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Sustained steroid release in pulmonary inflammation model
Biomaterials 31 (2010) 6050e6059
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Sustained steroid release in pulmonary inflammation model Harry Karmouty-Quintana a, Faleh Tamimi b, Toby K. McGovern a, Liam M. Grover c, James G. Martin a, Jake E. Barralet b, * a
Meakins-Christie Laboratories, McGill University, 3626 Rue St Urbain, Montreal, Quebec, H2X 2P2, Canada Faculty of Dentistry, Strathcona Anatomy & Dentistry, McGill University, 3640 University Street Montreal, Montreal, Quebec H3A 2B2, Canada c The Department of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham, UK b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 March 2010 Accepted 12 April 2010 Available online 15 May 2010
There is a need for particles which exhibit controlled release of therapeutic agents delivered via the inhalational route, for tissue specific applications such as anti-cancer, bronchodilators and antiviral agents as well as drugs for systemic action. The aim of this study was to assess the acute toxicity, distribution and capacity of the microspheres to exhibit controlled release properties in an in vivo model of airway inflammation. Calcium pyrophosphate nanofibrous microspheres were loaded with dexamethasone phosphate (Dex-P); the profile of drug release was studied in vitro and validated in vivo. Unloaded microspheres were administered intra-tracheally (i.t.) to rats to assess the tissue reaction. The anti-inflammatory properties of the Dex-P loaded microspheres against an inflammatory agent (compound 48/80), were evaluated in vivo. Unloaded microspheres did not cause an inflammatory response when given at doses below 3 mg, and appeared to be eliminated through mucus clearance mechanisms. Microspheres loaded with Dex-P but not Dex-P alone, were capable of inhibiting eosinophil and total inflammatory cell increases in bronchoalveolar lavage fluid for 42 h following a single application. These observations demonstrated that calcium pyrophosphate nanofibrous microspheres displayed in vivo controlled release properties, were well tolerated and did not accumulate in the lung. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Calcium pyrophosphate Microsphere Controlled-release Anti-inflammatory Inhalation drug delivery
1. Introduction Therapeutic agents are frequently delivered by inhalation, in particular for respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD). However, to date, there are no controlled release inhalation systems available clinically [1]. Treatments for asthma, COPD, cancer, infectious disease or for systemic action would benefit from improvements in inhalation controlled release systems [1,2]. The treatment of chronic asthma often relies on the delivery by inhalation of corticosteroids comparable to dexamethasone [3,4]. In addition to the potential advantages in some settings of reduction of dose cycling, sustained release is important because it reduces the dose required, improves biological efficacy, requires less patient compliance and is less irritating to the bronchial tissues [5]. For example asthma is worse at night because endogenous anti-inflammatory mechanisms are least effective at this time [6,7]. However, most patients do not comply with four times or even twice daily dosing [8,9] and the ability to achieve sustained release for up to 12 h and beyond may * Corresponding author. E-mail address: [email protected] (J.E. Barralet). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.04.025
be an important step forward in improving efficacy of several lung disease therapies. The use of aerosolised or dry powder inhalation therapy for asthma is common. The particle size is an important determinant of its site of deposition within the respiratory tree. However, the micronisation of drug particles often leads to large distribution in particle size, alteration of surface properties and renders the particles more cohesive leading to poor aerosolisation [10,11]. Furthermore, the use of lactose as a drug carrier and bulking agent for many drug formulations [11] can lead to poor inhalation performance [12]. Although used in many drug formulations, lactose is known to react with formoterol (a long-acting bronchodilator used in asthma), peptides and proteins [13]. Aerosolisation of particles is often difficult as it is necessary to dissolve them in a suitable vehicle, a growing problem concerning large organic molecular entities, monoclonal antibodies and silencing RNA approaches [11,14]. Thus inert vehicles for the controlled release of medication aimed at treating respiratory conditions are of great interest to the field. In this study we used an experimental model of lung inflammation induced by compound 48/80, a condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde. It is a basic secretagogue that causes mast cell degranulation
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[15] via direct activation of the Gi protein [16]. Compound 48/80 has been used extensively in vivo to induce inflammation in the lungs [17], rat paw edema [18], and inflammation of the skin [19]. We chose to use this stimulus and the subsequent inflammatory response as the model system to test the efficacy of newly developed porous nanofibrous microspheres composed of calcium pyrophosphate in the size range from 5 to 15 mm to act as sustained release depots to deliver dexamethasone phosphate.
a concentration of 30% w/w (Dex-P), 70 mg of pyrophosphate microspheres were mixed with 10 mg of calcium chloride (Sigma Aldrich, St. Louis, MO) in 1 ml of distilled water before adding 30 mg of Dex-P to the suspension and mixing thoroughly for 10 s. Afterwards, the suspension was left to dry overnight under vacuum at room temperature. In order to visualize the distribution of the microspheres within the lungs, after i.t. administration, fluorescent microspheres labelled by adsorption of fluorescent doxycycline. 100 mg of microspheres were mixed with 0.5 ml of a doxycycline solution (Sigma Aldrich, St. Louis, MO) (5% w/w), and left to dry under vacuum at room temperature.
2. Materials and methods
2.2. In vitro controlled release kinetics
2.1. Microsphere preparation and loading with dex, dex-P or fluorescent marker
To determine the in vitro release profiles of the microspheres, 10 mg of the drug loaded microspheres were incubated in 50 ml PBS solutions. At predetermined time points (1 h, 2 h, 4 h, 8 h, 12 h, 1 day, 2 days, 3 days) the whole volume of the PBS solution was refreshed and the withdrawn liquid was analyzed by UV spectrophotometer absorbance 290 nm (Cary5000 UVeviseNIR; Varian Inc., Palo Alto, CA). The amount of released drug was calculated against the calibration curves of Dex and Dex-P. The release profile was fitted by using both Hixon Crowell’s cubic root law equation to determine whether release was solubility controlled [21], and the Peppas equation [22] to assess whether Fickian diffusion was operative. The Peppas power law equation relates Mt/Mtotal is the cumulative amount of drug release at time t with the release exponent n and k a constant incorporating structural and geometrical characteristics of the drug dosage form.
5e15 mm diameter porous nanofibrous microspheres composed of calcium pyrophosphate were prepared as reported previously [20]. These spheres had a porosity of 68% and a density of 0.77 g cm3 with a hierarchical structure comprised of bundles of loosely assembled nanofibres yielding spheres with and specific surface area of approximately 200 m2 g1 (Fig. 1). Dexamethasone (Dex) and dexamethasone phosphate (Dex-P) were purchased from Sigma Aldrich, (St Louis, MO). For a 30% w/w concentration of Dex, 30 mg of the drug was dissolved in 1 ml of chloroform (Fisher Scientific, Ottawa, Ontario). Subsequently, 70 mg of pyrophosphate microspheres were suspended in the solution and left to dry under vacuum at room temperature until complete evaporation of the chloroform had occurred. For
Fig. 1. Scanning Electron Microscopy (SEM) images showing a calcium pyrophosphate nanofibrous microsphere (A), the nanofiber mesh (B) and the nanofibers (C) are shown at higher magnification using Transmission Electron Microscopy (TEM). Dexamethanone-loaded microspheres are shown (D), no change to their structure was apparent.
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Mt ¼ k tn Mtotal To assess the potential for sequestration of particles within macrophages in vivo, we performed in vitro analyses to determine whether macrophages were able to phagocytose microspheres. In these experiments, the murine macrophage cell line J774A.1 was plated at a density of 100 000 cells per well and approximately 1000 microspheres per well were added. The percentage of microspheres undergoing phagocytosis was determined for 4 consecutive days from three separate experiments performed on duplicate wells. Microspheres in the process of being phagocytosed by macrophages were recognizable under the microscope and were expressed as a percentage of the total microspheres present in each well. 2.3. In vivo studies Male Brown Norway (BN) rats, weighing 270e300 g, were purchased from Harlan Industries (Indianapolis, IN, USA). The rats were kept in a conventional animal facility and were supplied drinking water and food pellets ad libitum. All experiments were approved by the Animal Care Committee of McGill University. 2.4. Intra-tracheal administration For the administration of microspheres animals were lightly anaesthetised with 4% isoflurane. The microspheres were suspended in a solution of 2% bovine serum albumin (BSA) and 0.2 ml of this suspension was intra-tracheally (i.t.) instilled into each animal. An equal volume of BSA was used to treat the negative control groups. The same volume of fluid (0.2 ml) was used for administration of compound 48/80 (Sigma Aldrich, St Louis, MO). Saline was used as the vehicle for compound 48/80 and Dex-P. Immediately following i.t. administration of the microspheres or compound 48/ 80 the rats were assessed for signs of stress that included difficulty in breathing, a hunched posture and for lack of grooming following recovery from the anaesthesia. 2.5. Histological assessment of goblet cells and microsphere distribution Fluorescently-labelled microspheres were administered i.t., animals were sacrificed after 3, 6 and 24 h (n ¼ 6/group) by overdose of pentobarbital and the lungs were inflated with 5 ml of 10% phosphate-buffered neutral formalin via a cannula inserted into the trachea. The lungs were then removed from the thorax and fixed in inflation using 10% buffered formalin at a pressure of 25 cm H2O for 24e72 h. The lungs were sectioned sagittally through the left lobe, so as to include the main bronchi as well as the parenchyma, embedded in paraffin wax and sections of 5 mm thickness were cut. Doxycycline-labelled microspheres were visible following excitation at 470e490 nm and emission at 510 nm. We counted doxycycline-labelled microspheres in 8e10 fields per slide. Each field of view was approximately 300 mm2. 5 slides per animal were assessed. For assessment of goblet cell metaplasia, rats (n ¼ 6 per group) were sacrificed at 24, 48 and 72 h, and slides were stained with Periodic Acid Schiff (PAS).
received 50 ml of diluted biotinylated detection antibodies, and incubated for 3 h at room temperature. Following the incubation period, wells were washed and 100 ml of streptavidin-HRP was added to all wells and allowed to incubate for 30 min. Wells were then washed and 100 ml of TMB and were kept in the dark for 15 min for the colour to develop. To stop the reaction 100 ml of sulphuric acid was added and plates were measured at 450 nm. 2.8. Sustained release protocol For the in vivo evaluation of sustained release, BN rats were pre-treated, at time 0, with unloaded microspheres or microspheres loaded with Dex or Dex-P. At time 8 h or 18 h, BN rats were treated with the inflammatory agent, compound 48/80 and at time 32 h or 42 h, BALF and histological analyses were performed to assess the controlled release properties of the microspheres on compound 48/80-indcued airways inflammation. A schematic showing the timeline of treatments is shown in Fig. 2. 2.9. Statistical analysis For the in vivo studies, a one-way analysis of variance (ANOVA) with a NewmaneKeuls post-test was performed. Statistical significance was defined as p 0.05. All statistical tests were performed using GraphPad Prism (GraphPad Software La Jolla, CA 92037 USA).
3. Results 3.1. Release kinetics for dexamethasone-loaded microspheres The structure and morphology of the microspheres are illustrated in Fig.1. The spheres were between 5 and 15 mm and consisted of an open porous network of nanofibrous calcium pyrophosphate. The drug loading protocol did not appear to alter the microstructure. In vitro release measurements of dexamethasone and dexamethasone phosphate showed that microspheres loaded with dexamethasone, had an immediate burst release and the drug had dissolved entirely within 8 h of incubation. However, microspheres loaded with Dex-P showed a much more prolonged release, requiring up to 6 days to release the entirety of the drug (Fig. 3A). Hixon Crowell’s cubic root law equation achieved excellent fitting for the release pattern of the burst releasing Dex-loaded microspheres (R ¼ 0.98) whereas the fit for Dex-P was poorer (R ¼ 0.83, not shown), indicating a solubility controlled release ([21], Fig. 3C).
2.6. BAL fluid analysis The rats were sacrificed with an overdose of pentobarbital (65 mg/ml), a cannula was inserted in the trachea and the lung was lavaged with 5 5 ml of phosphatebuffered saline (PBS). The volume recovered was centrifuged and the supernatant stored for determination of tumour necrosis factor alpha (TNF-a) concentration. The pellet was re-suspended in 1 ml of cold (4 C) PBS for total cell counting and differentials. Total cell counts were performed following addition of 25 ml of trypan blue to 50 ml of the BALF sample and 25 ml of PBS. Following total cell counts the BALF fraction was diluted to a concentration of 60 000 cells per ml. Duplicate cytospins from the diluted cell samples were performed using a cytocentrifuge (Shandon, Shandon Scientific, Cheshire, England). Slides were stained using Diff-Quik stain (Dade Behring, Newark DE, USA) and cell differentials were determined from a count of 300 cells per slide by light microscopy. 2.7. Determination of BALF TNF-a concentration For determination of TNF-a concentration, the BALF from pre-sensitized rats and challenged with ovalbumin (OVA) was used as the positive control. Rats were sensitized with a subcutaneous injection of 1 mg of OVA (SigmaeAldrich) adsorbed in 100 mg of aluminium hydroxide (EM Industries Inc.) and dissolved in phosphatebuffered saline (PBS). Immediately after rats received 0.5 109 heat-killed Bordetella pertussis bacilli intraperitoneally (supplied by T. Issekutz, Dalhousie University, Halifax, Nova Scotia, Canada). Two weeks after the sensitization procedure, rats were challenged with an aerosol of 5% (wt/vol) OVA for 5 min intra-tracheally (i.t.) under pentobarbital anaesthesia. BALF was collected 48 h after OVA challenge. TNF-a levels from BALF supernatants were determined using a rat TNF-a Enzyme LinkedImmuno-Sorbent Assay (ELISA) kit (Diaclone, Besancon France). Briefly, 100 ml of the BALF supernatant samples were added in duplicates to 96-well coaster plates. Wells
Fig. 2. Diagrammatic representation illustrating the sustained efficacy in vivo of microspheres loaded with dexamethasone phosphate (Dex-P) on compound 48/80induced airways inflammation. The inflammation in the lungs observed 42 h after intra-tracheal (i.t.) instillation of the microspheres is inhibited as a result of the controlled release of Dex-P from the microspheres.
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Fig. 3. Dexamethasone and Dexamethasone phosphate (Dex-P) release from the pyrophosphate microspeheres as a function of time (A), Hixon Crowell fitting for the cumulative release of Dex-P (B). Peppas fitting for the cumulative Dex-P release (C).
While fit of the release pattern of Dex microspheres obtained by the Peppas equation was R ¼ 0.88 that for Dex-P was very strong (R ¼ 1.00) indicating that dissolution was not the dominant mechanism of release for this drug-material couple, (Fig. 3B). 3.2. Safety profile of varying doses of microspheres administered in vivo The safety profile of the microspheres was determined in vivo. In these studies, BN rats (n ¼ 6 per group) received increasing doses of microspheres i.t. ranging from 1, 3 or 10 mg per animal. Twentyfour hours after challenge, animals were sacrificed for BALF analysis. BALF cell counts showed an increase in the number of total cells following administration of 10 mg of spheres that was attributable to an increase in neutrophils and eosinophils (Fig. 4AeC). No changes in macrophage cell numbers were observed (Fig. 4D). TNF-a levels were not significantly different following administration of 1, 3 or 10 mg of microspheres compared to controls (Fig. 4E). Histological analyses demonstrated no increase in goblet cells in PAS stained sections at 24 h. However a slight increase was observed at 48 h that subsided 72 h after administration of 1 mg of microspheres (Fig. 4F). 3.3. Pattern of microsphere deposition and clearance To evaluate the deposition and clearance of the microspheres from the lung, BN rats (n ¼ 4 per time point) were administered
3 mg of fluorescently-labelled microspheres and were sacrificed at 3, 6 and 24 h after i.t. instillation. The number of microspheres present in the lung was determined at these time points. There was an increase in the microspheres at 3 and 6 h after instillation reaching a peak at 6 h. At 3 and 6 h a greater number of microspheres were observed in the main bronchi compared to the alveolar compartment (Fig. 5AeD). However at 24 h microspheres were most apparent in the alveolar compartment. At the later time points of 48 and 72 h, a reduction in the number of microspheres in the lung was observed (Fig. 5 B, D). It is of interest to note that clusters of fluorescent microspheres were apparent at 24 h after treatment, and histochemical staining with PAS revealed the presence of mucus enveloping these structures (Fig. 5E and F). In vitro studies aimed at studying the capacity of macrophages to phagocytose microspheres revealed that approximately 1% of microspheres were phagocytosed at day 1 increasing up to 2% on day 4 (Table 1). 3.4. A comparison of anti-inflammatory efficacy of dexamethasoneladen microspheres to free dexamethasone phosphate In subsequent experiments we aimed to explore the potential of Dex-P loaded microspheres in preventing lung inflammation induced by the mast cell secretagogue, compound 48/80. BN rats were pre-treated with saline (vehicle for compound 48/80), 1 mg of unloaded microspheres, standard Dex (without phosphate groups)-loaded microspheres, Dex-P loaded microspheres (30%
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Fig. 4. Bronchoalveolar lavage fluid (BALF) analyses of rats treated with a bolus of 1, 3 and 10 mg of microspheres (n ¼ 8 per dose) intra-tracheally (i.t.) and sacrificed 24 h after administration. The panels show total inflammatory counts (A), macrophage cell numbers (B), neutrophil (C), eosinophil (D) and tumour necrosis factor alpha (TNF-a) concentration (E). Goblet cell counts from histological sections stained with Periodic Acid Schiff (PAS) stain were performed in rats (n ¼ 3) treated with 1 mg of microspheres (or vehicle e2% bovine serum albumine)and sacrificed at 24, 48 and 72 h after i.t. treatment.
w/w), and 1 mg/kg of Dex-P (equivalent to 30% w/w Dex-P loaded MS). At 8 h after pre-treatment, experimental groups were challenged with the mast cell secretagogue, compound 48/80 (1 mg/kg i.t.). BALF analysis, 32 h after microsphere (or vehicle control) instillation, demonstrated an increase in inflammatory cells, neutrophilia and eosinophilia in the lungs following challenge with compound 48/80 compared to vehicle-treated animals (Fig. 6AeC). Pre-treatment with unloaded microspheres did not inhibit the inflammatory cell increase induced by the secretagogue (Fig. 6 A). However, only pre-treatment with Dex-P loaded microspheres resulted in an inhibition of neutrophilia, 32 h after microsphere instillation, which was not seen in Dex-P alone treated rats or following treatment of microspheres loaded with
standard Dex (devoid of phosphate groups, Fig. 6B). Nevertheless, both formulations using Dex-P (alone and loaded to MS) were able to significantly inhibit eosinophilic infiltration into the BALF at this time point. Standard Dex-loaded microspheres were able to inhibit eosinophilia however; this was not statistically significant (Fig. 6 C). When administered 42 h prior to the assessment of airways inflammation, Dex-P loaded microspheres were still able to significantly reduce the total inflammatory cells in the lungs and inhibit eosinophilia compared to rats treated with Dex-P alone which had no effect (Fig. 7 A, B). Administration of Dex-P alone or Dex-P loaded microspheres did not inhibit neutrophil influx to the lungs (Fig. 7C).
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Fig. 5. Fluoresencent image of a representative area of the lung showing a main bronchus at 6 h after intra-tracheal instillation of microspheres (A) and the number of microspheres counted in the bronchus at time points of 3, 6, 24, 48 and 72 h after instillation (B). Representative image of the lung taken 24 h after instillation of the microspheres (C) and the number of microspheres present in the alveolar compartment of the lung at 3, 6, 24, 48 and 72 h after instillation (D). A representative image of an airway showing a strong fluorescent area 24 h after instillation (E) that stained positive for Periodic Acid Shciff (F). The black arrows in F point at microspheres that were present in the airway.
4. Discussion 4.1. Control release kinetics At a molecular level, Dex-P differs from Dex by the presence of an additional phosphate group that increases its water solubility.
Phosphate groups are well known to interact with calcium containing compounds; therefore, Dex-P is probably more likely to interact with the calcium pyrophosphate microspheres. This factor proved to be crucial in controlling the release profile of the drug. Accordingly, in vitro assessment of the controlled release kinetics of the corticosteroid loaded microspheres demonstrated
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Table 1 Quantification of phagocytosis of nanofibrous calcium phosphate microspheres by a macrophage cell line in culture. Days in culture
Percent of microspheres undergoing phagocytosis by the macrophage cell line J774A.1 SEM
Day Day Day Day
0.2 0.04% 0.7 0.04% 1.3 0.13% 2.0 0.17%
1 2 3 4
Approximately 100,000 J774A.1 (a murine macrophage cell line) were co-cultured with 10,000 calcium pyrophosphate nanofibrous microspheres. The number of microspheres undergoing phagocytosis was counted from duplicate wells. The data is expressed as percentage of microspheres phagocytosed from 3 separate experiments.
that Dex-P loaded microspheres exhibited controlled drug release over a longer period of time than Dex-loaded microspheres. The analysis of the release profile for both drugs revealed that while Dex was released by simple dissolution, Dex-P release was likely to be controlled diffusion. However, the value of the exponent n obtained from Peppas equation was low (n ¼ 0.14) indicating that the drug diffusion was probably interfered with by adsorption of the drug to the calcium pyrophosphate matrix [23]. This was also apparent from the slow and constant release beyond 12 h.
4.2. Safety profile in vivo An essential concern in the development of controlled release inhalation systems is the toxicity profiles of the release modifying agents. The inflammatory potential and accumulation effects of novel agents are important concerns that are not always evaluated [1]. In the present study we demonstrated that doses of 1 mg of calcium pyrophosphate nanofiber microspheres had no significant effect on the influx of inflammatory cells to the lungs. At doses of 3 and 10 mg neutrophil and eosinophil counts in BALF were increased, however increases in macrophage activation (TNF-a) in BALF were not observed at any of the doses of microspheres tested. These results show that despite an increase of neutrophils, the microspheres are well tolerated at doses of up to 10 mg. Furthermore, it is important to note that the number of neutrophils increased from about 0.1 million cells in the vehicle to up to 0.3 million cells following 10 mg of microspheres. This increase in cells is statistically significant but may not be biologically relevant. Similar increases in BALF neutrophils have been demonstrated to be evoked by instillation of saline into the airways [24]. Furthermore, this level of neutrophilia was very mild compared to the level in the range of 2e3 million observed in this study following compound 48/80 instillation. The increase in goblet cell counts at 48 h may be related to the mechanism by which the microspheres are cleared by the lung, as indicated in our studies with fluorescentlylabelled microspheres (below). Furthermore, the increase in goblet cell counts appears to be transient returning to baseline levels at 72 h. Poly(lactic-co-glycolic) acid (PLGA), poly-lactic acid (PLA) and polyvinyl alcohol (PVA) are among the matrices explored for inhalation controlled release that have shown the greatest potential. However, PLGA and PLA can result in a significant reduction in cell viability in vitro [25], neutrophilia at sites of microparticle deposition [26] and a high probability of pulmonary accumulation [27]. While in vitro cell assays for PVA have shown that it appears to better tolerated than PLGA or PLA [1]. However no in vivo pulmonary toxicity studies have been reported.
4.3. Microsphere distribution and clearance In many studies, the distribution of particles for inhalation controlled release is assessed by non-invasive imaging techniques [28,29]. Although useful to image the entire thoracic cavity, these imaging techniques are not able to discriminate between bronchial and alveolar particle deposition that would is otherwise possible by conventional histology. In the present study, histological analysis following a single administration of microspheres revealed that microspheres eventually reached the alveolar compartments of the lungs. At 3e6 h post treatment a larger number of microspheres was found to be in the large conducting airways. At 6 h, an increase in microspheres was apparent in the alveolar compartments and microspheres present in large conducting airways tended to aggregate in groups of >10, possibly due to the adhesive effect of increased amounts of mucus. This observation correlates with the increased number of goblet cells observed at 48 h after i.t. microsphere instillation. Together, these results suggest that mucus clearance may have played an important role in the later removal of calcium pyrophosphate microspheres from the lung. Even though the microspheres are composed of nanofibre bundles with very high specific area, their size is large enough to allow them to be removed by mucus clearance, which would prevent alveolar retention if inhaled in large quantities [30,31]. On the other hand, our in vitro studies revealed that, only 2% of the microspheres were phagocytosed by macrophages after 4 days of exposure in cell culture. These results highlight the bioinert properties of the microspheres and correlate with our in vivo data showing no increase in TNF-a levels following i.t. administration of microspheres at doses up to 10 mg. The combination of these observations suggests that the primary mechanism for removal of microspheres from the lung is via mucus clearance and that phagocytosis by macrophages plays a discreet role. 4.4. Therapeutic and in vivo controlled release profile Compound 48/80 is a basic mast cell secretagogue that causes mast cell degranulation [15] via direct activation of Gi proteins leading to the release of histamine, 5-hydroxytryptamine and arachidonic acid metabolites implicated in the de novo synthesis of leukotrienes and prostaglandins [32,33]. It has been used extensively in vivo to induce inflammation in the lungs [34,17], rat paw edema [18] and inflammation of the skin [19]. Calcium pyrophosphate microspheres were loaded with dexamethasone, a corticosteroid commonly used in models of airways inflammation [35e37]. Dex-P, a more water soluble form of Dex was chosen for our in vivo studies, based on our in vitro controlled release kinetics study. In these studies, administration of microspheres (1 mg) on their own 32 h prior to compound 48/80-induced airways inflammation did not attenuate the influx of inflammatory cells to the lungs. However, Dex-P loaded microspheres was the only formulation capable of reducing total inflammatory cell influx including neutrophilia and eosinophilia, 32 h prior to the assessment of airways inflammation. Inhibition of eosinophils by Dex-P alone or Dex-P loaded microspheres was comparable; however, Dex-P loaded microspheres were significantly better at attenuating neutrophilia than administration of Dex-P alone. Pre-treatment with microspheres loaded with standard dexamethasone, devoid of phosphate groups, was not able to significantly inhibit total inflammatory cell infiltration, neutrophilia or eosinophilia induced by compound 48/80. Furthermore, Dex-P loaded microspheres were significantly better at reducing
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neutrophilia compared to Dex-loaded microspheres. These observations highlight the critical role of the phosphate groups in proving controlled release of dexamethasone from the microspheres. In experiments where animals were pre-treated with Dex-P alone or Dex-P loaded microspheres 42 h prior to the assessment of compound 48/80-induced lung inflammation, inhibition of eosinophilia in BALF, a hallmark of asthma, was significantly larger for Dex-P loaded microspheres compared to Dex-P alone or vehicle-treated animals. These observations demonstrate the controlled release properties of the microspheres. Thus it is likely that a small concentration of corticosteroid, released by the microspheres, is able to inhibit eosinophilia observed at 42 h when compared to rats treated with Dex-P only. The comparable inhibition of both Dex-P alone and Dex-P loaded microspheres on eosinophil influx at 32 h, can be explained by the increased sensitivity of eosinophils to corticosteroids [38,39] where drug levels are high enough in the Dex-P alone and Dex-P loaded groups to induce a comparable response. Similarly, the relatively greater resistance of neutrophils to steroids [40,41] would explain why neutrophilia is inhibited at 32 h with both drug delivery methods but is more prominent in Dex-P loaded microsphere groups compared to Dex-P alone groups and at 42 h neither Dex-P alone nor Dex-P loaded microspheres are inhibitory. Although some inhalation controlled release systems have been developed and tested in vitro, however very few of them have been tested in vivo for their therapeutic efficacy, (for a recent review see [1]). Furthermore, proof of controlled release properties in vitro does not necessarily equate with success in vivo, as has been reported with salbutamol acetonide loaded solid lipid nanoparticles of glyceryl behenate [42]. Controlled release inhalation systems that have been tested in vivo include PLGA or PLA that have shown increased levels of insulin or testosterone lasting up to 96 h for insulin or 24 h for testosterone [1]. However, as discussed previously the toxicological effects of these agents are of important concern. Similar studies by Yamada et al. [43], have demonstrated that polysaccharide gels containing iota or kappa carrageenan are capable of enhancing absorption of fluticasone and theophylline systemically up to 6 h following i.t. delivery. These studies have shown that iota and kappa carrageenan gels do not appear to induce lung inflammation in BALF 5 and 24 h after delivery [43]. However, no therapeutic improvement on airways inflammation or physiology following an inflammatory insult was demonstrated by this formulation. Thus, in comparison with recent developments in inhalation controlled release systems, calcium pyrophosphate nanofibrous microspheres do not show signs of toxicity or pulmonary accumulation, demonstrate controlled release properties in vitro and in vivo, and possess therapeutic efficacy 42 h after a single dose. The clinical implications of this work suggest that use of these microspheres as novel matrix for pulmonary drug delivery has the capacity to significantly improve drug efficacy and compliance in patients being treated with corticosteroids for asthma or COPD as a result of its long lasting controlled release properties.
Fig. 6. Total inflammatory counts (A), neutrophil (B), eosinophil (C) observed from bronchoalveolar lavage fluid (BALF) analyses of rats sacrificed 32 h after treatment with vehicle for the microspheres (0.2 ml intra-tracheallye i.t. of a 2% bovine serum albumin eBSA), 1 mg of microspheres (i.t.), dexamethasone-loaded micropheres (DexMS 30% w/w i.t.), dexamethasone phosphate alone (1 mg/kg i.t.) or 1 mg of dexamethasone phosphate-loaded microspheres (30% w/w i.t.). Compound 48/80 (1 mg/kg i.t.) or saline (vehicle for compound 48/80, 0.2 ml i.t.), was administered 8 h after microsphere or vehicle control. (N ¼ 6 for all groups). BALF was performed 32 h after the microsphere or vehicle instillation.
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5. Conclusion In the current study we explored the potential of calcium pyrophosphate microspheres as an alternative matrix for the administration of therapeutic agents for pulmonary delivery and demonstrated the controlled release behaviour of Dex-P loaded microspheres in vitro. The safety profile of calcium pyrophosphate nanofibres microspheres in vivo showed that the microspheres appeared to be well tolerated and did not accumulate in the lung. Finally we demonstrated that microspheres loaded with Dex-P also exhibited controlled release properties in vivo and were capable of inhibiting compound 48/80-induced airway inflammation at 32 and 42 h after microsphere instillation. These new inorganic microspheres likely have many applications in sustained release applications. Appendix Figures with essential color discrimination. Figs. 3 and 5 of this article have parts that are difficult to interpret in black and white. The full color images can be found in the on-line version, at doi:10. 1016/j.biomaterials.2010.04.025. References
Fig. 7. Total inflammatory counts (A), eosinophil (B) and neutrophil (C) observed from bronchoalveolar lavage fluid (BALF) analyses of rats sacrificed 42 h after treatment with vehicle for the microspheres (0.2 ml intra-tracheallye i.t. of a 2% bovine serum albumin eBSA, n ¼ 6), dexamethasone phosphate alone (1 mg/kg i.t., n ¼ 10) or 1 mg of dexamethasone-loaded microspheres (30% w/w i.t., n ¼ 17). Compound 48/80 (1 mg/i. t.) was administered 18 h after microsphere or vehicle treatment.
[1] Salama R, Traini D, Chan HK, Young PM. Recent advances in controlled release pulmonary therapy. Curr Drug Deliv 2009;6(4):404e14. [2] Thompson DC. Pharmacology of therapeutic aerosols. In: Hickey AJ, editor. Pharmaceutical inhalation aerosol technology. New York, New York: Informa Health Care; 2003. p. 31e6. [3] Barnes PJ. Inhaled glucocorticoids for asthma. N Engl J Med 1995;332:868e75. [4] Schramm CM, Carroll CL. Advances in treating acute asthma exacerbations in children. Curr Opin Pediatr 2009;21:326e32. [5] Byron PR. Prediction of drug residence times in regions of the human respiratory tract following aerosol inhalation. J Pharm Sci 1986;75:433e8. [6] Ballard RD, Irvin CG, Martin RJ, Pak J, Pandey R, White DP. Influence of sleep on lung volume in asthmatic patients and normal subjects. J Appl Phys 1990; 68:2034e41. [7] Kraft M, Vianna E, Martin RJ, Leung DY. Nocturnal asthma is associated with reduced glucocorticoid receptor binding affinity and decreased steroid responsiveness at night. J Allergy Clin Immunol 1999;103:66e71. [8] Malo JL, Cartier A, Merland N, Ghezzo H, Burek A, Morris J, et al. Four-times-aday dosing frequency is better than a twice-a-day regimen in subjects requiring a high-dose inhaled steroid, budesonide, to control moderate to severe asthma. Am Rev Respir Dis 1989;140:624e8. [9] Chmelik F, Doughty A. Objective measurements of compliance in asthma treatment. Ann Allergy 1994;73:527e32. [10] Rasenack N, Muller BW. Micron-size drug particles: common and novel micronization techniques. Pharm Dev Technol 2004;9:1e13. [11] Chow AH, Tong HH, Chattopadhyay P, Shekunov BY. Particle engineering for pulmonary drug delivery. Pharm Res 2007;24:411e37. [12] Kawashima Y, Serigano T, Hino T, Yamamoto H, Takeuchi H. A new powder design method to improve inhalation efficiency of pranlukast hydrate dry powder aerosols by surface modification with hydroxypropylmethylcellulose phthalate nanospheres. Pharm Res 1998;15:1748e52. [13] Steckel H, Bolzen N. Alternative sugars as potential carriers for dry powder inhalations. Int J Pharm 2004;270:297e306. [14] Rytting E, Nguyen J, Wang X, Kissel T. Biodegradable polymeric nanocarriers for pulmonary drug delivery. Expert Opin Drug Deliv 2008;5:629e39. [15] Koibuchi Y, Ichikawa A, Nakagawa M, Tomita K. Histamine release induced from mast cells by active components of compound 48/80. Eur J Pharmacol 1985;115:163e70. [16] Shefler I, Seger R, Sagi-Eisenberg R. Gi-mediated activation of mitogen-activated protein kinase (MAPK) pathway by receptor mimetic basic secretagogues of connective tissue-type mast cells: bifurcation of arachidonic acidinduced release upstream of MAPK. J Pharmacol Exp Ther 1999;289:1654e61. [17] Karmouty-Quintana H, Blé FX, Cannet C, Zurbruegg S, Fozard JR, Page CP, et al. In vivo pharmacological evaluation of compound 48/80-induced airways edema by MRI. Br J Pharmacol 2008;154:1063e72. [18] Maling HM, Webster ME, Williams MA, Saul W, Anderson Jr W. Inflammation induced by histamine, serotonin, bradykinin and compound 48e80 in the rat: antagonists and mechanisms of action. J Pharmacol Exp Ther 1974; 191:300e10. [19] James MP, Kennedy AR, Eady RA. A microscopic study of inflammatory reactions in human skin induced by histamine and compound 48/80. J Invest Dermatol 1982;78:406e13.
H. Karmouty-Quintana et al. / Biomaterials 31 (2010) 6050e6059 [20] Grover L, Tamimi F, Bassett D, Karmouty-Quintana H, Martin J, Barralet J. Porous Calcium Phosphate Microspheres for Sustained Release. In: 36th Annual Meeting and Exposition of the Controlled Release Society, July 18e22 Copenhagen, Denmark; 2009. [21] Lee RW, Shaw JM, McShane J, Wood RW. Particle size reduction. In: Liu R, editor. Water-insoluble drug formulation. Boca Raton FL: CRC Press; 2002. p. 461e2. [22] Peppas NA. Analysis of Fickian and non-Fickian drug release from polymers. Pharm Acta Helv 1985;60:110e1. [23] Ginebra MP, Traykova T, Planell JA. Calcium phosphate cements as bone drug delivery systems: a review. J Control Release 2006;113:102e10. [24] Haley PJ, Muggenburg BA, Rebar AH, Shopp GM, Bice DE. Bronchoalveolar lavage cytology in cynomolgus monkeys and identification of cytologic alterations following sequential saline lavage. Vet Pathol 1989; 26:265e73. [25] Muller RH, Maassen S, Weyhers H, Specht F, Lucks JS. Cytotoxicity of magnetite-loaded polylactide, polylactide/glycolide particles and solid lipid nanoparticles. Int J Pharm 1996;138:85e94. [26] Armstrong DJ, Elliott PN, Ford JL, Gadsdon D, McCarthy GP, Rostron C, et al. Poly-(D,L-lactic acid) microspheres incorporating histological dyes for intrapulmonary histopathological investigations. J Pharm Pharmacol 1996; 48:258e62. [27] Dunne M, Corrigan I, Ramtoola Z. Influence of particle size and dissolution conditions on the degradation properties of polylactide-co-glycolide particles. Biomaterials 2000;21:1659e68. [28] Mizuno T, Mohri K, Nasu S, Danjo K, Okamoto H. Dual imaging of pulmonary delivery and gene expression of dry powder inhalant by fluorescence and bioluminescence. J Control Release 2009;134:149e54. [29] Elfinger M, Geiger J, Hasenpusch G, Uzgün S, Sieverling N, Aneja MK, et al. Targeting of the beta(2)-adrenoceptor increases nonviral gene delivery to pulmonary epithelial cells in vitro and lungs in vivo. J Control Release 2009;135:234e41. [30] Martonen TB. Mathematical model for the selective deposition of inhaled pharmaceuticals. J Pharm Sci 1993;82:1191e9. [31] Griffith Edward J. What is a safe fibre? In: Griffth Edward, editor. Phosphate fibres. New York: Plenum Press; 1994. p. 18e27.
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[32] Stevens RL, Austen KF. Recent advances in the cellular and molecular biology of mast cells. Immunol Today 1989;10:381e6. [33] Gordon JR, Burd PR, Galli SJ. Mast cells as a source of multifunctional cytokines. Immunol Today 1990;11:458e64. [34] Fingar VH, Taber SW, Wieman TJ. A new model for the study of pulmonary microcirculation: determination of pulmonary edema in rats. J Surg Res 1994;57:385e93. [35] Renzi PM, Olivenstein R, Martin JG. Effect of dexamethasone on airway inflammation and responsiveness after antigen challenge of the rat. Am Rev Respir Dis 1993;148(4):932e9. [36] El-Hashim AZ, Banner KH, Paul W, Page CP. Effects of dexamethasone on airway hyper-responsiveness to the adenosine A1 receptor agonist cyclo-pentyl adenosine in an allergic rabbit model. Br J Pharmacol 1999;126:1513e21. [37] Eum SY, Maghni K, Hamid Q, Eidelman DH, Campbell H, Isogai S, et al. Inhibition of allergic airways inflammation and airway hyperresponsiveness in mice by dexamethasone: role of eosinophils, IL-5, eotaxin, and IL-13. J Allergy Clin Immunol 2003;111:1049e61. [38] Sabag N, Castrillon MA, Tchernitchin A. Cortisol-induced migration of eosinophil leukocytes to lymphoid organs. Experientia 1978;34:666e7. [39] Sale R, Sabatini F, Silvestri M, Serpero L, Petecchia L, Rossi GA. Concentrationdependent activity of mometasone furoate and dexamethasone on blood eosinophils isolated from atopic children: modulation of Mac-1 expression and chemotaxis. Int Immunopharmacol 2004;4:1687e96. [40] Green RH, Brightling CE, Woltmann G, Parker D, Wardlaw AJ, Pavord ID. Analysis of induced sputum in adults with asthma: identification of subgroup with isolated sputum neutrophilia and poor response to inhaled corticosteroids. Thorax 2002;57:875e9. [41] Barnes PJ. New molecular targets for the treatment of neutrophilic diseases. J Allergy Clin Immunol 2007;119:1055e62. [42] Jaspart S, Bertholet P, Piel G, Dogné JM, Delattre L, Evrard B. Solid lipid microparticles as a sustained release system for pulmonary drug delivery. Eur J Pharm Biopharm 2007;65:47e56. [43] Yamada K, Kamada N, Odomi M, Okada N, Nabe T, Fujita T, et al. Carrageenans can regulate the pulmonary absorption of antiasthmatic drugs and their retention in the rat lung tissues without any membrane damage. Int J Pharm 2005;293:63e72.
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Sustained steroid release in pulmonary inflammation model Harry Karmouty-Quintana a, Faleh Tamimi b, Toby K. McGovern a, Liam M. Grover c, James G. Martin a, Jake E. Barralet b, * a
Meakins-Christie Laboratories, McGill University, 3626 Rue St Urbain, Montreal, Quebec, H2X 2P2, Canada Faculty of Dentistry, Strathcona Anatomy & Dentistry, McGill University, 3640 University Street Montreal, Montreal, Quebec H3A 2B2, Canada c The Department of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham, UK b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 March 2010 Accepted 12 April 2010 Available online 15 May 2010
There is a need for particles which exhibit controlled release of therapeutic agents delivered via the inhalational route, for tissue specific applications such as anti-cancer, bronchodilators and antiviral agents as well as drugs for systemic action. The aim of this study was to assess the acute toxicity, distribution and capacity of the microspheres to exhibit controlled release properties in an in vivo model of airway inflammation. Calcium pyrophosphate nanofibrous microspheres were loaded with dexamethasone phosphate (Dex-P); the profile of drug release was studied in vitro and validated in vivo. Unloaded microspheres were administered intra-tracheally (i.t.) to rats to assess the tissue reaction. The anti-inflammatory properties of the Dex-P loaded microspheres against an inflammatory agent (compound 48/80), were evaluated in vivo. Unloaded microspheres did not cause an inflammatory response when given at doses below 3 mg, and appeared to be eliminated through mucus clearance mechanisms. Microspheres loaded with Dex-P but not Dex-P alone, were capable of inhibiting eosinophil and total inflammatory cell increases in bronchoalveolar lavage fluid for 42 h following a single application. These observations demonstrated that calcium pyrophosphate nanofibrous microspheres displayed in vivo controlled release properties, were well tolerated and did not accumulate in the lung. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Calcium pyrophosphate Microsphere Controlled-release Anti-inflammatory Inhalation drug delivery
1. Introduction Therapeutic agents are frequently delivered by inhalation, in particular for respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD). However, to date, there are no controlled release inhalation systems available clinically [1]. Treatments for asthma, COPD, cancer, infectious disease or for systemic action would benefit from improvements in inhalation controlled release systems [1,2]. The treatment of chronic asthma often relies on the delivery by inhalation of corticosteroids comparable to dexamethasone [3,4]. In addition to the potential advantages in some settings of reduction of dose cycling, sustained release is important because it reduces the dose required, improves biological efficacy, requires less patient compliance and is less irritating to the bronchial tissues [5]. For example asthma is worse at night because endogenous anti-inflammatory mechanisms are least effective at this time [6,7]. However, most patients do not comply with four times or even twice daily dosing [8,9] and the ability to achieve sustained release for up to 12 h and beyond may * Corresponding author. E-mail address: [email protected] (J.E. Barralet). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.04.025
be an important step forward in improving efficacy of several lung disease therapies. The use of aerosolised or dry powder inhalation therapy for asthma is common. The particle size is an important determinant of its site of deposition within the respiratory tree. However, the micronisation of drug particles often leads to large distribution in particle size, alteration of surface properties and renders the particles more cohesive leading to poor aerosolisation [10,11]. Furthermore, the use of lactose as a drug carrier and bulking agent for many drug formulations [11] can lead to poor inhalation performance [12]. Although used in many drug formulations, lactose is known to react with formoterol (a long-acting bronchodilator used in asthma), peptides and proteins [13]. Aerosolisation of particles is often difficult as it is necessary to dissolve them in a suitable vehicle, a growing problem concerning large organic molecular entities, monoclonal antibodies and silencing RNA approaches [11,14]. Thus inert vehicles for the controlled release of medication aimed at treating respiratory conditions are of great interest to the field. In this study we used an experimental model of lung inflammation induced by compound 48/80, a condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde. It is a basic secretagogue that causes mast cell degranulation
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[15] via direct activation of the Gi protein [16]. Compound 48/80 has been used extensively in vivo to induce inflammation in the lungs [17], rat paw edema [18], and inflammation of the skin [19]. We chose to use this stimulus and the subsequent inflammatory response as the model system to test the efficacy of newly developed porous nanofibrous microspheres composed of calcium pyrophosphate in the size range from 5 to 15 mm to act as sustained release depots to deliver dexamethasone phosphate.
a concentration of 30% w/w (Dex-P), 70 mg of pyrophosphate microspheres were mixed with 10 mg of calcium chloride (Sigma Aldrich, St. Louis, MO) in 1 ml of distilled water before adding 30 mg of Dex-P to the suspension and mixing thoroughly for 10 s. Afterwards, the suspension was left to dry overnight under vacuum at room temperature. In order to visualize the distribution of the microspheres within the lungs, after i.t. administration, fluorescent microspheres labelled by adsorption of fluorescent doxycycline. 100 mg of microspheres were mixed with 0.5 ml of a doxycycline solution (Sigma Aldrich, St. Louis, MO) (5% w/w), and left to dry under vacuum at room temperature.
2. Materials and methods
2.2. In vitro controlled release kinetics
2.1. Microsphere preparation and loading with dex, dex-P or fluorescent marker
To determine the in vitro release profiles of the microspheres, 10 mg of the drug loaded microspheres were incubated in 50 ml PBS solutions. At predetermined time points (1 h, 2 h, 4 h, 8 h, 12 h, 1 day, 2 days, 3 days) the whole volume of the PBS solution was refreshed and the withdrawn liquid was analyzed by UV spectrophotometer absorbance 290 nm (Cary5000 UVeviseNIR; Varian Inc., Palo Alto, CA). The amount of released drug was calculated against the calibration curves of Dex and Dex-P. The release profile was fitted by using both Hixon Crowell’s cubic root law equation to determine whether release was solubility controlled [21], and the Peppas equation [22] to assess whether Fickian diffusion was operative. The Peppas power law equation relates Mt/Mtotal is the cumulative amount of drug release at time t with the release exponent n and k a constant incorporating structural and geometrical characteristics of the drug dosage form.
5e15 mm diameter porous nanofibrous microspheres composed of calcium pyrophosphate were prepared as reported previously [20]. These spheres had a porosity of 68% and a density of 0.77 g cm3 with a hierarchical structure comprised of bundles of loosely assembled nanofibres yielding spheres with and specific surface area of approximately 200 m2 g1 (Fig. 1). Dexamethasone (Dex) and dexamethasone phosphate (Dex-P) were purchased from Sigma Aldrich, (St Louis, MO). For a 30% w/w concentration of Dex, 30 mg of the drug was dissolved in 1 ml of chloroform (Fisher Scientific, Ottawa, Ontario). Subsequently, 70 mg of pyrophosphate microspheres were suspended in the solution and left to dry under vacuum at room temperature until complete evaporation of the chloroform had occurred. For
Fig. 1. Scanning Electron Microscopy (SEM) images showing a calcium pyrophosphate nanofibrous microsphere (A), the nanofiber mesh (B) and the nanofibers (C) are shown at higher magnification using Transmission Electron Microscopy (TEM). Dexamethanone-loaded microspheres are shown (D), no change to their structure was apparent.
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Mt ¼ k tn Mtotal To assess the potential for sequestration of particles within macrophages in vivo, we performed in vitro analyses to determine whether macrophages were able to phagocytose microspheres. In these experiments, the murine macrophage cell line J774A.1 was plated at a density of 100 000 cells per well and approximately 1000 microspheres per well were added. The percentage of microspheres undergoing phagocytosis was determined for 4 consecutive days from three separate experiments performed on duplicate wells. Microspheres in the process of being phagocytosed by macrophages were recognizable under the microscope and were expressed as a percentage of the total microspheres present in each well. 2.3. In vivo studies Male Brown Norway (BN) rats, weighing 270e300 g, were purchased from Harlan Industries (Indianapolis, IN, USA). The rats were kept in a conventional animal facility and were supplied drinking water and food pellets ad libitum. All experiments were approved by the Animal Care Committee of McGill University. 2.4. Intra-tracheal administration For the administration of microspheres animals were lightly anaesthetised with 4% isoflurane. The microspheres were suspended in a solution of 2% bovine serum albumin (BSA) and 0.2 ml of this suspension was intra-tracheally (i.t.) instilled into each animal. An equal volume of BSA was used to treat the negative control groups. The same volume of fluid (0.2 ml) was used for administration of compound 48/80 (Sigma Aldrich, St Louis, MO). Saline was used as the vehicle for compound 48/80 and Dex-P. Immediately following i.t. administration of the microspheres or compound 48/ 80 the rats were assessed for signs of stress that included difficulty in breathing, a hunched posture and for lack of grooming following recovery from the anaesthesia. 2.5. Histological assessment of goblet cells and microsphere distribution Fluorescently-labelled microspheres were administered i.t., animals were sacrificed after 3, 6 and 24 h (n ¼ 6/group) by overdose of pentobarbital and the lungs were inflated with 5 ml of 10% phosphate-buffered neutral formalin via a cannula inserted into the trachea. The lungs were then removed from the thorax and fixed in inflation using 10% buffered formalin at a pressure of 25 cm H2O for 24e72 h. The lungs were sectioned sagittally through the left lobe, so as to include the main bronchi as well as the parenchyma, embedded in paraffin wax and sections of 5 mm thickness were cut. Doxycycline-labelled microspheres were visible following excitation at 470e490 nm and emission at 510 nm. We counted doxycycline-labelled microspheres in 8e10 fields per slide. Each field of view was approximately 300 mm2. 5 slides per animal were assessed. For assessment of goblet cell metaplasia, rats (n ¼ 6 per group) were sacrificed at 24, 48 and 72 h, and slides were stained with Periodic Acid Schiff (PAS).
received 50 ml of diluted biotinylated detection antibodies, and incubated for 3 h at room temperature. Following the incubation period, wells were washed and 100 ml of streptavidin-HRP was added to all wells and allowed to incubate for 30 min. Wells were then washed and 100 ml of TMB and were kept in the dark for 15 min for the colour to develop. To stop the reaction 100 ml of sulphuric acid was added and plates were measured at 450 nm. 2.8. Sustained release protocol For the in vivo evaluation of sustained release, BN rats were pre-treated, at time 0, with unloaded microspheres or microspheres loaded with Dex or Dex-P. At time 8 h or 18 h, BN rats were treated with the inflammatory agent, compound 48/80 and at time 32 h or 42 h, BALF and histological analyses were performed to assess the controlled release properties of the microspheres on compound 48/80-indcued airways inflammation. A schematic showing the timeline of treatments is shown in Fig. 2. 2.9. Statistical analysis For the in vivo studies, a one-way analysis of variance (ANOVA) with a NewmaneKeuls post-test was performed. Statistical significance was defined as p 0.05. All statistical tests were performed using GraphPad Prism (GraphPad Software La Jolla, CA 92037 USA).
3. Results 3.1. Release kinetics for dexamethasone-loaded microspheres The structure and morphology of the microspheres are illustrated in Fig.1. The spheres were between 5 and 15 mm and consisted of an open porous network of nanofibrous calcium pyrophosphate. The drug loading protocol did not appear to alter the microstructure. In vitro release measurements of dexamethasone and dexamethasone phosphate showed that microspheres loaded with dexamethasone, had an immediate burst release and the drug had dissolved entirely within 8 h of incubation. However, microspheres loaded with Dex-P showed a much more prolonged release, requiring up to 6 days to release the entirety of the drug (Fig. 3A). Hixon Crowell’s cubic root law equation achieved excellent fitting for the release pattern of the burst releasing Dex-loaded microspheres (R ¼ 0.98) whereas the fit for Dex-P was poorer (R ¼ 0.83, not shown), indicating a solubility controlled release ([21], Fig. 3C).
2.6. BAL fluid analysis The rats were sacrificed with an overdose of pentobarbital (65 mg/ml), a cannula was inserted in the trachea and the lung was lavaged with 5 5 ml of phosphatebuffered saline (PBS). The volume recovered was centrifuged and the supernatant stored for determination of tumour necrosis factor alpha (TNF-a) concentration. The pellet was re-suspended in 1 ml of cold (4 C) PBS for total cell counting and differentials. Total cell counts were performed following addition of 25 ml of trypan blue to 50 ml of the BALF sample and 25 ml of PBS. Following total cell counts the BALF fraction was diluted to a concentration of 60 000 cells per ml. Duplicate cytospins from the diluted cell samples were performed using a cytocentrifuge (Shandon, Shandon Scientific, Cheshire, England). Slides were stained using Diff-Quik stain (Dade Behring, Newark DE, USA) and cell differentials were determined from a count of 300 cells per slide by light microscopy. 2.7. Determination of BALF TNF-a concentration For determination of TNF-a concentration, the BALF from pre-sensitized rats and challenged with ovalbumin (OVA) was used as the positive control. Rats were sensitized with a subcutaneous injection of 1 mg of OVA (SigmaeAldrich) adsorbed in 100 mg of aluminium hydroxide (EM Industries Inc.) and dissolved in phosphatebuffered saline (PBS). Immediately after rats received 0.5 109 heat-killed Bordetella pertussis bacilli intraperitoneally (supplied by T. Issekutz, Dalhousie University, Halifax, Nova Scotia, Canada). Two weeks after the sensitization procedure, rats were challenged with an aerosol of 5% (wt/vol) OVA for 5 min intra-tracheally (i.t.) under pentobarbital anaesthesia. BALF was collected 48 h after OVA challenge. TNF-a levels from BALF supernatants were determined using a rat TNF-a Enzyme LinkedImmuno-Sorbent Assay (ELISA) kit (Diaclone, Besancon France). Briefly, 100 ml of the BALF supernatant samples were added in duplicates to 96-well coaster plates. Wells
Fig. 2. Diagrammatic representation illustrating the sustained efficacy in vivo of microspheres loaded with dexamethasone phosphate (Dex-P) on compound 48/80induced airways inflammation. The inflammation in the lungs observed 42 h after intra-tracheal (i.t.) instillation of the microspheres is inhibited as a result of the controlled release of Dex-P from the microspheres.
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Fig. 3. Dexamethasone and Dexamethasone phosphate (Dex-P) release from the pyrophosphate microspeheres as a function of time (A), Hixon Crowell fitting for the cumulative release of Dex-P (B). Peppas fitting for the cumulative Dex-P release (C).
While fit of the release pattern of Dex microspheres obtained by the Peppas equation was R ¼ 0.88 that for Dex-P was very strong (R ¼ 1.00) indicating that dissolution was not the dominant mechanism of release for this drug-material couple, (Fig. 3B). 3.2. Safety profile of varying doses of microspheres administered in vivo The safety profile of the microspheres was determined in vivo. In these studies, BN rats (n ¼ 6 per group) received increasing doses of microspheres i.t. ranging from 1, 3 or 10 mg per animal. Twentyfour hours after challenge, animals were sacrificed for BALF analysis. BALF cell counts showed an increase in the number of total cells following administration of 10 mg of spheres that was attributable to an increase in neutrophils and eosinophils (Fig. 4AeC). No changes in macrophage cell numbers were observed (Fig. 4D). TNF-a levels were not significantly different following administration of 1, 3 or 10 mg of microspheres compared to controls (Fig. 4E). Histological analyses demonstrated no increase in goblet cells in PAS stained sections at 24 h. However a slight increase was observed at 48 h that subsided 72 h after administration of 1 mg of microspheres (Fig. 4F). 3.3. Pattern of microsphere deposition and clearance To evaluate the deposition and clearance of the microspheres from the lung, BN rats (n ¼ 4 per time point) were administered
3 mg of fluorescently-labelled microspheres and were sacrificed at 3, 6 and 24 h after i.t. instillation. The number of microspheres present in the lung was determined at these time points. There was an increase in the microspheres at 3 and 6 h after instillation reaching a peak at 6 h. At 3 and 6 h a greater number of microspheres were observed in the main bronchi compared to the alveolar compartment (Fig. 5AeD). However at 24 h microspheres were most apparent in the alveolar compartment. At the later time points of 48 and 72 h, a reduction in the number of microspheres in the lung was observed (Fig. 5 B, D). It is of interest to note that clusters of fluorescent microspheres were apparent at 24 h after treatment, and histochemical staining with PAS revealed the presence of mucus enveloping these structures (Fig. 5E and F). In vitro studies aimed at studying the capacity of macrophages to phagocytose microspheres revealed that approximately 1% of microspheres were phagocytosed at day 1 increasing up to 2% on day 4 (Table 1). 3.4. A comparison of anti-inflammatory efficacy of dexamethasoneladen microspheres to free dexamethasone phosphate In subsequent experiments we aimed to explore the potential of Dex-P loaded microspheres in preventing lung inflammation induced by the mast cell secretagogue, compound 48/80. BN rats were pre-treated with saline (vehicle for compound 48/80), 1 mg of unloaded microspheres, standard Dex (without phosphate groups)-loaded microspheres, Dex-P loaded microspheres (30%
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Fig. 4. Bronchoalveolar lavage fluid (BALF) analyses of rats treated with a bolus of 1, 3 and 10 mg of microspheres (n ¼ 8 per dose) intra-tracheally (i.t.) and sacrificed 24 h after administration. The panels show total inflammatory counts (A), macrophage cell numbers (B), neutrophil (C), eosinophil (D) and tumour necrosis factor alpha (TNF-a) concentration (E). Goblet cell counts from histological sections stained with Periodic Acid Schiff (PAS) stain were performed in rats (n ¼ 3) treated with 1 mg of microspheres (or vehicle e2% bovine serum albumine)and sacrificed at 24, 48 and 72 h after i.t. treatment.
w/w), and 1 mg/kg of Dex-P (equivalent to 30% w/w Dex-P loaded MS). At 8 h after pre-treatment, experimental groups were challenged with the mast cell secretagogue, compound 48/80 (1 mg/kg i.t.). BALF analysis, 32 h after microsphere (or vehicle control) instillation, demonstrated an increase in inflammatory cells, neutrophilia and eosinophilia in the lungs following challenge with compound 48/80 compared to vehicle-treated animals (Fig. 6AeC). Pre-treatment with unloaded microspheres did not inhibit the inflammatory cell increase induced by the secretagogue (Fig. 6 A). However, only pre-treatment with Dex-P loaded microspheres resulted in an inhibition of neutrophilia, 32 h after microsphere instillation, which was not seen in Dex-P alone treated rats or following treatment of microspheres loaded with
standard Dex (devoid of phosphate groups, Fig. 6B). Nevertheless, both formulations using Dex-P (alone and loaded to MS) were able to significantly inhibit eosinophilic infiltration into the BALF at this time point. Standard Dex-loaded microspheres were able to inhibit eosinophilia however; this was not statistically significant (Fig. 6 C). When administered 42 h prior to the assessment of airways inflammation, Dex-P loaded microspheres were still able to significantly reduce the total inflammatory cells in the lungs and inhibit eosinophilia compared to rats treated with Dex-P alone which had no effect (Fig. 7 A, B). Administration of Dex-P alone or Dex-P loaded microspheres did not inhibit neutrophil influx to the lungs (Fig. 7C).
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Fig. 5. Fluoresencent image of a representative area of the lung showing a main bronchus at 6 h after intra-tracheal instillation of microspheres (A) and the number of microspheres counted in the bronchus at time points of 3, 6, 24, 48 and 72 h after instillation (B). Representative image of the lung taken 24 h after instillation of the microspheres (C) and the number of microspheres present in the alveolar compartment of the lung at 3, 6, 24, 48 and 72 h after instillation (D). A representative image of an airway showing a strong fluorescent area 24 h after instillation (E) that stained positive for Periodic Acid Shciff (F). The black arrows in F point at microspheres that were present in the airway.
4. Discussion 4.1. Control release kinetics At a molecular level, Dex-P differs from Dex by the presence of an additional phosphate group that increases its water solubility.
Phosphate groups are well known to interact with calcium containing compounds; therefore, Dex-P is probably more likely to interact with the calcium pyrophosphate microspheres. This factor proved to be crucial in controlling the release profile of the drug. Accordingly, in vitro assessment of the controlled release kinetics of the corticosteroid loaded microspheres demonstrated
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Table 1 Quantification of phagocytosis of nanofibrous calcium phosphate microspheres by a macrophage cell line in culture. Days in culture
Percent of microspheres undergoing phagocytosis by the macrophage cell line J774A.1 SEM
Day Day Day Day
0.2 0.04% 0.7 0.04% 1.3 0.13% 2.0 0.17%
1 2 3 4
Approximately 100,000 J774A.1 (a murine macrophage cell line) were co-cultured with 10,000 calcium pyrophosphate nanofibrous microspheres. The number of microspheres undergoing phagocytosis was counted from duplicate wells. The data is expressed as percentage of microspheres phagocytosed from 3 separate experiments.
that Dex-P loaded microspheres exhibited controlled drug release over a longer period of time than Dex-loaded microspheres. The analysis of the release profile for both drugs revealed that while Dex was released by simple dissolution, Dex-P release was likely to be controlled diffusion. However, the value of the exponent n obtained from Peppas equation was low (n ¼ 0.14) indicating that the drug diffusion was probably interfered with by adsorption of the drug to the calcium pyrophosphate matrix [23]. This was also apparent from the slow and constant release beyond 12 h.
4.2. Safety profile in vivo An essential concern in the development of controlled release inhalation systems is the toxicity profiles of the release modifying agents. The inflammatory potential and accumulation effects of novel agents are important concerns that are not always evaluated [1]. In the present study we demonstrated that doses of 1 mg of calcium pyrophosphate nanofiber microspheres had no significant effect on the influx of inflammatory cells to the lungs. At doses of 3 and 10 mg neutrophil and eosinophil counts in BALF were increased, however increases in macrophage activation (TNF-a) in BALF were not observed at any of the doses of microspheres tested. These results show that despite an increase of neutrophils, the microspheres are well tolerated at doses of up to 10 mg. Furthermore, it is important to note that the number of neutrophils increased from about 0.1 million cells in the vehicle to up to 0.3 million cells following 10 mg of microspheres. This increase in cells is statistically significant but may not be biologically relevant. Similar increases in BALF neutrophils have been demonstrated to be evoked by instillation of saline into the airways [24]. Furthermore, this level of neutrophilia was very mild compared to the level in the range of 2e3 million observed in this study following compound 48/80 instillation. The increase in goblet cell counts at 48 h may be related to the mechanism by which the microspheres are cleared by the lung, as indicated in our studies with fluorescentlylabelled microspheres (below). Furthermore, the increase in goblet cell counts appears to be transient returning to baseline levels at 72 h. Poly(lactic-co-glycolic) acid (PLGA), poly-lactic acid (PLA) and polyvinyl alcohol (PVA) are among the matrices explored for inhalation controlled release that have shown the greatest potential. However, PLGA and PLA can result in a significant reduction in cell viability in vitro [25], neutrophilia at sites of microparticle deposition [26] and a high probability of pulmonary accumulation [27]. While in vitro cell assays for PVA have shown that it appears to better tolerated than PLGA or PLA [1]. However no in vivo pulmonary toxicity studies have been reported.
4.3. Microsphere distribution and clearance In many studies, the distribution of particles for inhalation controlled release is assessed by non-invasive imaging techniques [28,29]. Although useful to image the entire thoracic cavity, these imaging techniques are not able to discriminate between bronchial and alveolar particle deposition that would is otherwise possible by conventional histology. In the present study, histological analysis following a single administration of microspheres revealed that microspheres eventually reached the alveolar compartments of the lungs. At 3e6 h post treatment a larger number of microspheres was found to be in the large conducting airways. At 6 h, an increase in microspheres was apparent in the alveolar compartments and microspheres present in large conducting airways tended to aggregate in groups of >10, possibly due to the adhesive effect of increased amounts of mucus. This observation correlates with the increased number of goblet cells observed at 48 h after i.t. microsphere instillation. Together, these results suggest that mucus clearance may have played an important role in the later removal of calcium pyrophosphate microspheres from the lung. Even though the microspheres are composed of nanofibre bundles with very high specific area, their size is large enough to allow them to be removed by mucus clearance, which would prevent alveolar retention if inhaled in large quantities [30,31]. On the other hand, our in vitro studies revealed that, only 2% of the microspheres were phagocytosed by macrophages after 4 days of exposure in cell culture. These results highlight the bioinert properties of the microspheres and correlate with our in vivo data showing no increase in TNF-a levels following i.t. administration of microspheres at doses up to 10 mg. The combination of these observations suggests that the primary mechanism for removal of microspheres from the lung is via mucus clearance and that phagocytosis by macrophages plays a discreet role. 4.4. Therapeutic and in vivo controlled release profile Compound 48/80 is a basic mast cell secretagogue that causes mast cell degranulation [15] via direct activation of Gi proteins leading to the release of histamine, 5-hydroxytryptamine and arachidonic acid metabolites implicated in the de novo synthesis of leukotrienes and prostaglandins [32,33]. It has been used extensively in vivo to induce inflammation in the lungs [34,17], rat paw edema [18] and inflammation of the skin [19]. Calcium pyrophosphate microspheres were loaded with dexamethasone, a corticosteroid commonly used in models of airways inflammation [35e37]. Dex-P, a more water soluble form of Dex was chosen for our in vivo studies, based on our in vitro controlled release kinetics study. In these studies, administration of microspheres (1 mg) on their own 32 h prior to compound 48/80-induced airways inflammation did not attenuate the influx of inflammatory cells to the lungs. However, Dex-P loaded microspheres was the only formulation capable of reducing total inflammatory cell influx including neutrophilia and eosinophilia, 32 h prior to the assessment of airways inflammation. Inhibition of eosinophils by Dex-P alone or Dex-P loaded microspheres was comparable; however, Dex-P loaded microspheres were significantly better at attenuating neutrophilia than administration of Dex-P alone. Pre-treatment with microspheres loaded with standard dexamethasone, devoid of phosphate groups, was not able to significantly inhibit total inflammatory cell infiltration, neutrophilia or eosinophilia induced by compound 48/80. Furthermore, Dex-P loaded microspheres were significantly better at reducing
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neutrophilia compared to Dex-loaded microspheres. These observations highlight the critical role of the phosphate groups in proving controlled release of dexamethasone from the microspheres. In experiments where animals were pre-treated with Dex-P alone or Dex-P loaded microspheres 42 h prior to the assessment of compound 48/80-induced lung inflammation, inhibition of eosinophilia in BALF, a hallmark of asthma, was significantly larger for Dex-P loaded microspheres compared to Dex-P alone or vehicle-treated animals. These observations demonstrate the controlled release properties of the microspheres. Thus it is likely that a small concentration of corticosteroid, released by the microspheres, is able to inhibit eosinophilia observed at 42 h when compared to rats treated with Dex-P only. The comparable inhibition of both Dex-P alone and Dex-P loaded microspheres on eosinophil influx at 32 h, can be explained by the increased sensitivity of eosinophils to corticosteroids [38,39] where drug levels are high enough in the Dex-P alone and Dex-P loaded groups to induce a comparable response. Similarly, the relatively greater resistance of neutrophils to steroids [40,41] would explain why neutrophilia is inhibited at 32 h with both drug delivery methods but is more prominent in Dex-P loaded microsphere groups compared to Dex-P alone groups and at 42 h neither Dex-P alone nor Dex-P loaded microspheres are inhibitory. Although some inhalation controlled release systems have been developed and tested in vitro, however very few of them have been tested in vivo for their therapeutic efficacy, (for a recent review see [1]). Furthermore, proof of controlled release properties in vitro does not necessarily equate with success in vivo, as has been reported with salbutamol acetonide loaded solid lipid nanoparticles of glyceryl behenate [42]. Controlled release inhalation systems that have been tested in vivo include PLGA or PLA that have shown increased levels of insulin or testosterone lasting up to 96 h for insulin or 24 h for testosterone [1]. However, as discussed previously the toxicological effects of these agents are of important concern. Similar studies by Yamada et al. [43], have demonstrated that polysaccharide gels containing iota or kappa carrageenan are capable of enhancing absorption of fluticasone and theophylline systemically up to 6 h following i.t. delivery. These studies have shown that iota and kappa carrageenan gels do not appear to induce lung inflammation in BALF 5 and 24 h after delivery [43]. However, no therapeutic improvement on airways inflammation or physiology following an inflammatory insult was demonstrated by this formulation. Thus, in comparison with recent developments in inhalation controlled release systems, calcium pyrophosphate nanofibrous microspheres do not show signs of toxicity or pulmonary accumulation, demonstrate controlled release properties in vitro and in vivo, and possess therapeutic efficacy 42 h after a single dose. The clinical implications of this work suggest that use of these microspheres as novel matrix for pulmonary drug delivery has the capacity to significantly improve drug efficacy and compliance in patients being treated with corticosteroids for asthma or COPD as a result of its long lasting controlled release properties.
Fig. 6. Total inflammatory counts (A), neutrophil (B), eosinophil (C) observed from bronchoalveolar lavage fluid (BALF) analyses of rats sacrificed 32 h after treatment with vehicle for the microspheres (0.2 ml intra-tracheallye i.t. of a 2% bovine serum albumin eBSA), 1 mg of microspheres (i.t.), dexamethasone-loaded micropheres (DexMS 30% w/w i.t.), dexamethasone phosphate alone (1 mg/kg i.t.) or 1 mg of dexamethasone phosphate-loaded microspheres (30% w/w i.t.). Compound 48/80 (1 mg/kg i.t.) or saline (vehicle for compound 48/80, 0.2 ml i.t.), was administered 8 h after microsphere or vehicle control. (N ¼ 6 for all groups). BALF was performed 32 h after the microsphere or vehicle instillation.
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5. Conclusion In the current study we explored the potential of calcium pyrophosphate microspheres as an alternative matrix for the administration of therapeutic agents for pulmonary delivery and demonstrated the controlled release behaviour of Dex-P loaded microspheres in vitro. The safety profile of calcium pyrophosphate nanofibres microspheres in vivo showed that the microspheres appeared to be well tolerated and did not accumulate in the lung. Finally we demonstrated that microspheres loaded with Dex-P also exhibited controlled release properties in vivo and were capable of inhibiting compound 48/80-induced airway inflammation at 32 and 42 h after microsphere instillation. These new inorganic microspheres likely have many applications in sustained release applications. Appendix Figures with essential color discrimination. Figs. 3 and 5 of this article have parts that are difficult to interpret in black and white. The full color images can be found in the on-line version, at doi:10. 1016/j.biomaterials.2010.04.025. References
Fig. 7. Total inflammatory counts (A), eosinophil (B) and neutrophil (C) observed from bronchoalveolar lavage fluid (BALF) analyses of rats sacrificed 42 h after treatment with vehicle for the microspheres (0.2 ml intra-tracheallye i.t. of a 2% bovine serum albumin eBSA, n ¼ 6), dexamethasone phosphate alone (1 mg/kg i.t., n ¼ 10) or 1 mg of dexamethasone-loaded microspheres (30% w/w i.t., n ¼ 17). Compound 48/80 (1 mg/i. t.) was administered 18 h after microsphere or vehicle treatment.
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