Achieving ultrasensitive in vivo detection of bone crack with polydopamine-capsulated surface-enhanced Raman nanoparticle

Biomaterials 114 (2017) 54e61

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Achieving ultrasensitive in vivo detection of bone crack with polydopamine-capsulated surface-enhanced Raman nanoparticle Chunhuan Jiang a, 1, Ying Wang a, b, 1, Jiawei Wang a, Wei Song c, **, Lehui Lu a, * a

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China University of Chinese Academy of Sciences, Beijing 100049, China c State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun 130012, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 April 2016 Received in revised form 28 October 2016 Accepted 7 November 2016 Available online 9 November 2016

The timely diagnosis and intervention of bone microdamage are essential for preventing its accumulation and further diminishing the risk of skeletal fracture. Although staining methods have been adopted to better depict the microdamage on bone, their clinical impacts are still limited because they are based on histological sections which are inherently destructive. Here, highly sensitive and non-invasive in vivo detection of bone crack can be achieved by surface-enhanced Raman scattering (SERS) technique using a carefully chosen polydopamine (PDA)-SERS nanoparticle tag. The bone crack can be specifically labeled by PDA-SERS tags taking advantage of high affinity of PDA towards calcium exposed on the damaged bone, and identified by an intense featured Raman signal both in vitro and in vivo benefiting from the surface-enhanced resonance Raman scattering effect. As a preliminary in vivo application, it is found that PDA-SERS tags present no serious adverse effect in mouse model and hold good biocompatibility. This work may help the development of SERS technique for ultrasensitive in vivo detection of bone crack in clinic. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Surface-enhanced Raman scattering Polydopamine Bone crack Ultrasensitive In vivo detection

1. Introduction The timely diagnosis and intervention of bone microdamage are essential for preventing its accumulation and further diminishing the risk of skeletal fracture [1e3]. Conventional detection strategies, such as bulk staining and electron microscopy, are commonly adopted for imaging bone microdamage [2,4]. However, these methods are based on histological sections [5e7], which are inherently destructive and tedious, and hence are impossible for seeking bone microdamage in clinical setting. To date, it is still a great challenge to image the bone microdamage non-destructively. Therefore, searching for non-invasive strategy capable of in vivo bone microdamage detection is of significant importance. Up to now, just few studies have been reported regarding in vivo detection of bone microdamage, and are mostly based on positron emission tomography (PET) [1,8,9]. However, PET usually requires radioactive tracer, which is specific to the activity of surrounding

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (W. Song), [email protected] (L. Lu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biomaterials.2016.11.007 0142-9612/© 2016 Elsevier Ltd. All rights reserved.

cells but not the crack site [10]. On the other hand, the low resolution (1e2 mm) of PET is also a vital hindrance for its practical application [10,11]. Computed tomography imaging (CT) with high resolution is suitable for imaging space occupying lesions in clinic and has become the front-line method for viewing bone injuries. Recently, we found that gemstone spectral computed tomography with the ability to extract the difference between the intrinsic X-ray attenuation characteristics of healthy bone and ytterbium-based contrast agent targeted damaged bone would efficiently image the bone crack [12,13]. However, the application of conventional CT imaging incapable of material differentiation for in vivo detection of bone microdamge remains unreported. The major hurdle may be that the microdamge, performed as microcrack, is usually small in size (25e300 mm in length) [7], and has little difference in the mineral composite from that of adjacent health bone which exceeds the sensitivity of CT. Besides, the radiation hazard from CT is also a pivotal hindrance that should not be neglected. Surface-enhanced Raman scattering (SERS) as an optical technology has been proved to be a powerful and safe analytical tool, because it can produce featured fingerprint spectrum of molecular with high sensitivity [14e17]. This high sensitivity can be further improved to orders-of-magnitude higher by using Raman active

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molecules with electronic transitions matchable to the incident laser, known as surface-enhanced resonance Raman scattering (SERRS) effect [18,19], resulting in the most desirable sensitive feature for biomedical detection. In the last decade, several pioneer works have been reported to evidence the capability of SERS technology for in vivo sensing application, in which Au NPs prelabeled by Raman active molecules were capsulated by a protective layer to obtain “SERS tag” with a definite fingerprint Raman spectrum [19e21]. However, in spite of its widespread use in analytical domain and the booming interest in biomedical area, the utility of SERS focused mainly on the field of oncology. Thus, extending the application of SERS into other arena has become an orientation for researchers. Here, we explore the application of SERS in bone crack detection taking advantage of its high sensitivity, and also introduce a dual functional protective material (Fig. 1a). For conventional procedures, polyethylene glycol and silica are usually used as the capsulation layer, playing a vital role in improving the units' stability and biocompatibility [22e24]. This coating layer also offers a platform to gain further functionalization by additional chemical modification, which, however, is time- and reagent-cost [25]. Here, we introduce polydopamine (PDA) as the coating layer for synthesis of SERS tag to overcome the abovementioned shortcomings. PDA, a bio-inspired material [26], has been proved to have good biocompatibility both as an individual nanoparticle and as the coating layer outside gold nanoparticles [22,27,28]. Most importantly, PDA can offer calcium ions binding sites that facilitate the mineralization of hydroxyapatites (HA) on the substrate surface [29]. In this study, the as-synthesized SERS nanoparticle, denoted as PDA-SERS tag, exhibits high affinity towards calcium exposed in crack site of bone without any further modification for this SERS nanoparticle, hence enabling the sensitive and selective detection of the bone crack by Raman

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spectroscopy. In vivo experimental results confirm that bone crack can be effectively labeled and yields intense signature Raman signal after intravenous injection of PDA-SERS tag. Furthermore, PDASERS tag is verified to present no serious adverse effect in mouse model and hold good biocompatibility. 2. Materials and methods 2.1. Materials 3,3’-diethyl-thiatricarbocyanine iodide (DTTC), thiazolyl tetrazolium and sodium citrate tribasic dehydrate were purchased from Sigma-Aldrich. Dopamine was purchased from Alfa Aesar. Chloroauric acid (HAuCl4$4H2O) was purchased from Shanghai Reagents Co., Ltd. Hydroxylammonium chloride was purchased from Beijing Yili Fine Chemicals Co., Ltd. High-glucose DMEM medium and RPMI 1640 medium were purchased from Thermo. Fetal bovine serum was purchased from Gibco. All the reagents were used as received without any purification. Ultrapure water (18.2 MU) used throughout this work was prepared by Milli-Q Academic (Millipore). 2.2. Characterization Transmission electron microscopy (TEM) images were recorded on TECNAI G2 high-resolution transmission electron microscope. Scanning electron microscopy (SEM) images were obtained on a FEI/Philips XL30 ESEM FEG field-emission scanning electron microscope with an acceleration voltage of 20 kV. X-ray photoelectron spectroscopy (XPS) measurements were carried out on a VG ESCALAB MKII spectrometer. UVeVis absorption analysis was performed with a cary 50 UVeViseNIR spectrophotometer (Varian).

Fig. 1. (a) Schematic illustration of the synthesis of PDA-SERS tag: (i) assembling the Raman reporter molecules (DTTC) on the surface of Au NPs; (ii) polymerization of dopamine on the surface of Au-DTTC nanoparticles. Transmission electron microscopy (TEM) (b) and energy-dispersive spectroscopy (EDS) mapping (c) images of PDA-SERS tag. The inset in b is the magnified TEM image of PDA-SERS tag.

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Zeta potential was measured on Malvern Zetasizer Nano ZS (Malvern Instruments Ltd, UK). Inductively coupled plasma-optical emission spectrometer (ICP-OES) analysis was conducted on a PerkinElmer ICP instrument. Raman spectra were excitated using a 785 nm diode laser (12 mW) controller and collected on a LabRAM ARAMIS Raman spectroscope system (Scientific HORIBA) equipped with a charge-coupled device (CCD) detector. The acquisition time of Raman spectra for solid/powder/solution and animal samples was 10 s and 30 s respectively. CT imaging test was carried out on a 64-row MDCT scanner (Discovery HD750, GE) with the imaging parameters as follows: 80 kVp, 500 mA; slice thickness, 1.25 mm; pitch, 0.18; gantry rotation time, 0.35s; field of view, 250 mm. 2.3. Preparation of PDA-SERS tag 55 nm Au nanoparticels (Au NPs) were prepared using seedmediated growth method. Au seed was synthesized according to Frens' method [30]. Briefly, gold chloride aqueous solution (94 mL, 1 mM) was heated to boiling under vigorous stirring; then freshly prepared sodium citrate aqueous solution (9 mL, 1 wt%) was quickly injected to the above solution. The reaction was allowed to proceed for additional 30 min and cooled down to room temperature. After three rounds of growth from the seeds as previously reported with minor modification, Au NPs with mean diameter about 55 nm were obtained [31,32]. The seed-mediated growth was carried out as follows: gold chloride solution (x mL, 2 wt%) was added drop by drop to a vigorously stirred solution containing water (y mL), hydroxylamine aqueous solution (z mL, 25 mM), and Au seed solution (t mL). The reaction took 30 min at 25
C. The obtained Au NPs from each ground were used as the seeds for the next ground. The practical values of x, y, z and t in each ground were given in Table 1. The final obtained Au NPs were dialyzed against water for 7 days to remove excess sodium citrate before use. To encode Au NPs with Raman reporters, DTTC ethanol solutions (3 mL, 2 mM) was added to Au NPs colloid solution (20 mL) by syringe pump at a rate of 50 mL min1. Then, Tris-HCl buffer (20 mL, 10 mM, pH 8.5) containing dopamine (4 mg) was added into the above solution to initiate the capsulation process. After 3 h, PDASERS tag was separated by centrifugation and washed with ultrapure water. 2.4. Cell culture and MTT assay Hela cells/L929 cells and 4T1 cells were cultured with highglucose DMEM medium and RPMI 1640 medium supplemented with fetal bovine serum (10%) respectively, and were maintained at 37
C in a incubator with the atmosphere of CO2 (5%). MTT assay was used to evaluate the cytotoxicity of PDA-SERS tag towards cells. In a typical process, cells planted in 96 cell plate were allowed to grow to >70% confluence. After washing with D-hanks buffer, cells were cultured with serum-free medium with different concentration of PDA-SERS tag for 4 h or 24 h. Then, serum-free medium (100 mL) containing thiazolyl tetrazolium (0.5 mg mL1) was added after twice washing with D-hanks buffer, and the cells were further cultured for 4 h. After medium removing, dimethyl sulfoxide (200 mL) was used to dissolve the purple crystal converted Table 1 The values of x, y, z and t in each ground of the synthesis process.

from thiazolyl tetrazolium by alive cells. Finally, the absorption was measured at 570 nm by a microplate reader (M200-PRO, Tecon). 2.5. Long-term toxicity evaluation of PDA-SERS tag Healthy female ICR mice (~20 g) were intravenously injected with PDA-SERS tag (100 mL, 1.32 mg mL1 calculated by gold mass). Mice as control group were fed with the same condition but without administration of nanoparticles. Their body weight and daily behavior, including eating, drinking, sleeping, urination, climbing and exploratory behavior were monitored every 3 days. For investigating the biodistribution of PDA-SERS tag, the mice were sacrificed at 4 h, 24 h, 7 d, 15 d and 30 d (3 mice for each group). The major organs were collected for measuring the gold amount by ICP-OES. In brief, the organs were treated by lyophilization, weighting and digestion in sequence. The %ID per gram of tissue (x) was calculated as follows:

x¼

mAu  100% mAu:o  mtissue

where mAu was the amount of gold from the tissue; mAu.0 was the initially injected amount of PDA-SERS tag (calculated with gold mass), and m tissue was the amount of tissue. For biochemistry and histopathology assessment, the mice were sacrificed 30 days after administration with PDA-SERS tag. The blood was collected for blood biochemistry analysis. The major organs were fixed and used for histological evaluation (H&E staining). 2.6. In vitro bone crack affinity test Affinity of PDA-SERS tag towards calcium: To test the affinity of PDA-SERS tag towards calcium ions, we incubated it with saturated calcium chloride solution for 24 h. The resulted nanoparticle was thoroughly washed to remove the uncombined ions, and was then used for XPS analysis after freeze-drying. Preparation of in vitro bone crack model: Fresh porcine cortical bone slice with the size of 8  5  2 mm was polished by 1200 mesh abrasive paper, and dipped in calcein solution (0.5 mM) for 1 h to mask mechanical damage induced during preparation. After several rounds of wash, a crack was then scratched manually by scalpel, and the bone slice was immerged in the solution of PDASERS tag for 24 h for labeling the crack. The bone slice was vertically placed to avoid gravity induced deposition. The labeled bone was allowed to dry under ambient condition after gently washed by water, and used for further Raman and SEM characterization. In vitro detection of bone crack: SERS mapping was conducted on a LabRAM HR800 confocal Raman microscope (Horiba Jobin Yvon) equipped with an electronic specimen holder. 785-nm laser (100 mW) was used for Raman spectra excitation. A long working distance objective (50, NA ¼ 0.45) was used for laser-focusing, and the diameter of the laser spot was ~2 mm. The Raman mapping was performed by recording Raman/SERS spectrum from each spot oriented by the electronic specimen holder. The acquisition time was 10s for each point (50% laser power). The final detection image was formed by LabSpec 6 Spectroscopy Suite based on the intensity of 508 cm1 band. 2.7. In vivo detection of bone crack

Ground

x [mL]

y [mL]

z [mL]

t [mL]

1st 2nd 3rd

0.243 0.546 0.910

28.0 80.0 144.5

12.0 9.0 5.5

10 10 100

Healthy female ICR mice (~22 g) were used to establish the conceptual mouse model of bone crack. After anesthetized with chloral hydrate solution (10 wt%), the skin surrounding the surgical site was unhaired and disinfected with iodophor. The tibia was

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exposed through a longitudinal skin incision on the inner side of right hind limb. A crack (5 mm in length) was created on the top third of the tibia with a scalpel. This surgical operation was conducted through the intermuscular space of adductor longus muscle and gracilis muscle, avoiding muscle injury. Then, the muscle attachment was recuperated and the skin was closed layer by layer. The major blood vessels and nerve were avoided, and no substantial bleeding occurred during and after the surgery. The mice were kept in individual cages freely and treated with regular disinfection. Food and water were given ad libitum. No death and abnormal behavior was observed for the mice after surgery. For detection of bone crack, PDA-SERS tag (100 mL, 1.32 mg mL1 calculated by gold mass) was administrated via tail vein or adjacent muscle post surgery. 2 mice were used in intramuscular injection group for in vivo detection of bone crack, and another 2 mice were used in intravenous injection group. The acquisition time was 30 s for each Raman spectrum (laser power: 100 mW). All the animal experiments were conducted in agreement with the guidelines of the Regional Ethics Committee for Animal Experiments. All efforts were made to minimize suffering of the mice. 3. Results and discussion 3.1. Synthesis and characterization of PDA-SERS tag PDA-SERS tag was designed containing two components: gold core encoded with Raman reporters for affording the signature SERS spectrum and a biocompatible PDA coating layer for protecting gold core and providing binding sites towards calcium ions. As shown in the transmission electronic microscopy (TEM) image (Fig. 1b), PDA-SERS tag was observed with a uniform coating layer outside the gold core and exhibited good monodispersity. This core-shell structure was also verified by energy-dispersive spectroscopy (EDS) mapping (Fig. 1c) and scanning electron microscopy (SEM, Fig. S1). Zeta potential analysis presented that the tags after PDA coating gave a negative surface charge of 29.7 mV (Fig. S2). The extremely negative charged surface derived from the exposed functional moiety of PDA [28], such as carboxy and phenol groups, offered their long term stability (maintained stable in water more than six months) by the aid of intense electrostatic interaction between the charged nanoparticles. Here, 3,3’-diethyl- thiatricarbocyanine (DTTC) was chosen as the Raman reporter to encode the gold nanoparticles. DTTC was an infrared dye with a wide absorption in 700e800 nm range (Fig. S3), which would resonate with irradiation laser (785 nm) and invoke high sensitive SERRS signals [33]. Here, we also found that the SERS spectrum excitated by 785 nm laser showed more complete information than that excitated by 632.8 nm laser (Fig. S4). As seen in Fig. 2a and Fig. S5, PDA-SERS tag presented a feature Raman spectrum, revealing the successful assembling of DTTC molecules on the gold surface facilitated by their positive charge and sulfur moieties [33]. The most intense band (508 cm1) was chosen as the representative of the SERS signal of PDA-SERS tag in this work. Generally, to prevent the leaking of the Raman active molecules and disturbance from hash environment, a reasonable thickness of the protective layer is required to completely protect the SERS tag. In addition, the thickness should be as thin as possible to minimize the interference on the SERS signal intensity from the capsulation layer. In view of the above two points, three PDA-SERS tags with the thickness of PDA layer ranging from 6 nm, to 8 nm and 12 nm (Fig. 2c) were prepared by simply tailoring the dopamine concentration from 0.05 mg mL1, to 0.1 mg mL1 and 0.2 mg mL1 (denoted as Tag-1, Tag-2, and Tag-3, respectively). Remarkably, the increased thickness of PDA layer not only caused a gradual red-shift of the gold featured plasmonic absorption (Fig. 2b), but also

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enervated the SERS intensity (calculated from 508 cm1, Fig. 2d), probably due to the shielding effect of PDA coating derived from its concentration-dependent absorption of the excitation laser. Nevertheless, it should be noted that PDA-SERS tag with a thin coating layer was more susceptible to harsh condition and tended to aggregate. Based on the above results, Tag-2 was selected for the following experiments to achieve a higher sensitivity and gain well protection of SERS tags simultaneously. 3.2. In vivo distribution and long-term toxicity of PDA-SERS tag To evaluate the biological effects of PDA-SERS tag, its cytotoxicity was firstly examined by MTT assay towards cancerous cells (Hela cells and 4T1 cells) and non-cancerous cells (L929 cells), respectively. After incubating cells with different concentration of PDA-SERS tag, no perturbation on the proliferation ability of three cell lines was observed over the assessing time of 4 h and 24 h (Fig. S6). Moreover, the cell viability remained above 80% within the tested concentration range, suggesting its good biocompatibility. To investigate the biodistribution of PDA-SERS tag in mice, its amount in major organs was measured by inductively coupled plasma-optical emission spectrometer (ICP-OES) after intravenous injection. Similar to most nanoparticle-based agents, PDA-SERS tag mainly accumulated in reticuloendothelial system (RES): liver and spleen (Fig. 3b, Fig. S7) [34e36]. Moreover, it was found that the amount of gold in liver presented a slow and gradual decline trend over time. By contrast, in the case of spleen, it was in a fluctuation form by firstly reaching a plateau at 15 days and decreasing subsequently. All above data implied that PDA-SERS tag could be eliminated from mice through hepatobiliary metabolism. Furthermore, the long-term toxicity of PDA-SERS tags was assessed by analyzing and comparing several important health indicators between treated and wild-type mice. Over the monitoring period of 30 days, both types of mice didn't show any abnormality in their daily behaviors, such as eating, drinking, sleeping, urination, climbing, exploratory behavior, etc. Besides, the body weight of the treated group increased with the same trend of the control group (Fig. 3a). At 30 days post-injection, the mice were sacrificed. The corresponding blood and major organs, including heart, liver, spleen, kidney, and lung, were collected for biochemistry and histopathology assessment. As shown in Fig. 3c, the hematoxylin and eosin (H&E) stained slices of these organs from treated mouse exhibited normal histological morphology, comparable with that of non-treated group. Furthermore, the similar values of liver function related indexes, such as total protein, albumin, aspartate aminotransferase, alkaline phosphatase and aline transferase (Fig. 3e), revealed that no dysfunction happened on liver of treated mice. Similarly, blood analysis also indicated that the concentrations of red blood cells, white blood cells, hemoglobin, platelets and hematokrit of treated mice were all in reasonable levels (Fig. 3d). Based on these results, it could be concluded that PDA-SERS tag did not show distinct adverse effect on living body, and thus held good biocompatibility. We speculated that this property may originate from the coating material, PDA, which had been confirmed by our previous work and Ji's research, in which PDA nanoparticles and gold nanoparticles with PDA surface layer showed no notable toxicity in vivo [22,27]. 3.3. Evaluation of the Raman analysis capability of PDA-SERS tag Motivated by the good biocompatibility of PDA-SERS tag, we sought to assess its performance in living animals. Generally, in vivo optical imaging commonly suffers from noise and background signals because of the autofluorescence and bio-absorption of living tissues, which greatly hindered its bio-medical application.

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Fig. 2. Characterization of the SERS performance of PDA-SERS tag. (a) The SERS spectra of DTTC and PDA-SERS tag (found SERS bands assignment in Table S1, calculated by Gaussian 09 B3LYP/LANL2DZ model as reference article [38]). Laser power, 12 mW. Extinction spectra (b), digital graphs and TEM images (c) of PDA-SERS tag prepared with different dopamine concentration. All scale bars in TEM images represent 50 nm. (d) The thickness of the PDA layer accompanied with the responding SERS intensity at Raman shift value of 508 cm1 of PDA-SERS tag with different initiating dopamine (DPA) concentration.

Fig. 3. Long-term toxicity assessment of PDA-SERS tag. (a) Body weight changes of mice treated with PDA-SERS tag and wild type mice. (b) Biodistribution of gold in liver and spleen of mice intravenously injected with PDA-SERS tag. (c) H&E stained slices of major organs from mice treated with and without PDA-SERS tag. (d) Blood analysis data of control and treated groups. (e) Liver-function test of control and treated groups.

To overcome this limitation, both the radiation laser and the scattering light in this work were located in the scope of near-infrared (700e900 nm), which was known as a “clear window” because of the low absorption from biological tissue [19,37]. Obviously, this

spectral region permitted deeper penetration of light into living tissues. To determine whether the Raman spectra of PDA-SERS tag could be recorded in vivo, the SERS nanoparticles were administrated through subcutaneous and deeper intramuscular injection.

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Fig. 4. Raman spectra collected from the skin of a mouse before administration with PDA-SERS tag (orange line), PDA-SERS tag solution (green), and different sites of the mouse after administration with PDA-SERS tag (pink and blue lines). Laser power, 12 mW. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

As shown in Fig. 4, intense SERS signals were collected from both the injected sites, with identical spectra to those of pure PDA-SERS tags in vitro. Hence, PDA-SERS tags with high sensitivity possessed a great potential for in vivo application. 3.4. Application of PDA-SERS tag in detection of bone crack 3.4.1. In vitro detection of bone crack Affinity of PDA-SERS tag towards calcium: Benefited from its good biocompatibility and high sensitivity, the feasibility of PDASERS tag as a marker for bone crack detection was further examined taking advantage of the inherent affinity of PDA towards calcium ions. Here, we found that PDA-SERS tag could concentrate calcium ions from calcium chloride solution (Fig. S8), which would facilitate the accumulation of PDA-SERS tag on the crack site. In vitro detection of bone crack: To evaluate the ability of PDASERS tag for detection of bone crack, an isolated porcine bone slice was used as an in vitro model. Briefly, a fresh porcine cortical bone was polished and dipped in calcein solution to mask the mechanical damage induced during sample preparation (found detail data of masking effect in Fig. S9). A crack was then scratched manually by scalpel, and the bone slice was immerged in the solution of PDASERS tag for 24 h to label the crack. To verify the labeling effect, a region (labeled in Fig. 5a) containing the crack site and the adjacent undamaged site was scanned

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by SERS mapping (168 points at an interval of 2 mm in x and y directions). As shown in Fig. 5b, a remarkable boundary was observed between the crack region (bright red color) and undamaged region (dark red-black color) in the mapping image, in which the red pseudo color represented the signal intensity of 508 cm1. The bone crack with the exposure of calcium site was identified by the intensively enhanced Raman signal (Fig. 5c, found blank control in Fig. S10). Conversely, only weak signal could be observed from the undamaged sites outside the crack, where less exposed calcium sites existed (Fig. 5c). To unveil the origin of the different SERS signals between the crack and the undamaged site, SEM was employed to image the distribution of the PDA-SERS tag. As shown in Fig. S11, there were much more tags at the crack sites rather than the sites located outside the crack. These results implied that PDASERS tag possessed good specificity towards the bone crack. To further validate the specificity, we pre-incubated the bone slice with a fresh crack in calcein solution, and then dipped into the PDASERS tag solution. As a result, the control crack pre-masked by calcein could not be labeled by PDA-SERS tag (Figs. S12 and S13), implying that the accumulation was guided by the exposed conjugating sites but not the confined space. To investigate whether calcium from biofluid influenced the specificity of bone crack detection, the labeling experiment was conducted in 10% serum solution spiked with normal amount of calcium (1.25 mM, the concentration of ionized calcium in human blood [39]). As anticipated, PDA-SERS tag could also successfully labeled the bone crack (Fig. S14). Although in vitro bone crack screening has been successfully achieved by many strategies, it remained a great challenge for most of the current clinical methods. For example, CT imaging, reputed for its high resolution, has become the first the front-line method for viewing bone injuries. But, CT imaging also did not have enough sensitivity for identifying minimal damage of bone (Fig. S15), owing to the extremely small difference of the mineral density between the healthy bone and bone crack. Comparatively, SERS technology with a notable virtue of high sensitivity performed perfectly in detection of the minimal damage of bone taking PDA-SERS tag as the targeting agent (Fig. 5). Noteworthily, the SERS intensity from the crack site was much higher than that of undamaged sites (Fig. 5c), enabling the above highly sensitive analysis.

3.4.2. In vivo detection of bone crack Encouraged by the above results, the ability to screen bone crack in vivo with PDA-SERS tag was then examined in a simple conceptual experiment. A crack on tibia bone was conducted with a health ICR mouse (found details in the supporting information). PDA-SERS tag was administrated by intravenous injection (i. v.). As

Fig. 5. In vitro detection of bone crack. (a) The micrograph of a bone slice with the crack labeled by PDA-SERS tag (10  objective). (b) SERS mapping image (50  objective) of the region labeled by blue square in a. The red pseudo-color was generated from LabSpec 6 Spectroscopy Suite based on the intensity of 508 cm1 band. (c) Raman spectra of the two regions (2 mm  2 mm) labeled in b: i was the region away from the crack; ii was the region on the crack. Laser power, 50 mW. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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anticipated, the crack was detected after 24 h injection by evidence of the presence of the featured Raman spectrum of PDA-SERS tag (Fig. 6b). On the contrary, no signal was collected from the adjacent undamaged bone (Fig. 6b), suggesting specific affinity of PDA-SERS tag towards damaged bone. To further validate this, another control experiment was conducted through administration of PDA-SERS tag to a mouse with healthy bone system. As expected, no detectable signal was obtained (Fig. S16) from the similar site on tibia. Then, the signal from the crack site was continuously monitored by Raman spectroscopy for the following 10 days. As shown in Fig. 6c and d, the intensity of Raman signal maintained constant after 48 h, and subsequently declined to negligible level. The decrease in SERS signal might originate from the decreased available concentration of PDA-SERS tag in the peripheral circulation and also the decrease of the exposed calcium sites during bone healing process. As a second administration route, intramuscular injection (i. m.) was also evaluated with the same dosage. Similarly, the characteristic Raman spectrum could only be collected from the crack site rather than surrounding undamaged bone, and the signal intensity changed with a similar tendency with that of intravenous injection, but with a longer period. As shown in Fig. 6c and e, the bone crack could also be detected after 24 h. Unlike intravenous injection, a maximal signal was collected 4 days after intramuscular administration, and the signal had a 2-fold higher intensity than that of intravenous injection. This difference was speculated leading by the different pharmacokinetics corresponding to intravenous and intramuscular administration methods. Since a longer period of time was required to reach the circulating system for PDA-SERS tag, the sustained release of PDA-SERS tag from the depot providing by intramuscular injection would be the origin of its more intense signal over intravenous injection. Obviously, both administration routes proved the capability of PDA-SERS tag for in vivo detection of bone crack. To our knowledge, this was for the first time using SERS

tag for detection of bone crack both in vitro and in vivo. In this work, the traveling of PDA-SERS tag to the bone crack site might be guided by both the active targeting and passive targeting means. i) Active targeting: the high binding affinity of PDA layer towards calcium ions exposed by bone crack would facilitate and lead to the specific accumulation of PDA-SERS tag on the crack site [29]. ii) Passive targeting: as reported by Yadav [40], the crack site would induce an ion gradient oriented outwards which could direct the migration of negatively charged nanoparticles towards the crack site. Thereby, this ion-gradient-driven local electric field greatly improved the probability and efficiency of the negatively charged PDA-SERS tag for reaching the crack site. Even though we suggested two possible traveling means, the exact mechanism has not been clearly understood due to the complex and mysterious biological system. The exact mechanism will be further investigated in our lab. 4. Conclusion We have demonstrated the utility of SERS in detection of bone crack both in vitro and in vivo using a newly developed SERS nanoparticle, PDA-SERS tag. The PDA-SERS tag, presenting high affinity towards calcium exposed site of damaged bone, was used as targeting agent for bone crack detection. Here, taking advantage of the high sensitivity of SERS technique and the high resolution of Raman microscopy, bone microcrack was easily identified from the SERS mapping image using PDA-SERS tag. Moreover, the ability of SERS technique for in situ detection of bone microcrack was also investigated and achieved in a preliminary conceptual mouse model. To our knowledge, this was for the first time using SERS tag for detection of bone crack in vivo, which both extended the application of SERS technique and provided new horizon for in vivo detection of bone crack. By conjugation of well designed SERS tag

Fig. 6. In vivo detection of bone crack with PDA-SERS tag. (a) Schematic illustration of the detection sites. (b) Raman spectra obtained from bone crack and healthy bone. (c) The signal intensity of bone crack at different time. Raman spectra recorded from bone crack treated by intravenous injection (d) and intramuscular injection (e) of PDA-SERS tag at different time after administration. Laser power, 100 mW. All the spectra in d and e shared the same intensity scale.

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and novel developed Raman imaging technology, highly sensitive and 3D Raman imaging of bone crack can be expected in the further. [19]

Acknowledgements Financial support by NSFC (21635007) and National Key Research and Development Program of China (2016YFA0203200) is gratefully acknowledged. We thank Mengchao Zhang (China-Japan Union Hospital of Jilin University) for the assistance with animal experiments.

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Appendix A. Supplementary data [23]

Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2016.11.007.

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