Abstract
Background/Aim: In vivo models of tuberculosis are effective tools for developing new drugs. The objective of this study was to prepare in vivo models for tuberculosis by utilizing nanocomposite particles (NCPs) containing imiquimod-loaded poly(lactic-co-glycolic acid) nanoparticles. Materials and Methods: NCPs were prepared from dichloromethane with imiquimod and poly(lactic-co-glycolic acid) using a spray dryer. Mice were treated with NCPs in the lungs by inhalation, and then infection with Mycobacterium bovis bacille Calmette-Guerin was performed (treatment groups). The concentrations of the pro-inflammatory cytokines, tumor necrosis factor-α and interferon-γ were measured in bronchoalveolar lavage fluid using an enzyme-linked immunosorbent assay. Results: When animals were treated with NCPs, the concentrations of tumor necrosis factor-α and interferon-γ in bronchoalveolar lavage fluid were significantly higher than in animals not treated with NCPs. In addition, high bacterial counts and circular granuloma were observed. Conclusion: NCPs prepared in this study enhanced the level of inflammation in the lungs and support the preparation of in vivo models of tuberculosis.
Although three-dimensional culture systems have aroused substantial interest worldwide, in vivo models remain an essential evaluation system (1-5). In vivo models for diseases can provide information on pathological conditions, drug effects, or side-effects in vivo (6). Tuberculosis is one of the most important health problems to be solved because approximately 10 million people developed tuberculosis, and 1.5 million patients died of it in 2018 (7). Mycobacterium tuberculosis, the leading cause of tuberculosis, proliferates in alveolar macrophages (8). Based on this phenomenon, to prepare in vivo models of tuberculosis, alveolar macrophages should be infected with M. tuberculosis. However, M. tuberculosis is virulent and its use requires specialized facilities compatible with Animal Bio Safety Level 3 (9). Due to this limitation, attenuated M. bovis Bacille Calmette-Guerin (BCG) is often used as an alternative (10). However, the level of inflammation induced using BCG is too low to prepare reproducible in-vivo models of tuberculosis (11). Therefore, technology capable of enhancing the level of inflammation is essential.
Pulmonary drug delivery can be used to treat lung diseases, such as tuberculosis (12). Use of such systems can increase the drug concentration in the lung or reduce the frequency of medication required compared to oral administration, which leads to the enhancement of drug effects and reduction of side-effects (8). For the efficient therapy of tuberculosis, in our previous studies, inhalable nanocomposite particles (NCPs) containing rifampicin-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles were prepared (13). The inhalable NCPs are delivered to the alveolar macrophages and efficiently kill M. tuberculosis because the macrophages can recognize the substance with micro-size (14-16). In our previous study, the results of in vivo experiments with fluorescent materials suggested the sustained release of rifampicin from the NCPs in the lungs (17).
This study aimed to increase the level of inflammation in the lung using inhalable NCPs and prepare in vivo models of tuberculosis. Firstly, we prepared NCPs containing imiquimod-loaded PLGA nanoparticles to promote induction of alveolar macrophages. Imiquimod can stimulate macrophages, leading to the secretion of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ) (18). To investigate the effects of NCPs on in vivo models of tuberculosis, we evaluated the concentration of inflammatory cytokines and the bacterial count in bronchoalveolar lavage fluid (BALF).
Materials and Methods
Materials. PLGA with a molecular weight of 10,000 and a DL-lactic acid/glycolic acid monomer composition of 75/25 (PLGA7510) was purchased from Taki Chemical Co., Ltd. (Kakogawa, Japan). Imiquimod (C14H16N4, purity >98.0%) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Acetonitrile [for high-performance liquid chromatography (HPLC), purity ≥99.8%] and L(+)-arginine (purity ≥98%) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Dry BCG vaccine was purchased from Japan BCG Laboratory (Tokyo, Japan). Freund’s adjuvant, complete H37Ra, was purchased from Sigma–Aldrich Co. LLC., (St. Louis, MO, USA). Isoflurane was purchased from Mylan Inc. (Pittsburgh, PA, USA). All other chemicals were of the highest grade commercially available.
Preparation of imiquimod-loaded PLGA nanoparticles. Imiquimod-loaded PLGA nanoparticles were prepared using the emulsion solvent evaporation method. Briefly, 90 mg of PLGA7510 and 10 mg of imiquimod were dissolved in 10 ml of dichloromethane. The solution was added to 40 ml of a 2.0% aqueous solution (w/v) of poly(vinyl) alcohol (pH 7). It was emulsified using a probe sonicator (Digital Sonifier S-250D; Branson Ultrasonics Corp., Danbury, CT, USA) at 200 W of energy output for 20 s. The resulting oil-in-water emulsion was stirred overnight on a magnetic stir plate at room temperature to evaporate the dichloromethane. The nanoparticles were collected by ultracentrifugation at 25,000×g for 10 min (Himac 80WX; Hitachi Koki Co. Ltd., Tokyo, Japan). Following centrifugation, the precipitated nanoparticles were rinsed with purified water in an ultrasonic bath sonicator to remove residual poly(vinyl) alcohol. Centrifugation and rinsing were repeated for a total of three cycles. The mean volume diameters of imiquimod-loaded PLGA nanoparticles were measured using a particle size analyzer (ELSZ-2; Otsuka Electronics Co., Ltd., Osaka, Japan). Samples were dispersed in purified water and measured at 25°C. The imiquimod content in the nanoparticles was measured using HPLC (SIL20A prominence, SPD-20 A prominence, LC-20AD prominence, CTO10ASvp, DGU-20A3 prominence; Shimadzu Corp., Kyoto, Japan) at 245 nm. The isocratic mobile phase used was acetonitrile, acetate buffer (pH 4.0), and dimethylamine with a volume ratio of 30:69.85:0.15. The samples were dissolved in 10 ml of the solution. HPLC measurements were carried out 40°C (flow rate: 1 ml/min), and 20 μl of sample solution was applied. All HPLC measurements were carried out under the same conditions.
Preparation of NCPs. The imiquimod-loaded PLGA nanoparticles were redispersed in 50 ml of purified water containing arginine and leucine with a mixture ratio of 1:6. Then the suspensions were spray-dried to prepare NCPs using a spray dryer (B-290; BÜCHI Corp., Flawil, Switzerland). Spray drying was carried out under the following conditions: Inlet temperature of 37-40°C, air volume of 20 m3/h, and a pump flow rate of 2.5 ml/min.
The size of NCPs in the air was measured using a a laser diffraction analyzer (LDSA-3500A; Nikkiso Co., Ltd., Tokyo, Japan). The mean volume diameters of imiquimod-loaded PLGA nanoparticles released from the NCPs were measured using a particle size analyzer (ELSZ-2) at 25°C. The imiquimod content of the NCPs was measured using HPLC. Surface properties of NCPs were analyzed using scanning electron microscopy (JSM-6060LA; JEOL Ltd., Akishima, Japan). The fine particle fraction (FPF) value was measured using a cascade impactor (Andersen nonviable impactor MODEL AN-200; Tokyo Dylec Co., Ltd., Tokyo, Japan) with a dry powder inhaler (DPI) (Jethaler; Hitachi, Ltd., Tokyo, Japan). One hydroxypropyl methylcellulose capsule containing 5 mg of NCPs was placed in the DPI, and the DPI was attached to the inlet of the cascade impactor. The measurement was carried out at a steady flow rate of 28.3 l/min for 5 s at a relative humidity of 90%. The amount of particles left in each stage of cascade impactor was measured using HPLC.
Animal experiments. Mice (ICR, 6 weeks old, male) were housed in stainless-steel cages under standard environmental conditions (23±1°C, 55%±5% humidity, and a 12/12 h light/dark cycle) and maintained with free access to water and as standard laboratory diet (carbohydrates: 30%; proteins: 22%; lipids: 12%; vitamins: 3%) ad libitum (Nihon Nosan Kogyo Co., Yokohama, Japan). They were used in accordance with the Guidelines for Animal Experimentation of Tokyo University of Science, which are based on the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science (approval numbers: Y21028 and Y21051). Firstly, H37Ra (1.0 mg/mouse) was injected subcutaneously for sensitization 7 days before BCG infection. Then 1 day before BCG infection, each animal was treated with or without (control group) administration of 5 mg of NCPs to the lungs using an animal DPI (Dry Powder Insufflator Model DP-4M; Penn Century, Inc., Glenside, PA, USA) under isoflurane anesthesia. Finally, the mice were infected with BCG at a cell number of 1×107 by intratracheal instillation.
Concentration of inflammatory cytokines in BALF. At 1, 2, 3, 4, 5, 6, 7, 14, and 21 days after BCG infection, the lungs were lavaged seven times with 1.5 ml phosphate-buffered saline, and the BALF was collected. The concentrations of TNFα and IFNγ in the BALF were measured using an enzyme-linked immunosorbent assay kit (BioLegend Inc., San Diego, CA, USA) (standard range: 7.8-500 pg/ml for TNFα and 15.6-1,000 pg/ml for IFNγ).
Determination of bacterial load in lung tissues and BALF. At 1, 3, 7, 14, and 21 days after BCG infection, mice (n=5) were sacrificed by cervical dislocation, and the lungs were obtained. The lungs were washed using physiological saline, and then they were cut into small parts to obtain fine pieces. The obtained tissues were suspended in physiological saline of four times the volume. The suspension was homogenized (Physcotron Microhomogenizer NS-310E; Microtec Co., Nition Ltd., Japan), and the homogenates were diluted with water of twice the volume. Lung homogenates and BALF were incubated on plates with Middle brook 7H10 agar (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) containing 10 μg/ml amphotericin B (Bristol-Myers Squibb Co., New York, NY, USA) and 200 U/ml polymyxin B (Wako Pure Chemical Industries, Ltd., Osaka, Japan) at 37°C. After 3 weeks, the number of colony-forming units (CFU) were counted from visible colonies and calculated considering the dilution value (diluted eight times), and the data were expressed as logarithmic values (base 10) according to a method reported elsewhere (9).
Histological examination of lungs. At 14 days after BCG infection, mice (n=5) were sacrificed by cervical dislocation, and the lung was obtained. The lungs were fixed in 4%-paraformaldehyde and embedded in carboxymethyl cellulose gels, and then frozen. Sections of lungs 10 μm-thick were then stained with hematoxylin and eosin to assess granuloma formulation.
Statistical analysis. All the data are expressed as the mean±the standard error of the mean. The data were analyzed by Student’s t-test or Tukey’s test to determine statistically significant differences, while the significance was accepted at p<0.05. Experiments for each sample were performed three times independently unless otherwise mentioned.
Results
The size distribution of the nanoparticles is shown in Figure 1. The mean volume diameter of imiquimod-loaded PLGA nanoparticles was 210.0±44.5 nm. The imiquimod content in the nanoparticles and the entrapment efficiency were 6.3±0.1% and 62.6±0.4%, respectively.
Size distribution of imiquimod-loaded poly(lactic-co-glycolic acid) nanoparticles.
Figure 2A show the size distribution of imiquimod-PLGA-bearing NCPs. The diameter of NCPs containing imiquimod-PLGA nanoparticles was 4.19±1.92 μm. The imiquimod content and the entrapment efficiency were 2.7±0.2% and 53.9±0.9%, respectively. In addition, we confirmed that the FPF value was 37.6±0.9%. Scanning electron microscopy confirmed that the NCPs for inhalation were non-spherical (Figure 2B).
A: Size distribution of nanocomposite particles (NCPs). B: Scanning electron microscopy images of NCPs taken at an accelerating voltage of 10 kV (magnification: 3,000×). Scale bar=5 μm.
Figure 3 shows the concentrations of TNFα and IFNγ in BALF. The concentrations of these two cytokines in BALF were significantly higher in the animals treated with NCPs compared with the NCP-free control mice. In addition, higher concentrations of these cytokines were observed every day for 21 days in the treated with NCPs, while the concentration was not maintained in the case of the NCP-free control mice.
Concentration of tumor necrosis factor-α (TNFα) (A) and interferon-γ (IFNγ) (B) in bronchoalveolar lavage fluid after Bacillus Calmette-Guerin infection in mice treated with (■) and without (control □) nanocomposite particles. *Significantly different from the corresponding control at p<0.05.
We evaluated the bacterial count for the lung tissues (Figure 4A) and BALF (Figure 4B). The bacterial counts in lung tissue were not different between the NCP-treated and the NCP-free control groups. On the other hand, in BALF, when the mice were treated with NCPs, the bacterial counts were significantly higher than in the NCP-free control group on days 7, 14, and 21.
Bacterial count (log10 scale) as colony-forming units (CFU) in lung tissues (A) and bronchoalveolar lavage fluid (B) at 1, 3, 7, 14, and 21 days after Bacillus Calmette-Guerin infection in mice treated with (■) and without (control □) nanocomposite particles. *Significantly different from the corresponding control at p<0.05.
Figure 5 shows the hematoxylin and eosin staining of lungs from mice with and without NCP treatment. A circular granuloma was observed when mice were treated with NCPs (Figure 5B), while the cells only aggregated and a circular granuloma was not observed without NCP treatment (Figure 5A).
Hematoxylin and eosin staining of lungs from control (A) and nanocomposite particle-treated (B and C) mice 14 days after Bacillus Calmette-Guerin infection. Scale bar=200 μm. Cell aggregates are indicated by arrowheads. A circular granuloma was observed in the lungs from nanocomposite particle-treated mice.
Discussion
The aim of using NCPs prepared in this study was to enhance the level of inflammation in the lungs, leading to the efficient preparation of in vivo models for tuberculosis. The mean FPF value for NCPs was 37.6±0.90, which means this was a suitable formulation for delivering the bioactive materials to the lungs (19, 20). To evaluate the effect of inhalable NCPs on the level of inflammation in the lungs, the concentration of the representative inflammatory cytokines TNFα and IFNγ in BALF were investigated. The data in Figure 3 suggest that the NCPs increased inflammation. Next, we evaluated the bacterial count in lung tissues and BALF. There was a significant difference in bacterial count in BALF with and without NCP treatment on 7, 14, and 21 days (Figure 4). Studies have shown that macrophages are major components in BALF (21, 22). Macrophages in BALF secrete several cytokines or chemokines and have a great role in the immune response and induction of inflammation due to foreign substances (14). Therefore, we interpret the data from Figure 3 and Figure 4 as showing that NCPs promote the secretion of inflammatory cytokines from macrophages.
Granuloma is the formation of aggregates composed of macrophages (23), and is the pathological hallmark of tuberculosis. From Figure 3 and Figure 5, it is suggested that the enhancement of inflammation by NCPs enabled granuloma formation.
Macrophages are polarized to M1 and M2 phenotypes, responding to the environmental conditions (24-26). M1 macrophages (pro-inflammatory) induce inflammation by the secretion of inflammatory cytokines, such as TNFα and IFNγ (27, 28). On the other hand, M2 macrophages have the capacity for wound-healing and tissue regeneration (29, 30). The balance of the M1/M2 ratio determines the level of tissue inflammation (31). To prepare in vivo models, it is important to develop technologies capable of inducing the M1 phenotype. From Figure 3 and Figure 4, it is apparent that there are many M1 macrophages in BALF. We suggest that circular granuloma was efficiently formed due to the M1-rich condition. These findings indicate that the NCPs produced in this study can promote the secretion of inflammatory cytokines by alveolar macrophages and support the toxicity of BCG for the preparation of in vivo models for tuberculosis.
Acknowledgements
The Authors are grateful to Professor Makiya Nishikawa of Tokyo University of Science for his cooperation in conducting animal experiments.
Footnotes
Authors’ Contributions
T. Nii, S. Takizawa, T. Akita, C. Yamashita, and I. Takeuchi designed the study and wrote the initial draft of the article. K. Makino contributed to the analysis and interpretation of data and assisted in preparing the article. All Authors approved the final version of the article and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Conflicts of Interest
The Authors declare that they have no potential conflicts of interest.
- Received April 14, 2022.
- Revision received June 6, 2022.
- Accepted June 8, 2022.
- Copyright © 2022 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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