1. Introduction
Pulmonary drug delivery is increasingly gaining attention as a desirable and non-invasive method for treating various medical conditions, particularly those affecting the lungs [
1]. The pulmonary route exhibits superiority over both oral and parenteral routes, as the lungs provide a large surface area for drug absorption, avoid first-pass metabolism, and provide high local concentration and quick onset of action [
2,
3]. Inhaled drug delivery helps deposit drugs directly in the target tissue, resulting in comparable therapeutic efficiency with a lower amount of drug compared to systemic administration. This reduces exposure of drugs to non-target sites and, consequently, reduces undesirable side effects [
4].
Various critical pulmonary diseases such as lung cancer, asthma, cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), and severe infections, including COVID-19, can impact the lungs, often resulting in significant mortality rates [
5]. Lung cancer (LC) is one of the most commonly diagnosed cancers and ranks as the top contributor to cancer-related fatalities globally, accounting for approximately 2 million new cases and 1.8 million deaths annually [
6,
7]. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer cases and is often associated with poor prognosis owing to its tendency for late detection, high incidence of metastasis, and an increased likelihood of relapse [
8]. Therefore, surgical intervention is not feasible at these late stages, which makes chemotherapy the primary treatment option [
9]. Despite the pivotal role of chemotherapy in the treatment of lung cancer, particularly in advanced scenarios, its application faces notable challenges, including the development of multidrug resistance (MDR), severe and potentially life-threatening side effects, and the prohibitive costs associated with chemotherapeutic agents [
10].
Ivermectin (IVM) is a macrolide anti-parasite, officially approved by the FDA, and is commonly administered orally to treat river blindness, elephantiasis, and scabies. Clinically, it holds significant importance as a well-tolerated and safe broad-spectrum antiparasitic medication. IVM demonstrates antiviral effects as well as the ability to regulate inflammatory diseases by reducing cytokine levels [
11]. Moreover, recent studies have highlighted IVM’s potential to inhibit tumor cell growth and overcome drug resistance by modulating various signaling pathways [
12,
13]. Given the adverse impact of chemotherapeutics on healthy organs and the challenges associated with chemotherapeutic resistance, it becomes imperative to consider drug repurposing as a strategic approach to surmount these obstacles [
10]. Despite the promising anti-cancer effects of IVM, it has poor water solubility, which limits its bioavailability and, therefore, its biological activity [
14]. In this case, nanoparticles (NPs) have emerged as a promising strategy for enhancing the bioavailability of drugs [
15] and increasing drug delivery efficiency by evading clearance pathways in the lungs [
16].
Nanotechnology has unlocked new prospects for controlled and targeted drug delivery for cancer therapy. Compared to conventional formulations, NPs offer several advantages: enhanced tissue targeting due to facile surface functionalization, improved tumor distribution through EPR effects, controlled drug release, the ability to deliver multiple drugs with different chemical properties simultaneously, the ability to evade innate biological impediments, and enhanced pharmacokinetic and pharmacodynamic properties, which all in all leads to better therapeutic efficiency and reduced side effects [
17]. Recent research has highlighted the advantages of using inhaled nanoparticle delivery as an efficient drug delivery approach to obtain therapeutic benefits at a very low dose of drugs. Nanotechnology offers benefits such as evading clearance pathways and establishing a sustained release pattern. It also enables targeted delivery of drugs to cancer tissues in the lungs, thereby enhancing treatment efficiency [
16].
Liposomes and polymeric nanoparticles are widely utilized as nanocarriers due to their favorable properties. Polymeric nanoparticles composed of natural or synthetic polymers offer superior structural integrity of NPs’ storage stability and sustained release of the encapsulated drugs; therefore, these are considered potential candidates for various biomedical applications, including diagnostic or therapeutic delivery [
18,
19]. Liposomes, on the other hand, exhibit high biocompatibility, resembling biological membranes and seamlessly integrating with the pulmonary surfactant layer in the lungs [
20]. To combine the advantages of liposomes and polymeric nanoparticles, lipid–polymer hybrid nanoparticles (LPHNPs) have been developed. The structure of LPHNPs comprises a polymer core encapsulating the drug, a phospholipid shell ensuring biocompatibility, and an outer layer of stabilizer aimed at enhancing in vivo circulation time and providing steric stabilization [
21]. These characteristics have made LPHNPs a promising drug delivery platform, especially for pulmonary delivery [
20,
22]. As reported in the literature, LPHNPs have been used to deliver various therapeutics, such as small molecules, nucleic acids, or a combination of both, as inhalable formulations for pulmonary diseases and achieved the desired outcome [
23,
24,
25,
26,
27,
28,
29,
30,
31].
Given the potential of IVM as an anti-cancer agent and recognizing the advantages of lipid–polymer hybrid nanoparticles (LPHNPs) for pulmonary delivery, the aim of this study is to develop inhalable IVM-loaded LPHNPs and evaluate their characteristics and suitability for pulmonary delivery. For this purpose, we used (1) polycaprolactone, serving as the polymer core, which provides a sustained release profile, structural integrity, and stability; (2) lecithin, forming the lipid shell to improve biocompatibility by mimicking the surfactant lining of the lung; along with (3) Pluronic F127 as the outer stabilizer/surfactant. The ability of Pluronic F127 to attenuate the binding of NPs to mucin and increase mucus penetration has been reported in previous studies [
32,
33]. Therefore, for pulmonary delivery, where mucus presents a barrier to nanoparticle transport, using such a stabilizer in developing drug delivery vehicles can enhance pulmonary delivery efficiency. Developed formulations were evaluated in terms of physicochemical characteristics, entrapment efficiency and drug loading, release profile, solid phase characteristics, flow, and aerosolization properties to achieve an optimized DPI formulation.
To the best of our knowledge, LPHNPs have never been studied for delivering IVM through the pulmonary route as a DPI formulation. Therefore, developed powder formulations were characterized properly to determine their suitability for pulmonary delivery as a DPI formulation.
2. Materials and Methods
2.1. Materials
Ivermectin, Pluronic® F127, polycaprolactone (Mn 70,000–90,000 g/mol), and dialysis bags (12,000 Da) were purchased from Sigma-Aldrich, St. Louis, MO, USA. Soybean lecithin was obtained from Merck Millipore, Bangalore, India. HPLC-grade acetonitrile and methanol were supplied from RCI Labscan, Bangkok, Thailand and Fisher Chemical, Couva, Trinidad, respectively. Dichloromethane and ethanol were provided from Thermo Fisher Scientific, Waltham, MA, USA. Deionized double distilled water (Milli-Q water) was used in all experiments.
2.2. IVM-Loaded LPHNP Preparation
IVM-loaded LPHNPs were prepared using a single-step emulsion solvent evaporation method with polycaprolactone (PCL) as the polymeric compartment, lecithin as the lipid compartment, and Pluronic F127 as a surfactant (stabilizer). Different formulations of LPHNPs were developed as described by Godara et al. [
34] with slight modifications. In this method, appropriate amounts of PCL, lecithin, and IVM were dissolved in dichloromethane (DCM), as indicated in
Table 1. This organic phase was added dropwise at a speed of 1 mL/min to an aqueous phase containing different concentrations of Pluronic F127 under continuous stirring at room temperature, followed by homogenization in an ice bath (2–8 °C) at 10,000 rpm for 3 min using a high-speed homogenizer (IKA ULTRA-TURRAX
® T25 (
Figure 1) (Staufen im Breisgau, Germany). The nanopreparation was then stirred overnight for solvent evaporation. Developed nanoparticle suspensions were centrifuged at 14,000×
g rpm for 30 min, washed three times with deionized water, and then freeze-dried (Freeze Dryer Alpha 1–4 LD plus (Christ, Osterode am Harz, Germany)) to obtain powder formulation.
2.3. Physicochemical Characterization
The particle size, polydispersity index (PDI), and zeta potential of developed LPHNPs before and after freeze drying were determined by dynamic light scattering (DLS) technique using Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). For samples before freeze drying, fresh NP suspensions were used for size analysis with appropriate dilution using deionized water [
35]. Moreover, freeze-dried powder (3 mg) was suspended in 5 mL of deionized water and sonicated for 15 min before DLS analysis. All measurements were performed in triplicate at room temperature.
2.4. Morphology of Nanoparticles
The morphological examination of the developed LPHNPs was observed by scanning electron microscopy (SEM) using Zeiss Sigma Field Emission. Prior to freeze-drying, a 10 μL drop of the LPHNP suspension was applied to a silicon wafer and allowed to air-dry. The freeze-dried LPHNP powder was then separately mounted on an aluminum stub using carbon adhesive tape. Excess particles on the adhesive tape were removed by blowing with nitrogen gas. Both samples were subsequently coated with a conductive sputtered gold layer.
2.5. Drug Loading and Entrapment Efficiency
Drug loading and entrapment efficiency were analyzed by HPLC through an indirect method. In this method, the non-entrapped drug (free drug) was collected by centrifugation at 14,000×
g rpm for 30 min. An aliquot of supernatant was extracted, diluted with methanol (1:20), and filtered by a 0.22 syringe filter to be injected into the system. The amount of IVM in the supernatant was determined with reference to the standard calibration plot. Each sample was repeated 3 times. After achieving the amount of IVM, drug loading and entrapment efficiency were calculated using Equations (1) and (2) below:
2.6. In Vitro Drug Release Study
To examine the release behavior of developed IVM-loaded NPs, the dialysis bag technique was employed in triplicate, followed by a previously reported method with little modifications [
35]. To prepare the dialysis membrane bag (MW 12,000 Da, Sigma-Aldrich, St. Louis, MO, USA) for the release study, it was immersed in deionized water for a duration of 24 h to ensure its readiness and appropriateness for the experimental procedure. A certain amount of IVM-loaded LPHNP formulations (equivalent to 2 mg IVM) was dispersed and placed in a dialysis membrane bag. A dialysis bag was then placed in a beaker containing 50 mL of ethanol/water (50:50) media under continuous magnetic stirring with a speed of 100 rpm at a controlled temperature of 37 °C. At different time intervals (0.5, 1, 1.5, 2, 4, 6, 12, 24, 48, 72, and 96 h), a 1 mL sample was withdrawn from the receiver media and immediately replaced with equal amounts of fresh mixture to keep the media at a constant volume to maintain the sink condition. Samples were analyzed by HPLC at 245 nm.
2.7. Kinetics of IVM Release from LPHNPs
Furthermore, the kinetics of IVM release from LPHNPs was mathematically modeled by calculating the correlation coefficient (r) value for each kinetic model, namely zero-order, first-order, Higuchi, and Hixon–Crowell. The Korsmeyer–Peppas model was also used to characterize the release mechanism of IVM from the LPHNP formulation by calculating the release exponent “n”.
2.8. Solid-Phase Characterization
2.8.1. Differential Scanning Calorimetry (DSC)
To evaluate the thermal behavior of the formulations, pure IVM, blank NPs, and IVM-loaded NPs were studied by DSC on TA Instruments, model Q100 DSC (New Castle, DE, USA)). Developed powder formulations were accurately weighed (3 ± 0.1 mg) and placed in a hermetic aluminum pan, sealed, and heated. An empty sealed pan was used as a reference. Both pans underwent heating within a specified temperature range of 25–300 °C at a constant heating rate of 10 °C/min.
2.8.2. Thermogravimetric Analysis (TGA)
A NETZSCH Simultaneous Thermal Analyzer (STA) 449 F3 Jupiter was used to evaluate the thermogravimetric behavior and decomposition of pure IVM, blank NPs, and IVM-loaded NPs. For this purpose, 5–8 mg of IVM, blank NPs, and IVM-loaded NPs were placed in TGA alumina crucibles and heated with a heating rate of 10 °C/min from 50 to 800 °C. An empty alumina crucible was used as the reference.
2.8.3. Attenuated Total Reflection–Fourier Transform Infrared (ATR–FTIR)
ATR-FTIR spectroscopy was performed using a Thermo is5 FTIR spectrometer (Nicolet, Madison, WI, USA) to analyze the chemical composition and structure of the nanoparticles. A small amount of powder samples was placed on top of the diamond crystal and secured with a high-pressure clamp. Spectra were obtained within the range of 400−4000 cm−1 with a resolution of 8 cm−1 and 64 scans. Data were analyzed using OMNIC 8.0 software.
2.8.4. Powder X-ray Diffraction (PXRD)
IVM, Pluronic F127, and lecithin were measured in capillary (internal diameter 0.8 mm) transmission geometry using a Rigaku SmartLab X-ray diffractometer (Tokyo, Japan). A focusing Goebel mirror in a CBO-E module was used to converge the X-ray beam from a Cu X-ray tube (λ = 1.54059 Å, 40 kV 40 mA), followed by a height limiting slit of 15 mm. Soller slits of 2.5° were used on both primary and secondary beam paths. A Hypix3000 detector (Rigaku, Tokyo, Japan) collecting diffraction signals in 1D mode with a PSD opening of 20 mm after an extended 6.6 mm anti-scattering slit and a 12 mm receiving slit. The capillary samples were spun at 15 rpm during XRD pattern collection from 3 to 70 °2θ at 0.02° step size in 1 h.
The polycaprolactone polymer beads were melted into a piece of self-standing foil (0.8 mm thickness), and their XRD pattern was taken in foil transmission geometry. IVM-loaded LPHNPs were held between two Kapton foils and measured in foil transmission geometry. The blank Kapton foil background was also collected. Both foil transmission XRD data were collected using the same focusing X-ray beam optics and the same measurement scheme described for capillary transmission.
2.9. Particle Density and Flow Property
The flow properties of the developed powder were determined using Carr’s index (CI), Hausner ratio (HR), and angle of repose (θ) according to the relevant equations (Equations (3) and (4)). The bulk density and tapped density of the nanoparticle powder were measured using a graduated cylinder in a tapped density tester (ERW-SVM101202, ERWEKA, Langen, Germany). Certain amounts of NP powder (500 ± 0.5 mg) were placed in a 5 mL graduated cylinder to record the initial volume (V
0). The cylinder was then subjected to 500 mechanical taps in the density tester to establish the new volume (V
1). Using V
0 and V
1, Carr’s index and the Hausner ratio were calculated according to Equations (3) and (4) [
35]. Each measurement was performed in triplicate.
The angle of repose is a crucial indicator for evaluating the flow characteristics of nanoparticle powders. It defines the maximum angle relative to the horizontal plane of a conical heap of particles. To measure the angle of repose, 250 ± 0.5 mg of nanoparticle powder was gradually poured through a funnel into a beaker situated roughly 3 cm beneath the funnel’s tip. Once the particles settled, the height (h) and base diameter (d) of the resulting cone were recorded. The angle of repose was then calculated using these measurements [
35] according to the following Equation:
2.10. In Vitro Aerosolization Study
Aerosolization performances of the developed NP powder formulations were evaluated by a twin-stage impinger (TSI) following the protocol outlined in the British Pharmacopeia. A Breezehaler® (Novartis Pharmaceuticals Pvt Ltd., Macquarie Park, NSW, Australia) was used as the DPI device. Then, 7 mL and 30 mL of water/ethanol 50:50 were poured into stage 1(S1) and stage 2 (S2) of the TSI, respectively. The airflow through the TSI was set to 60 L/min, regulated by a vacuum pump (D-63150, Erweka, Langen, Germany), and monitored through a calibrated digital flow meter (Fisher and Porter, Model 10A3567SAX, London, UK).
In the context of aerosol characterization studies, 20 ± 0.5 mg of the nanoparticle powder samples were loaded into size 3 hypromellose capsules (Vcaps@ Plus, Capsugel, Lonza, Basel, Switzerland). These capsules were then placed in a Breezehaler® dry powder inhaler (DPI) and twisted using the DPI device. Actuation of the apparatus was performed by the vacuum pump for 5 s at 60 ± 5 L/min to disperse the powder formulations in different stages of the TSI device. This procedure was conducted 5 times for each formulation (n = 5). Following each experimental run, all stages of TSI underwent separate washing with ethanol/water (50:50), and the quantity of IVM was determined by both HPLC assay and gravimetric analysis.
A validated method developed in our laboratory was employed for the gravimetric analysis. For this analysis, filter paper (orifice 0.20 μm, Phenomenex, Torrance, CA, USA) that had been dried and weighed was utilized to filter washings from each stage of the TSI device. After filtration, the particles that had accumulated on the filter paper were dried at 60 °C for 24 h until the filter paper reached a constant weight. This weight was then used to gravimetrically determine the mass of NPs. For chromatographic analysis, washings from each stage were gently stirred (100 rpm) for 96 h at 37 °C to ensure drug was released. The HPLC method was then used to measure the amount of IVM. Thus, both gravimetric and chromatographic methods contributed to determining the quantity of IVM deposited into stage 2 from the IVM-loaded LPHNPs [
36].
Aerosolization performance of the prepared formulations was determined by measuring recovered dose (RD), emitted dose (ED), and fine particle fraction (FPF) using Equations (6) and (7). RD is the total amount of particles collected from the inhaler, S1 and S2. ED is the fraction of RD delivered from the inhaler into S1 and S2. FPF is defined as the fraction of RD deposited in the S2 of TSI.
2.11. HPLC Assay
IVM analytical assay was carried out using HPLC, as previously reported [
37]. For this purpose, the calibration plot was produced using a 1 mg/mL Ivermectin stock solution by dissolving 5 mg Ivermectin powder in 5 mL methanol in a volumetric flask. Stock solution was diluted with methanol to achieve various concentrations (2.5, 5, 10, 25, and 50 µg/mL) for developing a calibration plot. For HPLC analysis, an Agilent HPLC Series 1100 (Santa Clara, CA, USA) was used to perform the analytical experiment. A mixture of acetonitrile (53.0%), methanol (27.5%), and ultra-pure water (19.5%) was used as the mobile phase and a Varian Microsorb 100 C18 column (4.6 × 250 mm) as the stationary phase (Palo Alto, CA, USA). Solvents were mixed properly, filtered through 0.22 µm filters, and degassed by putting them in ultrasonic bath for 10 min. Flow rate and injection volume were set at 2 mL/min and 20 μL, respectively, with a detection wavelength set at 245 nm. A linear plot of the area under the curve (AUC) versus concentration with a coefficient of determination was obtained with limit of detection (LOD) and limit of quantification (LOQ) of 0.60 and 1.83 µg/ml, respectively, calculated based on the calibration curve [
38].
2.12. Statistical Analysis
Each experiment was conducted in triplicate, and the results are presented as mean ± SD. Statistical differences between samples were assessed using one-way ANOVA followed by Tukey’s post hoc test, with significance set at p < 0.05. This analysis was performed using GraphPad Prism software, version 10.0.2.