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Effects of drying methods on the preparation of dexamethasone-loaded chitosan microspheres

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Biomedical Materials Biomed. Mater. 9 (2014) 055003 (9pp)

doi:10.1088/1748-6041/9/5/055003

Effects of drying methods on the preparation of dexamethasone-loaded chitosan microspheres Fei Xu1,2, Miao Yin2, Yanru Wu2, Huifen Ding2, Fangfang Song2 and Jiawei Wang2,3 1

  Department of Stomatology, Xiangya Hospital, Central South University, Changsha, 410008, People’s Republic of China 2   Hubei-MOST KLOS & KLOBM, School and Hospital of Stomatology, Wuhan University, 430079, People’s Republic of China E-mail: [email protected] Received 1 December 2013, revised 10 May 2014 Accepted for publication 15 July 2014 Published 18 August 2014 Abstract

The purpose of this study was to investigate the effects of drying methods on the preparation of dexamethasone- (Dex-) loaded chitosan microspheres. Two drying methods, namely, air drying and freeze drying, were adopted. The physical properties of the beads were first investigated and then the loading and release of Dex were characterized. Finally, the bioactivity of released Dex was evaluated. The data showed that, compared with freeze-dried beads, air-dried beads were denser and smaller, and had lower swelling ratios, slower degradation rate and greater Rockwell hardness. In terms of drug delivery, air-dried beads had lower encapsulation efficiency and a slower release rate of Dex. Regarding bioactivity, both groups prompted cell differentiation without significant differences. However, Dex released from freeze-dried beads inhibited cell proliferation, while Dex released from air-dried beads did not. Based on these results, we conclude that incorporation of Dex enhanced the osteogenic potential of chitosan microspheres and drying methods did affect the physical properties of the chitosan microspheres, which further influenced the drug loading and release. At the moment, the airdrying method is more appropriate to prepare Dex-loaded chitosan microspheres. Keywords: chitosan microsphere, dexamethasone, air drying, freeze drying, bioactivity (Some figures may appear in colour only in the online journal)

1. Introduction

In this way, they can be implanted directly in vivo with immobilized cells, which offers additional attractive features for tissue engineering, such as ease of handling and improved cell viability [3]. Finally, microspheres can be used to carry drugs, proteins and genes, which further enhances their bioactivity [4]. Chitosan, the polysaccharide deacetylated from chitin that exists in the shells of crab and shrimp, has been widely used in bone tissue engineering due to its excellent biocompatibility, enzyme-regulated degradation and similarity to the extracellular matrix [5, 6]. However, with regard to the complexity of the bone tissue environment [7], the osteogenic potential of chitosan scaffold alone is limited and bioactive drugs are always needed. As a drug carrier, chitosan possesses several special advantages

Microspheres have attracted increasing attention and proven to be versatile as scaffolds in bone tissue engineering. Although there are some possible disadvantages of using microspheres, for example, their tendency to move from the defect site and their need of a stabilization device, the advantages far outweigh the disadvantages. First of all, microspheres can be injected into bone defects through minimally invasive procedures, thus avoiding excessive surgical trauma [1]. Second, the size and morphology of microspheres allow them to fill various defect shapes with close packing [2]. Third, microspheres can serve as cell carriers. 3

  Author to whom all correspondence should be addressed.

1748-6041/14/055003+9$33.00

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© 2014 IOP Publishing Ltd  Printed in the UK

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Biomed. Mater. 9 (2014) 055003

chitosan powder (medium molecular weight, Brookfield viscosity 200 cps, Sigma-Aldrich, USA) was dissolved in 2% (v/v) acetic acid solution to form a 3% chitosan solution (w/v), which was dripped slowly into a 2% (m/v) NaOH solution containing 80% ethanol (v/v) and stirred for 1 h to precipitate spherical beads. The formed beads were washed in deionized water to a neutral pH. To achieve crosslink, genipin solution was obtained by dissolving genipin powder (Wako, Japan) in 60% (v/v) ethanol. The microspheres were then cross-linked in 1% genipin solution at room temperature. Twenty-four h later, the cross-linked wet beads were placed on Petri dishes after removing residual genipin and divided into two parts according to the design: one was followed by air drying and the other was pre-frozen at −80 °C overnight and then placed in a freeze dryer for 24 h. The end point of freeze drying was observed to be 0.1 MPa and the temperature was −52 °C.

compared with other drug delivery systems. For example, it carries a positive charge and thus in turn can react with negatively charged mucosal surfaces and polymers [8]. Additionally, chitosan has various functional groups, such as amino and hydroxyl groups, that can be easily reacted and functionalized [6]. When preparing drug-loaded chitosan microspheres, it is necessary to transform the formulation of chitosan from the liquid state to the solid state for long-term preservation. At present, air drying and freeze drying are two commonly used drying methods [9, 10]. The air-drying process is easy to operate without using special equipment. However, the resulting microspheres are dense and significantly shrunken [11]. Freeze drying is a method that consists of removing water from a tissue by sublimation. It will keep the diameter of the dried microspheres the same as that of the wet beads. Particularly, it can generate matrices with high porosity [12]. However, this process is relatively complicated and time-consuming [11]. Studies have shown that the drying process can exert a strong influence on the physical properties of scaffolds [12]. For instance, Gomez-Carracedo et al reported that the microstructural, morphological and mechanical properties of the pellets can be modulated by controlling the drying step [10]. Soares et al also found that samples dried by freeze drying showed higher porosity and a higher swelling ability than the samples dried in an oven [13]. On the other hand, drying methods can affect the drug delivery. For instance, Reves et al found that the freeze-dried microspheres could enhance the loading capacity of the alkaline phosphatase and bone morphogenetic protein-2 compared with the non-freeze-dried microspheres [14]. Kumari et  al reported that the rate of drug release from freeze-dried beads was much faster than that from air-dried beads [9]. Since both the physical properties and drug delivery of scaffolds can influence the ability of cells to attach, produce extracellular matrix and form a complex tissue construct [15], the drying methods should be carefully examined. Dex is a synthetic glucocorticoid and has been widely accepted as a stimulatory factor for osteogenic differentiation [16]. It can not only increase the alkaline phosphatase activity, the expression of osteocalcin and bone sialoprotein of mesenchymal stem cells [17], but also plays an important role in the Runx-2 pathway by enhancing the expression of Cbfa1 at both the gene and protein levels [18, 19]. So far, some vehicles have been designed to deliver Dex, such as poly(D,L-lactide-co-glycolide) microspheres [20] and polycaprolactone nanofibers [16]. However, little research has been focused on the Dex-loaded chitosan microspheres. Also, there is little research to study the effects of drying methods on the preparation of Dex-loaded chitosan microspheres. Therefore, in this study, two kinds of drying methods, namely, air drying and freeze drying, were used to prepare chitosan microspheres. The differences in the physical properties of beads were characterized. The loading and release of Dex was evaluated. Finally, the bioactivity of released Dex was observed.

2.2.  Diameter and morphological properties

The overall morphology of the prepared microspheres was observed under a light microscope (Leica, Wetzlar, Germany) and photographed. At least 50 images were randomly picked out and examined to measure the average diameter using Image J software (National Institutes of Health, USA [22]). The surface topography and cross section  of the beads were further examined by a scanning electron microscope (SEM, FEI Sirion 200, USA). For cross-section analysis, the microspheres were quick-frozen in liquid nitrogen and sectioned. Then the samples were sputter coated with gold and observed. 2.3.  Swelling ratio, degradation rate and mechanical strength

The swelling test was carried out as follows: microspheres were first weighed (W0) and immersed in phosphate buffered saline (PBS, pH 7.4, 37 °C) for 24 h, then at a specific time point the hydrated samples were collected and weighed again after the excess surface water was removed using filter paper (W1). It was calculated by swelling ratio (%) = [(W1‒W0)/W0] × 100%. To monitor the degradation rate, samples with a known dry weight (W0) were incubated in PBS (pH 7.4, 37 °C) containing 500 μg mL−1 lysozyme over 3 weeks (n = 6). After each week the microspheres were removed, dried and weighed (W1). The degradation rate was calculated by the weight remaining: weight remaining (%) = W1/W0  ×  100%. To measure the Rockwell hardness, beads were embedded in resin and polished gradiently to form a smooth surface, then loaded with 10 g of force for 15 s via a tensile tester (HXD‒1000, Taiming, China).

2.  Materials and methods

2.4.  Specific surface area

The specific surface areas of the microspheres were determined using the Brunauer–Emmett–Teller (BET) nitrogen sorption– desorption measurement as described before (Micromeritics ASAP2020, USA) [23].

2.1.  Preparation of chitosan microspheres

The microspheres were prepared using a coacervate precipitation method, as we have reported before [21]. Briefly, 2

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Biomed. Mater. 9 (2014) 055003

2.5.  Porosity and weight

2.8  . Cell proliferation

Porosity was calculated using the liquid intrusion method. Briefly, dry beads were weighed and immersed in 100% ethanol until all bubbles were removed. Then, scaffolds were taken out from ethanol, gently wiped to remove excess ethanol and weighed again. The porosity (P (%)) was calculated as follows: P% = Vp/Vs = [(Ww‒Wd)/ρeth]/Vs = (Ww‒Wd)/ (Wd‒Wo), where Vp was the pore volume, Vs was the total volume of pores and solid matrix, Ww was the wet weight of the scaffold after immersion in ethanol, Wd was the dry weight of the scaffold, ρeth was the density of ethanol, Wo was the weight of the scaffold in ethanol, Ww‒Wd is equal to Vp × ρeth and Wd-Wo is equal to the buoyancy. To calculate the dry weight of the spheres, 100 beads were randomly weighed and the average weight for each bead was calculated (n = 6).

Cell proliferation was determined using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratory, Japan) test, which measures metabolic activity and is an indirect measure of cell number [24]. However, this method is useful for biomaterial tests, since it is not necessary to digest cells from the microspheres and the test can be continued without changing samples. In this study, after cells were seeded in 48 culture plates for 4 hours (1   ×   105 cells cm−2), Dex-loaded or -unloaded microspheres (1 mg/well) were added. After 1, 3, 5 and 7 d, the old medium was removed and fresh medium supplemented with CCK-8 was added to each well. Three hours later, the optical density (OD) of supernatant solution at 450 nm was measured. Five samples per time point were characterized in each group. 2.9.  Cell differentiation

2.6.  Drug loading and release

To further evaluate the released Dex, we observed the differentiation status of rat BMSCs. It was assessed from four aspects: cell morphology, alkaline phosphatase (ALP) activity, calcium deposition capacity and gene expression.

Blank spheres (100 mg) of each group were added into a 1 mL PBS containing 1 mg Dex and gently shaken at room temperature. 24  hours later, drug-loaded samples were acquired, rinsed with water to remove loosely bound drugs and left to dry according to the design (air drying or freeze drying). The amount of Dex adsorbed into the microspheres was determined by taking the difference between the initial 1 mg mL−1 of Dex in solution and the final concentration of the solution, which was measured by a UV spectrophotometer at 242 nm and calculated according to the standard curve of Dex. The drug encapsulation efficiency was calculated as the actual drug content divided by the theoretical drug content multiplied by 100. All samples were measured in triplicate. Then, the release test of Dex was conducted. Specifically, 30 mg of loaded chitosan microparticles were placed in 5 mL PBS (pH 7.4, 37 °C). At predetermined time points, 2 mL aliquots were taken out and replaced with 2 mL fresh PBS solution in order to maintain the initial volume. The concentrations in the samples were quantified by UV analysis at 242 nm. All the release experiments were carried out in triplicate.

2.9.1.  Cell morphology.  To observe the changes in cell morphology, cells (1   ×   105 cells cm−2) were seeded on glass coverslips in 24 culture plates for 4 h, then drug-loaded and -unloaded microspheres were added (2 mg/well). After being cultured for 4 h (day 0) and 7 d (day 7), cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Cell morphology was visualized by using Alexa Fluor phalloidin to stain for F-actin. Nuclei were visualized by staining with Hoechst and observed under fluorescence microscopy (Leica, Wetzlar, Germany). 2.9.2. Alizarin red staining.  To observe the mineralization nodules, cells (1  ×  105 cells cm−2) were seeded and cultured using the same procedure as in the cell morphology test. At day 14, cells were fixed in 10% formalin, washed with PBS and stained using 2% Alizarin Red S. 2.9.3. ALP activity.  For evaluating the ALP activity, cells (1   ×   105 cells cm−2) were seeded, cultured and lysed at day 7 and day 14. Briefly, the samples were washed twice with PBS and 0.2% Triton X-100 at 4 °C was added. Four hours later, the cell lysates were collected, sonicated and centrifuged. Then, 50  μL cell homogenate was mixed with  150 μL p-nitrophenyl phosphate substrate solution (Sigma-Aldrich, USA) and incubated at 37 °C for 30 min. Absorbance was measured at a wavelength of 405 nm. The ALP activity was expressed as the value of the p-nitrophenol quantity divided by the reaction time and the cell protein content, as measured by a BCA protein assay kit (Pierce, UK).

2.7.  Cell culture

Rat bone-marrow-derived mesenchymal stem cells (BMSCs) derived from the bone marrow of male, six-week-old SD rats were used in this study. Briefly, the femora and tibiae were dissected and bone marrow was flushed out with modified Eagle medium- (MEM-)a (Hyclone, USA) containing 10% fetal bovine serum (FBS, Hyclone, USA) and 100 units mL−1 penicillin/streptomycin (Sigma, USA). Passage 2–4 cells were used. According to the design, cells were seeded in the culture plate and 4 h later, drug-loaded or -unloaded microspheres that were sterilized by ultraviolet light for 1 h were also placed in the plate. For all the in vitro studies, cells were cultured in Dex-absent osteogenic differentiation media (basal medium supplemented with 50 mg mL−1 ascorbic acid and 10 mM β-glycerophosphate).

2.9.4 . Reverse-transcriptase polymerase chain reaction.  To determine the gene expression of osteogenic mark-

ers, cells (2.5  ×  105 cells cm−2) were seeded and cultured in

3

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Table 1.  Primer sequences for RT-PCR.

Gene

Forward primer (5’ − 3’)

Reverse primer (5’ − 3’)

Alkaline phosphatase (ALP) Collagen type I (COL I) Osteopontin (OPN) GADPH

CAATTAACGGCTGACACTGC TCTGCGACACAAGGAGTCTG GCAGAGAGCGAGGATTCTGT AACGACCCCTTCATTGAC

TTTCAGGGCATTTTTCAAGG GGGACCATCAACACCATCTC AGGTCCTCATCTGTGGCATC TCCACGACATACTCAGCAC

Figure 1.  Light microscope images of microspheres. (a) Air-dried microspheres. (b) Freeze-dried microspheres. Scale bar = 200 µm. Table 2.  The physical properties of air-dried and freeze-dried chitosan microspheres.

Sample

Porosity (%)

Diameter (μm)

Weight (mg)

BET surface area (m2 g−1)

Air-dried microspheres Freeze-dried microspheres

12.14  ±  3.98 93.13  ±  2.07***

232.17  ±  41.76 389.37  ±  69.85***

0.045  ±  0.0043 0.041  ±  0.003

0.0032 3.2332

***: p 

Effects of drying methods on the preparation of dexamethasone-loaded chitosan microspheres.

The purpose of this study was to investigate the effects of drying methods on the preparation of dexamethasone- (Dex-) loaded chitosan microspheres. T...
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