Dual-Functionalized Graphene Oxide Based siRNA Delivery System

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Dual-Functionalized Graphene Oxide Based siRNA Delivery System for Implant Surface Bio-modification with Enhanced Osteogenesis Li Zhang, Qing Zhou, Wen Song, Kaimin Wu, Yumei Zhang, and Yimin Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12079 • Publication Date (Web): 19 Sep 2017 Downloaded from http://pubs.acs.org on September 20, 2017

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ACS Applied Materials & Interfaces

Dual-Functionalized Graphene Oxide Based siRNA Delivery System for Implant Surface Bio-modification with Enhanced Osteogenesis Li Zhang,†,# Qing Zhou, ‡,# Wen Song,† Kaimin Wu,§ Yumei Zhang†,* and Yimin Zhao†,*

†

The State Key Laboratory of Military Stomatology, National Clinical Research

Center for Oral Diseases, and Shaanxi Key Laboratory of Oral Diseases, Department of Prosthodontics, School of Stomatology, The Fourth Military Medical University, Xi’an 710032, China. ‡

Department of Pharmaceutical Analysis, School of Pharmacy, The Fourth Military

Medical University, Xi’an 710032, China. §

Department of Stomatology, 401 Military Hospital, Qingdao 266071,China.

ACS Paragon Plus Environment


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Abstract Surface functionalization by siRNA is a novel strategy for improved implant osseointegration. Gene delivery system with safety and high transfection activity is a crucial factor for siRNA-functionalized implant to exert its biological function. To this end, polyethylene glycol (PEG) and polyethylenimine (PEI) dual-functionalized graphene oxide (GO) (nGO-PEG-PEI) may present a promising siRNA vector. In this study, nano-sized nGO-PEG-PEI was prepared and optimized for siRNA delivery. Titania nanotubes (NT) fabricated by anodic oxidation were bio-modified with nGO-PEG-PEI/siRNA by cathodic electrodeposition, designated as NT-GPP/siRNA. NT-GPP/siRNA possessed benign cytocompatibility, as evaluated by cell adhesion and proliferation. Cellular uptake and knockdown efficiency of the NT-GPP/siRNA were assessed by MC3T3-E1 cells, which exhibited high siRNA delivery efficiency and sustained target gene silencing. Casein kinase-2 interacting protein-1 (Ckip-1) is a negative regulator of bone formation. siRNA targeting Ckip-1 (siCkip-1) was introduced to the implant and a series of in vitro and in vivo experiments were carried out to evaluate the osteogenic capacity of NT-GPP/siCkip-1. NT-GPP/siCkip-1 dramatically improved the in vitro osteogenic differentiation of MC3T3-E1 cells in terms of improved osteogenesis-related gene expression, and increased alkaline phosphatase (ALP) production, collagen secretion and extracellular matrix (ECM) mineralization. Moreover, NT-GPP/siCkip-1 led to apparently enhanced in vivo osseointegration, as indicated by histological staining and EDX line scanning. Collectively, these findings suggest that NT-GPP/siRNA represents a practicable and promising approach for implant functionalization, showing clinical potential for dental and orthopedic applications.

Keywords:

dual-functionalized

graphene

oxide,

siRNA

bio-modification, osteogenesis

1


delivery,

implant

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1. INTRODUCTION The metallic implant or scaffold is extensively used as biomedical materials in clinic, among which the titanium based implant is the mostly applied due to its unique capacity of osseointegration.1 However, in some pathological conditions, such as osteoporotic and diabetic patients, the osseointegration may be impaired due to the poor bone quality.2 In order to improve the implant bioactivity, the surface modification process is a common method. Titania nanotube (NT) is a widely studied surface modification technique to optimize the titanium implant for its excellent osteogenic performance. In addition, it could serve as an excellent platform for loading and delivering of a variety of bioactive agents.3,4 In recent years, the biofunctionalization of titanium implant surface with bioactive molecules has attracted favorable attention because that these molecules may directly participate in the osteogenesis around implant.5-7 The addition of biomolecules for implant decoration may help to reshape the interface between cells and implant, which is believed to be an effective method for faster and reliable osseointegration, especially in compromised clinical scenarios such as osteoporosis.8,9 The RNA interference (RNAi) is a powerful tool to modulate gene expression efficacy. By silencing some specific gene with small interfering RNA (siRNA), the cell functions may be manipulated conveniently. Consequently, the siRNA shows great therapeutic potential with intrinsic biological response, great targeting ability and high specificity.10 In fact, the siRNAs have been investigated by us and other researchers as targeted therapeutics biomolecules to facilitate the bone regeneration on implant surface.11-13 Delivery system is a key parameter for siRNA biofunctionalized implant to exert its function. It is well known that the naked siRNA molecules have to overcome various barriers, including rapid excretion by kidney, easily degradation by enzymes, inefficient cellular uptake and difficulty in escaping from endosomes/lysosomes.14 Therefore, the effective and safe siRNA delivery systems are highly required and always in pursue. Nanoparticles have recently emerged as versatile platforms with appealing properties for drug/gene delivery applications.15-17 They can be modified by various organic and/or inorganic molecules, 2



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resulting in functionalized nanoparticles with superior drug/gene protecting, delivery and targeting efficiency. Graphene oxide (GO) has generated great interest in biomedical applications due to its unique electrical, thermal, and mechanical properties. The presence of epoxides, carbonyls and hydroxyls on the surface of GO enables the facile chemical and biological modification, obtaining GO derivatives as delivery systems for biomolecules such as proteins, drugs and nucleic acids.18 Owing to their unique characteristics including capability to condense genetic material, protection of nucleic acid from enzymatic degradation and efficient cellular internalization, Go derivatives for plasmid DNA (pDNA) and siRNA delivery have recently become an attractive research field.19 Among various cationic polymers that have been investigated to interact with GO for pDNA /siRNA delivery, polyethylenimine (PEI) was the most widely studied one. Reported researches have revealed that PEI based GO derivatives prepared via covalent interaction exhibited high gene transfection efficiency and biocompatibility.20-22 However, these PEI based GO nanoparticles were mainly used as gene carriers for cancer cells previously, and studies focusing on these nanoparticles for siRNA delivery to osteoblasts, especially on material surface have not been reported to the best of our knowledge. Another important parameter for siRNA biomodification of implant is how to immobilize transfection complex onto implant surface. In our previous study, the thermo-alkali treatment was used to obtain superhydrophilic and mesoporous surface and the siRNA complex was directly absorbed.13 In order to control the siRNA loading profiles, the layer-by-layer self-assembly technique is the most commonly used, which can control siRNA loading amount by accumulating different layers.23, 24 However, these methods are either uncontrollable or less efficient, and may not suitable for large-scale production. The cathodic electrodeposition (CED) technique is a mature surface coating method to formulate homogeneous layer on metal surface, in the manners of highly efficient, loading controllable and economic.25-27 Moreover, it has also been tried to immobilize siRNA complex onto implant surface.28 Therefore, the CED technique may also be suitable for GO based siRNA complex loading. 3


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Here, a dual-modified GO-based nanovector was prepared for efficient siRNA delivery on implant surface (Scheme 1). GO was first covalently conjugated with polyethylene glycol (PEG) and polyethyleneimine (PEI) to prepare the nGO-PEG-PEI which can be used as a promising carrier of siRNA. Titania nanotubes (NT) samples were fabricated via anodization and acted as a platform for implant biomodification. After optimizing the related parameters, the nGO-PEG-PEI condensed siRNA complex was deposited onto the pre-prepared NT surface

via cathodic

electrodeposition, obtaining the nGO-PEG-PEI/siRNA biofunctionalized implant as NT-GPP/siRNA. NT-GPP/siRNA with high biocompatibility and siRNA delivery activity could induce sustained and efficient gene silencing. Furthermore, NT-GPP/siRNA with siCkip-1 exhibited significantly promoted in vitro osteogenic differentiation and in vivo osseointegration, implying the potential of NT-GPP/siRNA for implant surface biomodification. 2. EXPERIMENTAL SECTION 2.1. Materials Commercial pure Ti foils (10× 10× 1 mm3) and Ti rods (ø1mm×2 mm) were provided by Northwest Institute for Nonferrous Metal Research (China). Branched PEI, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were purchased from Sigma–Aldrich (USA). Amine-terminated six-armed PEG (10 kDa) was purchased from Sun-bio (Korea). Fetal bovine serum (FBS), penicillin/streptomycin, α minimum essential

medium

(α-MEM),

phosphate

buffered

saline

(PBS),

4′6-diamidino-2-phenylindole (DAPI) and Lipofectamine®2000 were purchased from Invitrogen (USA). The real-time polymerase chain reaction (real-time PCR) primer were bought from Sangon Biotech Co., LTD (China). Sirius red, saturated picric acid and

alizarin

red,

paraformaldehyde,

β-glycerophosphate,

ascorbic

acid,

dexamethasone and pelltobarbitalum natricum were obtained from Sigma (USA). Nine-week-old female C57BL mice were purchased from Animal Center of The Fourth Military Medical University. PrimeScript RT reagent kit and SYBR Premix Ex Taq™ II were obtained from TaKaRa (Japan). Cell counting kit-8 (CCK-8) and 4



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BCIP/NBT ALP color development kit were bought from Beyotime (China). SiCkip-1 (sense:

5′-CUACUCGAGACAGAGCAAATT-3′,

antisense:

5′-UUUGCUCUGUCUCGAGUAGTT′) labeled with FAM or not, negative control siRNA duplex (siNC, sense: 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense: 5′-ACGUGACACGUUCGGAGAATT′) labeled with Cy3 or not and siRNA duplex targeting

enhanced

green

fluorescent

protein

(EGFP)

5′-UCAAGGAGGACGGCAACAUTT-3′,

(siGFP,

sense:

antisense:

5′-AUGUUGCCGUCCUCCUUGATT-3′) were synthesized by GenePharma Co., LTD (China). 2.2. Preparation and Characterization of nGO-PEG-PEI. nGO-PEG-PEI was synthesized according to previously reported method.29 Briefly, GO was prepared using modified Hummers method first. For preparation of nGO-PEG conjugate, GO solution (0.5 mg/mL) was mixed with 6-armed amine-terminated PEG (0.5 mg/mL) and sonicated for 10 min. EDC (1.0 mg/mL) was then added following 10 min sonication and 2 h stirring respectively. After that, nGO-PEG conjugate was sonicated with PEI (2.5 mg/mL) for 10 min. EDC was added again under sonication and the mixture was stirred at room temperature for 12 h. The obtained mixture was washed with deionized water by 100 nm Milli-Q membrane filter for several times, obtaining nGO-PEG-PEI aqueous solution. The concentration of nGO-PEG-PEI were calculated by UV-Vis spectrometer (PerkinElmer) at absorbance of 230 nm. The nGO-PEG and nGO-PEG-PEI conjugates were also confirmed by atomic force microscope (AFM, Agilent 5500), transmission electron microscope (TEM, Tecnai Spirit, FEI Co) and FTIR spectrometer (IRAffinity-1, Shimadzu). The hydrodynamic size and the zeta potential of GO, GO-PEG and nGO-PEG-PEI were examined by dynamic light scattering (DLS) using a Malvern zeta sizer (Malvern Instrumentation Co). The element content of nGO-PEG and nGO-PEG-PEI was measured by vario Micro cube (Elementar). The PEG and PEI content of nGO-PEG-PEI were estimated by elemental and thermo-gravimetric analysis (TGA, USA) respectively. The solubility of GO, nGO-PEG and nGO-PEG-PEI conjugates dissolved in PBS, α-MEM and FBS was analyzed after 5


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centrifugation with a speed of 5000 rmp for 5 min. 2.3. The nGO-PEG-PEI/siRNA Complexes preparation and validation Assigned amounts of nGO-PEG-PEI conjugate were mixed with 20 µmol siRNA (1 OD siRNA dissolved in 125 µL RNA-release water) at different N/P ratios (5, 10, 20, 40 and 80) and incubated at room temperature for 20 min to form nGO-PEG-PEI/siRNA complexes. After that, the as-prepared complexes were analyzed by 0.8% agarose gel electrophoresis running in Tris-EDTA (TE) buffer. The gel was imaged by a gel imaging analysis system (Peiqing, Shanghai, China). The murine osteoblast cell line MC3T3-E1 (ATCC, USA) was cultured in α-MEM supplemented with 10% FBS, streptomycin (100 U/mL) and penicillin (100 U/mL) at 37 °C in a humidified atmosphere containing 5% CO2. To determine the cell viabilities, the MC3T3-E1 cells were seeded in a 24-well plate. After one day culturing, nGO-PEG-PEI/siRNA of indicated N/P ratios were added to the cell culture, and cell viability was evaluated by a cell counting kit-8 assay at 1 and 3 days after incubation. Cells culturing without nGO-PEG-PEI/siRNA was used as the control group. For siRNA transfection, cells were plated at the density of 20, 000 cells/cm2 in a 24-well plate one day prior to the transfection. On the transfection day, the complexes with FAM labeled siRNA at different N/P ratios at a concentration of 200 nM siRNA/well were incubated with MC3T3-E1 cells for 6 h. Cells incubated with only nGO-PEG-PEI was named NO siRNA group and the lipofectamine 2000 with siRNA (lipo2000/siRNA) was used as positive vector control. Afterwards, cells were washed with PBS three times, fixed with 4% formaldehyde and imaged by a confocal laser scanning microscope (CLSM, Olympus, FV1200). In the meanwhile, the cells were collected to run the flow cytometry (FACScalibur, BD Biosciences) to determine the FAM fluorescence. For further comparison of the transfection efficiency, siRNA targeting Ckip-1(siCkip-1) was applied and the mRNA level of Ckip-1 was assessed by real-time PCR 48 h after transfection. Lipofectamine 2000 with siRNA (lipo2000/siRNA) was used as positive vector control and siRNA was transfected according to the manufacturer’s protocols. 6



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2.4. Fabrication of NT Titania nanotubes arrays were prepared by anodization as previously reported.30 Ti samples were wet polished with the SiC paper to mesh 3000 (designated as PT), followed by ultrasonic cleaning sequentially in acetone, absolute ethanol and deionized water and dried at 37 °C. After that, PT specimen was anodized in 0.5 wt% HF at 20 V for 1 h using a DC power supply and then ultrasonically washed with acetone, absolute ethanol and deionized water thoroughly. Finally, the specimens were dried at 37 °C and sterilized by Cobalt 60 irradiation, obtaining NT. 2.5. Cathodic electrodeposition of nGO-PEG-PEI/siRNA Complexes The nGO-PEG-PEI/siRNA solution was firstly ultrasonicated to form a homogeneous

nGO-PEG-PEI/siRNA suspension

solution.

For

the

cathodic

electrodeposition, nGO-PEG-PEI/siRNA solution was used as the electrolyte, NT specimens were connected to the cathode as the working electrode, and platinum dices were connected to the anode as the counter electrode. The applied voltage ranged from 1 V to 20 V, and the distance of the electrode was 0.8 cm. After 1 min of electrodeposition, specimens were rinsed in deionized water and dried at 4 °C, obtaining

nGO-PEG-PEI

(GPP)/siRNA

modified

NT

implant,

named

NT-GPP/siRNA. 2.6. The siRNA loading and release profiles Cy3 labeled siRNA (Cy3-siRNA) was used to formulate the NT-GPP/siRNA samples under different voltages. After the electrodeposition, the specimens were observed with a fluorescence microscopy (OLYMPUS, IX70) to evaluate the siRNA loading efficiency. The Cy3 fluorescence intensity of the solution before and after the electrodeposition was measured by a spectral scanning multimode reader (Varioskan Flash, Thermo Scientific) to quantify the siRNA deposited on the implant surface. To analyze the release behavior of NT-GPP/siRNA, samples with Cy3 labeled siRNA were immersed in 1 mL phosphate buffered saline (PBS) at pH 7.4 and kept in an incubator at 37 °C. At predetermined time slots, the samples were taken out for images by a fluorescence microscopy, then the supernatant was collected and replaced with new PBS. For quantitatively measurement of siRNA release, the fluorescence 7


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intensity of the collected supernatant was read by a spectral scanning multimode reader. 2.7. Surface characterization of NT-GPP/siRNA The morphology of PT, NT and NT-GPP/siRNA was observed by a field-emission scanning electron microscopy (FE-SEM, HITACHI S-4800). 3D reconstruction images of the samples and surface roughness average (Sa) were further characterized by tapping mode AFM. The surface wettability of the samples was evaluated by contact angle measurement. Briefly, deionized water drop on the samples surface, then images of the droplet were obtained by an imaging analysis microscope (Camscope, Sometech Inc.) 5 seconds after contacting, and the contact angle was quantitatively measured by DSA1 software (KRUSS). 2.8. Cell morphology and actin cytoskeleton arrangement The MC3T3-E1 cells were seeded on the Ti samples with different surfaces at the density of 20,000 cells/cm2. After culturing for 24 h, the cells were rinsed in PBS and fixed in 2.5% glutaraldehyde overnight. After dehydrated sequentially in 50%, 70%, 80%, 90%, and 100% ethanol, the substrates were dried at 37 °C and sputter-coated with gold, and then cell morphology was observed by FE-SEM. For actin cytoskeleton arrangement, the cells were fixed with 4% paraformaldehyde and then rinsed gently by PBS. The actin cytoskeleton was stained in dark by 150 µg/mL rhodamine-phalloidin in PBS for 40 min, and nuclei was then stained with 4,6-diamidino-2-phenylindole (DAPI) for 15 min. Finally, the samples were observed by CLSM and the images were captured. 2.9. Implant cytotoxicity evaluation The cells were seeded on the samples at a density of 20,000 cells/cm2. At 1, 3 and 7 days, the cell viability was evaluated by a cell counting kit-8 (CCK-8) assay. Briefly, the substrates were transferred to a new plate and the cells were rinsed slightly with PBS. Solution mixed with 360 µL medium and 40 µL CCK-8 was added to each well and incubated for 2 h at 37 °C. Then, the supernatant was collected to determine the absorbance at 450 nm using a spectrophotometer (Bio-Tek). 2.10. Cell uptake from NT-GPP/siRNA surface 8



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MC3T3-E1 cells were seeded on NT-GPP/siRNA samples with FAM labeled siRNA (FAM-siRNA) at a density of 20,000 cells/cm2. After culturing for 24 h, the cells were rinsed in PBS and fixed with 4% formaldehyde for 20 min. Lipofectamine 2000 with siRNA (lipo2000/siRNA) was transfected according to the manufacturer’s protocols as a control. The actin cytoskeleton and nucleus were labeled as described above. Then the samples were examined by CLSM. 2.11. Knockdown efficiency of NT-GPP/siRNA The GFP+-MC3T3-E1 cells were prepared to assess the knockdown efficiency of the NT-GPP/siRNA samples. Lentiviral plasmids encoding GFP was designed and produced by Genechem (Shanghai, China). MC3T3-E1 cells were plated in 6-well plates and incubated overnight. Then, the culture medium was replaced with transduction enhancing solution containing lentivirus with a multiplicity of infection (MOI) of 80 and 50 µg/ml polybrene. After 12 h, the medium was replaced with complete medium, and the cells were cultured for 72 h, obtained GFP+-MC3T3-E1 cells. Then, the cells were cultured on the NT-GPP/siRNA surfaces functionalized with the siRNA targeting GFP and NT-GPP/siNC with non-targeted siRNA as control. GFP expression of the cells was observed by inverted fluorescence microscope (OLYMPUS, IX70) at the green channel 2 days post cell culture. To assess the silencing efficiency of the NT-GPP/siCkip-1 samples on the target Ckip-1mRNA level. The Ckip-1mRNA expression of NT-GPP/siCkip-1 and NT-GPP/siNC was evaluated by real-time PCR 3 and 7 days post-seeding. Briefly, cells were lysed with Trizol reagent (Invitrogen, California, USA) and total RNA was extracted. Then RNA was reverse transcribed to complementary DNA (cDNA) by a PrimeScript RT reagent kit (TaKaRa) and a specific RT primer (Ribobio). The qRT-PCR analysis was performed on the CFX96™ Real Time RT-PCR System with SYBR PremixEx Taq™ II (TaKaRa). The expression of Ckip-1 was analyzed and normalized to that of the housekeeping gene GAPDH. 2.12. In vitro osteogenesis of MC3T3-E1 cells MC3T3-E1 cells were shifted to and cultured in the osteogenic medium (a-MEM supplemented with 10% FBS, 50 mg/mL Vc (Sigma), 0.1 mM dexamethasone, and 10 9


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mM b-glycerophosphate) 2 days post-seeding. At pre-set time points, the alkaline phosphatase (ALP) production, collagen secretion, extracellular matrix (ECM) mineralization as well as osteogenesis-related gene (ALP, COl-1, BMP2 and Runx2) expression on the samples were evaluated. 2.12.1. ALP production, collagen secretion and ECM mineralization After 7 days of osteogenic induction, the samples with cells were washed with PBS 3 times and fixed with 4% paraformaldehyde. The ALP production was stained by the BCIP/NBT ALP color development kit and the images were obtained by a stereomicroscopy. To quantitatively analysis the ALP level on the substrates, alkaline phosphatase colorimetric assay kit (BioVision, USA) was used. Collagen secretion was assessed 14 days after the osteogenic induction, the samples were washed with PBS, fixed with 4% paraformaldehyde and stained with 0.1 wt% Sirius red (sigma) in saturated picric acid for overnight. Images was taken after unbound stain was washed with 0.1 M acetic acid. Then, the stain was dissolved by 0.2 M NaOH/methanol (1:1) and the absorbance at 540 nm was measured. ECM mineralization analysis was carried out after 21 days of incubation in the osteogenic medium. The specimens were fixed and then stained using 0.1% Alizarin Red S solution (pH 4.2) for 10 min to display the Calcium nodule. 10% cetylpyridinium chloride and 10 mM sodium phosphate mixed solution was used to dissolve the stain and quantify the nodules, and then absorbance values at 620 nm were determined by a spectrophotometer. 2.12.2. Osteogenesis related gene expression Real Time RT-PCR was carried out for the evaluation of osteogenic gene expression including ALP, type I Collagen (Col-1), bone morphogenetic protein 2 (BMP2) and runtrelated transcription factor-2 (Runx2) after 7 days culturing in the osteogenic medium. The purified gene specific primers of the genes were synthesized and listed in Table S1. 2.13. In vivo osseointegration 2.13.1. Implant surgery The animal experiment was approved by the Animal Research Committee of the 10



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Fourth Military Medical University (No.2016-kq-012) and conducted in accordance with the international standards on animal welfare. The C57BL/6J mice were randomly divided into 5 groups, and each group contained 5 animals. The femur model was applied for implantation in this study and the procedure of the surgery was similar to that described before.31 Briefly, before the surgery, the mice were anesthetized by pelltobarbitalum natricum. After incision of the skin and gently dissection of the muscle, the distal femurs were exposed. Then, a cylindrical implant hole with 1 mm diameter were drilled with cooling by saline solution. Finally, the prepared Ti samples were randomly pressed into the cavity and the muscle tissue and skin was sutured gently. 2.13.2. Van-Gieson staining Van-Gieson staining was applied for the histomorphometric observation 1 month after implantation. The femurs with implants in were harvested, fixed with 4% paraformaldehyde,

dehydrated

with

graded

alcohol

and

embedded

in

methylmethacrylate. Afterwards, the embedded femurs were cut by a macro-cutting and grinding system (SP1600&SP2600, Leica) and sections parallel to the long axis of the implants with about 120 µm-thick were obtained. All sections were then ground, and stained with mixed solution of 1% acid fuchsine and 0.5% saturated picric acid. Finally, the sections were observed by stereomicroscopy and images were taken. 2.13.3. Line-scanning of the bone-to-implant interface Line-scanning by energy dispersive X-ray spectroscopy (EDX, Hitachi) was applied for more detailed analysis of the bone-to-implant interface. After fixed with 4% paraformaldehyde,

dehydrated

with

graded

alcohol

and

embedded

in

methylmethacrylate, the bone-to-implant interfaces of the embedded samples were scanned by SEM and the line-profiles of C, O, Ca, P and Ti elements from the implant side to medullary cavity side were analyzed by EDX. 2.14. Statistical analysis Data were analyzed by SPSS 19.0 software (SPSS, USA). All data were presented as mean±standard deviation (SD) from at least three independent experiments and analyzed by one-way ANOVA combined with Student-Newmane-Keuls post hoc test. 11


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P< 0.05 was considered statistically significant.

Scheme 1. Schematic illustration of the formation of nGO-PEG-PEI/siRNA, cathodic electrodeposition of nGO-PEG-PEI/siRNA on NT, and the in vitro and in vivo osteogenesis.

3. RESULTS AND DISCUSSION 3.1. Synthesis and characterization of nGO-PEG-PEI nanoparticles GO was fabricated and covalently conjugated with hydrophilic PEG polymer and the positively charged polymer PEI via amide bond at a weight ratio of GO:PEG:PEI = 1:1:5, obtaining nGO-PEG-PEI. The successful synthesis of nGO-PEG-PEI was evidenced by a series of experiments. The AFM and TEM images (Figure 1A) showed that compared to GO, nGO-PEG and nGO-PEG-PEI exhibited apparently increased sheet thickness and decreased sheet sizes. The hydrodynamic diameters of GO, nGO-PEG and nGO-PEG-PEI were assessed by dynamic light scattering (DLS) measurement (Figure 1B), being about 561.8 nm, 46.3 nm and 53.9 nm respectively. 12



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The polydispersity index (PI) of GO, nGO-PEG and nGO-PEG-PEI were 0.224, 0.164 and 0.126 respectively. The significantly decreased size of nGO-PEG and nGO-PEG-PEI may mainly result from the lacerating effect of multi-ultrasonic reaction. In addition, curl and fold of GO during the modification process increase the thickness of the sheets, which may also contribute to the size decrease. Surface charge of gene carrier directly affect its interaction with negatively charged siRNA.32 Therefore, the zeta potential was measured as shown in Figure 1C. GO possessed highly negatively charge (-40.2 mV) due to large amount of carboxyl and hydroxyl groups on the surface. After conjugation with PEG and PEI that are rich in amino groups by amide linkage, the zeta potential dramatically increased, resulting in positive charged surface of nGO-PEG-PEI (30.5 mV). The high positive charge of nGO-PEG-PEI enables the noncovalently conjugate to negatively siRNA via electrostatic interactions. In addition, the element content analysis by vario Micro cube (Figure 1D) showed an obvious increase of N element from 0.84% of GO-PEG to 12.84% of nGO-PEG-PEI, indicating the abundant binding of PEI. PEI content in nGO-PEG-PEI conjugate was evaluated by elemental analysis of nitrogen, being about 36.9%. The PEG content was then assessed by the thermo-gravimetric analysis (TGA) and was estimated to be about 15.7% (Figure S1). Furthermore, IR spectrum (Figure S2) showed an obvious C-H vibration band (2800 cm-1) in nGO-PEG-PEI, all demonstrating the successful synthesis of nGO-PEG-PEI conjugate. Stability of nanoparticles in physiological conditions is crucial for in vivo application. Therefore, the fabricated nanoparticles were incubated with PBS, ɑ-MEM and FBS to study their stability (Figure S3). GO showed poor solubility owing to the protein adsorption effect, aggregated in all the solutions after centrifuged at 5000 rmp for 5 min. However, the nGO-PEG and nGO-PEG-PEI complexes were well dispersed in these solutions. Since PEG is a kind of hydrophilic polymer that could prevent non-specific binding of proteins and improve the biocompatibility of nanoparticles,

33

the improved solubility of GO-PEG and GO-PEG-PEI could be

attributed to the conjugation of PEG. 13


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Figure 1. Characterization of nGO-PEG-PEI. (A) AFM and TEM images of GO, nGO-PEG, and nGO-PEG-PEI. (B) Dynamic light scattering (DLS) measured size of GO, nGO-PEG, and nGO-PEG-PEI. (C)Zeta potential of GO, nGO-PEG, and nGO-PEG-PEI in water. (D) The percentage of C, H and N element of nGO-PEG and nGO-PEG-PEI assessed by vario Micro cube.

3.2. Optimization of nGO-PEG-PEI nanoparticles for siRNA delivery The siRNA binding ability of synthesized nGO-PEG-PEI was evaluated by the agarose gel electrophoresis assay with different N/P ratios. As shown in Figure 2A, the band of unbound siRNA could only be found when the N/P ratio is below 10:1, indicating that nGO-PEG-PEI could completely bind siRNA via electrostatic interaction at N/P ratios above 10. To evaluate the biocompatibility of nGO-PEG-PEI at different N/P ratios, relative viabilities of MC3T3-E1 cells were determined by CCK-8 assay after culturing with nGO-PEG-PEI for 1 day and 3 days (Figure 2B). No apparent difference of viabilities was observed among N/P 20, 30 and 40 groups at 14



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both 1 day and 3 days, whereas the N/P 50 group showed obvious decreased cell viabilities at both time slots. This indicates that the N/P ratio less than 50 may be safe for cell transfection. PEI is an extensively studied gene vector, however high cytotoxicity, especially at high molecular weight and high N/P ratios compromised its application.34 Therefore, the decreased biocompatibility of N/P 50 may originate from the increased concentration and cell toxicity of PEI in nGO-PEG-PEI. Next, the delivery efficiency of nGO-PEG-PEI for siRNA transfection at N/P ratio 20, 30 and 40 was tested using MC3T3-E1 cells under serum-free conditions. The CLSM images (Figure 2C) of intracellular FAM-siRNA showed that the siRNA uptake was increased with the N/P ratios. To quantitatively determine the delivery efficiency, flow cytometry analysis on the FAM signals was conducted (Figure 2D). In consistence with the CLSM results, the ratios of FAM cells increased significantly from about 30% to nearly 100% with the N/P ratio from 20 to 40. In addition, it was notable that there was no significant difference of siRNA transfection between N/P 40 group and lipofectamine 2000 group. Consequently, nGO-PEG-PEI/siRNA at N/P ratio of 40 would be the most optimal condition for siRNA delivery and was chosen for following implant deposition. Afterwards, the intracellular level of mRNA was further measured 48 h after the nGO-PEG-PEI/siRNA transfection with Ckip-1siRNA (Figure S4). The intracellular Ckip-1 amount decreased from 73% to about 28% with the increase of N/P ratio. The N/P 40 group generated the best knockdown effect which

was

similar

to

that

of

lipo2000/siRNA

group.

Consequently,

nGO-PEG-PEI/siRNA at N/P ratio of 40 would be the most optimal condition for siRNA delivery and was chosen for following implant deposition. In fact, there is often a balance between the carriers and siRNA, which is indicated by appropriate N/P ratio.35, 36 In order to condense siRNA to protect and deliver into cells, the carrier amount must be enough. However, excess carrier often leads to cytotoxicity. Therefore, the optimal N/P ratio must give consideration to both efficient siRNA delivery and cell viability. From our observation, the siRNA binding ability and delivery efficiency were increased with carrier amount, which is similar to other cationic delivery systems.15,37 This may because that with increase of carrier, the 15


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positive charge density is also enhanced, which is beneficial for siRNA interaction as well as getting through the negatively charged cell membrane. However, there might be a threshold that when siRNA binding is saturated, the excess carrier may result in cell apoptosis.

Figure 2. siRNA transfection of nGO-PEG-PEI. (A) Agarose gel retardation study of nGO-PEG-PEI/siRNA mixtures at indicated N/P ratios. (B) Relative cell viability data of MC3T3-E1 cells treated by nGO-PEG-PEI/siRNA at indicated N/P ratios for 24 h as determined by CCK-8 assay. Cells without nGO-PEG-PEI/siRNA were used as control. *p NT-GPP/siNC = NT-GPP> NT>PT. Additionally,

the

osteogenic

differentiation

was

also

determined

by

osteogenesis-related genes expression (Figure 7B). Relative expression of ALP, COl-1, BMP2 and Runx2 were assessed by RT-PCR after 7 days incubation in osteogenic medium. The overall trend of gene expression was in line with the osteogenic staining. NT-GPP /siCkip-1 gave rise to dramatically higher expression for all these osteogenesis related genes. A proper gene silencing target is crucial for satisfactory osteogenic activity for NT-GPP/siRNA. Based on previous researches, we choose casein kinase 2-interacting protein 1 (CKIP-1), which is a negative regulator for bone formation by upgrading the E3 ligase activity of Smurf1, and inhibiting the bone morphogenetic protein (BMP) signaling pathway and the differentiation of osteoblasts. Zhang et al53,54 have done many work about Ckip-1siRNA, and they revealed that knockdown of Ckip-1 by siRNA dramatically promoted in vitro osteogenic differentiation and in vivo osseointegration in both healthy and osteoporotic condition. In addition, in our previous work, siCkip-1 biofunctionalized implant was developed and exhibited notably enhanced implant performance.13,28 All these previous research, together with our osteogenic staining and PCR results indicate that siCkip-1 is a promising choice for implant surface bio-modification. It is interesting from the above results that while no apparently difference in cell proliferation was observed, NT-GPP /siNC and NT-GPP generated increased osteogenic differentiation than NT, suggesting the osteogenic potential of nGO-PEG-PEI. GO-based materials receive a great share of interest for promoting the osteogenic differentiation of cells.55 It is hypothesized that the ability of GO to 25


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promote the binding of osteogenic enhancing molecules/proteins on material surface via π–π interactions with the carbon lattice, or through electrostatic interactions with functional groups on GO, may contribute to its ostogenic properties. However, the osteogenic activity of nGO-PEG-PEI has not been reported. The unique physical properties of the implant surface may be one factor for the enhanced osteogenic differentiation. Compared to NT, nGO-PEG-PEI modified samples exhibited increased surface roughness and hydrophobicity, which enhance the interactions with specific biological molecules and the following osteogenic activity. Moreover, surface chemistry

modulates

osteoblastic

differentiation

and

matrix

mineralization

independently from alterations in cell proliferation. Amine group (-NH2) functionalized on biomaterial can induce favorable protein adsorption and up-regulate osteoblast-specific gene expression, ALP activity, and matrix mineralization.56,57 Hence, abundant NH2 groups of nGO-PEG-PEI may be another factor for the enhanced osteogenic differentiation. However, further studies are required to verify our hypothesis and clarify the osteogenic mechanism in the future.

Figure 7. Osteogenic differentiation on NT-GPP/siCkip-1. (A) Staining and 26



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quantification of ALP production (a), collagen secretion(b), and ECM mineralization (c) on the different Ti samples after culturing for 7, 14, and 21 days. (B) The mRNA expression level of the osteogenesis-related genes ALP, Col-1, BMP-2 and Runx2 on different samples determined by real time PCR 7 days after incubation. Data represent the mean expression relative to that of GAPDH. The experiments were repeated independently at least 3 times. **and ***p< 0.01 and 0.001 vs PT. ## and ###p< 0.01 and 0.001 vs NT. &&&p< 0.001 vs NT-GPP. ^^^p< 0.001 vs NT-GPP/siNC.

3.8. In vivo Osseointegration of NT-GPP/siCkip-1 Osseointegration of an implant could be evaluated by various methods including histological observations and EDX line scanning. To assess the in vivo osseointegration, the samples were implanted into the mouse femurs and allowed for 1 month healing. Then the femurs with implants were harvested to conduct a series of tests (Figure 8A). Van Gieson staining is a central hoistological test for examining new bone formation and the bone-implant contact. As shown in Figure 8B(a), new bone structure on the implant surface were stained into red. Compared to PT group with thin and discontinuous new bone, NT induced better bone formation, which was further enhanced on NT-GPP, NT-GPP/siNC and NT-GPP/siCkip-1 surfaces. NT-GPP/siCkip-1 generated the highest bone volume and bone continuity than the other groups. Quantitative measurement of bone-to-implant contact (BIC) of the samples (Figure 8B (b)) showed the similar trend with the above results. The percent of bone-to-implant contact increased from PT about 36% to NT-GPP /siCkip-1 about 96%. For more detailed information of the bone-to-implant interface, the samples were subjected to cross-section for SEM observation (Figure 8C(a)) and elements including C, O, Ca, P and Ti across the interface were analyzed by EDX line scanning in order to distinguish new bone formation (Figure 8C(b)). The formulated new bone thickness on NT-GPP/siCkip-1 surface was significantly improved. The thickness of new bone rich in Ca and P was measured to be about 30 µm, 52 µm, 72 µm, 78 µm and 104 µm for PT, NT, NT-GPP, NT-GPP/siNC and NT-GPP/siCkip-1 respectively. It can also be 27


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observed from the Van Gieson staining and EDX line scanning images that the bonding between new bone and the NT-GPP/siCkip-1 implant surface is directly and tightly with no fibrous or other tissue could be found. All the above results indicate the satisfactory osseointegration of NT-GPP/siCkip-1.

Figure 8. In vivo osseointegration of NT-GPP/siCkip-1. (A) Schematic illustration of the implant process and in vivo osseointegration evaluation. (a) An implant hole with 1 mm diameter was drilled in the distal femur. (b) The as-prepared Ti samples were randomly pressed into the implant cavity. (c) New bone formed around the implants (red) 1 month after the implantation. (d) Osseointegration evaluated by Van-Gieson staining and EDX line scanning of the bone-to-implant area (blue frame). (B) VG staining of the hard tissue sections after 1 month of implantation (a) and the corresponding analysis of BIC (bone-to-implant contact) from the histomorphometric measurements (b). New bone on implant surface was stained into red. ***p

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