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sodium alginate in situ gel combination delivery system
Colloids and Surfaces B: Biointerfaces 95 (2012) 162–169
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Preparation and characterization of rifampicin-PLGA microspheres/sodium alginate in situ gel combination delivery system Chunhui Hu, Hanzhou Feng, Chunyan Zhu ∗ Department of Drug Delivery Research Center, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 151 Malianwa North Road, Haidian District, Beijing 100193, PR China
a r t i c l e
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Article history: Received 8 November 2011 Received in revised form 21 February 2012 Accepted 21 February 2012 Available online 3 March 2012 Keywords: Alginate PLGA Biodegradable polymers Microspheres Controlled release Drug delivery system
a b s t r a c t We prepared a complex drug delivery system consisted of rifampicin-poly(lactic-co-glycolic acid) (PLGA) microspheres in combination with sodium alginate in situ gel. The microspheres were obtained by using a solvent evaporation method, the mean diameter was 1.748 m and the span of particle distribution was 0.78. The combination delivery system was obtained by adding microspheres to sodium alginate solution followed by physically mixing. In an in vitro study of drug release monitored for 11 days, the release of rifampicin from combination delivery system was slower than microspheres. The cumulative release percent of rifampicin from combination delivery system was 91.83 ± 1.26%, which was lower than 97.36 ± 3.41% of rifampicin released from microspheres. An in vivo fluorescence imaging study suggests that the gel adhered to lungs within 24 h, and microspheres stayed in lungs at least for 504 h (21 days). In vivo drug release study indicates that the maximum local rifampicin concentration in lungs was 48.60 ± 15.67 g mL−1 5 h after administration. After 21 days, the local rifampicin concentration was 0.81 ± 0.14 g mL−1 , which was above the minimum inhibitory concentration of rifampicin. The combination delivery system significantly prolonged RFP release compared to microspheres, from which RFP released could only be detected for 10 days. This approach to control the release of rifampicin using PLGA microspheres/in situ gel combination delivery system in conjunction with interventional technology is useful for improving anti-tuberculosis treatment effectiveness for patients. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Tuberculosis (TB) is a common and in many cases lethal infectious disease caused by various strains of Mycobacteria, usually Mycobacterium tuberculosis. Globally, there were an estimated 13.7 million chronic active cases, 9.27 million incident cases of TB and 1.8 million deaths. This is an increase from 9.24 million cases in 2006, 8.3 million cases in 2000 and 6.6 million cases in 1990. One-third of the world’s population is thought to be infected with M. tuberculosis and new infections occur at a rate of about one per second [1]. Tuberculosis usually attacks the lung, so improving drug pulmonary concentration has been one of the goals in anti-tuberculosis study areas. There are reported studies that focus on improving drug concentration in the lung, such as lung-targeting microspheres [2,3], nanoparticles [4,5], aerosolized liposome [6] and aerosols for inhalation administration [7–9]. However, a possibility has not been well studied. Anti-tuberculosis
Abbreviations: CDS, combination delivery system; MS, microspheres; HPLC, high-performance liquid chromatography; BAL, bronchoalveolar lavage. ∗ Corresponding author. Tel.: +86 10 57833276; fax: +86 10 57833276. E-mail addresses: [email protected], [email protected] (C. Zhu). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2012.02.030
drug administered in these ways could distribute to other nontargeted organs and may cause adverse and side effects. Hence, it is of importance to seek a substituted administration method. Interventional technology has been applied commonly in clinical treatment of tuberculosis because drug can be delivered directly to the lung instead of through blood vessel system. But the retention property of liquid or suspension, two commonly used formulations in interventional technology, is not satisfying because they cannot persistently stay in the lung [10]. Owing to this limitation, tuberculosis patients have to receive interventional operation frequently in order to maintain a therapeutic drug concentration. The objective of the present work was to improve patient compliance by developing a new drug delivery system that can be successfully used in interventional technology. To attain our purpose, the delivery system should include some necessary abilities: First, materials used with good biocompatibility and biodegradability. Second, good syringeability. It should be injectable for interventional use [11]. Third, a quick phase shift. After administration, it must adhere to lungs by transforming from a liquid state to semisolid. Fourth, sustained release. It is favorable to reduce the administration frequency by restricting drug release. For above concerns, we chose ion-sensitive in situ gel, sodium alginate, combined with poly(lactic-co-glycolic acid) (PLGA) microspheres as an
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appropriate drug carrier in this study [12–14]. In the presence of calcium, sodium alginate can form resilient gel, it has been widely used for encapsulation of drug in pharmaceutical areas. PLGA is a copolymer with demonstrated safety used in a host of Food and Drug Administration (FDA) approved therapeutic devices [15]. PLGA has been successfully used as a biodegradable polymer because it undergoes hydrolysis in the body to produce the original monomers, lactic acid and glycolic acid [16]. Under normal physiological condition, they are by-products of various metabolic pathways in the body. Since the body effectively deals with the two metabolic products, there is very minimal systemic toxicity associated with using PLGA for drug delivery or biomaterial applications. There are already a lot of reports that focus on using PLGA to prepare microspheres loaded with rifampicin (RFP), a semisynthetic bactericidal antibiotic drug that was also selected as model drug in our study. Ito et al. prepared RFP-PLGA microspheres by solvent evaporation method with a membrane emulsification technique using Shirasu Porous Glass (SPG) membranes [17]. Doan et al. applied solvent evaporation method with premix membrane homogenization to prepare RFP-PLGA microspheres with 80% RFP released from 12 h to 4 days [18]. Keiji Hirota’s study demonstrated that PLGA microspheres loaded with RFP are bio-safe due to their “silent” nature when taken into macrophage cells and are promising for treating tuberculosis [19]. Although previous reported studies have investigated many aspects of RFP-PLGA microspheres, such as preparation process, in vitro release and related delivery efficiency to alveolar macrophage cells, few studied preparing combination delivery system (CDS) containing RFP and its related properties. In the present work, RFP was encapsulated in PLGA microspheres in the first step. Thereafter, RFP-PLGA microspheres were dispersed in sodium alginate gel to form CDS as the final formulation. Fluorescence imaging study was conducted to explore its actual in vivo retention property. In vitro and in vivo drug release of CDS was studied to investigate its drug release-restricting ability. 2. Materials and methods 2.1. Reagents and chemicals PLGA with a molecular weight of 55,300 and having a monomer composition of lactic acid: glycolic acid of 50:50 was purchased from Birmingham Polymers company (USA). Rifampicin standard was purchased from Sigma–Aldrich (USA). Rifampicin sample was purchased from NOVO (USA) and used as an anti-tuberculosis drug. Polyvinyl alcohol (PVA) was purchased from Sinopharm Chemical Reagent Co. (China). Sodium alginate was purchased from FMC BioPolymer (USA). CaCl2 was purchased from Beijing Fengli Jingqiu Trade Co. (China). CY5.5 (excitation and emission wavelength = 670 and 760 nm) was purchased from Beijing Fanbo Biochemicals CO. (China). DIR (excitation and emission wavelength = 790 and 830 nm) was purchased from Invitrogen (USA). Other chemicals were of the highest grade commercially available.
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laboratory animal use, the guide for the care and use of laboratory animals (Institute of Laboratory Animal, Washington, D.C., National Academy Press, 1996). 2.3. Preparation of RFP-loaded PLGA microspheres RFP-PLGA microspheres were prepared using a solvent evaporation method [20,21]. 1 mg RFP was dispersed in 10 mL methylene chloride containing 10% (w/v) PLGA by sonication. The organic phase was then emulsified with magnetic stirring in 200 mL aqueous phase containing 1% (w/v) polyvinyl alcohol (PVA). Stirring was continued for 4 h until the methylene chloride was completely evaporated. The system was protected from light. The resultant dispersion was centrifuged at 12,000 rpm for separation of the microspheres. The obtained sediments were washed four times with de-ionized water, and collected by filtration, then subjected to freeze drying overnight. 2.4. Particle size analysis A light scattering particle size analyzer (Mastersizer 2000, London, England) was used to determine the mean particle diameter. Approximately 50 mg of microspheres were dispersed in 5 mL of hexane by sonication. 1 mL of the dispersion was used for particle size analysis. All measurements were conducted in triplicate. 2.5. Microsphere morphology Microsphere morphology and shape were characterized using a scanning electron microscopy (SEM). Samples were mounted on carbon taped aluminum stubs and gold coated in a sputter coater for 1 min. The samples were analyzed using a JSM-6510 SEM (JEOL, Japan) at an accelerating voltage of 5.0 kV. 2.6. Differential scanning calorimetry of microspheres RFP sample, blank PLGA microspheres, RFP and PLGA physical mixture and RFP-PLGA microspheres were examined by a DSC analyzer (Q200, TA, USA), respectively. The heating rate was 10 ◦ C min−1 , heating temperature range was 30–300 ◦ C, nitrogen flow velocity was 50 mL min−1 . 2.7. Preparation of sodium alginate in situ gel loaded with PLGA microspheres A certain amount of sodium alginate was added to de-ionized water and dissolved by stirring for 24 h [22]. The microspheres/gel combination delivery system (CDS) was obtained by adding microspheres to the in situ gel solution followed with physically mixing well. The CDS prepared in this way was a solution containing suspended microspheres. After gelation with calcium ions, it transformed from liquid state to semisolid gel. 2.8. Studies of rheological properties of RFP-microspheres/gel mixture
2.2. Animals Nu/Nu mice (male, 20 g) and SD rats (male, 250 g) were obtained from Vital River Laboratory Animal Technology Co. (Beijing, China). Mice and rats weighing about 20 g and 250 g respectively were randomly assigned to each experimental group. They were allowed free access to water but forbidden to food 12 h before experiments. The experiments were carried out after approval by the ethical committee at Institute of Medicinal Plant Development in accordance with internationally accepted guidelines on
The rheological property of microspheres/gel CDS was studied using a Physica MCR300 rheometer (Physica, Germany). The sample volume was about 20 mL. The measurements were made at room temperature. Different amounts (5 × 10−5 mol, 1 × 10−4 mol, 2 × 10−4 mol, 3 × 10−4 mol) of CaCl2 solution were added to the mixture and mixed well before being measured. Viscosity measurements were made at room temperature for shear rates between 0.1 and 100 s−1 [23]. Moreover, we compared the microspheres/gel CDS with blank sodium alginate in situ gel (after gelation with
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Fig. 1. SEM pictures of RFP-PLGA microspheres A – 2700×; B – 5000×.
calcium ions) to examine whether microspheres affect the viscosity of the delivery system. The flow behavior of the CDS was assessed in terms of their syringeability by withdrawing 1 mL of the CDS through a hypodermic needle of dimensions 0.45 mm × 19 mm (26G) [24]. 2.9. HPLC determination of RFP The concentration of RFP was determined using a pump L7100 and a Shimadzu SPD-10AVP UV-detector (Shimadzu, Japan). A SLC-10AVP (Shimadzu, Japan) equipped with a Hypersil ODS (4.6 mm × 150 mm, 5 m) analytical column was used. The column was kept at 30 ◦ C throughout the elution process, which used a mobile phase consisting of methanol:acetonitrile:0.075 mol L−1 KH2 PO4 :1 mol L−1 citric acid (30:30:26:4%, v/v/v/v) at a flow rate of 1.0 mL min−1 , and the detection wavelength was set to 254 nm. The linear calibration curve (y = 1455x + 2714.8; R2 = 0.9993; standard solution 0.27–34.2 g mL−1 ). 2.10. In vitro drug release We compared the release behaviors of RFP from microspheres and microspheres/gel CDS, respectively. RFP-microspheres (30 mg) were dispersed in blank gel solution (10 mL). Thereafter, CaCl2 solution was added to gelatinize sodium alginate. The gelatinized CDS was put in a group of dialysis tubes which were separating the CDS from the dissolution medium. Another group of dialysis tubes contained only RFP-PLGA microspheres (30 mg). 3 mL dissolution medium (1%EDTA–2Na + 1%Na2 SO3 ) was added all tubes, then they were put into stirring baskets. The study was carried out in 200 mL dissolution medium, the system was temperature controlled at (37 ± 0.5) ◦ C with a stirring rate at 100 rpm. 1 mL samples was withdrawn and replaced with fresh medium at predetermined time intervals. The samples were analyzed using HPLC as described in Section 2.9. All release experiments were performed as triplicates. The release behavior of RFP from microspheres and microspheres/gel CDS were analyzed based on various mathematical models.
(CY5.5-CDS) and DIR-PLGA microspheres combined with sodium alginate gel (DIR-CDS). After being anesthetized by pentobarbital sodium, two groups of Nu/Nu nude mice (about 20 g) were administered with two formulations by endotracheal intubation operation. The gel was gelatinized by pulmonary injection of 0.1 mol L−1 CaCl2 solution right after administration of CDS. The retention property of two marked formulations was measured by observing mice in the in vivo imaging system at different pre-determined time intervals [29]. 2.12. Studies of in vivo release SD Rats were divided into two groups. The control group was administered with 0.2 mL RFP-PLGA microspheres suspension (microspheres dispersed in physiological saline), the other group was administered with 0.2 mL RFP-CDS. Both two formulations theoretically contained 10 mg microspheres. After anesthetized by pentobarbital sodium (60 mg kg−1 ), two groups rats were administered with different formulations separately by endotracheal intubation operation. Rats were sacrificed at pre-determined time intervals, and then tracheae and lung were carefully separated. Afterward, free drug was extracted by bronchoalveolar lavage (BAL) [30]. We injected 2.5 mL physiological saline into the lung and withdrew it immediately, the process was repeated four times. The lavage fluid was collected and vortex mixed 30 s with methylene chloride (2/1, v/v), then the organic layer was separated by centrifuge at 12,000 rpm for 3 min. After dryness with nitrogen, the sample was re-dissolved by adding 0.2 mL acetonitrile before being analyzed by HPLC. 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
2.11. Studies of in vivo retention
3.1. Microsphere formulation
For the purpose to examine the retention property of gel and microspheres [25], a Kodak in vivo multispectral imaging system was applied. A fluorescent agent CY5.5 was used to mark the in situ gel formulation [26], which was added to sodium alginate solution and physically mixed well. Another fluorescent marker DIR was used to replace RFP as the model drug to prepare DIR-PLGA microspheres by the same method as we described in the part of Section 2.3 [27,28]. So there were two formulations: blank PLGA microspheres combined with sodium alginate gel marked by CY5.5
Fig. 1 shows SEM photographs of RFP-loaded PLGA microspheres. The microspheres were spherical. The particle distribution of RFP-PLGA microspheres, shown in Fig. 2, was as follows: D10% = 1.372 m, D50% = 1.748 m and D90% = 2.727 m, respectively (D10, D50 and D90 mean 10%, 50% and 90% of the total microspheres we prepared, respectively). The mean diameter of the microspheres was 1.748 m and the span of particle distribution was 0.78. This result shows that it is feasible to use the O/W emulsion solvent evaporation method to prepare RFP-PLGA
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Fig. 2. Particle distribution of RFP-PLGA microspheres. D10, D50 and D90 means 10%, 50% and 90% microspheres detected respectively of the total microspheres we prepared.
microspheres. Although there are new methods to prepare PLGA microspheres, such as spray drying [31,32] and SPG membrane emulsification [33,34], emulsion solvent evaporation is a more simple way to apply in this study. In addition, Fig. 3 shows that there are similar exothermic peaks at 280 ◦ C for RFP sample and its physical mixture with PLGA, while there is no similar peak for RFP-PLGA microspheres. This result suggests that the RFP was encapsulated in PLGA microspheres [35]. RFP-PLGA microspheres with diameter 1.748 m were investigated in this current study, microspheres with other various diameters will be examined in future. We will study the effect of different diameters on in vitro and in vivo drug release. 3.2. Preparation of sodium alginate in situ gel and combination delivery system By forming a viscous skeleton carrier after gelation, in situ gel can effectively delay the touch between microspheres and body liquid, which is beneficial to restrict drug release. Adding calcium ions to sodium alginate solution was used to attain this purpose and we investigated the relation between the amount of calcium ions added and the viscosity of the delivery system [36,37]. The combination delivery system had the strongest viscosity when adding 2 × 10−4 mol CaCl2 . Its viscosity lowered when we kept adding CaCl2 . As a result, using 10−5 mol CaCl2 to gelatinize 1 mL mixture was the best ratio for our further study. Fig. 4 shows that the viscosity of the gel decreased as the shearing rate increased, it demonstrates that the CDS was a pseudoplastic fluid. The result of syringeability study indicates that the CDS (before gelation) can be easily injected through 26G syringe needle. Some reported studies used in situ gel as a mucoadhesive skeleton carrier to load drug, aimed to control drug release [38,39]. But burst effect was a common problem if gel acted as the only drug carrier. There was a lag time during the gelation process, which means that the gel failed to transform from liquid state to semisolid
Fig. 3. DSC measurement of (A) physical mixture of RFP and PLGA; (B) RFP-PLGA microspheres; (C) blank PLGA microspheres; (D) PLGA sample.
Fig. 4. Viscosity change of microspheres/hydrogel combination delivery system. (A) Blank sodium alginate in situ gel; (B) adding 5 × 10−5 mol CaCl2 ; (C) adding 1 × 10−4 mol CaCl2 ; (D) adding 2 × 10−4 mol CaCl2 ; (E) adding 3 × 10−4 mol CaCl2 .
instantly and thus drug could be promptly released from the carrier. Consequently, we applied PLGA microspheres to control the drug release in order to avoid burst effect. The materials we used in preparing the delivery system were all pharmaceutical adjuvant approved by FDA, because good safety is the prerequisite to develop a formulation aims to be used in clinical treatment. 3.3. In vitro RFP release from microspheres and combination delivery system In vitro release profiles of RFP in microspheres (RFP-MS) and the combination delivery systems (RFP-CDS) were monitored for 11 days. By comparing two release profiles (Fig. 5), we found that there was significant difference between them. The total release percent of RFP-PLGA microspheres and RFP-CDS were 97.36 ± 3.41% and 91.83 ± 1.26%, respectively. Fig. 6 shows that from 0 h to 24 h, the cumulative release percent of RFP-MS and RFP-CDS were 22.83 ± 2.14% and 15.15 ± 0.74%, respectively. The release percent of RFP-MS was significantly higher than that of RFP-CDS. But from 24 h to 72 h, the cumulative release percent of RFP-MS and RFPCDS were 22.93 ± 4.73% and 22.98 ± 3.66%, respectively. There was no significant difference between them as the same result for the release from 24 h to 264 h (1–11 days). In our previous erosion study, which is not contained in this paper, sodium alginate gel (after gelation with calcium ions) completely eroded within 24 h. Thus, it can be reasonably inferred that the release profiles for CDS was consisted of two parts: The first step, in situ gel eroded gradually and microspheres were released from this skeleton carrier at the same time. The sustained release of drug from PLGA microspheres was the second step. The difference between two release profiles of the two formulations got explained in this way. At first, the gel blocked the touch between RFP-MS and the release medium. As the gel eroded gradually, increasing amount of microspheres released. After the gel completely eroded at 24 h, all microspheres
Fig. 5. In vitro release of RFP from MS (microspheres) and CDS (combination delivery system) (mean ± SD, n = 3).
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Fig. 6. Comparison of in vitro RFP release from microspheres and combination delivery system (mean ± SD, n = 3). *P < 0.05 versus in vitro release of drug from microspheres (MS). Table 1 The release equation of RFP from microspheres and CDS. Model
Zero-order First-order Higuchi
Microspheres
CDS 2
Equation
R
Equation
R2
y = 0.3034x + 34.524 y = −0.0137x + 4.3839 y = 5.8933x + 13.416
0.7538 0.9375 0.9808
y = 0.2982x + 27.731 y = −106.63x + 470.16 y = 0.1701x − 1.0407
0.8099 0.9632 0.9725
were released and they had a similar release rate with RFP-MS because the release behavior of microspheres was based on a diffusion mechanism (by fitting the release data to Higuchi equation, shown in Table 1). So, this was the reason why there was no significant difference in two release profiles from 24 h to later time. In addition, the 0–24 h cumulative release percent of microspheres was 12.23 ± 0.70%, which indicates that there was no burst effect. 3.4. In vivo retention property In vivo imaging in general allows a non-invasive insight into living organisms and helps to understand metabolic processes and
disease related changes [40,41]. By using this technology, we can focus on the same subject investigated and avoid individual difference. CY5.5 and DIR were commonly used fluorescent markers [42], their emission wavelengths belong to infrared wavelength zone [43], which means good penetrability. Hydrophilic CY5.5 was used to mark water soluble sodium alginate gel, while RFP was replaced by a hydrophobic fluorescent agent DIR in order to mark microspheres. Fig. 7 shows strong fluorescence occurred at the lung of mice that were administered CY5.5-CDS immediately. The fluorescence had gradually intensified since 0 h, and it obviously weakened at 24 h. The fluorescence disappeared when we observed the mice at the 36 h after administration. The results indicate that the sodium alginate gel was able to adhere to lungs within 24 h. Besides, we observed fluorescence with increasing intensity at hypogastric regions of all tested mice since 1 h. We infer that the regions were bladders, because hydrophilic CY5.5 was metabolized by kidney and the metabolic products were excreted in the urine. Accordingly, we can observe increasing fluorescence there while it was decreasing at lungs. The mice administered DIR-CDS were monitored for 672 h (28 days). Fig. 8(1) and (2) shows that strong fluorescence immediately appeared at lungs of all tested mice right after administration. The fluorescence had weakened from 336 h to 504 h (14th to 21st day), and we almost observed nothing at the final pre-determined time 672 h (28th day). The results suggest DIR-PLGA microspheres may retain at lungs within at least 21 days because we observed weak fluorescence till 504 h. But the microspheres may adhere to lungs even after all DIR was released, e.g. longer than 672 h. To clarify this problem, we went a step further to investigate in vivo release of drug. Besides, we observed no fluorescence at hypogastric regions of mice after administration of DIR-CDS. A possible reason to account for this is that DIR is hydrophobic and its metabolic products are easily reabsorbed by kidney tubule, thereby excreted into intestinal tract in the bile. As a result, DIR is likely eliminated from the body as excrement and herein we failed to observe any fluorescence at the expected locations of mice. To truly explain this problem, determination of them in excrement and urine would be needed, but it is not covered by this current study.
Fig. 7. In vivo fluorescence imaging: the retention property of mice after administration of CY5.5-CDS for 0–36 h (n = 3).
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Fig. 8. (1) In vivo fluorescence imaging: the retention property of mice after administration of DIR-CDS for 0–36 h (n = 3). (2) In vivo fluorescence imaging: the retention property of mice after administration of DIR-CDS for 72–672 h (n = 3).
3.5. In vivo release Although tissue homogenization is a normal method to extract drug in organs [44–47], there is a problem if using it in our study. The process of homogenization will destroy microspheres adhered to lungs, which will cause leakage of drug encapsulated in the microspheres and thus increase drug’s concentration determined. To avoid the possible inaccuracy resulted from traditional tissue homogenization method, we used a modified bronchoalveolar lavage (BAL) to extract free drug in lungs of rats. We modified BAL in two ways: First, in vivo lung lavage was changed to in vitro way.
We separated lungs from the body of rats, this process is necessary for collecting more lavage fluids in order to improve the yield. Second, we squeezed the lungs gently at the end of lavage and more lavage liquids could be collected. Owing to these two modifications, the yield of lavage liquids was improved up to 90%. Fig. 9 shows that the combination delivery system significantly prolonged RFP in vivo release than microspheres. RFP released from microspheres could only be detected within 240 h (10 days) while it was 0.81 ± 0.14 g mL−1 after 504 h (21 days) after administration of RFP-CDS. A possible reason to account for this difference could be that microspheres were unable to retain in lungs if
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References
Fig. 9. Comparison of pulmonary RFP concentration–time for rats after administration of microspheres and combination delivery system (mean ± SD, n = 6).
administered in water without the fixation help provided by in situ gel. The mean maximum RFP concentration in lungs of rats was 48.60 ± 15.67 g mL−1 at 5 h. RFP’s minimum inhibitory concentration (MIC) against M. tuberculosis is 0.75 g mL−1 [48], it can be inferred that RFP released from our CDS could be able to inhibit M. tuberculosis effectively for at least 21 days in lungs. Considering the 90% yield of our modified BAL method, the factual concentration of RFP in lungs was even higher. In virtue of this sustained release property, administration frequency could be reduced while the drug concentration will still be maintained at a therapeutic level. In the meantime, patient compliance will be improved if using this drug delivery system. According to our in vitro release study, the time for a total release of RFP from CDS was 11 days. We infer that it will take longer time for RFP to release from CDS in vivo because of two possible situations. First, PLGA is degraded to lactic acid and carbon dioxide by a mechanism of reaction with H2 O and oxygen. In vitro release medium provided sufficient H2 O and oxygen for this reaction, so the degrading process should be faster than in vivo release in lungs of mice, the little body fluids of which failed to assist the reaction. Second, after erosion of sodium alginate gel, PLGA microspheres got released from the combination delivery system. Microspheres possibly congregated and adhered to each other, the total release surface area of microspheres was reduced a lot, hence slowing down RFP’s release. In vitro release medium is able to disperse the microspheres and thereby avoiding this situation. However, the narrow space in lungs may aggravate this congregated problem.
4. Conclusions In this study, we showed that it is feasible to prepare a complex drug delivery system consisted of PLGA microspheres loaded with RFP in combination with the natural polymer sodium alginate ion-sensitive in situ gel. The in situ gel significantly delayed drug release from microspheres and adhered in lungs within 24 h. The microspheres successfully controlled drug release and avoided the potential problem of burst effect. In vivo release study result suggested that this combination delivery system is able to inhibit M. tuberculosis for 21 days. These results indicate that the combination delivery system is useful for interventional therapy of TB in clinical treatment.
Acknowledgments This work was supported by the foundation of 11th FiveYear Important National Science & Technology Specific Projects (2008ZX10003-016). The financial support from National Science &Technology Major Special Project on Major New Drug Innovation (Item No. 2009ZX09301-003) is gratefully acknowledged.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Preparation and characterization of rifampicin-PLGA microspheres/sodium alginate in situ gel combination delivery system Chunhui Hu, Hanzhou Feng, Chunyan Zhu ∗ Department of Drug Delivery Research Center, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 151 Malianwa North Road, Haidian District, Beijing 100193, PR China
a r t i c l e
i n f o
Article history: Received 8 November 2011 Received in revised form 21 February 2012 Accepted 21 February 2012 Available online 3 March 2012 Keywords: Alginate PLGA Biodegradable polymers Microspheres Controlled release Drug delivery system
a b s t r a c t We prepared a complex drug delivery system consisted of rifampicin-poly(lactic-co-glycolic acid) (PLGA) microspheres in combination with sodium alginate in situ gel. The microspheres were obtained by using a solvent evaporation method, the mean diameter was 1.748 m and the span of particle distribution was 0.78. The combination delivery system was obtained by adding microspheres to sodium alginate solution followed by physically mixing. In an in vitro study of drug release monitored for 11 days, the release of rifampicin from combination delivery system was slower than microspheres. The cumulative release percent of rifampicin from combination delivery system was 91.83 ± 1.26%, which was lower than 97.36 ± 3.41% of rifampicin released from microspheres. An in vivo fluorescence imaging study suggests that the gel adhered to lungs within 24 h, and microspheres stayed in lungs at least for 504 h (21 days). In vivo drug release study indicates that the maximum local rifampicin concentration in lungs was 48.60 ± 15.67 g mL−1 5 h after administration. After 21 days, the local rifampicin concentration was 0.81 ± 0.14 g mL−1 , which was above the minimum inhibitory concentration of rifampicin. The combination delivery system significantly prolonged RFP release compared to microspheres, from which RFP released could only be detected for 10 days. This approach to control the release of rifampicin using PLGA microspheres/in situ gel combination delivery system in conjunction with interventional technology is useful for improving anti-tuberculosis treatment effectiveness for patients. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Tuberculosis (TB) is a common and in many cases lethal infectious disease caused by various strains of Mycobacteria, usually Mycobacterium tuberculosis. Globally, there were an estimated 13.7 million chronic active cases, 9.27 million incident cases of TB and 1.8 million deaths. This is an increase from 9.24 million cases in 2006, 8.3 million cases in 2000 and 6.6 million cases in 1990. One-third of the world’s population is thought to be infected with M. tuberculosis and new infections occur at a rate of about one per second [1]. Tuberculosis usually attacks the lung, so improving drug pulmonary concentration has been one of the goals in anti-tuberculosis study areas. There are reported studies that focus on improving drug concentration in the lung, such as lung-targeting microspheres [2,3], nanoparticles [4,5], aerosolized liposome [6] and aerosols for inhalation administration [7–9]. However, a possibility has not been well studied. Anti-tuberculosis
Abbreviations: CDS, combination delivery system; MS, microspheres; HPLC, high-performance liquid chromatography; BAL, bronchoalveolar lavage. ∗ Corresponding author. Tel.: +86 10 57833276; fax: +86 10 57833276. E-mail addresses: [email protected], [email protected] (C. Zhu). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2012.02.030
drug administered in these ways could distribute to other nontargeted organs and may cause adverse and side effects. Hence, it is of importance to seek a substituted administration method. Interventional technology has been applied commonly in clinical treatment of tuberculosis because drug can be delivered directly to the lung instead of through blood vessel system. But the retention property of liquid or suspension, two commonly used formulations in interventional technology, is not satisfying because they cannot persistently stay in the lung [10]. Owing to this limitation, tuberculosis patients have to receive interventional operation frequently in order to maintain a therapeutic drug concentration. The objective of the present work was to improve patient compliance by developing a new drug delivery system that can be successfully used in interventional technology. To attain our purpose, the delivery system should include some necessary abilities: First, materials used with good biocompatibility and biodegradability. Second, good syringeability. It should be injectable for interventional use [11]. Third, a quick phase shift. After administration, it must adhere to lungs by transforming from a liquid state to semisolid. Fourth, sustained release. It is favorable to reduce the administration frequency by restricting drug release. For above concerns, we chose ion-sensitive in situ gel, sodium alginate, combined with poly(lactic-co-glycolic acid) (PLGA) microspheres as an
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appropriate drug carrier in this study [12–14]. In the presence of calcium, sodium alginate can form resilient gel, it has been widely used for encapsulation of drug in pharmaceutical areas. PLGA is a copolymer with demonstrated safety used in a host of Food and Drug Administration (FDA) approved therapeutic devices [15]. PLGA has been successfully used as a biodegradable polymer because it undergoes hydrolysis in the body to produce the original monomers, lactic acid and glycolic acid [16]. Under normal physiological condition, they are by-products of various metabolic pathways in the body. Since the body effectively deals with the two metabolic products, there is very minimal systemic toxicity associated with using PLGA for drug delivery or biomaterial applications. There are already a lot of reports that focus on using PLGA to prepare microspheres loaded with rifampicin (RFP), a semisynthetic bactericidal antibiotic drug that was also selected as model drug in our study. Ito et al. prepared RFP-PLGA microspheres by solvent evaporation method with a membrane emulsification technique using Shirasu Porous Glass (SPG) membranes [17]. Doan et al. applied solvent evaporation method with premix membrane homogenization to prepare RFP-PLGA microspheres with 80% RFP released from 12 h to 4 days [18]. Keiji Hirota’s study demonstrated that PLGA microspheres loaded with RFP are bio-safe due to their “silent” nature when taken into macrophage cells and are promising for treating tuberculosis [19]. Although previous reported studies have investigated many aspects of RFP-PLGA microspheres, such as preparation process, in vitro release and related delivery efficiency to alveolar macrophage cells, few studied preparing combination delivery system (CDS) containing RFP and its related properties. In the present work, RFP was encapsulated in PLGA microspheres in the first step. Thereafter, RFP-PLGA microspheres were dispersed in sodium alginate gel to form CDS as the final formulation. Fluorescence imaging study was conducted to explore its actual in vivo retention property. In vitro and in vivo drug release of CDS was studied to investigate its drug release-restricting ability. 2. Materials and methods 2.1. Reagents and chemicals PLGA with a molecular weight of 55,300 and having a monomer composition of lactic acid: glycolic acid of 50:50 was purchased from Birmingham Polymers company (USA). Rifampicin standard was purchased from Sigma–Aldrich (USA). Rifampicin sample was purchased from NOVO (USA) and used as an anti-tuberculosis drug. Polyvinyl alcohol (PVA) was purchased from Sinopharm Chemical Reagent Co. (China). Sodium alginate was purchased from FMC BioPolymer (USA). CaCl2 was purchased from Beijing Fengli Jingqiu Trade Co. (China). CY5.5 (excitation and emission wavelength = 670 and 760 nm) was purchased from Beijing Fanbo Biochemicals CO. (China). DIR (excitation and emission wavelength = 790 and 830 nm) was purchased from Invitrogen (USA). Other chemicals were of the highest grade commercially available.
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laboratory animal use, the guide for the care and use of laboratory animals (Institute of Laboratory Animal, Washington, D.C., National Academy Press, 1996). 2.3. Preparation of RFP-loaded PLGA microspheres RFP-PLGA microspheres were prepared using a solvent evaporation method [20,21]. 1 mg RFP was dispersed in 10 mL methylene chloride containing 10% (w/v) PLGA by sonication. The organic phase was then emulsified with magnetic stirring in 200 mL aqueous phase containing 1% (w/v) polyvinyl alcohol (PVA). Stirring was continued for 4 h until the methylene chloride was completely evaporated. The system was protected from light. The resultant dispersion was centrifuged at 12,000 rpm for separation of the microspheres. The obtained sediments were washed four times with de-ionized water, and collected by filtration, then subjected to freeze drying overnight. 2.4. Particle size analysis A light scattering particle size analyzer (Mastersizer 2000, London, England) was used to determine the mean particle diameter. Approximately 50 mg of microspheres were dispersed in 5 mL of hexane by sonication. 1 mL of the dispersion was used for particle size analysis. All measurements were conducted in triplicate. 2.5. Microsphere morphology Microsphere morphology and shape were characterized using a scanning electron microscopy (SEM). Samples were mounted on carbon taped aluminum stubs and gold coated in a sputter coater for 1 min. The samples were analyzed using a JSM-6510 SEM (JEOL, Japan) at an accelerating voltage of 5.0 kV. 2.6. Differential scanning calorimetry of microspheres RFP sample, blank PLGA microspheres, RFP and PLGA physical mixture and RFP-PLGA microspheres were examined by a DSC analyzer (Q200, TA, USA), respectively. The heating rate was 10 ◦ C min−1 , heating temperature range was 30–300 ◦ C, nitrogen flow velocity was 50 mL min−1 . 2.7. Preparation of sodium alginate in situ gel loaded with PLGA microspheres A certain amount of sodium alginate was added to de-ionized water and dissolved by stirring for 24 h [22]. The microspheres/gel combination delivery system (CDS) was obtained by adding microspheres to the in situ gel solution followed with physically mixing well. The CDS prepared in this way was a solution containing suspended microspheres. After gelation with calcium ions, it transformed from liquid state to semisolid gel. 2.8. Studies of rheological properties of RFP-microspheres/gel mixture
2.2. Animals Nu/Nu mice (male, 20 g) and SD rats (male, 250 g) were obtained from Vital River Laboratory Animal Technology Co. (Beijing, China). Mice and rats weighing about 20 g and 250 g respectively were randomly assigned to each experimental group. They were allowed free access to water but forbidden to food 12 h before experiments. The experiments were carried out after approval by the ethical committee at Institute of Medicinal Plant Development in accordance with internationally accepted guidelines on
The rheological property of microspheres/gel CDS was studied using a Physica MCR300 rheometer (Physica, Germany). The sample volume was about 20 mL. The measurements were made at room temperature. Different amounts (5 × 10−5 mol, 1 × 10−4 mol, 2 × 10−4 mol, 3 × 10−4 mol) of CaCl2 solution were added to the mixture and mixed well before being measured. Viscosity measurements were made at room temperature for shear rates between 0.1 and 100 s−1 [23]. Moreover, we compared the microspheres/gel CDS with blank sodium alginate in situ gel (after gelation with
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Fig. 1. SEM pictures of RFP-PLGA microspheres A – 2700×; B – 5000×.
calcium ions) to examine whether microspheres affect the viscosity of the delivery system. The flow behavior of the CDS was assessed in terms of their syringeability by withdrawing 1 mL of the CDS through a hypodermic needle of dimensions 0.45 mm × 19 mm (26G) [24]. 2.9. HPLC determination of RFP The concentration of RFP was determined using a pump L7100 and a Shimadzu SPD-10AVP UV-detector (Shimadzu, Japan). A SLC-10AVP (Shimadzu, Japan) equipped with a Hypersil ODS (4.6 mm × 150 mm, 5 m) analytical column was used. The column was kept at 30 ◦ C throughout the elution process, which used a mobile phase consisting of methanol:acetonitrile:0.075 mol L−1 KH2 PO4 :1 mol L−1 citric acid (30:30:26:4%, v/v/v/v) at a flow rate of 1.0 mL min−1 , and the detection wavelength was set to 254 nm. The linear calibration curve (y = 1455x + 2714.8; R2 = 0.9993; standard solution 0.27–34.2 g mL−1 ). 2.10. In vitro drug release We compared the release behaviors of RFP from microspheres and microspheres/gel CDS, respectively. RFP-microspheres (30 mg) were dispersed in blank gel solution (10 mL). Thereafter, CaCl2 solution was added to gelatinize sodium alginate. The gelatinized CDS was put in a group of dialysis tubes which were separating the CDS from the dissolution medium. Another group of dialysis tubes contained only RFP-PLGA microspheres (30 mg). 3 mL dissolution medium (1%EDTA–2Na + 1%Na2 SO3 ) was added all tubes, then they were put into stirring baskets. The study was carried out in 200 mL dissolution medium, the system was temperature controlled at (37 ± 0.5) ◦ C with a stirring rate at 100 rpm. 1 mL samples was withdrawn and replaced with fresh medium at predetermined time intervals. The samples were analyzed using HPLC as described in Section 2.9. All release experiments were performed as triplicates. The release behavior of RFP from microspheres and microspheres/gel CDS were analyzed based on various mathematical models.
(CY5.5-CDS) and DIR-PLGA microspheres combined with sodium alginate gel (DIR-CDS). After being anesthetized by pentobarbital sodium, two groups of Nu/Nu nude mice (about 20 g) were administered with two formulations by endotracheal intubation operation. The gel was gelatinized by pulmonary injection of 0.1 mol L−1 CaCl2 solution right after administration of CDS. The retention property of two marked formulations was measured by observing mice in the in vivo imaging system at different pre-determined time intervals [29]. 2.12. Studies of in vivo release SD Rats were divided into two groups. The control group was administered with 0.2 mL RFP-PLGA microspheres suspension (microspheres dispersed in physiological saline), the other group was administered with 0.2 mL RFP-CDS. Both two formulations theoretically contained 10 mg microspheres. After anesthetized by pentobarbital sodium (60 mg kg−1 ), two groups rats were administered with different formulations separately by endotracheal intubation operation. Rats were sacrificed at pre-determined time intervals, and then tracheae and lung were carefully separated. Afterward, free drug was extracted by bronchoalveolar lavage (BAL) [30]. We injected 2.5 mL physiological saline into the lung and withdrew it immediately, the process was repeated four times. The lavage fluid was collected and vortex mixed 30 s with methylene chloride (2/1, v/v), then the organic layer was separated by centrifuge at 12,000 rpm for 3 min. After dryness with nitrogen, the sample was re-dissolved by adding 0.2 mL acetonitrile before being analyzed by HPLC. 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
2.11. Studies of in vivo retention
3.1. Microsphere formulation
For the purpose to examine the retention property of gel and microspheres [25], a Kodak in vivo multispectral imaging system was applied. A fluorescent agent CY5.5 was used to mark the in situ gel formulation [26], which was added to sodium alginate solution and physically mixed well. Another fluorescent marker DIR was used to replace RFP as the model drug to prepare DIR-PLGA microspheres by the same method as we described in the part of Section 2.3 [27,28]. So there were two formulations: blank PLGA microspheres combined with sodium alginate gel marked by CY5.5
Fig. 1 shows SEM photographs of RFP-loaded PLGA microspheres. The microspheres were spherical. The particle distribution of RFP-PLGA microspheres, shown in Fig. 2, was as follows: D10% = 1.372 m, D50% = 1.748 m and D90% = 2.727 m, respectively (D10, D50 and D90 mean 10%, 50% and 90% of the total microspheres we prepared, respectively). The mean diameter of the microspheres was 1.748 m and the span of particle distribution was 0.78. This result shows that it is feasible to use the O/W emulsion solvent evaporation method to prepare RFP-PLGA
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Fig. 2. Particle distribution of RFP-PLGA microspheres. D10, D50 and D90 means 10%, 50% and 90% microspheres detected respectively of the total microspheres we prepared.
microspheres. Although there are new methods to prepare PLGA microspheres, such as spray drying [31,32] and SPG membrane emulsification [33,34], emulsion solvent evaporation is a more simple way to apply in this study. In addition, Fig. 3 shows that there are similar exothermic peaks at 280 ◦ C for RFP sample and its physical mixture with PLGA, while there is no similar peak for RFP-PLGA microspheres. This result suggests that the RFP was encapsulated in PLGA microspheres [35]. RFP-PLGA microspheres with diameter 1.748 m were investigated in this current study, microspheres with other various diameters will be examined in future. We will study the effect of different diameters on in vitro and in vivo drug release. 3.2. Preparation of sodium alginate in situ gel and combination delivery system By forming a viscous skeleton carrier after gelation, in situ gel can effectively delay the touch between microspheres and body liquid, which is beneficial to restrict drug release. Adding calcium ions to sodium alginate solution was used to attain this purpose and we investigated the relation between the amount of calcium ions added and the viscosity of the delivery system [36,37]. The combination delivery system had the strongest viscosity when adding 2 × 10−4 mol CaCl2 . Its viscosity lowered when we kept adding CaCl2 . As a result, using 10−5 mol CaCl2 to gelatinize 1 mL mixture was the best ratio for our further study. Fig. 4 shows that the viscosity of the gel decreased as the shearing rate increased, it demonstrates that the CDS was a pseudoplastic fluid. The result of syringeability study indicates that the CDS (before gelation) can be easily injected through 26G syringe needle. Some reported studies used in situ gel as a mucoadhesive skeleton carrier to load drug, aimed to control drug release [38,39]. But burst effect was a common problem if gel acted as the only drug carrier. There was a lag time during the gelation process, which means that the gel failed to transform from liquid state to semisolid
Fig. 3. DSC measurement of (A) physical mixture of RFP and PLGA; (B) RFP-PLGA microspheres; (C) blank PLGA microspheres; (D) PLGA sample.
Fig. 4. Viscosity change of microspheres/hydrogel combination delivery system. (A) Blank sodium alginate in situ gel; (B) adding 5 × 10−5 mol CaCl2 ; (C) adding 1 × 10−4 mol CaCl2 ; (D) adding 2 × 10−4 mol CaCl2 ; (E) adding 3 × 10−4 mol CaCl2 .
instantly and thus drug could be promptly released from the carrier. Consequently, we applied PLGA microspheres to control the drug release in order to avoid burst effect. The materials we used in preparing the delivery system were all pharmaceutical adjuvant approved by FDA, because good safety is the prerequisite to develop a formulation aims to be used in clinical treatment. 3.3. In vitro RFP release from microspheres and combination delivery system In vitro release profiles of RFP in microspheres (RFP-MS) and the combination delivery systems (RFP-CDS) were monitored for 11 days. By comparing two release profiles (Fig. 5), we found that there was significant difference between them. The total release percent of RFP-PLGA microspheres and RFP-CDS were 97.36 ± 3.41% and 91.83 ± 1.26%, respectively. Fig. 6 shows that from 0 h to 24 h, the cumulative release percent of RFP-MS and RFP-CDS were 22.83 ± 2.14% and 15.15 ± 0.74%, respectively. The release percent of RFP-MS was significantly higher than that of RFP-CDS. But from 24 h to 72 h, the cumulative release percent of RFP-MS and RFPCDS were 22.93 ± 4.73% and 22.98 ± 3.66%, respectively. There was no significant difference between them as the same result for the release from 24 h to 264 h (1–11 days). In our previous erosion study, which is not contained in this paper, sodium alginate gel (after gelation with calcium ions) completely eroded within 24 h. Thus, it can be reasonably inferred that the release profiles for CDS was consisted of two parts: The first step, in situ gel eroded gradually and microspheres were released from this skeleton carrier at the same time. The sustained release of drug from PLGA microspheres was the second step. The difference between two release profiles of the two formulations got explained in this way. At first, the gel blocked the touch between RFP-MS and the release medium. As the gel eroded gradually, increasing amount of microspheres released. After the gel completely eroded at 24 h, all microspheres
Fig. 5. In vitro release of RFP from MS (microspheres) and CDS (combination delivery system) (mean ± SD, n = 3).
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Fig. 6. Comparison of in vitro RFP release from microspheres and combination delivery system (mean ± SD, n = 3). *P < 0.05 versus in vitro release of drug from microspheres (MS). Table 1 The release equation of RFP from microspheres and CDS. Model
Zero-order First-order Higuchi
Microspheres
CDS 2
Equation
R
Equation
R2
y = 0.3034x + 34.524 y = −0.0137x + 4.3839 y = 5.8933x + 13.416
0.7538 0.9375 0.9808
y = 0.2982x + 27.731 y = −106.63x + 470.16 y = 0.1701x − 1.0407
0.8099 0.9632 0.9725
were released and they had a similar release rate with RFP-MS because the release behavior of microspheres was based on a diffusion mechanism (by fitting the release data to Higuchi equation, shown in Table 1). So, this was the reason why there was no significant difference in two release profiles from 24 h to later time. In addition, the 0–24 h cumulative release percent of microspheres was 12.23 ± 0.70%, which indicates that there was no burst effect. 3.4. In vivo retention property In vivo imaging in general allows a non-invasive insight into living organisms and helps to understand metabolic processes and
disease related changes [40,41]. By using this technology, we can focus on the same subject investigated and avoid individual difference. CY5.5 and DIR were commonly used fluorescent markers [42], their emission wavelengths belong to infrared wavelength zone [43], which means good penetrability. Hydrophilic CY5.5 was used to mark water soluble sodium alginate gel, while RFP was replaced by a hydrophobic fluorescent agent DIR in order to mark microspheres. Fig. 7 shows strong fluorescence occurred at the lung of mice that were administered CY5.5-CDS immediately. The fluorescence had gradually intensified since 0 h, and it obviously weakened at 24 h. The fluorescence disappeared when we observed the mice at the 36 h after administration. The results indicate that the sodium alginate gel was able to adhere to lungs within 24 h. Besides, we observed fluorescence with increasing intensity at hypogastric regions of all tested mice since 1 h. We infer that the regions were bladders, because hydrophilic CY5.5 was metabolized by kidney and the metabolic products were excreted in the urine. Accordingly, we can observe increasing fluorescence there while it was decreasing at lungs. The mice administered DIR-CDS were monitored for 672 h (28 days). Fig. 8(1) and (2) shows that strong fluorescence immediately appeared at lungs of all tested mice right after administration. The fluorescence had weakened from 336 h to 504 h (14th to 21st day), and we almost observed nothing at the final pre-determined time 672 h (28th day). The results suggest DIR-PLGA microspheres may retain at lungs within at least 21 days because we observed weak fluorescence till 504 h. But the microspheres may adhere to lungs even after all DIR was released, e.g. longer than 672 h. To clarify this problem, we went a step further to investigate in vivo release of drug. Besides, we observed no fluorescence at hypogastric regions of mice after administration of DIR-CDS. A possible reason to account for this is that DIR is hydrophobic and its metabolic products are easily reabsorbed by kidney tubule, thereby excreted into intestinal tract in the bile. As a result, DIR is likely eliminated from the body as excrement and herein we failed to observe any fluorescence at the expected locations of mice. To truly explain this problem, determination of them in excrement and urine would be needed, but it is not covered by this current study.
Fig. 7. In vivo fluorescence imaging: the retention property of mice after administration of CY5.5-CDS for 0–36 h (n = 3).
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Fig. 8. (1) In vivo fluorescence imaging: the retention property of mice after administration of DIR-CDS for 0–36 h (n = 3). (2) In vivo fluorescence imaging: the retention property of mice after administration of DIR-CDS for 72–672 h (n = 3).
3.5. In vivo release Although tissue homogenization is a normal method to extract drug in organs [44–47], there is a problem if using it in our study. The process of homogenization will destroy microspheres adhered to lungs, which will cause leakage of drug encapsulated in the microspheres and thus increase drug’s concentration determined. To avoid the possible inaccuracy resulted from traditional tissue homogenization method, we used a modified bronchoalveolar lavage (BAL) to extract free drug in lungs of rats. We modified BAL in two ways: First, in vivo lung lavage was changed to in vitro way.
We separated lungs from the body of rats, this process is necessary for collecting more lavage fluids in order to improve the yield. Second, we squeezed the lungs gently at the end of lavage and more lavage liquids could be collected. Owing to these two modifications, the yield of lavage liquids was improved up to 90%. Fig. 9 shows that the combination delivery system significantly prolonged RFP in vivo release than microspheres. RFP released from microspheres could only be detected within 240 h (10 days) while it was 0.81 ± 0.14 g mL−1 after 504 h (21 days) after administration of RFP-CDS. A possible reason to account for this difference could be that microspheres were unable to retain in lungs if
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References
Fig. 9. Comparison of pulmonary RFP concentration–time for rats after administration of microspheres and combination delivery system (mean ± SD, n = 6).
administered in water without the fixation help provided by in situ gel. The mean maximum RFP concentration in lungs of rats was 48.60 ± 15.67 g mL−1 at 5 h. RFP’s minimum inhibitory concentration (MIC) against M. tuberculosis is 0.75 g mL−1 [48], it can be inferred that RFP released from our CDS could be able to inhibit M. tuberculosis effectively for at least 21 days in lungs. Considering the 90% yield of our modified BAL method, the factual concentration of RFP in lungs was even higher. In virtue of this sustained release property, administration frequency could be reduced while the drug concentration will still be maintained at a therapeutic level. In the meantime, patient compliance will be improved if using this drug delivery system. According to our in vitro release study, the time for a total release of RFP from CDS was 11 days. We infer that it will take longer time for RFP to release from CDS in vivo because of two possible situations. First, PLGA is degraded to lactic acid and carbon dioxide by a mechanism of reaction with H2 O and oxygen. In vitro release medium provided sufficient H2 O and oxygen for this reaction, so the degrading process should be faster than in vivo release in lungs of mice, the little body fluids of which failed to assist the reaction. Second, after erosion of sodium alginate gel, PLGA microspheres got released from the combination delivery system. Microspheres possibly congregated and adhered to each other, the total release surface area of microspheres was reduced a lot, hence slowing down RFP’s release. In vitro release medium is able to disperse the microspheres and thereby avoiding this situation. However, the narrow space in lungs may aggravate this congregated problem.
4. Conclusions In this study, we showed that it is feasible to prepare a complex drug delivery system consisted of PLGA microspheres loaded with RFP in combination with the natural polymer sodium alginate ion-sensitive in situ gel. The in situ gel significantly delayed drug release from microspheres and adhered in lungs within 24 h. The microspheres successfully controlled drug release and avoided the potential problem of burst effect. In vivo release study result suggested that this combination delivery system is able to inhibit M. tuberculosis for 21 days. These results indicate that the combination delivery system is useful for interventional therapy of TB in clinical treatment.
Acknowledgments This work was supported by the foundation of 11th FiveYear Important National Science & Technology Specific Projects (2008ZX10003-016). The financial support from National Science &Technology Major Special Project on Major New Drug Innovation (Item No. 2009ZX09301-003) is gratefully acknowledged.
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