Enhanced Macrophage Uptake of Spray-Dried Phosphatidylserine-Loaded Microparticles for Pulmonary Drug Delivery Applications

Abstract

Macrophages are an integral part of the innate immune system and act as a first line of defense to pathogens; however, macrophages can be reservoirs for pathogens to hide and replicate. Tuberculosis, influenza virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are common diseases whose pathogens are uptaken into macrophages. Current treatments for diseases such as these are limited by the therapeutic delivery method, which typically involves systemic delivery in large, frequent doses. This study aims to overcome this limitation via the development of an inhalable dry powder microparticle (MP) formulation capable of targeted drug delivery to alveolar macrophages in addition to controlled release of a therapeutic. A simple one-step spray drying method was used to synthesize acetalated dextran (Ac-Dex) MP loaded with the model therapeutic, curcumin, and 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), which is a phospholipid that induces ligand-receptor mediated macrophage phagocytosis. The resulting MP exhibited significantly more uptake by RAW 264.7 macrophages in comparison to MP without DPPS, and it was shown that DPPS-mediated uptake was macrophage specific. The particles exhibited pH-responsive release and in vitro aerosol dispersion analysis confirmed the MP can be effectively aerosolized for pulmonary delivery. Overall, the described MP has the potential to improve treatment efficacy for macrophage-associated diseases.

Graphical Abstract

1. INTRODUCTION

Pulmonary drug delivery is a rapidly growing field of interest in medicine as targeted delivery to the lungs provides several advantages compared to other delivery routes. The lungs contain between 200 and 600 million alveoli, presenting a massive surface area with a thin single cellular layer for aerosolized therapeutics to deposit [1]. The large surface area of the lungs can lead to a rapid on set of therapeutic actions, while avoiding the first-pass effect, leading to higher bioavailability of drugs [2]. Furthermore, targeted delivery to the lungs can reduce off-site side effects and improve treatment efficacy, challenges normally associated with systemic delivery routes [3]. Spray drying has been employed to create a variety of dry powder formulations for pulmonary delivery applications. Spray drying is a one-step, scalable process with tunable instrument parameters that can achieve particle characteristics suitable for specific formulation requirements. For effective pulmonary delivery, particle size is an important factor that dictates aerosol particle deposition [4]. For example, particles > 10 μm deposit in the oropharyngeal region, resulting in a large portion of the dose never reaching the lungs [5], whereas particles between 1 and 5 μm are capable of depositing in the alveolar regions of the lungs [4,6,7]. Compared to liquid formulations, dry powder MP produced via spray drying can enhance the stability, solubility, and bioavailability of therapeutics in addition to enhancing the chemical and physical stability of the MP themselves [8,9].

The lungs have several mechanisms to clear particles, where particles deposited in the upper lungs are primarily cleared through mucociliary clearance [4,10]. However, the thickness of the mucus layer decreases dramatically in the peripheral regions of the lung, which decreases mucociliary clearance [11–14]. In this region, alveolar macrophages become the predominant mechanism for particle clearance via phagocytosis [7,15]. As the predominate phagocytic cell type in the lungs, alveolar macrophages are responsible for clearing inhaled microorganisms, cellular debris, particles, and other environmental toxins [15–17]. An important function of macrophages is the clearance of apoptotic cells which express signaling pathways and markers that initiate phagocytosis [18]. While macrophages play an important role in the innate immune system, they can become sources for pathogens associated with infectious diseases to reside and replicate. Tuberculosis (TB), influenza virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and human immunodeficiency virus (HIV) are several examples of infections that utilize macrophages [16,17,19]. Treatment efficacies can be improved by directly targeting pathogen-loaded macrophages, resulting in increased localized delivery and reduced off-site side effects.

To date, several methods have been implemented to target macrophages based on receptor-ligand interactions [20]. A common target for enhanced macrophage uptake is the mannose receptor, CD206, a carbohydrate-recognition domain highly expressed on alveolar macrophages [21]. Other studies have focused on targeting tumor-associated macrophages through the use of peptide-functionalized particles that carry anti-cancer therapies [22–24] or by targeting different receptors on macrophages. Another method for macrophage targeting is the use of phosphatidylserine (PS) in particle formulations. PS is an anionic phospholipid that is normally located on the inner membrane of cells but will transition to the outer cell membrane during apoptosis [25–27]. Once PS transitions to the outer membrane, phagocytes such as macrophages recognize PS via surface receptors (receptor-ligand interactions) and begin phagocytosis [27]. Numerous particle-based formulations have been developed to take advantage of PS-mediated macrophage phagocytosis by coating drug-loaded particles with PS to enhance macrophage uptake [18]. PS-mediated phagocytosis, particularly for 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), has been shown to be concentration dependent, therefore, a high concentration of PS coating on particles can be sufficient to induce phagocytosis [18,28]. DPPS coatings have been used to increase macrophage uptake or as a binding site for the treatment of cancer, HIV, and atherosclerosis, and in imaging applications [17,29–33].

Acetalated dextran (Ac-Dex) is a pH-responsive, biocompatible, and biodegradable polymer with a simple synthesis process, as seen in Figure S1 [34]. Ac-Dex can be utilized as a polymeric carrier for therapeutics and be formulated to provided targeted delivery to the lungs. Furthermore, the degradation rate of Ac-Dex is easily tunable by adjusting the reaction time during its synthesis [34,35]. Drug release from Ac-Dex formulations is dictated by the environmental pH where Ac-Dex formulations degrade faster in lower pH conditions in comparison to physiological pH [36]. This pH sensitivity minimizes drug release until the Ac-Dex-based formulation reaches a lower pH environment, where it can deliver a localized payload. Overall, Ac-Dex formulations can provide sustainable release of therapeutics while reducing off-target side effects [37].

In the present study, a one-step spray drying process was implemented to formulate dry powder microparticle (MP) formulations consisting of Ac-Dex (bulk polymer), curcumin (a fluorescent model drug), and DPPS. This simple formulation process was designed to create aerosol MP formulations capable of pulmonary deposition with enhanced macrophage uptake due to the presence of DPPS in the MP. Furthermore, Ac-Dex provides controlled release of the therapeutic, exhibiting sustained release at physiological pH and rapid release at the lower pH once phagocytized by macrophages.

2. MATERIALS AND METHODS

2.1. Materials

Dextran from Leuconoctoc mesenteroides (9–11 kDa), phosphate buffered saline (PBS) powder, pyridinium p-toluenesulfonate (PPTS, 98%), D-mannitol (≥ 98%), 2-methoxypropene (2-MOP, 97%), triethylamine (TEA, ≥ 99%), anhydrous dimethyl sulfoxide (DMSO, ≥ 99.9%), curcumin (CUR), resazurin powder, and chloroform (HPLC grade, ≥ 99%) were obtained from Sigma-Aldrich (St. Louis, MO). 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt, DPPS) was obtained from Avanti Lipids (Alabaster, AL). Dulbecco’s Modified Eagle Medium (DMEM), penicillin-streptomycin (Pen-Strep), and Fungizone^® were purchased from Life Technologies (Norwalk, CT). Trypsin-EDTA, sodium pyruvate, Dulbecco’s PBS, and CellMask^™ plasma membrane stain were purchased from Fisher Scientific (Waltham, MA). Fetal bovine serum (FBS) was purchased from Atlanta Biologics (Flowery Branch, GA). Paraformaldehyde (32% aqueous solution) was purchased from Electron Microscopy Sciences (Hartfield, PA). A549 and RAW 264.7 cells were obtained from American Type Culture Collection (ATTC, Manassas, VA).

2.2. Synthesis of Acetalated Dextran

Acetalated dextran was synthesized as previously described [34,38], where 1 g of lyophilized dextran and 30 mg of PPTS were dissolved in 10 mL of anhydrous DMSO in the presence of nitrogen gas. 2-MOP (5 mL) was added to the solution and the reaction was carried out for 3 hours before being quenched with TEA. Following quenching, the polymer was precipitated in a basic water (pH 9.0), vacuum filtered, lyophilized, and stored at −20 °C in a desiccator.

2.3. Preparation of Dry Powder Microparticle Formulations via Spray Drying

DPPS/CUR-loaded Ac-Dex microparticles (MP) were prepared via spray drying using a Büchi B-290 spray-dryer (Büchi Labortechnik, AG, Flawil, Switzerland) in closed mode. CUR, DPPS, and Ac-Dex were dissolved in chloroform and sonicated for 10 minutes prior to spray drying at the following conditions: 0.7 mm nozzle diameter, atomization gas flow rate of 414 L/h using ultra high purity dry nitrogen, aspiration rate of 28 m³/h, pump rate of 6 mL/min, and nozzle cleaner rate of 2. The ratio of DPPS, CUR, and Ac-Dex were adjusted to maintain 5% w/w CUR while varying the DPPS concentration to 1%, 2% and 5% w/w, as seen in Figure 1. MP loaded with 5% w/w DPPS and no CUR was also fabricated (5% DPPS Blank). The resulting MP were separated in a high-performance cyclone, collected, and stored in amber glass vials at −20 °C for later analysis.

Figure 1.

Schematic of (Top) the formation of microparticles (MP) comprised of 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), curcumin (CUR), and acetalated dextran (Ac-Dex) via spray drying; (Center) the MP formulations developed for the study; and (Bottom) the delivery of dry powder MP to alveolar macrophages and epithelia.

2.4. Scanning Electron Microscopy (SEM) and Particle Size Analysis

Images of the formulations were acquired using a field emission-scanning electron microscope (FE-SEM, Zeiss Sigma VP, Germany) to elucidate particle morphology. Prior to imaging, samples were sputter coated with a film of gold/palladium alloy using a BIO-RAD system at a 20 μA for 10 seconds under argon. All images were captured at 3 kV. The geometric diameter (d_g) of the particles was determined using ImageJ software by averaging the diameter of at least 100 particles.

2.5. Differential Scanning Calorimetry (DSC)

Thermal phase transitions of the formulations and their raw components were analyzed via differential scanning calorimetry (DSC) using a TA Q10 DSC system (TA Instruments, New Castle, DE) connected to an RSC-90 cooling accessory. Each sample (3–5 mg) was hermetically sealed in an aluminum pan and an empty pan of similar mass was used as a reference. Samples were heated from 0 to 300 °C at 10 °C/min.

2.6. Powder X-Ray Diffraction (XRD)

The crystallinity of the formulations and raw materials were evaluated using a Rigaku Multiflex X-ray diffractometer (The Midlands, TX) with a Cu Kα source set to 40 kV and 44 mA. Samples were placed in a 3 mm horizontal quartz glass holder for analysis and scans were performed from 5–60 ° 2θ scan angle with a step of 0.1 and a scan rate of 2 °/min.

2.7. Fourier Transform Infrared (FTIR) Spectroscopy

Infrared activity of the particles and raw materials were investigated via Fourier transform infrared (FTIR) spectroscopy using a Shimadzu IRAffinity-1S spectrometer (Shimadzu, Japan). Samples were analyzed from 4000 to 700 cm⁻¹ and each spectrum is the average of 24 scans.

2.8. Surface Charge Analysis

The surface charge (e.g., zeta potential) of the formulations was evaluated using a Malvern Nano Zetasizer (Malvern Instruments, Worcestershire, UK). The MP were diluted to 0.1 mg/ml in distilled water and analyzed at 25 °C.

2.9. Quantification, Loading, and Encapsulation Efficiency of DPPS in Formulations

The amount of DPPS in each formulation was quantified as previously described [39]. Ferric chloride hexahydrate (2.7 g) and ammonium thiocyanate (3 g) were dissolved in 100 mL of distilled water. MP samples were dissolved in chloroform (1 mg/mL) and mixed with the ammonium ferrothiocyanate solution at a 2:1 (v/v) ratio for 2 minutes. After mixing, the solution was allowed to separate and the DPPS content in the chloroform was measured via UV-vis spectroscopy at 452 nm using a Jasco V-780 Spectrophotometer (Easton, MD). DPPS encapsulation efficiency and loading were calculated using the following equations:

$$\mathit{Encapsulation}\mathit{Efficiency}\left(EE\right)=\frac{\mathit{actual}\mathit{mass}\mathit{of}\mathit{DPPS}\mathit{in}MP}{\mathit{theorectical}\mathit{mass}\mathit{of}\mathit{DPPS}\mathit{in}MP}\times 100\mathrm{\%}$$ [1]

$$\mathit{DPPS}\mathit{Loading}=\frac{\mathit{mass}\mathit{of}\mathit{DPPS}\mathit{in}MP}{\mathit{mass}\mathit{of}MP}$$ [2]

2.10. Evaluation of Curcumin Loading in Microparticle Formulations

The amount of CUR in the MP systems was evaluated via fluorescence spectroscopy using a Biotek Cytation 3 Microplate Reader (Winooski, VT) as previously described [40]. MP were dissolved in DMSO (1 mg/ml) and the fluorescence of the solutions was measured at an excitation of 420 nm and emission of 520 nm. The encapsulation efficiency (EE) and loading of CUR in the MP were calculated as follows:

$$\mathit{Encapsulation}\mathit{Efficiency}\left(EE\right)=\frac{\mathit{actual}\mathit{mass}\mathit{ofCUR}\mathit{in}MP}{\mathit{theorectical}\mathit{mass}\mathit{of}\mathit{CUR}\mathit{in}MP}\times 100\mathrm{\%}$$ [3]

$$\mathit{CUR}\mathit{Loading}=\frac{\mathit{mass}\mathit{of}\mathit{CUR}\mathit{in}\mathit{MP}}{\mathit{mass}\mathit{of}MP}$$ [4]

2.11. In Vitro Aerosol Performance of MP

In vitro aerosol performance of the MP systems was evaluated using a Next Generation Impactor (NGI, Copley Scientific, Nottingham, UK) equipped with a stainless-steel induction port and stainless-steel gravimetric insert cups attached to a Copley HCPS vacuum pump. To model the breathing flow rate of a healthy adult, the pump flow rate was set to 60 L/min. Particle bounce and re-entrapment was minimized by placing glass fiber filters (55 mm, Type A/E, Pall Life Sciences, PA) in the gravimetric cups. Prior to analysis, 4 to 6 mg of each MP system was added to hydroxypropyl methylcellulose capsules (HPMC, size 3, Quali-V, Qualicaps Inc., Whitsett, NC). A human dry powder inhaler device (HandiHaler, Boehringer Ingelhelm Pharmaceuticals, CT) attached to a rubber mouthpiece was used to deliver the MP from the capsules to the NGI. Filter mass was recorded before and after each run to determine particle mass deposited on each stage and each measurement consisted of three HPMC capsules run in triplicate. The NGI was run with a 10 s delay time and a 10 s running time. For a flowrate of 60 L/min, each stage has the following effective cutoff diameter: stage 1 (8.06 μm), stage 2 (4.46 μm), stage 3 (2.82 μm), stage 4 (1.66 μm), stage 5 (0.94 μm), stage 6 (0.55 μm), and stage 7 (0.34 μm). The fine particle dose (FPD), fine particle fraction (FPF), respirable dose (RD), and emitted dose (ED) of the MP systems were calculated using the following equations:

$$FineParticleDose\left(FPD\right)=MassofMP<4.4\mu m$$ [5]

$$\mathit{Fine}\mathit{Particle}\mathit{Fraction}\left(FPF\right)=\frac{FPD}{\mathit{Total}\mathit{MP}\mathit{Mass}\mathit{on}\mathit{Filters}}\times 100\mathrm{\%}$$ [6]

$$\mathit{Respirable}\mathit{Dose}\left(RD\right)=\frac{\mathit{Total}\mathit{MP}\mathit{Mass}\mathit{on}\mathit{Filters}}{\mathit{Initial}\mathit{MP}\mathit{Mass}\mathit{in}\mathit{Capsules-Final}\mathit{MP}\mathit{Mass}\mathit{in}\mathit{Capsules}}\times 100\mathrm{\%}$$ [7]

$$\mathit{Emitted}\mathit{Dose}\left(ED\right)=\frac{\mathit{Initial}\mathit{NP}\mathit{Mass}\mathit{in}\mathit{Capsules-Final}\mathit{NP}\mathit{Mass}\mathit{in}\mathit{Capsules}}{\mathit{Initial}\mathit{NP}\mathit{Mass}\mathit{in}\mathit{Capsules}}\times 100\mathrm{\%}$$ [8]

The experimental mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated using a Mathematic^® program written by Dr. Warren Finlay [41].

2.12. In Vitro Drug Release from Microparticle Formulations

To demonstrate the pH sensitivity of Ac-Dex, release studies were carried out in 1X PBS (pH 7.4) and sodium acetate buffer (pH 5.2), both supplemented with 0.2% Tween^® 80 to facilitate CUR solubility. The samples were dispersed in buffer (1 mg/ml) and incubated at 37 °C and 100 rpm. At pre-determined time points, samples were centrifuged at 23,000 × g for 15 minutes and 200 μL of supernatant was removed and frozen at −20 °C for later analysis. Fresh media (200 μL) was added to each sample to maintain sink conditions. Prior to fluorescence analysis, samples were diluted with an equal volume of DMSO to buffer solution and a calibration curve was made using the same ratio for each buffer. Release samples were analyzed via fluorescence spectroscopy as described in Section 2.10. The release profiles for CUR were fit to several semi-empirical release models including zero order, first order, Korsmeyer-Peppas, Higuchi, and Hixon-Crowell models [42,43], as seen in supplemental Table A1. The fit to these models was determined using an adjusted coefficient of determination:

$$R_{\mathit{adjusted}}^2=1-\frac{(n-1)}{(n-p)}\left(1-R^2\right)$$ [9]

where $\mathrm{n}$ is the number of dissolution data points, $\mathrm{p}$ is the number of parameters in the model, and ${\mathrm{R}}^2$ is the original coefficient of determination determined by fitting the data to each semi-empirical model.

2.13. Cell Culture

Murine macrophage cells (RAW 264.7) and A549 human adenocarcinoma cells were maintained at 37 °C and 5 % CO₂ in DMEM supplemented with 10 % v/v FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, Fungizone^® (0.5 μg amphotericin B, 0.41 μg/mL sodium deoxycholate), and 1 mM sodium pyruvate.

2.14. In vitro Cytotoxicity Analysis

A resazurin assay was used to determine the cytotoxic effect of the MP on RAW 264.7 and A549. Cells were seeded in a 96-well plate (5000 cells/well) and incubated overnight at 37 °C prior to being exposed to varying concentrations of MP, where the 5% DPPS Blank NP were normalized to the concentration of 5% DPPS MP; untreated cells served as a negative control. After 48 hours, resazurin (60 μM) was added to the samples and incubated for 3 hours. The resorufin fluorescence intensity was analyzed at an excitation at 544 nm and emission of 590 nm. The following equation was used to calculate relative cell viability:

$$RelativeViability=\frac{\mathit{Sample}\mathit{Fluorescence}\mathit{Intensity}}{\mathit{Control}\mathit{Fluorescence}\mathit{Intensity}}\times 100\mathrm{\%}$$ [10]

2.15. Analysis In Vitro Cellular Uptake of MP via Spectroscopy

Uptake of the MP systems by RAW 267.4 and A549 cells was determined via fluorescence spectroscopy. Cells were seeded in a 96-well plate (10,000 cells/well) and incubated overnight at 37 °C. The cells were then exposed to 0.01 mg/mL of MP solutions and equivalent concentrations of raw CUR for 3 hours. After incubation, the media was removed from the wells and the cells were washed three times with 200 mM glycine to remove any unbound MP. The fluorescence of the CUR within the cells was analyzed via fluorescence spectroscopy at 420 nm (excitation) and 520 nm (emission).

2.16. Statistics

All measurements were performed in at least triplicate. Statistical differences for in vitro cell studies were determined using one-way or two-way ANOVA with Dunnettt’s and Sidak’s multiple comparison test (GraphPad Prism Version 9). Student’s t-test was used to determine statistical significance for cumulative drug release. A p-value < 0.05 was considered statistically significant.

3. RESULTS AND DISCUSSION

3.1. Project Overview and Purpose

Curcumin-loaded microparticles (MP) comprised of the biodegradable polymer acetalated dextran (Ac-Dex) loaded with varying amounts of 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) were developed as solid dry powder aerosol formulations, with the goal of increasing the uptake of MP into macrophages. A one-step spray drying process was used to fabricate the MP with varying amounts of DPPS (1, 2, and 5 wt %) at constant a CUR loading (5 wt %), in addition to a control CUR-only MP system (Figure 1). DPPS has been used extensively to coat particles for targeted delivery of payloads to macrophages [17,19]; however, most methods used to fabricate particles require multiple processing steps leading to batch-to-batch variability. This work aims to add to the field by providing a simple approach to enhance macrophage uptake via DPPS-loaded spray-dried MP capable of aerosol pulmonary deposition, which can be beneficial for pulmonary drug delivery applications.

3.2. Drug Loading and Encapsulation

Curcumin was chosen as a model drug owing to its fluorescence properties, low water solubility, and potential therapeutic applications [19,36,44]. DPPS-mediated macrophage uptake has been shown to be concentration dependent [28], therefore, formulations with varying amounts of DPPS were developed to investigate this phenomenon. As shown in Table 1, the MP formulations displayed high CUR loading and encapsulation efficiency (EE), and the systems exhibited similar CUR loading amounts, allowing them to be easily comparable for this study. DPPS-loaded MP displayed high DPPS EE in addition to concentration-dependent loading of DPPS. The high EE values for the formulations indicate that spray drying is a viable method for effectively loading a hydrophobic drug and phospholipid into dry powder MP.

Table 1.

Microparticle (MP) formulations loaded with curcumin (CUR) and 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), where CUR MP contain no DPPS and 1, 2, and 5% DPPS MP have theoretical DPPS loadings of 1, 2, and 5 wt %, respectively. The characteristics of the MP include DPPS loading and encapsulation efficiency (EE), CUR loading and EE, geometric diameter (d_g), experimental mass median aerodynamic diameter (MMAD_E), geometric standard deviation (GSD), and spray drying yield.

Formulation	CUR Loading (μg CUR/mg MP)	CUR EE (%)	DPPS Loading (μg DPPS/mg MP)	DPPS EE (%)
CUR MP	41.7 ± 3.2	83 ± 6	---	---
1% DPPS MP	45.7 ± 3.5	92 ± 7	8.5 ± 0.9	86 ± 3
2% DPPS MP	45.7 ± 4.0	93 ± 8	17.0 ± 1.7	82 ± 9
5% DPPS MP	42.5 ± 0.6	89 ± 1	41.2 ± 2.3	86 ± 5
Formulation	dg (μm)	Zeta (ζ) itential (mV)	MMAD (μm)	GSD (μm)	Yield (%)
CUR MP	0.80 ± 0.27	−13.9 ± 1.9	3.7 ± 0.3	1.8 ± 0.1	52
1% DPPS MP	0.82 ± 0.31	−37.8 ± 1.4	3.8 ± 0.1	1.8 ± 0.1	50
2% DPPS MP	0.83 ± 0.28	−41.2 ± 1.0	4.0 ± 0.2	2.0 ± 0.3	55
5% DPPS MP	1.01 ± 0.28	−41.2 ± 1.1	3.6 ± 0.3	1.8 ± 0.1	50

3.3. Particle Morphology and Geometric Diameter

The MP formulations displayed geometric diameters (d_g) ≤ 1 μm, which is within the range that macrophages effectively internalize particles (e.g., 0.5–2 μm), indicating that the formulations are the appropriate size for macrophage uptake [4,45,46]. Scanning electron microscopy showed that the MP systems are spherical with raisin-like surface morphology (Figure 2), which can be attributed to the spray drying conditions used in this study that likely result in a high Péclet (Pe) number. The Pe number relates the solvent evaporation rate to the diffusion rate of solutes during spray drying [47,48]. For Pe numbers ≤ 1, the solute diffusion rate is faster or similar to the evaporation rate, resulting in a solid spherical particle, whereas when Pe < 1, the evaporation rate is faster than the solute diffusion rate, leading to enrichment of the solutes at the droplet surface, which results in the formation of hollow particles where eventually the spherical shell will collapse and wrinkle during solvent evaporation [48]. Particles possessing a hollow, wrinkled morphology have been shown to be advantageous for pulmonary delivery by improving aerosol dispersion characteristics and reducing particle-particle interaction [6,47].

Figure 2.

Representative scanning electron microscopy (SEM) images of CUR MP, 1% DPPS MP, 2% DPPS MP, and 5% DPPS MP. Scale bar = 5 μm for all systems. CUR = curcumin, MP = microparticles, and DPPS = 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine.

3.4. Particle Surface Charge

As seen in Table 1, MP systems containing DPPS exhibited large negative zeta (ζ) potential values, indicating a negative surface charge for these formulations, while CUR MP had a ζ potential of −14 mV. Particle systems incorporating phospholipid coatings have reported similar large negative ζ potentials [19,41,47,49,50] and DPPS itself is negatively charged [27,51]. These results indicate that the DPPS incorporated into the MP systems is present on the surface of the particles to some extent. Furthermore, the surface charge of particles is an important characteristic that can influence their behavior, and the negative surface charge of the DPPS MP can help prevent particle aggregation, which is beneficial for aerosol dispersion. Moreover, particles with large negative zeta potential have also been shown to experience enhanced macrophage uptake [52].

3.5. Particle Thermal Transitions and Crystallinity

Spray drying imparts the advantage of transitioning crystalline materials to their amorphous state, which is advantageous as amorphous therapeutics exhibit higher aqueous solubility than their crystalline form, thereby increasing their bioavailability [53]. Differential scanning calorimetry (DSC) was used to investigate the thermal transitions of the formulations (Figure 3). Raw DPPS and raw CUR displayed sharp endothermic melting peaks at 118 and 179 °C, respectively, which are indicative of their crystalline structures in agreement with previously reported results [19,36]. Raw Ac-Dex displayed a broad endothermic peak centering at 157 °C, indicating an amorphous melting peak. Similar to raw Ac-Dex, the spray-dried MP formulations displayed broad endothermic peaks around 150 °C but did not have the crystalline melting peaks associated with raw DPPS and raw CUR, indicating that the DPPS and CUR in the MP systems are in the amorphous state. Notably, no raw materials used in the fabrication of the formulations displayed degradation in the tested temperature range (0–300°C). As a result, material degradation should not be experienced at the spray drying temperatures used in this study.

Figure 3.

Solid-state characterization of CUR MP, 1% DPPS MP, 2% DPPS MP, 5% DPPS MP, 5% DPPS MP Blank, and their raw components, including (Left) differential scanning calorimetry thermograms, (Middle) x-ray dispersion diffractograms, and (Right) Fourier transform infrared spectroscopy. CUR = curcumin, MP = microparticles, DPPS = 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine, and Ac-Dex = acetalated dextran.

The microstructure of the raw materials and MP systems was evaluated via powder X-ray diffraction (XRD). As seen in Figure 3, raw CUR exhibits sharp diffraction peaks from 10–30 °, while raw DPPS displayed several smaller peaks from 20–25 ° due to the crystalline microstructure each possess. Raw Ac-Dex and the MP systems exhibited amorphous behavior, displaying broad diffraction without any sharp peaks. The combination of the DSC and XRD confirm that spray drying successfully transitioned CUR and DPPS from a crystalline state to an amorphous state in the dry powder MP formulations.

3.6. Structural Analysis of Microparticles via FTIR Spectroscopy

The structure of the MP systems was investigated further using Fourier transform infrared (FTIR) spectroscopy. As seen in Figure 3, several peaks of interested were observed for Ac-Dex, including C-H vibrational stretching at 2990 cm⁻¹ and 2830 cm⁻¹ and a large peak at 1373 cm⁻¹ associated with C-H bending vibrations from the acetal groups [54,55]. Distinct peaks were observed for raw DPPS from 3000–2800 cm⁻¹ and 1800–500 cm⁻¹. The peaks at 2916 cm⁻¹ and 2850 cm⁻¹ can be attributed to CH₂ stretching vibrations of the fatty acid chains in DPPS [56], whereas the peak at 1737 cm⁻¹ is a result of symmetric C=O stretching vibrations from the ester groups [56,57]. Raw CUR displayed a distinct peak at 3508 cm⁻¹, corresponding to the O-H stretching vibration of the phenol group, whereas the peak at 1626 cm⁻¹ is from the C=C stretching vibration of an aromatic group [57–59]. Raw CUR displayed another high intensity peak at 1512 cm⁻¹ due to various stretching and bending vibrations [60].

The raw Ac-Dex and MP formations displayed similar FTIR spectra (see Figure S2), with distinct peaks at 2990, 2830, and 1373 cm⁻¹, indicating that the MP are comprised predominantly of Ac-Dex, which is as expected. The characteristic peaks for DPPS almost entirely disappear for the DPPS MP sample (see Figure S3), however, small peaks at 2916, 2850, and 1737 cm⁻¹ are clearly present in the 5% DPPS MP formulations, with decreasing intensity with the reduction of DPPS loaded in the particle systems. CUR-loaded MP formulations exhibited characteristic peaks associated with CUR (e.g., 1626 and 1512 cm⁻¹), whereas the peak at 3508 cm⁻¹ for raw CUR is not seen for these formulations (Figure S4). The significant decrease in peak intensity at 1626 cm⁻¹ and small peak shift around 1512 cm⁻¹ can be attributed to loading of CUR throughout the particles and the transition of CUR to an amorphous state, which is also responsible for the disappearance of the peak at 3508 cm⁻¹ [61]. Similar to CUR, the disappearance/minimization of the peaks for DPPS in the MP can be attributed to a phase transition to its amorphous state and due to interactions with the bulk Ac-Dex. In conjunction with CUR loading, it can be inferred CUR is incorporated throughout the particle and is not forming a CUR rich coating on the surface of an Ac-Dex core [62,63]. However, the same cannot be inferred for DPPS.

3.7. In Vitro Aerosol Dispersion of Dry Powder Microparticles

A Next Generation Impactor was used to evaluate the in vitro aerosol dispersion properties of the MP systems (Figure 4). The MP formulations displayed particle deposition on all NGI stages, indicating that the systems are able to be delivered as dry powder aerosols to the lungs. The mass median aerodynamic diameter (MMAD) is an important value that describes how particles behave in air flow, and the MMAD values ranged from 3.6 to 4.0 μm, with no dependency on the particle conditions (see Table 1). Dry powder aerosolized particles with an MMAD between 0.5–5 μm have been shown to effectively deposit in the alveolar regions of lungs [2,4,6], the target region for this study. The fine particle fraction (FPF), which shows the percentage of particles with an MMAD < 4.4 μm that can pass through the larynx and conducting airways to reach the alveolar region [4], was greater than 80% for all MP. Additionally, the MP systems displayed respirable fraction (RF) and emitted dose (ED) values above 30% and 80%, respectively. The GSD for the MP formulations indicated that the spray-dried particles are hetero dispersed, which is consistent with SEM imaging. The spray drying yield ranged from 50–55% for the MP systems, which is consistent with previously reported results and is considered an acceptable yield for a bench top spray dryer [64–66]. Overall, the MP systems displayed favorable aerosol dispersion properties for effective pulmonary delivery to the alveolar region.

Figure 4.

In vitro aerosol dispersion performance of the microparticle formulations, including: (Left) particle deposition represented as percentage (%) of particles deposited on each stage of the Next Generation Impactor (NGI). The effective cutoff diameters (D₅₀) for each stage are the following for an air flow rate of 60 L/min: Stage 1 = 8.06 μm, Stage 2 = 4.46 μm, Stage 3 = 2.82 μm, Stage 4 = 1.66 μm, Stage 5 = 0.94 μm, Stage 6 = 0.55 μm, and Stage 7 = 0.34 μm. (Right) Fine particle fraction (FPF), respirable fraction (RF), and emitted dose (ED) for each particle system. FPF = percentage (%) of particles deposited on stages 2 through 7. RF = percentage (%) of emitted dose deposited on stages 2 through 7. ED = percentage (%) of particles emitted from dry powder inhaler. CUR = curcumin, MP = microparticles, and DPPS = 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine. CUR MP contain no DPPS, whereas 1, 2, and 5% DPPS MP initially contain these respective DPPS amounts by weight.

Upon aerosolization to the lungs, particulates will experience physiological pH (7.4) upon deposition in the alveoli and endosomal pH (5.2) once phagocytized by macrophages. Therefore, the release of CUR from MP systems was evaluated in two biorelevant release media. PBS at pH 7.4 was used to mimic the pH of lung tissue, while a sodium acetate buffer at pH 5.2 was used to mimic the endosomal pH of macrophages [6]. As seen in Figure 5, Ac-Dex provides sustained and pH-responsive release of CUR over the course of a week from all of the MP systems. As expected, MP exposed to lower pH experienced faster degradation (and thus CUR release) than those in the neutral pH release media, which can be attributed to the acid-catalyzed degradation mechanism for Ac-Dex [34,35]. The cumulative release of CUR from MP systems at pH 7.4 ranged from 38 to 52% after seven days, whereas the MP achieved a cumulative release of 100% at pH 5.2 for the same time period, representing a statistically significant difference for each MP system when comparing the pH conditions. The MP provided sustained release of CUR at physiological pH 7.4, which can allow CUR to remain in the particle when delivered to the epithelium and subsequently be released in the lower pH of the endosome upon uptake into macrophages.

Figure 5.

In vitro curcumin (CUR) release profiles from the microparticle (MP) formulations in various release media, including (Left) modified phosphate buffer solution (pH 7.4) and (Right) sodium acetate buffer (pH 5.2) at 37 °C. Data represent the mean ± standard deviation (n = 3) and *p < 0.05, **p < 0.005 when comparing each system for the cumulative release end points at pH 7.4 versus pH 5.2. DPPS = 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine. CUR MP contain no DPPS, whereas 1, 2, and 5% DPPS MP initially contain these respective DPPS amounts by weight.

3.8. In Vitro Release of the Model Therapeutic Curcumin

DPPS loading appeared to have an effect on the release of CUR from the MP systems. At pH 7.4, CUR release was statistically higher for the 5% DPPS MP at all time points in comparison to CUR MP, whereas at pH 5.0, CUR release was higher for both 2% and 5% DPPS MP from 2 to 72 hours. The increased release associated with DPPS can potentially be attributed to an increase in the amount of DPPS on the particle surface, leading to increased wettability of the MP system. DPPS is an amphiphilic molecule with a hydrophobic tail and hydrophobic head group [67]. This results in DPPS precipitating first at the particle surface during drying with the hydrophobic tails remaining in the drying particle while the head groups are exposed. However, further investigation is needed to confirm this hypothesis.

The release profiles of CUR from the MP systems were fit to semi-empirical release models, and the resulting release rate constants and coefficients of determination (${\mathrm{R}}^2$) are listed in Table A2. Based on the adjusted ${\mathrm{R}}^2$ values, the release data exhibited the best fit using the Higuchi release model. The Higuchi model is often used to study the release of poorly water-soluble drugs incorporated in solid matrices [68], which is appropriate for the given study. The Higuchi release rate constants (k_H) were significantly higher for pH 5.2 in comparison to pH 7.4 (Tables A2 and A3), indicating a faster release of CUR at pH 5.2, which is in agreement with the pH-sensitive degradation of Ac-Dex. Interestingly, the data fit to the Korsmeyer-Peppas exhibited similar adjusted ${\mathrm{R}}^2$ values at pH 5.2, with an increase in the Korsmeyer-Peppas release rate constant (k_KP) with increasing DPPS loading. This result is consistent with the release profile observed in Figure 5 for pH 5.2. A comparison of the k_H values for CUR MP versus DPPS-loaded MP, it was shown that the only significant difference was seen for CUR MP versus 5% DPPS MP at pH 7.4, showing that loading DPPS into the formulations had minimal impact on the release of CUR.

3.9. Particle Uptake in Pulmonary Epithelial Cells and Macrophages

It has been extensively shown that macrophages can readily clear foreign material from the lungs, including aerosolized particulates. In particular, macrophages can clear particles ranging from 0.1–10 μm, with greater clearance efficiency for particles ranging from 0.5–2 μm [4,46]. Since the MP formulations in this study exhibited geometric diameters (d_g) within the size range that can be effectively cleared by macrophages, it is likely that any change in particle uptake by macrophages can be attributed to the presence of DPPS. To investigate particle uptake, RAW 264.7 macrophages and A549 pulmonary epithelia cells were exposed to MP systems for 1 and 3 hours and analyzed via fluorescence spectroscopy (see Figure 6). CUR MP (d_g = 0.80 μm) without DPPS was used as a control to demonstrate whether macrophage uptake is DPPS-dependent rather than size dependent. After 1 hour, 5% DPPS MP (d_g = 1.01 μm) demonstrated the highest uptake in RAW 264.7 macrophages, which was significantly more than CUR MP uptake; however, there was no statistical significance when comparing 1% DPPS MP (d_g = 0.82 μm) and 2% DPPS MP (d_g = 0.83 μm) to CUR MP at this time point. After 3 hours, 5% DPPS MP exhibited significantly higher particle uptake in RAW 264.7 in comparison to the other MP systems and the 1% DPPS MP and 2% DPPS MP formulations displayed statistically significant higher particle uptake compared to CUR MP. Overall, these data show that the uptake of DPPS-loaded MP into macrophages is both concentration and time dependent.

Figure 6.

In vitro uptake analysis of raw curcumin (CUR) and microparticle (MP) formulations in A549 lung epithelial cells and RAW 264.7 macrophages after (Left) 1 hour and (Right) 3 hour incubation periods. DPPS = 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine. CUR MP contain no DPPS, whereas 1, 2, and 5% DPPS MP initially contain these respective DPPS amounts by weight. Data represent the mean ± standard deviation (n = 6) and *p < 0.05, **p < 0.005, ***p < 0.0005, and †p < 0.0001.

The uptake of the MP formulations into A549 lung epithelial cells was investigated to determine if the particle uptake was macrophage specific. After 1 and 3 hours, the internalization of DPPS MP formulations into RAW 264.7 was significantly more than A549, while there was no significant uptake for CUR MP or raw CUR, confirming that DPPS-mediated MP uptake is occurring and will not result in increased MP uptake in pulmonary epithelial cells, which is consistent with previous studies [19,28,69]. For MP uptake in A549, there was no statistical difference when between the 1 and 3 hour time points, whereas MP uptake in RAW 264.7 was statistically higher at 3 hours in comparison to 1 hour for the DPPS MP systems, confirming that the uptake of DPPS MP in macrophages is time dependent. Complete statistical analysis for cellular uptake can be seen in Table S4. Overall, these studies confirm that the uptake of DPPS-loaded MP is cell dependent (e.g., more uptake in RAW 264.7 than A549), as well as time- and DPPS concentration-dependent in macrophages.

3.10. Cytotoxicity Analysis of Drug-Loaded Microparticles

The in vitro cytotoxic effect of the MP systems on RAW 264.7 and A549 cells was evaluated to elucidate CUR-, DPPS-, or formulation-mediated toxicity (Figure 7). Relative viability was evaluated for the 5% DPPS MP system as it contains the highest concentration of DPPS and resulted in the highest macrophage uptake. CUR MP were used as a control to show the impact of DPPS loading on viability and DPPS MP without CUR (5% DPPS MP Blank) were used to show the impact of CUR on cell uptake, where the amount of 5% DPPS Blank was normalized to the mass of 5% DPPS MP.

Figure 7.

In vitro cytotoxicity analysis (relative viability) of (Top) RAW 264.7 macrophages and (Bottom) A549 epithelial cells exposed to microparticle (MP) formulations at various curcumin (CUR) concentrations for 48 hours in comparison to cells not exposed to particles (control). DPPS = 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine. 5% DPPS MP contain DPPS and CUR, 5% DPPS Blank contain only DPPS, and CUR MP contain only CUR. Data represent the mean ± standard deviation (n = 4) and *p < 0.05, **p < 0.005, ***p < 0.0005, and †p < 0.0001.

For RAW 264.7 cells, there was a significant decrease in cell viability upon exposure to 5% DPPS MP at 10 μM and 15 μM CUR and when exposed to an equivalent mass of the 5% DPPS Blank formulations. Macrophages exposed to CUR MP containing no DPPS did not exhibit a decrease in cell viability at the concentrations evaluated. These results show that the presence of DPPS impacts cell viability at higher concentrations, whereas CUR itself does not. While the current DPPS MP formulations are designed to specifically target macrophages, the pulmonary epithelia will also be exposed to the aerosolized MP. A549, cell line often used as an in vitro model for alveolar epithelium [70], was used to assess the in vitro cytotoxicity of the MP systems on epithelial cells. A549 exposed to the MP for 48 hours did not show any decrease in cell viability at the tested concentrations, exhibiting the biocompatibility of the MP systems with pulmonary epithelial cells. These data show the DPPS MP systems will not adversely affect lung tissue prior to macrophage clearance. Furthermore, it is likely that the decrease in viability for RAW 264.7 upon exposure to DPPS-loaded MP is due to the uptake of MP into the cells, as there is no impact on viability in A549 cells that do not significantly internalize the DPPS MP formulations.

4. CONCLUSIONS

Many disease-causing pathogens survive and multiply in the lungs, specifically in alveolar macrophages. Pathogens that reach the deep lung are phagocytized by alveolar macrophages as the first step of pathogenesis and, if left untreated, replicate in the macrophages. Therefore, the aim of this study was to use a simple one-step spray drying process to develop a microparticle-based drug delivery platform capable of pulmonary delivery that enhances macrophage uptake and provides controlled drug release. Such formulations have the potential to decrease off-site side effects and treatment times by providing high drug concentrations directly to the site of infection. In this study, DPPS- and CUR-loaded MP demonstrated controlled release of a hydrophobic therapeutic, enhanced macrophage uptake, and appropriate aerosol dispersion characteristics for effective MP delivery to the lower airways, thereby meeting the aforementioned objectives. Overall, these data suggest the DPPS MP systems in this study have the potential to effectively delivery therapeutics to the alveolar region of the lungs while increasing macrophage uptake and providing pH-responsive drug release.

Abbreviations:

Ac-Dex

acetalated dextran

CUR

curcumin

DSC

differential scanning calorimetry

ED

emitted dose

FPF

fine particle fraction

FTIR

Fourier transfer infrared spectroscopy

GSD

geometric standard deviation

MMAD_E

experimental mass median aerodynamic diameter

MMAD_T

theoretical mass median aerodynamic diameter

MP

microparticles

NGI

Next Generation Impactor

PBS

phosphate buffered saline

PS

phosphatidylserine

Pe

Péclet number

PPTS

pyridinium p-toluenesulfonate

RD

respirable dose

SEM

scanning electron microscopy

TAC

tacrolimus

TEA

trimethylamine

2-MOP

2-methoxypropene

XRD

X-ray diffraction