Thin Solid Films 519 (2011) 4658–4662

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Thin Solid Films

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t s f

Release behaviors of drug loaded chitosan/calcium phosphate coatings on titanium

Jiabei Zhou, Xinwei Cai, Kui Cheng, Wenjian Weng ⁎, Chenlu Song, Piyi Du, Ge Shen, Gaorong Han
Department of Materials Science and Engineering, State Key laboratory of Silicon Materials, Zhejiang University, Hangzhou 310027, China

a r t i c l e i n f o a b s t r a c t

Available online 12 January 2011 Implants with antibiotic drug loaded bioactive coatings have been increasingly applied in orthopedic
operations. Here we report the drug release behavior of gentamycin loaded chitosan/calcium phosphate
Keywords: coatings on titanium. Chitosan/calcium phosphate coatings with different component ratios and surface
Bioactive coating topographies were prepared by electrochemical deposition method. Our results showed that the drug release
Drug release behavior from these coatings was controlled by their component ratio and surface topography, and the former ratio
Chitosan/calcium phosphate
played a more significant role. The present coatings could provide an effective way to create both good
Surface topography
bioactivity and antibacterial activity.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction gentamycin is commonly used to evaluate the drug loading and

Coatings that exhibit with both bioactivity and anti-inflammation
properties are required in clinic surgery. Titanium implants with
bioactive calcium phosphate (Ca–P) coatings have been used in hard-
tissue replacement in orthopedics [1]. However, pure Ca–P coatings
are normally inefficient in drug loading and releasing due to the lack
of appropriate accommodating microstructure and chemical bonding
[2]. Different efforts have been directed at improving the drug loading
ability of these coatings [3,4]. The incorporation of biopolymers to the
coating has been proven to be an efficient way of improving their drug
loading capabilities [5].
Chitosan, a natural cationic polysaccharide, has been used in a
number of biomedical applications, including as a drug carriers [6].
Chitosan has been incorporated into the Ca–P coating to improve its
drug loading ability [5,7]. Chitosan/Ca–P scaffold could act as an ideal
drug delivery carrier of antibiotic drug [5,8]. Hence, the chitosan/Ca–P
coating is ideal for both bioactivity and drug release.
Among all available methods for chitosan/Ca–P coating, the
electrochemical deposition method demonstrates a variety of advan-
tages, such as low process temperature, low cost, a high degree of
thickness-control and straightforward deposition on irregular sub-
strates [8–10]. Besides Collagen/Ca–P coatings, a chitosan/octacalcium
phosphate coating was also prepared by the electrochemical
deposition process [11,12].
As one of the amino-glycosides, a diverse class of antibiotics
gentamycin is widely used in orthopedic applications [13]. Gentamycin
is effective against Gram-positive and Gram-negative aerobic organisms
[14,15], and can be quickly adsorbed and excreted. Therefore,
⁎ Corresponding author. Tel./fax: +86 0571 87953787.
E-mail address: wengwj@zju.edu.cn (W. Weng).
releasing ability of materials [4,16].
In this study, chitosan/Ca–P coatings with different component
ratio and surface topographies were prepared by an electrochemical
deposition process. The drug loading and releasing capacity and
releasing rate of the coatings were evaluated. The relationship
between release behaviors and the component ratio and surface
topography is briefly discussed.
2. Materials and methods
2.1. Coating preparation
A piece of titanium plate (10 mm × 10 mm) was treated in diluted
acid (mixture of nitric acid and hydrofluoric acid), then rinsed with
de-ionized water, and mounted as the working electrode. A platinum
plate was used as the counter electrode. The distance between the two
electrodes was fixed at 15 mm. The electrolyte solution was prepared by
adding 30–10 mM Ca(NO3)2 (Shanghai Chem. Co., China) and 10–0 mM
NaH2PO4 (Shanghai Chem. Co., China) into 0.5 g/100 mL chitosan
(Shandong Aokang Chem. Co., China). The pH was adjusted to 5.8. The
working voltage was set at 2.8 V and the deposition time was 30 min.
The as-deposited coatings were washed with de-ionized water and
dried. The experimental conditions of each coating are listed in Table 1.
2.2. Coating characterizations
The morphology and microstructure of the samples were charac-
terized by scanning electron microscopy (SEM, Hitachi, S4800) and
transmission electron microscopy (HRTEM, TECNAI, G2 F30 S-TWIN).
Composition and phase determination was done via X-ray diffraction
(XRD, X' Pert PRO, CuKα, 2°/min, 0.02 per step). Fourier transform-
infrared spectroscopy (FTIR, Thermofisher iS10) was employed to
reveal the chemical bonding of the deposited coatings.

0040-6090/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.tsf.2011.01.012
 J. Zhou et al. / Thin Solid Films 519 (2011) 4658–4662 4659

Table 1 examined by ultraviolet-visible spectrometry (TU1901) with an

List of the samples with different calcium and phosphate concentration in the indicator of Evans blue (CAS: 314-13-6 Shyysw Co).
electrolyte solutions.
The dynamic contact-angle measurement could be used to analyze
Sample Ca(NO3)2 mmol/L NaH2PO4 mmol/L the interface behavior of a drop of the drug on the coating surface [17].
Porous chitosan/Ca–P (PCP) coating 30 10 A camera was equipped to record the volume change of the droplet of
Micro porous chitosan/Ca–P (MCP) coating 10 10 the drug on the coating surface. In this study, 5 μL of GS solution was
Dense chitosan (DC) coating 30 0 dropped onto chitosan/Ca–P coating surface. During de-wetting, the
average rate of the drop volume loss, which could represent the
velocity of drug drop infiltrated into the sample coatings, could be
calculated. The drop volume left after de-wetting could represent the
2.3. Drug loading and releasing evaluations capacity of drug loading.
The absorption peak intensity of Evans blue decreased substan-
The drug loading properties were determined by dynamic contact- tially after the several milligrams of Gentamycin was added in to the
angle obtained through a contact-angle meter (Dataphysics, OCA20). assay. The reagent of Evans blue 1 × 10-4 mM has been proven to be
The release of gentamycin with quantitative drug loading was sufficient enough to detect antibiotic presence in the assays [18].
Standard solutions (1 to 10 mg/L) were prepared from a stock solution
of 40 mg/L gentamycin in the physiological saline, which generated
reliable standard curves.
Gentamycin sulfate (GS) solution (40 mg/mL) was prepared by
dissolving gentamycin sulfate powder (C21H43N5O7.H2SO4 CSA: 1405-
41-0 Shyysw Co.) into de-ionized water. The drug droplet was added
to the chitosan/Ca–P coating's surface carefully by a transferpette. The
addition of drug volume was controlled at 25 μL. The drug loading
surface was fixed at 0.5cm2 (5 mm × 10 mm). After loading, the
coatings were dried at 37 °C for 24 h.
The loaded coatings were immersed in 5 mL physiological saline,
whose concentration was 0.9% NaCl, and were kept at 36.7 °C, to
simulate body fluid in vivo. The physiological saline solution was
refreshed at 5 min, 10 min, 15 min, 20 min, 30 min, 45 min, 60 min,
75 min, 90 min, 180 min, 1 day, 2 day, and 3 day, to analyze both short
term and long term behavior. The concentration of gentamycin in the
physiological saline was determined by ultraviolet-visible spectrometry
(TU1901). Each group contained three parallel samples.
The coatings were soaked into the GS solution (40 mg/mL) for
10 min, then were taken out and dried for 24 h. The loaded coatings
were immersed in 10 mL of physiological saline solution for 30 min
for the evaluation of the release of gentamycin. Each group also
contained three parallel samples.

3. Results and discussion

3.1. Coating characterizations

The resultant coatings developed into a porous structure with the

increase of calcium and phosphate concentration in the electrolyte
solutions. As showed in Fig.1, the dense chitosan (DC) coating derived

Fig. 1. SEM images of (a) PCP coating; (b) MCP coating; (c) DC coating. Fig. 2. XRD spectra of (a) PCP coating; (b) MCP coating; (c) DC coating.
4660 J. Zhou et al. / Thin Solid Films 519 (2011) 4658–4662
Fig. 3. FTIR spectra of (a) PCP coating; (b) MCP coating; (c) DC coating.
from the solution with only calcium ions, presents a dense and smooth
surface. The micro porous chitosan/Ca–P (MCP) coating derived from
the solution with low calcium and phosphate concentration exhibits
micro porosity with 1–2 μm pore size. The porous chitosan/Ca–P (PCP)
coating derived from the solution with high calcium and phosphate
concentration shows a porous surface with 5–10 μm pore size.
XRD spectra in Fig. 2 indicate that the PCP coating consists of
dicalcium phosphate (DCPD, JCPDS 11-0293) and hydroxyapatite
(HA, JCPDS 09-0432) phases, and the MCP coating consists of another
Fig. 4. HRTEM image spectra of (a) PCP coating; (b) MCP coating.
crystalline phase of dicalcium phosphate (DPCD, JCPDS 09-0077)
phase. IR spectra in Fig. 3 shows that the DC coatings exhibit only the
characteristic bands of chitosan at 2930 cm-1 and 2860 cm-1[12]. The
PCP and MCP coatings have characteristic bands of both chitosan and
phosphate. The absorption intensity of phosphate bands at 950–
1200 cm-1 in the PCP coating is obviously stronger than that in the
MCP coating, implying that the former contains more phosphate.
Hence, the introduction of more calcium phosphate into chitosan
leads to a more porous morphology for the coatings.
HRTEM was applied to analyze the microstructure of calcium
phosphate crystals combined with chitosan in PCP and MCP coatings.
In Fig. 4, the PCP coatings contain homogeneously distributed calcium
phosphate nanocrystals (5–10 nm) on the chitosan matrix surface.
The lattice planes with spacing of 0.278 and 0.344 nm are assigned to
(112) and (002) planes of HA. The (002) plane was also detected in
the XRD spectrum. The selected area electron diffraction ring patterns
of the coating, match the HA phase. In MCP coatings, the calcium
phosphate nanocrystals are also found in the chitosan matrix, but do
not homogeneously cover on the chitosan surface.
3.2. Drug release behavior evaluation
The contact angle of GS solution with the coating surface was used
to evaluate their early interaction behavior and their loading capacity.
Fig. 5 shows that the PCP coating has the largest volume loss rate
during de-wetting, and the MCP coating has a moderate rate. These
could be attributed to the fact that there are more Ca–P phase in PCP
than in MCP due to the hydrophilicity of Ca–P phase, and also the
 J. Zhou et al. / Thin Solid Films 519 (2011) 4658–4662 4661

Fig. 4 (continued).

higher porosity. The remaining drop volume reflects the infiltration
ability of GS solution into the sample coating surface. This implies that
the PCP coating has a better ability to absorb GS solution. Meanwhile,
the dense surface in DC coating might have a negative impact on its
absorption. It has the smallest de-wetting rate, but the largest drop
volume remaining after de-wetting.
The cumulative release of gentamycin from the coatings is shown
in Fig. 6. The results show that most of the drugs in PCP and MCP
coatings are eluted for 30 min. The absorption curves can be fitted
with the following linear relations: (a) PCP coating, y = 80.46 + 1.78x;
(b) MCP coating: y = 79.60 + 1.64x; (c) DC coating: y = 70.67 + 0.92x.
The PCP coating has the fastest rate of drug delivery, while DC coating
has relatively low release ability in the early stage. This could be
attributed to the strong interaction of pure chitosan with the
gentamycin [5]. The fast rate of drug release of the PCP coating may
be due to the Ca–P nanocrystals attached to the chitosan surface, which
reduces the bonding of drug molecules with the coating, is suggesting
absorption of the drug was mainly affected by the surface attachment

Fig. 5. Dynamic contact angles of (a) PCP coating; (b) MCP coating; (c) DC coating. Fig. 6. Drug-loading release curve of (a) PCP coating; (b) MCP coating; (c) DC coating.
4662 J. Zhou et al. / Thin Solid Films 519 (2011) 4658–4662

of GS. The three coatings differed in porosity and Ca–P content. These
results show that drug release behavior varied with the component
ratio and surface topography. It could suggest that high surface
porosity would contribute to the drug loading, and the proper ratio of
chitosan composition would also favor drug loading. The present
coatings could give an alternative approach to an implant with both
favorable bioactive and anti-inflammation properties in orthopedic
operations.

Acknowledgment

This work is financially supported by the National Natural Science

Foundation of China (50872122), Research Fund of the Doctoral
Fig. 7. Calculated drug-releasing amounts by soaking methods (0.5 h). Program of Higher Education of China (200703351015) and the
research fund of Shenzhen Key Laboratory of Functional Polymers.

to the chitosan. These results show that different coating surfaces and
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4. Conclusions

Three chitosan/Ca–P coatings with different morphologies were

deposited and used to evaluate for drug loading and releasing ability