in vivo evaluation of Zn-pectinate microparticles reinforced with chitosan for the colonic delivery of progesterone.

http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–14 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2015.1028602

RESEARCH ARTICLE

Development and in vitro/in vivo evaluation of Zn-pectinate microparticles reinforced with chitosan for the colonic delivery of progesterone Hytham H. Gadalla, Ghareb M. Soliman, Fergany A. Mohammed, and Ahmed M. El-Sayed

Drug Delivery Downloaded from informahealthcare.com by Nyu Medical Center on 04/15/15 For personal use only.

Department of Pharmaceutics, Faculty of Pharmacy, Assiut University, Assiut, Egypt

Abstract

Keywords

The colon is a promising target for drug delivery owing to its long transit time of up to 78 h, which is likely to increase the time available for drug absorption. Progesterone has a short elimination half-life and undergoes extensive first-pass metabolism, which results in very low oral bioavailability (25%). To overcome these shortcomings, we developed an oral multiparticulate system for the colonic delivery of progesterone. Zn-pectinate/chitosan microparticles were prepared by ionotropic gelation and characterized for their size, shape, weight, drug entrapment efficiency, mucoadhesion and swelling behavior. The effect of crosslinking pH, cross-linking time and chitosan concentration on progesterone release were also studied. Spherical microparticles having a diameter of 580–720 mm were obtained. Drug entrapment efficiency of 75–100% was obtained depending on the microparticle composition. Microparticle mucoadhesive properties were dependent on the pectin concentration, as well as the cross-linking pH. Progesterone release in simulated gastric fluids was minimal (3–9%), followed by burst release at pH 6.8 and a sustained phase at pH 7.4. The in vivo study revealed that the microparticles significantly increased progesterone residence time in the plasma and increased its relative bioavailability to 168%, compared to the drug alone. This study confirms the potential of Zn-pectinate/chitosan microparticles as a colon-specific drug delivery system able to enhance the oral bioavailability of progesterone or similar drugs.

Chitosan, colon-targeting, microparticles, mucoadhesion, oral bioavailability, progesterone, Zn-pectinate

Introduction Colon-specific drug delivery systems have gained increasing interest in the last decades due to the well-recognized importance of this region of the GIT, both for local and systemic administration of conventional and labile drugs (Yang et al., 2002; Maestrelli et al., 2008a; Bai et al., 2015). Drugs which are destroyed by the stomach acid or metabolized by pancreatic enzymes are minimally affected in the colon (Vervoort et al., 1998). The colon is also a useful site for delivery of drugs where a delay in drug absorption is required from a therapeutic point of view, e.g. in case of nocturnal asthma, angina, rheumatoid arthritis, etc (Rubinstein, 1995; Vervoort et al., 1998; Sinha & Kumria, 2003; Ramasamy et al., 2013). Moreover, colonic delivery is beneficial for systemic absorption of some drugs, e.g. nifedipine, theophylline, isosorbide due to the unique metabolic activity of the colon that makes it an attractive organ for targeting with various drug delivery systems (Bode et al., 1996; Ishibashi et al., 1999; Semde´ et al., 2000).

Address for correspondence: Ghareb M. Soliman, Department of Pharmaceutics, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt. Tel: +201023091884. Email: [email protected]

History Received 31 January 2015 Revised 9 March 2015 Accepted 9 March 2015

Various approaches have been utilized for achieving colonspecific drug delivery. They can be categorized as: (a) Prodrug approach (Riley & Turnberg, 1990; McLeod et al., 1994); (b) Pressure-based systems (Muraoka et al., 1998; Jeong et al., 2001); (c) The temporal control of delivery (Steed et al., 1997; Del Curto et al., 2009); (d) pH-based systems (Klein et al., 2005; Basak & Adhikari, 2009; Sareen et al., 2014), and (e) Enzyme-based systems (Xi et al., 2005; Varshosaz et al., 2006; Kshirsagar et al., 2011; Ramasamy et al., 2013; Rai et al., 2014). Polysaccharides which undergo hydrolysis of their glycosidic bonds by the microflora of the colon appear to be the most promising because of their attractive properties, such as wide availability, low cost of production, low toxicity and biodegradability (Chourasia & Jain, 2004). Pectin is an anionic polysaccharide consisting of linear chains of a-(1-4) linked D-galacturonic acid residues (El-Gibaly, 2002) (Figure 1A). Pectin is non-toxic and not affected by gastric or intestinal enzymes while being completely degraded by pectinolytic enzymes produced by microflora in the colon (Bourgeois et al., 2006). The amidated low methoxy (LM) pectin, in which some of the carboxylic acid groups are amidated, is more tolerant to pH variations which makes it an appropriate candidate for the design of colon-targeted drug delivery systems (Oliveira et al., 2010). However, pectin high aqueous solubility and swelling

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H. H. Gadalla et al.

Figure 1. (A) Chemical structure of pectin showing its functional groups: (a) carboxyl; (b) amide; (c) ester groups. (B) Chemical structure of chitosan. The indices x and y represent the mole fractions of D-glucosamine and N-acetyl-D-glucosamine moieties, respectively. (C) Schematic illustration of the formation of PG-loaded microparticles. * Indicates continuation of the structure.

Drug Deliv, Early Online: 1–14

(A)

a O

COOH O

HO

b

OH

CONH2

O

O HO OH

c O

COOCH3 O

HO OH O


(B) HO * O

OH

NH2

HO O

O O OH

NHCOCH3 O

O

OH

*

O NH2

OH

y

x (C)

properties are considered the major drawbacks that lead to rapid drug release during transit in the upper GIT (Maestrelli et al., 2008a; Wei et al., 2014). This necessitates the combination of pectin with other polymers or targeting strategies and also highlights the need for cross-linking of pectin chains to maintain the integrity of the dosage form until it reaches the colon (Mura et al., 2003). Chitosan is a cationic polysaccharide obtained by alkaline N-deacetylation of chitin, the main component of the protective shells of crustaceans (Sinha et al., 2004). It is a co-polymer of D-glucosamine and N-acetyl-D-glucosamine (George & Abraham, 2006) (Figure 1B). Chemically, chitosan has one primary amino and two free hydroxyl groups in each D-glucosamine building block. Due to the availability of this amino group, chitosan carries positive charges at pH 6.5 which enable it to react with a variety of negatively charged polymers and surfaces (Sinha et al., 2004). However, chitosan microspheres have shown limited strength with very fast drug release in the stomach due to the high solubility of chitosan in acidic media (Risbud et al., 2000). Despite these results, the use of chitosan for colonic targeting offers a great advantage

in virtue of its insolubility at pH values above 6.5 that prevail in the jejunum and ileum parts of the small intestine while it gets soluble again at the colon pH (Wittaya-areekul et al., 2006). Thus, chitosan has to be combined with another polymer to prevent premature drug release in the stomach and ideally release it in the colon (Wittaya-areekul et al., 2006). Pectin/chitosan microparticles that are formed by the electrostatic interactions between the pectin carboxylate groups and chitosan amino groups could overcome chitosan shortcomings and prevent premature drug release in the stomach due pectin insolubility in acidic media. Further, the insolubility of chitosan at pH 6.5 overcomes the pectin high solubility in the small intestine and prevents premature drug release before reaching the colon. These microparticles have many other desirable properties as drug-delivery vehicles, such as biocompatibility, non-toxicity, mucoadhesiveness and biodegradability (Chen & Fan, 2007; Mennini et al., 2008; Jin et al., 2014). Pectin/chitosan microparticles can be further reinforced through ionic cross-linking between pectin and zinc ions with the formation of semi-interpenetrating polymer networks.

Zn-pectinate microparticles for colonic delivery


DOI: 10.3109/10717544.2015.1028602

Progesterone (PG) is a naturally-occurring female sex hormone, widely used for maintenance of pregnancy in case of habitual or threatened abortion. It is also used as an oral contraceptive, alone or in combination with estrogen in addition to many other uses as hormone replacement therapy (Sweetman, 2009). PG is currently administered by I.M. injection or as rectal and vaginal suppositories. Natural PG has a very short elimination half-life (19–95 min) and undergoes extensive pre-systemic elimination in the liver and gut wall which results in very low oral bioavailability (25%) (Dollery, 1999). Higher doses are then required to achieve effective plasma drug concentrations, which further increase the risk of GI side-effects and also augment the economic burden of treatment. These shortcomings could be overcome by developing a suitable colonic delivery system for PG. The current work was then undertaken to establish the feasibility of creating Zn-pectinate/chitosan microparticles (MPs) for the colonic delivery of PG. The MPs were prepared at different pectin/PG ratios and at different chitosan concentrations. They were evaluated for their size, shape, mucoadhesion strength and PG entrapment efficiency. PG release kinetics from the microparticles were also investigated. Selected MPs were tested in vivo for their ability to increase PG plasma residence time and enhance its oral bioavailability.

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Table 1. Preparation conditions of progesterone-loaded Zn-pectinate/ chitosan microparticles.

Formula Code F1 F2 F3 F4 F5 F6 F7 F8

Cross-linking pH

Chitosan concentration (% w/v)

Cross-linking time (min)

Pectin/ PG ratio

4.8 3 1.2 1.2 1.2 1.2 1.2 1.2

0.5 0.5 0.5 0.1 0.25 0.5 0.5 0.5

120 120 120 120 120 10 30 120

4:1 4:1 4:1 4:1 4:1 4:1 4:1 2:1

were allowed to cure in the cross-linking solution for 120 min. The MPs were separated, washed three times with deionized water and dried at room temperature for 48 h and subjected to further evaluation. Empty MPs were prepared following the same procedures and were used as control. Different batches were prepared by varying the formulation parameters, such as cross-linking time, cross-linking solution pH, pectin/PG ratio and chitosan concentration (Table 1). All batches were prepared in triplicates. Drug-polymer compatibility studies Fourier-transform infrared spectroscopy studies

Materials and methods Materials Micronized PG was a generous gift from Pharco Pharmaceuticals Inc. (Alexandria, Egypt). Genu pectin (type LM-104 AS-FS) with degree of esterification of 36% and degree of amidation of 14% was obtained from Pkelco, Atlanta, GA. Chitosan (average MW of 850 kDa) with degree of deacetylation of 90% was purchased from Industrial Manufacturing Co. (Kyoto, Japan). Mucin from porcine stomach, type II, with sialic acid content of 1%, acetonitrile and methanol (HPLC grade) were purchased from Sigma Chemical Co. (St. Louis, MO). Zinc acetate dihydrate, monobasic sodium phosphate, disodium hydrogen phosphate, hydrochloric acid, dehydrated ethanol and diethyl ether were obtained from Prolabo Chemicals (Cairo, Egypt). All other chemicals were of analytical grade and were used as received. Preparation of Zn-pectinate/chitosan MPs The MPs were prepared following the modified ionotropic gelation technique previously described by El-Gibaly (2002). In a typical experiment, pectin solution (4% w/v) in deionized water was prepared followed by dispersion of PG (1% w/v) under stirring until homogenous dispersion was obtained. Chitosan solution (1% w/v) was prepared by dissolving chitosan in 2% v/v aqueous acetic acid solution followed by mixing with the cross-linking agent solution (4% w/v Zn(CH3COO)2). The pH of this solution was adjusted to 1.2, 3 or 4.8. The homogenous, bubble-free pectin-PG mixture was then added drop-wise at an average rate of 1 ml/min with a disposable syringe (needle size 23 G) into 20 mL of a gently stirred mixture of chitosan/Zn(CH3COO)2. The falling distance was fixed at 5 cm. The produced MPs

Samples (3–4 mg) of PG, pectin, chitosan, empty and PG-loaded MPs were mixed with potassium bromide (IR grade), compressed into disks under vacuum and analyzed using JASCO FT-IR– 4200 type A (JASCO Co., Tokyo, Japan). Spectral scanning was done in the range between 4000 and 500 cm1. Differential scanning calorimetry studies Differential scanning calorimetric (DSC) analyses were performed using a computer-interfaced Shimadzu Calorimeter (Model DSC–50, Kyoto, Japan). Samples (3– 4 mg) of PG, pectin, chitosan, empty and PG-loaded MPs were placed in aluminum pans, sealed and continuously heated at a rate of 10 C/min in the range of 25–350 C under constant flow of nitrogen gas. Indium was sealed in an aluminum pan and used to calibrate the instrument. Characterization of microparticles Size and shape. Twenty MPs were randomly selected from each batch. Morphological analyses were performed on images captured by Leica EC3 camera connected to Leica optical microscope (Leica Microsystems, Heerbrugg Switzerland). The images were digitalized and the perimeter (P) and projected area (A) of the MPs were determined by image analyzing software (Imagej, ver. 1.48 Bethesda, MD). The pixel resolution was 8 mm per pixel. The length and breadth were taken as the longest and shortest diameter of each MP, respectively. The MP size was then calculated from equation (1) (Das et al., 2011):

Size ¼

ðlength þ breadthÞ 2

ð1Þ

4



The shape of MPs was shown in terms of elongation ratio (ER) and circularity (C) which were calculated from equations (2) and (3), respectively: Elongation ratio ðERÞ ¼

Circularity ðCÞ ¼

length breadth

4A p2

ð2Þ

ð3Þ


where A and P are the projected area and the perimeter of the MP as seen in the image, respectively (Berggren & Alderborn, 2001). According to Das et al. (2011), MPs are considered spherical when their corresponding ER values are less than 1.15. Also, the more spherical a particle, the closer its C to 1; while more elongated particles have lower observed circularity (Eun Shim et al., 2004). Surface topography of MPs was examined under scanning electron microscope (SEM). Samples of dried MPs were mounted onto stubs, sputter coated with gold in a vacuum evaporator and photographed using a scanning electron microscope (Jeol, JSM-5200, Tokyo, Japan) at 15 keV.

Morphological examination of MPs.

Fifty MPs were randomly selected from each batch and dried for 48 h at 25 C and relative humidity of 60%. Their weight before (WW) and after (WD) drying were accurately measured using an analytical balance (Sartorius TE-2145, Goettingen, Germany). Weight loss of MPs upon drying was calculated from the following equation:

Weight, weight loss and moisture content.

Weight loss ð%Þ ¼

WW WD 100 WW

ð4Þ

In order to completely remove moisture, the selected MPs were placed in oven at 60 C for 72 h or until no further weight change was observed and the weight of these completely dried MPs was recorded as (WF). The moisture content of MPs was calculated from the following equation (Das & Ng, 2010a): Mositure contentð%Þ ¼

WD WF 100 WD

ð5Þ

In vitro evaluation of the mucoadhesive properties of Zn-pectinate/chitosan MPs was carried out using the wash-off method previously described by Wittaya-areekul et al. (2006) with slight modifications. Mucin discs (200 mg) were prepared by direct compression using a single punch hydraulic press (Carver Inc. Wabash, IN) with 12-mm diameter flat-faced die tooling. The discs were obtained by applying a pressure of 2.5 tons for 1 min. Next, each disc was attached on a microscopic slide using cyanoacrylate glue and hydrated with 80 mL of enzyme-free simulated small intestine fluid (SSIF) (10 mM phosphate buffer pH 7.4, 0.02% Tween 80) for 1 min in order to obtain a gel-like adhesive surface. Twenty MPs from each batch were allowed to swell in SSIF for 3 min. The MPs were then attached to the mucin discs by placing a small stainless steel plug weighing 30 g over them for 2 min to force them to adhere to the pre-hydrated mucin disc. The slides were then placed horizontally in the glass cells of the USP XXV dissolution apparatus (Erweka, DT-D6, Heusenstamm, Germany) and MPs were forced to wash-off under the stirring motion of 50 rpm in 200 mL of SSIF (pH 7.4) at 37 C. The number of MPs remaining attached after 15, 30, 45 and 60 min was counted and mucoadhesion was expressed as the percent of MPs remaining attached at each time point.

In vitro mucoadhesion studies.

Swelling behavior of MPs. Swelling studies of MPs were

carried out in enzyme-free simulated gastric fluid (SGF, 0.1 M HCl, pH 1.2) and enzyme-free simulated small intestinal fluid (SSIF, pH 7.4). Constant weight of MPs (100 mg) was placed in the baskets of the USP XXV dissolution apparatus (Erweka, DT-D6, Heusenstamm, Germany) and immersed in 500 mL of these media. MPs were allowed to swell for the required period of time at 37 ± 0.2 C. At different time intervals, MPs were removed, dried with filter paper and their weight changes, after correction for drug loss were measured. In order to account for the drug loss, MPs were dried at each time point, weighed and a 5-mL sample was withdrawn from the swelling medium and assayed for drug content as described above. The amount of drug released into the medium was then added to the actual MPs weight to compensate for drug loss due to dissolution during the swelling period. The swelling index (SI) of MPs was then calculated from the following equation (Sharma et al., 2013): Swelling index ð%Þ ¼

Drug entrapment efficiency. MPs (25 mg) were crushed in a

mortar, extracted several times with dehydrated ethanol (50 mL), stirred for 2 h and then filtered through filter paper. The filtrate was properly diluted and the absorbance of an aliquot was measured at 242 nm using a Shimadzu, Double-Beam spectrophotometer 150-02, Kyoto, Japan. Empty MPs prepared under the same conditions were used as a control. The percent drug entrapment efficiency was calculated from the following equation: Entrapment efficiency ð%Þ ¼

AQ 100 TQ

ð6Þ

where AQ is the actual quantity of drug in the MPs and TQ is the total quantity of drug used in MP preparation.

WT W0 100 W0

ð7Þ

where WT and W0 are the weight of MPs at time t and at the beginning of test, respectively. The release of PG from Znpectinate/chitosan MPs was investigated using the in vitro USP XXV dissolution apparatus (Erweka, DT-D6, Heusenstamm, Germany) with the dissolution basket assembly at a rotational speed of 50 rpm and a temperature of 37 ± 0.2 C. In order to simulate pH changes along the GIT, the sequential pH change method reported previously was used with slight modification (Ribeiro et al., 2014). Thus, an accurately weighed amount of MPs containing 5 mg drug was immersed in 200 mL of SGF (0.1 M HCl, pH 1.2) for 2 h.

In vitro drug release studies.

DOI: 10.3109/10717544.2015.1028602

Subsequently, the baskets containing the MPs were removed and immediately transferred to 200 mL of fresh SSIF (10 mM phosphate buffer, pH 6.8). After 0.5 h, the baskets were removed and transferred to 200 mL of fresh SSIF (10 mM phosphate buffer, pH 7.4) for the rest of the study. All release media contained 0.02% Tween 80. At pre-determined intervals, 5-mL samples were withdrawn with an equal volume of fresh medium being added in order to maintain a constant volume of the release medium throughout the study. Samples were then assayed for PG content spectrophotometrically at 242 nm. The PG concentration was calculated from a previously constructed calibration curve. Empty MPs were used as a control. All the experiments were done in triplicates and the results were expressed as mean ± SD.


Kinetics of drug release. In order to find the best-fitting drug-

release model, the drug-release data was fitted into different models using linear regression analysis. The following models were used: Zero order: (Sood & Panchagnula, 1998) R ¼ k0 t First order: (Carbinatto et al., 2014) R ¼ 1 ek1 t Higuchi diffusion model: (Higuchi, 1963)


5

heparinized centrifuge tubes at the following time points: predose, 1, 2, 3, 4, 6, 8, 10, 12, 18, 24 and 30 h following drug administration (Andre´en et al., 2006). Sample preparation and assay method were performed as described previously with slight modifications (Wu et al., 2000). Briefly, blood samples were centrifuged at 5000 rpm for 15 min and plasma was separated. Liquid–liquid extraction with diethyl ether was performed to recover PG from plasma before analysis. Samples were then analyzed by high performance liquid chromatography (HPLC, Shimadzu 2010A, Kyoto, Japan) using a RP-C18 column (Phenomenex, 150 4.6 mm, 5 mm). The mobile phase consisted of filtered, degassed mixture of acetonitrile and water (70/30 v/v) pumped at a flow rate of 1 ml/min. The column was maintained at room temperature and the effluent was delivered directly into the UV-diode array detector (model K-2500; Knauer, Berlin, Germany). The detection wave length was set at 240 nm. The lower limit of detection for this assay method was found to be around 1 ng/ml. Drug recovery from plasma was found to be 492%. Serum concentrations versus time profiles were fit using onecompartment open model with oral input and first-order output. The peak plasma concentration (Cmax) and the time to attain peak concentration (Tmax) were obtained directly from the plasma concentration-time profile. Absorption rate constant (Ka), elimination half-life (t½), area under the curve (AUC) and other parameters were calculated according to conventional algorithms (Gibaldi, 1991).

R ¼ k2 t1=2 Baker–Lonsdale model: (Baker & Lonsdale, 1974) h i 3=2 1 ð1 Mt =M1 Þ2=3 Mt =M1 ¼ k3 t Hixson–Crowell cube root law: (Chawla et al., 2000) ðURÞ1=3 ¼ k4 t Korsmeyer–Peppas equation: (Ritger & Peppas, 1987) Mt =M1 ¼ k5 tn where R is the fraction of drug released at time t, k is the rate constant corresponding to each model, (UR) represent the unreleased fraction of drug, Mt/M1 denotes the fraction of drug released at time t and n is the diffusional exponent indicative of the release mechanism. In vivo evaluation of MPs. The in vivo study was designed as a randomized parallel study using twelve healthy male New Zealand white rabbits weighing 1.8–2 kg. All the experiments were done according to Animal Ethical Guidelines approved by Assiut University, Egypt. Rabbits were randomly divided into two groups, each consisted of six rabbits. They were fasted for 12 h prior to drug administration but had free access to water (Qureshi et al., 2014). Food was not allowed for further 12 h after drug administration. A dose of 10 mg/kg body weight of PG powder or equivalent dose of selected microparticle formulations (corresponding to 200 mg human dose) were filled into hard gelatin capsules of size 4 and then administered to rabbits by the aid of stomach tube. Blood samples of about 1–2 mL were withdrawn via an indwelling catheter inserted in the eye vein, into a 5-mL screw-capped

Statistical analysis of data All experiments were performed in triplicates and results were expressed as mean ± SD. Statistical analysis of data was performed using the Graph-Pad Prism version 5 software (La Jolla, CA). For statistical evaluation, the data was analyzed by one-way analysis of variance (ANOVA) with Newman–Keuls post-hoc test and differences were considered significant at p50.05.

Results and discussion Preparation of Zn-pectinate/chitosan microparticles Progesterone-loaded MPs were prepared by adding aqueous pectin solution containing dispersed drug to a mixture of chitosan and zinc acetate aqueous solutions. Gelled spheres were produced immediately due to the formation of intermolecular ionic cross-links between the negatively charged carboxyl groups of pectin and the divalent zinc cations. This bridging of pectin chains forms what is called the egg-box complex. These ionic cross-links were further reinforced by electrostatic interactions between the negatively charged carboxyl groups of pectin and the positively charged amino groups of chitosan (Figure 1C) (Marudova et al., 2004). Characterization of the prepared microparticles FT-IR studies The FT-IR spectra of PG, pectin, chitosan, empty and PGloaded Zn-pectinate/chitosan MPs are depicted in Figure 2. The PG spectrum shows two typical intense bands at 1662 and 1699 cm1 corresponding to stretching vibrations of the C¼O groups of PG (Liu et al., 2007). The spectrum of pectin


6



Figure 2. FT-IR spectra of (A) PG, (B) pectin, (C) chitosan, (D) empty Zn-pectinate/chitosan microparticles and (E) PG-loaded Zn-pectinate/chitosan microparticles.

(Figure 2B) shows a broad band between 3600 and 3000 cm1 due to stretching of its OH groups in addition to two important bands: low-intensity band at 1740 cm1 and a stronger one at 1639 cm1 which are attributed to the esterified and non-esterified carboxyl groups of pectin, respectively (Maestrelli et al., 2008a). Chitosan spectrum (Figure 2C) shows the characteristic bands of amidic C¼O stretching at 1652 cm1 and of amidic N-H bending at 1580 cm1 (Bigucci et al., 2008). The spectrum of empty MPs evidenced disappearance of pectin band at 1740 cm1 and strong reduction in the intensity of carboxylate ion band which was also shifted to lower wave number (1635 cm1), both indicative of Zn-pectinate formation (Maestrelli et al., 2008b). Further, the appearance of new band at 1749 cm1 in this spectrum indicates mutual interaction between the COO group of pectin and the +NH3 group of chitosan, thus confirming the electrostatic interactions between them (Marudova et al., 2005). However, these changes could not be clearly observed in the spectrum of PG-loaded MPs due to overlapping and masking of these changes by the strong absorption bands of PG, which were retained unchanged in all spectra, indicating no significant interactions with the MP components.

Figure 3(B) shows the thermogram of pectin where a forked peak appears at 190 C and 210 C due to its endothermic transition and melting process, respectively (Auriemma et al., 2013). Also, a small exothermic deviation from baseline at around 240 C was noticeable, indicating the start of pectin chains depolymerization (Maestrelli et al., 2008a). Chitosan (Figure 3C) shows a broad endothermic peak at 95 C due to the evaporation of absorbed water and a sharp exothermic peak starting at 280 C and gaining its maximum at 310 C due to chitosan degradation (Borges et al., 2005). The thermograms of both empty (Figure 3D) and PG-loaded MPs (Figure 3E) show disappearance of the degradation exothermic peaks of chitosan at 310 C and of pectin at 240 C, as well as pectin endothermic peaks which could be attributed to the electrostatic interactions between pectin and chitosan. Similar results were previously reported for chitosan-alginate polyelectrolyte complex (Simonoska Crcarevska et al., 2008). The thermogram of PG-loaded MPs (Figure 3E) shows the characteristic melting peak of PG with no change in the peak shape or position confirming the absence of physical or chemical interactions with the microparticle components. Reduction of PG peak intensity after loading into the microparticles might be attributed to the dilution effect.

Differential scanning calorimetry studies

Size and shape of microparticles

Differential scanning calorimetry (DSC) thermograms of PG, pectin, chitosan, empty and PG-loaded Zn-pectinate/chitosan MPs are presented in Figure 3. PG exhibited a characteristic sharp endothermic peak at 130 C corresponding to its melting (Figure 3A) (Memi¸sog˘lu et al., 2003).

The size of MPs from different batches ranged between 583.3 ± 30.0 and 718.3 ± 44.4 mm (Table 2). Statistical analysis showed no significant difference in size among formulations, except for the formula prepared at pectin/PG ratio of 2:1 (F8) which was very significantly smaller in


DOI: 10.3109/10717544.2015.1028602

7


Figure 3. DSC thermograms of (A) PG, (B) pectin, (C) chitosan, (D) empty Zn-pectinate/ chitosan microparticles and (E) PG-loaded Zn-pectinate/chitosan microparticles.

Table 2. Physical characteristics and entrapment efficiency of the prepared Zn-pectinate/chitosan microparticles. Formula code F1 F2 F3 F4 F5 F6 F7 F8

Size (mm)

Circularity (C)

Elongation ratio (ER)

Shape

Weight (mg)

Weight loss (%)

Moisture content (%)

%EE

663.7 ± 19.9 709.3 ± 19.4 718.3 ± 44.4 700.7 ± 10.1 681.3 ± 41.8 678.3 ± 60.1 708.3 ± 49.5 583.3 ± 30.0

0.94 ± 0.01 0.96 ± 0.01 0.95 ± 0.01 0.88 ± 0.03 0.88 ± 0.01 0.93 ± 0.01 0.90 ± 0.03 0.88 ± 0.02

1.12 ± 0.01 1.11 ± 0.02 1.14 ± 0.02 1. 2 ± 0.09 1.18 ± 0.08 1.15 ± 0.01 1.15 ± 0.07 1.16 ± 0.07

Spherical Spherical Spherical Non Spherical Non Spherical Spherical Spherical Non Spherical

27.2 ± 1.3 25.1 ± 1.3 22.4 ± 1.6 27.2 ± 3.4 23.9 ± 2.6 30.6 ± 1.6 30.9 ± 5 11.8 ± 0.6

92.07 ± 0.48 93.03 ± 0.12 94.08 ± 0.38 94.78 ± 0.37 94.68 ± 0.40 93.79 ± 0.43 94.04 ± 0.46 96.0 ± 0.39

6.09 ± 0.91 7.30 ± 1.90 9.65 ± 0.77 8.26 ± 1.81 6.21 ± 2.04 7.86 ± 1.39 8.35 ± 1.70 5.88 ± 1.01

76.7 ± 1.4 88.4 ± 5.2 101.2 ± 4.4 101.6 ± 6.3 97.9 ± 4.5 97.8 ± 7.2 98.7 ± 1.2 99.4 ± 2.37

Mean ± SD of three independent measurements.

size (p50.001). The smaller size of this formula might be attributed to its lower pectin content compared to other formulations. This produced a less viscous solution and yielded smaller droplets, which in turn resulted in smaller MPs (Jose et al., 2011). The elongation ratio (ER), along with circularity (C) provide an accurate indicator about the MP shape. MPs are considered spherical when their corresponding ER values are less than 1.15 and their C approaches unity (Eun Shim et al., 2004; Das et al., 2011). All formulations exhibited good sphericity as indicated by their quite acceptable ER and C values (Table 2). However, some formulations, namely those made with chitosan concentration50.5% or pectin/PG ratio of 2:1, possessed slightly higher ER and lower C values, presumably due to depression of one side of their weak surface during drying (Das et al., 2011). Maestrelli et al. (2008a) also demonstrated that lower degrees of cross-linking yielded microspheres with irregular shape. Weight, weight loss and moisture content The weight of 50 MPs from different batches after drying at room temperature for 48 h ranged from 11.8 to

30.9 mg (Table 2). Statistical analysis showed that the MP weight decreased very significantly (p50.001) with decreasing the cross-linking pH. For instance, the microparticle weight decreased from 27.2 ± 1.3 (F1) to 22.4 ± 1.6 (F3) when the pH decreased from 4.8 to 1.2, respectively. This could be attributed to the promotion of stronger cross-links at lower pH which resulted in contraction of polymeric chains with consequent squeezing of the entrapped water (Das et al., 2011). Moreover, MPs prepared at pectin/PG ratio of 4:1 were very significantly (p50.001) heavier than those prepared at pectin/PG of 2:1 (Table 2). This might be attributed to the two-fold increase in pectin concentration from 2 to 4% which facilitated the incorporation of more pectin into the MPs and thus increased their weight. There was no significant difference among formulations in their weight loss (WL) (Table 2), except for the formula F8 prepared at pectin/PG ratio of 2:1, which exhibited a very significantly higher WL (p50.001). This might be attributed to the lower pectin content of this formula and the lower cross-linking density leading to a more water permeable matrix (Maestrelli et al., 2008a). Moisture content (MC) of different batches after drying at 60 C for 72 h ranged between 5.88 and 9.65% (Table 2), which reflects good chemical and


microbiological stability of the formulations. MC increased significantly (p50.05) by reducing the cross-linking pH from 4.8 to 1.2 (6.09 and 9.65%, respectively). These results were not in accordance with those of Das et al. (2011) who reported a decrease in MC by decreasing the cross-linking pH. This contradiction may be related to differences in the characteristics of pectin used, such as its degree of amidation (DA) which affects its hygroscopicity. Further, the formula having pectin/PG ratio of 2:1 showed a significantly lower MC (p50.05) as compared to formula F3 made at a ratio of 4:1 (5.88 and 9.65%, respectively). This might be attributed to the lower polymer content of the former formula, in addition to its higher weight loss during the initial drying step (Table 2) (Das et al., 2011).


(A) 120

Microparticles attached to mucin (%)

8

F1 (pH 4.8) F3 (pH 1.2)

100 80 60 40 20 0 15

All the prepared formulations showed high PG entrapment efficiency of 88.4%, except for formula F1 in which the entrapment efficiency was 76.7% (Table 2). This high drug entrapment efficiency was attributed to the very limited aqueous solubility of PG, which prevented its diffusion from the MPs into the surrounding aqueous medium, in addition to the rapid formation of the cross-linked matrix (Mennini et al., 2012). Such high drug entrapment efficiency is advantageous for clinical applications where clinical response could be achieved with minimal amount of MPs. It was also noticed that the entrapment efficiency increased significantly (p50.001) from 76.7 to 101.2% upon decreasing the cross-linking pH from 4.8 to 1.2. Taking into account that the solubility of PG is unaffected by this pH change, one possible explanation is that the high cross-linking density at low pH leads to the formation of a more dense matrix with minimal drug leaching. This was further supported by a slight increase in the entrapment efficiency upon increasing the cross-linking time from 10 to 120 min (97.8 and 101.2%, respectively). Chitosan concentration had a minimal effect on the drug entrapment efficiency, possibly due to its low values in the formulations (0.1–0.5%) (Table 2). In vitro mucoadhesion studies Mucoadhesion offers the advantage of extending the dosage form residence time along the GIT, which could enhance the oral bioavailability and efficacy of treatment (Bigucci et al., 2008). Fortunately, pectin shows maximum mucoadhesion on the large intestinal tissue compared to other regions of the GIT, which represents an added advantage for colon targeting (Thirawong et al., 2007). It is noteworthy that the pressure applied to adhere MPs to the hydrated mucin discs is not relevant to the in vivo conditions but this test is helpful in screening the most promising formulation in terms of mucoadheisve strength. Figure 4(A) shows the effect of cross-linking pH on the mucoadhesion of Zn-pectinate/chitosan MPs. The MPs were prepared at chitosan concentration of 0.5%, pectin/PG ratio of 4:1 and cross-linking time of 120 min while the pH of crosslinking solution was either 4.8 (F1) or 1.2 (F3). Almost all the MPs fabricated at both pH values remained attached to the mucin discs for 30 min, after which the number of attached particles was dependent on the cross-linking pH. For instance,

60

(B) 120 Microparticles attached to mucin (%)


Drug entrapment efficiency of microparticles

30 45 Time (min)

Pecn/PG (4:1) (F3) 100

Pecn/PG (2:1) (F8)

80 60 40 20 0 15

30

45

60

Time (min)

Figure 4. (A) Effect of cross-linking pH on the mucoadhesive properties of Zn-pectinate/chitosan MPs prepared at pH 4.8 (F1) or pH 1.2 (F3) (Preparation conditions: chitosan concentration, 0.5%; pectin/PG, 4:1; cross-linking time, 120 min) (B) Effect of pectin/PG ratio on the mucoadhesive properties of pectin/chitosan MPs prepared at pectin/PG of 4:1 (F3) or 2:1 (F8) (Preparation conditions: pH, 1.2; chitosan concentration, 0.5%; cross-linking time, 120 min).

85 and 41.7% of MPs remained attached to the mucin discs after 1 h, for the MPs made at pH 4.8 and 1.2, respectively (Figure 4(A)). The ionization of pectin carboxyl groups (pKa 4.2) is higher at pH 4.8 compared to pH 1.2 which in turn, enhances its swelling and physical entanglement with mucin chains (Bigucci et al., 2008; Hagesaether et al., 2008). Figure 4(B) shows the effect of pectin/PG ratio on the mucoadhesive properties of MPs prepared at chitosan concentration of 0.5%, cross-linking time 120 min and pH 4.8. Reducing pectin/PG ratio from 4:1 to 2:1 adversely affected the microparticle mucoadhesive properties as indicated by a fall in the percent of adhering particles after 1 h from 41.7 to 5% (Figure 4B). This might be attributed to the lower pectin concentration employed along with the much smaller size of these MPs which make them easily washable from the mucin discs surface (Hagesaether et al., 2008). Microparticle swelling behavior Microparticles based on electrostatic interactions usually show pH dependent swelling behavior due to the pH effect on


DOI: 10.3109/10717544.2015.1028602

400 SGF

Swelling index (%)

350

SSIF

300 250 200 150 100 50 0 0

15

30

45

60

75

90

105

120

135

Time (min)


Figure 5. Swelling behavior of Zn-pectinate/chitosan MPs (F3) in enzyme-free simulated gastric fluid (SGF) and enzyme-free simulated small intestinal fluid (SSIF) as a function of time.

the ionization of the interacting species (Bigucci et al., 2008). Figure 5 shows that the MPs had a moderate swelling in SGF during the first 15 min where the swelling index was 150% and remained at that level for the rest of the study period. At the low pH of SGF, the carboxyl groups of pectin were not ionized, which reduced the repulsive forces among the polymer chains and decreased their swelling (Oliveira et al., 2010). Although chitosan was fully ionized in SGF and could increase the MP swelling, its lower concentration (0.5%) compared to that of pectin (4%) limited its effect on the MP swelling. Contrary to SGF, the MP swelling in SSIF was much higher. Thus, the microparticles continued to swell for up to 45 min where a swelling index of 330% was achieved which was very significantly higher than that observed in SGF (p50.001) (Figure 5). This higher swelling could be attributed to the ionization of pectin carboxylate groups in alkaline media, which increases the repulsion between the polymer chains and hence increases their swelling (Hiorth et al., 2006). The MP swelling index started to decrease after 1 h due to microparticle erosion and dissolution. This might be attributed to counter ion exchange between sodium and potassium ions in SSIF and zinc ions in the microparticles leading to the formation of water soluble pectin salts that facilitate microparticle erosion (Munjeri et al., 1997; Bajpai & Sharma, 2004). In vitro drug release In order to get an insight into the drug-release behavior from different formulations, all release studies were carried out under conditions simulating the different pH values encountered along the GIT. Thus, enzyme-free SGF (pH 1.2) was used for the initial 2 h, followed by enzyme-free SSIF (pH 6.8) for 0.5 h and finally enzyme-free SSIF (pH 7.4) until the end of release study. All the studied formulations showed a very significantly slower release in the enzyme-free SGF compared to the drug alone (p50.001) confirming the ability of the microparticles to prevent premature drug release in the stomach (Figure 6). Thus, PG release from Zn-pectinate/ chitosan microparticles in SGF was 3–9%, compared to 52% for the drug alone under identical conditions. Previous studies have shown that chitosan microparticles cross-linked with

9

tripolyphosphate released 60% of a lipophilic drug after 2 h in SGF (Wan et al., 2012). Thus, the minimal progesterone release from Zn-pectinate/chitosan microparticles in SGF confirms the ability of these microparticles to overcome the disadvantages of chitosan microparticles. Progesterone release rate rapidly increased upon increasing the pH to 6.8 and 7.4 (Figure 6). This pH-dependent drug release is presumably attributed to the pH-dependent swelling behavior of the microparticles as illustrated in Figure 5. High swelling and water uptake of the microparticles at pH 7.4 loosen the microparticle matrix giving a chance for the incorporated drug to be dissolved and released (Yu et al., 2009; Araujo et al., 2013). The effect of the cross-linking solution pH on the drugrelease pattern is illustrated in Figure 6(A). The microparticles prepared at a cross-linking pH of 1.2 pronouncedly released the drug at a slower rate (t1/2 ¼ 11.4 h) as compared to those cross-linked at pH 4.8 (t1/2 ¼ 7 h) (Figure 6A). This might be attributed to the reduced solubility and charge density of pectin at low pH which allowed its conformational transition from the two-fold into the more compact three-fold helical structure (Das & Ng, 2010b). In addition, non-ionic interactions between pectin and chitosan, such as hydrophobic interactions and hydrogen bonds are more favored at low pH (Lootens et al., 2003; George & Abraham, 2006; Bigucci et al., 2008). Contrary to the cross-linking pH, the crosslinking time did not affect the drug release rate. Thus, the MPs prepared at a cross-linking time of either 10 or 120 min have almost the same drug release rate (Figure 6B). This is in agreement with previous reports where an increase in the cross-linking time from 5 to 20 min did not appreciably affect the drug release rates (Maestrelli et al., 2008b; Mennini et al., 2008). This might be attributed to the rapid nature of ionic interactions where a minimum cross-linking time of 5 min was reported to be enough for complete formation of microspheres (Maestrelli et al., 2008b; Mennini et al., 2008). The effect of chitosan concentration on the drug release behavior is shown in Figure 6(C). Increasing the concentration from 0.1 to 0.5% w/v resulted in slowing the drug release rate in SSIF as evidenced by an increase in the t1/2 value from 8.5 to 11.4 h (Figure 6(C)). Higher concentration of chitosan might form a more rigid polymeric matrix through contraction of micro-voids which could retard the drug diffusion from MPs (Maestrelli et al., 2008a). Several studies have previously shown that a certain dosage form requires at least 2–4 h to reach the colon (Krishnaiah et al., 1998; Rubinstein, 2005). Taking into account that all the microparticles released a relatively small amount of progesterone during the first 3 h of the study, it could be assumed that most of the incorporated progesterone will be delivered to the colon. Kinetics of drug release In order to determine the exact mechanism of drug release from microparticles, the release data was analyzed according to various kinetic models (Table 3). A comparative evaluation of the obtained correlation coefficients showed that the Korsmeyer–Peppas model was the most appropriate to describe the kinetics of drug release from most microparticle formulations. Except for the formula prepared at pectin/PG

10



(B) 100 80 60 pH 4.8 40

pH 1.2

20

100

Cumulative drug released (%)


(A)

Free Drug

0

80 60 10 min. 40

120 min. Free Drug

20 0

0

5

10

15

20

25

30

0

5

10

15

20

25

30

35

Time (h)

Time (h)



(C) 100 80 60 0.1 %

40

0.5 % 20

Free Drug

0 0

5

10

15

20

25

30

Time (h)

Figure 6. (A) In vitro release profiles of PG powder and PG-loaded Zn pectinate/chitosan MPs prepared at a cross-linking pH of 4.8 (Formula F1) or pH 1.2 (Formula F3). (B) In vitro release profiles of PG powder and PG-loaded Zn pectinate/chitosan MPs cross-linked for 10 min (Formula F6) or 120 min (Formula F3). (C) In vitro release profiles of PG powder and PG-loaded Zn pectinate/chitosan MPs prepared at a chitosan concentration of 0.1% w/v (Formula F4) or 0.5% w/v (Formula F3). Release medium: enzyme-free SGF (pH 1.2) for the initial 2 h, followed by enzyme-free SSIF (pH 6.8) for 0.5 h and then enzyme-free SSIF (pH 7.4) until the end of release study (30 h).

Table 3. Kinetic assessment of drug release data from different Zn-pectinate/chitosan MPs according to various kinetic models. Correlation coefficient (R2) Formula code F1 F2 F3 F4 F5 F6 F7 F8

Zero order

First order

Higuchi-diffusion

Baker–Lonsdale

Hixson–Crowell

Korsmeyer–Peppas

Korsmeyer–Peppas release exponent (n)

0.806 0.807 0.795 0.788 0.794 0.807 0.786 0.712

0.917 0.885 0.865 0.882 0.877 0.881 0.854 0.724

0.906 0.903 0.895 0.890 0.895 0.904 0.887 0.833

0.955 0.936 0.924 0.933 0.933 0.941 0.914 0.765

0.881 0.859 0.841 0.850 0.849 0.856 0.831 0.720

0.953 0.957 0.963 0.953 0.969 0.941 0.947 0.924

1.32 1.20 1.51 1.81 2.19 1.54 1.58 0.88

ratio of 2:1 (F8), all formulations exhibited a Korsmeyer– Peppas release exponent (n) 41, indicating that the drug release from microparticles followed the so-called super caseII transport kinetics (Table 3). This is a type of non-Fickian diffusion, in which the system has an increased swelling at the relaxing front, usually occurring at long periods of time, i.e. diffusion driven by slow swelling of polymer chains (Langer & Peppas, 1981). On the other hand, the results obtained with formula (F8) suggested anomalous (non-Fickian) transport controlled by a combination of fickian diffusion and swelling due to relaxation of polymeric chains (n ¼ 0.88) (Ritger & Peppas, 1987).

Morphological examination of Zn-pectinate/chitosan microparticles Scanning electron microscopy images show mostly spherical particles with rough and rugged surfaces (Figure 7). Slight depression of one side of microparticle surface was noticed and was attributed to the drying process (Figure 7A). Drug crystals can be seen embedded in the microparticle matrix (indicated by arrows in Figure 7B). Microparticles prepared at a cross-linking pH of 4.8 (F1) show larger and deeper surface folds, able generate channels that would promote the diffusion of biological fluids into the matrix and facilitate drug


11


DOI: 10.3109/10717544.2015.1028602

Figure 7. Scanning electron microscope photographs (75 and 1000 magnifications) of different formulations of PG-loaded Zn pectinate/chitosan MPs. (A) Formula F1, (B) Formula F3 and (C) Formula F4. Left panels are taken at magnification 75 and right panels at 1000.

dissolution (Figure 7A, right panel). This might have contributed to the accelerated drug release observed from formula F1 (Figure 6A). In contrast, those prepared at pH 1.2 (F3) show a relatively smooth surface free from cracks and fissures (Figure 7B, right panel) that resisted the penetration

of release media and reduced drug release rate as shown in Figure 6(A). Microparticles prepared at lower chitosan concentration (0.1%) (F4) were slightly distorted in shape, in agreement with their higher elongation ratio (Table 2, Figure 7C). Rough texture, as well as numerous surface cracks


and pores could be clearly observed (indicated by arrows in Figure 7C, right panel). This may account for the higher drug release rate from this formula which was attributed to the lower degree of cross-linking in presence of lower chitosan concentration (Figure 6C) (Maestrelli et al., 2008b).


In vivo evaluation of PG-loaded microparticles Based on the higher drug-entrapment efficiency, acceptable mucoadhesive properties and greater ability to sustain drug release, PG-loaded microparticles (formula F3) were selected for further in vivo evaluation. The microparticles were filled into hard gelatin capsules of size 4 and orally administered to healthy rabbits. Equivalent dose of micronized PG powder was similarly treated and used as a control. The pharmacokinetic parameters obtained for the microparticles, in comparison with those of the free drug, are listed in Table 4. After oral administration of PG micronized powder, the plasma drug concentration increased progressively and achieved a peak concentration (Cmax) of 1.606 ± 0.037 mg/ml at a Tmax of 6 h. Later, it rapidly decreased and the drug was undetectable in plasma at 30 h post administration (Figure 8). In contrast, the increase in plasma PG concentration after administration of the drug-loaded microparticles (formula F3) was not as sharp as that observed for the drug alone. One hour post administration, the PG plasma concentration was relatively small (2.4 ± 1 ng/ml). Thereafter, the concentration increased gradually and a Cmax of 1.319 ± 0.008 mg/ml was achieved at a Tmax of 12 h, after which it started to slowly decrease. At 30 h post administration, the PG plasma concentration observed for the microparticles was 0.469 ± 0.013 mg/ml, which was very significantly higher than that achieved for the drug alone (p50.001). The drug mean residence time (MRT) in the plasma for the microparticles was more than two-fold higher than that obtained for the drug alone, confirming the ability of the microparticles to achieve a sustained drug effect (Table 4). Further, the drug clearance rate for the microparticles was 1.5 times less than that of the drug alone (Table 4). This significant prolongation of the drug MRT is presumably attributed to the slower drug release from the microparticles, which serve as a drug reservoir. The oral bioavailability of the drug from the microparticles was highly enhanced as indicated by the significantly higher AUC0–1 value of the microparticles compared to the free drug (p50.001). Thus, the relative bioavailability of formula (F3) was found to be 168.18% (Table 4). This much better performance of the drug-loaded microparticles might be attributed to their mucoadhesive properties, which increase the residence time in the GIT and hence the time available for drug dissolution and absorption. Further, drug-release studies (Figure 6) have confirmed that most of the microparticle-loaded drug was delivered to the colon, which might allow partial avoidance of pre-systemic drug elimination and enhance its bioavailability (Nappinnai et al., 2013).


Table 4. Pharmacokinetic parameters of PG after oral administration of micronized PG powder and Zn-pectinate/chitosan microparticles (formula F3) to rabbits. Pharmacokinetic parameters Cmax (mg/mL) Tmax (h)a Ka (h1) t½a (h) Kel (h1) AUC0–1 (mg h/mL) t½ (h) MRT (h)b ClT (mL/min) FR (%)c

Micronized PG powder

PG microparticles (Formula F3)

1.606 ± 0.037 6 0.296 ± 0.081 2.433 ± 0.668 0.063 ± 0.001 19.36 ± 0.845 10.92 ± 0.173 8.30 ± 0.106 16.81 ± 0.497 –

1.319 ± 0.008 12 0.201 ± 0.052 3.564 ± 0.913 0.050 ± 0.004 32.56 ± 0.723 13.89 ± 0.984 18.98 ± 0.312 11.07 ± 0.971 168.18

a

Tmax, time of maximum drug plasma concentration, observed from the plasma concentration-time profile. b Mean residence time (h) calculated as AUMC/AUC. c Relative bioavailability (%) calculated as AUC0–1 of microparticles 100/AUC0–1 of PG powder.

PG plasma concentration (µg/ml)

12

1.8 1.6

PG powder

1.4

F3

1.2 1 0.8 0.6 0.4 0.2 0 0

5

10

15

20

25

30

Time (h)

Figure 8. Mean plasma concentration-time profiles of PG after oral administration of selected Zn-pectinate/chitosan microparticle formula (F3) to healthy rabbits compared to the micronized PG powder.

preparation method. The mucoadhesion properties of the microparticles were dependent on the cross-linking solution pH and pectin concentration. Drug incorporation into the microparticles prevented its release in the stomach whereas burst release was observed at pH 6.8 followed by a sustained phase at pH 7.4. The cross-linking pH and chitosan concentration exhibited the major influences on drug release rate whereas the cross-linking time had a minimal effect. Drugloaded microparticles had a relative oral bioavailability of 168% and more than twice the plasma residence time of progesterone, compared to the drug alone. Taken together, these results confirm the potential of Zn-pectinate/chitosan microparticles as an effective colon-targeted system able to enhance the oral bioavailability of hydrophobic drugs, such as progesterone or similar drugs.

Declaration of interest Conclusion Colon-targeted Zn-pectinate/chitosan microparticles were successfully prepared and used to enhance the oral bioavailability of progesterone. Drug entrapment efficiency of 76.7% was achieved, confirming the efficacy of the

The authors report no conflicts of interest in this work.

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Formulation and characterization of atropine sulfate in albumin-chitosan microparticles for in vivo ocular drug delivery.

In vivo evaluation of vascular-targeted spheroidal microparticles for imaging and drug delivery application in atherosclerosis.

In vivo evaluation of thiolated chitosan tablets for oral insulin delivery.

Preparation and Evaluation of Enteric-Coated Chitosan Derivative-Based Microparticles Loaded with Salmon Calcitonin as an Oral Delivery System.

Production of microparticles with gelatin and chitosan.

Comparison of Simple Eudragit Microparticles Loaded with Prednisolone and Eudragit-Coated Chitosan-Succinyl-Prednisolone Conjugate Microparticles: Part II. In Vivo Evaluation of Efficacy, Toxicity, and Biodisposition Characteristics.

microparticles for cartilage regeneration.

Mucoadhesive microparticles for gastroretentive delivery: preparation, biodistribution and targeting evaluation.

Poly(γ-Glutamic Acid) Composite Microparticles.

Development of Novel Chitosan Microcapsules for Pulmonary Delivery of Dapsone: Characterization, Aerosol Performance, and In Vivo Toxicity Evaluation.

Development of a novel drug delivery system: chitosan nanoparticles entrapped in alginate microparticles.

Targeting delivery of tocopherol and doxorubicin grafted-chitosan polymeric micelles for cancer therapy: In vitro and in vivo evaluation.

Novel Pullulan-Eudragit® S100 blend microparticles for oral delivery of risedronate: formulation, in vitro evaluation and tableting of blend microparticles.

Bilayer mucoadhesive microparticles for the delivery of metoprolol succinate: Formulation and evaluation.

In vitro and in vivo evaluation of polyhydroxybutyrate and of polyhydroxybutyrate reinforced with hydroxyapatite.

chitosan microparticles containing the antituberculosis drug rifampicin.

pH-responsive thiolated chitosan nanoparticles for oral low-molecular weight heparin delivery: in vitro and in vivo evaluation.

Design, characterization and ex vivo evaluation of chitosan film integrating of insulin nanoparticles composed of thiolated chitosan derivative for buccal delivery of insulin.

Development and evaluation of thymol-chitosan hydrogels with antimicrobial-antioxidant activity for oral local delivery.

In vivo evaluation of biodegradable progesterone microspheres in mares.

Enzyme encapsulation in magnetic chitosan-Fe3O4 microparticles.

In vitro and in vivo evaluation of chitosan graft glyceryl monooleate as peroral delivery carrier of enoxaparin.

chitosan: microscopic, a nuclear imaging and an in vivo test analysis for burn treatment.

Targeted oral delivery of BmpB vaccine using porous PLGA microparticles coated with M cell homing peptide-coupled chitosan.