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Intrinsic ultrahigh drug/miRNA loading capacity of biodegradable bioactive glass nanoparticles towards highly efficient pharmaceutical delivery Meng Yu, Yumeng Xue, Peter X Ma, Cong Mao, and Bo Lei ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13874 • Publication Date (Web): 27 Feb 2017 Downloaded from http://pubs.acs.org on March 3, 2017

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ACS Applied Materials & Interfaces

Intrinsic ultrahigh drug/miRNA loading capacity of biodegradable bioactive glass nanoparticles towards highly efficient pharmaceutical delivery Meng Yu a,#, Yumeng Xue a, #, Peter X Ma c, Cong Mao b , Bo Lei a,* a

Frontier Institute of Science and Technology, State Key Laboratory for Mechanical Behavior of Materials,

State Key Laboratory for Manufacturing Systems Engineering, Xi’an Jiaotong University, Xi’an 710054, China b

Department of orthopedics, The Second Affiliated Hospital and Yuying Children Hospital of Wenzhou

Medical University, Wenzhou 325001, China c

Department of Biologic and Materials Sciences, University of Michigan, Ann Arbor, MI 48109-1078, USA

* Corresponding author: Bo Lei, [email protected], Tel. +86-29-83395361 #

These authors contributed equally to this work.

Keywords: Bioactive glass; Nanoparticles; Drug and gene delivery; Intrinsic properties; Cytotoxicity Abstract: The lack of safe and efficient drug/gene delivery vectors has become a major obstacle for the clinical applications of drug/non-viral gene therapy. To date, for non-viral gene vectors, most of studies are focused on cationic polymers, liposomes and modified inorganic nanoparticles which have shown high cellular toxicity or low transfection efficiency or non-degradation. Additionally, few biodegradable biomaterials demonstrate intrinsic high binding abilities to both drug and gene. Bioactive glasses (BGs) have achieved successful applications in bone regeneration due to their high biocompatibility and biodegradation. Here, for the first time, we demonstrate the intrinsic ultrahigh drug and miRNA binding ability of bioactive glass nanoparticles (BGN) without any cationic polymer modification. BGN demonstrates an over 45-fold improvement in hydrophilic drug loading (diclofenac sodium) and 7-fold

ACS Paragon Plus Environment


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enhancement in miRNA binding, over their corresponding silica nanoparticles (SN). The hydrophilic drug loading ability of BGN (> 45 wt% loading) is also higher than most of reported inorganic nanoparticles including mesoporous silica nanoparticles (MSN). BGN shows significantly low cytotoxicity, high cellular uptake and miRNA transfection efficiency, as compared to commercial transfection reagents polyethyleneimine (PEI 25 K) and lipofectamine 3000. Our results demonstrate that BGN may become a new competitive vehicle for drug and gene delivery applications. This study may also provide a new strategy to develop novel biomaterials with intrinsic drug and gene binding ability for disease therapy. 1. Introduction In past two decades, the gene and drug-based therapy has played an important role in treating or preventing various diseases 1, 2. However, due to various technical obstacles in drug and gene delivery, their successful clinical applications are usually limited, especially in cancer chemotherapy 3. In cancer therapy, conventional small molecule cytotoxic drugs are usually showing the high toxicity and low effectiveness 4. Employing nanomaterials or polymer nanocomposites to deliver the therapeutic drugs could reduce the toxicity 5, 6. Thus, development of biocompatible vectors with high drug loading ability has been one of the important research directions 7. On the other hand, the major challenge of gene therapy is the development of safe and highly efficient delivery vectors, because naked genes are difficult to penetrate cellular membranes and also easy undergo enzymatic degradation by serum nucleases 8. Viral-based vectors have shown their success in gene delivery in vitro and in vivo 9. However, the broad applications of viral carriers are limited due to the concern of safety and immune response, as well as their high cost

10

. Non-viral

vectors, such as cationic polymers and liposomes, have been widely investigated as safe alternatives for gene delivery and shown increased interests in past decades, due to their safety, stability and low cost 11–13. Non-viral carriers can efficiently deliver genes into cells and release them. The major drawbacks of


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non-viral vectors lie on their low transfection efficiency and cytotoxicity at the high concentration 14. The development of non-viral vectors with high biocompatibility and transfection efficiency still remains a challenge 15, 16. As an outstanding representative in cationic polymer, branched polyethyleneimine (PEI 25K) shows the high transfection efficiency for various genes but exhibits high cytotoxicity even at low concentration 17. Cationic liposomes also demonstrate good transfection efficiency but potential immunogenicity in vivo applications 18. Compared with polymers and liposomes, inorganic materials usually show high biological stability and good potential for gene delivery. For example, gold, magnetite, quantum dots, carbon-based materials and mesoporous silica have been studied as non-viral vectors for gene delivery

19–25

. However,

the most of inorganic nanoparticles show the low gene or drug loading efficiency and high accumulation toxicity in vivo due to their nondegradation

26

. The development of new biodegradable inorganic vectors

with high drug or gene binding efficiency, transfection ability and enhanced biocompatibility may be a promising strategy for in vivo applications. Bioactive glasses (BG) have achieved successful clinic applications in bone tissue repair and regeneration, due to their high biocompatibility and osteoconductive ability and gene-activation properties 27-30

. Sol-gel derived BG shows significantly enhanced porosity and specific surface area, as compared to

conventional melt-derived BG 31. For example, the sol-gel derived BG had a specific surface area of 80.1 m2 g -1, which was significantly higher than melting derived BG (1.1 m2 g-1) 32. Based on these advantages, sol-gel derived BG nanoparticles (BGNs) have shown promising drug and gene delivery capacities, along with high cellular biocompatibility 33-36. In previous studies, BGN has been successfully used in controlling stem cell proliferation and differentiation, drug and gene delivery, as well as living cell imaging applications 30, 37-40. However, current studies of BGN for drug and gene delivery usually showed low drug



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or gene loading ability and poor transfection efficiency, because their drug loading was based on the physical adsorption process (dependent on the specific surface area) and gene binding was relied on the surface modification of cationic polymer. For instance, hydrophilic or hydrophobic drugs were loaded on mesoporous BG through soaking BG particles in the drugs solution and released via a diffusion process 41, 42

. Moreover, to achieve a desired loading efficiency of gene, BG was usually modified by various positive

charged functional groups. For example, amino modified mesoporous BGN has been reported as gene vectors for the delivery of DNA and siRNA in previous work

33-35

. The inherent drug loading and gene

binding (especially the miRNA) performance of BGN are rarely reported. Here, we demonstrate the intrinsic high drug/gene binding ability and gene transfection efficiency of sol-gel derived BGN, using conventional silica nanoparticles (SN) as a control. It is well known that calcium ions have strong interactions with phosphate, carboxylate or sulphate groups in nucleic acid or drugs

43, 44

. Based on this mechanism, calcium phosphate nanoparticles have been widely studied for gene

delivery, but the low transfection efficiency and complicated fabrication limit their successful in vivo applications

45

. In this study, it is hypothesized that BGN could bind drug and gene through the novel

calcium-drug or gene complex mechanism (Figure 1). We investigate the effects of BGN and SN on the diclofenac sodium (drug) or miRNA-5106 (gene) binding (loading) ability, controlled release behavior, cytotoxicity, cellular uptake and gene transfection. 2. Materials and methods 2.1. Materials. Dodecylamine (DDA), tetraethyl orthosilicate (TEOS), triethylphosphate (TEP), calcium nitrate tetrahydrate (CN), diclofenac sodium (DS), ethyl alcohol (99% ETOH), agarose, tris, boric acid were purchased from Sigma-Aldrich. 4-(2-hydroxyethyl)-1-piperazine-N'-2-ethanesulfonic acid (HEPES), Dulbecco's modified eagle medium (DMEM), Fetal bovine serum (FBS), Opti-MEM, AlamarBlue cell


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viability kit, LIVE/DEAD® mammalian cell viability stain kit, NucBlue Live ReadyProbes Reagent (Hoechst 33342) were purchased from Thermo Fisher Scientific. Normal miRNA and FAM labeled miRNA (miR-5106) were purchased from Genepharma (China). All chemicals were used as received without further purification. 2.2. Synthesis of BGN and SN. BGN were synthesized via a sol-gel process by using DDA as a catalyst and template agent according to our previous study 39. Typically, DDA was dissolved in ethyl alcohol/water solution (2/1= v/v) at 40 °C, followed by adding TEOS dropwise under magnetically stirring for 30 min. Then, TEP and CN were added and the mixed solution was further reacted for 3 h. The resulted product was collected by a centrifuge, and the remaining reactants and templates were removed by washing with ETOH and deionized water for several times. The obtained product was dried overnight in a freeze drying machine (Alpha 2-4 LDplus,CHRIST) and heated at 650 °C for 3 h under air condition (CWF 11/13, Carbolite). The molar composition of BGN was 80SiO2:16CaO:4P2O5. The SN was synthesized in the same way as BGN but without the incorporation of CaO and P2O5. 2.3. Physicochemical structure characterizations of BGN and SN. The nanoscale morphology of BGN and SN was analyzed by a transmission electron microscope (TEM, HT-7700, Hitachi) at an acceleration voltage of 100 kV. The X-ray diffraction (XRD) patterns of samples were obtained from an X-ray Diffractometer (D/MAX-RB, Rigaku). Fourier transform infrared spectroscopy (FTIR) spectra of samples were measured by a KBr pellet method (Nicolet 6700, Thermo Sci.) in the range of 2000–400 cm-1. The specific surface area, pore structure of samples were measured through a nitrogen adsorption-desorption method (Quadrasorb SI-3, Quantachrome). The mean pore size distribution of SN and BGN was calculated based on a Barrett–Joyner–Halenda (BJH) model. The samples were degassed at 120 °C and 104 Pa for 12 h prior to the test. UV-Vis absorption spectra were collected from a spectrophotometer (Lambda 35,



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PerkinElmer) at room temperature. The chemical composition of samples was obtained by an X-ray energy dispersive spectroscopy (EDS, Quanta 250 FEG, FEI). The zeta potential of samples was measured by a dynamic light scattering (Nano-ZS, Malvern). 2.4. Drug loading and release assay. 5-Fluorouracil (5-Fu, anti-cancer drug) and diclofenac sodium (DS, anti-inflammation drug) were employed as the model drugs respectively to evaluate drug loading and release behavior of BGN and SN. In brief, BGN/SN was soaked in 5-Fu or DS solution (3 mg/mL) and the mixture was stirred vigorously at room temperature for 24 h. After loading, the mixture solution was centrifuged and washed with deionized water carefully to get the drugs-loaded samples. The loading amount was analyzed by measuring drug concentrations in the supernatant before and after loading process through the UV-Vis spectra analysis at a wavelength of 276 nm (DS) and 267 nm (5-Fu). The drug loading efficiency was expressed as the drug weight per sample weight (mg/g). The morphology, chemical structure and crystalline phase composition of BGN/SN after drug loading were also evaluated through TEM, FTIR and XRD. The evaluation of drug releasing behaviors was carried out by soaking the drug-loaded samples in 1 mL phosphate buffer saline (PBS, pH 7.4) solution in a dialysis bag (5 KDa). The dialysis bag was immersed in 20 mL PBS solution and placed in a shaker table (100 rpm) at 37 °C. At predetermined time intervals, 5 mL medium solution was taken out for testing and replaced with another 5 mL of fresh PBS. For the release of DS, the release medium solution was taken out after a 15 min interval in the first 4 h, and then the interval time was increased to 30 min, 1 h and 2 h. For the release of 5-Fu, the interval time was 10 min in the first 1 h and gradually extended to 20 min, 1 h and 2 h. At least 3 species per sample were used in the drug release tests. 2.5. Ethidium bromide exclusion assay. Ethidium bromide (EB) exclusion assay was carried out to verify


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the interaction between miRNA and BGN. 5µg miRNA were dissolved in 20 µL HEPES buffer (pH=6.5) in a 96-well microplate, then EtBr solution were added to the wells and incubated for 15 min at a room temperature. BGN/SN were added to the EB-miRNA mixture at different weight ratios (sample: miRNA=8:1 and 16:1) and further incubated for 20 min. The fluorescence intensity of EB was measured by a microreader (SpectraMax@, Molecular Services) at 510 nm excitation and 590 nm emission wavelength. EB-miRNA complex without samples were used as a control. EB-samples without miRNA were used as a background. 2.6. miRNA loading and stability evaluation. The loading ability and stability of BGN and SN with miRNA (Genepharma, Shanghai) were analyzed using gel retardation assay. Briefly, miRNA (0.26 mg/mL) were added to the BGN (10 mg/mL) and SN solution (10 mg/mL) at different weight ratios. After 30 min incubation at 37 °C, the BGN/SN-miRNA complexes were obtained through centrifugation and washing. The miRNA loading ability was tested according to the 2% agarose gel retardation assay (110V for 15 min) using an electrophoresis system (HE120, Tanon). Commercial transfection reagents polyethyleneimine (PEI 25K) and Lipofectamine™ 3000 (LIPO) were used as controls. In all miRNA loading and transfection experiments, miRNA (0.26 mg/mL) loading in the initial step among BGN/SN, PEI 25K and LIPO was identical. The concentration of LIPO was according to the manufacturer’s instruction. The stability of BGN/SN-miRNA complexes was carried out through soaking samples into a heparin and serum solution for different times. Briefly, 25% volume of fetal bovine serum (FBS) was added into the BGN/SN/PEI complexes solution to degrade miRNA. At pre-determined time points, the samples were collected by adding a ribonuclease inhibitor to inhibit further miRNA degradation (RRI, Tiangen). After all samples were collected, 25 µg/mL heparin were added into samples. The stability of complexes in serum and heparin solution were analyzed using 4% agarose gel (110V for 20 min) and the miRNA bands were



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detected using a gel imaging system (Gel Doc XR+, Bio-rad). The average intensity of intact miRNA bands were evaluated using the quantity one software (Bio-Rad) and the intact percentage was calculated according to the followed equation:

IR = − )⁄( − × 100%

(1)

where IR is the intact percentage of miRNA complexes; is the intensity of intact gene bands in each wells contained samples; is the intensity of intact gene bands in control well without sample; and is the intensity of background. 2.7. Cellular biocompatibility investigation. The bone mesenchymal stromal cells (BMSCs, MT-BIO) were used to investigate the cytotoxicity of BGN/SN/PEI25K. Briefly, BMSCs were seeded on 24-well plate at a density of 1×105 cells/mL, cultured with Dulbecco’s Modified Eagle Medium (DMEM) containing 20% FBS and 1% penicillin/streptomycin (Thermo Fisher Scientific) at 37 °C and 5% CO2 atmosphere. After seeded for 24 h, BMSCs were exposed to BGN/SN/PEI25K at different concentrations for 3 days. The alamar blue assay (Thermo Fisher Scientific) was to detect the cytotoxicity of samples according to the manufacturer’s instructions. The cytotoxicity was also analyzed using LIVE/DEAD® assay after incubation for 24 h according to the manufacturer’s instructions. At least 5 species per sample were measured to get the mean value and standard deviation. PEI 25K and LIPO were used as controls. 2.8. Transfection efficiency analysis. The transfection efficiency of FAM labeled miRNA by BGN/SN was characterized through the intracellular fluorescent expression of miRNA and cellular uptake analysis. Briefly, BMSCs were grown on 24-well plate at a density of 1×105 cells/mL, cultured with DMEM containing 20% FBS and 1% penicillin/streptomycin at 37 °C and 5% CO2 atmosphere. 0.26 µg miRNA (0.26 mg/mL) were added to the BGN/SN, PEI 25K and LIPO, followed by further incubation for 40 min at 37 °C. The weight ratio of BGN/SN and miRNA was 40:1. PEI 25K (weight ratio of 20:1 with miRNA)


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and LIPO (volume ratio of 1:1 with miRNA) were used as controls. After washed by PBS for 2 times, the various complexes were incubated with BMSCs for 24 h, then the culture medium changed with a fresh culture medium. After further incubation for 48 h, the transfected cells were observed by a Laser Confocal Microscopy (FV1200, Olympus). The cellular uptake and transfection efficiency of BGN/SN/PEI/LIPO with miRNAs were evaluated using a flow cytometer (CytoFLEX, Coulter Beckman). Briefly, the transfected BMSCs were washed by Dulbecco’s PBS (Thermo Fisher Scientific) for two times, then trypsinized, centrifuged and suspended with 100 µL PBS containing 2% FBS, added with 100 µL 4% paraformalclehyde. After incubated at 4 °C for 2 h, the fluorescent intensity of samples were analyzed based on 10,000 cells through CytExpert software (Beckman Coulter). 2.9. Statistical Analysis. All data are expressed as mean ± standard deviation (SD). All statistical analyses were performed with a SPSS software (version 13.0). Significant differences between two groups were determined by a Student’s t test. *P < 0.05 and **P < 0.01 was considered statistically significant. All experiments were performed at least in triplicate. 3. Results and discussion 3.1. Synthesis and characterizations of BGN and SN In this work, monodispersed BGN and SN were successfully synthesized through a template-assisted sol-gel process 39. Figure 2 shows the physicochemical structure characterizations of samples. BGN and SN exhibited a representative spherical morphology with a uniform size of 200–300 nm (Figures 2A–D, Figure S1 in supporting information). The mean pore size of SN and BGN calculated by a nitrogen adsorption-desorption analysis was 3.136 nm and 3.541 nm respectively (Figures 2C-D). SN showed a significantly high specific surface area (568.596 m2 g-1), as compared to BGN (332.087 m2 g-1) (Figure S2 and Table S1 in supporting information). The chemical structure, element composition and phase structure



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of BGN and SN were analyzed by FTIR, EDS and XRD, as shown in Figures 2E–H. BGN/SN has an absorbance at 1600 cm-1 due to the bending vibration of -OH, and the broad band at 1060 cm-1 and the peaks at 800 cm-1 and 480 cm-1 were attributed to the stretch vibration and bending vibration of Si-O-Si bond (Figure 2E). The typical element composition of BGN (Si and Ca) and SN (Si) was identified by EDS analysis (Figures 2F–G). The XRD pattern for both BGN and SN showed a broad peak at 24°, indicating the representative amorphous structure of synthesized BGN and SN (Figure 2H). The zeta potential of BGN and SN in physiological buffer environment (PBS) was -19.5 mV and -18.27 mV respectively, suggesting their similar surface potential (Figure S3 in supporting information). 3.2. Intrinsic high drug loading capacity and controlled release behavior To demonstrate the intrinsic drug binding ability of BGN, two different types of drugs DS and 5-Fu were used. After DS drug loading, the UV-Vis absorbance intensity of drug solution at 276 nm was decreased significantly, implying their successful loading by BGN and SN (Figure 3A). The absorbance intensity of DS drug after loaded by BGN was significantly weaker than that loaded by SN, suggesting that BGN may have the higher DS binding ability than SN (Figure 3B). The quantitative analysis demonstrated that BGN showed exceptionally high DS loading efficiency (487.3 mg/g) which was significantly high than that of SN (10.5 mg/g) ((Figure 3C). To eliminate the effect of specific surface area, the drug loading efficiency as mg/m2 was also determined. BGN exhibited an over 100-fold high DS loading capacity (1.467 mg/m2) compared with SN (0.0183 mg/m2) (Figure S4 in supporting information). Figure 3D shows the cumulative release profile of DS from BGN and SN. DS exhibited sustained release behavior for both BGN and SN in 12 h. Importantly, it can be clearly observed that drugs released faster from SN than BGN, demonstrating the sustained release ability of DS from BGN. Compared with DS, 5-Fu demonstrated very different loading and controlled release behavior on BGN and SN (Figure 3E–H). After loaded by samples,


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the UV-Vis absorbance intensity of 5-Fu solution at 267 nm only showed a weak decrease and drug-loaded samples solution also exhibited low absorbance (Figures 3E–F), suggesting that BGN and SN may have poor binding ability for 5-Fu. The drug loading efficiency results confirmed that BGN and SN showed rather low binding amount (below 3 mg/g) (Figure 3G). No significant difference on 5-Fu loading and release behavior was observed between BGN and SN (Figure 3H). Figures 3I-J present the different loading and releasing process of DS and 5-Fu through BGN. There are no any possible chemical groups in 5-Fu to bind with BGN, and the poor physical adsorption between BGN and 5-Fu may be the main driving force to load drug (Figure 3I). This can also explain why 5-Fu only showed the low loading efficiency and fast release from both BGN and SN. Different from 5-Fu, DS possesses a typical carboxyl group in their structure which can efficiently bind with calcium in BGN, then DS showed the exceptionally high loading by BGN and sustained release behavior (Figure 3J and Figure S5 in Supporting Information) 44, 45. Because no calcium was exited in SN, the poor interaction between drugs (5-Fu and DS) and SN may result in their low drug loading and release behavior. Moreover, the specific surface area of BGN was significantly lower than SN, but BGN presented significantly high loading ability of DS compared with SN. It was well known that the chemophysical properties of drug carriers played an important role on their drug loading and delivery. Specially, the surface area, pore structure and surface property for conventional inorganic nanoparticles significantly influence the drug loading capacity 46. Generally large surface area and pore size and strong host-guest interaction would induce the high drug loading. In our case, BGN showed a significantly low surface area and a little high pore size (332 m2/g and 3.5 nm) compared with SN (568 m2/g and 3.1 nm), but demonstrated almost 40 times high drug loading efficiency. A little high pore size and low surface area for BGN should not induce the more than 10 times improvement of drug loading, relative to SN. Vallet-Regí and co-workers found that mesoporous silica nanoparticles (MCM-41) only



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showed a 0.5 times improvement when the pore size was changed from 3.3 to 3.6 nm 46. In addition, the surface could also influence the drug loading through controlling the interaction between drug and nanoparticles 47. In our study, BGN and SN presented a similar surface potential (-19 mV and -18 mV), and the DS drug also had a negative potential in PBS medium. That means that surface properties were also not the reason of high drug loading capacity of BGN. These results demonstrate that the specific surface area and surface charges did not determine the high drug loading capacity of BGN. The high drug loading capacity of BGN could be attributed to the strong host-guest interaction between the drugs and BGN. The physicochemical interactions between drugs and vectors (BGN and SN) were characterized in Figure 4. After drug loading, the surface of BGN-DS was coated by a new layer, indicating the highly efficient DS loading (Figure 4A), no any change was observed for 5-Fu loading (Figure 4B). The size and surface morphology of SN did not show any change after loading DS and 5-Fu (Figures 4C–D). FTIR analysis also showed the presence of DS and 5-Fu in BGN (Figures 4E–F). DS showed characteristic peaks of C=C and C=O bonds at about 1507 cm-1 and 1575 cm-1 (Figure 4E, Figure S6 in supporting information). After loaded by BGN, the peaks corresponded to carboxyl group was shifted to 1571 cm-1, which may be due to the interaction between carboxyl group in DS and Ca2+ in BGN (Figure S6 in supporting information). No significant characteristic peaks of DS were detected in DS loaded SN, indicating the low loading amount of DS. The characteristic peaks of 5-Fu could be observed in the spectra of BGN-5-Fu and SN-5-Fu, however no any shifting of peaks positions was found, suggesting poor interaction between 5-Fu and BGN/SN (Figure 4F). In addition, compared with SN, the significant XRD pattern was observed from DS-loaded BGN, indicating the high DS binding ability of BGN (Figure 4H). Except the characteristic peaks of DS, no other patterns related with crystalized calcium carboxylate were detected. The result of XRD suggested that the interaction between BGN and DS did not result in the formation of calcium


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carboxylate crystalline. Actually it was difficult to form crystalized calcium carboxylate under our condition (PH=7.4, 37 °C). The FTIR and XRD analysis demonstrated that the high DS loading on BGN was probably attributed to the complexation interaction between calcium and drugs. As compared to SN, the only difference lies on Ca and P presence in BGN, and different DS loading and release was observed. The remarkable DS loading ability of BGN may be ascribed to the incorporation of Ca. On one hand, in the structure of BGN (SiO2-CaO-P2O5), calcium is the network modifier and uniformly distributed in Si-O-Si network

48, 49

. Compared with 5-Fu, DS possesses the typical carboxylate structure which is very easy to

chelate with Ca-contained materials. Therefore, BGN and SN may bind with 5-Fu through a physical adsorption, and then showed a similar 5-Fu loading efficiency and release behavior (Figure 3). The DS showed a significantly high loading efficiency on BGN through a interaction of calcium and carboxylate group. This strong interactions between DS and BGN also prevented the initial burst release (Figure 3D). BGN may have a great potential as a highly efficient vector to deliver calcium-responsive drugs for tissue regeneration and cancer therapy. There were no significant differences between the FTIR spectra and XRD patterns of SN-miRNA and BGN-miRNA. After loading miRNA, the peak at 962 cm-1 attributed to P-O vibration in miRNA was observed clearly on SN and BGN, indicating the presence of miRNA (Figure 4G). SN-miRNA and BGN-miRNA still showed the representative amorphous XRD patterns (Figure S6 in supporting information). In order to verify the interaction between BGN and miRNA, an EtBr (EB) exclusion assay was carried out (Figure 4J). The EB exclusion evaluation was proved to be efficient at testing the interaction between inorganic ions (such as Ca, Zn) and genes 50, 51. The effective complexation between Ca from BGN and phosphate group from miRNA should lead to the exclusion of intercalating EtBr from miRNA, resulting in the quenching of EB fluorescence. From the results it was found that increasing the



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concentration of BGN, the significant fluorescence quenching could be observed, suggesting a strong complex interaction between BGN and miRNA. However, no significant fluorescence quenching was observed for SN, indicating that no complex interaction happened. 3.3. miRNA loading ability and stability evaluation The miRNA loading ability and binding stability evaluation of BGN are shown in Figure 5. After binding miRNA, the surface of BGN was changed to be a loose and porous morphology, suggesting a strong surface interaction between BGN and miRNA (Figure 5A). Compared with BGN, there was no significant surface change for SN after loading miRNA, indicating the poor interaction of miRNA-SN (Figure 5B). Figure 5C shows a miRNA condensation of BGN at various weight ratios (w/w). The miRNA migrations in agarose gel were completely retarded through BGNs when the w/w ratio was higher than 4:1, indicating that BGN can efficiently bind miRNA. The miRNA mobility could not be retarded by SN even a w/w ratio of 64, suggesting their poor miRNA binding ability. The quantitative analysis indicated that BGN showed a significantly high miRNA binding amount (~200 µg/mg) at various nanoparticle concentrations (40~320 µg/mL) as compared to SN (~20 µg/mg) (Figure 5D). The binding stability of miRNA in serum was determined by incubating naked miRNA and BGN/SN nanocomplexes in 25% FBS (Figures 5E–F). After 3 h incubation, the naked miRNA and SN-miRNA was almost completely degraded by nuclease in FBS. Moreover, above 82% of the intact miRNA was still observed for BGN/miRNA and PEI/LIPO groups after incubated for 24 h in nuclease and heparin, suggesting that BGN could protect miRNA from enzymolysis compared with SN (Figure 5F). The miRNA binding mechanism of SN was mainly through a physical adsorption, and the miRNA may be desorbed easily in the delivery process (Figure 5G). Different from SN, there was strong interactions between BGN and miRNA through the calcium in BGN and phosphate group in miRNA


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(Figure 5H). The BGN-miRNA nanocomplex could be taken by cells by endocytosis and the miRNA probably was released from BGN after degradation (Figure 5H). The mechanism of BGN binding miRNA was also calcium-responsive process which was similar with that of BGN and DS. In the structure of various nucleic acids, there were many phosphate residues which may have strong interaction with calcium in BGN

50-52

. Therefore, here, BGN demonstrated an excellent miRNA binding and protection ability,

compared to SN. 3.4. Cellular biocompatibility and miRNA transfection assays Both BMSCs and BG have shown good potential for bone tissue regeneration applications. We anticipate that BGN-based miRNA delivery could be employed to enhance BMSC osteogenic differentiation and bone formation in vivo. Therefore, in this study, we choose BMSCs as model to evaluate the biocompatibility and miRNA transfection of BGN. Figure 6 shows the cellular biocompatibility of BGN and SN, using PEI 25K and LIPO as controls. BMSCs exhibited a good cell attachment morphology after incubated with BGN at different concentrations (30-240 µg/mL) (Figure 6A). As compared to BGN, PEI 25K and LIPO demonstrated a significantly low live cells attachment (Figure 6A). After culture for 72 h, the cells incubated with 30 and 60 µg/mL BGN demonstrated a significantly high cell viability, as compared to PEI 25K and LIPO (Figure 6 B). Additionally, BMSCs also showed a significantly high proliferation after cultured with BGN for 24 h and 72 h (~120 µg/mL), suggesting the high cellular biocompatibility of BGN compared with commercial PEI 25K and LIPO. The FAM-labelled miRNA transfection efficiency was examined by confocal fluorescent microscopy and flow cytometry analysis (Figure 7). BGN group showed significantly strong green fluorescence of miRNA, as compared to SN, PEI 25K and LIPO groups (Figure 7A). The magnified fluorescent images exhibited that miRNA in BGN group was uniformly distributed around cell nucleus (Figure 7B). The



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quantitative results also demonstrated that BGN group presented a significantly high miRNA fluorescent intensity, as compared to SN, PEI 25K and LIPO group (Figure 7 C). The cellular uptake ability and transfection of BGN was further examined through a flow cytometry analysis (Figures 7D–E). BGN group showed a significantly high fluorescent intensity of miRNA, as compared to other groups. Significantly high miRNA transfection efficiency for BGN group (~45%) was observed compared with commercial PEI (25%) and LIPO group (35%) (Figure 7E). These results demonstrated that BGN possessed excellent cellular biocompatibility and highly efficient ability to transfect miRNAs into cells. In our study, during the transfection process, the initial concentration of BGN/SN and miRNA was the same. On the other hand, the cell uptake ability of pure BGN and SN without any surface modification should be similar because they have similar chemical structure and surface potential. The only reason of high transfection efficiency lies on the high miRNA loading of BGN, because the transfection efficiency was expressed as the fluorescent intensity of miRNA in cells. Additionally, as compared with SN, BGN transfected miRNAs significantly enhanced the osteogenic differentiation of BMSCs through upregulating their ALP activity, suggesting the promising bone regeneration application via a BGN-based miRNA delivery (Figure S7 in supporting information). The detailed transfection process, mechanism and in vivo bone regeneration through BGNs will be studied in further work. 4. Conclusion In summary, for the first time, the inherent high drug and miRNA delivery ability of BGN were successfully demonstrated. As compared to SN, BGN showed a significantly high drug (DS) loading ability (45 times higher than SN) and sustained release behavior. BGN also exhibited a significantly high miRNA loading ability (7 fold improvement compared with SN) and serum stability relative to LIPO. Additionally, BGN shows significantly low cytotoxicity, high cellular uptake and gene (miRNA) transfection efficiency,


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over the commercial transfection reagents polyethyleneimine (PEI 25KD) and lipofectamine 3000. Apart from surface properties and pore structure, the calcium-complex-based host-guest interaction could be a novel pathway for BGNs to highly efficient drug and gene loading. The in vivo drug/gene delivery ability and potential applications for BGN will be demonstrated in further study. The excellent drug/gene binding ability and good biocompatibility make BGN a promising biomaterial towards a new-generation pharmaceutical delivery for biomedicine. Our study may also provide a new strategy to develop novel nanomaterials with good biocompatibility and highly efficient drug/gene delivery ability for tissue regeneration and cancer therapy.

Acknowledgements We acknowledge the valuable comments of potential reviewers. This work was supported by State Key Laboratory for Mechanical Behavior of Materials, the Scientific Research Starting Foundation from Xi’an Jiaotong University (Grant No. DW011798N3000010), the Fundamental Research Funds for the Central Universities (Grant No. XJJ2014090), the Natural Science Basic Research Plan in Shaanxi Province of China (Grant No. 2015JQ5165), National Natural Science Foundation of China (Grant No. 51502237). Supporting Information Zeta potential analysis, DS and 5-Fu drug loading efficiency dependent on surface area, Alp activity evaluation. References [1] Chen, Y.; Gao, D.-Y.; Huang, L. In Vivo Delivery of miRNAs for Cancer Therapy: Challenges and Strategies. Adv. Drug Delivery Rev. 2015, 81, 128-141. [2] Sun, T.; Zhang, Y. S.; Pang, B.; Hyun, D. C.; Yang, M.; Xia, Y. Engineered Nanoparticles for Drug



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Figure captions Figure 1. Illustrations of drug and gene loading and delivery process through BGN and SN. (A) Chemical structure and drug/gene delivery process of SN; (B) Chemical structure of BGN and their drug/gene delivery based on the calcium-organic group coordination mechanism. SN shows a complete Si-O-Si network structure, and BGN exhibits a defective Si-O-Si network structure due to the incorporation of Ca and P. Figure 2. Physicochemical structure of BGN and SN. (A–B) TEM images and microstructure of BGN (A) and SN (B); (C–D) Pore size distributions of SN (C) and BGN (D); (E) FTIR spectra presenting the chemical structure of BGN and SN; (E–F) EDS analysis exhibiting the chemical composition of BGN (F) and SN (G); (H) XRD patterns demonstrating the phase structure of samples. Figure 3. Intrinsic high drug loading capacity and controlled release evaluations for BGN. (A) UV-Vis spectra of DS solution before and after loaded by BGN and SN; (B) UV-Vis spectra of DS-loaded BGN and SN solution; (C) Loading efficiency (mg/g) of DS through BGN and SN; (D) Controlled release behavior of DS from BGN and SN; (E) UV-Vis spectra of 5-Fu solution before and after loaded by BGN and SN; (F) UV-Vis spectra of 5-Fu-loaded BGN and SN solution; (G) Loading efficiency (mg/g) of 5-Fu through BGN and SN; (H) Controlled release behavior of 5-Fu from BGN and SN; (I–J) Loading and release process of 5-Fu (I) and DS (J) in BGN. **P

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