A general strategy for template-free and low-cost synthesis of inorganic hollow spheres

A general strategy for template-free and low-cost synthesis of inorganic hollow spheres

    A general strategy for template-free and low-cost synthesis of inorganic hollow spheres Tao Qin, Peng Zhang, Ishtiaq Hassan Wani, Yua...

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    A general strategy for template-free and low-cost synthesis of inorganic hollow spheres Tao Qin, Peng Zhang, Ishtiaq Hassan Wani, Yuanyuan Han, Klaus Leifer, Fredrik Nikolajeff, H˚akan Engqvist PII: DOI: Reference:

S0032-5910(17)30506-5 doi:10.1016/j.powtec.2017.06.051 PTEC 12627

To appear in:

Powder Technology

Received date: Revised date: Accepted date:

25 February 2017 14 June 2017 17 June 2017

Please cite this article as: Tao Qin, Peng Zhang, Ishtiaq Hassan Wani, Yuanyuan Han, Klaus Leifer, Fredrik Nikolajeff, H˚ akan Engqvist, A general strategy for templatefree and low-cost synthesis of inorganic hollow spheres, Powder Technology (2017), doi:10.1016/j.powtec.2017.06.051

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ACCEPTED MANUSCRIPT A General Strategy for Template-free and Low-cost

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Synthesis of Inorganic Hollow Spheres

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Tao Qin,a Peng Zhangb, Ishtiaq Hassan Wani,a Yuanyuan Han,a Klaus Leifer,a Fredrik

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Nikolajeff a and Håkan Engqvist a

The Division for Applied Material Science, Department of Engineering Science, Uppsala

University, Sweden

The Division for Nanotechnology and Functional Materials, Department of Engineering

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Science, Uppsala University, Sweden

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Corresponding author: Tao Qin, [email protected]

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Abstract

Inorganic hollow spheres have a great potential in many fields, such as calcium phosphate (Ca3(PO4)2) as carriers of active ingredients and local delivery. They are typically synthesized

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by the methods that reply on template-based strategies. However, the template residue and energy consumption during template removal are drawbacks. Currently developed templatefree methods remain challenges such as time, cost and complicated procedures. In this paper, we introduce a general low-cost and template-free precipitation method with simple procedure. A series of inorganic hollow spheres, including calcium phosphate, calcium fluoride, strontium phosphate, strontium fluoride, barium phosphate and barium fluoride via magnesium were successfully synthesized, respectively. Based on these experimental results, a new model is proposed to explain the mechanism of the hollow inorganic spheres formation. This paper provides a general method to synthesize inorganic hollow spheres, which may have an important indication to other systems. Key words: inorganic, spheres, hollow, mechanism

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Introduction

Inorganic hollow spheres have very wide applications in batteries,[1] gas sensors,[1] catalysis[2–4], solar device coating[5] and biomedical devices.[6–9] Among these applications,

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calcium phosphate hollow spheres have been investigated for drug carrier for bone applications,[10] cell carrier for tissue engineering,[11] raw material for composite materials[12] and raw material for 3D printing.[13] For example, it has been reported that

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spherical tricalcium phosphate particles could enhance injection ability of a calcium phosphate cement paste.[14] However, the current technologies for the synthesis of calcium

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phosphate spheres are mainly focused on spraying,[15,16] self-assemble using surfactants and biomolecules.[17,18] The inherent disadvantages of these technologies include complicated

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process, energy consuming and template residue. The template or surfactant free strategy will

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be promising in synthesis of hollow spheres for future application.[19]

Template-free methods have been developed[20,21], but the high-cost and complicated procedures make large-scale production impossible. By using microwave-assisted rapid

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synthesis,[20] creatine phosphate (CP) was used as phosphate source to synthesize calcium phosphate hollow spheres without surfactants. However, as an excellent biocompatible organic phosphorus source, CP is extremely expensive compared to inorganic phosphorous sources. Moreover, high temperature and pressure have to be maintained in autoclaves during synthesis process, which limited its large-scale production.

A hydrothermal method to

synthesize calcium phopshate hollow spheres via a surfactant-free biomineralisation process has been described,[21] but it has to be based on supersaturated phosphate buffer solution. The solution demands large proportions of sodium chloride and/or potassium chloride. Mechanism of several hollow nanostructures synthesized without templates has been studied by Ostwald ripening[22–24] or Kirkendall effect.[25–28] Co-solvent solution of magnesium and sodium ions were used to synthesize calcium phosphate hollow spheres[21],

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ACCEPTED MANUSCRIPT but the role of ions cannot be well identified. Until now, mechanism of calcium phosphate spheres formation without templates has not been well explained.

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In this paper, low-cost and template-free method is developed to synthesize inorganic

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hollow spheres. The particular effect of magnesium ions on the formation of inorganic hollow spheres is demonstrated. Based on our experimental results, a new model is proposed

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to explain the mechanism of magnesium in the formation of the calcium phosphate hollow spheres.

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Experimental Section Materials

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Disodium phosphate, monosodium phosphate, barium chloride, strontium nitride, magnesium chloride, sodium fluoride, potassium sulphate and calcium chloride used in this study were

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used without further purification.

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purchased from Sigma-Aldrich. All chemicals were received as analytical grade reagents and

Method

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The method is a general strategy for synthesis of inorganic spheres. In the reaction solutions, the active ions in the formation of inorganic spheres are three ions: one of cations (Ca 2+, Sr2+ or Ba2+), one of anions (HPO42-, F-) and the third ions (Mg2+). The details about ions and their

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ratios between the ions were described in Table S1. The control group was in absence of the third ions (Mg2+).

One solution (containing one of cations and Mg ions) was mixed with another solution (containing one of anions) to form a clear solution at room temperature, as seen in Figure 1. The two solutions were of the same volume before mixing. The obtained clear solutions were kept in tightly covered glass bottles at 100℃ for a period of time sufficient for the formation of the desired particles. The formation time could take from 1 hour to 24 hours in static conditions. Calcium chloride, magnesium chloride, barium chloride, strontium nitrite, potassium sulphate disodium phosphate and sodium fluoride were used to prepare the clear solution. The obtained

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ACCEPTED MANUSCRIPT clear solution had an initial pH value of 6.0 to 8.0. The concentrations of cations, anions and Mg ions were in the range of 0.1-20 mM.

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Dissolution experiments

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10 mg of calcium phosphate microspheres were immersed in 100 mL solutions of 0.1 M HCl at 37 ℃ with constant shaking (120 rpm). Supernatant (0.5 mL) was withdrawn to measure

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the concentrations of Ca, Mg and P elements after completely dissolution, using inductively coupled plasma atomic emission spectroscopy (ICP-AES; Kleve, Germany). 30 mg of calcium phosphate microspheres were soaked in 30 mL solutions of HCl with

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different pH values (4 and 7.4) at 37 ℃ with constant shaking (120 rpm). Supernatant (0.5 mL) was withdrawn to measure the concentrations of Ca, P and Mg elements at given time

Drug loading and releasing

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same volume and the same pH value.

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intervals for the ICP analysis. The withdrawn was replaced with fresh HCl solution with the

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5.7 g calcium phosphate microspheres were soaked and stirred in 23 ml of vancomycin solution (10 mg/ml) for 24 hours in dark condition. The percentage of drug loaded into the

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spheres (MS) was calculated according to following equation: MS = (M1-M2)/M ×100%. Where M1 is the amount of drug in the solution before loading, M2 the amount of drug in the

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solution after loading, and M the amount of spheres. Vancomycin-loaded calcium phosphate spheres were re-suspended in phosphate buffer solution (PBS) at different pH values (4.0 and 7.4) for various times at 37℃. Supernatant (0.5 mL) was withdrawn to measure the concentrations of vancomycin at given time intervals, using an 1800 UV spectrophotometer (Shimadzu, Kyoto, Japan). The withdrawn was replaced with fresh PBS solution with the same volume and the same pH value. At each time point, the amounts of released antibiotic in solutions were measured at a wavelength of 280 nm. Analysis The morphologies of the particles were imaged by field emission scanning electron microscopy (SEM, LEO 1550, Germany). In order to increase conductivity, the samples were sputtered with Au/Pd before imaging the particles. Energy dispersive X-ray (EDX) analysis

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ACCEPTED MANUSCRIPT was also performed for the particle composition. D8 Advanced (Bruker, USA) was performed to collect XRD-pattern of particles in a theta-theta setup with Cu-k irritation and nickel filter. Calcium phosphate spheres were deposited on silicon substrate with ethanol. When the

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sample was dry, FIB/SEM dual beam system (FEI Strata DB235) was used to carry out cross

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sectioning/imaging. Approximately 2 µm platinum layer deposited on the sample using ion

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beam induced deposition (IBID) technique. Cross sectioning was achieved using focused beam of Gallium ions at 50 pA and 30kV. Imaging was performed using E-beam at 10kV and spot size 3.

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The inorganic spheres were examined by transmission electron microscopy (TEM, Tecnai)

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under bright filed. The accelerating voltage is 300 kV and the extraction voltage is 4.5 kV.

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Results

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Influence of Mg/Ca ratio on the formation of Ca/P spheres The study found that the reaction temperature, pH value, Ca/P ratio and Mg/Ca ratio were the key factors affecting the morphology and composition of the precipitates.

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The molar ratio of Ca/P was fixed in the ratio of 4.5. When Mg/Ca=0.11, calcium phosphate particles were debris, see Figure 2 (A). When Mg/Ca=0.45, calcium phosphate particles were porous spheres made of needle-like shell, see Figure 2 (B). When Mg/Ca=1.1, calcium phosphate particles were spheres with flake-like shell, see Figure 2 (C). When Mg/Ca=3.3, calcium phosphate particles were spheres with perfect smooth surfaces, see Figure 2 (D). When Mg/Ca=6.6, smooth spheres with defect on the shell could be formed, see Figure 2 (E). The TEM image of the smooth spheres is shown in Figure 2 (F). When Mg/Ca=13.2, there was no precipitates from clear solution. With Ca/P ratio varying from 0.0045 to 9 as shown in Table S2, calcium phosphate precipitated as spheres with Mg/Ca greater than 0.34. The conclusion could be made that with suitable ratio of Mg/Ca hollow Calcium phosphate spheres could precipitate at any ratio of Ca/P. 5

ACCEPTED MANUSCRIPT Composition and size of Calcium phosphate spheres XRD patterns of calcium phosphate debris were similar at any ratio of Ca/P showed in Figure S1, which indicate they are hydroxyapatite. XRD patterns of calcium phosphate spheres are

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showed in Figure 3. When ratio of Mg/Ca is less than 2, the particles were magnesium

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substituted calcium phosphate, see Figure 3 (a). When ratio of Mg/Ca was greater than 2, the

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particles tended to be amorphous, see Figure 3 (b and c). The size range of Calcium phosphate spheres was from 400nm to 900nm, as seen in Figure 4. Dissolution study

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After complete dissolution and dilution, ion concentrations of Ca, Mg and P are 5.4 ppm, 0.6

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ppm and 9.1 ppm in the diluted supernatant respectively, as seen in Figure 5. The ion concentrations of Ca, Mg and P elements in the pH 7.4 solution are very low (below 3 ppm) (Figure 6), while the ion concentrations of Ca, Mg and P elements in pH 4 solution

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reach 8, 2 and 16 ppm, respectively (Figure 7). The ion concentrations of Ca, P and Mg

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elements significantly increase with decreasing pH value from 7.4 to 4, as seen in Figure 6

Drug release

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and 7.

The vancomycin-loading capacity of the calcium phosphate microspheres reached approximately 38 wt%. Figure 8 shows the release behaviours of vancomycin from the

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microspheres over a 24 h period under pH 4 and 7.4 at 37 °C, respectively. At pH 4.0, approximately 70% of the drug was released within first 24 hours. At pH 7.4, approximately 40% of the drug was released within first 24 hours. General influence of Mg/Ca ratio on the formation of inorganic spheres Precipitates from calcium ions and fluoride ions were cuboids, see Figure 9 (A-0Mg). Precipitates from calcium ions, fluoride ions and magnesium ions were spheres and cuboids, see Figure 9 (A-Mg) and EDX-7 in Table S3. Without magnesium, strontium phosphate precipitates were particles with multiple shapes including self-assembled structure made of needles and spheres, see Figure 9 (B-0Mg). With magnesium, however, spheres were observed, see Figure 9 (B-Mg) and EDX-8 in Table S3.

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ACCEPTED MANUSCRIPT Reaction of clear solution containing strontium ions and fluoride ions resulted in cuboids, see Figure 9 (C-0Mg). Reaction of clear solution containing strontium ions, fluoride ions and magnesium ions resulted in hollow spheres, see Figure 9 (C-Mg) and EDX-9 in Table S3.

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Without magnesium, barium phosphate particles were stars with six angles, see Figure 9 (D-

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0Mg). With magnesium, however, spheres were observed, see Figure 9 (D-Mg) and EDX-10

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in Table S3.

Reaction of clear solution containing barium ions and fluoride ions resulted in cuboids and leaf-like particles, see Figure 9 (E-0Mg). Heat treatment of clear solution of barium ions,

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fluoride ions and magnesium ions resulted in spheres, see Figure 9 (E-Mg) and EDX-11 in

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Table S3.

The structure of the spheres

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High-resolution images of calcium phosphate spheres in the sample show the hollow structure

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of the spheres, as shown in Figure 10 (A and B). The hollow structure of the spheres was confirmed by FIB/SEM, as shown in Figure 10 (C, D, E and F). From C to F, the sphere’s

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cross-section pointed by black arrow varied. The shell and hollow part are pointed at by white arrows in Figure 10 (C).

The hollow structure of strontium phosphate, strontium fluoride and barium phosphate

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spheres was observed by SEM pointed at by black arrows, see Figure 9 (B-Mg, C-Mg and DMg). The hollow structure was confirmed by TEM from calcium fluoride, strontium phosphate, strontium fluoride, barium phosphate and barium fluoride spheres, see Figure 11 (A-Mg, B-Mg, C-Mg, D-Mg and E-Mg). In the images, a light contrast of the inner core and a dark contrast of the outer layer indicate the hollow structure of the spheres.

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ACCEPTED MANUSCRIPT Discussion

The vancomycin-loaded microspheres exhibited a sustained release property within 24 hours,

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which was useful for clinical cases. The drug release efficiency was much higher at pH 4 (70%) than at pH 7.4 (40%). This might be due to the obvious pH-responsive dissolution

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performance of calcium phosphate spheres, which can be used in pH-dependent drug delivery systems. The pH-sensitive drug release might be beneficial at the reduced pH in tumours and inflammatory tissues[29,30].

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The method reported in this paper proved that only three ions were necessary to form the spherical hollow particles. They are one of cations (Ca2+, Sr2+ or Ba2+), one of anions (HPO42-,

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F-) and the third ions (Mg2+). The ratios of these three ions, pH and temperature are key factors in controlling the morphologies.

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The key of the method in this paper was to make a clear solution before hydrothermal reaction. Therefore, a modified procedure and concentrations range should be followed. If directly

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dissolving calcium chloride and disodium phosphate in distilled water, the solution gave precipitates immediately at room temperature, which were non-spherical calcium phosphate

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particles. Moreover, the concentration of phosphate ions should decrease as concentration of calcium ions increases, and vice versa. The previous study[21] provided a supersaturated phosphate buffer solution demanding high concentration of sodium or potassium ions and

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chloride ions in the solution. The method in this paper reduces significantly the cost of production compared to the previous study[21] because clear solutions containing calcium ions and phosphate ions were achieved without extra sodium chloride and potassium chloride. In this paper, spherical calcium phosphate particles could form under any ratios of Ca/P. The functions of magnesium in the formation of calcium phosphate hollow spheres could be revealed. Under a certain ratio of Ca/P, the morphologies of the final products could be determined by the concentration of magnesium ions. With a low concentration of magnesium ions (Mg/Ca0.34), the precipitates were calcium phosphate debris. With a high magnesium ion concentration (0.34Mg/Ca2.73), the precipitates were calcium phosphate spheres. With abundant magnesium (Mg/Ca2.73), the precipitates were a mixture of calcium phosphate spheres and magnesium phosphate micro particles. As Mg/Ca increased, the amount of calcium phosphate particles became less and less. 8

ACCEPTED MANUSCRIPT This method could be successfully applied to synthesize other inorganic spheres. In absence of magnesium ions in the clear solution, precipitates of calcium fluoride, strontium fluoride, strontium phosphate, barium fluoride and barium phosphate could not form spherical

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structures, as seen in Figure 9 (A-0Mg, B-0Mg, C-0Mg, D-0Mg and E-0Mg). All the

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precipitates end up with spherical structure in the presence of magnesium, as seen in Figure 9

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(A-Mg, B-Mg, C-Mg, D-Mg and E-Mg).

It has been well studied that hollow spheres could form due to surface capping effect of

surfactants.[31–34] Our study demonstrated that hollow spheres could also be generated without the presence of surfactant or template once magnesium ions were introduced into the

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precipitation system. In this paper, three ions “virtual” micelle effect was proposed to explain the function of magnesium. Three ions “virtual” micelle could function similarly as a micelle

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due to three ions interactions. Figure 12 (a) shows structure of micelle which led to the formation of nano-spheres up to 50 nm31 and micro-spheres up to 500 nm32 by ABC triblock copolymers. In this paper, we hypothesized that Ca ions, HPO4 ions and Mg ions could

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function as ABC triblock copolymers and form the “three ions virtual micelle”, as seen in

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Figure 12 (b). Via our precipitation method, the “three ions virtual micelle” effect could lead

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to formation of inorganic spheres, of which size range is from 300 nm to 1200 nm. Interaction Mg-Ca: inhibitory effect of Mg ions to Ca ions in formation of calcium phosphate, depending on molar ratio of Mg/Ca. Magnesium ions need higher concentration and longer time to combine with phosphate ions, compared to calcium ions. For example, the binding

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energy of calcium ions (2P3/2) in CaHPO4 is about 347.5 eV,[37] while the binding energy of magnesium ion (2P) is around 50 eV.[38] Interaction Ca-P: attractive effect between Ca ions and phosphate ions to form calcium phosphate, depending on molar ratio of Ca/P. Interaction Mg-P: attractive effect between Mg ions and phosphate ions, depending on Ca/P and Mg/Ca. The formation of hollow spheres by calcium, phosphate and magnesium could be explained due to items of a and b below, referring to micelles[39]. a) The molar ratios of Mg/Ca are larger than the critical ratio (CR, approximately 0.34) and the temperature of reaction is higher than the critical temperature (CT, approximately 60℃).

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ACCEPTED MANUSCRIPT CR0.3,CT 60,6 pH 8 Ca2  HPO42  Mg 2   spherical Cax Mg y ( PO4 ) z

Once the ratio of Mg/Ca is lower than that of CR, there is no “three ions virtual micelle”

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effect because magnesium’s inhibitory effect is not enough.

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After the critical ratio and critical temperature were reached, inhibitory effect of magnesium and interaction of three ions led to three ions virtual micelle effect, as can be seen in Figure 12

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(b). It revealed that calcium ions, phosphate ions and magnesium ions were dominating in inner circle (dot area), middle ring (diagonal area) and outside (blank area), respectively.

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b) Ca ions and Mg ions are repelling each other and competing to combine with phosphate ions. Calcium ions are much easier to combine with phosphate ions than magnesium ions. This could be explained by the higher binding energy of calcium ions with phosphate ions

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compared to magnesium ions. However, abundant magnesium ions could exist with phosphate ions in aqueous solution and could inhibit the formation of calcium phosphate, according to

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my observation and previous studies.[40–42] The inhibitory effect of Mg2+ in the formation of

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calcium phosphate[40] is driving force in the thermodynamic balance. Following this model, we could explain the magnesium function in synthesis of spheres of

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calcium fluoride, strontium phosphate, strontium fluoride, barium phosphate, and barium fluoride. The previous study[35] proved that cationic and anionic micelle was formed due to extra H+ and OH-, respectively. In this paper, the three ions virtual micelles were cationic due

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to extra Mg2+.

The proposed model of “three ions virtual micelle effect” may explain the mechanism of hollow spheres formation in other ionic and aqueous system without surfactants. This will give us a new understanding of synthesis mechanism in inorganic chemistry. Inorganic spheres synthesized in this paper could have many important applications. In biomedical field, calcium phosphate hollow spheres could be applied as delivery carriers and bone filler. In optical field, calcium fluoride, strontium fluoride and barium fluoride spheres could be used as optical materials, for instance as retroreflecting layers or focusing elements.

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ACCEPTED MANUSCRIPT Conclusion

In the paper, we developed a general low-cost and template-free method to synthesize

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inorganic hollow spheres including calcium phosphate, calcium fluoride, strontium phosphate, strontium fluoride, barium phosphate and barium fluoride. We revealed that magnesium is a

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key factor in formation of these spheres. A new model was proposed to explain the function of magnesium.

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Acknowledgement

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The authors would like to thank the CSC for providing financial support to this project.

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Conflict of interest

[1]

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[41] F. Abbona, M. Franchini-Angela, Crystallization of calcium and magnesium

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phosphates from solutions of low concentration, J Crys Growth. 104 (1990) 661–671.

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[42] F. Abbona, A. Baronnet, A XRD and TEM study on the transformation of amorphous

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calcium phosphate in the presence of magnesium, J Crys Growth. 165 (1996) 98–105.

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Figure.1 The diagram of the synthesis method in the study. The cations include Ca2+, Sr2+ and Ba2+.The anions include HPO42- and F-.

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Figure.2 Electron images of particles synthesized at molar ratio of Ca/P=4.5 , the molar ratio of Mg/Ca: (A) 0.11, (B) 0.45, (C) 1.1, (D) 3.3, (E) 6.6 and (F) TEM image at 100℃.

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Figure.3 XRD patterns of calcium phosphate spheres synthesized at molar ratio of Ca/P= 4.5, the molar ratio of Mg/Ca: (a) 1.2 (b) 3.3 and (c) 6.6 at 100℃. The composition of crystalline particles is magnesium substituted calcium phosphate.

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Numbers of the spheres

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400-449450-499500-549550-599600-649650-699700-749750-799800-849

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Size (nm)

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Figure.4 Size distribution of calcium phosphate spheres synthesized by the general method.

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Figure.5 The ion concentration of Ca, P and Mg elements measured by ICP analysis after soaking calcium phosphate microspheres in 0.1M HCl solution.

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Figure.6 The ion concentration of Ca, P and Mg elements measured by ICP analysis after soaking calcium phosphate microspheres in HCl solution at pH 7.4.

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Figure.7 The ion concentration of Ca, P and Mg elements measured by ICP analysis after soaking calcium phosphate microspheres in HCl solution at pH 4.

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Figure.8 Release profile of vancomycin from vancomycin-loaded calcium phosphate microspheres in PBS at pH=4 and pH=7.4 (n=6).

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Figure.9 SEM images of particles synthesized by control groups and standard groups (magnesium induced in precipitation process). (A-0Mg) control with Ca/F=1.8, Mg/Ca=0 (A-Mg) standard with Ca/F=1.8, Mg/Ca=0.55. (B-0Mg) control with Sr/P=0.09, Mg/Sr=0 (B-Mg) standard with Sr/P=0.09, Mg/Sr=0.55. (C-0Mg) control with Sr/F=0.2, Mg/Sr=0 (C-Mg) standard with Sr/F=0.2, Mg/Sr=1.06. 24

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(D-0Mg) control with Ba/P=0.09, Mg/Ba=0 (D-Mg) standard with Ba/P=0.09, Mg/Ba=0.55. (E-0Mg) control with Ba/F=0.2, Mg/Ba=0 (E-Mg) standard with Ba/F=0.2, Mg/Ba=1.04.

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Figure.10 SEM images and cross-sections of calcium phosphate spheres (Ca/P=4.5) observed by FIB/SEM. (A) and (B) the high-resolution SEM images of observed hollow spheres in the sample. (C) This position was set as starting position, 0 nm. (D), (E) and (F) were acquired at cutting depth of approximately 50 nm, 100 nm and 200 nm with respect to position A, respectively.

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Figure.11 TEM images of inorganic spheres synthesized via magnesium. (A-Mg) standard with Ca/F=1.8, Mg/Ca=0.55. (B-Mg) standard with Sr/P=0.09, Mg/Sr=0.55. (C-Mg) standard with Sr/F=0.2, Mg/Sr=1.06. (D-Mg) standard with Ba/P=0.09, Mg/Ba=0.55. (E-Mg) standard with Ba/F=0.2, Mg/Ba=1.04.

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Figure.12 Diagrammatic sketch for explaining mechanism of (a) micelle effect in previous reference[31,32](b)three ions virtual micelle via Ca, HPO4 and Mg ions interactions

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Graphical abstract

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Calcium phosphate (CaP) spheres can be synthesized at any Ca/P ratio via sufficient magnesium. Calcium, phosphate and magnesium are the only active ions in the sphere formation. A range of inorganic hollow spheres were synthesized by the same strategy. A new model is proposed to explain the mechanism of the hollow sphere formation.

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