TetraethylenepentamineâGraphene Hollow Microspheres as

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CaCO3/Tetraethylenepentamine-graphene Hollow Microspheres as Biocompatible Bone Drug Carriers for Controlled Release Jie Li, Hongkun Jiang, Xiao Ouyang, Shihui Han, Jun Wang, Rui Xie, Wenting Zhu, Ning Ma, Hao Wei, and Zhongyi Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b10697 • Publication Date (Web): 18 Oct 2016 Downloaded from http://pubs.acs.org on October 22, 2016

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ACS Applied Materials & Interfaces

CaCO3/Tetraethylenepentamine-graphene Hollow Microspheres as Biocompatible Bone Drug Carriers for Controlled Release Jie Lia,b,c, Hongkun Jianga, Xiao Ouyanga, Shihui Hana, Jun Wanga, Rui Xied, Wenting Zhud, Ning Ma*a, Hao Wei*a and Zhongyi Jiangb,c a

Key Laboratory of Superlight Material and Surface Technology of Ministry of Education,

College of Material Science and Chemical Engineering, Harbin Engineering University, Harbin 150001, China. b

Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical

Engineering and Technology, Tianjin University, Tianjin 300072, China. c

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin

300072, China. d

Tumor Hospital of Harbin Medical University, Harbin 150081, China.

KEYWORDS: bone drug carriers, drug delivery, rGO-TEPA, CaCO3, doxorubicin

ACS Paragon Plus Environment

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ABSTRACT: CaCO3 is one kind of important biological mineral, which widely exists in coral, shell and other organisms. Since similar to bone tissue elements and good biocompatibility, it was very suitable for candidates as bone drug carriers. In this paper, we used tetraethylenepentamine-graphene

(rGO-TEPA)

sheet

matrices

induction

of

CaCO3

mineralization and successfully constructed CaCO3/rGO-TEPA drug carriers with a hollow structure and rough surface. As potential drug carriers, doxorubicin (DOX) loading and release measurements were carried out. It showed that load efficiency was 94.7% and the release efficiency were 13.8% and 91.7% by the pH value of 7.4 and 5.0. The as-prepared drug carriers showed some appealing advantages, such as the pH-sensitive release characteristics and the mild storage-release behaviors. The excellent biocompatibility and nontoxicity of CaCO3/rGO-TEPA hybrid microspheres were tested by the cell viability of the mouse pre-osteoblast cells (MC3T3E1). And cytotoxicity with human osteosarcoma cells (MG-63) was carried out to demonstrate the drug release effect in cells system. Therefore, the CaCO3/rGO-TEPA hybrid microspheres would be a competitive alternative in bone drug carriers.

Introduction Currently, drug delivery systems (DDS) have caused widespread concern in contemporary medication and pharmaceutical fields.1-2 Different from traditional medicine, DDS have even more advantages, such as high drug efficacy, low toxicity, convenience, etc.3-5 Up to now, various methods have been used to formulate the types of materials as drug carriers for DDS.6-11 Unlike polymer-based micro/nano-spheres carriers, organic-inorganic composite microspheres provide a mild preparation process, which have a great competitive advantage.12-16


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In DDS research area, the bone drug carriers is an important part since bone is one such kind of special and important organs. The research of bone drug carriers is a very important way to the treatment of bone diseases, which via loading different drugs, including analgesic, antibiotic, and anti-cancer. Calcium carbonate is one kind of important biological mineral, which widely exist in coral, shell and other parts of organisms and similar to bone tissue elements.17-18 In addition, CaCO3 is natural pH-sensitive while the cargo can be response to the weak acidic environment of tumor tissues. As a nontoxic natural biomineral, CaCO3 could be suitable candidates as bone drug carriers. In recent years, it has been reported that CaCO3 was used in orthopaedic surgery bone grafts19-21, gene delivery22-24 and drug delivery of anticancer drugs for the internal organs25. A few studies have reported on CaCO3/polymer hybrid particles as bone drug carriers.26-27 However, the morphologies of these CaCO3/polymer hybrid particles were usually flake or solid sphere.The further research of biomineral materials can help us to find more excellent drug carrier materials. The hollow microspheres, especially with microporous surface structure, would have a large BET surface area. And this specific morphology and structure with large BET surface area could improve the capacity of loading drug molecules regardless of their surface charge and hydrophilicity, in which the drug molecules absorbed to the surface of composite crystals or permeated to the inside of composite crystals.28-29 Recently, many scientists used hollow hybrid microspheres in drug delivery systems and made encouraging progress.30-32 Therefore, hollow hybrid microspheres possibly represent an ideal drug carrier. Furthermore, the study of CaCO3 hollow microspheres as a drug carrier especially the bone drug carrier has rarely been reported. In this paper, we chose tetraethylenepentamine-graphene (rGO-TEPA) sheet matrices for induction of calcium carbonate mineralization. rGO-TEPA has the similar structure to graphene33


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with one-atom-thick of two-dimensional layered structure, bearing amino (-NH2) groups on the basal planes, which possibly act as an ideal mineralization matrix.34 It has been reported that polymer bearing amino (-NH2) groups was suitable to be utilized as drug carriers.35 The asprepared CaCO3/rGO-TEPA microspheres exhibited both porous surface structure and hollow inner structure in comparison with other CaCO3/polymer hybrid particles.36-41 The excellent biocompatibility and nontoxicity of CaCO3/rGO-TEPA hybrid microspheres were tested by the cell viability of the mouse pre-osteoblast cells (MC3T3-E1) 42. And cytotoxicity with human osteosarcoma cells (MG-63) 26 was carried out to demonstrate the drug release effect in cells system. Then, the drug loading capacity of CaCO3/rGO-TEPA hybrid microspheres was discussed through loading conventional anti-cancer drug of doxorubicin (DOX). The release properties of DOX-loaded CaCO3/rGO-TEPA hybrid microspheres revealed the pH-responsive drug-release behaviour, which could reduce the drug toxicity to the normal tissue. Therefore, the CaCO3/rGO-TEPA hybrid microspheres would be a competitive alternative in bone drug carriers. Experimental Chemicals and CaCO3/rGO-TEPA synthesis. Tetraethylenepentamine-graphene (rGO-TEPA) (purity ~99.8 wt%, thickness ~1 nm, diameter > 0.5 µm, nitrogen content 9.34 wt%) were from XFNANO Materials Technology Company Ltd (Nanjing, China). All of the inorganic reagents (CaCl2, Na2CO3, NaOH, HCl, ethanol) were analytical grade and obtained from Sinopharm Chemical Reagent Company Ltd (Shanghai, China). For cell culture, the mouse pre-osteoblast cells (MC3T3-E1) and human osteosarcoma cells (MG-63) were obtained from Shanghai Institutes for Biological Sciences Cell Resource Center (Shanghai, China). CaCl2 and Na2CO3 aqueous solutions were prepared immediately before use.


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Preparation of CaCO3/rGO-TEPA hollow microspheres. The CaCO3/rGO-TEPA hybrid carriers were obtained through the mineralization of CaCO3 crystals in solution with rGO-TEPA. In a typical synthesis, 4 mg rGO-TEPA and a CaCl2 aqueous solution (50 mM, 40 mL) were mixed, and 200 rpm stirred at 35°C water bath heating for 10 min. Then, a Na2CO3 aqueous solution (50 mM, 40 mL) was slowly added to the CaCl2/rGO-TEPA solution, and the mixture solution was kept stirring for 1 h. The sample was repeatedly centrifuged with ultrapure water, absolute ethanol, and then the centrifugal products were dried at 40°C in a vacuum oven. Preparation of CaCO3/rGO-TEPA solid composites. For CaCO3/rGO-TEPA cube bulks, 4 mg rGO-TEPA and a CaCl2 aqueous solution (100 mM, 40 mL) were mixed and 200 rpm stirred at 25°C water bath heating for 10 min. Then, a Na2CO3 aqueous solution (100 mM, 40 mL) was slowly added to the CaCl2/rGO-TEPA solution, and the mixture solution was kept stirring for 1 h. For CaCO3/rGO-TEPA solid spheres, the concentration of CaCl2 aqueous solution was 10 mM instead of 100 mM and other experiment remains the same with the method of CaCO3/rGOTEPA cube bulks. The sample was repeatedly centrifuged with ultrapure water, absolute ethanol, and then the centrifugal products were dried at 40°C in a vacuum oven. DOX Loading and Release Test. DOX loading and release test were referring to the published report.42-43 30 mg of CaCO3/rGO-TEPA samples were added into 5mL ultrapure water and dispersed via ultrasound in the dark. Then, 2.5 mg DOX was added and slowly shaken at 37°C for 24 h in the dark by an oscillator. The mixture solution was centrifuged and collected the supernatant solution for UV−vis test. The as-prepared DOX@CaCO3/rGO-TEPA microspheres carriers were kept in the dark during cryopreservation, to ensure the long term storage and release behavior of DOX. In the DOX release test, 10 mL of fresh phosphate buffered saline (PBS) was added and set in the oscillator with slowly shaking at 37°C for 15 min, and collected


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the supernatant solution. Then, repeated the process with different release time, 30 min, 45 min, 60 min, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h, respectively. PBS solutions with different pH value of 7.4 and 5.0 were provided for pH-sensitive release. Figure S2 showed the standard curve of DOX concentration with the UV absorbance in PBS. The loading capacity ( ), loading efficiency (%) and release efficiency ( %) were received referring to following formulas

=

O − R

% =

O − R

% =

∑ M

in which , , and represent the concentration of original DOX, residual DOX, the CaCO3/rGO-TEPA carriers and DOX in the supernatant, respectively. Cell viability assay of CaCO3/rGO-TEPA hollow microspheres. The cell viability of CaCO3/rGO-TEPA hollow carriers was evaluated using the MTT cell assay according to the literature26, 42. A 5 mg/mL of MTT solution was prepared with phosphate buffered saline (PBS). 5 × 104 MC3T3-E1/MG-63 cells were seeded on 96-well plates and incubated in 0.1 mL αDMEM medium with 5% CO2 at 37°C for 24 h to make the cells proliferate to wells, and eight wells were left empty for blank controls. The CaCO3 and CaCO3/rGO-TEPA hollow microspheres were disinfected through UV irradiation before use. And then, the carriers were added onto plates with different concentrations of 25, 50, 100, and 200 µg/mL, respectively. The MTT assay of MG-63 cells with DOX@CaCO3/rGO-TEPA drug carriers was carried out under


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the same condition, to demonstrate drug release effect in cells system. Then the 96-well plates were incubated for 24 h and 48 h. The averages and standard deviations of cell viability were performed in triplicate, and the control experiment which received without CaCO3/rGO-TEPA carriers and DOX was regarded as 100% growth. Characterization. The CaCO3/rGO-TEPA particles were coated with Au and their morphologies were used a scanning electron microscope (SEM) (QUANTA 200). X-ray diffraction images were measured by Rigaku TTR-III. The transmission electron microscopy (TEM) was acquired on JEM-2200FS. The concentration of DOX was carried out on UV−vis spectrophotometer (UV-1601). N2 adsorption/desorption isotherms were carried out with Micromeritics ASAP 2010 apparatus. Results and discussion Synthesis and characterization of CaCO3/rGO-TEPA hollow microspheres. CaCO3 is one of such kind biological mineral, which widely exist in coral, shell and other organisms. On account of its varied morphology structure, good biocompatibility, biodegradability and similar to bone tissue elements, CaCO3 has unique advantages as bone drug carriers. To make a high surface area and the high capability of loading sundry drugs of CaCO3, the organic matrices play an important role. Many organic matrices have been discussed and successfully formed the different morphology of CaCO3 composite crystals.36-40, 44-45 In recent years, graphene and functionalized graphene were reported to control the crystallization of CaCO3 crystals and have made encouraging progress.18, 46-47 rGO-TEPA has the similar structure to graphene with one-atom-thick of two-dimensional layered structure, bearing amino (-NH2) groups on their basal planes, which possibly acts as an ideal mineralization matrix. Here, we


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used rGO-TEPA sheet matrices induction of calcium carbonate mineralization. The CaCO3/rGOTEPA hybrid microspheres were obtained through the mineralization of CaCO3 crystals in solution with rGO-TEPA. The morphology of obtained particles was characterized by SEM measurement. Figure 1 showed the SEM images of CaCO3/rGO-TEPA particles obtained at 35°C water bath heating for 1 h. Figure 1a revealed spherical and a few of cubic shape crystals with approximately 2~4 µm in size. Further observation of Figure 1a, there were a small amount of damage crystals in the sample of the spherical crystals. High-magnification SEM image (Figure 1b) of a damage crystal indicated a hollow structure and rough surface. Figure 1c showed the CaCO3/rGO-TEPA particles obtained under ultrasonic oscillation destroy; it was further found that multiple damage crystals were hollow spherical structure. High-magnification SEM image (Figure 1d) of a damage crystal indicated a hollow structure, which the surface was rough and contained a great deal of nano-pores and nano-channels. It was found that the destroyed section consisted many of nanometer particles and a sheet throughout the microsphere. The element ratios in different positions of (e) and (f) in Figure 1d were carried out on SEM-EDX spectra test (Figure 1e and 1f). The C, N, Ca and O atomic ratios in positions (e) and (f) were 23.80, 23.98, 10.99, 41.23 and 27.98, 18.67, 13.02, 40.33, respectively. The content of nitrogen in sites (e) was higher than that in sites (f), while the content of calcium was lower, which could speculate that the sheet in sites (e) was rGO-TEPA. Figure S1 shows the SEM images of pure calcium carbonate particles without rGO-TEPA, got from the precipitation of 50 mM CaCl2 and Na2CO3 at 35°C, which was under the same conditions with the preparation of CaCO3/rGO-TEPA hollow microspheres. It revealed that diamond particles were approximately 2~5 µm in size. And the corresponding XRD diffraction pattern and the FT-IR spectrometry verified that the pure CaCO3 crystals under this


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condition were CaCO3 calcite crystals. In comparison with the hollow hybrid microspheres, it was speculated that the matrix of rGO-TEPA was very important to control the morphology and crystal form of CaCO3 particles.

Figure 1. SEM images of CaCO3/rGO-TEPA composite crystals: (a, b) CaCO3/rGO-TEPA composite crystals, (c, d) CaCO3/rGO-TEPA composite crystals with ultrasound destruction. Figure (e) and (f) were corresponding SEM-EDX spectra in figure (d)


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To characterize the further morphology and crystal form, the representative TEM and electron diffraction of rGO-TEPA and CaCO3/rGO-TEPA composite crystals were carried out (Figure 2). In Figure 2a and 2b, the large nanosheets of rGO-TEPA were clearly presented and the electron diffraction of rGO-TEPA was the amorphous diffraction ring. The TEM image of the CaCO3/rGO-TEPA composite crystals (Figure 2c and 2d) revealed large number of nanometersized blocks. The electron diffraction (Figure 2e) revealed new crystals formation in addition to an amorphous concentric ring structure of rGO-TEPA, which stand for the (118), (114), (104), (211) diffraction planes of CaCO3 calcite and vaterite crystals. The lattice distance of CaCO3/rGO-TEPA composite crystals in HRTEM image (Figure 2f) was about 0.36 nm and indexed as (110) planes of CaCO3 vaterite crystals.


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Figure 2. TEM images of rGO-TEPA (a and b), CaCO3/rGO-TEPA composite crystal (c and d). The inset (b) is the corresponding SAED pattern of rGO-TEPA. Images (e) and (f) are the electron diffraction and HRTEM image of CaCO3/rGO-TEPA composite crystals, respectively.


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The XRD pattern was consistent with the TEM results (Figure 3a), which the diffraction peaks of CaCO3/rGO-TEPA composite crystals (004), (110), (112) and (114) were for CaCO3 vaterite crystals. And the weak diffraction peak of calcite is 29.40, which correlated with the (hkl) indices (104). The FTIR spectra of rGO-TEPA and CaCO3/rGO-TEPA composite crystals were illustrated in Figure 3b. The absorption bands at 2930/2850 and 3400 cm−1 were ascribed to the CH- stretching in -CH2- and the -NH- stretching in -NH2 groups in rGO-TEPA. The identification of the CaCO3/rGO-TEPA hybrids were verified by the vibration bands, that 1421, 1082, 874, 712 cm−1 for CaCO3 calcite crystals and 1421, 1082, 870, 750 cm−1 for CaCO3 vaterite crystals. The specific morphology and structure of CaCO3/rGO-TEPA particles could improve the capacity of loading drug molecules which absorbed to the surface of composite crystals or permeated to the inside of composite crystals.

Figure 3. (A) Typical XRD diffraction pattern of rGO-TEPA (a), CaCO3/rGO-TEPA composite crystals (b); (B) FT-IR spectrometry of rGO-TEPA (a), CaCO3/rGO-TEPA composites (b) DOX release properties of CaCO3/rGO-TEPA hollow microspheres


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DOX, cytotoxicity anthracycline-based member of the antibiotics, is the most common anticancer drug and can successfully inhibit the progression of many types of tumour and relieve symptom. However, DOX has high cytotoxicity since it not only kills the cancer cells but also damages the normal tissue cells and is difficult to be loaded due to the high water solubility. Up to now, various methods have been used to reduce cytotoxicity and increase the DOX loading efficiency.48 Herein, doxorubicin drug was chosen as a model to study on the drug-loaded properties of the CaCO3/rGO-TEPA hollow microspheres. To further investigate the drug-loaded formation of the hollow microspheres carriers, the UV-vis spectroscopy was carried out. As shown in Figure 4a (inset was related digital photo of the supernatant solutions), the results observed in DOX-loaded hollow microspheres carriers, bare CaCO3/rGO-TEPA hollow microspheres and free DOX of 120µg mL-1. The peak of DOX absorption spectrum at 480 nm was chosen as the standard estimated solution absorbance. The standard curve between the DOX concentration and UV-vis absorbance was showed in Figure S2. In DOX loading test, 2.5 mg of DOX was added to 5mL rGO-TEPA hollow microspheres PBS mixed solution and the initial drug concentration was 500µg mL-1. As for the UV-vis absorbance curve of DOX@CaCO3/rGOTEPA hollow microspheres carriers, there is an obvious difference between the free DOX and CaCO3/rGO-TEPA hollow microspheres. In addition, DOX was almost convergent to the CaCO3/rGO-TEPA hollow microspheres with the drug loading capacity and efficiency of 78.9 mg g-1 and 94.7%, respectively. It was speculated that the drug carriers could associate with DOX molecules through several types of interactions; the π−π stacking among the aromatic rings of rGO-TEPA and the conjugated rings of DOX molecules, and the hydrogen bonding and Van der Waals force among the surface hydroxyl of CaCO3 and the hydroxyl/amine groups of DOX.49 Moreover, the CaCO3/rGO-TEPA composite crystals have rough surface and nano-


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pores, which could offer a high performance of loading drug molecules, regardless of their hydrophilicity and surface charge.28-29

Figure 4. (A) UV absorbance spectra: (a) bare CaCO3/rGO-TEPA hollow microspheres, (b) DOX-loaded hollow microspheres carriers, (c) 120µg mL-1 free DOX in PBS aqueous solution. (B) DOX release efficiencies of DOX@CaCO3/rGO-TEPA carriers at pH values of 7.4 and 5.0 In different bone tissues, the physiological pH values are different, which the environments in tumors tissue and normal cells were approximately 5.0 and 7.4, respectively. In the DOX release test (Figure 4b), the pH values 5.0 and 7.4 of PBS solution were selected to simulate the environments of intracellular endosomal/lysosomal acidic environments inside cancer cells and normal tissue cells, respectively. The DOX release efficiencies presented two obviously different behaviors with release time, i.e. the burst release and sustained release. When the pH value was 7.4 (the normal tissue environment), the DOX showed an initial burst or fast release of 9.1% within 4 h due to the part of DOX affixed to the DOX@CaCO3/rGO-TEPA carriers surface and secondly the persistent released of 13.8% followed up to 48 h, indicating that DOX@CaCO3/rGO-TEPA carriers were stable loading DOX molecules during 48 h. It was


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conjectured that the interactions of DOX with carrier microspheres could limit the drug release to solution. Therefore, when the pH value is 5.0 (the cancer cells environment), the DOX showed a notably faster release of 59.3% within 4 h compared with the pH value of 7.4. Followed up to 48 h, the drugs sustained release reach up to 91.7%. It was attributed to the destruction of the CaCO3/rGO-TEPA hollow microspheres structure and a higher DOX drug molecules solubility under the weak acidic medium. The release properties of DOX-loaded CaCO3/rGO-TEPA hybrid microspheres presented outstanding pH-responsive release behavior, which could overcome the high cytotoxicity of DOX to the normal tissue. The property of controlled releasing by low pH could be benefit for responsive releasing of the physiological acidic environment in tumour tissue. In particular, the CaCO3/rGO-TEPA hollow carriers provide a great potential application in drug delivery system. In vitro cytotoxicity of CaCO3/rGO-TEPA hollow microspheres. As potential drug carriers, the cytotoxicity of the CaCO3/rGO-TEPA hollow microspheres should be discussed. Only nontoxic carriers can be used in drug delivery systems. In a previously study, graphene–biomineral hybrid particles had a low cytotoxicity in vivo40. The MTT assay was proceed with MC3T3-E1 cell lines to test the cell viability of CaCO3 and CaCO3/rGO-TEPA hollow drug carriers. Figure 5a showed the MC3T3-E1 cells viability with different concentrations of CaCO3 and CaCO3/rGO-TEPA hollow drug carriers, which incubated for 24 h. It was clearly presented that the MC3T3-E1 cells viability of CaCO3 in all dosages was up to 91.4~114.5%. When added the rGO-TEPA sheet matrices, the viability of the CaCO3/rGOTEPA hollow microspheres in all dosages still kept between 91.9% and 108.6%. When incubated for 48 h (Figure 5b), the viability of the as-prepared CaCO3 in all dosages was up to 96.2~105.1%. The viability of the CaCO3/rGO-TEPA hollow microspheres still kept between


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97.4% and 106.3%. It was demonstrated that the CaCO3 and CaCO3/rGO-TEPA hollow drug carriers exhibited very low cytotoxicity, even in the high amount of 200 µg/mL after 2 days.

Figure 5. In-vitro viability study of MC3T3-E1 cells after 24 h (a) and 48 h (b) exposure to CaCO3 and CaCO3/rGO-TEPA drug carriers; In-vitro cytotoxicity test of MG-63 cells after 24 h (c) and 48 h (d) exposure to CaCO3, CaCO3/rGO-TEPA and DOX@CaCO3/rGO-TEPA drug carriers In-vitro cytotoxicity test of MG-63 osteosarcoma cells was carried out to demonstrate drug release effect in cells system. We had carried out the MTT assay of CaCO3, CaCO3/rGO-TEPA and DOX@CaCO3/rGO-TEPA drug carriers and incubated for 24 and 48 hours. Without the


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DOX drug, CaCO3 and CaCO3/rGO-TEPA carriers exhibited very low cytotoxicity in all dosages, even after 48 h (Figure 5c and 5d). After loading the DOX drug, the cell viability of the DOX@CaCO3/rGO-TEPA drug carriers was reduced to 90.5~64.4%. Increasing the incubation time to 48 h, it was found that the cell viability with DOX@CaCO3/rGO-TEPA drug carriers significantly reduced in all dosages because of the increased and accumulated of the drug concentrations.

Figure 6. SEM images of CaCO3/rGO-TEPA composite crystal with different morphologies; (a) solid spheres, (c) cube bulks, (b) and (d) high magnification of (a) and (c) To further prove the DOX-release excellent properties of hollow structure of the CaCO3/rGOTEPA microspheres, the CaCO3/rGO-TEPA solid composites were used rather than the CaCO3/rGO-TEPA hollow microspheres to carry out the same experiment. As shown in Figure 6,


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SEM observation of the CaCO3/rGO-TEPA solid composites revealed different morphologies of solid spheres (Figure 6a) and cube bulks (Figure 6c) and approximately 1~4 µm in size which was consistent with the CaCO3/rGO-TEPA hollow microspheres (Figure 1). High-magnification SEM image (Figure 6b) of the CaCO3/rGO-TEPA solid spheres indicate a rough surface, while the cube bulks showed a smooth surface (Figure 6d). After oscillating continuously for 24 h, the drug was convergent to CaCO3/rGO-TEPA solid spheres and CaCO3/rGO-TEPA cube bulks with the drug loading capacity of 62.6 mg g-1 and 52.1 mg g-1, loading efficiencies of 87.9% and 78.1%, respectively. This was much lower than the drug loading performance of the CaCO3/rGO-TEPA hollow microspheres which were 78.9 mg g-1 and 94.7%. The cumulative DOX release efficiencies curves of DOX@CaCO3/rGO-TEPA solid carriers were depicted in Figure 7. When the pH value was 7.4 (the normal tissue environment), CaCO3/rGO-TEPA cube bulks and CaCO3/rGO-TEPA solid spheres have similarly DOX release behaviors which the release efficiencies for 48 h were 7.9% and 10.4%, respectively. At the pH value of 5.0 (the cancer tissue environment), these two solid carriers showed much faster DOX release behaviors than those at pH value of 7.4. The release efficiencies also had few difference between CaCO3/rGO-TEPA cube bulks (43.9%) and CaCO3/rGO-TEPA solid spheres (49.5%), which was much lower than the CaCO3/rGO-TEPA hollow microspheres with a release efficiency of 91.8%. It is well accepted that the CaCO3/rGO-TEPA hollow microspheres have an excellent DOX loading capacity and release properties than CaCO3/rGO-TEPA solid spheres and CaCO3/rGO-TEPA cube bulks.


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Figure 7. DOX release efficiencies of DOX@CaCO3/rGO-TEPA with different morphologies at pH values of 5.0 and 7.4 To better verify the structure and property between the different CaCO3/rGO-TEPA composites, N2 adsorption/ desorption of CaCO3/rGO-TEPA samples were carried out and the results were shown in Figure 8. It was found that the CaCO3/rGO-TEPA hollow microspheres (Figure 8a) presented the H1 hysteresis loops and typical IV-type isotherm, which was the feature of the mesoporous samples. And the BET surface area was 13.98 m2/g and the single pore volume (P/P° = 0.97) was 0.083 cm3/g. From the corresponding pore-size distribution curve, it was observed that the narrow pore-size of the microspheres was 2.1 nm. However, the N2 adsorption/desorption isotherms of CaCO3/rGO-TEPA solid spheres (Figure 8b) and CaCO3/rGO-TEPA cube bulks (Figure 8c) were almost overlapped and have no hysteresis loops.


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In Figure 7, it seemed that CaCO3/rGO-TEPA in the form of cube and microsphere exhibit similar release efficiency, which might be due to their similar solid structure. The BET surface area and the single pore volume of CaCO3/rGO-TEPA solid spheres were calculated to be 5.44 m2/g and 0.026 cm3/g, while the CaCO3/rGO-TEPA cube bulks were 0.96 m2/g and 0.006 cm3/g, respectively. And the bigger BET surface area of the CaCO3/rGO-TEPA solid spheres could contribute to the higher DOX loading and release property than the CaCO3/rGO-TEPA cube bulks. It should be noted that the BET surface area of the CaCO3/rGO-TEPA solid spheres and the CaCO3/rGO-TEPA cube bulks were much smaller than those of the CaCO3/rGO-TEPA hollow microspheres. However, the drug loading capacity and loading efficiencies of the CaCO3/rGO-TEPA cube bulks were still 52.1 mg g-1 and 78.1%, respectively. For clear contrast, DOX loading and release property of DOX@CaCO3/rGO-TEPA with different morphologies were summarized in Table 1. That could be speculated that the CaCO3/rGO-TEPA composites were good for drug delivery, since the well drug loading capacity and loading efficiencies. The pH-responsive drug-release behavior was good for targeted drug release to the physiological acidic environment in tumour tissues. There is no doubt that the drug loading and releasing property of the CaCO3/rGO-TEPA hollow microspheres is outstanding.


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Figure 8. N2 adsorption/desorption isotherm (left) and the corresponding pore diameter distribution curve (right) of CaCO3/rGO-TEPA hollow microspheres (a), CaCO3/rGO-TEPA solid spheres (b) and CaCO3/rGO-TEPA cube bulks (c)


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Table 1. DOX loading and release property of DOX@CaCO3/rGO-TEPA particles with different morphologies the morphologies of drug carriers hollow microspheres solid spheres cube bulks

the BET

DOX loading and release property loading

loading

release efficiency

release efficiency

capacity

efficiencies

(pH=7.4, 48 h)

(pH=5.0, 48 h)

13.98 m2/g

78.9 mg/g

94.7%

13.8%

91.8%

5.44 m2/g

62.6 mg/g

87.9%

10.4%

49.5%

52.1 mg/g

78.1%

7.9%

43.9%

surface area

2

0.96 m /g

Conclusions In summary, we used rGO-TEPA sheet matrices induction of calcium carbonate mineralization and obtained the CaCO3/rGO-TEPA hybrid microspheres. The as-prepared microspheres exhibited both porous surface structure and hollow inner structure. As potential drug carriers, the biocompatibility of the CaCO3/rGO-TEPA hybrid microspheres was evaluated through the standard MTT assay and showed nontoxic. The DOX loading and release measurements showed that the load efficiency of CaCO3/rGO-TEPA hybrid carriers is 94.7% and the release efficiency are 13.8% and 91.7% by the pH value of 7.4 and 5.0. The as-prepared CaCO3/rGO-TEPA hybrid microspheres showed some appealing advantages, such as the pH-sensitive release characteristics and the mild storage-release behaviors. Therefore, the CaCO3/rGO-TEPA hybrid microspheres would be a competitive alternative in bone drug carriers. ASSOCIATED CONTENT Supporting Information. The SEM images of pure CaCO3 crystals, XRD diffraction pattern of pure CaCO3 crystals, FT-IR spectrometry of pure CaCO3 crystals, and the standard curve of


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DOX concentration. This material is available free of charge via the Internet at http://pubs.acs.org.

Corresponding Author: E-mail address: [email protected] (H. Wei). E-mail address: [email protected] (N. Ma). Acknowledgements This work was supported by National Natural Science Foundation of China (21374009), and Fundamental Research Funds of the Central University (HEUCFJ161003). References 1

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