Nanostructured lipid carriers-loaded dry powder inhaler for pulmonary delivery of amphotericin B in treatment of allergic bronchopulmonary aspergillosis

Abstract

Amphotericin B (AmB) is used to treat Allergic Bronchopulmonary Aspergillosis (ABPA) caused by Aspergillus fumigatus. AmB is available in oral and injectable forms but has limitations, resulting in low levels of AmB in the lungs. We formulated AmB using Nanostuctured Lipid Carriers (NLCs) and Dry Powder Inhaler (DPI) systems to improve the effectiveness of AmB delivery to the lungs. Oleic acid and Compritol® 888 ATO were used as liquid lipid and solid lipid materials for the preparation of NLCs. Characterization tests were conducted to determine the best formula, followed by in vivo pharmacokinetic studies. Characterization results of NLCs-AmB showed that AmB was encapsulated in NLCs in high amounts (21.37 %), with a particle size of 199.95 ± 18.97 nm. This value is better than NLCs-AmB 1 and NLCs-AmB 2. In vitro study showed the release of AmB from NLCs-AmB was 94.38 ± 5.68 %, while pure AmB was only 11.43 ± 1.83 %. Characterization of NLC-AmB-DPI showed good flow properties for NLCs-AmB-DPI 3, supporting delivery to the lungs. In vivo pharmacokinetic studies resulted in a high concentration in the lungs of 7.33 ± 0.56 µg/mL. Overall, this study offers proof-of-concept that the developed NLCs-AmB-DPI are good, safe, and can be used as an alternative for AmB delivery to the lungs, especially in the treatment of ABPA.

Introduction

Allergic Bronchopulmonary Aspergillosis (ABPA) is a disease caused by Aspergillus fumigatus in the respiratory tract. ABPA often occurs in patients with asthma or cystic fibrosis. According to the World Health Organization (WHO) with the Global Asthma Network, the number of asthma cases worldwide reached 334 million (Herman et al., 2023), and globally the prevalence of ABPA among asthmatics is 2.5% (Agarwal et al., 2023). Aspergillus fumigatus has a small size of about 2-3 µm, which allows it to easily enter the lungs and evade the immune defense mechanisms in the lung (Nywening et al., 2020). As a result, the fungus Aspergillus fumigatus that enters the upper respiratory tract is difficult to remove and wouldl persist in the inner airway and cause ABPA (Roboubi et al., 2023).

One of the treatments for ABPA disease involves using a polyene antifungal, Amphotericin B (AmB) (Roboubi et al., 2023). The mechanism of action of AmB is to inhibit fungal growth by inhibiting ergosterol synthesis in fungal cell membranes (Sen et al., 2022). AmB preparations for ABPA treatment are available in the form of oral and injection. Oral AmB supplies have limitations in terms of bioavailability. The oral bioavailability of AmB is very low, ranging from 0.2-0.9 % (Fairuz et al., 2022) because AmB is included in the Class IV Biopharmaceutical Classification System (BSC), characterized by extremely low solubility and permeability properties (Nimtrakul et al., 2020). In addition to the low bioavailability problem, the oral preparation of AmB also presents other drawbacks, such as potential gastrointestinal disturbances during its preparation and susceptibility to first-pass metabolism. It can also cause a lack of concentration of the drug to reach the target, so the effect of the treatment is reduced. AmB injectable preparations are also available for ABPA treatment. However, this preparation also has a limitation, it can cause nephrotoxicity, especially if used for a long time. The mechanism of AmB nephrotoxicity is the high concentration of AmB that reaches the kidneys via endocytosis, causing nephrotoxicity (Carolus et al., 2020). Various restrictions in oral preparation and injection are reasons to look for alternatives to AMB delivery in ABPA treatment. The inhalation route is one of the alternatives that can be used for the treatment of a disease, including ABPA. The inhalation route can deliver drugs directly into the lungs and produce high drug concentrations. This route can also decrease problems in the gastrointestinal tract, avoiding systemic toxicity and nephrotoxicity (Nainwal, 2022). Previous studies have developed lipid-based AmB such as Liposomal-AmB (AmBisome®), Amphotericin B lipid complex (Abelcet®), and Amphotericin B colloidal dispersion (Amphotec®/Amphocil®). These formulations of AmB are administered parenterally. However, this can lead to systemic exposure, infusion-related side effects, and less exposure time of the drug at the site of infection, such as in the lungs. Therefore, the formulation is administered via the lungs. Lipid-based AmB is nebulised to produce an aerosol that can be inhaled (Faustino and Pinheiro, 2020). However, this still has the disadvantage that nebulisation takes longer and requires a pressure source device that also needs to be maintained and cleaned (DE and Geller, 2005). In addition, the liposome system also still has weaknesses as a carrier system for AmB. AmB is one of the poorly soluble drugs. The entrapment of poorly soluble drugs into liposomes results in a limited amount that can be loaded. It can also lead to destabilisation of the double layer which makes the liposomes prone to membrane leakage (Mennini et al., 2016). As a result, the amount of drug inside the liposome may be reduced before reaching the target site of treatment. Therefore, another system is needed for a more optimised delivery of AmB to the lungs. Other research used Solid Lipid Nanoparticles (SLNs). This system uses solid lipids in its formulation, but its dense structure leads to a lack of drug loading for SLN (Yeganeh et al., 2020).

Nanostructured Lipid Carriers (NLCs) are a new system developed to overcome the problems of the previous system, liposome and SLN, which has been used for AmB delivery. Unlike SLNs, which only use solid lipid materials, NLCs combine solid lipid and liquid lipid materials in the formulation process. The combination of the two materials results in an imperfect matrix in the NLCs structure, thus having more space for drug loading. This is a major advantage of the NLCs system as it increases the drug loading capacity and also improves its stability during storage. Moreover, the drug release profile of NLCs is faster and more controlled than SLN. The particle size, zeta potential, and polydispersity index of NLCs are also much better than SLN (Viegas et al., 2023, Dudhipala et al., 2018, Das et al., 2012). These NLC systems are suitable for formulations that use a lipophilic active substance. The ratio between solid and liquid lipids affects the encapsulation efficiency and drug release profile. The ratio of solid and liquid lipids commonly used in formulations of NLCs is 70:30 to 99,9:0,1 (Elmowafy and Al-Sanea, 2021). Previously, NLC systems have been used for AmB delivery in liquid form (Nimtrakul et al., 2020). Dry Powder Inhaler (DPI) is a form of drug made in dried powder and can be circulated through the mouth and directly reach the lungs (Gaikwad et al., 2023). Dry powder inhaler is widely used in formulations of drugs targeting the lung such as salbutamol sulfate (Kumaresan and Sathishkumar, 2016), sunitinib (Taymouri et al., 2019), and ciprofloxacin (Almurshedi et al., 2021). Dry Powder Inhaler preparations are better than conventional preparations for the lungs such as nebulizers and Metered-Dose Inhalers (MDI). The DPI is easy to use, and propellant-free, and the stability is better than the dispersed shape (Magramane et al., 2023). The combination of NLCs and DPI systems has been applied in the formulation of several drugs made to reach the lungs. Examples of drugs that have been made in the preparation are phenofibrate (Xia et al., 2016). This form of preparation offers easy drug use, more drug loading, and produces a high concentration of the drug (He et al., 2022).

From our knowledge, no research has been conducted to formulate AmB-based Nanostructured Lipid Carriers in the form of a Dry Powder Inhaler preparation. Therefore, for the first time in the research we have done, we have developed a formulation of Nanostructured lipid carriers as a dry powder inhaler as a new strategy to deliver AmB in the ABPA treatment process. Nanostructured lipid carriers are made using stearate acid and oleic acid as solid lipids and liquid lipids. This preparation is evaluated by characterized, in vitro, ex vivo, and in vivo tests.

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Materials

Amphotericin B (AmB) (purity, ≥ 98 %), chitosan (MW medium, 190.000–310.000 Da), oleic acid, and Tween® 80 were obtained from Sigma-Aldrich Pte Ltd. (Singapore, Singapore). Compritol® 888 ATO was provided by Gattefossé (Lyon, France).

B. Florensia Dana Carla, Ananda Fachriza, Muh. Thaufik Umar, Johusua Entho Unawekla, Waode Ainun Anggraini, Maria Mir, Ahmad R. Alsayed, Andi Dian Permana, Nanostructured lipid carriers-loaded dry powder inhaler for pulmonary delivery of amphotericin B in treatment of allergic bronchopulmonary aspergillosis, International Journal of Pharmaceutics, 2025, 125926, ISSN 0378-5173, https://doi.org/10.1016/j.ijpharm.2025.125926.

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