One-step synthesis of platinum nanoparticles loaded in alginate bubbles

Composite particles with multifunctions have been extensively utilized for various applications. Bubble particles can be applied for ultrasound-mediated imaging, drug delivery, absorbers, cell culture, etc. This study proposes a one-step strategy to obtain Pt nanoparticles loaded in alginate bubbles. A needle-based droplet formation was used to generate uniform alginate particles about 2 mm in diameter. The hydrolysis reaction of NaBH4 was utilized to produce gaseous hydrogen and then trapped within alginate particles to form bubbles. The Pt4+ mixed with alginate solution was dropped into the reservoir to react with reducing NaBH4 and hardening CaCl2 to form Pt nanoparticles-alginate composite bubbles. Results indicate that the size of bubbles decreases with the CaCl2 concentration (1% ~ 20%), and size of bubbles increases with the NaBH4 concentration (1 ~ 20 mM). The advantages for the present approach include low cost, easy operation, and effective production of Pt nanoparticles-alginate composite bubbles.


Background
The metal nanoparticles (NPs) are powerful products of nanotechnology, providing broad variety of applications in life science [1,2]. For example, drug delivery, cellular imaging, and biosensing have been extensively described [3][4][5][6]. The chemical versatility of metal NPs holds the potential to outclass in a number of applications [2]. These unique properties and applications of metal NPs are well reviewed [7][8][9]. Platinum is used in various applications such as catalysts in many organic reactions [10,11], preparation of organic dyes [12], and biomedical applications [13,14]. For example, the Pt NPs were employed for successful photothermal treatment of Neuro 2A cancer cell by using irradiation with 1,064 nm near-infrared pulse wave and the Nd YAG laser set at 3 W for 480 s. The Pt NPs increased 9°C in temperature leading to effective photothermal killing of cancer cells [15].
The Pt composite materials have gained much attention due to their good multifunctions [16,17]. Pt NPs-chitosan composite particles have been extensively studied over the last decade [18,19], and Pt NPs-chitosan composite bubbles are one of the most emerging and intriguing topics [20,21]. Bubble particles have import features entrapping air bubbles inside. Due to their low density, bubble particles can float on liquid surface for specific applications. They can also be applied as novel vehicles for ultrasound-mediated imaging and targeted drug delivery followed by burst release [22][23][24][25][26][27]. Besides, bubble particles can be utilized as absorbers to facilitate adsorption of substrates due to a high-surface area. Pt NPs-chitosan composite bubbles can be applied in controlled release and tissue engineering; however, chitosan carrier substrates will disintegrate and dissolve in acid solution such as gastric juices. Therefore, Pt NPs-chitosan composite bubbles are limited in acidic condition. Fortunately, alginate polymer provides a solution to overcome this problem. Alginate polymer has a dense structure to pass the acid solution. To our best knowledge, Pt NPs-alginate composite (Pt NPs@alginate) bubbles are seldom reported in literatures, and they can provide applications for wide pH ranges.
By extending our previous works to prepare uniform alginate particles [28][29][30][31] and alginate bubbles [32], this work further develops a novel one-step method to fabricate composite Pt NPs@alginate bubbles through a simple chemical reaction. The Pt NPs and bubbles within alginate particles are investigated and characterized. The manufactured alginate products will provide great promise for multifunctional applications.

Mechanism of bubbles formation
The gas source is from the hydrolysis of NaBH 4 as following reaction [33]: The NaBH 4 hydrolysis is spontaneous, and gaseous H 2 generation continues with the hydrolysis reaction. Due  to the density difference, generated H 2 bubbles move upwards in the reservoir solution. After a dropwise addition of Na-alginate solution into the reservoir, gas bubbles were entrapped within alginate particles to be alginate bubbles. One alginate particle can hold many numbers of bubbles by random. After 30 min, alginate bubbles were collected by filter, washed twice with 30 mL dd-H 2 O, and finally collected and characterized.

Preparation of Pt NPs@alginate bubbles
Na-alginate (0.08 g dissolved in 2 mL of deionized water) and 2 mM platinum salt solution from dihydrogen hexachloroplatinate hexahydrate were mixed homogenously to be Pt 4+ mixed Na-alginate (Pt 4+ -Na-alginate) solution. As shown in Figure 1, Pt 4+ -Na-alginate solution loaded in the syringe (TERUMO® syringe, 3 mL; Terumo, Tokyo, Japan) was extruded from the needle tip by a KDS230 syringe  pump (KD Scientific Inc., Holliston, MA, USA). Under a constant injection rate, Pt 4+ -Na-alginate solution was broken up to form a series of isolatable Pt 4+ -Na-alginate droplets at the tip of the needle. The liquid in the receiving collector was filled with CaCl 2 and NaBH 4 for crosslinking alginate (by CaCl 2 ) and generating Pt NPs (by NaBH 4 ) and bubbles (by NaBH 4 ), respectively. Pt 4+ -Na-alginate droplets are gelled in situ to be Ca-alginate particles when contacting Ca 2+ ions. The NaBH 4 plays a dual functional. One is a reducing agent to reduce Pt 4+ to be Pt NPs by a chemical reduction reaction. The other one is gaseous H 2 generation by a hydrolysis reaction. When the Pt 4+ -Na-alginate droplets immersed in the receiving collector, the Pt NPs@alginate bubbles are generated.
Characterization An optical microscope system (TE2000U, Nikon, Lewisville, TX, USA) and a USB digital microscope (UPG621, UPMOST Technology Corp., Taipei, Taiwan) were utilized to observe the morphology of the collected particles. To minimize selection bias, a total of more than 50 individual particles were analyzed to ensure statistical representation. X-ray diffraction (XRD, D2 Phaser, Bruker AXS Gmbh, Germany) patterns were obtained at room temperature by using Cu K-α radiation (30 kV/10 mA) with a range of 2θ = 20°~80°, and a scanning rate of 4°min −1 . Laser Raman spectroscopy was obtained using a Renishaw Microscope Raman Spectrometer (Renishaw plc., Gloucestershire, UK) from 200 to 1,100 cm −1 at room temperature. The 785-nm line of the laser was used as the excitation source, with the capability of supplying 300 mW. The morphology of the particle composites was analyzed using a scanning electron microscope (SEM, S-3400, Hitachi Ltd, Tokyo, Japan) and

Cell viability test
The viability of the control and the treated cells were evaluated using 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay with human breast adenocarcinoma MCF-7 cells (1 × 10 4 /well) seeded in a 96-well microtiter plate with a 100 μL culture medium treated with various amounts of Pt NPs@alginate bubbles. After 1 day exposure, a 200-μL MTT solution was added to react with the cells for 4 h. After removal of the medium, 100 μL DMSO was added and examined at 595 nm using a microplate reader (Multiskan Ascent, Thermo Electron Corporation, Vantaa, Finland). The control group in the untreated well was considered to be 100%.

Pt NPs@alginate bubbles
Alginate is a kind of polysaccharide from marine brown algae. A variety of fundamental properties such as excellent biodegradability and biocompatibility make alginate a very attractive material for applications. Alginate has been applied in diverse areas [34][35][36] including serving biomedical materials for drug delivery and tissue engineering, and being adsorbent materials for elimination of heavy metals or organic pollutants [37]. Due to acid dissolution, conventional Pt NPs@chitosan bubbles have constraint applications for limited pH conditions. Therefore, it is needed to develop Pt NPs@alginate bubbles for wide pH applications. Figure 2 shows the effects of CaCl 2 concentration on Pt NPs@alginate bubbles. Results indicate that the size of the bubbles decreases with the CaCl 2 concentration. The difference between the two alginate materials with distinct viscosities was not significant. The size of bubbles reaches 1 mm at 1% CaCl 2 , but only 0.4 mm at 20% CaCl 2. The reason may be attributed to a lower crosslinking rate of alginate in a low CaCl 2 concentration. The alginate pregel allows entrapped small bubbles merging into lager bubbles before gel network (solidification) formation in a low CaCl 2 concentration. Figure 3 shows the effects of NaBH 4 concentration on Pt NPs@alginate bubbles. The results indicate that the number of bubbles within an alginate particle increases with NaBH 4 concentration, but there is no significant difference between two alginate materials. There are no obvious bubbles in the low 1 mM NaBH 4 due to the little amount of entrapped gas. Furthermore, the bubbles are well dispersed within the particle, but their size is not significantly varied with different NaBH 4 concentrations. This phenomenon resulted from the high viscous alginate matrix to retard the fusion of bubbles.
Reduction reaction of Pt salts by reducing agents such as borohydrides and citrates is one of the convenient methods to prepare Pt NPs [38]. This study demonstrates a proof-of-concept approach for encapsulating the Pt NPs and bubbles into alginate particles utilizing simple reduction and hydrolysis reactions. Produced Pt NPs@alginate bubbles combined the characteristics of Pt NPs and bubbles. The composite bubble particles can provide wide applications, such as smart vehicles for ultrasoundmediated imaging and targeted drug delivery, and effective absorbers and catalysts for decomposing pollutants. In the future, this proposed strategy to formulate Pt NPs@alginate bubbles can also be applied for synthesis of other composite materials. Characterization Figure 4 shows SEM images of Pt NPs@alginate bubbles. The exterior and interior morphologies of alginate particles obtained from different NaBH 4 concentration are compared. In absence of NaBH 4 , there is no bubbles formation and the morphology is smooth and intact. For 10 and 20 mM NaBH 4 , ridges and cavities are found at particle surface and interior, showing entrapped bubbles.
The TEM images shown in Figure 5 with different magnifications reveal that synthesized Pt NPs were nearly spherical and well dispersed in the Ca-alginate particle. The electron diffraction pattern of Pt NPs were indexed as (111), (220), and (222), indicating the polycrystalline characteristic. Figure 6 shows the XRD pattern of synthesized Pt NPs. Four distinct peaks at 39.6, 46.1, and 67.9 correspond to the crystal planes (111), (200), and (220) of cubic Pt NP structure, respectively. This result agrees with the finding in the electron diffraction data. Figure 7 is the Raman spectrum of different Pt substrates. There are different Raman patterns for Pt 4+ and Pt. Compared to nonionic Pt, ionic Pt 4+ shows more splits between 300 cm −1 and 350 cm −1 . The Raman pattern of Pt NPs agrees with Pt NPs@alginate bubbles, and Pt 4+ is consistent with Pt 4+ @alginate solution.
Cytotoxicity Figure 8 shows the cytotoxicity test of Pt NPs@alginate bubbles. The MCF-7 cells were used to test the cytotoxicity. Four kinds of alginate particles varying from alginate viscosity and CaCl 2 concentration were tested. After a 24-h exposure to alginate particles ranging from 5 to 1,000 μg/mL, the cell viability was assayed. Results show that there was no significant difference among the control (without adding alginate particles) and the samples. Furthermore, the differences among the four kinds of alginate particles were rather indistinguishable. These results ensure the low cytotoxicity of prepared particles on the MCF-7 cells. Therefore, Pt NPs@alginate bubbles obtained in this study can be safely applied for biomedical applications in the future, such as the scaffold for cartilage tissue engineering [39]. Table 1 shows the particle morphology of chitosan and alginate materials in different pH conditions. The three particles, chitosan, alginate, and NPs@alginate bubbles, were compared along the immersion time. The results indicate that chitosan particles disintegrated in acid solution after 1 h immersion but the alginate material still had an entire particle shape. Although alginate displayed swelling in alkaline solution, the particles still remained. Therefore, NPs@alginate bubbles can provide more applications for wide pH ranges than conventional NPs@chitosan bubbles.

Conclusions
This paper developed a facile method to synthesize platinum nanoparticles within alginate bubbles. Sodium borohydrate was utilized to generate platinum NPs and gaseous hydrogen by reduction reaction and hydrolysis reaction, respectively. Bubbles entrapped within around 2-mm alginate particles increased with the borohydrate concentration and alginate viscosity. This proposed one-step method to prepare Pt NPs@alginate bubbles has advantages of low cost, easy operation, and effective pore formation. Compared with conventional Pt NPs@chitosan bubbles, Pt NPs@alginate bubbles provide more applications for wide pH ranges.