Quantitative Detection Method of Hydroxyapatite Nanoparticles Based

Oct 23, 2015 - The Eu3+ fluorescent labeling method is attempted to trace the intracellular nHAP in Bel-7402 cells and tissue distribution in rat (det...
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Quantitative Detection Method of Hydroxyapatite Nanoparticles Based on Eu3+ Fluorescent Labeling in Vitro and in Vivo Yunfei Xie,†,‡,§ Thalagalage Shalika Harshani Perera,‡,†,⊥ Fang Li,‡,§ Yingchao Han,*,‡,§ and Meizhen Yin*,# ‡

State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, Wuhan 430070, P.R. China ⊥ Faculty of Applied Sciences, Sabaragamuwa University of Sri Lanka, 70140 Belihuloya, Sri Lanka # School of Medicine, Hubei Institute of Technology, Huangshi, Hubei 435003, China § Biomedical Materials and Engineering Research Center of Hubei Province, Wuhan University of Technology, Wuhan 430070, P.R. China S Supporting Information *

ABSTRACT: One major challenge for application of hydroxyapatite nanoparticles (nHAP) in nanomedicine is the quantitative detection method. Herein, we exploited one quantitative detection method for nHAP based on the Eu3+ fluorescent labeling via a simple chemical coprecipitation method. The trace amount of nHAP in cells and tissues can be quantitatively detected on the basis of the fluorescent quantitative determination of Eu3+ ions in nHAP crystal lattice. The lowest concentration of Eu3+ ions that can be quantitatively detected is 0.5 nM using DELFIA enhancement solution. This methodology can be broadly applicable for studying the tissue distribution and metabolization of nHAP in vivo. KEYWORDS: hydroxyapatite, nanoparticles, Eu3+ ions, fluorescent labeling, quantitative detection

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human and environment, and should be carried out under rigorous experimental conditions; the organic fluorescent labels will be escaped from HAP along with the dissolving of HAP and cannot continue to monitor HAP. Recently, based on the fluorescent signal of rare earth ions with narrow emission bandwidths, high photochemical stability, and long fluorescence lifetime (up to several milliseconds),15 the rare earth doped fluorescent nHAP has attracted more attention as high-quality cell labels.16 Rare earth ions are doped into crystal lattice of nHAP and located at Ca2+ sites, not adsorbed on the surface of nHAP. Therefore, it can be predicted that rare earth ions in crystal lattice of nHAP can provide a continuous monitoring for nHAP even though nHAP is gradually dissolving. Herein, we aim to develop a quantitative detection method for tracing nHAP based on the Eu3+ fluorescent labeling. By determining the concentration of Eu3+ ions in nHAP (using acid to dissolve nHAP completely) according to the linear relationship between fluorescence intensity and concentration, the amount of nHAP in cells and tissues can be obtained on the basis of definite labeling content of Eu3+ on nHAP. It is different from the trace method by means of determining the fluorescent intensity of Eu doping

s the main inorganic component of bone and tooth, hydroxyapatite (Ca10(PO4)6(OH)2, HAP) possesses good biocompatibility and bioactivity, and has been extensively used in biomedical applications.1 In particular, nanosize HAP shows many functions differing from bulk HAP and attracts more attentions. For instance, HAP nanoparticles (nHAP) can deliver and escape molecules (gene, drug, protein) in cells because of their good adsorption capacity and increasing solubility in the acidic pH environment of cells;2−4 nHAP displays more accumulation in cancer cells and higher inhibitory effect on cancer cells than normal cells by depressing protein synthesis.5 However, one major challenge for application of nHAP in nanomedicine is the quantitative detection in tissues and organs. So, it is important to seek a suitable method to monitor the delivering and degrading progressions of nHAP in vitro and in vivo. Signal labeling is a normal way to trace HAP particles. The radio-labeling is a sensitive and effective technology and has been used for monitoring HAP micro and nanoparticles in tissues and organs by the radioactive elements labeling such as 45 Ca, 177Lu, Tc-99m, 90Y, 166Ho, 125I, [18F]NaF, and 68GaNO2APBP.6−12 Fluorescent labeling is an alternative route to trace HAP particles. Organic fluorescent labels such as green fluorescent protein and fluorescent dye can be adsorbed on the surface of nHAP for studying their location and biodistribution.13,14 However, the radio-labeling might exist some risk to © XXXX American Chemical Society

Received: September 16, 2015 Accepted: October 23, 2015

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DOI: 10.1021/acsami.5b08767 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Letter

ACS Applied Materials & Interfaces

Figure 1. Effects of Eu3+ labeling parameters on the dissolution property of nHAP. (A) Effect of Eu3+ content (∼4%) on the dissolution property of nHAP (121 °C) in neutral and acidic media. (B) Dissolution profile of Eu(2%)-nHAP (121 °C) in acidic media. (C) Effects of synthesized temperature and pH value on RCa/Eu of Eu(2%)-nHAP.

nHAP under fluorescence imaging equipment. This method can realize the quantitative detection of nHAP in cells and tissues. To the best of our knowledge, this should be a new proposal for monitoring nHAP. Our study preliminarily verifies that nHAP can be quantitatively traced in vitro and in vivo via Eu3+ fluorescent labeling. Chemical coprecipitation method was utilized to realize the Eu3+ labeling on nHAP (Eu-nHAP). The crucial point for the labeling of nHAP using rare earth ions is the thermal diffusion of rare earth ions to the Ca2+ sites in HAP crystal lattice.17 By elevating the temperature from 37 to 121 °C at a constant Eu molar content of 2%, the luminescent intensity of Eu-nHAP is improved to some extent because of the enhanced thermal diffusion of Eu3+ ions to the Ca2+ sites (Figure S1). The emission peaks at 618 nm (5D0 → 7F2), 593 nm (5D0 → 7F1), 700 nm (5D0 → 7F4), and 654 nm (5D0 → 7F3) demonstrates that the Eu3+ ions are thermally diffused into the high C3 symmetry Ca2+ I position of HAP.17 The XRD patterns and FT-IR spectra display the characteristics of crystalline HAP, suggesting that the Eu3+ labeling does not change the crystalline phase composition (Figure S2). The crystallinity degree and crystallite size of Eu-nHAP are increased along with the increase of temperature. The labeling amount of rare earth ions on HAP is another crucial parameter. The Eu3+ labeling with content of 0.1−4% does not result in the change of crystalline phase composition. However, the increasing Eu3+ leads to the decreases of crystallinity degree and crystalline size (D002) of nHAP (Figure S3). Additionally, the intensity of Eu(2%)nHAP at 618 nm was enhanced about 1370, 209, and 109% compared to those of 0.1, 0.5, and 1% Eu-nHAP, respectively, indicating that more Eu3+ ions are located at the Ca2+ I site of nHAP. However, the further increase of Eu3+ (4%) generates some decrease in luminescent intensity of Eu-nHAP (Figure S4). It can be inferred that a large proportion of Eu3+ ions are not located at Ca2+ sites of nHAP due to the excessive decrease of crystallinity degree of nHAP. Thus, it can be concluded that the 2% Eu3+ is the feasible labeling amount. HRTEM images (Figure S5) further display that Eu(2%)-nHAP (14.4 ± 4.9 nm × 65.2 ± 13.6 nm) maintains the rod-like shape similar to nHAP (14.3 ± 3.2 nm × 90.7 ± 25.1 nm). Only the size in length of Eu-nHAP is decreased, which is in good accordance with the calculated crystalline size in XRD patterns. EDS result demonstrates the elements of Ca, P, O, Eu with (Ca+Eu)/P molar ratio of about 1.67 and Eu/(Ca+Eu) molar ratio of about 1.30 (Figure S6). The ICP analysis also shows the similar

Table 1. Comparation of Properties of HAP and Eu(2%)HAP characterization a

lattice constants (Å) unit-cell volumea (Å3) crystallite sizesa (nm) crystallinity degreea crystal sizeb (nm) aspect ratiob ZAvec (nm) PDIc zeta potential (mV) dissolutiond (mM)

HAP

Eu(2%)-HAP

a = b = 9.430, c = 6.880 530.14 D002 = 79, D310 = 14.6 0.549 14.3 ± 3.2 × 90.7 ± 25.1 6.3 91.0e; 111.8f; 133.7g 0.203e; 0.428f; 0.339g −39.1e; −9.0f ; −18.0g 0.026h; 2.457i; 5.345j

a = b = 9.430, c = 6.876 529.47 D002 = 42.4, D310 = 12.8 0.352 14.4 ± 4.9 × 65.2 ± 13.6 4.5 85.1e; 123.5f; 123.3g 0.198e; 0.459f; 0.345g −45.7e; −9.3f ; −21.5g 0.025h; 2.362i; 4.663j

a

XRD characterization. bTEM observation. cDLS analysis (nanoparticles are stabilized by SH). dDissolved Ca2+ ions determined in acidic solution. eDilution in water (0.2M). fDilution in DMEM media (0.2M). gDilution in 40 g/L BSA solution (0.2M). hpH 7.4. ipH 5.0. j pH 4.0.

results of (Ca+Eu)/P molar ratio of about 1.59 and Eu/(Ca +Eu) molar ratio of about 1.95. It is known that nHAP is more stable in alkaline conditions and considered as pH-responsive drug carriers in intracellular acidic environment. Reasonably, Eu-nHAP displays very small dissolution similar to nHAP in pH 7.4 buffer. In acidic environment, their dissolutions are enhanced about 100−200 times. However, Eu(∼2%)-nHAP shows little change on its dissolution and Eu(4%)-nHAP displays a little decrease in its dissolution (Figure 1A). So, the Eu3+ labeling of ∼2% does not influence the dissolution property of nHAP in acidic media. Moreover, the dissolution profile in acidic media of Eu(2%)nHAP displays the simultaneous dissolving out of Ca2+ and Eu3+ from nHAP, indicating that the dissolution of nHAP can be traced by determining the dissolved Eu3+ (Figure 1B). However, it should be noted that Eu(2%)-nHAP shows some differences in the molar ratios of dissolved Ca2+ to dissolved Eu3+ (RCa/Eu) depending on the synthesized temperature and the pH value of medium (Figure 1C). In the acidic environment of pH 5.0, the RCa/Eu value for Eu(2%)-nHAP is increased a little from 37 to 80 °C and dropped drastically at 121 °C, corresponding to deviation rates of −17.7%, 9.3, −2.4, and −33.4% from the calculated value, respectively. With the pH value of 4.0, the RCa/Eu value is increased from 37 to 60 °C and declined drastically from 60 to 121 °C, corresponding to deviation rates of −6.8, 12.1, −40.6, and −68.5% respectively. Therefore, under specific conditions, the real RCa/Eu value B

DOI: 10.1021/acsami.5b08767 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Letter

ACS Applied Materials & Interfaces

Figure 2. Comparation of FLM and ICP for quantitative detection of Eu3+ concentration dissolved out from nHAP. (A) Eu(2%)-nHAP (121 °C) versus dissolution time in acidic media. (B) Eu(2%)-nHAP synthesized at different temperature in acidic media (6 h). (C) Eu-nHAP with different Eu3+ content (121 °C) in acidic media (6 h).

Figure 3. Quantitative detection of nHAP by FLM in cells and tissues, and TEM observation of intracellular nHAP. (A) Total amount of nHAP in Bel-7402 cancer cells at different cultured time. (B) Averaged amount of nHAP in single Bel-7402 cancer cell at different cultured time. (C) Tissue distribution of nHAP in rat. (D−F) TEM observations of intracellular nHAP in Bel-7402 cancer cells at (D) 2, (E) 5, and (F) 48 h of cultured time.

should be tested first for amending the determined results in the tracing study of nHAP. For nanomaterials, the physicochemical properties such as size, shape, surface charge, adsorption ability of proteins and dispersity in media play crucial effects on their interaction with biological building blocks.18 So, the influence of Eu3+ ions labeling on the intrinsic property of HAP should be paid attentions. Our study shows that the labeling of Eu3+ ions on HAP generates slight changes in some HAP’s characteristics (Table 1). The substitution of Ca2+ (99 pm) of HAP by smaller

Eu3+ (95 pm) results in a little decrease in lattice constant c as well as unit cell volume, which corresponds to some decline of crystal’s size in length. However, the shape is still rod-like and only aspect ratio is diminished from 6.3 to 4.5. HAP and EuHAP all can be ultrasonically dispersed in water to obtain stable suspensions by adding 0.6 mg/mL sodium heparin (SH), showing similar averaged particle size (ZAve), polydispersity index (PDI) and stability. This demonstrates that the dispersity of HAP is not influenced and agglomeration is not elevated after Eu3+ ions labeling. Additionally, the replace of Eu3+ for C

DOI: 10.1021/acsami.5b08767 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Ca2+ is bound to cause the rise of positive surface charge of HAP as well as the adsorption ability via the electrostatic attraction, which can be indicated by the decreased zeta potential value due to larger adsorption of negatively charged SH on more positively charged surface (Figure S9). However, the ZAve and PDI values of SH stabilized HAP and Eu-HAP suspensions diluted in DMEM media and 40 g/L BSA solution are similar (Figure S10), implying the varied surface charge does not cause significant difference for them in biological environment. They display similar adsorption property for protein and component of media. In our opinion, the slight changes of HAP’s characteristics resulted from Eu3+ ions labeling will not significantly influence its interaction with biological building blocks. The further hemolysis and cytotoxicity tests prove that the biocompatibility of HAP is not significantly changed after the labeling of Eu3+ ions (Figures S11 and S12). For the determination of infinitesimal Eu3+ ions, the DELFIA Enhancement Solution is used to enhance the fluorescence intensity of Eu3+ ions. The fluorescence signal of Eu3+ ions can be detected at the lowest concentration of 0.5 nM and the fluorescence intensity of Eu3+ ions displays good linear relationship between 0.5 nM and 5.75 nM (Figure S7). The trace amount of nHAP can be quantitatively detected based on the quantitative detection of Eu3+ by the fluorescent method (FLM). The Eu3+ concentration detected by FLM is compared with the results of ICP. Results show that the Eu 3+ concentrations detected by FLM are mainly close to those obtained by ICP (Figure 2A−C). This demonstrates that the FLM is feasible for quantitative detection of Eu3+ ions as well as nHAP. The Eu3+ fluorescent labeling method is attempted to trace the intracellular nHAP in Bel-7402 cells and tissue distribution in rat (detailed experiment in the Supporting Information). Results demonstrate that nHAP in cells and tissues can be quantitatively detected by this method. During a very short culture time of 15 min, a small amount of nHAP (3.7 μM) are detected in cells. Along with the increase in culture time, the amount of intracellular nHAP is gradually increased (Figure 3A). However, the amount of intracellular nHAP per cell displays a decline after culture time of 4 h (Figure 3B). nHAP shows highest accumulation in spleen tissue, higher accumulation in liver and pancreas tissues, lower accumulation in lung, heart and lowest accumulation in kidney, which is similar to the reported results11,13 (Figure 3C). The higher accumulation of nHAP in some organs can be attributed to the reticuloendothelial system. Also, the NanoEL effect might result in the entering of nHAP to organs.19,20 The cell proliferation of Bel7402 is not significantly inhibited by Eu(2%)-nHAP (0.1 mM) during 3 days (Figure S13). This indicates that the intracellular nHAP may be dissolved in the acidic environment of cells and released out from cells. Also, TEM observations provide some evidence for verifying the dissolution of nHAP in Bel-7402 cells. Seemingly, the intracellular amount of nHAP is first increased along with the increase of culture time from 2 h (Figure 3D) to 5 h (Figure 3E) and then decreased along with the further increase of culture time to 2 day (Figure 3F). In summary, Eu3+ fluorescent labeling can be used as a quantitative detection method for nHAP in cells and tissues. The lowest concentration of Eu 3+ ions that can be quantitatively detected is 0.5 nM and several microliters sample is enough for detection. This study provides an easy and

feasible method for studying the tissue distribution and metabolization of nHAP in vivo.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b08767. Materials and methods,21−23 photoluminescence property, XRD and FT-IR, TEM images, size distribution and stability, hemolysis, and cytotoxicity (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions †

Y.F. and T.S.H.P. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (51002109, 81190133, 51172171), the HongKong, Macro and Taiwan Science & Technology Cooperation Program of China (2015DFH30180), the Fundamental Research Funds for the Central Universities (WUT: 2014-IV-121, 2014-VII-028, 2015-zy-017), and the project supported by State Key Laboratory of Advanced Technology for Materials Synthesis and Processing (Wuhan University of Technology).

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DOI: 10.1021/acsami.5b08767 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX