Formulation and characterization of ORMOSIL particles loaded with budesonide for local colonic delivery

International Journal of Pharmaceutics 484 (2015) 75–84

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Formulation and characterization of ORMOSIL particles loaded with budesonide for local colonic delivery Vesna Petrovska-Jovanovska a,b , Nikola Geskovski a , Maja Simonoska Crcarevska a , Oya Memed a,b , Gjorgji Petruševski b , Marina Chachorovska b , Marija Petrusevska c , Ana Poceva-Panovska d , Kristina Mladenovska a , Sonja Ugarkovic b , Marija Glavas-Dodov a, * a Institute of Pharmaceutical Technology, Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, University Ss. Cyril and Methodius, Majka Tereza 47, 1000 Skopje, Macedonia b Research & Development, Alkaloid AD-Skopje, Blv. Aleksandar Makedonski 12, 1000 Skopje, Macedonia c Institute of Preclinical and Clinical Pharmacology and Toxicology, Faculty of Medicine, University Ss. Cyril and Methodius, 50 Divizija 6, 1000 Skopje, Macedonia d Institute of Applied Chemistry and Pharmaceutical Analyses, Faculty of Pharmacy, University Ss. Cyril and Methodius, Majka Tereza 47, 1000 Skopje, Macedonia

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 October 2014 Received in revised form 15 January 2015 Accepted 17 February 2015 Available online 20 February 2015

In this study, hybrid silica xerogel particles were developed as carriers of budesonide (BDS) for efficient local treatment of inflammatory bowel diseases (IBD). Organically modified silica particles (ORMOSILs) were prepared by co-condensation of 3-aminopropyltriethoxysilane (APTES) and tetraethyl orthosilicate (TEOS) by an ambient temperature acid catalysed sol–gel process followed by spray-drying. Formulation for preparation of BDS-loaded particles was optimized and their physicochemical parameters and drug release profiles were evaluated in vitro. Optimal formulation had a small particle size (mean diameter of 1.45
0.02 mm) with unimodal narrow size distribution and high encapsulation efficiency (98.0
1.85%). Due to the positive surface charge originated from amino group of APTES, ORMOSILs showed excessive mucoadhesiveness in comparison to native TEOS particles. The drug release decreased with increasing pH from 2.0 to 7.4. In order to avoid undesirable erroneous performance in the upper GI tract, particles were additionally coated with Eudragit1 FS 30D, as a barrier to the drug release at pH range from 2.0 to 7.0. After Eudragit1 FS 30D coating, the release of BDS in acidic media was sustained, while no significant differences in drug release were observed at pH 7.4. In conclusion, pH-responsive ORMOSILs showed great potential for efficient BDS delivery to the colon region. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Colonic delivery Budesonide Organically modified silica particles Sol–gel processes

1. Introduction Inflammatory bowel diseases (IBDs), such as Crohn’s disease and ulcerative colitis, are one of the most common gastrointestinal disorders. Treatment, in particular via the oral route, is confronted with many obstacles such as premature drug release, ineffective drug concentration at the site of action, systemic absorption and poor drug bioavailability, thus, resulting in lower therapeutic value and enhanced probability for occurrence of strong adverse and side effects (Park, 2013).

* Corresponding author. Tel.: +389 23126032; fax: +389 23123054. E-mail address: [email protected] (M. Glavas-Dodov). http://dx.doi.org/10.1016/j.ijpharm.2015.02.044 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.

The potential of particulate drug delivery systems to enhance IBD therapy was recognized by Lamprecht and his co-workers, and till now different micro/nano-carriers were designed and evaluated (Collnot et al., 2012; Lamprecht et al., 2000, 2001b). Particulate carrier systems intended for oral administration and efficient IBD treatment should have particle size below 10 mm (preferably below 5 mm), positive surface charge, specific muco/bioadhesive characteristics and controlled release properties thus allowing targeting and retention of the carrier system as well as high local drug concentration at the desired site of action (Crcarevska et al., 2009; Glavas-Dodov et al., 2013; Mladenovska et al., 2007). In recent years, the preparation of silica based particles has attracted much attention due to their unique properties and numerous applications especially in the field of biomedical engineering/medicine. By careful selection of sol–gel reaction

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terms such as concentration of reactants, type of catalyst, reaction temperature, pH value, etc., particles with tunable physicochemical and biopharmaceutical properties could be engineered (Kim et al., 2013; Zhang et al., 2010). Aside from biocompatibility, favorable structural characteristics, high loading capacity for different drug moieties and controlled drug release, silica particles can be also modified aiming at specific biomedical application (Tzankov et al., 2013). On the basis of the above-mentioned considerations, particulated delivery systems that combine pH-sensitivity, controlled release properties and site-specificity to inflamed mucosal tissue could be promising candidates for drug targeting in IBD. The aim of this work was to develop silica based particles loaded with budesonide (BDS) as a targeted oral delivery system intended for efficient IBD treatment. The carrier system was prepared using sol–gel chemistry followed by spray-drying process. In order to ensure localization of the particles at the desired site, tetraethoxysilane (TEOS) based inorganic particles were tailored with organic functional groups using an organoalkoxysilane containing amino groups such as 3-aminopropyltriethoxysilane (APTES) via direct co-condensation synthesis method. For further control of the bio-stability, organically modified silica particles (ORMOSILs) were coated with pH sensitive polymethyl (methacrylate) copolymer (Eudragit1 FS-30D). The prepared formulations were characterized for particle size and morphology, surface charge, drug-polymer and polymer–polymer interactions, drug loading, muco/bioadhesiveness and in vitro drug release properties. 2. Materials and methods 2.1. Materials Tetraethoxysilane, 98% (TEOS) and aminopropyltriethoxysilane, 99% (APTES) were purchased from Sigma–Aldrich, Germany. Ethanol, 96%, acetic acid, 100%, triethyl citrate, Polisorbate1 20 and Polysorbate1 80 were all obtained from Merck, Germany. Budesonide (BDS) was purchased from Crystal Pharma, Spain. Eudragit1 FS 30D was supplied from Evonik, Germany. All other chemicals were of analytical reagent grade and were used as received. 2.2. Methods 2.2.1. Preparation of inorganic silica particles (ISP) BDS loaded ISPs particles were prepared by acetic acidcatalyzed hydrolysis and polycondensation of TEOS in ethanol/ water mixture at room temperature, followed by spray-drying. Briefly, BDS was dissolved in ethanol on an ultrasonic bath (Ultrawave Limited, Cardif, UK) and distilled water was slowly dropped into the above solution. Afterwards, TEOS and acetic acid

were added and the solution was mechanically stirred at room temperature (500 rpm, 3 h; Jenway, UK). The resulting solution was spray-dried (Buchi 190, Mini Spray Dryer, Switzerland) using the following process parameters: inlet temperature 135  C, flow rate 4 mL/min, atomizer pressure 600 NL/h and aspirator pressure 100%. To optimize the formulation in respect to its physicochemical and biopharmaceutical properties, two different formulation groups of ISPs were prepared at a TEOS:water molar ratio of 0.022:5.55 (ISPA) and 0.022:11.1 (ISPB) and a constant water: ethanol molar ratio of 5.55:1.712 (ISPA) and 11.1:3.424 (ISPB). In each formulation group, TEOS:BDS molar ratio was varied in range of 0.226–1.375 (Table 1). Blank formulations (samples ISPA0 and ISPB0)were also prepared as comparison. 2.2.2. Preparation of organically modified silica particles – ORMOSILs Formulation group of ISPs prepared with TEOS:water molar ratio of 0.022:11.10 (samples ISPB1–4) with mean particle size of 1 mm and narrow unimodal particle size distribution was chosen for further modification (Table 3). ORMOSILs (HSPs) were prepared by substituting 2.5 mol% of the initial amount of TEOS with APTES using same preparation procedure as described for ISPs. The molar ratios of TEOS:APTES:water:ethanol:acetic acid:BDS of the prepared HSPs are given in Table 2. Blank formulation (HSP0) was also prepared for comparison. 2.2.3. Preparation of pH-sensitive ORMOSIL particles Based on the physicochemical and biopharmaceutical properties suitable for effective delivery of BDS to colon region via oral administration, HSP2 was selected for further coating with Eudragit1 FS 30D. This anionic copolymer, based on methyl acrylate, methyl methacrylate and methacrylic acid, is insoluble in acidic media, but dissolves by salt formation above pH 7.0, thus allowing targeted colon delivery. Before coating, polymer dispersion of Eudragit1 FS 30D was prepared as following: distilled water (0.8 g) was heated up to 70–80  C and triethyl citrate (0.3 g) was dissolved under mechanical stirring (700 rpm, 10 min; Jenway, UK). Eudragit1 FS 30D (4.0 g) was dispersed into the above solution under stirring (700 rpm, 10 min; Jenway, UK). Then, 400 mg of spray-dried BDS loaded HSP2 were dispersed in 20 mL of distilled water with the aid of sonication for 15 min (Ultrawave Limited, Cardif, UK). The dispersed particles were slowly added into polymer dispersion of Eudragit1 FS 30D and the resulting dispersion was stirred for additional 10 min (300 rpm; Jenway, UK). Afterwards, obtained dispersion was spray-dried (Buchi 190, Mini Spray Dryer, Switzerland) using the following process parameters: inlet temperature 135  C, flow rate 0.5 mL/min, atomizer pressure 600 NL/h, aspirator pressure 100%. The coat to core mass ratio was 3:1.

Table 1 ISPs formulations prepared with different molar ratio water/TEOS and BDS as drug substance. Formulation code

TEOS (mol)

ISPA0 ISPA1 ISPA2 ISPA3 ISPA4

0.022 0.022 0.022 0.022 0.022

ISPB0 ISPB1 ISPB2 ISPB3 ISPB4

0.022 0.022 0.022 0.022 0.022

Water (mol) 5.550 5.550 5.550 5.550 5.550 11.10 11.10 11.10 11.10 11.10

Ethanol (mol)

Acetic acid (mol)

BDS (mol)

1.712 1.712 1.712 1.712 1.712

1.7  102 1.7  102 1.7  102 1.7  102 1.7  102

/ 1.6  102 3.2  102 6.5  102 9.7  102

3.424 3.424 3.424 3.424 3.424

1.7  102 1.7  102 1.7  102 1.7  102 1.7  102

/ 1.6  102 3.2  102 6.5  102 9.7  102

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Table 2 Formulations of HSPs loaded with BDS. Formulation code

TEOS (mol)

APTES (mol)

Water (mol)

Ethanol (mol)

Acetic acid (mol)

BDS (mol)

HSP0 HSP1 HSP2 HSP3 HSP4

0.021 0.021 0.021 0.021 0.021

5.35  104 5.35  104 5.35  104 5.35  104 5.35  104

11.10 11.10 11.10 11.10 11.10

3.424 3.424 3.424 3.424 3.424

1.7  102 1.7  102 1.7  102 1.7  102 1.7  102

/ 1.6  102 3.2  102 6.5  102 9.7  102

2.3. Physical and chemical characterization of particles 2.3.1. Particles morphology The shape and surface morphology of the particles were examined by SEM. The samples were coated with gold under an argon atmosphere using a gold sputter module in a high-vacuum evaporator. The coated samples were randomly scanned and photomicrographs were taken with scanning electron microscope (Jeol SEM 6400, Japan). The porous structure of the samples was characterized using TEM. The samples were prepared by dispensing the particles in MilliQ water followed by vigorous mixing. Then, a small quantity of the particles dispersion (2 mL) was placed on coated copper grid (100 mesh, Sigma–Aldrich, Belgium) with additional carbon film (Jeol, JEC-530, Japan). The grid was placed on filter paper (WhatmanTM 42, Austria) in order to absorb excess solvent and air dried at ambiental temperature. Then, it was loaded into the TEM (JEM-1400, Jeol, Japan) attached to a digital camera (Veleta TEM Camera, Olympus, Germany) and controlled by the iTem software v.5.2. 2.3.2. Particle size determination Prepared particles were characterized in terms of particle size and particle size distribution by laser diffractometry using Mastersizer 2000, Malvern Instruments Ltd., UK, equipped with Hydro 2000S, Malvern Instruments Ltd., UK, for wet dispersions. Ten measurements for each batch were performed. Wet dispersions for particle size analysis were prepared by dispersing the particles (15–20 mg) in 2 mL 2% (w/w) solution of Polisorbate1 20, by means of ultrasonic bath (Ultrawave Ltd., Cardif, UK) for 10 min. A small aliquot of the resulting suspension was transferred to the optical measurement cell containing the blank dispersing medium, distilled water. Measurements were performed while the sample was in the cell under stirring (2520 rpm/stirrer rate) and ultrasound (50%), previously applied for 5 min. The obscuration was set between 10% and 12%. The particle size distribution was also expressed in terms of SPAN factor determined as: SPAN ¼

d90  d10 d50

where d90,d10 and d50 are the diameter sizes and the given percentage value is the percentage of particles smaller than that size. A high SPAN value indicates a wide size distribution (Gavini et al., 2008). 2.3.3. Zeta potential measurements Zeta potential measurements were carried out using a Zetasizer Nano Series (Nano-ZS, Malvern Instruments Ltd., UK) at 25  C on dispersions of the particles in low molarity buffer solutions (Ph. Eur. 7) with pH 2.0 (9  107 M, ionic strength 2.7  103 M) and 7.4 (phosphate buffer, 9.7  104, ionic strength 9  104 M), respectively. Six measurements for each batch were performed. 2.3.4. Fourier transform infrared (FT-IR) spectroscopy The FT-IR spectra were recorded on a Varian-660 series FT-IR spectrometer at an ambient temperature. Spectral range 4000–400 cm1 was selected and the samples were prepared

according to the KBr disc method. The acquisition resolution was set at 4 cm1 and 16 averaged scans per spectrum were used in order to obtain good quality spectra. 2.3.5. Differential scanning calorimetry (DSC) Differential scanning calorimetry experiments were conducted on a Netzsch DSC 204 F1 Phoenix instrument. Weighed samples between 2 and 3 mg were scanned in aluminum pans with a perforated lid, applying speed of 10 K/min, from 25  C to 280  C in dry nitrogen atmosphere. 2.3.6. Drug content Accurately weighted samples (5 mg) were dispersed in 5 mL acetonitrile and vigorously mixed in order to dissolve the polymers and to extract the drug substance (5 min; Heidolph, Germany). Afterwards, the samples were centrifuged at 4000 rpm for 10 min (Tehtnika Centric 322B, Slovenia), supernatants were withdrawn, filtered through 0.45 mm membrane filter (Ministar RC 25, Sartorius, Germany) and assayed by HPLC, as previously reported in details (Simonoska Crcarevska et al., 2008). Commercial BDS is an epimeric mixture of two isomers which have the same pharmacological activity patterns. The two epimers can be separated under the conditions described below. Analyses were performed on Agilent 1100 Series HPLC system, equipped with Binary pump SL, Dioda Array Detector (DAD). The column used was Lichrosper1 100 RP-18 5 mm, 125  4 mm, Merck, Germany. Chromatographic conditions for this method were: flow rate 1 mL/min, column temperature 25  C, injection volume 100 mL. The mobile phase was acetonitrile/water (40:60). Results were calculated from linear regression of the external standard of BDS. The drug encapsulation efficiency (EE) was calculated from the following equation (Sairam et al., 2006): EEð%Þ ¼

Actual drug loading  100 Theoretical drug loading

Blank particles were also analyzed, in the same manner and at the same time as particles loaded with BDS. All experiments were performed in triplicate. Method validity was established from the specificity, linearity and accuracy. 2.4. Biopharmaceutical characterization of particles 2.4.1. In situ mucoadhesion studies Mucoadhesive potential of the prepared ISPs and HSPs was determined in situ, according to the modified method of Santos et al. (1999). The animals used in our study received humane care, and the study protocols were approved by the Ethical Committee of the Faculty of Pharmacy, University of “Ss. Cyril and Methodius” – Skopje, Republic of Macedonia, in accordance to national and institutional guidelines. Unfasten Wistar rats (mass approximately 400
20.8 g, female) were sacrificed and colons were removed and flushed with 10 mL phosphate buffer saline pH 7.4 containing 2 g/L glucose (PBSG). Six centimeter segments were everted using a stainless steel rod and lightly washed with PBSG to remove the contents. Ligatures were

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placed at both ends of the segment and the sac was filled with 2 mL of PBSG. The sacs were introduced into a 15 mL glass tubes containing 40 mg of prepared particles in 5 mL PBSG. After incubation for 30 min (100 rpm, 37  C; Shaker Unitronik OR, Selecta, Spain), the sacs were removed and detached (unbound) particles suspension was collected and freeze-dried (47  C, 0.055 mBar 24 h, Labconco, FreeZone 2.5 I Freeze Dry System, USA). Blank samples, sacs immersed in 5 mL PBSG without particles were also investigated. The tests were performed in triplicate for each sample. Mucoadhesive potential of the particles, expressed as % of binding was calculated as follows:

not been taken into account in all release models. Huang and Brazel (2001) have introduced a modified power law model that includes an additional burst parameter, a: At/A1 = ktn + a. The release profile is shifted vertically by a accounting for a rapid jump in concentration at t = 0. The initial release phase may also be slower than the longer lasting main release phase, i.e., lag time is sometimes observed. Both burst and lag time differ from the main release phase and lag time may also last a relatively long time. In order to take this initial release, either burst or lag time, into account we suggest a modified power law model:

initial weight of particles  weight of unbound particles  100 initial weight ofparticle

where At is the cumulative release at time t, A2 is the amount released within the initial release phase (that differs from the main release phase), A1 is the cumulative drug release at infinite time, k is the constant depending on the structural and geometrical characteristics of the device, t2 is the end point time of the initial release and nb is the release exponent characteristics for the mechanism of drug release after the initial release. By plotting the ln (Mt  M2/M1) versus ln (t  t2), the nb can be calculated from the slope of this curve. The values of nb are interpreted in the same way as the values of n (in power law), which are characteristic for the drug release mechanism, but nb describes the release, which occurs after the initial release (Viitala et al., 2007).

%binding ¼

2.4.2. In vitro drug release studies In vitro release of BDS from prepared particles was investigated in pH dissolution media simulating the human GIT conditions. For that purpose experiments were performed in acid buffer at pH 2.0 (Ph. Eur. 7) for 2 h to simulate fasted stomach and in phosphate buffer at pH 7.4 (Ph. Eur. 7) to simulate ileo-colon conditions for 24 h (Kalantzi et al., 2006; Mladenovska et al., 2007; Sonaje et al., 2009). Accurately weighted BDS loaded particles, corresponding to 100 mg of BDS were placed in closed plastic tubes and were incubated in 40 mL of above stated buffer solutions on horizontal shaker water-bath (100 rpm; Shaker Unitronik OR, Selecta, Spain) at 37  C. At appropriate time intervals, 2 mL samples were withdrawn, replaced by 2 mL of fresh buffer, and assayed by HPLC method described above. The studies were performed under sink conditions and in triplicate for each batch/formulation to calculate the mean values and standard deviations. Obtained results were plotted as the cumulative and percentage of the content into dissolution medium versus time. Drug release constants were determined by different kinetic models (Higuchi, power law and modified power law). The in vitro release patterns were evaluated to check the goodness of fit to the Higuchi’s square root of time equation (Higuchi, 1963): A ¼ k  t1=2

(1)

where A is the amount of drug dissolved in time t, k is the Higuchi dissolution constant, and t is the release time. Although the power law has its limitations, it is considered to be more useful in the comparative studies providing explanations also for matrices that do not fulfill the presumptions of the Higuchi model (Harland et al., 1988; Siepmann and Peppas, 2001). The general form of the power law is: At ¼ k  tn A1 which includes At – the cumulative drug released at time t, A1 – the cumulative drug release at infinite time, k – constant, depending on the structural and geometrical characteristics and the release exponent n – indicating the mechanism of drug release. Higher n values (0.85–1.0) are typical for drug transport in swelling polymer devices. n = 1.0 indicates actually zero order drug release kinetics, which can be achieved also with matrix erosion controlled systems (Siepmann and Peppas, 2001). The n values between 0.5 and 1.0 are indicative of anomalous transport behavior including both diffusion and swelling or erosion (Siepmann and Peppas, 2001). Peppas has suggested that the n values below 0.50 are indicative of porous matrix structure. The burst effect which is the initial, short-time release of large amount of drug before a more stable and slower release phase, has

At  A2 ¼ kðt  t2 Þnb A

2.5. Stability studies The optimal formulation coated with Eudragit1 FS 30D (EHSP2) was sealed in glass vials and was stored under controlled ambient conditions (25
2  C, 60
5% RH). Periodical testing of different parameters (particle size, particle size distribution and encapsulation efficiency) during 12 months real time stability studies was performed. 3. Results and discussion 3.1. Physicochemical characterization of particles 3.1.1. Morphological analysis The shape and morphology of the prepared ISPs and HSPs were analyzed by SEM and TEM. Obtained micrographs showed that ISPs were uniform in size with well-formed spherical shape and smooth surfaces (Fig. 1a and c, ISPB2). By contrast, HSPs were slightly larger, spherical particles but with a porous-like structure (Fig. 1b and d, HSP2). The morphology could be attributed to the aminopropyl-group in APTES, because compared to TEOS, APTES shows a lower cross-linking density in the network (Digigow et al., 2014), which also affects the particle size. 3.1.2. Particle size analysis Former investigations (Lamprecht et al., 2001a,b) of the size dependent accumulation of particulate carriers in a preclinical animal model showed that localization of particles in the inflamed areas can be used as a novel targeting strategy for efficient IBD treatment, if particles of an appropriated size (100–1000 nm) are employed. However, later study (Schmidt et al., 2013) showed that the effect of nanoparticles observed in small animal models was not observed in human IBD patients. Microparticles with size of 2 mm showed better accumulation to the inflamed areas in the intestine and colon, with no absorption across the epithelial barrier. Also, several in vivo studies reiterate that the potential toxicity through oral delivery is highly dependent on the size of the carrier and that silica particles with diameters of 1 mm do not show toxicity and organ abnormalities after oral administration to mice (Jaganathan and Godin, 2012). Having this in regard, we

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79

Fig. 1. SEM (a and b) and TEM (c and d) images of ISPB2 (a and c) and HSP2 (b and d) samples. The bar presents 0.5 mm.

intended to prepare BDS loaded inorganic silica particles (ISPs) with mean diameter of 1–2 mm. Particle size and particle size distribution (presented as SPAN factor value) of inorganic silica particles prepared in this study using different water/TEOS ratio (samples ISPA1–4 and ISPB1–4) and ORMOSILs (samples HSP1–4) are given in Table 3. From the obtained results one can see that the mean diameters d50 (mm) of the prepared ISPs ranged from 2.1 to 2.5 mm. ISPs prepared with lower water/TEOS molar ratio (5.55:0.022, samples

Table 3 Particle size and particle size distribution of ISPs s and HSPs (n = 10). Formulation code

d10(mm)
SD d50(mm)
SD d90(mm)
SD SPAN
SD

ISPA0 ISPA1 ISPA2 ISPA3 ISPA4

0.91 0.83 0.99 0.95 0.92








0.202 0.274 0.039 0.040 0.004

2.19 2.21 2.11 2.21 2.23








0.151 0.051 0.165 0.101 0.010

7.68 7.76 7.86 7.98 7.84








0.328 0.588 0.625 0.660 0.510

3.09 3.14 3.26 3.18 3.1








0.151 0.161 0.131 0.176 0.003

ISPB0 ISPB1 ISPB2 ISPB3 ISPB4

0.49 0.51 0.53 0.49 0.53








0.020 0.013 0.013 0.017 0.020

1.00 1.10 1.06 1.09 1.08








0.050 0.065 0.058 0.086 0.053

1.81 1.85 1.89 1.87 1.83








0.028 0.030 0.026 0.059 0.020

1.32 1.22 1.28 1.27 1.20








0.027 0.046 0.024 0.067 0.025

HSP0 HSP1 HSP2 HSP3 HSP4

0.59 0.68 0.71 0.67 0.72








0.090 0.050 0.043 0.045 0.066

1.38 1.41 1.45 1.40 1.40








0.017 0.060 0.020 0.018 0.013

2.00 2.14 2.10 2.20 2.19








0.020 0.040 0.066 0.035 0.030

1.02 1.04 0.96 1.09 1.05








0.020 0.027 0.071 0.008 0.005

E-HSP2

2.57
0.113

3.9
0.033

5.53
1.013

0.76
0.007

ISPA1–4) were with mean diameters of 2.1 mm and SPAN factor higher than 4, indicating a wide particle size distribution (Gavini et al., 2008). On the other hand, ISPs prepared with a higher water/ TEOS molar ratio (11.1:0.022, samples ISPB1–4) had mean diameters of 1 mm and SPAN factor less than 1.3. Obtained results are in accordance with the previous findings suggesting that particle size of ISPs depends on the process of hydrolysis (rate of nucleation) and process of condensation (rate of growth) (Dingsoyr and Christy, 2000; Sinkó, 2010). Lower water/ TEOS molar ratio decrease the rate of hydrolysis of TEOS and the subsequent formation rates of ISPs. Thus, the preferential particle growth to nucleation occurs, and larger ISPs are formed (Schaefer et al., 1990). Contrary, higher water/TEOS molar ratio leads to formation of smaller particles with a very narrow size distribution (Rahman et al., 2007). Partial substitution of TEOS with APTES implicated significant increase in the mean diameter of the particles (samples HSP1–4) in comparison with the same formulation group of ISPs (samples ISPB1–4) (Table 3). This is probably due to the presence of NH2 groups in APTES that leads to lower cross-linking density formation on the gel network due to the decrease in the degree of orderliness of the functionalized matrix, thus, inducing formation of larger particles (Rahman et al., 2009; Xu et al., 2006). Also, it is important to notice that in hybrid silica particles there are less free silanol groups in comparison to inorganic silica particles, resulting in changes in the pore diameter, hidrophobicity/hidrophilicity of the system and chemical reactivity (Lev et al., 1995; Mah and Chung, 1995), as factors important for their performance in vivo. No significant differences in mean particle size were observed between blank and BDS loaded HSPs.

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pH-sensitive hybrid silica particles (sample E-HSP2) had a mean diameter of 3.9
1.23 mm and SPAN factor value of 1.35
0.16. It is obvious that the coated particles have larger diameter, probably due to the surface adherence of the coating polymer. Considering that dissolution of Eudragit1 FS 30D coating starts at pH higher than pH 7.0 (in correlation with the in vivo surface pH of human colonic mucosa in a range 7.1–7.5), particle size of pH-sensitive hybrid silica particles was not a key criterion for this delivery system. In order to confirm our presumption, particle size analysis of E-HSP2 after 30 min incubation in phosphate buffer (pH 7.4) was performed. No significant differences in the mean diameter (1.47
0.23 mm) relative to the uncoated HSP2 were observed (p < 0.05), which verifies the complete dissolution of the pH dependent coating in relevant ileo-colon conditions.

Transmitance (arbitrary units)

80

4000

3.1.3. Zeta potential measurements As the surface charge and surface functional groups are important for the stability (agglomeration and fusion phenomena) and outcome of the particles in vivo (Andrade et al., 2013), zeta potential measurements of ISPs and HSPs suspended in buffer solutions at pH 2.0 and pH 7.4 were made in order to simulate their behavior in GIT and to confirm that there is a charge change in polymeric particles after modification with APTES. ISPs (samples ISPA1–4 and ISPB1–4) have zeta potential values of 6.5–8.5 mV and 30.4 to 35.4 mV in buffer solutions at pH 2.0 and 7.4, respectively. Obtained results are in accordance with the fact that in aqueous solutions, surface silanol groups can be protonated or deprotonated depending on the solution pH, which results in the corresponding changes of the surface charge (Wu et al., 2006). At pH 2, for instance, the particles are protonated, i.e., positively charged. Contrary, at pH 7.4 due to the dissociation of the silanol groups, ISPs became negatively charged (McNamee et al., 2001). The partial substitution of TEOS by APTES introduces amine groups into the silica matrix, changing the nature of the particles from a purely inorganic entity to a mixed organic–inorganic hybrid. As a result of existence of additional functional (amine) groups (pKa = 10.6), a considerable number of amino groups will be protonated at pH below 10.0 (Digigow et al., 2014; Yokoi et al., 2012). Therefore, prepared ORMOSILs (samples HSP1–4) have positive surface charge in range of 34.2–39.0 mV and 30.4–32.8 mV in buffer solutions with pH 2.0 and pH 7.4, respectively (Asouhidou et al., 2009; Yoncheva et al., 2014). The zeta potential of E-HSP2 was 25
0.98 mV and 12.3
0.76 mV in buffer solutions at pH 2.0 and 7.4, respectively. It is expected that the coating of the ORMOSIL particles will induce changes in their surface characteristics as a result of the effective covering of the exposed amine groups. In addition, the effect of the coating on the zeta potential was more pronounced at pH 7.4 due to the anionic character of Eudragit1 FS 30D. However, when the coated particles were incubated for 30 min at pH 7.4, the zeta potential rose back to 30.8 mV, thus, confirming the complete dissolution of the pH sensitive coating. Presented surface properties of prepared HSPs are of great importance, since positive charge is necessary for the interaction with negatively charge mucus and cell membranes, and consequently, muco/bioadhesion (Glavas Dodov et al., 2009). 3.1.4. Fourier transform infrared (FT-IR) spectroscopy The infrared spectra of crystalline and spray-dried BDS have been previously studied in details (Naikwade et al., 2009; Tajber et al., 2009). The spray-drying procedure introduces negligible spectral alterations (Fig. 2a and b), mostly manifested as band broadening (e.g., O—H stretching mode at about 3500 cm1). In the FT-IR spectra of the studied blank ORMOSILs (HSP0) (Fig. 2c), absorption bands arising from the antysimmetric

a

b

c d e

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber/cm

Fig. 2. FT-IR spectra of crystalline BDS (a), spray-dried BDS (b), blank HSPs, HSP0 (c), binary mixture spray-dried BDS-blank HSPs, HSP0 (d), and BDS loaded HSPs, HSP2 (e).

stretching mode of the Si—O groups (1090 cm1), antysimmetric stretches from the Si—OH groups (940 cm1), and symmetric stretching modes originating from the Si—O group (800 cm1) can be clearly identified. The wide band that appears in the high frequency region between 3300 cm1 and 3500 cm1 is assigned to the O—H stretching mode from the absorbed water molecules. The bands that can be observed in the region 1550–1400 cm1 originate from the deformation vibrations of the CH2 and NH2 groups from the alkyl-amino chain. According to the literature data, the appearance of these particular bands in the FT-IR spectrum is attributed to the incorporation of the APTES in the silica framework (Shurvell et al., 2001). In the FT-IR spectrum of the binary mixture of spray-dried BDS and HSP0 (ratio 1:1, w/w) (Fig. 2d), most of the prominent bands originating from the active ingredient can be easily identified (Naikwade et al., 2009; Tajber et al., 2009). On the contrary to the spectroscopic observations for the binary mixture, the FT-IR spectrum of the HSP2 (Fig. 2e) reveals that there are no observable characteristic absorption bands from BDS. This result strongly implies that the active ingredient is firmly incorporated in polymer matrix during the spray-drying procedure. 3.1.5. Differential scanning calorimetry (DSC) The DSC analysis of crystalline BDS confirmed the crystalline character of the starting material, exhibiting characteristic melting endotherm at 260.3  C (DH = 83.9 J/g) (Fig. 3a and Table 4). This result is in good agreement with the literature data (Tajber et al., 2009; Velaga et al., 2002). In comparison, in the DSC curve of the spray-dried BDS, exothermic event (190.9  C) originating from the cold crystallization of the amorphous phase can be identified, followed by an endothermic melting peak at about 250  C (Fig. 3b and Table 4). The DSC curve of the HSP0 does not exhibit any thermal phenomena in the studied temperature range (Fig. 3c). Observed interactions in physical mixtures can be considered as strong indication that interaction also, occurs in the system prepared by drug substance and corresponding excipients (Forster et al., 2001; Gonzalez Novoa et al., 2005). In order to check the possible interaction between BDS and TEOS/APTES matrix, binary mixture (ratio 1:1, w/w) of spray-dried BDS and HSP0 was prepared. The obtained DSC data for this binary mixture (Fig. 3d and Table 4) showed that the cold crystallization exothermic peak of the spray-dried BDS was shifted at higher temperature. This thermal phenomenon is followed by an endothermic event at 239  C, most probably associated with solid–solid phase transition of some metastable form of BDS to more stable modification, that

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81

Table 5 BDS content in ISPs and HSPs, n = 3.

DSC (endo up)

a

b

Formulation code

BDS content (mg/g particles)

EE(%)
SD

ISPB1 ISPB2 ISPB3 ISPB4 HSP1 HSP2 HSP3 HSP4 E-HSP2

5.85
0.80 11.72
0.65 21.42
1.10 31.82
1.40 5.54
0.60 10.35
0.95 22.85
1.20 30.65
0.85 2.73
0.48

91.0 88.1 85.7 80.0 95.0 98.0 92.1 89.0 97.3












1.25 0.26 0.53 0.95 0.86 1.85 0.40 2.64 0.94

c

drug substance was completely entrapped in the polymer matrix during the spray-drying process (Wong et al., 2006). This result is in complete accordance with the comprehensive FT-IR spectroscopy study.

d

e

50

100

150

200

250

0

Temperatur e ( C) Fig. 3. DSC curves of crystalline BDS (a), spray-dried BDS (b), blank HSPs, HSP0 (c), binary mixture of spray-dried BDS-blank HSPs, HSP0 (d) and BDS loaded HSPs, HSP2 (e).

melts at 249.7  C, producing strong endotherm almost identical with the corresponding endotherm of the spray-dried BDS (Fig. 3b). When the drug substance is in higher concentration, shifting of the exothermic peak of spray-dried BDS toward higher temperature is most probably result of solid-state interaction between BDS and organic modified matrix. Naikwade et al. showed that hydroxyl groups of BDS are potential hot-spot for cross-linking via amine groups of chitosan (Naikwade et al., 2009). On the other side, Bandi et al. reported the formation of intermolecular hydrogen bonds between carbonyl groups of the BDS and hydroxyl groups of hydroxypropyl b-cyclodextrin (Bandi et al., 2004). Having in regard the structure of BDS, as well as the structures of the silica matrix and the organic modified matrix, it can be assumed that there is real possibility for formation of intermolecular bonds of different nature between the BDS and HSP0. Neither exothermic nor endothermic peaks of spray-dried BDS were observed in the DSC curve of HSP2 (Fig. 3e). Subsequently, it can be concluded that the absence of typical BDS thermal phenomena in the DSC trace of the loaded particles suggest that

Table 4 DSC data of BDS, binary mixture, blank (HSP0) and BDS loaded HSPs (HSP2). Sample

Tonset ( C)

Tmax ( C)

DH (J g1)

Crystalline BDS

257.8

260.3

83.9

Spray-dried BDS

179.2 243.0

189.9 250.1

45.5 71.8

HSP0

ND 206.1

ND 214.0

ND 71.5

Binary mixture BDS/HSP0

235.7 248.5

239.0 249.7

8.2 16.6

HSP2

ND

ND

ND

ND: not detected.

3.1.6. Drug content Drug content (mg/g particles) and calculated BDS encapsulation efficiency (EE) in prepared formulations is presented in Table 5. Different initial polymer:drug molar ratios affect the drug content into ISPs and HSPs, respectively. Namely, the drug loading significantly increased when the polymer:drug molar ratio was increased. EE(%) was found to be quite high for both formulation groups (>86%) and there was a significant difference in EE of inorganic particles and comparable ORMOSILs (one way ANOVA with the Fischer least square difference (LSD) multiple comparison test, p < 0.05). ISPs showed lower encapsulation capacity for BDS in comparison to its HSPs analog probably due to the formation of hydrogen bonds between hydroxyl groups of BDS and amino groups of the APTES-modified particles (Yoncheva et al., 2014). 3.2. Biopharmaceutical characterization of particles 3.2.1. Mucoadhesion study In situ mucoadhesive potential of selected formulations (ISPB2, HSP2 and E-HSP2) was determined using everted sac technique in phosphate buffer solution at pH 7.4. As it was expected, TEOS particles showed significantly lower binding potential to colon mucosa segments (20%) in comparison to hybrid (85%) and Eudragit1 FS 30D coated hybrid particles (76%). The adhesive capacity of ORMOSIL particles could be attributed to the interactions between the moieties within the mucus, such as sulfonic and sialic acid substructures and the particle surface. Thus, we may expect that in phosphate buffer at pH 7.4 electrostatic interactions between the positively-charged surface and the negatively charged mucus exist, promoting particle adhesion. Also, possibility of formation of secondary chemical bonds, such as hydrogen bonding and van der Waals’ forces could contribute to the mucoadhesive potential of the designed hybrid particles (Xu et al., 2006). The coated formulation E-HSP2 demonstrated slightly lower adhesive potential relative to its uncoated counterpart HSP2, probably because of the initial lag time needed for dissolution of the pH dependent coating thus shortening the exposure time of the positive charged ORMOSIL surface to the colon mucosa. It is important to notice that particle mucoadhesion could be considered a desired property prolonging the contact with the mucus layer, enhancing drug absorption and/or improving local therapy (Varum et al., 2008). Mucoadhesion is one of the drug delivery concepts that we used during the formulation (functionalization of the silica particles by APTES), besides size-dependent targeting as another rational drug delivery strategy for IBD treatment. We tried to avoid nonspecific adherence to the substrate and premature adherence to the intestinal mucus or

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cumulative drug release (%)

100 90

ISPB1 ISPB2 ISPB3 ISPB4 HSP1 HSP2 HSP3 HSP4

80 70 60 50 40 30 20 10 0 0

5

10

15

20

25

time (h) Fig. 4. In vitro release profiles of BDS from ISPs and HSPs in buffer solution at pH 2.0 (mean
SD, n = 3).

surfaces of other part of the gut by coating the hybrid silica particles with pH-sensitive polymer Eudragit1 FS 30D. 3.2.2. In vitro drug release studies In order to explore the capability of the designed particles for local colon delivery of BDS and efficient IBD treatment, prepared formulations of ISPs (samples ISPB1–4), HSPs (samples HSP1–4), as well as pH-sensitive coated HSPs (sample E-HSP2) were examined for in vitro drug release in buffer solutions with pH values 2.0 and 7.4, that are typically applied in order to mimic the in vivo GIT conditions. The drug release showed bi-phasic profile with an initial burst release and a slower release period. The initial burst release may be owned to the BDS molecules localized in the external pores of the particles, which are more accessible and are rapidly released while the slower release phase may be a result of BDS encapsulation into the designed matrix structures (Doadrio et al., 2004). The cumulative drug release in pH 2.0 from ISPs ranged from 35.78% to 53.33%, while from HSPs ranged from 53.59% to 99.85% over 24 h time period (Fig. 4). The cumulative drug release in pH 7.4 from ISPs ranged from 19.40% to 29.90%, while from HSPs ranged from 55.12% to 72.20% over 24 h time period (Fig. 5). Significant differences in the BDS release were observed inside the formulation groups (ISPs and HSPs), but it is important to notice that drug loading was not a key factor influencing the release process, having in mind that for performed studies accurately weighted particles corresponding to 100 mg BDS were

cumulative drug release (%)

100 90 ISPB1 ISPB2 ISPB3

80 70 60

ISPB4 HSP1 HSP2 HSP3 HSP4

50 40 30 20 10 0 0

5

10

15

20

25

time (h) Fig. 5. In vitro release profiles of BDS from ISPs and HSPs in buffer solution at pH 7.4 (mean
SD, n = 3).

used. Consequently, drug release process was governed mainly by the functionality of the carrier (polymer-matrix structure and properties). Considering the effect of the pH, it can be clearly seen that the release of BDS from both formulation groups decreased with increasing pH from 2.0 to 7.4 (Figs. 4 and 5). These findings on the release of BDS can basically be explained as follows: (i) at pH 2.0, occurrence of repulsive electrostatic interactions between partially protonated silanol groups and protonated BDS (for ISPs) or repulsive electrostatic interactions between highly protonated amino groups and hydroxyl groups of BDS (for HSPs) could trigger the drug release to a higher extend and (ii) at pH 7.4, occurrence of attractive intermolecular interactions, as possible hydrogen bonding between hydroxyl groups of BDS and dissociated silanol groups (for ISPs), and hydrogen interactions between protonated amino group of HSP and carbonyl and hydroxyl groups present in BDS structure (for HSPs), could retain the drug release (Yoncheva et al., 2014). Obtained results are in accordance with measured surface charge of ISPs and HSPs in tested buffer solutions. Also, significant different drug release profiles were observed for ISPs and HSPs-analogs in tested buffers over 24 h time period (Figs. 4 and 5). Irrespectively of the pH, BDS release form HSPs was faster compared with dissolution rate of BDS from ISPs analog. The apparent differences in drug release can be explained by the changes in the nature of the matrix with incorporation of organically modified alkoxide. Namely, in the direct co-condensation method, the amino-organosilane was introduced into the mother gel with TEOS simultaneously, leading to a homogeneous distribution of amino-organic moieties in the silica framework though amino groups are partly unavoidably present inside the silica wall (Yokoi et al., 2012), thus, decreasing the degree of orderliness of the matrix and leading to extensive cross-linking and the formation of ramified, three-dimensional species (Kamarudin et al., 2013). Consequently, the modification with amino groups of TEOS particles led to pores enlargement (in accordance with increase in particle size and TEM images) and cannot hinder the BDS release by diffusion (Szegedi et al., 2011). The cumulative drug release in pH 2.0 from HSP2 was 40.55% and from E-HSP2 was 2.20%. The cumulative drug release in pH 7.4 from HSP2 was 66.39% and from E-HSP2 was 66.47% (Fig. 6). When comparing the drug release from pH-sensitive coated hybrid particles (sample E-HSP2) with uncoated ones (HSP2), it can be clearly seen that Eudragit1 FS 30D coating successfully sustained the release of BDS in acidic media, while no significant differences in BDS release were observed at pH 7.4 (Fig. 6). In order to understand the drug release mechanisms, the release data were analyzed using Higuchi’s square root of time equation, power law and modified power law. The main parameter values are listed in Table 6. The goodness of fit was evaluated using the correlation coefficient r. Comparing the values of the r of different kinetics models it can be clearly seen that release data showed best fitting to the modified power law. The modified power law takes into account an initial release phase, either burst or lag time that differ from the main release phase. Low values of n (below 0.5) are indicative for porous matrix structure, where diffusion is the main mechanism controlling the release. 3.2.3. Stability studies After 12 months storage at controlled ambient conditions, no significant differences (p < 0.05) in particle size, particle size distribution, encapsulation efficacy and drug release rate of the optimal formulation coated with Eudragit1 FS 30D (E-HSP2) were observed.

83

100 E-HSP2 (pH 7.4) HSP2 (pH 7.4)

90 80

cumulative drug release (%)

cumulative drug release (%)

V. Petrovska-Jovanovska et al. / International Journal of Pharmaceutics 484 (2015) 75–84

70 60 50 40 30 20

100

E-HSP2 (pH 2.0) HSP2 (pH 2.0)

80 60 40 20 0 0

1

2

time (h)

10 0 0

5

10

15

20

25

time (h) Fig. 6. In vitro release profiles of BDS from HSP2 and E-HSP2 in buffer solution at pH 2.0 and 7.4 (mean
SD, n = 3). Table 6 Comparison of different dissolution kinetics models in phosphate buffer at pH 7.4. Formulation code

HSP1 HSP2 HSP3 HSP4 E-HSP2

Higuchi

Power law

Modified power law

k (h1)

r2

k (h1)

r2

n

k (%h1/2)

r2

n

17.68 17.45 20.22 20.20 17.85

0.766 0.881 0.744 0.727 0.775

0.375 0.323 0.286 0.269 0.338

0.981 0.990 0.913 0.956 0.989

0.186 0.198 0.202 0.177 0.191

0.078 0.055 0.100 0.069 0.074

0.994 0.998 0.993 0.998 0.999

0.436 0.497 0.289 0.340 0.426

4. Conclusion Based on all experimental results, it can be concluded that Eudragit coated ORMOSIL particles loaded with BDS could be suitable candidates for oral delivery of BDS with controlled release properties, making them new potential carriers for local treatment of IBD. The preparation procedure used in this study, two-step process, offers simplicity, reproducibility and stability of the prepared formulations during one year storage period at controlled room temperature. Eudragit coating successfully sustains BDS release in the upper GIT while providing potential for efficient release in the colon. Results from the physicochemical and biopharmaceutical characterization of the prepared particles are in favor of their localization and prolonged residence time in the colon, which can reduce the systemic toxic effects and improve the therapeutic efficacy of the drug. Acknowledgment The authors would like to acknowledge Professor Maja Cvetkovska from Faculty of Technology and Metallurgy, UKIMSkopje, Macedonia for scientific contribution in this study. References Andrade, G.F., Soares, D.C.F., dos Santos, R.G., Sousa, E.M.B., 2013. Mesoporous silica SBA-16 nanoparticles: synthesis, physicochemical characterization, release profile, and in vitro cytocompatibility studies. Microporous Mesoporous Mater. 168, 102–110. Asouhidou, D.D., Triantafyllidis, K.S., Lazaridis, N.K., Matis, K.A., 2009. Adsorption of Remazol Red 3BS from aqueous solutions using APTES- and cyclodextrinmodified HMS-type mesoporous silicas. Colloids Surf. A: Physicochem. Eng. Asp. 346, 83–90. Bandi, N., Wei, W., Roberts, C.B., Kotra, L.P., Kompella, U.B., 2004. Preparation of budesonide- and indomethacin-hydroxypropyl-beta-cyclodextrin (HPBCD) complexes using a single-step, organic-solvent-free supercritical fluid process. Eur. J. Pharm. Sci. 23, 159–168.

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