alginate microparticles with promising cytocompatibility and antibacterial properties

Colloids and Surfaces A 585 (2020) 124081

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Synthesis and characterization of silver nanoparticles-doped hydroxyapatite/alginate microparticles with promising cytocompatibility and antibacterial properties

T

Qiuju Zhoua,1, Tianwen Wangb,c,1, Can Wangb,c, Zheng Wangb, Yanan Yangb,c, Pei Lib, Ruihua Caib, Meng Sunb,c, Hongyu Yuanb,c, Lei Nieb,c,⁎ a b c

Analysis & Testing Center, Xinyang Normal University, Xinyang 464000, China Henan Key Laboratory of Tea Plant Biology, College of Life Sciences, Xinyang Normal University, Xinyang 464000, China Institute for Conservation and Utilization of Agro-Bioresources in Dabie Mountains, Xinyang 464000, Henan, China

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO

A B S T R A C T

Keywords: Silver nanoparticles Microparticles Biomaterials Cytocompatibility Antibacterial activity

In this paper, a facile double-emulsion approach was reported to fabricate the silver nanoparticles (AgNPs)doped hydroxyapatite/alginate nanocomposite microparticles. First, hydroxyapatite nanoparticles (HApNPs) were prepared using coprecipitation method, then, HApNPs were involved in sodium alginate (Alg) network to form HAp/Alg microparticles via emulsion process. Next, silver nitrate was involved into HAp/Alg microparticles using second emulsion, which ascorbic acid was used as a trigger to form in situ-AgNPs-doped HAp/Alg microparticles. Physicochemical properties of the nanocomposite microparticles were characterized by Transmission Electron Microscopy (TEM), Fourier Transform Infrared (FT-IR) spectra, Dynamic Light Scattering (DLS), Ultraviolet-Visible (UV–vis) Absorption Spectra, and Thermogravimetric analysis (TG). It was demonstrated that AgNPs and HApNPs were evenly distributed in entire microparticles (diameter ∼550 nm). Furthermore, the AgNPs-doped HAp/Alg microparticles showed a good cytocompatibility by culturing with A549 cells. Finally, the microparticles displayed an excellent antibacterial activity against Gram-negative E. coli and Gram-positive S. aureus. Above results manifested the significance of the final microparticles in diverse biomedical applications.

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Corresponding author at: Henan Key Laboratory of Tea Plant Biology, College of Life Sciences, Xinyang Normal University (XYNU), Xinyang 464000, China. E-mail address: [email protected] (L. Nie). 1 Qiuju Zhou and Tianwen Wang contributed equally to this work and should be considered co-first authors. https://doi.org/10.1016/j.colsurfa.2019.124081 Received 23 March 2019; Received in revised form 8 August 2019; Accepted 6 October 2019 Available online 11 October 2019 0927-7757/ © 2019 Elsevier B.V. All rights reserved.

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1. Introduction

2. Experimental section

The overuse and inappropriate consumption of antibiotics have driven the rapid emergence of multidrug-resistant pathogens, which were currently considered as an emergent global disease [1]. Antimicrobial resistance, which leads to the rapid increases in the morbidity, mortality, length of hospitalization and healthcare costs, has become one of the most serious threats to human health today. Regarding that the antimicrobial resistance is a response to antimicrobial exposure, so it is challenged to develop new antibiotic without emerging resistance [2–4]. Developing novel and effective bactericidal agents, with avoiding potential health risks associated with the emergence of multidrug-resistant bacteria and/or incidences of cross contamination, remains highly desirable. Many metals, such as silver (Ag), copper (Cu), and Zinc (Zn), have been used to against bacteria for a long history before the antibiotics [5]. Since antiquity, the antibacterial effects of Ag salts had been noticed, and Ag was considered as the most popular antibacterial or antifungal agent [6,7]. Now Ag was employed to treat infections or wounds in a variety of applications, including dental work, catheters, and burn wounds, and so on [8,9]. It is well known that both Ag ions and Ag-based compounds are toxic to microorganisms. The Ag nanoparticles (AgNPs) have been reported to be strong and broad-spectrum antimicrobial materials and the antibacterial activity is a thousand times higher than silver ions, due to severe DNA damage and free radical generation caused by AgNPs [10]. Recently, the notable antibacterial activity of AgNPs against various class of bacteria including Salmonella, Staphylococcus, and Pseudomonas, etc, has been observed. However, both antibacterial activity and stability of AgNPs are closely related to their size and distribution, and small-sized AgNPs tend to aggregate, severely sacrificing their antibacterial activities. To overcome such shortcomings, one popular approach was to prepare the large-size nanoparticles with diameter ca. at 70 nm [11–13]. In addition, the AgNPs was immobilized on a proper support material such as SiO2, zinc oxide, and graphene, to improve the physicochemical stability of AgNPs exposed to the aqueous medium [14]. It was confirmed that AgNPs immobilized on various substrates exhibited an enhanced antibacterial performance over long term of use [15]. AgNPs could be adsorbed on the surface of hydroxyapatite (HAp) nanoribbon spherites to form nanocomposite spheres, which were used as coating on metal substrate, displaying a good balance between the biocompatibility and antibacterial properties [16,17]. Hydroxyapatite, as the main inorganic component of natural bone, was widely used for biomedical applications [18,19]. HAp hybridized with AgNPs showed an excellent antibacterial activity, which expanded its applied range [20–23]. In addition, AgNPs could also be incorporated into hybrid materials, e.g. biomimetic HAp-collagen matrix, to provide more binding sites for drug delivery applications [23,24]. However, the AgNPs distribution in hybrid nanocomposite, and the regulation on nanocomposite morphology and nanostructure need to be further improved to ameliorate the balance between cytocompatibility and antibacterial properties for a longer lasting period [25,26]. As a result, we hypothesized that nanostructured hybrid microparticles with incorporating AgNPs possess specific interesting multifunctional characteristics, e.g. cytocompatibility and antibacterial ability [27]. To demonstrate the feasibility of our idea, we first synthesized multifunctional hydroxyapatite/alginate (HAp/Alg) microparticles by following previous reports with minor modifications [28]. Then, AgNPs were in situ incorporated into HAp/Alg microparticles to form the final nanocomposite microparticles. The synthesis mechanism of the AgNPs-doped HAp/Alg microparticles was displayed in Scheme 1. The physicochemical properties of the prepared microparticles were characterized with TEM, FTIR, DLS, UV–vis, and TG. Additionally, cytocompatibility and antibacterial activity were systematically evaluated.

2.1. Materials Calcium chloride (CaCl2), disodium phosphate dodecahydrate (Na2HPO4·12H2O), sodium alginate and sodium hydroxide (NaOH) were supplied by Macklin Co., Ltd. Ascorbic acid, and silver nitrate (AgNO3) were purchased from Aldrich Co., Ltd. All the chemical regents used in this work were in analytical reagent level, and without further purification. 2.2. Synthesis of hydroxyapatite nanoparticles The hydroxyapatite nanoparticles (HApNPs) was prepared by a coprecipitation method [29]. In a typical synthesis, 100 mL of Na2HPO4·12H2O (0.12 M) solution was added to 250 mL of CaCl2 (0.08 M) solution under mechanical stirring at room temperature. Meanwhile, the pH value of the solution was maintained at pH 11 by slowly adding the NaOH solution (1 M). Then, the react solution was continued to stir mechanically for 10 h at 70 °C using water bath. The product was collected by the suction filter way, cleaned with Millipore water, and vacuum filtrated, and dried at 60 °C for 4 h, finally the HApNPs were obtained. 2.3. Synthesis of nanocomposite microparticles In a 50 mL Eppendorf tube, 150 mg of powdered HApNPs with 12 mL of 1% (w/v) sodium alginate was emulsified (8000 rpm) for 10 min first. Then, the obtained product was centrifuged (3000 rpm, 3 min) and washed with Millipore water 3 times. Afterward, 10 mL of AgNO3 (0.01 M) was injected into above product. The mixture was sequentially emulsified (8000 rpm, 5 min), centrifuged (3000 rpm, 3 min), and washed with Millipore water 3 times. Finally, 2 mL of 0.1 M ascorbic acid was added. The final microparticles were centrifuged (3000 rpm, 3 min) and washed with Millipore water 3 times again. Regarding that the influence of AgNO3 molar concentration on the physiochemical properties of final nanocomposite microparticles, three molar concentrations were used and reported in this paper. The designation of MP1, MP2, and MP3 represented that 0.01 M, 0.05 M, and 0.1 M of AgNO3 were used for the preparation of microparticles. 2.4. Microparticles characterization The morphology of prepared AgNPs-doped HAp/Alg microparticles was observed by Cold Field Emission-Scanning Electron Microscopy (SEM, S 4800), and FEI-Transmission Electron Microscopy (TEM, Tecnai G2 F20) equipped with EDS and elemental mapping accessories, the microparticles were deposited on the copper grids. The FT-IR spectra of the samples in the range of 500-4000 cm−1 were recorded on KBr pellets using Fourier Transform Infrared (FT-IR, PerkinElmer, Spectrum 2). The size distribution of microparticles was examined by Dynamic Light Scattering (DLS, Malvern Zetasizer 3000E). The UV–vis spectra of the samples were investigated by using Ultraviolet-visible spectroscopy (UV–vis, Lambda 950). Thermogravimetric analysis (TG) was conducted with TG TA Q600 instrument, heating samples from room temperature to 850 °C at the heating rate of 10 °C min−1 in a normal air to study the thermal behaviour. 2.5. Cytotoxicity assay A549 cell (ATCC® CCL-185™) was used in this paper. According to ATCC instructions, cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich) supplemented with 10% of fetal bovine serum, 100 U mL−1 of penicillin, and 100 μg mL−1 of streptomycin under a humidified atmosphere of 95% air and 5% CO2 at 37 °C. The culture medium was replaced every other day. The cells were 2

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Scheme 1. Synthesis of silver nanoparticles-doped hydroxyapatite/alginate microparticles by double-emulsion method. Fig. 1. Morphology of prepared AgNPs-doped HAp/Alg microparticles (MP2). (a) SEM micrographs showed microparticles were roundlike, and the surface of microparticles was coarse (insets at the top-right corner, b). (c) TEM images showed HApNPs for microparticles preparation were need-like and < 100 nm in length. Elemental mapping of the prepared microparticles (d) revealed the presence of silver (Ag, red), calcium (Ca, green), phosphorus (P, yellow), oxygen (O, purple) and carbon (C, blue-green), indicating that AgNPs and HApNPs evenly distributed in the entire microparticles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

2.6. Bacterial culture and antibacterial assay

passaged by trypsinization, and cells at passage 5 were used for following experiments. The prepared AgNPs-doped HAp/Alg microparticle with different concentration were added into cells solution. The cytocompatibility of prepared microparticles was characterized by using CCK-8 assay. At the same time, the phalloidin-FITC (50 μg/mL, Invitrogen) and 4,6-diamidino-2-phenylindole (DAPI, 10 μg/mL, Invitrogen) were used to stain A549 cells to investigate the proliferation, the green color was used to indicate cytoskeleton, blue color was used to indicate nucleus.

Strain specific antibacterial activity of AgNPs-doped HAp/Alg microparticles was determined against bacteria Gram-negative E. coli (ATCC 25922) and Gram-positive S. aureus(ATCC 6538). A single colony of E. coli and S. aureus on the Luria-Bertani (LB) agar plate was transferred to liquid LB culture medium to make seed culture by growing at 37 °C overnight with 180 rpm rotation. Then, the seed culture was diluted into fresh LB medium and cultured under the same 3

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Fig. 2. FT-IR spectra (a) of the s AgNPs-doped HAp/Alg microparticles. (b) DLS indicated microparticles exhibited very narrow size distributions and centered at round (540∼590 nm, in Millipore water). (c) UV–vis DRS of the prepared microparticles confirmed the presence of AgNPs. (d) TG analysis showed that the molar concentration of AgNO3 could influence the organic/inorganic composition of microparticles.

Supplementary Material). 2.7. Statistical analysis All data were given as mean ± SD (n = 5). PASW statistics program was employed for analysis with significance reported at a p value of < 0.05 for 95% confidence. 3. Results and discussion 3.1. Physicochemical properties of nanocomposite microparticles Though the molar concentration of AgNO3 varied during the preparation of AgNPs-doped HAp/Alg microparticles, it was also difficult to observe the differences between MP1, MP2, and MP3, by naked eyes. Fig. 1a–b displayed the SEM images of AgNPs-doped HAp/Alg microparticles with using 0.05 M AgNO3 (MP2). The SEM images showed that the prepared nanocomposite microparticles were roughly circular in shape and had a rough surface. The SEM images of MP1 and MP3, which were not shown in the paper, which illustrated a very close morphology compared to MP2. X-ray diffraction (XRD) affirmed the success of HApNPs preparation, as shown in Fig. S1. Due to the needlelike HApNPs were used, confirmed by TEM analysis (Fig. 1c), the round-like AgNPs-doped HAp/Alg microparticles disclosed a coarse surface (Fig. S2). In addition, the elemental mapping analysis showed that silver, calcium, phosphorus, oxygen, and carbon were evenly distributed in the entire nanocomposite microparticles, indicating that AgNPs and HApNPs had been completely incorporated into alginate polymer network, as shown in Fig. 1d. A variety of silver

Fig. 3. In vitro cytotoxicity of the AgNPs-doped HAp/Alg microparticles with different silver nitrate molar ratio during the preparation after incubation with A549 cells for different hours confirmed by CCK-8 assay. Without adding nanoparticles as Control Group (CK).

condition. When an OD600nm at 0.6 was reached, the broth was diluted to 105 CFU mL−1 with sterile 0.9% NaCl solution. Cell suspension (50 μL) was spread onto 90 mm LB agar plate. Wells were made with a hole puncher (diameter 4 mm). 30 μL of AgNPs-doped HAp/Alg microparticles of different concentrations was added into the wells. Then, the plates were kept in an incubator (37 °C) for 24 h. The antibacterial activity of microparticles with liquid culture was also tested (Electronic 4

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Fig. 4. Florescence images of A549 cells after incubation with the AgNPs-doped HAp/Alg microparticles with different silver nitrate molar ratio (MP1, MP2 and MP3) at 120 h. Without adding nanoparticles as Control Group (CK), the scale bar is 50 μm.

microparticles was characterized by UV–vis analysis, as shown in Fig. 2c. The UV–vis absorption spectra of microparticles in the region of 325–800 nm showed that microparticles had strong and stable optical absorption capability, which was mainly attributed to good dispersibility of AgNPs in microparticles. The band at 440 nm was due to the surface plasmon resonance of silver, which confirmed that AgNPs were successfully immobilized into microparticles. Furthermore, the formation of HAp/Alg hybrid nanoparticles might cause a red shift (from 235 to 320 nm) in the extinction peak of HApNPs, which was possibly caused by the strong interfacial coupling between immobilized AgNPs and HApNPs [32,33]. The composition of AgNPs-doped HAp/ Alg microparticles was further analysed by TG analysis, as exhibited in Fig. 2d, the curves indicated that the process of weight loss took place in three main steps (30–200 °C, 200–450 °C and 450–900 °C). The thermal and oxidative decomposition of alginate took place in the final step, illustrating that molar ratio of silver nitrate could influence the composition of microparticles. Some silver irons were chelated with alginate network to solidify the microparticles during the emulsion process, thus, the thermal stability of AgNPs-doped HAp/Alg microparticles increased with increasing silver nitrate molar ratio, and the saturation of silver nitrate resulted in the stabilization of final microparticles.

nanocomposite microparticles, such as symmetric nanoporous microparticles and hierarchical silver microparticles, were developed, this was firstly investigating the AgNPs evenly distributed in nanocomposite microparticles [30,31]. The FT-IR analysis was used to study the chemical interaction between different components for AgNPs-doped HAp/Alg microparticles, as shown in Fig. 2a. The band at 1035 cm−1 was due to COe stretching vibrations, and bands at 1611 cm−1 and 1416 cm−1 was corresponded to the asymmetric stretching of carboxylate OeCOe vibration and symmetric stretching vibration of carboxylate group, respectively. Broad band centered at 3440 cm−1 was due to hydrogen bonded OeH stretching vibrations. A PO4-3 stretching peak appeared at 1035 cm−1, and PO4-3 bending vibrations appeared at 602 cm−1 and 564 cm−1. The FT-IR results proved an interaction occurred between AgNPs, and HApNPs and alginate by forming chemical bond or electrostatic attraction. In addition, DLS was applied to measure the size distribution of the prepared microparticles in Millipore water, as shown in Fig. 2b. The DLS analysis confirmed that the microparticles had a good colloidal stability in Millipore water. The mean diameter of MP1, MP2, and MP3 were about 541, 562, and 587 nm, means that the microparticles increased with increasing the molar concentration of AgNO3 from 0.01 M to 0.1 M. Because that UV–vis spectroscopy was a simple and sensitive method for analysing AgNPs, the prepared AgNPs-doped HAp/Alg 5

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Fig. 5. Optical density (OD600nm) of cultured E. coli (a) and S. aureus (b) in the Luria-Bertani (LB) medium supplemented with the AgNPsdoped HAp/Alg microparticles (500 μg mL−1). Insets in the a and b showed the significant antibacterial activity of microparticles in the actual culture tubes (left: control, without microparticles; right: test, supplemented with the indicated microparticles). In addition, antibacterial activities were also confirmed with E. coli (c) and S. aureus (d) grown on nutrient agar plates.

for inorganic antibacterial materials, MP2 still showed an excellent antibacterial activity. The inhibition zones (over 1 mm) caused by the microparticles confirmed that MP1 and MP2 had good antibacterial activities, which was well matched with dose-dependent manner antibacterial tests (Fig. 5c–d, Fig. S4) [36].

3.2. Cytocompatibility of nanocomposite microparticles In vitro cytotoxicity of the AgNPs-doped HAp/Alg microparticles with different silver nitrate molar ratio during the preparation were investigated by CCK-8 assay, as shown in Fig. 3. For all samples, the A549 cells proliferated with increasing culture time from 60 to 120 h, the cell viability decreased with increasing the microparticles concentration, especially that sample MP1 showed a higher cell viability compared to MP2 and MP3. The morphology of A549 cells at hour 120 could be clearly viewed by treated fluorescence assay (DAPI/FITC), the fluorescence images of cells were shown in Fig. 4. The cytoskeleton was represented by green colour, and the nucleus was represented by blue colour. The results displayed that the number of A549 cells in incubation with MP1 was relatively higher than that of MP2 and MP3, which was corresponding to optical microscopy investigation (Fig. S3) and CCK-8 analysis. The above results indicated that the composite microparticles have a good cytocompatibility.

4. Conclusion In summary, a simple and facile method was successfully developed to synthesize AgNPs-doped HAp/Alg nanocomposite microparticles. The AgNPs could be in situ formed in HAp/Alg microparticles, and distributed in entire microparticles evenly. In addition, the AgNPsdoped HAp/Alg microparticles displayed a good cytocompatibility in incubation with A549 cells. Furthermore, the microparticles displayed an effective antibacterial activity against Gram-negative E. coli and Gram-positive S. aureus. The prepared nanocomposite microparticles inhered with a good balance between cytocompatibility and antibacterial activity, might be used in diverse biomedical applications.

3.3. Antibacterial activity of the composite microparticles

Declaration of Competing Interest

Finally, the antibacterial activity of the prepared AgNPs-doped HAp/Alg microparticles was tested against bacteria E. coli (Gram negative) and S. aureus (Gram positive). The optical density at 600 nm (OD600) of bacterial suspension after a growth for 12 h in presence of microparticles indicated that the prepared microparticles could efficiently inhibited the proliferation of both E. coli and S. aureus as compared with control groups (Fig. 5a–b). Regarding that the peptidoglycan layers of bacteria cell wall for E. coli and S. aureus, which prevents the penetration of silver nanoparticles inside the cytoplasm, the antibacterial effect of prepared microparticles on different bacteria using different silver nitrate during the preparation. However, the prepared AgNPs-doped HAp/Alg microparticles still showed good antibacterial activity while bacteria growing on agar plates, especially sample MP2 showed an excellent effect against on both bacteria E. coli and S. aureus [34,35]. Even at a low concentration (i.e., 100 μg mL−1) which is well below the standard minimum inhibitory concentration (800 μg mL−1)

None. Acknowledgements This research was funded by the Nanhu Scholars Program for Young Scholars of XYNU, the Natural Science Foundation of China (31700840), and the Key Scientific Research Project of Henan Province (18B430013, 18A150049) and Key Scientific and Technological Project of Henan Province (182400410166). The assistance in the measurement of TEM and UV–vis DRS by Zongwen Zhang, technician of Analysis & Testing Center of XYNU, was highly appreciated. Appendix A. Supplementary data Supplementary material related to this article can be found, in the 6

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