Chitosan-Polyphenols Nanostructured Matrices Drug Release Kinetics Studies

SCIENTIFIC ANNALS OF “ALEXANDRU IOAN CUZA DIN IAŞI” UNIVERSITY Tomul I, s. Biomaterials in Biophysics, Medical Physics and Ecology 2008

CHITOSAN-POLYPHENOLS NANOSTRUCTURED MATRICES DRUG RELEASE KINETICS STUDIES Ana Gârlea 1, V. Melnig1, M. I. Popa2, G. Lisa2 KEYWORDS: tannic acid, chitosan, drug release, kinetic study. The chitosan-polyphenols nanostructured matrices obtained by phase inversion method in concentration range of 2–3% chitosan and 5–10 mm tannic acid stands as structured drug nanocontainers matrices. The nanostructured matrices were investigated by Scanning Electron Microscopy and X-Ray Diffraction, showing that the nanocontainers with dimensions in the range of 16.35–98.45 nm are congregating in clusters settled in layers having dimensions in the domain of 50–300 nm. The layers resulted from these nanostructures arrangement constitute a tannic acid–surfactant–chitosan matrix suitable for drug controlled release. The drug release kinetics performed in liquid models at pH 2, 5.2 and 9.3 by spectrophotometric analysis shown a zero order kinetic for a long time.

1.

INTRODUCTION

From previous studies [1 - 3] it was observed that the chitosan can be used in combination with a cationic surfactant (cetyl trimethylammonium bromide - CTAB) to form matrices for drugs entrapment. Considering the fact that both chitosan and CTAB are cationic components it is obvious that the matrix self-assembling in aqueous solution is dictated by the presence of the drug. Polyphenols are antioxidants characterized by the presence of several phenol functional groups. In human health, they are thought to be useful in combating oxidative stress, a syndrome causative of neurodegenerative and cardiovascular diseases. The main source of polyphenol antioxidants is nutritional, since they are found in most legumes and fruits. As polyphenol model we used tannic acid. In this paper are presented results related to the self-assembled membranes obtained using two matrices of 2% and 3% chitosan solutions in combination with the 6mm cationic surfactant (CTAB) and different concentrations of drugs (1, 2, 3, 5, 7 and 10mm). 2.

MATERIALS AND METHODES

The chitosan, purchased from Sherbrooke University (Quebec, Canada), has the N-deacetylation degree of 88%, the average molecular weight number of 150000, the average molecular weight of 350000 and the polydispersity index of 2.33. 1 2

Faculty of Physics, “Al.I. Cuza” University, Carol I Blvd., No.11, 700506, Iasi, Romania Faculty of Chemistry, “Gh. Asachi” University, D. Mangeron, 71A, 700050, Iasi, Romania

26

Ana Gârlea, V. Melnig, M. I. Popa, G. Lisa

The cationic surfactant, cetyl trimethylammonium bromide (C19H42BrN) (CTAB), was purchased from Chemapol. The tannic acid, with a molecular weight of 1701.20 was acquired from Sigma Aldrich. All solutions were made using a 1% (wt/wt) acetic acid (with a purity of 99.5%, from Chemical Company) in distilled water solution. Two matrices of 2% and 3% chitosan solutions were prepared by mixing the 6mm cationic surfactant (CTAB) with different concentrations of drug (1, 2, 3, 5, 7 and 10mm). The mixtures were stirred at room temperature for 24 hours, and the solutions were centrifuged at 4000 rpm/30 min for degassing. For the preparation of the investigated chitosan membranes it was used the dry phase inversion method: initially, the solutions were poured into Teflon moulds and then, they were left for evaporation in a thermostat chamber at 50°C for 24 hours. The microphase structure was investigated by X-Ray diffraction (XRD) performed on a DURON-2 diffractometer, employing nickel-filtered Cu Kα radiation (1.54182 Å) at 25 kV operational voltage. The order degree was evaluated by computing the interplanar distances, D, using the Bragg relation:

2 D sin θ = nλ ,

(1)

where n is an integer, θ is the incidence angle of X-rays, λ is the wavelength of diffracted X-rays and D is the interplanar distance of sample. The particle size was determined from X-ray diffractograms according to Scherrer relationship [4]: L=

Kλ , β cos θ

(2)

where L is the diameter of nanoparticle, K is a constant (0.89) and β is the half maximum line width. The membranes morphology was investigated by SEM on a VEGA TESCAN microscope. The kinetic studies were made using a NanoDrop-1000 spectrophotometer, for which the sample quantity required is of the order of microliters. This device is very convenient for this kind of studies because the analysis time is extremely small, and by extracting small quantities the process and the eluent concentration can be considered unchanged. The drug release kinetics from the 2 and 3% chitosan matrices was studied in three different elution media: hydrochloric acid (pH = 2), phosphate buffer solution (pH = 5.2) and urea (pH = 9.3). The drug release kinetic studies were made by submerging 20 mg of membrane in 50 mL eluent, taking samples at different times and measuring the drug absorbance. The samples were kept at the constant temperature of 37°C. The measurements were carried out in the UV range, at the wavelength of 277 nm where the tannic acid spectrum presents a maximum. At this wavelength, neither the solvent nor the polymer absorbs UV light.

CHITOSAN-POLYPHENOLS NANOSTRUCTURED MATRICES DRUG RELEASE …

3.

27

RESULTS AND DISCUSSIONS

From previous studies [5] it was observed that the X-ray diffractogram of pure chitosan shows an almost amorphous structure while, the membranes with CTAB show an increasing in crystallinity with the increasing of CTAB concentration. The chitosan presents a certain crystallinity degree, known from speciality literature, the most intense maximum being at 2θ = 27.25, for which D = 0.32 nm and the nanometric domains have a dimension of L = 17.96 nm. It was observed that especially in the case of CTAB the membranes present a high crystallinity degree with a complex interplanar structure and nanocrystals with dimension in the range 6.4–34.29 nm. 160 Tannic acid 3mm 7mm 10mm

Intensity (a.u.)

120

80

40

0 10

20

30

40

Difraction angle (2θ)

60

(a)

160

Tannic acid 3mm 7mm 10mm

120

Intensity (a.u.)

50

80

40

0 10

20

30

40

Difraction angle (2θ)

50

60

(b) Fig. 1 X-ray diagrams for 2% (a) and 3% (b) chitosan membranes with CTAB 6mm and different concentration of tannic acid.

28

Ana Gârlea, V. Melnig, M. I. Popa, G. Lisa

The X-ray diffractograms (Fig. 2) reveal an increasing in the crystallinity with the increasing of tannic acid concentration and a decreasing of mesophasic ordering due to the wide range of cluster dimensions. The nanodomains dimensions, L, computed by Scherrer relation (2) are 3 to 5 times bigger than in the case of CTAB clusters, varying from 16.35 to 98.45 nm while the interplanar distances, D, are not sensible diminished (Table 1). Comparing these values with those of pure CTAB nanocrystals (L = 27–34 nm) and with those of chitosan (L = 17.96 nm) it can be concluded that surfactant in association with chitosan dictates a certain ordering degree of the domains which determine the complex mesophasic structure. Table 1. Crystalline characteristics of chitosan with tannic acid-CTAB membranes obtained from the X-ray diffractograms. Sample Chitosan 2%_CTAB 6mm_tannic acid 3mm Chitosan 2%_CTAB 6mm_tannic acid 7mm Chitosan 2%_CTAB 6mm_tannic acid 10mm Chitosan 3%_CTAB 6mm_tannic acid 3mm Chitosan 3%_CTAB 6mm_tannic acid 7mm Chitosan 3%_CTAB 6mm_tannic acid 10mm

2θ

Intensity (a.u.)

D (nm)

L (nm)

27.33

29.93

0.32

26.96

27.26

32.83

0.32

24.88

27.26

22.62

0.32

26.99

27.22

26.56

0.32

21.17

27.29

17.97

0.32

27.68

27.08

12.43

0.32

16.35

(a) (b) Fig. 2 The SEM micrographs of transversal section (a) and air-facing surface (b) of 2% chitosan and 6mm CTAB membranes in combination with 10mm tannic acid.

CHITOSAN-POLYPHENOLS NANOSTRUCTURED MATRICES DRUG RELEASE …

29

The SEM images (Fig. 2) demonstrate an ordering of the tannic acid – CTAB containers in horizontal planes, which means that these nanocontainers will control release the active substance (tannic acid, in this case) as the chitosan matrix is subject of the swelling process. The air-facing surface SEM images indicate that while at low tannic acid concentration the nanocontainers are distributed as islands, with the concentration increasing these planes become better distributed. Moreover, it seems that with the tannic acid concentration increasing there is a tendency of the containers to homogenize (dissolute) in chitosan. This fact means that over a concentration of 10mm tannic acid the containers formation is diminuend. The drug release kinetics from the 2 and 3% chitosan matrices are shown in (Fig. 3). 0,7

Absorbance (a.u.)

0,6 pH 2 pH 5.2 pH 9.3

0,5 0,4 0,3 0,2 0,1 0,0

0

5

10

15

20

25

30

35

Time (hour)

(a)

Absorbance (a.u.)

0,5 0,4

pH 2 pH 5.2 pH 9.3

0,3 0,2 0,1 0,0

0

5

10

15

20

25

30

35

Time (hour)

(b) Fig. 3 The kinetic curves for tannic acid release from the 2% (a) and 3% (b) chitosan membranes in combination with 6 mm CTAB and 10 mm acid tannic.

30

Ana Gârlea, V. Melnig, M. I. Popa, G. Lisa

The precise quantities of membranes submerged in the three different eluents, where they were kept for 32 hours, release the tannic acid in a different way. The membrane degradation in hydrochloric acid (pH = 2) took place in 3 hours, while the membranes from the eluents with pH = 5.2 and pH = 9.3 remained partially degradated during those 32 hours. It was observed an erosion and diffusion complex process, the diffusion process being faster in acid medium than in the others. It can be said that in hydrochloric acid (pH = 2), after a relatively short time, the kinetic is of zero order, while in pH 5.2 and 9.3 the kinetic becomes of zero order after approximately 7 hours. We can affirm that this kind of nanostructured matrix is suitable for releasing both in stomach and intestine, the diurnal cycle allowing the required time for releasing. The zero order kinetic is governed by the erosion and diffusion complex process, being known that the erosion process is rate restrictive because is slower. 4.

CONCLUSIONS

The investigation of X-ray diffractograms reveals that the crystallinity of the membranes with tannic acid – CTAB nanocontainers increases with the active substance concentration increasing and the order at mesophasic level decreases because of the cluster formations. From the analysis data it was concluded that the nanocontainers are orderly structured in horizontal layers and present a vertical gradient. By visual analysis of the SEM micrographs it can be observed that the nanocontainers formed have dimensions in the range of 50 – 300 nm for tannic acid. Conclusively, from de release kinetic curves it was observed that as the chitosan is swelled and the surface is subject to erosion and diffusion, the drug - CTAB containers are dissolved and will release the active substance (tannic acid) in a zero order kinetic controlled manner diffusion process. Acknowledgements This study was financially supported by the CEEX MOD I Nr. 1927/2006 (NANOCOFARM) and PN II TD 17/2008 scientific research project in the frame of the Romanian MEC Program. 1. 2. 3. 4. 5.

REFERENCES Ana Gârlea, V. Melnig, M. I. Popa, G. Rusu, Tannic acid as polyphenol model entrapped in chitosan based nanostructure matrices, Proceeding of The 1st International Conference on Polymers Processing in Engineering, Universitatea „Dunarea de Jos”, p. 155-162, 2007. Ana Gârlea, Alina Manole, M. I. Popa, V. Melnig, Nanostructured chitosan - surfactant matrices as polyphenols nanocontainers template, Biomaterials National Symposium “Biomaterials and Medical-Surgical Aplications” the VIth edition, Cluj-Napoca, October 18-20, 2007. Ana Gârlea, V. Melnig, M. I. Popa, G. Rusu, Entrapment of tannic acid in chitosan based nanostructure matrices, Materiale Plastice, vol. 45, nr. 2, p. 193-197, 2008. P. Scherrer, Bestimmung der Größe und der inneren Struktur von Kolloidteilchen mittels Röntgenstrahlen, Göttinger Nachr, .2, 98 (1918). A. Manole, L. Obreja, M.I. Popa, V. Melnig, Proceedings of the 12th International Conference “The Knowledge Based Organization Proceedings”, p. 272, 2007.