (Fig. 1B). Lower cytotoxicity of GNP was shown by SPSS result as compared to GTU and TPP under 1% significance level (P = 0.01). Our experimental result was similar to those reported literatures [44,45]. Owing to its better safety and higher biocompatibility, GNP was used as the crosslinking agent to prepare CPM in the next step.
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Fig. 2. SEM observations of chitosan microspheres.
3.2. Preparation of complex microspheres The cumulative release percentage of RBT released from noncrosslinked CTS microspheres was 68.46 ± 2.87% in 24 h, which decreased to 58.25 ± 3.45% after crosslinking by GNP. Although the significant sustained release effect that GNP exerted on CTS microspheres, it was insufficient to attain our expectation: cumulative drug release percentage lower than 50% in 168 h (7 days). Similar results were observed even by alternating the weight ratio of GNP/CTS from 1/20 to 1/5. Therefore, developing a drug delivery system that can be used in intra-tracheal treatment, meanwhile carrying the functions of restricting drug release and bio-adhesion was the challenge of this study. For these considerations, CPM were prepared in the way that EC functioned like a “core”, encapsulating the hydrophobic model drug RBT and controlling the drug release rate; CTS functioned as the bio-adhesive “Shell”, promoting pulmonary retention property of the delivery system.
3.3. Encapsulation of rifabutin The permeation of RBT from organic phase to water phase during the emulsion process caused part of the drug to be encapsulated in CTS layer. Due to the hydrophilic swelling of CTS, drug would be quickly released from CTS layer as compared to that from EC layer. Theoretically, biphasic drug release would be expected to observe. The quick release phase increased pulmonary drug concentration to therapeutic level in a short time, while sustained release phase helped to maintain the concentration level for an extended period. Factors include different viscosities of EC, EC and CTS at different concentrations and different RBT/EC weight ratios were used to optimize the preparation process of CPM by studying their possible influences on the encapsulation efficiency (EE) and drug loading capacity (LC), as reported in Table 1. Higher EE was observed at 20 cp viscosity and 1% (w/v) concentration of EC, with the EC/drug weight ratio of 5/1. Drug permeation from EC layer to CTS solution was impeded as the viscosity of EC increased from 10 cp to 45 cp, theoretically increasing EE and LC of CPM. Nevertheless, EC with a higher viscosity (45 cp) was difficult to be completely dissolved in ethyl acetate, causing inaccuracy to the preparation process. The inaccuracy possibly accounts for the results that CPM prepared with 20 cp EC had higher EE and LC than that of CPM prepared with 45 cp EC. The results of higher LC were observed in groups of CPM prepared with EC at 4% (w/v) concentration, as compared to that of CPM prepared with EC at 1% (w/v). The different drug inputting amounts, 0.16 g RBT for 4% (w/v) EC and 0.04 g RBT for 1% (w/v) EC, accounted for the results. The optimized parameters for the preparation of CPM were as followed: 20 cp and 4% (w/v) EC, RBT/EC ratios of 1:5 and 1% (w/v) CTS.
The crosslinking effect of GNP significantly improved EE and LC of CPM, which can be also reflected in Table 1. Maintaining all the other parameters constant, EE and LC were increased for CPM crosslinked by GNP. It is thought that the viscosity of CTS solution increased after crosslinking, preventing RBT permeation from EC into CTS. To demonstrate this supposition, the percentages of RBT encapsulated in CTS layers were compared between non-crosslinked and crosslinked CPM, which decreased from 27.43 ± 2.47% to 17.62 ± 2.05% after crosslinking. GNP crosslinking effect on CTS was investigated in pilot test and it was observed that the viscosity of CTS solution gradually increased as the GNP crosslinking reaction continued and the solution finally transformed to a semi-solid gel. This phenomenon was similar to the report of Cao et al. [46]. Therefore, 40 ◦ C was set as the crosslinking temperature to prepare CTS microspheres and CPM. Furthermore, EE and LC of CTS microspheres were higher as GNPcrosslinking time prolonged. The crosslinking degree was inferred to increase from 0.5 h to 4 h, promoting the amount of drug encapsulated in CTS. The maximum crosslinking time for CPM was 2 h, in order to maintain the proper viscosity of the solution for the emulsion process. 3.4. Characterization of complex microspheres 3.4.1. SEM observation CTS microspheres were observed under SEM as a contrast. It can be seen in Fig. 2, both groups of non-crosslinked (Fig. 2A) and GNP crosslinked (Fig. 2B) CTS microspheres had uniform and spherical shape. Shown in Fig. 3, all CPM were spherical, but instead of plicated features of CTS microspheres, only compact layers were observed on surfaces of non-crosslinked (Fig. 3A) CPM, while the surfaces of GNP crosslinked CPM (Fig. 3B) were relatively smoother. This can be attributed to the theoretical “shell” structure of CTS formed on the surfaces of EC “core”. 3.4.2. Particle size distribution As shown by Fig. 4A, the decreased mean particle diameter of CPM was inferred as the result of crosslinking effect, tightening intermolecular distance of CTS. The increased span of particle distribution was possibly as a result of higher viscosity of CTS solution after crosslinking, lowering the uniformity of emulsion droplets when sprayed from the nozzle. Similar results were observed in characterization of CTS microspheres, which can be seen in Fig. 4B. 3.4.3. Zeta potentials Due to the electrostatic interaction between its positive amino groups and anionic substructures in mucous layer or epithelial cells, CTS has been extensively studied as a bio-adhesive material. In this study, zeta potentials of EC microspheres were measured as contrast, which was −19.73 ± 0.64 mV. The presence of CTS at the
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Table 1 Effects of polymers viscosity and concentration (w/v), polymers/drug ratios (w/w) and crosslinking agent GNP on encapsulation efficiency (EE) and loading capacity (LC) of complex microspheres prepared by spray drying. EC viscosity (cp)
EC concentration (%, w/v)
10 10 10 20 20 20 20 20 20 45 45 45
4 1 4 4 4 1 4 4 4 4 1 4
EC/drug ratios (w/w) 5:1 5:1 5:1 5:1 5:1 5:1 10:1 20:1 5:1 5:1 5:1 5:1
CTS concentration (%, w/v)
GNP (g)
EE (%, w/w)
1 1 1 0.5 1 1 1 1 1 1 1 1
– – 0.1 – – – – – 0.1 – – 0.1
45.10 47.06 59.40 52.45 72.63 75.53 54.72 30.42 78.65 61.21 64.68 70.08
± ± ± ± ± ± ± ± ± ± ± ±
3.71 3.95 2.59 2.77 3.22 2.55 2.85 0.96 2.69 2.86 3.85 3.14
LC (%, w/w) 3.66 1.53 4.61 5.75 5.95 2.44 2.33 0.66 6.11 5.00 2.07 5.44
± ± ± ± ± ± ± ± ± ± ± ±
0.46 0.32 0.62 1.06 0.84 0.42 0.37 0.03 0.76 0.63 0.28 0.87
Fig. 3. SEM observations of complex microspheres.
surface of the CPM could be indicated by the positively charged CPM. The crosslinking effect markedly decreased the zeta potential of CPM from 33.60 ± 3.32 mV to 28.97 ± 1.37 mV. Similar results were observed in the study of non-crosslinked and GNP crosslinked CTS microspheres, demonstrating that free amine groups of CTS being bound by GNP. Moreover, zeta potential of CPM was lower than that of CTS microspheres, indicating the possible presence of negative charged EC on the surface. 3.4.4. DSC measurement DSC curve of the CPM loading RBT was drawn in comparison to that of simple EC, CTS, RBT and their physical mixture (Fig. 5). Exothermic peaks were observed around 241DSC curve of the CPM loading RBT was drawn in comparison to for RBT, 313 ◦ C for CTS, 199 ◦ C and 389 ◦ C for EC, attributing to their chemical degradation, respectively. The two exothermic peaks of EC could be explained by its stepwise degradation. All typical exothermic peaks of three pure samples corresponded well with physical mixture. However, only typical exothermic peaks of CTS and EC samples were observed in the curve of CPM loading RBT, indicating the formation of microspheres and its encapsulation effect on RBT. 3.5. In vitro release of complex microspheres
Fig. 4. Particle size distribution.
RBT was demonstrated to be stable in PBS for 14 days at 37.5 ◦ C under static condition, however, its degradation was observed after 48 h in vitro release test. Situations such as different release medium (PBS or de-ionized water), different temperature (25 ◦ C or 37.5 ◦ C) and adding different antioxidants (sodium citrate or sodium sulfite) were examined. No significant improvement to RBT stability was observed. According to the study of Sangshetti et al. [47], RBT was unstable under oxidation condition. Shaking action
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produced by the bath instrument was assumed to cause a more oxidized environment by increasing the contact area between air and release medium. This caused more oxygen to dissolve in the medium than a static system. Similar result was also observed in release study using paddle method with similar action mechanism of stirring. As a result, a modified in vitro release method was performed. This method involved the release medium being replaced with fresh PBS every 24 h, within which RBT was proved to be stable even under shaking action. At the end of the test, microspheres in the dialysis tubes were collected and the encapsulating RBT was extracted for quantification. The sum of drug amount detected in release medium and extracted from microspheres corresponded well with theoretical drug loading capacity. In vitro drug release of RBT-loaded CPM was studied for 192 h at pH 7.4 PBS. Viscosity was chosen as 10 cp, 20 cp and 45 cp while the concentration was 4% and 1% (w/v) respectively. In this study, we compared the release rate of CPM made of EC with each combination (Fig. 6A). In the first four hours, 3.38% of RBT was released for CPM made of 10 cp EC. The percentage of RBT released increased to 15.57% in 24 h followed by a gradual and sustained release up to 20.27% in 192 h. The drug release behavior showed a biphasic pattern indicated by its initial quick release, followed by a very slow release. The initial quick release could be explained by the hydrophilic swelling of CTS, facilitating the drug diffusion from CTS layer to the medium. In 24 h to 192 h, release may have occurred due to the diffusion of drug through CTS corrosion and EC matrix of CPM. This biphasic release profile was also observed in CPM made of 20 cp and 45 cp EC. The release behavior was similar to the biphasic profile discussed above from the curves. The drug release rate of CPM made of 4% EC concentration was significantly faster, due to its higher drug loading rate. To meet the potential clinical requirement, CPM made of 10 cp EC was selected as the subject of in vivo release study. It was demonstrated that in vitro release rate of CPM crosslinked by GNP was significantly slower than non-crosslinked CPM. As shown in Fig. 6B, cumulative release percentages of GNP crosslinked and non-crosslinked CPM were compared, with a result of 1.16% to 3.38% in the first 4 h, 7.89% to 15.57% in 24 h and 10.72% to 20.27% in 192 h, respectively. It is reasonable to infer that the crosslinking effect restricted the drug release by
Fig. 5. DSC study.
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Fig. 6. In vitro release study.
tightening the CTS molecular chain segments, forming more stable heterocyclic-intra-/intermolecular structures. This results in a limitation of hydrophilic pathways in CTS layers thus obstructing the penetration of the medium to trigger swelling action. Also the drug release is decelerated. 3.6. In vivo release Bronchoalveolar lavage (BAL) method was used to extract free RBT from the lungs of administered rats [48,49]. After administration of CPM, lungs were separated to be injected with PBS. The lavage fluid was collected and free drug was extracted by dichloromethane. It is interesting to note that in vivo release curves of noncrosslinked and GNP crosslinked CPM both had double peaks characteristics (Fig. 7). In first 4 h, GNP crosslinking effect significantly slowed the in vivo drug release rate, which corresponded well with in vitro release study result. The initial burst occurred as expected and the RBT load in CTS external layer released
Fig. 7. In vivo release study.
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quickly. In 4–24 h, there was no significant difference between noncrosslinked and GNP crosslinked CPM. The crosslinking effect of GNP on CTS could be weakened by taking the more complicated in vivo environment into account such as enzymes in lungs [50]. It has been reported that CTS mainly be degraded by lysozyme, an enzyme which is synthesized and secreted by surface epithelial cells and macrophages in human airways [51–55]. In 24–192 h, the drug was released faster from GNP crosslinked CPM. The mechanism of diffusion through EC layer following concentration gradient was considered to be the leading role in this stage of in vivo release, therefore GNP crosslinked CPM had a faster release rate for its higher LC. In 596 h (24th day), the local concentration of RBT in lungs was 0.68 ± 0.15 g mL−1 and 1.54 ± 0.23 g mL−1 for noncrosslinked and GNP crosslinked CPM, respectively. They were both higher than the minimal inhibitory concentration (MIC) of RBT, which is 0.015–0.06 g mL−1 [56]. CPM were observed adhering to lungs in bronchoalveolar lavage process through the whole in vivo release time line, demonstrating its good pulmonary retention property. Two peaks – in vivo release curves of CPM indicated that the microspheres exerted such a long pulmonary retention. This was because free drug would quickly be absorbed by pulmonary alveolar and capillaries and then transferred to systemic circulation. The time range of increased drug concentration, 0–1 h and 72–360 h, indicated the drug continuously released from microspheres. As shown by the time range 4–72 h and 360–576 h, decreased drug concentration manifested that the drug absorption rate was faster than drug release rate. The twopeaks in vivo release profile strongly demonstrated that microspheres led to lung pulmonary retention. This data permitted us to conclude that the CPM delivery system could effectively sustain the RBT release and help maintain the local drug concentration at a therapeutic level at length. 4. Conclusion This study has demonstrated that it was feasible to prepare the RBT-loaded CPM made of ethyl cellulose and CTS with spray drying method. The influences of various specifications of ethyl cellulose, CTS and different amount of model drug on encapsulation efficiency and loading capacity were studied. The CPM exhibited sustained release behaviors over an extended period and its initial burst release was restricted by the crosslinking effect of genipin. The result of in vivo release study further demonstrated that the CPM possessed biphasic release and long time pulmonary retention features. We hope this study will be fruitful for the clinical treatment for pulmonary tuberculosis. Acknowledgements This work was supported by the Foundation of 12th Five-Year Important National Science & Technology Specific Projects. Financial support from National Science &Technology Major Special Project on Prevention and Cure of Acquired Immune Deficiency Syndrome and Virus Hepatitis. (Item No. 2012ZX10003009-001002) is gratefully acknowledged. Special thanks to Chunhui Hu whose work paved the way for our research. References [1] P. Nahid, D. Menzies, Update in tuberculosis and nontuberculous mycobacterial disease 2011, Am. J. Respir. Crit. Care Med. 185 (2012) 1266–1270. [2] Short-course chemotherapy in pulmonary tuberculosis, A controlled trial by the British Thoracic and Tuberculosis Association, Lancet 1 (1975) 119–124. [3] V.I. Chukanov, O.G. Komissarova, V. Maishin, R. Abdullaev, A.S. Kononets, Efficiency of a new standard chemotherapy regimen in the treatment of patients with recurrent pulmonary tuberculosis, Probl. Tuberk. Bolezn. Legk. 8 (2006) 9–13.
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