Characteristics of functionalized nano-hydroxyapatite and internalization by human epithelial cell
- Zhao Yan-zhong†1, 2, 3,
- Huang Yan-yan†1,
- Zhu Jun2,
- Zhu Shai-hong1, 3Email author,
- Li Zhi-you2 and
- Zhou Ke-chao2, 3Email author
© Yan-zhong et al; licensee Springer. 2011
Received: 13 June 2011
Accepted: 23 November 2011
Published: 23 November 2011
Hydroxyapatite is the main inorganic component of biological bone and tooth enamel, and synthetic hydroxyapatite has been widely used as biomaterials. In this study, a facile method has been developed for the fabrication of arginine-functionalized and europium-doped hydroxyapatite nanoparticles (Arg-Eu-HAP). The synthesized nanoparticles characterized by transmission electron microscopy, X-ray diffractometry, Fourier transform infrared, and Zeta potential analyzer. Its biological properties with DNA binding, cell toxicity, cell binding and intracellular distribution were tested by agarose gel electrophoresis assay, flow cytometry, and fluorescence microscope and laser scanning confocal microscope. The synthesized Arg-Eu-HAP could effectively bind DNA without any cytotoxicity and be internalized into the cytoplasm and perinuclear of human lung epithelial cells.
To date, one of the main barriers for gene therapy to achieve substantial breakthrough is probably due to the lack of high efficacy and safe gene delivery vector. The death of several clinical trials with viral-based gene delivery systems, especially the one using a retrovirus system, leads to more concerns for the future of gene therapy. The US Food and Drug Administration had suspended gene therapy trials [1, 2]. In recent years, some nonviral-based gene delivery systems, such as functional cationic polymers [3–5] and nano-carriers [6–8], circumvent some of the problems occurring with viral vectors such as endogeneous virus recombination, oncogenic effects, and unexpected immune response, but their gene transfer efficiency is inferior to viral vectors. In addition, the cytotoxicity of cationic polymers is an essential problem in the polyplex-based gene transfer field. Therefore, to develop a novel gene delivery system with safe, non/low-toxic, non-immunogenicity, and easy-assemblage has recently received intensive attention.
Among nanoparticles with different materials composition, inorganic nanoparticles composed of calcium phosphate have numerous advantages including ease of synthesis, control of physicochemical properties, strong interactions with their payload, and biocompatibility. As the main inorganic component of biological bone and tooth enamel, hydroxyapatite shows excellent biocompatibility and bioactivity [9, 10]. It has been widely used as an implant biomedical material in orthopedic and dental treatments [11, 12]. Moreover, hydroxyapatite nanoparticles (HAP) are low crystalline with highly active surfaces and used as carrier in drug delivery systems as well as for protein separation as an absorptive material [13, 14]. Interestingly, HAP can inhibit some cancer cells growth . Our previous study reported  that HAP-incorporating pEGFP-N1 are able to deliver DNA into gastric cancer cells without any significant cytotoxicity, which transfer efficiency of is equal to 50% of liposome's under the equivalent conditions. Tan  discovered that after being modified by protamine, gene transfer efficiency of HAP can be enhanced more times. Sun  successfully used HAP to delivery NT-3 gene into the cochlear neurons of guinea pig both in vitro and in vivo. The demonstrating HAP may be a potential effective and safe material as a gene delivery agent. However, the low gene transfer efficiency limits their applications.
Nanoparticles with well-defined inner and outer surfaces that can be easily functionalized for biological application have attracted intensive attention recently in biotechnological studies [19, 20]. To optimize the efficacy in gene delivery, the authors conjugated the hydrophilic arginine with a guanidyl group onto the surface of HAP in a previous study . The result demonstrated that arginine-modified HAP had good biocompatibility and gene binding property. Meanwhile, some research revealed that arginine with guanidyl group can facilitate the cellular uptake of nanoparticles , but the mechanism of their uptake is disputed . These physicochemical properties of HAP that provide for intracellular penetration of drug molecules have great importance for gene delivery.
In this article, the authors report a facile method for the fabrication of arginine-functionalized and europium-doped hydroxyapatite nanoparticles (Arg-Eu-HAP). Almost nontoxic and more stable inorganic europium is selected as fluorescent bioimaging probes [24–27]. Europium doping was performed to enable photoluminescence of HAP. The characterization of physicochemical and photoluminescence properties of Arg-Eu-HAP were examined. Preliminary studies on gene binding, cell toxicity, and cell uptaking in vitro were carried out. The results suggest that Arg-Eu-HAP with unique biological properties make them suitable for the next research as a gene delivery agent.
Materials and methods
Calcium nitrate, ammonium phosphate, arginine (Sigma Corporation, St. Louis, MO, USA), pEGFP-N1 plasmid (Wuhan Genesil Biotechnology Co., Ltd., Wuhan, China) and other materials were used in this research. All reagents were of the highest analytical grade available. Cell culture media, fetal bovine serum, was obtained from American Type Culture Collection (Rockville, Maryland, USA). Ham's F-12 medium with L-glutamine was purchased from Fisher Scientific (Logan, UT, USA). Trypsin-EDTA (×1) and Hank's balanced salt solution were purchased from Invitrogen (Carlsbad, CA, USA). Phosphate buffer salt solution (PBS) and penicillin-streptomycin were obtained from Sigma-Aldrich (Logan, UT, USA). Ultrapure deionized water was prepared using a Milli-Q system (Millipore, Bedford, MA, USA).
Synthesis of Arg-Eu-HAP
Arg-Eu-HAP were synthesized by hydrothermal method. Aqueous solution with calcium nitrate Ca(NO3)2·4H2O and europium nitrate Eu(NO3)3 was added dropwisely into ammonium dibasic phosphate (NH4)2HPO4 and arginine solution, and then were completely stirring and the mole ratio of Ca/P should be 1.67. The reaction temperature should be 60°C. During the reaction, the solution pH was maintained at 9.5 by using ammonia solution or urea. After calcium and phosphate solution was stirred evenly, the solution was transferred into an autoclave. Then the reaction was continued under the set solution temperature until completion. At the end of the experiment, the solids were collected by centrifugation (10,000 rpm/min) and filtration and then were washed thoroughly by using ethanol and deionized water. The product was dried overnight at the vacuum condition.
Characterization of Arg-Eu-HAP
The nanoparticles samples were characterized by a transmission electron microscope (JEOL., Tokyo, Japan) to analyze the nanoparticle crystalline appearance and the particle size, X-ray diffractometry to have phase analysis on Arg-Eu-HAP (Rigaku D-Max/2550VB+, Tokyo, Japan, Cu Ka radiation, λ = 1.54178 Å, 40 Kv, 30 mA), where the scanning angle and speed should apply 25° to approximately 55°, 2.4°/min, or 5° to approximately 75°, 5°/min and the Fourier infrared spectrometer is Nicolet Nexus470, KBr flaking. The excitation and emission spectra of Arg-Eu-HAP were determined by a RF-5301pc spectrofluorometer (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan).
Zeta potential measurement of Arg-Eu-HAP
Under the condition of neutral pH value (pH = 7.4), British Malvern Instrument Corporation's (Malvern, UK) Zetasizer 3000 HS nano size and potential analyzer was used to measure the electrophoretic mobility of Arg-Eu-HAP, thus obtain the Zeta potential. Eight samples were taken respectively, sample measurement was repeated three times, and their mean value was taken.
DNA binding of Arg-Eu-HAP
Plasmid DNA (1 μg) was mixed with the solution of Arg-Eu-HAP suspension at various HAP/DNA mass ratios (0:1, 10:1, 30:1, 50:1, 70:1, and 90:1) and allowed to incubated at room temperature for 20 min before loading into the agarose gel. The solution was centrifuged at 12,000 rpm/min for 10 min and then its supernatant was taken to have electrophoresis on 0.7% (w/v) agarose gel (80 V) for 45 min and stained with ethidium bromide for 10 min. The staining results were investigated under UV transilluminator.
Cell toxicity of Arg-Eu-HAP
The cytotoxicity of Arg-Eu-HAP was evaluated using flow cytometry in human lung epithelial (A549) cell line. In brief, cells were seeded in six-well tissue culture plates at a density of 1 × 105 cells per well. Three different concentrations of samples (20, 100, 200 μg/mL) were added to cell culture wells. After the cells were exposed to nanoparticles for 4, 8, 24, or 48 h, the experiments were terminated by flow cytometry (ChemoMetec, Allerød, Denmark) and the manufacturer's instructions were followed.
Cell binding and cellular internalization of Arg-Eu-HAP
To track the internalization of Arg-Eu-HAP, A549 cells were seeded in 12-well plates at 1 × 105 cells per well and incubated. Subsequently, cells were rinsed twice with serum media (F-12K without FBS, pH 7.0) and replenished with 1 mL serum-free media containing Arg-Eu-HAP at a final concentration of 30 μg/mL. After incubation for 2 h at 37°C, test samples were aspirated. Cells were then washed twice with ice-cold phosphate-buffered saline (PBS) before they were fixed with fresh 4% paraformaldehyde for 3 min at room temperature. Finally, the fixed cells were counterstained to visualize nuclei by 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). The intracellular localization of nanoparticles was visualized under a laser scanning confocal microscope (Bio-Rad MRC 1024, Tokyo, Japan) equipped with Argon (488 nm) and HeNe (543 nm) lasers.
All experiments were repeated at least three times, and the values are expressed as means ± standard deviations. Statistical analysis was performed using student's t test, with the significant level with a p value of less than 0.05.
Results and discussion
Synthesis of Arg-Eu-HAP
Characterization of Arg-Eu-HAP
Zeta potential of Arg-Eu-HAP
DNA binding of Arg-Eu-HAP
Cell toxicity of Arg-Eu-HAP
Cellular uptake studies of Arg-Eu-HAP
Despite the unique advantages of HAP in biomedical applications, exploration of their interactions with biological systems remains at a very early stage. To effectively develop these systems for application, it is necessary to systematically delineate its functional properties about cellular uptake and interactions after arginine functionalized and europium doped. The majority of uptake studies in vitro have been performed in buffers devoid of protein. In physiological fluids, however, a protein corona could be formed on a particle surface and affect its interaction with cells [28, 29]. We performed uptake studies in cell culture medium with free serum. Cellular uptake of Arg-Eu-HAP was investigated in A549 cell line.
In conclusion, nontoxic Arg-Eu-HAP have been prepared and characterized in vitro by various physicochemical means. As arginine surface functionalization changes HAP surface electron, its Zeta potential is changed from the unmodified (-10.6 ± 4.2 mV) into the functionalized (30.1 ± 6.3 mV). Meanwhile, arginine-functionalized and europium-doped hydroxyapatite nanoparticles with positive zeta potential can effectively bind negative plasmid DNA, and can be visualized in the cytoplasm and perinuclear of A549 cells by fluorescence microscope and laser scanning confocal microscope.
This work was partly supported by Project (no. 81071869) supported by the National Natural Science Foundation of China (NSFC), Scholarship Program (no. 2009637526) supported by China Scholarship Council and Project (no. 2010QZZD006) supported by the Key Program of Central South University Advancing Front Foundation.
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