Arbekacin-Loaded Inhalable Nanocomposite Particles Specific to Pseudomonas aeruginosa Prepared Using a Two-Solution Mixing-Type Spray Nozzle

Abstract

Hospital-acquired pneumonia is an important infectious disease that requires special management and therapy for patients with compromised immunity, as opportunistic infections with microorganisms such as Pseudomonas aeruginosa can be fatal. Nanoparticle-based drug delivery to lung tissue provides several advantages in the treatment of respiratory diseases. In the current study, inhalable nanocomposite particles consisting of microparticles containing solid-state arbekacin (ABK) nanoparticles coated with hydrophobic surfactant (ABK-SD nanoparticles) were prepared using a spray dryer equipped with a two-solution mixing-type spray nozzle we previously developed. ABK-SD/mannitol (MAN) nanocomposite particles were obtained from ABK-SD nanoparticles by varying the amounts of hydrophobic surfactant and ABK. The aerosol performance of ABK-SD/MAN nanocomposite particles was superior to that of ABK-MAN microparticles in terms of the fine particle fraction (28.4 ± 5.4%, ABK-SD/MAN nanocomposite particles; 11.4 ± 7.6%, ABK-MAN microparticles). These results suggest that ABK-SD/MAN nanocomposite particles are suitable for use in inhalation drug formulations and useful for the treatment of lung infections involving Pseudomonas aeruginosa.

INTRODUCTION

Nosocomial pneumonia, also known as hospital-acquired pneumonia, is an important nosocomial infectious disease caused by infection with Gram-positive or Gram-negative bacteria. In the United States, Pseudomonas aeruginosa is a Gram-negative organism frequently isolated from patients hospitalized with pneumonia and from patients with ventilator-associated pneumonia.¹⁾ Pseudomonas aeruginosa is a typical opportunistic pathogen that does not exhibit pathogenicity against healthy subjects but can cause fatal infections in immunocompromised patients or patients with underlying lung diseases such as cystic fibrosis. Once Pseudomonas aeruginosa is established in the host, complete eradication of the bacteria is difficult due to the organism’s high level of intrinsic antibiotic resistance associated with high membrane permeability and bacterial efflux systems.^2,3) The bacteria can also escape the host immune defenses mediated by immunocompetent cells by altering the expression of pathogen-associated molecular pattern molecules and bacterial components associated with initiating inflammatory responses (e.g., flagellin and lipopolysaccharides).^4,5) Additionally, Pseudomonas aeruginosa is capable of forming a biofilm that enhances drug resistance.⁶⁾ Bronchoscope is a risk factor of outbreak of multi-drug resistance Pseudomonas aeruginosa infection.⁷⁾ Currently, few agents remain broadly effective against the Pseudomonas aeruginosa strains frequently isolated from patients with pneumonia.⁸⁾ Patients hospitalized in the intensive care unit (ICU) typically exhibit poorer drug responses than those in non-ICU settings⁹⁾ due to the antibiotic selection pressure in ICU, resulting in the emergence of antibiotic-resistant Pseudomonas aeruginosa.¹⁰⁾ Thus, proper management to prevent infections and development of effective treatments are essential to combat hospital-acquired pneumonia.

Inhalation formulations are effective dosage forms that can directly deliver intended drugs to disease sites in the lung. The drug delivery efficacy of topical applications is higher than that of ordinary treatments involving oral administration or intravenous drip infusion. The aerodynamic diameter of an inhaled drug formulation particle is closely related to its deposition in the lung during aspiration.^11,12) Single-micron size powders (i.e., dry powder inhalers, pressurized metered-dose inhalers, and breath-actuated inhalers) and mists (e.g., Soft Mist Inhaler™, and nebulizers) are suitable for depositing drugs in lung tissue. Although most inhalation formulations are used in the treatment of asthma and chronic obstructive pulmonary disease, a few inhalation antibiotic formulations are currently available, such as TOBI^® (tobramycin inhalation solution for Pseudomonas aeruginosa infection in patients with cystic fibrosis¹³⁾) and ARIKAYCE^® (amikacin liposome inhalation suspension for the treatment of Mycobacterium avium complex lung disease¹⁴⁾). Dry powder formulation is useful to reduce the treatment (time) burden of nebulizing of antibiotics solution to prevent bacterial contamination.¹⁵⁾ Then, the inhalation formulations of four anti-tuberculosis drugs were in clinical trial, and the secretion of proinflammatory cytokines was investigated to evaluate the safety and adverse events.¹⁶⁾

Arbekacin (ABK) is an aminoglycoside antibiotic exhibiting broad-spectrum activity against both Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, and Gram-negative bacteria, including organisms associated with pneumonia in hospitalized patients.^1,17,18) As the active pharmaceutical ingredient is available for injection formulation in Japan, a clinical trial examined the efficacy of ABK inhalation solution (ME1100 regimen) or the treatment of patients with hospital-acquired and ventilator-associated bacterial pneumonia.¹⁹⁾

In this study, we developed powder-based inhalable microparticles containing ABK-loaded nanoparticles using a spray drying method. Nanoparticulate drug delivery systems have received considerable attention because they offer several advantages for pulmonary delivery, including sustained drug release,²⁰⁾ prolonged lung retention,²¹⁾ drug penetration of biofilms,^22,23) and delivery of high amounts of drug to target sites through encapsulation. In a previous study, we developed nanocomposite particles containing sugar derivative (OCT313, anti-tuberculosis compound^24,25))–loaded nanoparticles consisting of solid-state drug nanoparticles coated with a hydrophobic surfactant (SD nanoparticles), with the aim of targeting alveolar macrophages. The SD nanoparticles released from the nanocomposite particles exhibited remarkably high cellular uptake by macrophage-like cells due to their hydrophobicity.²⁶⁾ To extend our previous study, this preparation method was applied in the present study to generate nanocomposite particles containing ABK-SD nanoparticles. We then investigated the effect of drug composition on the formation of ABK-SD nanoparticles and evaluated the in vitro aerosol performance, drug release, and antimicrobial effects of the nanocomposite particles.

MATERIALS AND METHODS

Reagents

Mannitol (MAN), o-phthalaldehyde (OPA), boric acid, and sodium dodecyl sulfate (SDS) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). A sucrose-erucic acid ester, ER-290, was obtained from Mitsubishi-Kagaku Foods Corporation (Tokyo, Japan). 2-Mercaptoethanol was purchased from Bio-Rad Laboratories (Hercules, CA, U.S.A.). ABK was kindly donated by Meiji Seika Pharma (Tokyo, Japan). Cyclohexane, isopropanol, and methanol were purchased from FUJIFILM Wako Pure Chemical Corporation.

Preparation of ABK-SD Nanoparticles

ABK-SD nanoparticles were prepared using a solid-in-oil emulsion method, as described previously.²⁶⁾ In brief, as a typical condition, 2.4 mL of ER-290 cyclohexane solution (0.5, 1, 2.5, 5, 10, or 20%) and 1.2 mL of ABK aqueous solution (1, 2, 4, 6, or 8%) were added to a glass tube, and the mixture was sonicated for 2 min using a probe-type ultrasonic dispersion machine (output, 6; amplitude, 40 µm: SMT Corppration, Tokyo, Japan) to generate a water/oil emulsion. The sample was placed in a deep freezer at −80 °C for rapid freezing and then stored overnight. Finally, the sample was lyophilized overnight using a freeze dryer (FD-1; EYELA, Tokyo, Japan) to obtain ABK-SD. A schematic illustration of the process for preparing the ABK-SD is shown in Fig. 1A.

Fig. 1. (A) Schematic Illustration of the Preparation of ABK-SD; (B) Schematic Illustration of the Preparation of Nanocomposite Particles Using a Two-Solution Mixing-Type Spray Nozzle

Spray Drying and Preparation of Nanocomposite Particles

ABK-SD/MAN nanocomposite particles (microparticles containing ABK-SD nanoparticles) were prepared using a CNL-3 spray dryer (Ohkawara Kakohki Co., Ltd., Kanagawa, Japan) equipped with a two-solution mixing-type spray nozzle (Twin Jet Nozzle RJ10 TLM1; Ohkawara Kakohki Co., Ltd.). A schematic illustration of the process for preparing the nanocomposite particles is shown in Fig. 1B. First, 3% MAN aqueous solution and 0.3% ABK-SD solution dissolved in isopropanol were prepared. The MAN aqueous solution and ABK-SD solution were passed into the spray nozzle at a rate of 0.54 and 0.18 kg/h (180 mL of MAN solution and 60 mL of ABK-SD solution), respectively. The two solutions were mixed in the mixing part of nozzle and immediately spray dried (inlet temperature, 145 °C; spray air pressure, 0.1 MPa). The resulting ABK-SD/MAN nanocomposite particles were collected, and the powdered sample was stored in desiccator until further use.

As a control to compare to the property of ABK-SD nanoparticles, ABK-MAN particles (MAN microparticles containing ABK molecule) were prepared using a CNL-3 spray dryer equipped with a conventional spray nozzle. Two-hundred milliliters of 0.2% ABK and 3% MAN aqueous mixture was passed into the spray nozzle at 0.54 kg/h and then spray dried (inlet temperature, 145 °C; spray air pressure, 0.1 MPa).

Determination of ABK Concentration

ABK concentration was determined by measuring the fluorescence of ABK derivative reacted with OPA, as described previously for the determination of gentamicin,²⁷⁾ with modifications. In brief, OPA solution was prepared by mixing 0.1 g of OPA, 500 µL of methanol, 5 mL of 0.4 M borate buffer (pH 10.4), and 100 µL of 2-mercaptoethanol. The pH of the OPA solution was adjusted to 10.4, and the solution was transferred to a light-impermeable container, stored at 4 °C, and used within 3 d.

Next, 500 µL of ABK solution at an intended concentration and 300 µL of methanol were added to a 1.5-mL microtube and mixed for 3 s using a vortex mixer. Then, 50 µL of OPA solution and 300 µL of methanol were added to the microtube sequentially and mixed for 3 s using a vortex mixer. Finally, 150 µL of each sample solution was transferred to the wells of a 96-well black plate, and the fluorescence was measured using a microplate reader (Nivo plate reader; PerkinElmer, Inc., Waltham, MA, U.S.A.; Ex/Em = 340/450 nm).

Scanning Electron Microscopy (SEM)

Each powdered sample was placed in an ion sputtering apparatus (E-102, Hitachi, Tokyo, Japan) and coated with Pt-Pd prior to observation by SEM on an S-4300 instrument (Hitachi).

Transmission Electron Microscopy (TEM)

The appearance and inner structure of ABK-SD/MAN nanocomposite particles were analyzed by TEM on a JEM-1400 plus instrument (JEOL, Tokyo, Japan). A sample of spray-dried ABK-SD/MAN nanocomposite particles prepared as described in “Spray Drying and Preparation of Nanocomposite Particles” was suspended in small amount of water not to dissolve completely. Then 20 µL of sample was dropped on the grid and allowed to stand for 5 min. The sample was then treated with phosphotungstic acid as negative staining. TEM images were obtained at 100 kV.

Measurement of Particle Size and Size Distribution of ABK-SD Nanoparticles

As a typical experiment, a sample of ABK-SD nanoparticles prepared as described in “Preparation of ABK-SD Nanoparticles” was dispersed to cyclohexane. Then the sample was transferred into a disposal cuvette for the measurement. In contrast, a sample of spray-dried ABK-SD/MAN nanocomposite particles prepared as described in “Spray Drying and Preparation of Nanocomposite Particles” was dispersed with pure water to generate a dispersion of ABK-SD nanoparticles. The mean diameter (Z-Ave) and poly dispersity index (PDI) of the nanoparticles at 20 °C were measured using a ZetaSizer (ZetaSizer Nano-ZS; Malvern Instrument Ltd., Malvern, U.K.). The material refractive index of all particles was estimated to be 1.45.

Encapsulation Efficiency of ABK

ABK-SD/MAN nanocomposite particles prepared as described in “Spray Drying and Preparation of Nanocomposite Particles” were dispersed in water to a concentration of 50 mg/mL and then ultracentrifuged at 200000 × g for 30 min using an Optima MAX-XP ultracentrifuge (Beckman Coulter Inc., Brea, CA, U.S.A.). The ABK concentration in the supernatant was measured as described in “Determination of ABK Concentration.” Encapsulation efficiency was calculated as following using equation (1):

  

(1)

In Vitro Aerosol Performance

In vitro aerosol performance was evaluated using an Andersen cascade impactor (ACI) as described previously,²⁸⁾ with modifications. In brief, a powder sample (approximately 40 mg) was placed in a gelatin capsule (size 2; Qualicaps Co., Ltd., Nara, Japan), and the capsule was placed in a Jethaler^® dry powder inhaler (Reverse type; Tokico System Solutions, Ltd., Kanagawa, Japan). The inhaler device was connected to the assembled ACI connected to a vacuum pump maintained at a flow rate of 28.3 L/min. The powder sample was then released from the capsule, and vacuum pump was turned off after 10 s.¹¹⁾ The powder present in each stage of the ACI was collected by dissolving in water (for ABK-MAN nanoparticles) or 70% methanol (for ABK-SD/MAN nanoparticles) because ABK and MAN had high aqueous solubility and ABK-SD needs methanol for its degradation. The concentration of ABK was determined as described in “Determination of ABK Concentration.” The fine-particle fraction was calculated as the total amount of ABK from stage 3 to stage 7 in the ACI.

Drug Release Study

Fifty milliliters of phosphate-buffered saline (PBS) or 0.3% SDS-PBS solution was added to a glass vial and mixed at 400 rpm. A cellulose tube for dialysis (MWCO, 14000; EIDIA Co., Ltd. Tokyo, Japan) containing 1 mL of PBS (or 0.3% SDS-PBS solution) and 0.1 g of powder sample was then placed into the vial. At appropriate time points (0, 3, 5, 10, 20, 30, 60, 120, 240 min), 1 mL of PBS was taken from the glass vial and replaced with the same volume of PBS. The ABK concentration was determined as described in “Determination of ABK Concentration.”

Antimicrobial Effect (Agar Disk Diffusion Analysis)

Antimicrobial effects were evaluated as described previously.²⁹⁾ Pseudomonas aeruginosa NBRC 3445 was purchased from NITE Biological Resource Center (Chiba, Japan). A suspension of cultured bacteria (100 µL; 1 × 10⁹ cells/mL) was spread on an agar medium plate prepared by Soybean-Casein Digest Agar DAIGO (Nihon Pharmaceutica; Tokyo, Japan). A small piece of wet filter paper (1 × 1 cm) was placed on the center of the plate, and powder sample and solution sample containing 40 µg of ABK was loaded onto the paper. After 24 h of incubation at 35 °C, the diameter of the growth inhibition zone was measured using a digital caliper.

Statistical Analysis

Statistical analysis was conducted by using MATLAB^® software (Mathworks, Natick, MA, U.S.A.; version, R2022b). One-way ANOVA with a Dunnett’s post hoc test was used for the comparison of the average of inhibition zone diameter of MAN-treated group. p < 0.05 was considered statistically significant.

RESULTS AND DISCUSSION

In the current study, inhalable nanocomposite particles containing ABK-SD nanoparticles were developed using a spray drying method with a special spray nozzle. The steps of nanocomposite particle preparation using a two-solution mixing-type spray nozzle are shown in Fig. 1B. This nozzle system is composed of a primary passage for aqueous solution containing the drug carrier (MAN) and subpassages for organic solvent containing hydrophobic compounds. These passages are joined in a mixing area, and then micromists containing the nanocomposite particles are immediately spray dried. In our laboratory, nanocomposite particles containing a poorly water-soluble drug were previously prepared using this spray nozzle approach, and these nanocomposite particles exhibited improved drug dissolution.^28,30) In this study, we focused on a water-soluble drug, ABK, which holds promise for the treatment of nosocomial pneumonia. ABK was encapsulated into SD nanoparticles prepared using a solid-in-oil emulsion method that efficiently encapsulates hydrophilic drugs, including proteins and genes.³¹⁾ After the preliminary preparation process, ABK-SD nanoparticles alone could be dispersible in organic solvents. However, viscous materials are difficult to disperse in aqueous solution, and the characteristic is not suitable for inhalation formulations. The spray-drying technique using a two-solution mixing-type nozzle allows for the dispersion of ABK-SD nanoparticles in MAN microparticles as nanocomposite particles, and the nanocomposite particles are useful in terms of handling characteristics. Both the ABK-SD nanoparticles and ABK-SD/MAN nanocomposite were characterized in the present study.

Development of a Novel Fluorometric Method to Determine ABK

In the beginning of the current study, a novel method to determine ABK was developed with reference to literature regarding the determination of gentamicin.²⁷⁾ As aminoglycoside antibiotics such as ABK exhibit poor UV and visible light absorption peaks and do not markedly fluorescence, derivatization of the chemical structure is necessary. For derivatization of the primary amine of ABK with high efficiency, o-phthalaldehyde was used. The derivatized ABK exhibited fluorescence in almost linear relationship over the concentration range 0–50 µg/mL, with an R² value of the standard curve of 0.996 (Fig. 2). This determination method was used to evaluate the encapsulation efficiency, in vitro aerosol performance, and drug release of ABK-SD/MAN nanoparticles, as shown below. This in vitro method is simple and effective, as it does not require the use of expensive equipment (e.g., HPLC; HPLC-mass spectrometry; HPLC-tandem mass spectrometry).

Fig. 2. Calibration Curve for ABK Concentration versus Fluorescence of ABK-Derivative

The fluorescence-based determination method was described in Materials and Methods.

Factors That Affect the Formation of ABK-SD Nanoparticles

Next, we investigated the factors affecting the preparation of ABK-SD nanoparticles, as shown in Fig. 3. The effect of ER-290 concentration on the size and PDI of ABK-SD nanoparticles was evaluated (Fig. 3A). Increasing the amount of ER-290, a lipophilic surfactant used as the coating material for ABK-SD, resulted in smaller nanoparticles with lower PDIs. Lowering the concentration of ER-290 tended to result in larger ABK-SD nanoparticles due to the lack of coating material. In contrast, when the amount of ER-290 in the preparation was increased, the total drug content in a given amount of ABK-SD nanoparticles decreased, with a proportion of the nanoparticles being empty. Thus, preparing ABK-SD nanoparticles with 5% ER-290 was optimal for obtaining stable nanoparticles.

Fig. 3. (A) Effect of ER-290 Concentration on the Size and PDI of ABK-SD Nanoparticles in the Preparation Step

ABK concentration was fixed at 2%. (B) Effect of ABK concentration on the size and PDI of ABK-SD nanoparticles in the preparation step. ER-290 concentration was fixed at 5%. The detailed preparation method was described in Materials and Methods. Data represent the mean ± standard deviation (n = 3).

The effect of ABK solution on the resulting particle size and PDI was also evaluated (Fig. 3B). Both the particle size and PDI increased with increasing ABK concentration in the preparation process. A lower ABK concentration (1%) resulted in larger PDI of ABK-SD particles. These results suggest that a lack of loaded drug results in partial failure of the nanoparticle self-assembly process. In contrast, a higher ABK concentration requires more ER-290 for adequate coating, and accordingly, the resulting particle size and PDI are increased. Thus, 2% ABK was set as the optimal concentration for generating ABK-SD nanoparticles.

Characterization of ABK-SD/MAN Nanocomposite Particles Prepared Using a Spray Dryer with a Two-Solution Mixing-Type Spray Nozzle

SEM images are shown in Fig. 4. ABK bulk powder exhibited a fragmented shape, whereas spray-dried ABK-MAN particles and ABK-SD/MAN nanocomposite particles were smooth and spherical in shape and approximately 2–3 µm in size, which is suitable for use in an inhalation formulation.

Fig. 4. SEM Images

(A) ABK bulk powder. (B) ABK-SD/MAN. (C) ABK-MAN.

Next, ABK-SD/MAN nanoparticles were partially dissolved in small amount of water to expose the ABK-SD nanoparticles in MAN microparticles for observation by TEM (Fig. 5A, partially exposed ABK-SD nanoparticles in MAN microparticles; Figs. 5B and C, exposed ABK-SD nanoparticles). The mean particle size and PDI of the ABK-SD nanoparticles were measured using a dynamic scattering method (Table 1). The mean size of ABK-SD nanoparticles after spray drying (272.1 ± 26.5 nm) was slightly larger than that before spray drying (203.9 ± 22.8 nm), as the standard deviation of PDI after spray drying (0.295 ± 0.222) was greater than that before spray drying (0.294 ± 0.033). The efficiency of ABK encapsulation was 82.2 ± 4.3% (drug contents, 0.566 ± 0.068%; mean ± standard deviation, n = 5) after preparation of the ABK-SD/MAN nanocomposite particles. These results suggest that the efficiency of ABK-SD encapsulation was high and that leakage after spray drying was probably minimal.

Fig. 5. TEM Images

(A) ABK-SD/MAN. (B, C) ABK-SD nanoparticles exposed within nanocomposite particles.

Table 1. Mean Particle Size and PDI of ABK-SD Nanoparticles before and after Spray Drying

The data represented the mean ± standard deviation (n = 3).

In Vitro Aerosol Performance of ABK-SD/MAN Nanocomposite Particles

The in vitro microparticle aerosol performance was compared between the ABK-MAN microparticles and ABK-SD/MAN nanocomposite particles (Fig. 6). The fine particle fraction of ABK-SD/MAN nanocomposite particles (28.4 ± 5.4%) was higher than that of ABK-MAN microparticles (11.4 ± 7.6%). ABK-MAN microparticles tended to adhere to the inhaler device (including the capsule) and the throat portion of the apparatus. One possible reason may be the charge characteristics of ABK molecules. The tribo-charging behavior of inhalation powders such as MAN and active pharmaceutical ingredient have been investigated.³²⁾ The charging characteristics of ABK-MAN microparticles in which ABK molecules are dispersed may differ from that of ABK-SD/MAN nanocomposite particles, in which ABK molecules are encapsulated by SD molecules. We used a Jethaler^® in which the agglomerates may be disintegrated into fine particles for inhalation.³³⁾ Disintegrated primary particles in which ABK-SD nanoparticles are exposed might prevent electrostatic adsorption between the wall of the device and the capsule.

Fig. 6. In Vitro Aerosol Performance and Fine-Particle Fraction Values as Determined Using ACI

Data represented the mean ± standard deviation (n = 3).

Analysis of ABK-SD/MAN Drug Release

The drug release profile is shown in Fig. 7. Drug release by ABK-SD/MAN particles was slower compared with ABK-MAN particles, with almost 20% of the drug released after 4 h. These results indicate that SD particles have a high drug retention property. Another study investigated drug release in the presence of SDS to assess the release of poorly water-soluble drugs for inhalation.³⁴⁾ We hypothesized that the use of a surfactant would partially imitate the lung environment due to the presence in the lungs of phospholipids and proteins that function as surfactants.³⁵⁾ Drug release was similar to that of ABK-MAN solution in the presence of the surfactant, indicating that the drug was almost completely released. The current results suggest that ABK-SD nanoparticles release ABK in lung tissue over time.

Fig. 7. Drug Release Profiles

The detailed method was described in Materials and Methods. Data represent the mean ± standard deviation (n = 3).

Antimicrobial Effect of ABK-SD Nanocomposite Particles

The antimicrobial effect of ABK solution, ABK-MAN microparticles, and ABK-SD/MAN nanocomposite particles is shown in Fig. 8. MAN alone and ER-290 alone (control) did not exhibit any antimicrobial effect (≈10 mm). Compared with the positive control (ABK solution), ABK/MAN exhibited almost the same effect. These results indicate that spray drying does not affect the stability or activity of ABK. ABK-SD/MAN nanocomposite particles exhibited a comparable antimicrobial effect, although the effect was less intense than that of ABK solution and ABK-MAN microparticles. This is due to the incomplete drug release from the ABK-SD formulation (Fig. 7). ABK-SD nanoparticles are neutral hydrophobic particles because the SD nanoparticles are coated with a non-ionic hydrophobic surfactant. Schneider et al. reported that neutral nanoparticles penetrate the mesh of human airway mucus ex vivo and are retained in the mucus.²¹⁾ Meers et al. reported the biofilm penetration characteristics of lipophilic nanoparticles.²²⁾ These reports support in part the usefulness of our current drug formulation.

Fig. 8. Antibacterial Effect (Agar Disk Diffusion Method)

Data represent the mean ± standard deviation (n = 3). ***, p < 0.005 compared to MAN, analyzed by one-way ANOVA with a Dunnett’s post hoc test.

CONCLUSION

ABK-loaded SD nanoparticles were successfully prepared by optimizing the experimental conditions, and ABK-SD nanocomposite particles were further prepared using a two-solution mixing nozzle with high drug encapsulation. The resulting nanocomposite particles exhibited good aerosol performance and comparable antimicrobial effects. The nanocomposite particles developed using the present unique spray-drying method, which is suitable for scale-up, could be useful for inhalation powder formulations. Although further experiments are necessary, the ABK-SD/MAN nanocomposite formulation holds promise for use in treating infections caused by Pseudomonas aeruginosa, including hospital-acquired pneumonia.

Acknowledgments

This research was conducted with funds from Nagoya City University.

Author Contributions

Nao Yamamoto: Conceptualization, Investigation, Methodology, Data curation, Formal analysis, Visualization, Project administration, Writing–original draft.

Tatsuaki Tagami: Conceptualization, Methodology, Project administration, Writing–original draft, Writing–review and editing, Supervision.

Koki Ogawa: Writing–review and editing, Supervision.

Tetsuya Ozeki: Resources, Supervision.

Conflict of Interest

The authors declare no conflict of interest.

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