International Journal of Biological Macromolecules 80 (2015) 636–643
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Silk sericin loaded alginate nanoparticles: Preparation and anti-inflammatory efficacy Thitikan Khampieng a,1 , Pornanong Aramwit b , Pitt Supaphol a,∗ a The Petroleum and Petrochemical College and Center of Excellence on Petrochemical and Materials Technology, Chulalongkorn University, Bangkok 10330, Thailand b Bioactive Resources for Innovative Clinical Applications Research Unit and Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
a r t i c l e
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Article history: Received 21 May 2015 Received in revised form 1 July 2015 Accepted 13 July 2015 Available online 16 July 2015 Keywords: Silk sericin Alginate nanoparticles Carrageenan-induced paw edema Anti-inflammation
a b s t r a c t In this study, silk sericin loaded alginate nanoparticles were prepared by the emulsification method followed by internal crosslinking. The effects of various silk sericin loading concentration on particle size, shape, thermal properties, and release characteristics were investigated. The initial silk sericin loadings of 20, 40, and 80% w/w to polymer were incorporated into these alginate nanoparticles. SEM images showed a spherical shape and small particles of about 71.30–89.50 nm. TGA analysis showed that thermal stability slightly increased with increasing silk sericin loadings. FTIR analysis suggested interactions between alginate and silk sericin in the nanoparticles. The release study was performed in acetate buffer at normal skin conditions (pH 5.5; 32 ◦ C). The release profiles of silk sericin exhibited initial rapid release, consequently with sustained release. These silk sericin loaded alginate nanoparticles were further incorporated into topical hydrogel and their anti-inflammatory properties were studied using carrageenan-induced paw edema assay. The current study confirms the hypothesis that the application of silk sericin loaded alginate nanoparticle gel can inhibit inflammation induced by carrageenan. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Silk sericin (SS) is a globular protein, consisting of 18 amino acids, that is obtained from silk cocoon. SS acts as an adhesive and holds two fibroin filaments together, leading to cocoon formation. Because of the tremendous amount of studies on the advantages and biological properties of SS, this protein has become attractive for use in many industries. SS provides a wide range of applications: antioxidant [1–5], anticoagulant [6], and anti-wrinkle [7]. This protein is also reported to suppress tumor growth [8,9] and to reduce oxidative stress [9]. Panilaitis et al. [10] concluded that SS itself does not significantly activate macrophage. In addition, SS has no ability to induce immune response in in vivo experiments and can be used in many biomedical applications [11,12]. SS accelerates cell proliferation and increases cell viability in different cell lines, i.e. hybridoma [13], insulinoma [14], and human skin fibroblast [15]. Moreover, UVB-induced apoptosis in human skin keratinocytes can be inhibited by SS [4].
∗ Corresponding author. E-mail address: [email protected] (P. Supaphol). 1 |Present address: Center for Research and Innovation, Faculty of Medical Technology, Mahidol University, Nakhon Pathom 73170, Thailand. http://dx.doi.org/10.1016/j.ijbiomac.2015.07.018 0141-8130/© 2015 Elsevier B.V. All rights reserved.
In wound applications, the proliferation and attachment of human skin fibroblasts can be promoted when cultured in SS media [15]. SS also increases collagen production [16]. According to Aramwit et al. [17], SS also plays a role in the suppression of carrageenan-induced inflammation which promotes wound healing. Regardless of many benefits associated with SS, SS solution is unstable and can degrade due to pH and temperature sensitivity. This instability makes the SS solution difficult to use. In the pharmaceutical industry, encapsulation is a technique that is widely used to sustain or prolong drug release, to stabilize drugs from heat, moisture, light or vaporization, and reduce drug toxicity. Alginate microparticles have been used for the encapsulation of abundant of biological and bioactive materials such as proteins [18–20], enzymes [21], antibodies [22], cells [23], and DNA [24]. Alginates are natural biodegradable polymers obtained from kelp (brown algae). The gelation of alginate can be simply induced by various divalent cations, especially Ca2+ , which interacts with two carboxyl groups on the neighboring alginate molecules resulting in ionic inter-chain connections [25]. Entrapment of protein into alginate microparticles has several advantages; the alginate matrix protects proteins from different environments, provides a mucoadhesive property, exhibits low toxicity, and low immunogenicity [26]. Hence, a mild protein
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encapsulation technique, emulsification followed by internal gelation, is a good way to fabricate SS loaded alginate nanoparticles without the degradation of SS. The aims of the current study was to prepare SS loaded alginate nanoparticles and investigate their morphology, size, chemical structure, thermal properties, and release profile of SS from the SS loaded alginate nanoparticles. A final aim of this study was to formulate a SS loaded alginate nanoparticle topical gel which will be beneficial for topical use and to further study the in vivo antiinflammatory efficacy. Therefore, SS loaded alginate nanoparticle gel formulations are proposed for this study, to obtain more stability and sustainable release of SS. It is hypothesized that the application of SS loaded alginate nanoparticle gel would inhibit inflammation induced by carrageenan. 2. Experiment 2.1. Materials Fresh white cocoons from Bombyx mori silkworms which were produced in a well-controlled environment were provided by Chul Thai Silk Co., Ltd. (Petchaboon, Thailand). SS was isolated from the cocoons using a high temperature and pressure degumming technique. Briefly, the silk cocoons were finely cut into small pieces and soaked in purified water (ratio of dry silk cocoon:purified water = 1 g:30 mL). Subsequently, the mixture was degummed using an autoclave (Tomy Seiko SS-320, Japan) at 120 ◦ C for 60 min. Then fibroin, which is insoluble, was removed from the obtained solution by filtration. The SS solution was concentrated until it reached the desired concentration, which was quantified by a Pierce® bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, USA). The obtained SS had a molecular weight of ∼25–150 kDa, as reported previously [27]. Sodium alginate was purchased from Aldrich (USA). Calcium carbonate (CaCO3 ) and Span® 80 were obtained from Fluka (USA). Paraffin oil was supplied from Carlo Erba Reagents (Italy). Glacial acetic acid, isopropyl alcohol, and sodium acetate were bought from Sigma-Aldrich (USA). Difelene gel® was purchased from Thai Nakorn Patana (Thailand). Carbopol® Ultrez 21, propylene glycol (PG), triethanolamine (TEA), carrageenan, sodium hydroxide (NaOH), carbon dioxide, corn cob were supported by Faculty of Pharmaceutical Sciences, Chulalongkorn University. Formalin buffer was provided by Department of Pathobiology, Faculty of Sciences, Mahidol University. All chemicals were used as received without further purification. Male Albino Wistar rats (4 weeks old, weighing 150–200 g) were used for the anti-inflammatory study by carrageenan-induced rat paw edema assay. Standard laboratory conditions of temperature (25 ± 2 ◦ C), a light/dark cycle of 12 h/12 h with free access to standard food (C.P. Company, Thailand) and water were provided for animals. The animals were acclimatized for at least 3–5 d before the experiments. At the end of the in vivo evaluation, the animals were euthanized using carbon dioxide inhalation. The investigation was carried out under aseptic conditions in agreement with local guidelines for the care of laboratory animals and approved by the Ethics committee of faculty of Pharmaceutical Sciences, Chulalongkorn University (No. 13-33-008). 2.2. Preparation of alginate and SS loaded alginate nanoparticles Alginate nanoparticles were successfully prepared by using an emulsification method and followed by internal crosslinking [28]. First, 0.2% (w/v) sodium alginate was completely dissolved in distilled water and then mixed with 5% (w/v) calcium carbonate
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aqueous solution with an ultra-turrax mixer for 5 min. The obtained solution was then poured into a mixture of paraffin oil and 1% (v/v) Span® 80 and mixed for another 15 min to allow for nanoparticle formation. Subsequently, paraffin oil and glacial acetic acid mixture was added to the oil phase to ionize calcium ion with continuous stirring for 30 min. At this step crosslinking of the alginate nanoparticles occurred. Finally, the alginate nanoparticles were harvested using centrifugation and washed with isopropanol to get the desired alginate nanoparticles. For the SS loaded alginate nanoparticles, the same procedure was carried out-except in the first step SS solution was added in the neat alginate solution to achieve SS ∼20, 40 and 80% (w/w) based on the weight of sodium alginate polymer. 2.3. Preparation of topical gel containing alginate or SS loaded alginate nanoparticles The topical gel containing alginate nanoparticles or SS loaded alginate nanoparticles were formulated by mixing the as-prepared nanoparticle with Carbopol® Ultrez 21 gel base. The formulation of gel base was 0.5% (w/w) Carbopol® Ultrez 21, 1% (w/w) PG, 0.5% (w/w) TEA, and distilled water. The alginate nanoparticle gel, 20% SS loaded alginate nanoparticle gel, and 80% SS loaded alginate nanoparticle gel were prepared by mixing gel base with alginate nanoparticle, 20% SS loaded alginate nanoparticle, and 80% SS loaded alginate nanoparticle, respectively. As the internal control, 20% and 80% SS gel were also prepared by mixing gel base with SS solution-to achieve the amount of SS equal to amount of SS within 20% and 80% SS loaded alginate nanoparticle, respectively. 2.4. Characterization and testing 2.4.1. Alginate and SS loaded alginate nanoparticles 2.4.1.1. Morphology observations. The morphology of the prepared alginate and SS loaded alginate nanoparticles were examined using a scanning electron microscope (SEM, Hitachi S-4800, Japan). Prior to SEM operation, the specimens were dispersed onto a conductive tape and sputter-coated to achieve a thin layer of platinum under vacuum. Then the specimens were imaged at a magnification of 50,000 and 100,000 times with incident electron beam energy of 10 kV. The average size of the alginate nanoparticles and SS loaded alginate nanoparticles was also determined by Semafore 5.21 software. At least 30 measurements on different nanoparticles were averaged to obtain a data point. 2.4.1.2. Fourier transform infrared spectroscopy (FTIR) study. To identify the interaction between alginate and SS in SS loaded alginate nanoparticles, an FTIR study was assessed using an FTIR spectrophotometer (NEXUS-870, Thermo Nicolet Corporation), equipped with a deuterated triglycine sulfate detector. Spectra were measured in the spectral region from 4000 cm−1 to 400 cm−1 in transmission mode at a resolution of ±4 cm−1 with scan frequency of 32 times. Optical grade potassium bromide was applied as the background measurement material. 2.4.1.3. Thermal analysis. Thermal behavior of the alginate nanoparticles and SS loaded alginate nanoparticles was analyzed through thermo-gravimetric analysis (Pyris Diamond TG-DTA, Perkin Elmer). Specimens (5 mg) were placed in aluminum crucibles then heated from ambient temperature to 650 ◦ C with a constant heating rate (10 ◦ C/min) under nitrogen atmosphere and the percentage of weight was collected over temperature. 2.4.1.4. In vitro release study. The release study of SS from the SS loaded alginate nanoparticles was performed in acetate buffer (pH 5.5) at skin temperature (32 ◦ C). To prepare the acetate buffer, 150 g
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of sodium acetate was dissolved in 250 mL of distilled water. Subsequently, 15 mL of glacial acetic acid was slowly added into the as-prepared sodium acetate solution. Eventually, the volume of the solution was then adjusted to 1000 mL with distilled water. For in vitro release characterization, experiments were carried out with the following procedures, approximately 300 mg of SS loaded alginate nanoparticles were incubated in 30 mL of citrate buffer that was maintained at 32 ◦ C and 200 rpm during the assay. At specific times, between 0 min to 7 d, 100 L of release medium was sampled to analyze the amount of SS released, using a BCA kit. Three parallel experiments were conducted and the results were presented as mean values. 2.4.2. Gel containing alginate and SS loaded alginate nanoparticles 2.4.2.1. Physical appearance of gel formulation. Formulations of the gel preparation were visually inspected for color, phase separation, and homogeneity. Texture and sensitivity when applying the SS loaded alginate nanoparticles gel onto the skin were also investigated. 2.4.2.2. Accelerated stability study. Selected gel formulations were subjected to an accelerated stability test. Briefly, the gel formulations were centrifuged at 3000 rpm or 10,000 rpm for 30 min to investigate any changes in appearance, which consequently affirmed the stability of the formulations. 2.4.2.3. Anti-inflammatory evaluation. Anti-inflammatory properties of the selected SS loaded alginate nanoparticle gels were evaluated using the carrageenan-induced edema model [29]. More specifically, forty-two Wistar rats were randomly distributed into seven groups of six rats for topical gel treatment. The rats in the control group were treated with gel base or alginate nanoparticle gel, while the rats that served as reference group were treated with commercial diclofenac gel (Difelene gel® (Thai Nakorn Patana, Thailand)). Two of the experimental groups received different formulations: 20% SS loaded alginate nanoparticle gel and 80% SS loaded alginate nanoparticle gel. The rats were treated with 20% SS gel, and 80% SS gel served as internal control group. Food was withheld from the rats for 6 h before the experiment; free access to water was provided. Each rat was weighed and marked on the right hind paw behind the tibiatarsal junction, and then a baseline measurement of the right hind paw volume was taken using a water displacement plethysmometer (Model 7140, Ugo Basile, Italy). Various SS loaded alginate gel formulations, reference gel (Difelene gel® ), or gel base (2.5 g) was applied topically onto the plantar surface of the right hind paw and rubbed for 15 s until completely absorbed. One hour after the treatment, 100 L of 1% (w/v) carrageenan solution was subcutaneously injected into the right hind paw. The paw volumes according to the inflammatory response were examined using a plethysmometer at 1 h time intervals until 6 h after the carrageenan injection. Percentage of edema inhibition was calculated in comparison to the control group using following formula: Percentage of Inhibition =
(Vt − Vo ) − (Vt − Vo )test × 100 (Vt − Vo )control
(1)
where Vt is paw volume at 1 h time intervals to 6 h after carrageenan injection, V0 is paw volume before the treatment. 2.4.2.4. Histological evaluation. After the carrageenan test, the animals were euthanized by carbon dioxide inhalation. The hind paws were removed and fixed by immersion in 10% neutral buffered formalin and then decalcified. The tissue samples from the paws were dissected and embedded in a paraffin block and thin sections (5 m) were prepared. Subsequently, the as-prepared
tissue sections were attached to glass slides and stained with Hematoxylin–Eosin (H&E). An Olympus BX50 optical microscope was used to capture digital images of the tissue samples. At least ten fields from the inflammatory site in each tissue section were randomly inspected. Morphology investigation of the inflammatory cell response-cellular infiltration, swelling, hemorrhage-was evaluated with a blind manner. 2.4.3. Statistical analysis Data were expressed as means ± standard errors of means (n = 6). Statistical analysis was performed based on ANOVA followed by LSD post hoc test. The statistical significance for the entire test was accepted at a 0.05 confidence level. 3. Results 3.1. Alginate and SS loaded alginate nanoparticles 3.1.1. Morphology observations The SEM images of the alginate nanoparticles and the SS loaded alginate nanoparticles are shown in Fig. 1. All particles were nearly spherical with some deflated surfaces and reasonable size uniformity in the range of 71.30–89.50 nm. The average grain size of SS loaded alginate nanoparticles was found to be slightly decreased with increasing amount of SS into the system, as shown in Table 1. 3.1.2. Fourier transform infrared spectroscopy (FTIR) analysis FTIR spectra were used to determine the chemical functional groups and molecular interactions between alginate and SS, as shown in Fig. 2. The strong and broad peaks in the 3500–3300 cm−1 range correspond to O H stretching and intermolecular hydrogen bonding. At the wave numbers of 1700–1400 cm−1 , the observed peaks belong to the C O stretching (amide). The FTIR spectrum of the alginate nanoparticles shows two distinctive absorption bands at ∼1637 cm−1 and 1412 cm−1 relative to both asymmetric and symmetric C O stretching absorption of the carboxylic acid salts, respectively. In spectra of SS loaded alginate nanoparticles, the absorption bands of Amide I, II, and III presented at 1625 cm−1 , 1506 cm−1 , and 1236 cm−1 , respectively, while characteristic peaks of alginate still observed. A slight difference can be observed between the alginate nanoparticles and the SS loaded alginate nanoparticles at various SS loadings. 3.1.3. Thermal analysis The TGA curves of the alginate nanoparticles and the SS loaded alginate nanoparticles are shown in Fig. 3. Two regions of degradation were observed: the first step, from 240 ◦ C to 270 ◦ C and the second rapidly decompose, from 280 ◦ C to 480 ◦ C. In addition, more thermal stability can be obtained with increasing SS loadings within the SS loaded alginate nanoparticles. 3.1.4. In vitro Release study The release characteristic of SS from SS loaded alginate nanoparticles was studied by the total immersion technique in acetate buffer at skin temperature. Fig. 4 shows the cumulative release amount of SS from these nanoparticles. During the first 8 h after Table 1 Average grain size of the as-prepared nanoparticles. Sample
Alginate: SS mass ratio
Average grain size (nm)
Alginate nanoparticles 20% SS loaded alginate nanoparticles 40% SS loaded alginate nanoparticles 80% SS loaded alginate nanoparticles
– 5:1 2.5:1 1.25:1
89.5 88.4 79.2 71.3
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Fig. 1. SEM micrographs of (A) alginate nanoparticles, (B) 20% SS loaded alginate nanoparticles, (C) 40% SS loaded alginate nanoparticles, and (D) 80% SS loaded alginate nanoparticles with a magnification of 100,000×.
Fig. 3. TGA curves of alginate nanoparticles and SS loaded alginate nanoparticles. Fig. 2. FTIR spectra of alginate nanoparticles and SS loaded alginate nanoparticles.
submersion in acetate buffer, the cumulative release of SS increased rapidly and then gradually after 8 h. As expected, the maximum amount of SS released from these materials increased with increasing SS loadings in the alginate nanoparticles. 3.2. Gel containing SS loaded alginate nanoparticles 3.2.1. Physical appearance of gel formulation Fig. 5 illustrates the as-prepared gel formulations. All formulations were rubbed on the back of the hand and no noticeable solid components were found, which can imply that all samples had uniform consistency. Also, no irritation was observed upon application of gel samples to the skin.
3.2.2. Accelerated stability study Stability studies of gel formulations were performed by centrifuge stress test. The results of the accelerated stability studies revealed that the selected gel formulations were stable under accelerated conditions. There were no significant alterations in the physical appearance-gel formulations remained the same and no phase separation occurred after subjecting the formulations to centrifugation at 3000 rpm and 10,000 rpm up to 30 min (the appearance of the selected gel formulations is available as Supplementary material). 3.2.3. Anti-inflammatory evaluation Pharmacological screening was performed by the carrageenaninduced edema model to evaluate the possible anti-inflammatory activity of the as-prepared gel formulations. Subcutaneous
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(Table 2). The results of paw edema assay were calculated and reported as the percentage of edema inhibition as shown in Fig. 6. As expected, the anti-inflammatory effect of reference gel achieved the highest percentage of edema inhibition, about 60.46%, 4 h after carrageenan injection. While the 80% and 20% SS loaded alginate nanoparticle gel formulations exhibited less anti-inflammatory effect when compared to reference gel. The 80% and 20% SS loaded alginate nanoparticle gel exhibited edema inhibition whereas the internal control groups hardly showed inhibition. In addition, edema inhibition of the 80% and 20% SS loaded alginate nanoparticle gel were comparatively better for longer periods compared to the 80% and 20% SS gel (internal control). Hence, the topical administration of SS loaded alginate nanoparticle gel was able to inhibit inflammation induced by carrageenan. The natural anti-inflammatory agent, SS loaded alginate nanoparticle gel formulations, can be encouraged as an alternative form for the treatment of acute inflammation. Fig. 4. Cumulative release profiles of SS from SS loaded alginate nanoparticles. The error bars represent the standard deviation based on three replication analysis (*p < 0.05).
administration of carrageenan into the right hind paw produced significant edema, with a maximum response being observed 3–4 h after injection, as shown in Table 2. In this study, carrageenan injection induced significant paw edema and reached maximum swelling at 3–4 h after injection
3.2.4. Histological evaluation According to the histological examination, paw biopsies revealed pathologic changes after carrageenan injection, including cellular infiltration and edema as shown in Fig. 7. In rats treated with reference gel, no sign of cellular infiltration or swelling was detected when compared to untreated rats. In the control groups treated with gel base and alginate nanoparticle gel, a large number of white blood cells, polymorphonuclear cells (PMN), which is
Fig. 5. Gel formulations: (A) gel base, (B) reference gel (Difelene gel® ), (C) alginate nanoparticle gel, (D) 20% SS loaded alginate nanoparticle gel, (E) 80% SS loaded alginate nanoparticle gel, (F) 20% SS gel, and (G) 80% SS gel.
Table 2 Average rats paw edema volume of various treatments in carrageenan-induced paw edema (n = 6). * p < 0.05 significantly different compared to gel base. # p < 0.05 significantly different compared to alginate nanoparticle gel. Treatments
Paw edema (mL) ± S.E.M. 1h
Gel base Reference gel (Commercial Diclofenac gel) 20% SS gel 80% SS gel Alginate nanoparticle gel 20% SS loaded alginate nanoparticle gel 80% SS loaded alginate nanoparticle gel
0.56 0.52 0.64 0.64 0.78 0.80 0.73
2h ± ± ± ± ± ± ±
0.08 0.11 0.05 0.07 0.09 0.05 0.06
0.61 0.50 0.96 0.90 0.95 0.90 0.73
3h ± ± ± ± ± ± ±
0.11 0.06 0.02 0.10 0.10 0.15 0.07
0.87 0.55 1.21 1.09 1.25 1.11 0.95
4h ± ± ± ± ± ± ±
0.07 0.07* 0.10 0.09 0.04 0.12 0.15
1.17 0.46 1.14 1.01 1.03 0.98 0.60
5h ± ± ± ± ± ± ±
0.12 0.09* 0.07 0.09 0.10 0.16 0.02#
0.94 0.65 1.14 1.13 1.08 0.61 0.68
6h ± ± ± ± ± ± ±
0.07 0.09* 0.08 0.10 0.11 0.09# 0.09#
0.94 0.65 1.16 0.98 0.79 0.72 0.45
± ± ± ± ± ± ±
0.07 0.07* 0.09 0.09 0.12 0.10 0.07#
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Fig. 6. The percentage of edema inhibition of various gel formulations. (Error bar represents the standard error of the mean (n = 6)).
crucial for cell inflammation, were observed. Swelling and hemorrhage were also noticed, which indicated high levels of inflammation. Whereas, there was less cellular infiltration and less edema in rats treated with the 80% and 20% SS loaded alginate nanoparticle gel, while the 80% SS gel and 20% SS gel treated tissues prior to carrageenan injection showed cellular infiltration in the dermal layer.
4. Discussions SS has been reported to be beneficial in accelerating cell proliferation and increasing cell viability in different cell lines [13–15]. Aramwit et al. [16,17] suggested that SS has ability to promote wound healing. In this study, we aimed to prepare SS loaded alginate nanoparticles which further formulated to a topical hydrogel, then proposed that SS loaded alginate nanoparticle gel could effectively decrease inflammation induced by carrageenan in rat model. Firstly, we observed the morphology of the nanoparticles and we found that the SS loaded alginate nanoparticles were uniformly spherical in shape (Fig. 1). According to the slightly decreasing in average grain size of SS loaded alginate nanoparticles when SS content increased (Table 1), this could relate to the amount of alginate in alginate/SS mass ratio which decreased from 5:1 to 1.25:1, less functional group to interact with Ca+ , resulting in the smaller size of as-prepared nanoparticles. To investigate the interaction between alginate and SS in nanoparticles, the FTIR spectrum of as-prepared nanoparticles were then analyzed, as shown in Fig. 2. Obviously, FTIR spectra of SS loaded alginate nanoparticles displayed all
the characteristic peaks of both alginate and SS. However, the absorption bands associated with C O stretching, O H stretching, and saccharide bands slightly shifted to a higher wave number and band intensities decreased as the SS content increased, which indicated the interaction between SS and alginate within the nanoparticles. The alginate nanoparticles and the SS loaded alginate nanoparticles showed thermal stability until 200 ◦ C, as shown in TGA thermograph (Fig. 3). Thermal stability above 240 ◦ C slightly increased with increasing SS loadings from 20% to 80%. This corresponded to Likitamporn et al. [30], the maximum decomposition temperatures of the sericin/PVA/bentonite scaffolds significantly increased with increasing sericin content. Additionally, we report the release profile of SS from SS loaded alginate nanoparticles (Fig. 4). At initial phase, SS rapidly diffused through the swollen alginate matrix. Subsequently, a sustained release was achieved. Both features would be favorable for the prolonged therapeutic effect of SS for transdermal application. To evaluate the in vivo anti-inflammatory property, the SS loaded alginate nanoparticles were first formulated as SS loaded alginate nanoparticle topical gel. The obtained gel formulations exhibited good homogeneity with absence of aggregates (Fig. 5). We also confirmed that all gel formulations were stable under accelerated conditions. The gel formulations were then tested in Albino Wistar rats for the anti-inflammatory study by carrageenan-induced rat paw edema assay which are widely used for evaluating anti-inflammatory activity at the local site. Carrageenan-induced paw edema consists of two phases. The early phase, mast cells secrete histamine and serotonin during 1 h after carrageenan injection [31,32]. The later phase involves the
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Fig. 7. Histopathological examination of paw tissue after 6 h with 1% carrageenan injection (100×, 400×). (A, B) Normal tissues. (C, D) Reference gel-treated tissues. (E, F) Gel base-treated tissues. (G, H) 20% SS gel-treated tissues and (I, J) 80% SS gel-treated tissues. (K, L) Alginate nanoparticle gel-treated tissues. (M, N) 20% SS loaded alginate nanoparticle gel-treated tissues and (O, P) 80% SS loaded alginate nanoparticle gel-treated tissues.
prominent release of prostaglandins. Interleukin-1 (IL-1) is also released from macrophage to induce PMNs infiltration at the inflammatory site. Subsequently, the activated PMNs secrete lysosomal enzyme as well as active oxygen species, resulting in debridement and edema which indicated the inflammation [33]. In this study, carrageenan injection induced significant paw edema and reached maximum swelling at 3–4 h after injection (Table 2). The results of paw edema assay were calculated and reported as the percentage of edema inhibition as shown in Fig. 6. The 80% and 20% SS loaded alginate nanoparticle gel exhibited edema inhibition whereas the internal control groups hardly showed inhibition. Even the inhibitory effect of the 80% SS loaded alginate nanoparticle gel formulations were found to be lower than those of reference gel, a natural anti-inflammatory property of SS also offer antiinflammatory effect with a sustainable action. We also performed histological examination in the paw tissue (Fig. 7) and our findings corresponded to the previous work [17]. Pathologic changes after carrageenan injection, including a large number of PMNs infiltration and edema which revealed localized inflammatory responses were clearly observed over the paw treated with gel base and alginate nanoparticle gel (control groups) as shown in Fig. 7 (E, F) and (K, L), respectively. However, the treatment of rats with reference gel as well as SS loaded alginate nanoparticle gel formulations prior to carrageenan injection exhibited a significant decrease in the number of cellular infiltration which implied that the inflammation can be suppressed by both treatments (Fig. 7 (C, D), (M, N), and (O, P)). Our findings suggest that the anti-inflammatory efficacy of SS loaded alginate nanoparticles gel in the carrageenan-induced
edema model of acute inflammation confirm its role as an effective natural anti-inflammatory agent which can be beneficial for the treatment of acute inflammation. 5. Conclusions The current study has shown that SS can be incorporated into alginate nanoparticles by a simple method. Various SS loadings slightly affected particle size and SS release. These as-prepared SS loaded alginate nanoparticles were further formulated into a topical gel. The gel preparations were stable under accelerated conditions. The topical application of SS loaded alginate nanoparticle gel formulation can achieve the inflammation inhibition induced by carrageenan. Acknowledgements The author would like to thank and show appreciation to Assistant Professor Flying Officer Dr. Pasarapa Towiwat, from the Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, for the valuable suggestions and support throughout of animal model in this work. The author also would like to acknowledge Assistant Professor Dr. Prasit Suwannalert, from the Department of Pathobiology, Faculty of Sciences, Mahidol University, for support and assistance. The author gratefully acknowledges the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund) and Grant for International Research Integration: Research Pyramid,
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Silk sericin loaded alginate nanoparticles: Preparation and anti-inflammatory efficacy Thitikan Khampieng a,1 , Pornanong Aramwit b , Pitt Supaphol a,∗ a The Petroleum and Petrochemical College and Center of Excellence on Petrochemical and Materials Technology, Chulalongkorn University, Bangkok 10330, Thailand b Bioactive Resources for Innovative Clinical Applications Research Unit and Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
a r t i c l e
i n f o
Article history: Received 21 May 2015 Received in revised form 1 July 2015 Accepted 13 July 2015 Available online 16 July 2015 Keywords: Silk sericin Alginate nanoparticles Carrageenan-induced paw edema Anti-inflammation
a b s t r a c t In this study, silk sericin loaded alginate nanoparticles were prepared by the emulsification method followed by internal crosslinking. The effects of various silk sericin loading concentration on particle size, shape, thermal properties, and release characteristics were investigated. The initial silk sericin loadings of 20, 40, and 80% w/w to polymer were incorporated into these alginate nanoparticles. SEM images showed a spherical shape and small particles of about 71.30–89.50 nm. TGA analysis showed that thermal stability slightly increased with increasing silk sericin loadings. FTIR analysis suggested interactions between alginate and silk sericin in the nanoparticles. The release study was performed in acetate buffer at normal skin conditions (pH 5.5; 32 ◦ C). The release profiles of silk sericin exhibited initial rapid release, consequently with sustained release. These silk sericin loaded alginate nanoparticles were further incorporated into topical hydrogel and their anti-inflammatory properties were studied using carrageenan-induced paw edema assay. The current study confirms the hypothesis that the application of silk sericin loaded alginate nanoparticle gel can inhibit inflammation induced by carrageenan. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Silk sericin (SS) is a globular protein, consisting of 18 amino acids, that is obtained from silk cocoon. SS acts as an adhesive and holds two fibroin filaments together, leading to cocoon formation. Because of the tremendous amount of studies on the advantages and biological properties of SS, this protein has become attractive for use in many industries. SS provides a wide range of applications: antioxidant [1–5], anticoagulant [6], and anti-wrinkle [7]. This protein is also reported to suppress tumor growth [8,9] and to reduce oxidative stress [9]. Panilaitis et al. [10] concluded that SS itself does not significantly activate macrophage. In addition, SS has no ability to induce immune response in in vivo experiments and can be used in many biomedical applications [11,12]. SS accelerates cell proliferation and increases cell viability in different cell lines, i.e. hybridoma [13], insulinoma [14], and human skin fibroblast [15]. Moreover, UVB-induced apoptosis in human skin keratinocytes can be inhibited by SS [4].
∗ Corresponding author. E-mail address: [email protected] (P. Supaphol). 1 |Present address: Center for Research and Innovation, Faculty of Medical Technology, Mahidol University, Nakhon Pathom 73170, Thailand. http://dx.doi.org/10.1016/j.ijbiomac.2015.07.018 0141-8130/© 2015 Elsevier B.V. All rights reserved.
In wound applications, the proliferation and attachment of human skin fibroblasts can be promoted when cultured in SS media [15]. SS also increases collagen production [16]. According to Aramwit et al. [17], SS also plays a role in the suppression of carrageenan-induced inflammation which promotes wound healing. Regardless of many benefits associated with SS, SS solution is unstable and can degrade due to pH and temperature sensitivity. This instability makes the SS solution difficult to use. In the pharmaceutical industry, encapsulation is a technique that is widely used to sustain or prolong drug release, to stabilize drugs from heat, moisture, light or vaporization, and reduce drug toxicity. Alginate microparticles have been used for the encapsulation of abundant of biological and bioactive materials such as proteins [18–20], enzymes [21], antibodies [22], cells [23], and DNA [24]. Alginates are natural biodegradable polymers obtained from kelp (brown algae). The gelation of alginate can be simply induced by various divalent cations, especially Ca2+ , which interacts with two carboxyl groups on the neighboring alginate molecules resulting in ionic inter-chain connections [25]. Entrapment of protein into alginate microparticles has several advantages; the alginate matrix protects proteins from different environments, provides a mucoadhesive property, exhibits low toxicity, and low immunogenicity [26]. Hence, a mild protein
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encapsulation technique, emulsification followed by internal gelation, is a good way to fabricate SS loaded alginate nanoparticles without the degradation of SS. The aims of the current study was to prepare SS loaded alginate nanoparticles and investigate their morphology, size, chemical structure, thermal properties, and release profile of SS from the SS loaded alginate nanoparticles. A final aim of this study was to formulate a SS loaded alginate nanoparticle topical gel which will be beneficial for topical use and to further study the in vivo antiinflammatory efficacy. Therefore, SS loaded alginate nanoparticle gel formulations are proposed for this study, to obtain more stability and sustainable release of SS. It is hypothesized that the application of SS loaded alginate nanoparticle gel would inhibit inflammation induced by carrageenan. 2. Experiment 2.1. Materials Fresh white cocoons from Bombyx mori silkworms which were produced in a well-controlled environment were provided by Chul Thai Silk Co., Ltd. (Petchaboon, Thailand). SS was isolated from the cocoons using a high temperature and pressure degumming technique. Briefly, the silk cocoons were finely cut into small pieces and soaked in purified water (ratio of dry silk cocoon:purified water = 1 g:30 mL). Subsequently, the mixture was degummed using an autoclave (Tomy Seiko SS-320, Japan) at 120 ◦ C for 60 min. Then fibroin, which is insoluble, was removed from the obtained solution by filtration. The SS solution was concentrated until it reached the desired concentration, which was quantified by a Pierce® bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, USA). The obtained SS had a molecular weight of ∼25–150 kDa, as reported previously [27]. Sodium alginate was purchased from Aldrich (USA). Calcium carbonate (CaCO3 ) and Span® 80 were obtained from Fluka (USA). Paraffin oil was supplied from Carlo Erba Reagents (Italy). Glacial acetic acid, isopropyl alcohol, and sodium acetate were bought from Sigma-Aldrich (USA). Difelene gel® was purchased from Thai Nakorn Patana (Thailand). Carbopol® Ultrez 21, propylene glycol (PG), triethanolamine (TEA), carrageenan, sodium hydroxide (NaOH), carbon dioxide, corn cob were supported by Faculty of Pharmaceutical Sciences, Chulalongkorn University. Formalin buffer was provided by Department of Pathobiology, Faculty of Sciences, Mahidol University. All chemicals were used as received without further purification. Male Albino Wistar rats (4 weeks old, weighing 150–200 g) were used for the anti-inflammatory study by carrageenan-induced rat paw edema assay. Standard laboratory conditions of temperature (25 ± 2 ◦ C), a light/dark cycle of 12 h/12 h with free access to standard food (C.P. Company, Thailand) and water were provided for animals. The animals were acclimatized for at least 3–5 d before the experiments. At the end of the in vivo evaluation, the animals were euthanized using carbon dioxide inhalation. The investigation was carried out under aseptic conditions in agreement with local guidelines for the care of laboratory animals and approved by the Ethics committee of faculty of Pharmaceutical Sciences, Chulalongkorn University (No. 13-33-008). 2.2. Preparation of alginate and SS loaded alginate nanoparticles Alginate nanoparticles were successfully prepared by using an emulsification method and followed by internal crosslinking [28]. First, 0.2% (w/v) sodium alginate was completely dissolved in distilled water and then mixed with 5% (w/v) calcium carbonate
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aqueous solution with an ultra-turrax mixer for 5 min. The obtained solution was then poured into a mixture of paraffin oil and 1% (v/v) Span® 80 and mixed for another 15 min to allow for nanoparticle formation. Subsequently, paraffin oil and glacial acetic acid mixture was added to the oil phase to ionize calcium ion with continuous stirring for 30 min. At this step crosslinking of the alginate nanoparticles occurred. Finally, the alginate nanoparticles were harvested using centrifugation and washed with isopropanol to get the desired alginate nanoparticles. For the SS loaded alginate nanoparticles, the same procedure was carried out-except in the first step SS solution was added in the neat alginate solution to achieve SS ∼20, 40 and 80% (w/w) based on the weight of sodium alginate polymer. 2.3. Preparation of topical gel containing alginate or SS loaded alginate nanoparticles The topical gel containing alginate nanoparticles or SS loaded alginate nanoparticles were formulated by mixing the as-prepared nanoparticle with Carbopol® Ultrez 21 gel base. The formulation of gel base was 0.5% (w/w) Carbopol® Ultrez 21, 1% (w/w) PG, 0.5% (w/w) TEA, and distilled water. The alginate nanoparticle gel, 20% SS loaded alginate nanoparticle gel, and 80% SS loaded alginate nanoparticle gel were prepared by mixing gel base with alginate nanoparticle, 20% SS loaded alginate nanoparticle, and 80% SS loaded alginate nanoparticle, respectively. As the internal control, 20% and 80% SS gel were also prepared by mixing gel base with SS solution-to achieve the amount of SS equal to amount of SS within 20% and 80% SS loaded alginate nanoparticle, respectively. 2.4. Characterization and testing 2.4.1. Alginate and SS loaded alginate nanoparticles 2.4.1.1. Morphology observations. The morphology of the prepared alginate and SS loaded alginate nanoparticles were examined using a scanning electron microscope (SEM, Hitachi S-4800, Japan). Prior to SEM operation, the specimens were dispersed onto a conductive tape and sputter-coated to achieve a thin layer of platinum under vacuum. Then the specimens were imaged at a magnification of 50,000 and 100,000 times with incident electron beam energy of 10 kV. The average size of the alginate nanoparticles and SS loaded alginate nanoparticles was also determined by Semafore 5.21 software. At least 30 measurements on different nanoparticles were averaged to obtain a data point. 2.4.1.2. Fourier transform infrared spectroscopy (FTIR) study. To identify the interaction between alginate and SS in SS loaded alginate nanoparticles, an FTIR study was assessed using an FTIR spectrophotometer (NEXUS-870, Thermo Nicolet Corporation), equipped with a deuterated triglycine sulfate detector. Spectra were measured in the spectral region from 4000 cm−1 to 400 cm−1 in transmission mode at a resolution of ±4 cm−1 with scan frequency of 32 times. Optical grade potassium bromide was applied as the background measurement material. 2.4.1.3. Thermal analysis. Thermal behavior of the alginate nanoparticles and SS loaded alginate nanoparticles was analyzed through thermo-gravimetric analysis (Pyris Diamond TG-DTA, Perkin Elmer). Specimens (5 mg) were placed in aluminum crucibles then heated from ambient temperature to 650 ◦ C with a constant heating rate (10 ◦ C/min) under nitrogen atmosphere and the percentage of weight was collected over temperature. 2.4.1.4. In vitro release study. The release study of SS from the SS loaded alginate nanoparticles was performed in acetate buffer (pH 5.5) at skin temperature (32 ◦ C). To prepare the acetate buffer, 150 g
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of sodium acetate was dissolved in 250 mL of distilled water. Subsequently, 15 mL of glacial acetic acid was slowly added into the as-prepared sodium acetate solution. Eventually, the volume of the solution was then adjusted to 1000 mL with distilled water. For in vitro release characterization, experiments were carried out with the following procedures, approximately 300 mg of SS loaded alginate nanoparticles were incubated in 30 mL of citrate buffer that was maintained at 32 ◦ C and 200 rpm during the assay. At specific times, between 0 min to 7 d, 100 L of release medium was sampled to analyze the amount of SS released, using a BCA kit. Three parallel experiments were conducted and the results were presented as mean values. 2.4.2. Gel containing alginate and SS loaded alginate nanoparticles 2.4.2.1. Physical appearance of gel formulation. Formulations of the gel preparation were visually inspected for color, phase separation, and homogeneity. Texture and sensitivity when applying the SS loaded alginate nanoparticles gel onto the skin were also investigated. 2.4.2.2. Accelerated stability study. Selected gel formulations were subjected to an accelerated stability test. Briefly, the gel formulations were centrifuged at 3000 rpm or 10,000 rpm for 30 min to investigate any changes in appearance, which consequently affirmed the stability of the formulations. 2.4.2.3. Anti-inflammatory evaluation. Anti-inflammatory properties of the selected SS loaded alginate nanoparticle gels were evaluated using the carrageenan-induced edema model [29]. More specifically, forty-two Wistar rats were randomly distributed into seven groups of six rats for topical gel treatment. The rats in the control group were treated with gel base or alginate nanoparticle gel, while the rats that served as reference group were treated with commercial diclofenac gel (Difelene gel® (Thai Nakorn Patana, Thailand)). Two of the experimental groups received different formulations: 20% SS loaded alginate nanoparticle gel and 80% SS loaded alginate nanoparticle gel. The rats were treated with 20% SS gel, and 80% SS gel served as internal control group. Food was withheld from the rats for 6 h before the experiment; free access to water was provided. Each rat was weighed and marked on the right hind paw behind the tibiatarsal junction, and then a baseline measurement of the right hind paw volume was taken using a water displacement plethysmometer (Model 7140, Ugo Basile, Italy). Various SS loaded alginate gel formulations, reference gel (Difelene gel® ), or gel base (2.5 g) was applied topically onto the plantar surface of the right hind paw and rubbed for 15 s until completely absorbed. One hour after the treatment, 100 L of 1% (w/v) carrageenan solution was subcutaneously injected into the right hind paw. The paw volumes according to the inflammatory response were examined using a plethysmometer at 1 h time intervals until 6 h after the carrageenan injection. Percentage of edema inhibition was calculated in comparison to the control group using following formula: Percentage of Inhibition =
(Vt − Vo ) − (Vt − Vo )test × 100 (Vt − Vo )control
(1)
where Vt is paw volume at 1 h time intervals to 6 h after carrageenan injection, V0 is paw volume before the treatment. 2.4.2.4. Histological evaluation. After the carrageenan test, the animals were euthanized by carbon dioxide inhalation. The hind paws were removed and fixed by immersion in 10% neutral buffered formalin and then decalcified. The tissue samples from the paws were dissected and embedded in a paraffin block and thin sections (5 m) were prepared. Subsequently, the as-prepared
tissue sections were attached to glass slides and stained with Hematoxylin–Eosin (H&E). An Olympus BX50 optical microscope was used to capture digital images of the tissue samples. At least ten fields from the inflammatory site in each tissue section were randomly inspected. Morphology investigation of the inflammatory cell response-cellular infiltration, swelling, hemorrhage-was evaluated with a blind manner. 2.4.3. Statistical analysis Data were expressed as means ± standard errors of means (n = 6). Statistical analysis was performed based on ANOVA followed by LSD post hoc test. The statistical significance for the entire test was accepted at a 0.05 confidence level. 3. Results 3.1. Alginate and SS loaded alginate nanoparticles 3.1.1. Morphology observations The SEM images of the alginate nanoparticles and the SS loaded alginate nanoparticles are shown in Fig. 1. All particles were nearly spherical with some deflated surfaces and reasonable size uniformity in the range of 71.30–89.50 nm. The average grain size of SS loaded alginate nanoparticles was found to be slightly decreased with increasing amount of SS into the system, as shown in Table 1. 3.1.2. Fourier transform infrared spectroscopy (FTIR) analysis FTIR spectra were used to determine the chemical functional groups and molecular interactions between alginate and SS, as shown in Fig. 2. The strong and broad peaks in the 3500–3300 cm−1 range correspond to O H stretching and intermolecular hydrogen bonding. At the wave numbers of 1700–1400 cm−1 , the observed peaks belong to the C O stretching (amide). The FTIR spectrum of the alginate nanoparticles shows two distinctive absorption bands at ∼1637 cm−1 and 1412 cm−1 relative to both asymmetric and symmetric C O stretching absorption of the carboxylic acid salts, respectively. In spectra of SS loaded alginate nanoparticles, the absorption bands of Amide I, II, and III presented at 1625 cm−1 , 1506 cm−1 , and 1236 cm−1 , respectively, while characteristic peaks of alginate still observed. A slight difference can be observed between the alginate nanoparticles and the SS loaded alginate nanoparticles at various SS loadings. 3.1.3. Thermal analysis The TGA curves of the alginate nanoparticles and the SS loaded alginate nanoparticles are shown in Fig. 3. Two regions of degradation were observed: the first step, from 240 ◦ C to 270 ◦ C and the second rapidly decompose, from 280 ◦ C to 480 ◦ C. In addition, more thermal stability can be obtained with increasing SS loadings within the SS loaded alginate nanoparticles. 3.1.4. In vitro Release study The release characteristic of SS from SS loaded alginate nanoparticles was studied by the total immersion technique in acetate buffer at skin temperature. Fig. 4 shows the cumulative release amount of SS from these nanoparticles. During the first 8 h after Table 1 Average grain size of the as-prepared nanoparticles. Sample
Alginate: SS mass ratio
Average grain size (nm)
Alginate nanoparticles 20% SS loaded alginate nanoparticles 40% SS loaded alginate nanoparticles 80% SS loaded alginate nanoparticles
– 5:1 2.5:1 1.25:1
89.5 88.4 79.2 71.3
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Fig. 1. SEM micrographs of (A) alginate nanoparticles, (B) 20% SS loaded alginate nanoparticles, (C) 40% SS loaded alginate nanoparticles, and (D) 80% SS loaded alginate nanoparticles with a magnification of 100,000×.
Fig. 3. TGA curves of alginate nanoparticles and SS loaded alginate nanoparticles. Fig. 2. FTIR spectra of alginate nanoparticles and SS loaded alginate nanoparticles.
submersion in acetate buffer, the cumulative release of SS increased rapidly and then gradually after 8 h. As expected, the maximum amount of SS released from these materials increased with increasing SS loadings in the alginate nanoparticles. 3.2. Gel containing SS loaded alginate nanoparticles 3.2.1. Physical appearance of gel formulation Fig. 5 illustrates the as-prepared gel formulations. All formulations were rubbed on the back of the hand and no noticeable solid components were found, which can imply that all samples had uniform consistency. Also, no irritation was observed upon application of gel samples to the skin.
3.2.2. Accelerated stability study Stability studies of gel formulations were performed by centrifuge stress test. The results of the accelerated stability studies revealed that the selected gel formulations were stable under accelerated conditions. There were no significant alterations in the physical appearance-gel formulations remained the same and no phase separation occurred after subjecting the formulations to centrifugation at 3000 rpm and 10,000 rpm up to 30 min (the appearance of the selected gel formulations is available as Supplementary material). 3.2.3. Anti-inflammatory evaluation Pharmacological screening was performed by the carrageenaninduced edema model to evaluate the possible anti-inflammatory activity of the as-prepared gel formulations. Subcutaneous
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(Table 2). The results of paw edema assay were calculated and reported as the percentage of edema inhibition as shown in Fig. 6. As expected, the anti-inflammatory effect of reference gel achieved the highest percentage of edema inhibition, about 60.46%, 4 h after carrageenan injection. While the 80% and 20% SS loaded alginate nanoparticle gel formulations exhibited less anti-inflammatory effect when compared to reference gel. The 80% and 20% SS loaded alginate nanoparticle gel exhibited edema inhibition whereas the internal control groups hardly showed inhibition. In addition, edema inhibition of the 80% and 20% SS loaded alginate nanoparticle gel were comparatively better for longer periods compared to the 80% and 20% SS gel (internal control). Hence, the topical administration of SS loaded alginate nanoparticle gel was able to inhibit inflammation induced by carrageenan. The natural anti-inflammatory agent, SS loaded alginate nanoparticle gel formulations, can be encouraged as an alternative form for the treatment of acute inflammation. Fig. 4. Cumulative release profiles of SS from SS loaded alginate nanoparticles. The error bars represent the standard deviation based on three replication analysis (*p < 0.05).
administration of carrageenan into the right hind paw produced significant edema, with a maximum response being observed 3–4 h after injection, as shown in Table 2. In this study, carrageenan injection induced significant paw edema and reached maximum swelling at 3–4 h after injection
3.2.4. Histological evaluation According to the histological examination, paw biopsies revealed pathologic changes after carrageenan injection, including cellular infiltration and edema as shown in Fig. 7. In rats treated with reference gel, no sign of cellular infiltration or swelling was detected when compared to untreated rats. In the control groups treated with gel base and alginate nanoparticle gel, a large number of white blood cells, polymorphonuclear cells (PMN), which is
Fig. 5. Gel formulations: (A) gel base, (B) reference gel (Difelene gel® ), (C) alginate nanoparticle gel, (D) 20% SS loaded alginate nanoparticle gel, (E) 80% SS loaded alginate nanoparticle gel, (F) 20% SS gel, and (G) 80% SS gel.
Table 2 Average rats paw edema volume of various treatments in carrageenan-induced paw edema (n = 6). * p < 0.05 significantly different compared to gel base. # p < 0.05 significantly different compared to alginate nanoparticle gel. Treatments
Paw edema (mL) ± S.E.M. 1h
Gel base Reference gel (Commercial Diclofenac gel) 20% SS gel 80% SS gel Alginate nanoparticle gel 20% SS loaded alginate nanoparticle gel 80% SS loaded alginate nanoparticle gel
0.56 0.52 0.64 0.64 0.78 0.80 0.73
2h ± ± ± ± ± ± ±
0.08 0.11 0.05 0.07 0.09 0.05 0.06
0.61 0.50 0.96 0.90 0.95 0.90 0.73
3h ± ± ± ± ± ± ±
0.11 0.06 0.02 0.10 0.10 0.15 0.07
0.87 0.55 1.21 1.09 1.25 1.11 0.95
4h ± ± ± ± ± ± ±
0.07 0.07* 0.10 0.09 0.04 0.12 0.15
1.17 0.46 1.14 1.01 1.03 0.98 0.60
5h ± ± ± ± ± ± ±
0.12 0.09* 0.07 0.09 0.10 0.16 0.02#
0.94 0.65 1.14 1.13 1.08 0.61 0.68
6h ± ± ± ± ± ± ±
0.07 0.09* 0.08 0.10 0.11 0.09# 0.09#
0.94 0.65 1.16 0.98 0.79 0.72 0.45
± ± ± ± ± ± ±
0.07 0.07* 0.09 0.09 0.12 0.10 0.07#
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Fig. 6. The percentage of edema inhibition of various gel formulations. (Error bar represents the standard error of the mean (n = 6)).
crucial for cell inflammation, were observed. Swelling and hemorrhage were also noticed, which indicated high levels of inflammation. Whereas, there was less cellular infiltration and less edema in rats treated with the 80% and 20% SS loaded alginate nanoparticle gel, while the 80% SS gel and 20% SS gel treated tissues prior to carrageenan injection showed cellular infiltration in the dermal layer.
4. Discussions SS has been reported to be beneficial in accelerating cell proliferation and increasing cell viability in different cell lines [13–15]. Aramwit et al. [16,17] suggested that SS has ability to promote wound healing. In this study, we aimed to prepare SS loaded alginate nanoparticles which further formulated to a topical hydrogel, then proposed that SS loaded alginate nanoparticle gel could effectively decrease inflammation induced by carrageenan in rat model. Firstly, we observed the morphology of the nanoparticles and we found that the SS loaded alginate nanoparticles were uniformly spherical in shape (Fig. 1). According to the slightly decreasing in average grain size of SS loaded alginate nanoparticles when SS content increased (Table 1), this could relate to the amount of alginate in alginate/SS mass ratio which decreased from 5:1 to 1.25:1, less functional group to interact with Ca+ , resulting in the smaller size of as-prepared nanoparticles. To investigate the interaction between alginate and SS in nanoparticles, the FTIR spectrum of as-prepared nanoparticles were then analyzed, as shown in Fig. 2. Obviously, FTIR spectra of SS loaded alginate nanoparticles displayed all
the characteristic peaks of both alginate and SS. However, the absorption bands associated with C O stretching, O H stretching, and saccharide bands slightly shifted to a higher wave number and band intensities decreased as the SS content increased, which indicated the interaction between SS and alginate within the nanoparticles. The alginate nanoparticles and the SS loaded alginate nanoparticles showed thermal stability until 200 ◦ C, as shown in TGA thermograph (Fig. 3). Thermal stability above 240 ◦ C slightly increased with increasing SS loadings from 20% to 80%. This corresponded to Likitamporn et al. [30], the maximum decomposition temperatures of the sericin/PVA/bentonite scaffolds significantly increased with increasing sericin content. Additionally, we report the release profile of SS from SS loaded alginate nanoparticles (Fig. 4). At initial phase, SS rapidly diffused through the swollen alginate matrix. Subsequently, a sustained release was achieved. Both features would be favorable for the prolonged therapeutic effect of SS for transdermal application. To evaluate the in vivo anti-inflammatory property, the SS loaded alginate nanoparticles were first formulated as SS loaded alginate nanoparticle topical gel. The obtained gel formulations exhibited good homogeneity with absence of aggregates (Fig. 5). We also confirmed that all gel formulations were stable under accelerated conditions. The gel formulations were then tested in Albino Wistar rats for the anti-inflammatory study by carrageenan-induced rat paw edema assay which are widely used for evaluating anti-inflammatory activity at the local site. Carrageenan-induced paw edema consists of two phases. The early phase, mast cells secrete histamine and serotonin during 1 h after carrageenan injection [31,32]. The later phase involves the
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Fig. 7. Histopathological examination of paw tissue after 6 h with 1% carrageenan injection (100×, 400×). (A, B) Normal tissues. (C, D) Reference gel-treated tissues. (E, F) Gel base-treated tissues. (G, H) 20% SS gel-treated tissues and (I, J) 80% SS gel-treated tissues. (K, L) Alginate nanoparticle gel-treated tissues. (M, N) 20% SS loaded alginate nanoparticle gel-treated tissues and (O, P) 80% SS loaded alginate nanoparticle gel-treated tissues.
prominent release of prostaglandins. Interleukin-1 (IL-1) is also released from macrophage to induce PMNs infiltration at the inflammatory site. Subsequently, the activated PMNs secrete lysosomal enzyme as well as active oxygen species, resulting in debridement and edema which indicated the inflammation [33]. In this study, carrageenan injection induced significant paw edema and reached maximum swelling at 3–4 h after injection (Table 2). The results of paw edema assay were calculated and reported as the percentage of edema inhibition as shown in Fig. 6. The 80% and 20% SS loaded alginate nanoparticle gel exhibited edema inhibition whereas the internal control groups hardly showed inhibition. Even the inhibitory effect of the 80% SS loaded alginate nanoparticle gel formulations were found to be lower than those of reference gel, a natural anti-inflammatory property of SS also offer antiinflammatory effect with a sustainable action. We also performed histological examination in the paw tissue (Fig. 7) and our findings corresponded to the previous work [17]. Pathologic changes after carrageenan injection, including a large number of PMNs infiltration and edema which revealed localized inflammatory responses were clearly observed over the paw treated with gel base and alginate nanoparticle gel (control groups) as shown in Fig. 7 (E, F) and (K, L), respectively. However, the treatment of rats with reference gel as well as SS loaded alginate nanoparticle gel formulations prior to carrageenan injection exhibited a significant decrease in the number of cellular infiltration which implied that the inflammation can be suppressed by both treatments (Fig. 7 (C, D), (M, N), and (O, P)). Our findings suggest that the anti-inflammatory efficacy of SS loaded alginate nanoparticles gel in the carrageenan-induced
edema model of acute inflammation confirm its role as an effective natural anti-inflammatory agent which can be beneficial for the treatment of acute inflammation. 5. Conclusions The current study has shown that SS can be incorporated into alginate nanoparticles by a simple method. Various SS loadings slightly affected particle size and SS release. These as-prepared SS loaded alginate nanoparticles were further formulated into a topical gel. The gel preparations were stable under accelerated conditions. The topical application of SS loaded alginate nanoparticle gel formulation can achieve the inflammation inhibition induced by carrageenan. Acknowledgements The author would like to thank and show appreciation to Assistant Professor Flying Officer Dr. Pasarapa Towiwat, from the Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, for the valuable suggestions and support throughout of animal model in this work. The author also would like to acknowledge Assistant Professor Dr. Prasit Suwannalert, from the Department of Pathobiology, Faculty of Sciences, Mahidol University, for support and assistance. The author gratefully acknowledges the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund) and Grant for International Research Integration: Research Pyramid,
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