International Journal of Pharmaceutics 465 (2014) 464–478

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Preparation and characterization of spray-dried powders intended for pulmonary delivery of Insulin with regard to the selection of excipients Seyed Salman Razavi Rohani a , Khalil Abnous b , Mohsen Tafaghodi a,c, * a

Student Research Committee, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Pharmaceutical Research Center, Department of Medicinal Chemistry, Mashhad University of Medical Sciences, Mashhad, Iran c Nanotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 18 November 2013 Received in revised form 12 February 2014 Accepted 15 February 2014 Available online 21 February 2014

The aim of this study was to produce microparticles with optimal aerodynamic diameter for deep lung delivery (i.e., 1–3 mm) of a protein drug intended for systemic absorption, using a combination of generally regarded as safe (GRAS) excipients. Based on the preliminary experiments, mannitol, L-alanine, sodium alginate, chitosan and dipalmitoylphosphatidilcholine (DPPC) were chosen as excipients and human insulin as a model protein drug. Dry powders were prepared by spray-drying. Powders with varying yields (29–80%) and low tapped densities (0.22–0.38 g/cm3) were obtained. Scanning electron microscopy (SEM) revealed distinctive particle morphologies among formulations from isolated spherical to highly folded particles. Aerodynamic properties were assessed by next generation impactor (NGI). Mass median aerodynamic diameter (MMAD) and fine particle fraction (FPF) ranged from 2.1 to 4.6 mm and 46 to 81%, respectively. A comparative study of protein release from microparticles was conducted in vitro using an open membrane system with more than 50% cumulative release in all formulations which followed different kinetic models. Insulin’s integrity was investigated by spectrofluorimetry and electrophoresis, and no tangible changes were observed in the structure of insulin. Of the formulations studied, the third, containing mannitol/sodium alginate/insulin/sodium citrate showed promising characteristics, optimal for systemic delivery of proteins via deep lung deposition. ã 2014 Elsevier B.V. All rights reserved.

PubChem Classifications: Recombinant human insulin (PubChem CID: 70678557) Mannitol (PubChem CID: 6251) Sodium citrate (PubChem CID: 6224) L-alanine (PubChem CID: 5950) Sodium alginate (PubChem CID: 6850754) Chitosan (PubChem CID: 21896651) DPPC (PubChem CID: 452110) Keywords: Spray-drying Dry powder inhaler Insulin Mannitol Sodium alginate Chitosan

1. Introduction Systemic delivery of therapeutic agents via pulmonary system, have been subject of rigorous researches in recent years (Klingler et al., 2009; Liu et al., 2008; Surendrakumar et al., 2003). A vast surface area (80–100 m2/adult) with high permeability awaits any drug which could reach respiratory zone of the lungs (Daniher and Zhu, 2008). In addition, low enzymatic activity and occurrence of transcytosis in the alveolar region allows efficient delivery of peptides and proteins into bloodstream (Newhouse, 2007). Particles must have an aerodynamic diameter in range of

* Corresponding author at: Nanotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran. Tel.: +98 511 8823255; fax: +98 511 8823251. E-mail address: [email protected] (M. Tafaghodi). http://dx.doi.org/10.1016/j.ijpharm.2014.02.030 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.

1–5 mm in order to be inhaled and efficiently deposited in the lungs, but only those with a diameter less than 3 mm, would have the chance of reaching the respiratory zone (Chow et al., 2007; Shoyele, 2008; Surendrakumar et al., 2003). Among different pulmonary drug delivery systems, dry powder platforms are most suitable for production of stable and readily dispersible particles within this narrow size distribution and can incorporate enough amount of an active ingredient to achieve desirable pharmacological effect (Timsina et al., 1994). Main advantages of dry powder inhalers (DPIs) over other pulmonary delivery systems (e.g., pMDIs and Nebulizers) are: (i) potential for delivering high mass of drug, (ii) physiochemical stability of materials in a dry state, (iii) greater possibility of particle engineering and (iv) suitability for delivery of delicate macromolecules such as peptides and proteins (Bosquillon et al., 2001; Maltesen and van de Weert, 2008; Tafaghodi and Rastegar, 2010).

S. Razavi Rohani et al. / International Journal of Pharmaceutics 465 (2014) 464–478

Spray-drying is a one-step process of converting an atomized feed solution into dried particles using hot air or nitrogen (Pilcer and Amighi, 2010). In this method of drying, beside formulation parameters (e.g., composition and nature of the ingredients, solid content, viscosity and type of solvent), properties of dried particles could be effectively controlled by process parameters including but not limited to the input temperature, feed flow rate, atomizing gas flow rate, aspirator capacity (i.e., flow rate of hot drying medium), nozzle diameter and cyclone performance (Baldinger et al., 2012; Chow et al., 2007; Patil et al., 2014). During this process, active ingredients would be subjected to the mechanical shear stress of atomization, exposure to the air-water interface and heat stress (Chan et al., 1997); the actual temperature which drug molecules experience, is significantly lower, close to the output temperature as a result of rapid drying and evaporation cooling, but these stresses could result in degradation of a sensitive drug such as a protein (Maltesen and van de Weert, 2008). Optimization of process parameters and addition of excipients are common methods of protecting proteins against denaturation during spray-drying (Baldinger et al., 2012; Schule et al., 2008; Silva et al., 2006). Excipients are essential in order to attain protein stability and desirable aerosolization properties (Andya et al., 1999; Minne et al., 2008; Seville et al., 2007b; Tewes et al., 2010). In this project mannitol was used as the primary excipient and the other excipients including L-alanine, sodium alginate, chitosan, and DPPC were utilized as modifying excipients to study their potential in minimizing crystallization of mannitol and improving aerosolization and formulation stability while human insulin was used as a model protein at a relatively low proportion (6%) (Bosquillon et al., 2004; Costantino et al., 1998; Learoyd et al., 2008; Sarmento et al., 2006; Silva et al., 2006). Safety of sodium alginate and chitosan in pulmonary delivery has previously been evaluated by Sivadas and co-workers (Sivadas et al., 2008). Phosphate buffered formulations have also shown to be safe and effective for preservation of lungs prior to transplantation in human subjects (Okada et al., 2012). Formulation and processing parameters were experimentally optimized during preliminary studies and five final formulations were spray-dried to prepare inhalable dry powders containing insulin. A comparative study of physiochemical properties of powders was performed including moisture content, tapped density, particle morphology and solid state analysis. Protein release was studied in vitro using an open membrane system (Learoyd et al., 2008; Salama et al., 2008; Sarmento et al., 2006; Silva et al., 2006; Sivadas et al., 2008). Aerodynamic properties of the powders including MMAD, size distribution and fine particle fraction were assessed using a next generation impactor (NGI) (Mitchell et al., 2003; Mohammed et al., 2012; Sarmento et al., 2006; Silva et al., 2006; Taki et al., 2010). Stability of insulin in the spray-dried powders was investigated by fluorescence spectroscopy and electrophoresis (Bekard and Dunstan, 2009; Murali and Jayakumar, 2005; Sarmento et al., 2006; Silva et al., 2006; Taki et al., 2010). In the present research the primary aim was to achieve a formulation with optimized characteristics for deep lnug delivery

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of a protein but practicality of the project was a major concern. The emphasize was on achieving high processing yield and using conventional and low priced excipients, so that the final formulation, in case of promising results, would have a chance of being put to the practical use in larger scales. The other main focus of this research was to do a characterization as completely as possible to minimize the possibility of unanswered questions and concerns. In addition, formulations were designed with a relatively low percentage of the active ingredient so the impact of the active ingredient on characteristics of the final powder (e.g., MMAD, FPF, size distribution, moisture content) would be minimal; as a result, proportion and combination of excipients in the most promising formulation, could be used to incorporate other potent drugs with only minor adjustments. This could in return speed up the preparation and formulation of other drugs to be evaluated for systemic delivery via pulmonary route. 2. Materials and methods 2.1. Materials Recombinant human insulin was obtained from Novo Nordisk A/S (Bagsvaerd, Denmark). Dipalmitoylphosphatidylcholine (DPPC) was purchased from Lipoid (Lipoid GMBH, Ludwigshafen, Germany). Sodium alginate from brown algae and 8-anilino-1-naphtalene sulfonic acid (ANS) was obtained from Sigma–Aldrich (St. Louis, MO, USA). Chitosan (Low-Viscous) was bought from Fluka (Japan). Dmannitol, L-alanine and sodium citrate were provided by Merck (Germany). All the other reagents used, were of analytical grade. 2.2. Formulation and spray-drying Based on the preliminary experiments, 5 formulations (F1–F5) were experimentally optimized for this study. Recombinant human insulin as the active ingredient at ratio of 6% w/w and sodium citrate at 4% w/w of solid content were kept constant through all the formulations and the basic formulation (F1) was designed by adding D-mannitol as the remaining 90%. A portion of mannitol was then replaced with other excipients to prepare the rest of formulations (Table 1). F1 and F2 were prepared in two steps: First, all excipients were dissolved in 100 ml of 0.05 M phosphate buffer (pH 7.4) and then insulin powder was added to the mixture and dissolved under gentle stirring until a transparent solution was obtained. F3 solution was prepared in three steps: First, mannitol and sodium citrate was dissolved in 100 ml 0.05 M phosphate buffer and then sodium alginate powder was added gradually under high speed stirring until a transparent solution was obtained. Finally insulin was dissolved under aforementioned conditions. F4 was prepared in two separate solutions; one 100 ml solution containing insulin, mannitol and sodium citrate which was prepared in the same way as in F1 and F2 solutions. The other solution was made by adding 0.4 ml of glacial acetic acid to 100 ml deionized water and then chitosan powder was gradually added. After complete dissolution,

Table 1 Composition of formulations (wt.: weight). Formulation Ingredients

F1 wt.%

F2 wt.%

F3 wt.%

F4 wt.%

F5 wt.%

Human insulin D-mannitol L-alanine Sodium alginate Chitosan (low-viscous) DPPC Sodium citrate

6 90 – – – – 4

6 50 40 – – – 4

6 60 – 30 – – 4

6 60 – – 30 – 4

6 15 55 – – 20 4

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a yellow, transparent, viscous solution was obtained. Finally, chitosan solution was added to the insulin solution drop by drop. The F5 solution was also prepared in two separate solutions. Mannitol, L-alanine and sodium citrate were dissolved in 60 ml of 0.033 M phosphate buffer to maintain a constant ratio of buffer salts and then insulin was added to the mixture. DPPC was dissolved in 140 ml of 99.8% ethanol then insulin solution was gradually added to the ethanolic solution. Powders were produced using a Büchi B290 mini spray dryer equipped with a high performance cyclone (Büchi Labortechnik AG, Flawil, Switzerland) and a standard 0.7 mm diameter nozzle tip. Processing parameters are presented in Table 2. The mini spray dryer was used in an open cycle, suction mode with air as atomizing and drying gas, except in the production of F5 which instead, was operated in a closed cycle, blowing mode with Inert Loop B-295 as a mediator and pure nitrogen as atomizing and drying medium. Each formulation was prepared in triplicate. Dried particles were filled in tightly closed containers and stored in2–8
C except for F5 which was stored in 20
C to meet the DPPC’s storage requirements. All Spray  drying yield ¼

7. Fine tuning of parameters and repeating the whole process to

achieve desired characteristics and the highest attainable yield. 2.3. Quantification method Intrinsic fluorescence of human insulin due to its four tyrosine and three phenylalanine residues was used as a highly sensitive and selective quantification method (Lakowicz, 2006). A 240 mg/ ml insulin solution in 0.05 M phosphate buffered saline sample was excited at 280 nm using Shimadzu RF-540 spectrofluorometer (Shimadzu, Japan); corresponding emission spectra was then collected in the range of 250–350 nm which had a maximum fluorescence intensity at 305 nm. 0.05 M phosphate buffered saline solution was used as blank sample. 2.4. Spray drying yield, tapped density and moisture content Since buffer salts would also dry during spray-drying, process yield should be calculated by the following equation:

Weight of collected powder  100% Total mass of all ingredients of each formulation þ anhydrate mass of buffer salts

the spray-dried powders were stored for 6 months prior to characterization so that the stability of these formulations could be accounted for. Blank powder of each formulation was prepared by physical mixture of all the ingredients including buffer salts and excluding insulin which were named B1–B5.

(2)

Powders were precisely weighed and filled in a measuring cylinder before tapping for 1000 times; the tapped density was then calculated by the Eq. (3) (Minne et al., 2008; Mollmann et al., 2006; Pilcer et al., 2009; Sarmento et al., 2006; Silva et al., 2006). Measurements were performed in triplicate. Tapped density

2.2.1. Optimization of formulation and processing parameters The principle of spray-drying provides a general understanding of how every processing and formulation parameter would affect the characteristics of the resultant powder; in addition to this, guidelines provided by conditions used in previous researches (Baldinger et al., 2012; Corrigan et al., 2006; Mollmann et al., 2006; Sarmento et al., 2006; Silva et al., 2006) were used to define a suitable range for the formulation and processing parameters to begin with and from this point on, results of the preliminary experiments was used to optimize these parameters by going through these steps: 1. Calculation of process yield. 2. Manual examination of adhesiveness and flowability of the

output powder. 3. Evaluation and estimation of average particle size by direct visualization under light microscopy. 4. Measurement of tapped density. 5. Estimation of MMAD by the following formula (Learoyd et al., 2008): rffiffiffiffiffiffi r da ¼ dp  (1)

r0

6. In which da = MMAD, dp = MMD (average physical diameter

obtained in the step 3), r = particle’s density (tapped density is an estimation of this value) and r0 = 1 g/cm3.

¼

Weight of the powderðgÞ Final volume of the powder after 1000 taps ðcm3 Þ

(3)

Moisture content of the powders was determined by loss on drying method. 250 mg of powder was precisely weighed and distributed as a thin layer in petri dishes and stored in an oven at 105
C. It was then weighed every 15 min until weight loss reached plateau. Moisture content reported as the percentage of weight loss using the Eq. (4) (Anon., 2007). Moisture content ¼

ðInitial weight  final weightÞ  100% Initial weight

(4)

2.5. Scanning electron microscopy (SEM) Morphological assessment of the spray-dried particles was performed using an Oxford S360 scanning electron microscope (Cambridge, UK). Samples were mounted on aluminum studs using silver glue and spotter coated with a thin layer of gold prior to scanning (Alamilla-Beltrán et al., 2005). 2.6. Differential scanning calorimetry (DSC) DSC was performed on spray-dried powders and physical mixture of each formulation plus a separate sample of each excipient. Every run was carried out under 20 ml/min nitrogen purge at heating rate of 10
C/min from 25
C to 350
C using a

Table 2 Processing parameters used in the spray-drying of F1–F5. Processing parameter

Input temperature (
C) Aspirator capacity (%) Atomizing gas flow (L/h) Peristaltic pump speed (%)

Formulation F1

F2

F3

F4

F5

130 100 473 3

130 100 601 5

130 100 670 10

135 100 601 4

105 100 601 10

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Mettler Toledo DSC 821e (Mettler Toledo, Switzerland). Mettler Toledo STARe software was used for analysis of thermal events. Crystallinity index (XC) of mannitol in spray-dried samples was calculated by Eq. (5). In this equation melting enthalpy of spraydried samples (DHfSD) and the melting enthalpy of physical mixture samples (DHfPM) were measured from area under the curve (AUC) of endothermic melting peak on DSC traces (GombÁs et al., 2002; Kong and Hay, 2003; Sarmento et al., 2006; Silva et al., 2006; de Waard et al., 2008).    DHf SD    100% XC ¼  (5) DHf FM  2.7. Aerodynamic particle size analysis Aerodynamic properties of the spray dried powders were analyzed with a next generation impactor (NGI, Copley Scientific Limited, Nottingham, UK). Since the formulations contained no coarse carrier, the NGI was utilized without pre-separator stage (Wong et al., 2010). The flow rate was adjusted to 60 L/min. Collecting cups of the NGI were coated using hydroxypropylmethylcellulose gel (20% w/v) before each test in order to minimize the particle bounce. A 50 mg sample powder of each formulation was drawn into the impactor using a specially modified spinhaler inhaler device. This modified inhaler acted simply as a mouthpiece adapter and powder de-agglomerator. Powders were weighed and strewed on a paraffin paper then placed near the entrance of the modified inhaler and then after starting the vacuum pump of the impactor, powders were sucked into the device through the inhaler which dispersed loose aggregates. Deposits from the device, throat and 7 stages of the impactor and micro orifice collector (MOC) were collected and analyzed for their insulin content. In order to recover these deposits for quantification, 10 ml of deionized water was used for each stage; the amount of input powder had to be increased so that weight of deposits even for those stages with minimal deposition could be accurately quantified. Preliminary tests showed that a 50 mg powder sample was needed for accurate measurement in all stages. Obtained data were inputted to the Copley inhaler testing data analysis software (CITDAS v3.0 Wibu) to calculate aerodynamic properties of the particles. Output data obtained from CITDAS were: Mass median aerodynamic diameter (MMAD), geometric standard deviation (GSD), fine particle fraction (FPF) which defined as fraction of particles with MMAD of less than 5 mm, recovered dose (RD) and emitted dose (ED) (Kong and Hay, 2003; Sarmento et al., 2006; Silva et al., 2006; Taki et al., 2010; Wong et al., 2010). Analysis was done in triplicate for each formulation. 2.8 In vitro protein release A Franz diffusion cell fitted with a nitrocellulose membrane filter, grade BA83 (Schleicher & Schuell BioScience GmbH, Dassel, Germany) with 0.2 mm pore size was used to evaluate in vitro protein release. The 25 ml receptor compartment was filled with 0.05 M phosphate buffered saline solution which magnetically stirred to maintain sink conditions at 37
C (Kong and Hay, 2003; Salama et al., 2008; Sarmento et al., 2006; Silva et al., 2006; Sivadas et al., 2008). Samples from each formulation (20 mg) along with equivalent amount of blank powder were distributed evenly on the membrane. At selected times, up to 6 h (0, 15, 30, 45, 60, 90,120, 150, 180, 210, 240, 270, 300, 330 and 360 min), 200 ml samples were taken from the receptor compartment and then replaced with isothermal buffer solution to keep the volume constant. The quantity of released insulin was determined using spectrofluorimetric method as described in Section 2.3. Measurements were performed in triplicate.

467

Methods used to compare release profiles were: 1. Statistical methods based on the analysis of variance; these

methods determine the difference between the means of two drug release data sets in single time point dissolution using ANOVA or t-student test (Costa and Sousa Lobo, 2001) including: a. T50% which is the time when 50% of active ingredient releases. b. Maximum cumulative drug release. 2. Model-independent methods : a. Mean dissolution time (MDT); this parameter is a measure of the rate of the dissolution process; a higher MDT indicates a slower release rate; this parameter is used to test the equivalence of two dissolution profiles or to compare different profiles statistically (Podczeck, 1993); Eq. (6) was used to calculate MDT. In this formula j is the sample number, n is the number of samples, t^j is the time at midpoint between tj and tj1 and DMi is the additional released drug between tj and tj1. Pn j¼1 t^j DMj MDT ¼ Pn (6) j¼1 DMj b. Difference factor (f1) and similarity factor (f2); f1 values

lower than 15 (0–15) and f2 values greater than 50 (50–100) are indicator of the similarity of the dissolution profiles (Gohel et al., 2009). The similarity factor (f2) was calculated using Eq. (7) and a modification of the difference factor’s formula was used to eliminate the need for defining a reference product in the calculation; this modified difference factor was named [TD$INLE] which was calculated by Eq. (8). In these equations, n is the number of sample, R and T are the percent release of the reference and test products at each time point of j (Costa and Sousa Lobo, 2001; Pillay and Fassihi, 1998).  0:5 1 Xn f 2 ¼ 50  log 1 þ jRj  T j j2  100g (7) j¼1 n

(8) 3. Model-dependent methods; these methods are based on [TD$INLE]

different mathematical formulas, which describe the dissolution profile. Once a suitable model has been identified, a prediction of release mechanism can be made depending on the model and calculated parameters. Mathematical models used for the study of dissolution profiles were kinetic models including Higuchi, zero order, first order and KorsmeyerPeppas which their equations are presented in Table 3 (Dash et al., 2010). In these equations, Q0 is the initial amount of released drug which is usually considered zero. Qt is the amount of Table 3 Mathematical models used to describe release profiles. Model

Equation K0 ¼

Qt  Q0 t

First order

K1 ¼

lnQ t  lnQ 0 t

Higuchi

Q K H ¼ ptffiffi t

Korsmeyer–Peppas

KK ¼

Zero order

mt =m1 tn

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released drug at the time t. In the Korsmeyer–Peppas model, mt/m1is the fractional release of drug at the time t which should only be used up to the 60% drug release (i.e., mt/ m1 < 0.6) (Korsmeyer et al., 1983); for consistency, data up to the 60% release were used in all models. The parameter n is the release exponent which defines the underlying release mechanism. Based on the coefficient of determination (i.e., R2) best fitting model for release profile of each formulation was identified.

2.10. Statistical data analysis IBM-SPSS Statistics v19.0 was used for dissolution model fitting studies. Data were statistically evaluated using one-way analysis of variance (ANOVA) with the Tukey-Kramer as posttest, using GraphPad Instat 3.0. A p-value < 0.05 was considered significant. 3. Results and discussion 3.1. Yield, tapped density and moisture content

2.9. Protein stability assessment 2.9.1. Intrinsic protein fluorescence spectroscopy Intrinsic fluorescence of insulin was used to evaluate structural stability of insulin in the formulations. Test sample solutions with nominal insulin concentration of 240 mg/ml were prepared from each formulation. A native insulin solution (240 mg/ml 0.05 M phosphate buffer) was used as positive control. A denatured insulin sample was prepared using low pH and thermal stress; HCl (1 M) was added to reduce the pH to 2, then the solution was heated at 70
C water bath for 1 h and finally addition of NaOH (0.1 M) increased the pH to 7.4. Since fluorescence intensity is dependent on solvent properties (e.g., polarity and viscosity), another native and denatured insulin solution was prepared specially for the F5 formulation using 70% v/v ethanolic solution as the solvent (Bekard and Dunstan, 2009; Hawe et al., 2008; Murali and Jayakumar, 2005; Sarmento et al., 2006; Silva et al., 2006). 2.9.2. Extrinsic protein fluorescence spectroscopy 8-annilino-1-naphtalene sulfonic acid (ANS) which is a fluorescence dye was used for protein characterization. Test sample solutions with nominal insulin concentration of 240 mg/ ml from each formulation plus native and denatured insulin solutions were used as control samples; for the evaluation of F5, all the samples including test, blank and controls were prepared using 70% v/v ethanolic solution as the solvent. After preparation, each sample was mixed with an equal volume of ANS 480 mg/ml and incubated for 30 min in darkness and then excited at 375 nm. Fluorescence emission spectra were collected in the range of 450–600 nm (Hawe et al., 2008; Sarmento et al., 2006; Silva et al., 2006; Stryer, 1965; Zierler and Rogus, 1978). 2.9.3. Native-polyacrylamide gel electrophoresis (native-PAGE) Structural integrity of insulin was assessed by native-PAGE. Test samples were prepared from each formulation with nominal insulin concentration of 1 mg/ml. Native-PAGE was done on the samples using Bio-Rad mini protean tetra (Bio-Rad, China) equipment and the resulting gel was developed using Coomassie Blue staining method (Anon., 2001). For eliminating the retarding effect of sodium alginate in progress of test and blank samples of F3 on the electrophoresis gel, another native-PAGE was run using 5 times diluted sample of this formulation and its blank along with native and denatured insulin samples as controls; the resulting gel was developed using silver nitrate staining method (Tye, 2005).

Spray-dried powders with suitable characteristics for pulmonary delivery of a protein (i.e., human insulin) in a dry powder platform were successfully prepared in this study. Application of the high performance cyclone as have previously been suggested by others, proved to be essential to achieve high yields when dealing with microparticles in the inhalable size range (Learoyd et al., 2008; Maltesen et al., 2008; Sarmento et al., 2006; Seville et al., 2007a; Silva et al., 2006). Yield of the spray-drying process varied from 29% in case of F1 up to more than 80% for F4 (Table 4). The basic formulation (i.e., F1) had the lowest yield and addition of modifying excipients significantly increased the process yield. In preliminary experiments, formulation and processing parameters were optimized to achieve the highest attainable yield for each formulation. For instance, a yield higher than 31% in case of the basic formulation wasn’t attainable; but when a portion of mannitol was replaced with other excipients, significantly higher yields became achievable; logically this increase must be attributed to the addition of new excipients. Following improvement of aerosolization properties (i.e., higher FPF, lower GSD and an optimal MMAD) the process yield increased significantly as seen in F3 (with more than 45% increase compared to F1). In case of F5, a very low MMAD (2.11 mm) with a relatively high GSD (2.66 mm) means that there was a tail of very small particles in its size distribution curve, which cannot be captured effectively, even with a high performance cyclone and resulted in the second lowest yield (i.e., 54.5%) (Rabbani and Seville, 2005). On the other hand F1, F2 and F4 had similar FPF (53, 52 and 46%, respectively) and even their particle morphologies were generally alike (Fig. 1), an almost linear correlation between MMAD and yields was seen (i.e., the higher MMAD, the higher the yield will be), this direct correlation can be easily explained based on the principle of particle recovery by the spray-dryer; separation of dried particles in a Büchi mini spray-dryer (which was used in this research) is done by cyclone separator. The efficiency with which the cyclone collects particles of a certain size is described by the grade efficiency, G(x); grade efficiency improves as the particle size increases so, larger particles will be separated and consequently collected more efficiently resulting higher yield (Rhodes, 2008). The tapped density of the spray-dried powders was in range of 0.22–0.38 g/cm3 (Table 4). It has been suggested that tapped density results can be used to predict aerosolization properties, and a low tapped density could be an indicator of a good aerodynamic behavior (You et al., 2007), but in this study F3 and F5 with the highest (0.382 g/cm3) and the lowest (0.218 g/cm3) tapped densities, respectively, were the best performing formulations in

Table 4 Dependent process parameters of the spray-dried powders (mean  SD, n = 3). Parameter formulation

Tout (
C)

Yield (%)

Tapped density (g/cm3)

Moisture content (%)

F1 F2 F3 F4 F5

74  1 61  1 63  1 65  1 60  3

29.4  0.7 55.2  1.4 75.4  2.2 80.5  1.6 54.5  1.4

0.314  0.005 0.261  0.002 0.382  0.007 0.303  0.006 0.218  0.002

2.9 4.6 6.0 11 5.6

[(Fig._1)TD$IG]

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469

Fig. 1. Scanning electron micrographs of F1 (A); F2 (B); F3 (C and D); F4 (E) and F5 (F). F1: mannitol/insulin/sodium citrate. F2: mannitol/L-alanine/insulin/sodium citrate. F3: mannitol/sodium alginate/insulin/sodium citrate. F4: mannitol/ chitosan/insulin/sodium citrate. F5: mannitol/L-alanine/DPPC/insulin/sodium citrate.

terms of aerosolization properties which suggests that other factors such as the nature of formulation ingredients, moisture content, particle size and morphology can play a significant role. For instance, it has been reported that incorporation of DPPC could result in powders with significantly lower densities as it was seen in F5 (Bosquillon et al., 2004; Minne et al., 2008; Sarmento et al., 2006; Seville et al., 2007a; Silva et al., 2006). The relatively high tapped density of F3 can be explained by the fact that spherical shape of particles in a variety of sizes allows better compaction and lowers the void volume following tapping (Bosquillon et al., 2001). This data suggest that tapped density measurements are not a reliable parameter to predict aerodynamic behavior of the studied powders. Measurement of loss on drying showed that moisture content of these powders were between 2.9% (in F1) and 11% (in F4) with a majority of the powders having a moisture content in range of 4–6% (Table 4). Previous reports showed that higher Tout results in a lower moisture content which was seen in case of F1 (Table 4)

when compared with the rest of formulations (Maa et al., 1998; Maltesen et al., 2008; Sarmento et al., 2006; Silva et al., 2006; Ståhl et al., 2002). But among these formulations despite experiencing a similar output temperature, F4 had a moisture content of almost twice the others, which could be due to formation of a gel matrix by chitosan and subsequent entrapment of water molecules in this matrix (Kristl et al., 1993). 3.2. Particle morphology Spray-dried particles were visualized by SEM and found to have two distinct morphologies; (1) spherical particles with a pitted surface in case of F3 and F5 with geometric diameter about 5 mm (Fig. 1C, D and F) and (2) crumpled and highly folded particles in case of F1, F2 and F4 (Fig. 1A, B and E). There was no visible crystalline particle in any of the samples. In the process of spray-drying, feed solution converts to a mist of tiny droplets which came in contact with hot drying air; solvent

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evaporates, almost instantaneously and a crust forms on the surface of these droplets which entraps the remaining solution and evaporation of this liquid core, leads to pressure buildup inside the sphere that needs a way out (Alamilla-Beltrán et al., 2005). When a formulation (e.g., F1 or F2) mostly comprised of low molecular weight ingredients, that crust will be unstable and fragile which would easily crushed by the escaping solvent vapor and result in deformed and crumpled particles (Fig. 1A and B). On the other hand in a viscous formulation like F4, droplets will be larger and consequently higher pressure buildup will occur, in this situation, formation of an impervious crust by the gel forming ingredient (i.e., chitosan) leads to collapse of the particle (Fig. 1E). In case of F3 and F5, this crust is stable and flexible enough (possibly due to high molecular weight ingredients such as alginate and DPPC) to let this vapor exit the sphere by creating a hole on its surface, while stability of the crust maintains the spherical shape of dried particles (Fig. 1C, D and F) (Bosquillon et al., 2001; Sarmento et al., 2006; Seville et al., 2007a; Silva et al., 2006; Ståhl et al., 2002). 3.3. Differential scanning calorimetry (DSC) Interaction of water and a protein in a molecular level stabilizes the native 3D structure of the protein (Levy and Onuchic, 2006). It has been proposed that when a protein drug formulated in a dry powder platform, addition of excipients such as mannitol could substitute water molecules and stabilize the protein but they must interact in a molecular level which can be achieved by spray-drying of a solution containing the protein drug and the excipients (Forbes et al., 1998; Sarmento et al., 2006; Schule et al., 2008; Silva et al., 2006; Ståhl et al., 2002). Spray-drying technique usually results in formation of mainly amorphous materials which is a favorable characteristics concerning the dissolution and subsequent absorption of the active ingredient but from the thermodynamic point of view, it is an unstable state and tends to convert to the stable crystalline form (Ambike et al., 2004). Crystallization of excipients in a molecular level has a detrimental effect on the stability of the protein due to loss of the interaction between excipients and the protein as a result of phase separation; further crystallization could deteriorate aerosol performance. Previous reports indicate that mannitol which was used as a primary excipient in this study, has a great tendency towards crystallization (Andya et al., 1999; Costantino et al., 1998; Maas et al., 2011; Sarmento et al., 2006; Silva et al., 2006). In this study amorphous or crystalline state of mannitol in the spray-dried powders was investigated by DSC in terms of crystallinity index (XC) as calculated by Eq. (2) which was described earlier in Section 2.6. Crystallinity Index ranged from near zero in case of F3 and F5 up to 77.8% in case of F1. Table 5 summarizes the melting temperature, melting enthalpy and

crystallinity index of mannitol in the studied formulations. The DSC thermograms of these formulations with marked thermal events along with DSC traces of each excipient are presented in Fig. 2. Mannitol as primary excipient in all of these formulations presented a distinctive endothermic peak of melting in the region of 150–160
C (Fig. 2A) which is in line with the literature (Armstrong, 2009). DSC traces of each excipient used in this study are presented separately on Fig. 2A and B. As it can be seen, none of these excipients have a noticeable thermodynamic event (either endothermic or exothermic) in the range of 150–170
C except for mannitol; so without interference of other excipient’s, melting enthalpy of mannitol could be traced back in all the formulations. Melting enthalpy increases as crystallinity of the material increases; it can be directly measured by calculating the area under the curve (AUC) of melting peak on the DSC thermograms (Youshia et al., 2012). A distinctive endothermic peak between 150–170
C can be seen in all DSC traces of physical mixture samples (Fig. 2C–G); comparing the shape and position of this peak with DSC trace of pure mannitol (Fig.2A) confirms that this endothermic event belongs to the melting enthalpy of mannitol. The proportion of mannitol in each formulation directly correlates with the DH values obtained from the calculation of the AUC of these peaks (Table 5). Onset, peak and Endset of melting temperature of mannitol in spray-dried powders along with their physical mixture counterparts are presented in Table 5 which facilitates identification of the melting peak on the DSC thermograms in Fig. 2C–G. As it can be seen on the DSC thermograms, melting enthalpy of mannitol in the samples significantly declined following spray-drying. Considering the 6 months storage prior to analysis, a good estimation of long term stability of the prepared powders could be obtained. The basic formulation (i.e., F1) containing 90% mannitol had a crystallinity index of more than 75% which dropped below 25% in F2 and F4 and even decreased to almost zero in F3 and F5.It has been suggested that the protein itself could inhibit crystallization of mannitol to some extent (Costantino et al., 1998); the addition of modifying excipients in this study, significantly limited the crystallization of mannitol either indirectly, by lowering the relative proportion of mannitol in the formulation or directly, by inhibiting the crystallization of mannitol due to molecular interactions with mannitol (Alfadhel et al., 2011). Since the relative proportion of mannitol in F3 and F4 was the same (i.e., 60%), the additional inhibition of crystallization in F3 must be attributed to a direct inhibitory effect of alginate on the crystallization of mannitol (Takeuchi et al., 2000). The crystallinity measured by DSC most probably occurred only in the molecular level, since there was no visible crystalline particle in SEM micrographs.

Table 5 Melting temperature, melting enthalpy and crystallinity index of mannitol in the spray-dried powders obtained from DSC thermograms. a: physical mixture, b: spray-dried; F1: mannitol/insulin/sodium citrate. F2: mannitol/L-alanine/insulin/sodium citrate. F3: mannitol/sodium alginate/insulin/sodium citrate. F4: mannitol/ chitosan/insulin/ sodium citrate. F5: mannitol/L-alanine/DPPC/insulin/sodium citrate. Sample

F1-PMa F1-SDb F2-PM F2-SD F3-PM F3-SD F4-PM F4-SD F5-PM F5-SD

Parameter Melting temperature (
C) Onset

Peak

Endset

141.84 145.61 140.99 138.46 141.76 153.46 142.45 143.47 146.67 –

156.15 154.61 153.28 145.05 158.50 157.15 158.35 153.21 154.02 –

161.39 160.67 157.47 149.55 164.23 163.08 162.57 156.78 156.90 –

DH (mJ)

XC (%)

-1079.76 -840.31 -611.68 -150.38 -742.99 -5.10 -733.40 -174.23 -78.09 –

77.8 24.6 0.7 23.8 0

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[(Fig._2)TD$IG]

471

Fig. 2. DSC thermograms of set 1 of excipients (A); set 2 of excipients (B); F1 (C); F2 (D); F3 (E); F4 (F) and F5 (G). F1: mannitol/insulin/sodium citrate. F2: mannitol/L-alanine/ insulin/sodium citrate. F3: mannitol/sodium alginate/insulin/sodium citrate. F4: mannitol/chitosan/insulin/sodium citrate. F5: mannitol/L-alanine/DPPC/insulin/sodium citrate.

3.4. Aerodynamic properties and particle size distribution Aerosolization properties of the spray-dried powders was evaluated in vitro, using next generation impactor (NGI) operated at 60 L/min. Deposition data are presented in Table 6.The recovered dose for all the formulations tested, defined as percentage of the loaded dose that recovered from inhaler and sampling apparatus

(i.e., NGI) was in pharmacopoeial acceptable range (i.e., 75–125%). The delivered dose, defined as percentage of the loaded dose that delivered into the apparatus i.e., excluding the inhaler deposition, ranged between 77.7 and 83.8%. The fine particle fraction (FPF), defined as fraction of delivered dose which has an aerodynamic diameter less than 5 mm, varied from less than 50% in case of F4 to more than 80% in case of F3. Calculated MMAD ranged between 2.1

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Table 6 Deposition data obtained from aerosolization analysis by NGI at 60 L/min (mean  SD, n = 3). F1: mannitol/insulin/sodium citrate. F2: mannitol/L-alanine/insulin/sodium citrate. F3: mannitol/sodium alginate/insulin/sodium citrate. F4: mannitol/ chitosan/insulin/sodium citrate. F5: mannitol/L-alanine/DPPC/insulin/sodium citrate. Formulation

F1 F2 F3 F4 F5

Deposition data Recovered dose (%)

Delivered dose (%)

FPF (%)

MMAD (mm)

GSD (mm)

83.52  3.38 84.78  3.74 87.44  0.94 81.10  3.53 82.61  2.21

81.69  3.24 80.61  3.34 83.75  1.44 77.68  3.21 79.65  2.06

53.62  0.54 52.87  1.52 81.13  0.57 46.27  1.13 77.78  0.38

3.55  0.07 4.00  0.12 2.51  0.02 4.61  0.11 2.11  0.03

3.49  0.15 3.68  0.27 1.77  0.02 1.81  0.06 2.66  0.05

and 4.6 mm with GSD values in range of 1.8–3.7 mm. Particle size distribution of the spray-dried powders expressed as weight of the deposited powder against the deposition site is shown in Fig. 3. An optimized aerodynamic behavior can be described as a powder with good flowability, a MMAD in range of 1–3 mm for systemic delivery or 3–5 mm for local delivery, high FPF and a narrow size distribution (i.e., low GSD values) (Sharma et al., 2013). Achieving a powder with an optimized aerodynamic behavior is one of the most challenging steps in the formulation design in pulmonary drug delivery (Davis, 1999). Aerosolization properties of the spray-dried powders in this study were determined in vitro using the NGI which is reported to be the most optimized and appropriately designed instrument for analysis of aerodynamic behavior of pharmaceutical aerosols (Taki et al., 2010). Obtained results indicate that all the formulations had MMAD values in the inhalable range (i.e., 1–5 mm). Surface tension and viscosity of the feed solution in the process of spray drying along with atomizing gas pressure are the main factors that determine the size of droplets during atomization step which subsequently controls the particle size. Incorporation of organic solvents, DPPC and amino acids in the formulations are reported to decrease the surface tension and result in formation of particles with low MMAD values which could explain the low MMAD of F5 (Arakawa et al., 2007; Bosquillon et al., 2004; Minne et al., 2008; Sarmento et al., 2006; Silva et al., 2006). On the other hand, higher viscosity of F4 solution due to chitosan is the most probable cause of its high MMAD. FPF is one of the key parameters in any aerosol which shows the respirable fraction and a good formulation should have the

highest FPF values possible. FPF values in this study were in range of 45% to more than 80% which were in acceptable range (Davis, 1999; Pilcer et al., 2009; Salama et al., 2008; Sarmento et al., 2006; Silva et al., 2006). Aerosols are polydispersed and particle size distribution could be best described by geometric standard deviation (GSD); larger GSD indicates wider particle size distribution, this value for aerosols usually falls between 1.3 and 3 mm (Musante et al., 2002). In this study F3 with GSD of 1.77 mm, had the narrowest particle size distribution but the GSD of other powders were also in acceptable range (Table 6). 3.5 In vitro protein release Drug release rate of the spray-dried particles studied as described in Section 2.8 and the cumulative release of insulin was plotted as a function of elapsed time (Fig. 4). Released insulin at the end of 6th hour varied from 57% in case of F1 and F2 up to 73% in case of F3 (Table 7). T50% ranged from 50 min for F1 to 202 min in F4 (Table 7). Mean dissolution time (MDT) was calculated for these formulations and ranged between 44 and 170 min (Table 7). Pairwise comparison of release profiles using modified difference factor (f1) and the similarity factor (f2) revealed no similarity among the five studied formulations (Table 8). Release profiles were analyzed further to determine the best describing kinetic model among zero order, first order, Higuchi and KorsmeyerPeppas models (Table 7). The best fitting model is the one with the highest coefficient of determination (R2) although it must be equal to or

[(Fig._3)TD$IG]

Fig. 3. Next generation impactor deposition profiles of spray-dried powders as the weight of deposited powder (mean  SD, n = 3). F1: mannitol/insulin/sodium citrate. F2: mannitol/L-alanine/insulin/sodium citrate. F3: mannitol/sodium alginate/insulin/sodium citrate. F4: mannitol/ chitosan/insulin/sodium citrate. F5: mannitol/L-alanine/DPPC/ insulin/sodium citrate.

S. Razavi Rohani et al. / International Journal of Pharmaceutics 465 (2014) 464–478

[(Fig._4)TD$IG]

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Fig. 4. In vitro release profiles of insulin from spray-dried microparticles obtained using Franz diffusion cell (mean  SD, n = 3). F1: mannitol/insulin/sodium citrate. F2: mannitol/L-alanine/insulin/sodium citrate. F3: mannitol/sodium alginate/insulin/sodium citrate. F4: mannitol/ chitosan/insulin/sodium citrate. F5: mannitol/L-alanine/DPPC/ insulin/sodium citrate.

Table 7 Parameters obtained from comparison and modeling of dissolution profiles of spray-dried formulations. Max. release (%)

T50% (min)

MDT (min)

Zero order K0

F1 F2 F3 F4 F5

57 57 73 64 68

50 167 134 202 62

44.1 82.2 117.7 170 58.5

4.6  0.5 5.3  0.3 12.2  0.3 11.5  0.5 11.6  1.5

First order R

2

K1

0.845 0.904 0.990 0.975 0.794

Table 8 0 Similarity factor (f2) and modified difference factor f 1 between release profiles of the formulations. Two formulations are considered as similar if f2 > 50 and f1