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Research Article CODEN: IJPRNK IMPACT FACTOR: 4.278 ISSN: 2277-8713 Amir Ibrahim Mohamed, IJPRBS, 2014; Volume 3(5): 177-193 IJPRBS

Available Online at www.ijprbs.com 177

GLYCERYL MONO OLEATE CUBOSOME / CA-ALGINATE MICROPARTICULATE

SYSTEM: A NEW MATRIX TO IMPROVE THE RELEASE PROPERTIES OF A WATER-

SOLUBLE MODEL DRUG

AMIR IBRAHIM MOHAMED1, OSAMA A. A. AHMED2, SUZAN AMIN3, OMAR ANWAR ELKADI4,

MOHAMED A. KASSEM5

1. Lecturer, Military Medical Academy, Cairo, Egypt. 2. Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia. 3. Medical services Department, Cairo, Egypt. 4. Faculty of Pharmacy, Cairo University, Egypt. 5. Department of Pharmaceutics, Faculty of Pharmacy, Cairo University, Cairo, Egypt.

Accepted Date: 11/09/2014; Published Date: 27/10/2014

Abstract: Objective: Alginate polymers suffer from rapid release of water soluble drugs in physiologic salt concentration. In this study, a new microsphere delivery system composed of GMO-cubosome embedded in Ca-alginate was designed to improve the release properties of a water-soluble model drug (Clindamycin). Methods: GMO-alginate based microspheres were prepared by w/o emulsion method, and formulation variables such as alginate molecular weight, concentration, surfactant ratio, and homogenization speed were fixed, while alginate:GMO ratio and drug concentration were investigated. Results: Alginate:GMO ratio significantly affected the particle size and morphology. Microparticles with low GMO contents (1:0.25) were spherical in shape with average diameter of 35.2 μm, with increasing GMO contents (1:0.5 to 1:1), microparticles become more flattened, collapsed and larger in size. Degradation study revealed that the erosion of microspheres decreased significantly by GMO-cubosomes addition as compared to blank Ca-alginate microspheres. 50% weight loss was reached at approximately 8hrs for 1:0.25 GMO containing microspheres as compared to 5.5hrs for blank microspheres without cubosomes, indicating that the presence of cubosomes increased the stability of Ca-alginate microspheres. Additionally, the extent of Clindamycin release from the Alginate:GMO(1:0.25) microspheres was 47% lower than the release from the conventional microspheres. Conclusions: These novel microparticles have a uniform structure, confined size distribution, satisfactory entrapment efficiency, and acceptable sustained release properties.

Keywords: Glyceryl mono-oleate (GMO); Cubosome dispersion; Ca-alginate; Clindamycin Hcl; Sustained release

microspheres; Water-soluble drug.

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Corresponding Author: DR. AMIR IBRAHIM MOHAMED

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Amir Ibrahim Mohamed, Osama A. A. Ahmed, Suzan Amin, Omar Anwar

Elkadi, Mohamed A. Kassem; IJPRBS, 2014; Volume 3(5): 177-193



INTRODUCTION

Lipids of various types have been extensively studied for drug delivery by different routes of

administration. Polar amphiphilic lipids such as glyceryl mono-oleate (GMO) Figure (1a) when

placed in water spontaneously forms thermodynamically stable lipid bilayers, and three types

of liquid crystalline phases (lamellar, reversed hexagonal and cubic phase) depending upon the

temperature and water content [1]. GMO–water cubic phase dispersions (Cubosomes) are

usually produced by combining water (concentrations from 55 to 85%) with 15–45% GMO at 40

°C. Then, the resultant cubic liquid crystalline gel is dispersed into particles via the application

of mechanical or ultrasonic energy. Finally, the cubosomes are stabilized against flocculation by

stabilizer poloxamer 407 (Pluronic F127) additions [2]. Structure of the cubic phase is unique

and consists of a curved bicontinuous lipid bilayer extending in three dimensions, separating

two congruent networks of water channels Figure (1c). The water pore diameter of the fully

swelled phase is about 5 nm and the phase is very viscous [1].

Monoglycerides are used in many food applications such as in bread and cake production for

improvement of shelf life and flavour retention [3]. The particular properties of monoglyceride-

based cubic phases, temperature stability, bicontinuous structure, high internal surface area,

and low cost raw materials, make them desirable for personal cure product and pharmaceutical

industry applications [4]. In addition, the stiffness and high viscosity make the GMO–water

cubic phase an excellent in situ forming biodegradable matrix type drug delivery system with

varying molecular weights and solubilities in water, such as Aspirin, vitamin E, Oxybutynine

hydrochloride, Metronidazole, Tetracycline, Timolol maleate, Chlorpheniramine maleate,

Propranolol HCl, Melatonin, and Haemoglobin [1]. Cubosomes have been shown to have a high

efficiency in entrapping antigens as well as providing extended antigen release. Sustain release

over an extended period of time may reduce the need for multiple vaccination, which will be a

benefit in terms of reduced costs and increased patient compliance [5]. Finally, bio-adhesive

nature of GMO cubic dispersion was found to be a gastro-retentive carrier system suitable for

both polar and as well as non-polar drugs. Cubic nanoparticles significantly enhanced oral

bioavailabity of Simvastatin, Cyclosporine, and Silymarin [6]. GMO formulations undergo

digestion in the gastrointestinal tract where ester bond of GMO is rapidly cleaved by pancreatic

lipase during the digestion to form oleic acid and glycerol [7].

Clindamycin [7(S)-chloro-7-deoxylincomycin] is a water soluble anti-biotic drug Figure (1d). It is

synthesized from microbially fermented lincomycin by replacing a hydroxyl group at the 7-

position of lincomycin by a chlorine group which significantly increases its activity [8].

Clindamycin is highly effective against Gram-positive and Gram-negative anaerobic pathogens,

as well as Gram-positive aerobes, the primary effect is exerted by its binding to the 50S

ribosomal subunit and the consequent inhibition of bacterial protein synthesis [9]. Clindamycin

is widely distributed throughout the body and has an average biological



half-life of 2.4 h. The major bioactive metabolites excreted in urine and feces are clindamycin

sulfoxide and N-desmethylclindamycin [10]. Clindamycin is mainly used in the treatment of

serious respiratory tract infection, serious skin and soft tissue infections, septicemia, intra-

abdominal infections and infections of the female pelvis and genital tract caused by susceptible

anaerobic bacteria [11].

Sodium alginate is a sodium salt of alginic acid, a naturally occurring, anionic, linear non-toxic

polysaccharide found in brown algae consisting of varying ratios of Guluronic and Mannuronic

acid units Figure (1b). Alginate has been widely used as food and pharmaceutical additives, such

as a tablet disintegrate and gelling agent. Alginate delivery systems are formed when

monovalent, water-soluble, salts of Guluronic, and Mannuronic acid residues undergo cross-

linking gelation with divalent cations, such as Ca++ into water-insoluble gel matrix. Each Ca++ ion

takes part in nine co-ordination link with an oxygen atom, resulting in three-dimensional

network of calcium-alginate, the so-called "egg-box" structure Figure (1e) [12, 13]. Alginate

polymers have been widely used in biomedical applications as they are biodegradable and

biocompatible, but suffer from the limitation of rapid drug release in physiologic salt

concentration. In the presence of monovalent (e.g. sodium) salts, insoluble calcium alginate

gets converted into soluble form (sodium alginate), resulting in rapid disintegration of the

delivery system and drug release [14].

Among the different approaches to achieve sustained release drug delivery, the use of

polymers, specifically biodegraded alginate, and monoglycerides holds great promise. In this

work, a new microsphere delivery system composed of GMO-cubosome embedded in Ca-

alginate was designed to improve the release properties of a water-soluble model drug

Clindamycin Hcl.

Figure 1 Molecular structure of the compounds under investigation; (a) Glyceryl mono-oleate

(GMO), (b) Na-alginate, (c) Cubic phase in three dimensions [1,4], (d) Clindamycin, (e) Ionic

interaction between alginate & divalent cations [15].



MATERIALS AND METHODS

1. Materials

Clindamycin hydrochloride, was purchased from Sigma-Aldrich Chemie GmbH (Riedstrasse,

Germany). Sodium Alginate was purchased from Sas chemicals (Mumbai, India). Monoolein was

obtained from Danisco Emulsifiers (DIMODAN® MO 90/D, Denmark). Sorbitane Monooleate

(Span80) was obtained from Loba Chemie (Mumbai, India). Pluronic© F127 was purchased

from Sigma-Aldrich Chemie (USA). Procine pancreatice lipase (59 IU/mg) was obtained from

AppliChem GmbH (Darmstadt, Germany). Methanol, Iso-propyl alcohol, Dichloromethne (DCM)

of analytical grade, and Calcium chloride dehydrate were purchased from El-Nasr chemicals Co.

(Cairo, Egypt).

2. Methods

2.1. Preparation and Characterization of GMO-Cubic Dispersion

Blank dispersions of GMO were prepared by conventional fragmentation method, which

involves mechanical dispersion of bulk cubic gel in presence of pluronics as stabiliser [4, 16]. In

details; 300mg GMO was weighed into 5mL glass vial, heated to 45 °C in a water bath, then

water (0.7ml) was gently dropped on the surface of lipids, and finally incubated at room

temperature 24hrs to allow formation and equilibration of the cubic gel phase. The resultant

viscous cubic gel was mixed with 1.5ml of 2% Pluronic F127 solution (10:1, GMO: F127 w/w) to

form a coarse dispersion. This dispersion was subsequently homogenised using a microtip

probe sonicator (4mm diameter tip, 25KHz frequency and 30 Watts power output, VCX series,

Sonics, USA) for 2 min at 25 °C. Particle size, morphology, distribution, and polydispersity (PDI)

were determined using Malvern Zetasizer 3000 (Malvern, UK), and Transmission electron

microscope (TEM), JEM-2100F (Jeol, Japan).

2.2. Preparation and Characterization Ca-alginate microspheres

Alginate microspheres were prepared by conventional water-in-oil (W/O) emulsion method

with some modifications [17-19]. Briefly, 10 ml of 4% (w/v) sodium alginate was poured into

250ml rounded bottom glass container which contained 50 ml DCM and 2 ml Span 80, and then

emulsified for 2 min at 15200 rpm using homogenizer. 2cm magnetic bare was placed into the

emulsion and the glass container tightly closed by a rubber closer. Microspheres were prepared

by adding 8 ml of 5% (w/v) CaCl2 (dissolved in 1:2 mixture of methanol and isopropyl alcohol) to

the emulsion drop by drop via 10ml syringe at1000 rpm and stirring for 60 min to assure

efficient cross-linking. In this step, microspheres were formed in suspension. Microsphere

suspension was allowed to stabilize on ice for about 10 min, and the microspheres were

collected by filtration in vacuum, washed with isopropyl alcohol twice and finally dried at room

temperature. The morphology and average particle diameter were characterized by Scanning

c) )



electron microscope (SEM), (Phnom-ProG2, Netherlands) after samples drying under vacuum,

and observed at 5 KV. For particle size analysis, the diameter of 50-100 particles was measured

and averaged from photos by a computer software (Particlemetric, Phenomworld) came

together with SEM equipment.

2.3. Preparation and Characterization GMO-Ca-alginate microspheres

Different volumes of blank GMO-dispersion were emulsified into 10 ml of 4% (w/v) sodium

alginate solution by vortexing at 1000 rpm (IKA Labortechnik, Germany) for 2 min to get a final

Alginate:GMO w/w ratios of 1:1, 1:0.75, 1:0.5, and 1:0.25. These primary emulsions were

further emulsified into 50 ml DCM/2ml Span 80, and the microspheres were prepared as

described before. The effect of GMO addition on Ca-alginate microspheres shape, size, and

degradation degree was studied then the optimum Alginate:GMO ratio was determined.

2.3.1. Morphology observation

The interior morphology of GMO-loaded Ca-alginate microspheres was examined using

scanning electron microscopy at an accelerating voltage of 5 kV.

2.3.2. In-vitro Degradation study

Degradation degrees of the free Ca-alginate microspheres and GMO-loaded Ca-alginate

microspheres were estimated gravimetrically from mass loss of microspheres (weight

remaining at a specific time relative to initial weight)[20]. In details, 50mg microspheres of each

formulations was placed in individual accurately weighed empty 2ml Eppendorf tube weighted

as We (Electronic Analytical Balance XB 220A, Precesia, Swaziland). 1.5 ml of Phosphate buffer

solution (PBS) (pH 7.4) was added to each tube and allow standing for 1 hour, then PBS was

removed from the Eppendorf tubes by centrifugation at 15000 rpm for 30 minutes (Z326K,

Hermle Labor Technik, Germany) and withdrawn by 3ml syringe. The tube containing swelling

microspheres was weighed as W0. Fresh 1.5 ml of PBS (pH 7.4) was added to each tube and

then all tubes were kept in a thermostated orbital shaker (Stuart, Bibby Scientific, United

Kingdom) that was maintained at 37°C and 100 rpm. At predetermined time intervals, the

degradation medium (PBS) was removed by centrifugation at 15000 rpm for 30 minutes and the

tubes containing microspheres residue were reweighed as Wt , then the degradation degree

calculated as follows:

Degradation % = W0 – Wt / W0 - We X 100 %

Where W0 is the initial swelling microspheres weight, Wt is the residue weight after time t, and

We is the individual Eppendorf tube weight.

New fresh 1.5 ml of PBS was added back to each tube and the experiment was continued until

no residue remained. All tests were performed in triplicate and the mean ±SD was calculated.



2.4. Preparation and Characterization Drug loaded Ca-alginate microspheres

Serial drug concentrations were used to prepare both Clindamycin-alginate and Clindamycin-

GMO-alginate microspheres. The effect of drug concentration on Ca-alginate microspheres

shape, size, and entrapment efficiency was studied then the optimum Drug:Polymer ratio was

determined.

2.4.1. Clindamycin-alginate microspheres preparation

Microspheres containing 1:0.25, 1:0.5, 1:0.75, 1:1, 1:2, and 1:3 w/w Alginate:Clindamycin ratios

were prepared by dissolving 100, 200, 300, 400, 800, and 1200mg Clindamycin into 10 ml of 4%

(w/v) sodium alginate solution by vortexing for 2 min. These primary mixtures were further

emulsified into 50 ml DCM/2ml Span 80 as described before.

2.4.2. Clindamycin-GMO-alginate microspheres preparation

Using the optimum GMO:Alginate ratio obtained before, the same Alginate:Clindamycin ratios

were prepared by dissolving 100, 200, 300, 400, 800, and 1200mg Clindamycin into 2 ml

distillate water then added to the molten GMO heated to 45 °C in a water bath. The resultant

viscous cubic gel incubated at room temperature 24hrs, mixed with 1.5ml of 2% Pluronic F127

solution, and homogenised using the sonicator for 2 min at 25 °C. Finally, Drug-GMO

dispersions were emulsified into 10 ml of 4% (w/v) sodium alginate solution by vortexing for 2

min and further emulsified into 50 ml DCM as described before.

2.4.3. Microspheres Characterization

The prepared Clindamycin-alginate and Clindamycin-GMO-alginate microspheres were

examined using SEM at an accelerating voltage of 5 kV.

2.4.4. Drug content determination

Accurately weighed 10mg microspheres were added to 10ml phosphate buffer (pH 7.4) in 15ml

falcon tube and left for 24hrs under orbital shaking 150rpm at 37 °C [17]. After 60 min

centrifugation at 6000 rpm, the supernatant liquid was collected and the concentration of

Clindamycin was analyzed by an UV spectrometer (Shumadzu 1700, Japan) against blank of Ca-

alginate microspheres to eliminate any polymer effect. The characteristic absorbance of

Clindamycin at 205 nm was recorded and compared with a standard curve generated from the

Clindamycin concentrations varying from 0 to 0.25mg/mL. The ratio of the actual to the

theoretical drug contents in microspheres was termed as entrapment efficiency. 1ml procine

pancreatice lipase (1500 IU/ml) digestion medium was added into phosphate buffer (pH 7.4) in

case of Clindamycin-GMO-Alginate microspheres to assure complete drug recovery.



2.5. In-vitro release studies

In vitro drug release from the optimum formulation microspheres was studied using the Dialysis

bag method[21]. 6cm long, 1cm wide dialysis bag molecular weight cut off 12000–14000

(Cellulose tubing membrane, Sigma-Aldrish, USA) was soaked in deionised water for 12hrs

before use. Then, accurately weighed amounts (10mg) of microspheres were filled into the

dialysis bag with the two ends fixed by thread and suspended into 500ml Stoppered Flask

containing 250ml phosphate buffers (pH 1.2, 4.5, and 7.4). The Flask was placed on a magnetic

stirrer (100 rpm) at 37 °C for 2 hrs with pH 1.2 buffer, then dialysis bag transferred into pH 4.5

buffer for another 2hrs, and finally, into pH 7.4 buffer to the end of the experiment. At fixed

time intervals of 30 minutes, 2ml samples were withdrawn and replaced with fresh medium.

The drug content was determined by using a UV Spectrophotometer at a λmax of 205nm

against blank of Ca-alginate microspheres. Since GMO in formulation was assumed to undergo

digestion in the gastrointestinal tract, in-vitro release test for Clindamycin-GMO-Alginate

microspheres was carried out in presence and absence of 1ml procine pancreatice lipase (1500

IU/ml) digestion medium. The measurements were performed triplet for each batch and the

cumulative percent released calculated by summing the drug amount in each sample with the

amounts of previous time point then the total divided by the microspheres drug content.

2.6. Statistical analysis

Tests including in-vitro degradation, entrapment efficiency, and in-vitro release were all

repeated in three separate experiments. Statistical analysis of the difference between each

group was tested by One-way ANOVA (SPSS software), and the results were considered

significant differences when P < 0.05.

RESULTS AND DISCUSSION

Cubosomes are self assemble liquid crystalline particles with a microstructure that provide

unique properties in a size range of 100 nm–10000 nm. There unique structure makes them

biologically compatible and capable to potentially retard release and protect the encapsulated

active against chemical and/or physiological degradation. An attempt has been made in the

present study to improve the controlled release properties of Ca-alginates microspheres using

Glyceryl mono-oleate (GMO) cubic phase dispersions containing a water-soluble model drug;

Clindamycin Hcl.

1. GMO-Cubic Dispersion Characterization

Cubic gel prepared Figure (2a) was clear, highly-viscous gel that does not flow under gravity.

After ultrasonic homogenisation, the resulting average particle size of GMO-dispersions was

between 126 - 150 nm and PDI ranged from 0.51 to 0.55 Figure (2b&c).



2. Ca-alginate Microsphere characterization

In the preparation of Ca-alginate gel microspheres by w/o emulsion method, the aqueous

emulsion droplets, containing polymer, transformed into solid microsphere upon addition of

CaCl2. The divalent calcium ions are bound in a highly cooperative manner to the guluronic

acids units of the alginate (cross-linking), leading to the formation of water-insoluble gel

particles. A SEM photograph of blank Ca-alginate microspheres was shown in Figure (3). As

seen from the photograph, the microspheres were almost of spherical in shape and have a

rough surface. The average diameter of microspheres was 24.8 μm ± 3.7μm; therefore, the

microspheres were considered an appropriate size for oral drug delivery [19]. This small particle

size was attributable to cooperative consequence of rapid diffusion of the water phase

(including alginate) into the external oil phase and further disrupted into smaller droplets

because of higher rapid homogenization speed [18].

Figure 2Photographs of GMO-Cubic Dispersion preparation; (a) Cubic gel, (b) Cubosome dispersion TEM photo, (c) Particle size distribution.



Figure 3 SEM photographs of Ca-alginate microspheres at different magnifications; (a) 140X

magnification, (b) 630X magnification, (c) 7800X magnification, (d) 13500X magnification.

2. GMO-Ca-alginate Microsphere characterization

2.1. Morphology observation

The microspheres prepared with GMO/alginate were examined as shown in SEM photographs

Figure (4). The Alginate:GMO ratio significantly affected the particle shape and morphology.

Particles with low GMO contents (1:0.25) were spherical in shape with white-doted surface and

average diameter of 35.2 μm ± 2.4μm Figure (4a,b&c). With increasing GMO contents (1:0.5,

0.75, &1), microparticles become more flattened, collapsed and larger in size (40-66 μm

average diameter) Figure (4d,e&f). This size increase may be related to higher alginate network

density produced by increased GMO concentration and thus reduced homogenization

efficiency.



Figure 4 SEM photographs of GMO-Ca-alginate microspheres with different Alginate:GMO

w/w ratios of 1:0.25 (a,b&c), 1:0.5 (d), 1:0.75 (e), 1:1 (f).

2.2. In-vitro Degradation study

When Ca++ crosslinked alginate hydrogels are placed in PBS 7.4, the ion-exchange process

between the Ca++ ions presenting in the ‘‘egg-box’’ cavity of polyguluronate blocks and Na+ ions

of buffer solution is mainly responsible for the swelling/degradation behaviour of Ca-alginate

microspheres [13]. The degradation of Ca-alginate microspheres formed with different GMO

concentration at constant cross-linking conditions, were studied and the percentage of weight

loss of microspheres as a function of degradation time is presented in Figure (5). We observed

that the weight loss was faster at the beginning, and then the degradation ratio slowly

increased with incubation time. The mass loss of the microspheres in the early stage may be a

result of a lower molecular part of the alginate polymer dissolving into the degradation

medium, while in the later stage of degradation, the mass loss may be due to the bulk

hydrolysis [22]. The erosion of microsphers decreased significantly by GMO-cubosomes

addition as compared to blank Ca-alginate microspheres. The degradation rate decreases

obviously with increasing Alginate:GMO w/w ratios from 1:0.25 to 1:0.5 . Further increase in

GMO concentration leads to a slight decrease of the degradation rate. 50% weight loss was



reached at approximately 8hrs for 1:0.25 GMO containing microspheres as compared to 5.5hrs

for blank microspheres without cubosomes, indicating that the presence of cubosomes

increased the stability of Ca-alginate microspheres. These results may relate to that, increased

alginate viscosity with increased GMO concentration, produced a tighter network matrix that is

more resistant to erosion and degradation.

Figure 5 Degradation profiles of blank and GMO-loaded

Ca-alginate microspheres.

3. Drug loaded Ca-alginate microspheres Characterization

The scanning electronic micrographs of Clindamycin-alginate and Clindamycin-GMO-alginate

microspheres are shown in Figures (6&7). The average diameter and entrapment efficiency of

these microspheres were summarized in Table (1).

Figure (6) shows morphological features of Alginate:Clindamycin microspheres at different drug

ratios. With low drug concentrations (1:0.25, and 1:0.5); alginate microspheres were generally

spherical, with average diameter ranged from 6.7 μm to 15.7 μm which obviously smaller than

those of the blank alginate microspheres (24.8 μm). A significant more reduction in

microsphere size (from 5.5 μm to 3.8 μm) was noted by increasing Clindamycin concentrations

(from 1:0.75 to 1:1) which a combined with the appearance of many sub-micron particles

Figure (6c&d). At higher drug ratios (1:2, and 1:3) the microspheres become fused, collapsed,

and irregular in presence of scattered aggregates of the sub-micron particles Figure (6e&f). This

could be related to hydrophilic-lipophilic properties of lincosamide antibiotics include

clindamycin [23], which may affect the surfactant (Span 80) activity.

Figure (7) shows the SEM images of Clindamycin-GMO-Alginate microspheres with different

drug ratios. At low Clindamycin concentrations (1:0.25) microspheres were generally oval in



shape, with average diameter of 27.4± 3.7μm Figure (7a), which smaller than those of the blank

GMO-alginate microspheres (35.2 μm). The microspheres with more drug ratios (1:0.5, 1:0.75

and 1:1) were less spherical in shape with collapsed centre and many aggregates of the sub-

micron particles Figure (7b,c,&d). Higher drug ratios (1:2, and 1:3) leaded to irregular and fused

microspheres formation, Figure (7e&f) which proved Clindamycin surface active agent

properties.

Figure 6 SEM photographs showing the effect of Alginate:Clindamycin ratio on microspheres

morphology (a) 1:0.25, (b) 1:0.5, (c) 1:0.75, (d) 1:1, (e) 1:2, (f) 1:3 w/w Alginate:Clindamycin.

Figure 7 SEM photographs of Clindamycin-GMO-Alginate microspheres; (a) 1:0.25, (b) 1:0.5,

(c) 1:0.75, (d) 1:1, (e) 1:2, (f) 1:3 w/w Alginate:Clindamycin ratio.



The values of entrapment efficiency of Clindamycin-alginate, and Clindamycin-GMO-

Alginate microspheres were shown in Table (1). The higher entrapment efficiencies were

observed with low drug ratios (1:0.25 & 1:0.5) in both formulations ranging from 85.57 to

87.46% for Clindamycin-Alginate and from 92.63 to 94.40% for Clindamycin-GMO-alginate

microspheres. These high entrapment efficiency can be explained by the method of

microsphere preparation, which involve the dispersion of hydrophilic drug solution in an

organic phase (DCM) to form a w/o emulsion and thus inhibit drug diffusion into preparation

medium[17]. Increasing drug ratios (1:0.75, 1:1, 1:2, & 1:3) decreased the entrapment

efficiency suggesting that the quantity of polymer present becomes insufficient to entrap the

hydrophilic drug. In addition, the porosity of alginate gel microspheres could be responsible of

the fast release of small molecular weight Clindamycin during the washing step [20]. The

entrapment efficiency of GMO-alginate microspheres was significantly higher than those of

conventional alginate microspheres at all Clindamycin-drug ratios. This result indicated that the

addition of GMO caused the formation of a core barrier that reduced drug leakage during

preparation and washing process. Moreover, GMO highly increase the internal structure

density of alginate matrix, and thus smaller pore channels for drug to diffuse out.

Table 1 The effect of Alginate/GMO:Clindamycin ratio on Particle Size and Entrapment

Efficiency.

Formulation

Composition (mg) Average diameter (μm)

Entrapment efficiency (%)

Alginate GMO Clindamycin

Blank Ca-alginate 400 -- -- 24.8 ± 3.7 -- Alginate – Clindamycin 1:0.25 1:0.50 1:0.75 1:1 1:2 1:3

400 400 400 400 400 400

-- -- -- -- -- --

100 200 300 400 800 1200

15.7± 2.1 6.7± 2.2 5.5± .2.3 3.8± 1.4 fused fused

85.57± 2.5 87.46± 1.3 83.72± 1.1 81.36± 1.6 79.76± 1.4 77.83± 2.1

Blank GMO-Ca-alginate 400 100 -- 35.2 ± 2.4 --

Alginate -GMO- Clindamycin 1:0.25 1:0.50 1:0.75 1:1 1:2 1:3

400 400 400 400 400 400

100 100 100 100 100 100

100 200 300 400 800 1200

27.4± 3.7 23.2± 2.6 irregular irregular fused fused

94.40± 5.4 92.63± 4.2 87.45± 3.7 83.15± 3.5 80.21± 4.6 79.42± 3.2



Considering the criteria of microspheres morphology, and entrapment efficiency; (1:0.25) w/w

Clindamycin-GMO-alginate microspheres was selected as optimum formulation for further

studies.

4. Drug-release studies

Figure (8) shows the release profiles of Clindamycin pure drug, Clindamycin-alginate, and

Clindamycin-GMO-alginate microspheres using the Dialysis bag method at pH 1.2, 4.5, and 7.4.

Being soluble in water, Clindamycin pure drug showed approximately total release (99.68%)

within 3hrs while, the conventional Ca-alginate microspheres showed 73.52% release within

3hrs. These quick releases could be attributed to drug hydrophilicity, and/or the removal of the

cross-linker bivalent cation, Calcium, from the alginate microspheres by monovalent cations,

such as Sodium or Potassium contained in phosphate buffers [24]. Addition of GMO dispersion

was shown to sustain drug release for a longer period of time (only 20.73% cumulative release

within 3hrs), which attributed to water uptake of GMO and formation of cubic phase that is

highly viscous and acts as a rate-limiting factor in drug release. Moreover, the release profile

from GMO-alginate microspheres showed a marked increase in drug release rate at pH (7.4) in

compared with very slow release at pH (1.2), which proved the pH effect on alginate release

properties. Many reports provided that Ca-alginate hydrogel shrinks at low pH, leading to a

much narrower pore size network for drug to diffuse out through; and swells at higher pH,

which broaden the network pore size and accelerate drug diffusion [20, 24]. Lipase digestion

medium has another effect on the release profile from GMO-alginate microspheres as shown in

Figure (8). A complete drug released (99.66%) was reached within 12.5 hrs in presence of lipase

enzyme compared to 99.58% cumulative release after 23hrs without lipase, which concluded

that digestive enzymes can obviously affected the drug release from our designing GMO-

alginate microspheres.

Figure 8 Release profiles of Clindamycin from GMO-Alginate microspheres.



CONCLUSION

In this study, a new kind of microparticle was prepared based on GMO- cubic phase dispersions

and Alginate using emulsification and cross-linking. These spherical particles had uniform

structure, confined size distribution, satisfactory entrapment efficiency, and acceptable

sustained release properties. (1:0.25) w/w Alginate-GMO microspheres showed a remarkable

entrapment efficiency (>94%) and prolonged Clindamycin release (>12hrs).This type of

microparticle matrix may have significant advantages for Ca-alginates microspheres controlled

release properties.

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