The respiratory tract is an attractive route for non-invasive drug delivery for the local treatment of lung diseases, such as asthma and cystic fibrosis. Specific lung cells can further be targeted for treating diseases such as tuberculosis and possibly even lung cancer [1
]. The fact that respiratory diseases rank among the top ten causes of death globally (four out of ten) [4
], makes this field of study highly important. In addition, as a result of the potential uptake of drugs across the respiratory epithelium, drug delivery through inhalation is also gaining importance for non-respiratory diseases, where the lungs are only considered for their potential as a portal of entry for pharmaceutically active compounds [5
]. The respiratory tract offers several advantages over conventional oral drug delivery [6
]. These include the highly vascularized and large surface area of the alveoli, which enables access to the microvascular circulation of the lungs, resulting in a faster drug adsorption, and also a possible circumvention of the first pass effect [8
]. The effectiveness of the drug uptake is related to the amount of drug deposited in and the distribution within the airways [9
Nanoparticles offer many unique advantages as therapeutic tools because of their design flexibility. In particular, silica nanoparticles have attracted much attention as being one of the most biocompatible drug carriers [10
], as amorphous silica has also been classified as a “generally recognized as safe” (GRAS) material by the food and drug administration (FDA). To date, drug delivery systems based on silica nanoparticles have primarily been studied through intravenous administration, but recently gained importance for use also through the pulmonary route [12
]. Mesoporous silica nanoparticles are bioerodible and have a high surface area that makes it possible to incorporate drug amounts equaling the particle’s own weight (1:1 drug:carrier) [13
]. Silica is a highly flexible platform that can be specifically designed, depending on the desired application and administration route [14
]. When particles enter the lungs, particle deposition occurs in the whole respiratory tract by different mechanisms. Particle size and geometry are the most important parameters for the deposition, next to the morphological characteristics and ventilation parameters of the lungs [15
]. Thus, the ease of particle size control, size uniformity, and flexible surface functionalization possibilities make silica nanoparticles ideal as pulmonary drug carriers. Nanoparticle-based carriers can improve the pharmacokinetic and pharmacodynamic profiles of conventional drugs, and may thus optimize the efficacy of the drug [16
]. Silica nanoparticles are hydrophilic, which is essential for any in vivo application, and the dispersibility of the particles in aqueous media can further be enhanced by the right surface functionalization [17
]. Consequently, mesoporous silica particles have recently been highlighted as ideal carriers for poorly water-soluble drugs [18
], which, despite a high potency, often have severely limited efficacy as a result of the fact that the amount of soluble drug is not high enough for reaching therapeutic concentrations.
Here, the possibility/feasibility of using mesoporous silica particles (MSPs) as carriers for anti-inflammatory drugs in the treatment of airway inflammation was investigated. Corticosteroids, such as dexamethasone (DEX), used in this study, are well-known to decrease the number of inflammatory cells in the airways, and to improve the respiratory function. However, DEX can also cause unwanted side effects, particularly when used at high doses [21
]. As DEX is practically insoluble in water (0.1 mg/mL) [25
], it would be expected that a silica particle-formulation can enhance the solubility and dissolution rate of the drug. Drug-loaded particles, compared to free drugs, also have a higher ability to reach the lower parts of the lungs [26
]. DEX has potent anti-inflammatory properties and is an ideal therapeutic agent for acute airway inflammation. By being able to load particles with DEX in order to improve airway distribution and to enable local treatment, side effects may be reduced, and it may also be possible to use higher doses locally in the lung. In our study, particle sizes of 200 nm and 1 µm were synthesized for comparison, and loading degrees exceeding 100 wt % (1:1 drug:carrier) of DEX were obtained. The particles were further coated with polyethylene glycol—polyethylene imine (PEG–PEI) copolymer to improve the dispersibility of the drug-loaded MSPs, and to potentially promote favorable and to suppress unfavorable interactions upon administration via the airways.
Two different mice models of airway inflammation were utilized, as follows: (1) chemical-induced airway inflammation provoked by exposure to the cytotoxic compound melphalan (MEL), and (2) endotoxin-induced pulmonary inflammation caused by exposure to lipopolysaccharide (LPS). Previous studies have shown that MEL is a possible surrogate for the chemical warfare agent sulphur mustard, causing a neutrophilic airway inflammation in the acute phase (1–2 days post exposure) [24
]. Inhalation of LPS induces an acute inflammation and has been studied extensively. One role of LPS is the activation and migration of blood leukocytes into the airways, the dominating type being neutrophils [30
]. The two models have in common that there is a rapid inflammatory process in the acute phase, which peaks within 24 h. In both models, we have earlier demonstrated that therapeutic treatment with DEX 1 h (intraperitoneal injection) after exposure is effective for the prevention of the acute inflammation [24
]. In this study, animals were treated with aerosolized free DEX or MSPs with and without loaded DEX, 1 h after exposure to MEL or LPS. Mice were evaluated for treatment effects 24 h after exposure; whereby the inflammatory cells and pro-inflammatory mediators (keratinocyte chemoattractant (KC), Matrix metalloproteinase-9 (MMP-9), and Mouse Myeloperoxidase (MPO)) were analyzed in bronchoalveolar lavage fluid (BALF).
2. Materials and Methods
2.1. Preparation of the Large (L-MSP) and Small (S-MSP) Mesoporous Silica Particles
The L-MSP and S-MSP were synthesized according to a procedure reported by D. Kumar et al. [32
], with slight modifications. Briefly, 2 g hexadecylamine (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was dissolved in 200 mL isopropanol, 180 mL milli-Q water, and 2.4 mL NH3
(33 wt %). For the synthesis of L-MSP, 11.6 mL tetraethyl orthosilicate (TEOS) was added to the solution as a silica source, while a mixture of 11 mL TEOS and 0.6 mL aminopropyl triethoxysilane (APTES) was added to the synthesis solution of S-MSP to reduce the resulting particle size. Both of the synthesis solutions were left for overnight reaction under stirring, whereafter the particles were separated by centrifugation (3360 g), and the surfactant template was removed by extraction two times for 1 h in slightly acidic ethanol (0.1 M). During extraction, the solutions were stirred and shaken, with no sonication involved, so as to preserve the porous structure of the particles. Finally, the particles were washed with ethanol and were vacuum-dried at room temperature (RT).
2.2. Loading of Drug to the Particles
20 mg of dexamethasone (DEX) was dissolved in 9 mL of anhydrous cyclohexane (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) by sonication. Then, 20 mg of vacuum-dried L-MSP or S-MSP were added, and the drug-particle suspension was ultrasonicated and vortexed repeatedly three times. The suspension was left under stirring overnight. The drug-loaded particles were separated by centrifugation (630 g), washed by cyclohexane, and vacuum-dried at RT.
2.3. Copolymer-Adsorption on the Particles
The particles were coated with an earlier developed PEG–PEI copolymer (mPEGlow
] as follows. 10 mg of drug-loaded particles were dispersed in 1 mL of HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) buffer (25 mM, pH 7.2) by careful sonication so as to not leach out the drug. For the same reason, the particle-concentration was kept high. Then, 10 mg of the PEG–PEI copolymer was dissolved in 1 mL HEPES, and was added to the particle-suspension during sonication. The reaction was left for 3 h under stirring. The copolymer-coated particles were separated by centrifugation (3629 g), washed with a small amount of milli-Q water, and then vacuum-dried at RT.
Empty particles (without drug) were also coated with copolymer to later serve as the control particles in the in vivo studies.
2.4. Determination of the Loaded DEX Amount
The amount of DEX inside the pores of the PEG–PEI-coated particles was determined by elution of the drug in methanol. A particle concentration of 0.1 mg/mL in the methanol was repeatedly sonicated and vortexed for 1 h. The particles were centrifuged, and the supernatant was measured on a Ultraviolet–visible (UV-Vis) Spectrophotometer (NanoDrop 2000c, Thermo Fisher Scientific Inc., Waltham, MA, USA) at a wavelength of 242 nm. The amount of DEX was calculated by using a standard curve of DEX in methanol.
2.5. Animal Models
Female C57BL/6OlaHsd mice (9–10 weeks old) obtained from Envigo RMS B.V, Netherlands, were used in this study. The animals were fed with standard chow and water ad libitum. The care of the animals and the experimental protocols were approved by the regional ethics committee on animal experiments in Umeå, Sweden (A69-15, 20/11/15).
Two different mice models for airway inflammation were utilized, as follows:
Melphalan (MEL)-induced airway inflammation: The mice were briefly anaesthetized with isofluran and melphalan (4-(bis(2-chlorethyl)amino)-l
-phenylalanine) (Sigma-Aldrich, St Louis, MO, USA) administered by intratracheal instillation in a volume of 50 µL (1 mg/kg). Melphalan was dissolved in acidic ethanol (30 µL concentrated HCl in 1 mL 99.5% ethanol) to a concentration of 100 mg/mL, and further diluted in phosphate buffered saline (PBS) to a final concentration just before administration [24
]. The control mice received only the solvent.
Lipopolysaccharide (LPS)-induced airway inflammation: The mice were exposed to an aerosol of Lipopolysaccharide (LPS; Escherichia coli
O128:B12; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) for 15 min using a nose-only Battelle exposure chamber. The aerosol was generated by a compressed-air Collison six-jet nebulizer at an airflow of 7 liters/min using a nebulizer concentration of 0.1 mg/mL of LPS dissolved in water [31
]. The control mice were exposed to an aerosol of solvent alone.
The samples with MSPs with and without DEX were placed in an ultrasonic bath for about 15 min before administration and were shaken every 5 min. All of the samples were diluted in a HEPES buffer to a volume of 200 µL, and were administered as an aerosol using Aeroneb™ PRO, SCIREQ®
. The mice were randomly allocated into different groups (listed in Table 1
= 6 animals/group) and were placed in individual nose-only containers and via a connecting tube exposed to aerosolized droplets (diameter of 4–6 µm) of MSPs with and without DEX in various concentrations and sizes 1 h after the exposure to MEL or LPS. One group of animals were administered free DEX (5 mg/mL in HEPES buffer) without MSPs as a positive control group.
After 24 h post exposure, the animals were tracheostomized and the BALF was collected. The lungs were lavaged four times via a tracheal tube, with a total volume of 1 mL + 3 × 1 mL Ca2+/Mg2+ free ice cold Hank’s Balanced Salt Solution (HBSS; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). The BALF was centrifuged (524 g, 10 min, and 4 °C) and the supernatant was stored at −80 °C for later analysis. The cells in the pellets were dissolved in PBS and the number of leukocytes were counted in a Bürker chamber using trypan blue staining. There were 20,000 cells per animal that were fixed on duplicate slides using a Cytospin® centrifuge (Shandon© cytospin 3 cyto-centrifuge, cell preparation system, Runcorn, UK) and were stained with May–Grünwald–Giemsa reagents (Merck Millipore, VWR International, Spånga, Sweden) before a differential count was performed in a blinded manner, counting 200 cells per slide. The total cells in the BALF and the percentage of neutrophils are presented in this study.
The BALF analysis was performed using ELISA kits, as follows: (1) Mouse CXCL1/KC DuoSet ELISA, (2) Mouse Myeloperoxidase (MPO) DuoSet ELISA, and (3) Matrix Metalloproteinase-9 (MMP-9; Mouse Total MMP-9 DuoSet ELISA), according to the manufacturer’s instructions (R&D systems™, Abingdon, UK), and were analyzed using an ELISA reader (Thermo Scientific Mutilskan FC, Thermo Fischer Scientific Oy, Vantaa, Finland). The analysis of the ELISA data was performed using the software program for the ELISA reader (SkanIt for Multiskan FC 3.1. Inc., Thermo Fischer Scientific Oy, Vantaa, Finland).
2.5.2. Statistical Analysis of the Animal Data
The results are presented as the means ± standard error of means (SEM). The statistical significance was assessed by parametric methods using a one-way analysis of variance (ANOVA) to determine the differences between the groups, followed by a Bonferroni post hoc test. A statistical result of p < 0.05 was considered significant. The statistical analyses were carried out and graphs were prepared using the GraphPad Prism program (version 6.0 GraphPad software Inc., San Diego, CA, USA).
In this study, mesoporous silica particles (MSPs) of two different sizes have been produced and characterized, and further evaluated as carriers for the corticosteroid dexamethasone (DEX). Their feasibility as delivery vehicles for treating airway inflammations in mice, which in the present case was induced by melphalan (MEL) or lipopolysaccharide (LPS), was investigated. The results showed that the MEL-induced neutrophilic airway inflammation could be treated by aerosolized MSP-encapsulated DEX, at least to the same extent as free DEX. Interestingly, in the MEL-induced inflammation model, inhaled empty particles had no apparent effect on inflammation; while, in the LPS-induced inflammation model, inhaled particles significantly down-modulated the inflammatory response regardless of the presence of DEX or not. The results from the MEL-exposed mice indicate that MSPs are viable drug carriers for pulmonary delivery of corticosteroids in the anti-inflammatory treatment of chemical-induced lung injury. The mechanism by which empty MSPs reduced the LPS-induced airway inflammation is presently not known, but should be the focus of future studies. Hypothetically, this observation could be explained by different magnitudes of inflammation in the two models, and, potentially, also by interactions of MSPs with proteins and cells in BALF. By using mouse models for the in vivo treatment of toxic chemical- and endotoxin-induced lung injury by MSPs loaded with corticosteroids, we have shown that inhaled MSPs can exert therapeutic action in inflammatory conditions. The results presented here provide a foundation for future studies aimed at identifying new concepts for treatment using MSP-based drug delivery.