# Drug Release Profile from Calcium-Induced Alginate-Phosphate Composite Gel Beads

Authors: Yoshifumi Murata; Youko Kodama; Takashi Isobe; Kyoko Kofuji; Susumu Kawashima
Journal: International Journal of Polymer Science
 (2009)

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## Abstract

Calcium-induced alginate-phosphate composite gel beads were prepared, and model drug release profiles were investigated in vitro. The formation of calcium phosphate in the alginate gel matrix was observed and did not affect the rheological properties of the hydrogel beads. X-ray diffraction patterns showed that the calcium phosphate does not exist in crystalline form in the matrix. The initial release amount and release rate of a water-soluble drug, diclofenac, from the alginate gel beads could be controlled by modifying the composition of the matrix with calcium phosphate. In contrast, the release profile was not affected by the modification for hydrocortisone, a drug only slightly soluble in water.


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## Body

## 1. Introduction

The calcium salt of
phosphoric acid (CP) is distributed widely in nature; several types of CP are
constituents of animals. For example, hydroxyapatite is a component of bones and
teeth in humans. In general, CP is formed by the coexistence of phosphate ions
with calcium ions. CP is a harmless material and is used as an additive in foods
and medicines; for example, as an active ingredient to protect against dental
caries. The suitability
of CP as an injectable material has been investigated because of its
biocompatibility [1, 2], and CP-polymer composites have been studied as
functional biomaterials [3–7]. Natural polysaccharides, which have also been
used widely in the food industry, serve as dietary fiber and are not absorbed
in the gastrointestinal tract. An anionic polysaccharide, alginic acid, has
been used as a medicine for stomach ulcers as well as a food additive because
of its protective effect on the gastric mucosa when given orally. Alginic acid undergoes
a sol-gel transformation in response to cations; for example, a solution of
sodium alginate immediately forms a cured gel matrix (Alg-Ca) in the presence
of calcium ions. The Alg-Ca matrix is used not only as a vehicle for drug
delivery, but also as a material in biomedical engineering [8–10].An investigation of drug release
profiles of Alg-Ca showed that the release rate could
be controlled by coating and modifying the composition of the gel matrix [11–13].
However, the controlled release of a water-soluble drug from Alg-Ca is difficult
because the drug immediately diffuses through the matrix to the gel surface.
Therefore, a modification of the gel matrix structure is necessary to control
drug release from the Alg-Ca [14]. In the present study, calcium
alginate-CP composite gel beads (CAPs) containing a drug were prepared by a
simple method in aqueous solution and the rheological properties of CAP were tested,
followed by the investigation of the drug release profile from dried CAP in vitro.


## 2. Materials and Methods

### 2.1. Materials

Sodium alginate and diammonium
hydrogen phosphate (AP) were obtained from Nacalai Tesque, Inc. (Kyoto, Japan;
300 cps). Sodium diclofenac (DF), hydrocortisone (HC), Ca(H2PO4)2,
CaHPO4,Ca3(PO4)2, and hydroxyapatite
(particle size 46–149 μm) were purchased from Wako Pure Chemical Industries (Osaka, Japan).
All other reagents used were of analytical grade.


### 2.2. Preparation of CP Powder

CP was prepared by the reaction between calcium and ammonium
phosphate in aqueous solution as
follows. Equal volumes of 8% AP and 9.5% CaCl2 were mixed, resulting
in the immediate  appearance of a white precipitate. The suspension was
stirred with a magnetic stirrer for one day at room temperature and then centrifuged at 3000 rpm for 5
minutes. The precipitate was collected and washed three
times with water, then dried on a dish. It was desiccated under vacuum
in the presence of P2O5.


### 2.3. Preparation of CAP

CAP was prepared as
follows: 1.5 (w/w)% or 2.0 (w/w)% sodium alginate solution was prepared with
demineralized water and AP was added to the solution with agitation. The model
drug was then added to 10 g of solution and mixed until homogeneity. Two grams
of this solution then combined with 10 mL of 0.1–0.4 M CaCl2 solution and left to stand for 1 hour at room temperature, after which
spherical hydrogel beads were obtained. The beads were washed twice with 50 mL distilled
water and dried at  37°C for 24 hours on a
culture dish, and then
desiccated under vacuum in the presence of P2O5. The
morphology of Alg-Ca was observed with a digital microscope (VHX-200, Keyence
Co., Osaka, Japan). The dried Alg-Ca or CAP
prepared as described above was ground by electromotive mill (WB-1, Osaka
Chemical Co., Osaka, Japan), and a sample of fine powder was used for X-ray
diffractometry.


### 2.4. X-Ray Diffractometry

Powder X-ray diffractometry was conducted using an automatic diffractometer
(MXP3; MAC Science Ltd., Yokohama,
Japan) with a
voltage of 40 kV and a current of 20 mA. The scanning rate was  2°min−1 over a 2 theta range of 5–60°. The results of X-ray diffraction were
interpreted using the XPRESS computer program (Bruker AXS K.K., Yokohama, Japan).


### 2.5. Rheological Test of Hydrogel Beads

The load weight (g) was
measured three times with a rheometer (Rheo Tex, Sun Scientific Co. Ltd., Tokyo,
Japan) at room temperature and a strain of hydrogel beads pressed with a flat
adapter (diameter: 15 mm) reached 0.5 mm.


### 2.6. Drug Release Test from CAP

The
release profile of the drug incorporated in CAP was determined in 500 mL of physiological saline using a
JP XIII dissolution test apparatus (Toyama Sangyo Co., Tokyo; paddle method,37±0.5°C, 150 rpm). A 5 mL aliquot of test
solution was removed periodically and 5 mL of new medium (37°C) were added to
maintain  a constant volume. Absorbance was measured at 275 nm (for DF) or 248 nm
(for HC) using an  ultraviolet spectrometer (UV-1200, Shimadzu, Kyoto, Japan).
All dissolution tests were performed  in triplicate.


## 2.1. Materials

Sodium alginate and diammonium
hydrogen phosphate (AP) were obtained from Nacalai Tesque, Inc. (Kyoto, Japan;
300 cps). Sodium diclofenac (DF), hydrocortisone (HC), Ca(H2PO4)2,
CaHPO4,Ca3(PO4)2, and hydroxyapatite
(particle size 46–149 μm) were purchased from Wako Pure Chemical Industries (Osaka, Japan).
All other reagents used were of analytical grade.


## 2.2. Preparation of CP Powder

CP was prepared by the reaction between calcium and ammonium
phosphate in aqueous solution as
follows. Equal volumes of 8% AP and 9.5% CaCl2 were mixed, resulting
in the immediate  appearance of a white precipitate. The suspension was
stirred with a magnetic stirrer for one day at room temperature and then centrifuged at 3000 rpm for 5
minutes. The precipitate was collected and washed three
times with water, then dried on a dish. It was desiccated under vacuum
in the presence of P2O5.


## 2.3. Preparation of CAP

CAP was prepared as
follows: 1.5 (w/w)% or 2.0 (w/w)% sodium alginate solution was prepared with
demineralized water and AP was added to the solution with agitation. The model
drug was then added to 10 g of solution and mixed until homogeneity. Two grams
of this solution then combined with 10 mL of 0.1–0.4 M CaCl2 solution and left to stand for 1 hour at room temperature, after which
spherical hydrogel beads were obtained. The beads were washed twice with 50 mL distilled
water and dried at  37°C for 24 hours on a
culture dish, and then
desiccated under vacuum in the presence of P2O5. The
morphology of Alg-Ca was observed with a digital microscope (VHX-200, Keyence
Co., Osaka, Japan). The dried Alg-Ca or CAP
prepared as described above was ground by electromotive mill (WB-1, Osaka
Chemical Co., Osaka, Japan), and a sample of fine powder was used for X-ray
diffractometry.


## 2.4. X-Ray Diffractometry

Powder X-ray diffractometry was conducted using an automatic diffractometer
(MXP3; MAC Science Ltd., Yokohama,
Japan) with a
voltage of 40 kV and a current of 20 mA. The scanning rate was  2°min−1 over a 2 theta range of 5–60°. The results of X-ray diffraction were
interpreted using the XPRESS computer program (Bruker AXS K.K., Yokohama, Japan).


## 2.5. Rheological Test of Hydrogel Beads

The load weight (g) was
measured three times with a rheometer (Rheo Tex, Sun Scientific Co. Ltd., Tokyo,
Japan) at room temperature and a strain of hydrogel beads pressed with a flat
adapter (diameter: 15 mm) reached 0.5 mm.


## 2.6. Drug Release Test from CAP

The
release profile of the drug incorporated in CAP was determined in 500 mL of physiological saline using a
JP XIII dissolution test apparatus (Toyama Sangyo Co., Tokyo; paddle method,37±0.5°C, 150 rpm). A 5 mL aliquot of test
solution was removed periodically and 5 mL of new medium (37°C) were added to
maintain  a constant volume. Absorbance was measured at 275 nm (for DF) or 248 nm
(for HC) using an  ultraviolet spectrometer (UV-1200, Shimadzu, Kyoto, Japan).
All dissolution tests were performed  in triplicate.


## 3. Results and Discussion

When 1.5%
sodium alginate containing 0.8% AP was dropped into 0.2 M CaCl2 solution, a cured gel bead approximately 3.5 mm in diameter was formed. As
shown in Figure 1, the formation of CP in alginate gel matrix was observed visually.
A change in Alg-Ca also was observed when another phosphate, such as Na2HPO4 or K2HPO4, was added to the alginate solution. For 1.6%
AP addition, aggregation of the gel beads occurred in the solution. The results
show that formation of calcium-induced alginate gel matrix and that of CP
progressed simultaneously.Figure 1
Pictures of Alg-Ca (left) and CAP (right) prepared with 0.8% AP.Figure2
shows the  mass (g) required for deformation of the hydrogel bead. The beads
quickly stiffened in CaCl2 solution and a consistent hardness was achieved
at about 1 hour. Addition of 0.8% AP did not affect the rheological properties
of Alg-Ca prepared with 1.5% alginate, and the CAP showed same profile as that obtained
from Alg-Ca.Figure 2
Change in hardness of hydrogel beads (mean ± S.D.,n=5).
Open circles: Alg-Ca; closed circles: CAP (0.8% AP).Figure3 shows the X-ray diffraction
data of CPs and the powder prepared by grinding dried Alg-Ca. Three species of
calcium phosphate, Ca(H2PO4)2,
CaHPO4, and Ca3(PO4)2, each
showed a different diffraction pattern.
CP obtained from mixing the AP solution with a CaCl2 solution produced
several X-ray diffraction peaks with a pattern compatible with CaHPO4.
In addition, the powder prepared by grinding Alg-Ca containing CP also showed the
same X-ray diffraction pattern as CaHPO4. The crystal structure of
CP was maintained even after Alg-Ca (hydrogel) containing CP was dried and
ground using an electromotive mill. However, the diffraction pattern of ground CAP resembled that of ground
Alg-Ca (free CAP), which produced a “halo” pattern.
These results indicate
that CP does not exist in a crystalline form in CAP.Figure 3
X-ray diffractograms.
(1) Ca(H2PO4)2      (2)  CaHPO4  (3)  Ca3(PO4)2
(4) CP
(5) ground CAP (6) ground Alg-Ca containing CP.Figure4 shows the release profile of DF from dried Alg-Ca or CAP. Approximately 20%
of DF incorporated in Alg-Ca prepared with 1.5% alginate was released at 10 minutes.
The initial release decreased according to the increment of AP added to the sodium
alginate for the preparation of CAP; for example, release was 4.0±0.2%
at 10 minutes for CAP prepared with 1.5% alginate containing 0.8% AP. However, the
DF release profile from Alg-Ca containing 0.8% hydroxyapatite was the same as
that of Alg-Ca (data not shown). Thus, CP formed in Alg-Ca was
assumed to act as a part of the dried gel matrix. A change in release rate was not clearly recognized unless CaCl2 concentration did not change. When Alg-Ca was prepared with 2.0% alginate,
similar results were obtained.Release profiles of DF in physiological saline.
(a) Prepared with 1.5% alginate and 0.1 M CaCl2.
(b) Prepared with 2.0% alginate and 0.1 M CaCl2.
Open circles: Alg-Ca; closed triangles: CAP (0.2% AP);
closed squares: CAP (0.5% AP); closed circles: CAP (0.8% AP).
(a)(b)DF
is a water-soluble drug and is released immediately through routes formed by
permeation of the dissolution medium into the dried alginate gel matrix.
Calcium ions act as a raw material for formation of the alginate gel matrix, CP,
and/or alginate-phosphate complex.Table1 shows the release parameters of DF from Alg-Ca or CAP prepared at various
CaCl2 concentrations. For Alg-Ca (CP free), the
amount of DF incorporated increased, and the release rate decreased, based on
CaCl2 concentration (although the initial release amount did not
change greatly). The effect was more remarkable for CAP prepared with 0.8% AP, which
produced a release rate of 0.19% min−1.Table 1
Release parameters of DF from Alg-Ca or CAP prepared with 0.8% AP.
SampleCaCl2Release*Release rate**Incorporation(M)(%)(%min−1)(%)Alg-Ca0.119.0±2.10.94±0.0168Alg-Ca0.218.9±1.90.65±0.0588Alg-Ca0.416.8±1.80.59±0.0290CAP0.14.0±0.20.78±0.0174CAP0.21.2±0.10.29±0.0095CAP0.41.0±0.20.19±0.0194Measured* at 10 minutes, calculated** at 10–60
minutes.
Each release and release rate value includes mean ± S.D. (n=3).Figure5 shows HC release profiles from Alg-Ca. The initial release of HC was minor
and the drug was released gradually from Alg-Ca (CP-free). Neither alginate nor
CaCl2 concentration affected drug release rate. Modification of
Alg-Ca with CP also did not affect the HC release profile. HC is slightly
soluble in water, and the release rate, therefore, appears to be governed by
the dissolution of the drug as the solvent permeates the pores of the gel
matrix.Figure 5
Release profiles of HC from Alg-Ca or CAP in physiological saline.
Open circles: Alg-Ca (1.5% Alg, 0.1 M CaCl2);
open squares: Alg-Ca (1.5% Alg, 0.2 M CaCl2);
open triangles: Alg-Ca (2.0% Alg, 0.1 M CaCl2);
closed circles: CAP (1.5% Alg, 0.5% AP, 0.1 M CaCl2);
closed squares: CAP (1.5% Alg, 0.8% AP, 0.2 M CaCl2);
closed triangles: CAP (2.0% Alg, 0.5% AP, 0.1 M CaCl2).


## 4. Conclusions

In this study, CAP was
prepared and used for the investigation of drug release from a drug delivery
vehicle. Drug release occurs when the Alg-Ca surface comes in contact with the
dissolution medium. The initial release profile of drugs contained in Alg-Ca is
especially affected by the surface structure of the gel matrix. A water-soluble
drug DF was immediately released from the intact Alg-Ca, and the drug release
rate could be controlled by modifying the composition of the matrix with CP. However,
CP and/or calcium alginate are good candidates as implanted materials for bone
tissue engineering. Therefore, work is continuing to investigate the
biocompatibility of CAP.



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*Source: 729057-2009-01-25.xml*