A general strategy for designing NIR-II emissive silk for the in vivo monitoring of an implanted stent model beyond 1500 nm

Zhiming Deng , Junqing Huang , Zhenluan Xue , Mingyang Jiang , Youbin Li and Songjun Zeng *
School of Physics and Electronics, Key Laboratory of Low-dimensional Quantum Structures and Quantum Control of Ministry of Education, and Key Laboratory for Matter Microstructure and Function of Hunan Province, Hunan Normal University, Changsha, 410081, P. R. China. E-mail: songjunz@hunnu.edu.cn

Received 27th November 2019 , Accepted 4th April 2020

First published on 6th April 2020


Silk fibroin-based materials spun by silkworms present excellent biocompatible and biodegradable properties, endowing them with broad applications for use in in vivo implanted devices. Therefore, it is highly desirable to explore functionalized silk with additional optical bioimaging abilities for the direct in situ monitoring of the status of implanted devices in vivo. Herein, a new type of silk material with a second near-infrared (NIR-II, 1000–1700 nm) emission is explored for the real-time observation of a biological stent model using a general route of feeding larval silkworms with lanthanide-based NaYF4:Gd3+/Yb3+/Er3+@SiO2 nanocrystals. After being fed lanthanide nanocrystals, the silk spun by silkworms shows efficient NIR-II emission beyond 1500 nm. Moreover, NIR-II bio-imaging guided biological stent model monitoring presents a superior signal-to-noise (S/N) ratio compared to the traditional optical imaging by utilizing the upconversion (UC) region. These findings open up the possibility of designing NIR-II optically functionalized silk materials for highly sensitive and deep-tissue monitoring of the in vivo states of the implanted devices.


Introduction

Natural biological materials have attracted considerable interest because of their superior properties and ecological friendliness.1,2 Silk made by the domesticated silkworm Bombyx mori has been used for more than 5000 years for making cotton materials in ancient China.3 In recent years, silkworm silk, as a type of mass-produced natural protein resource, has emerged as an ideal bioapplication material owing to its various advantages, such as the superior physical and chemical properties as well as the biocompatibility and biodegradability.4–12 Optical silk fibers have a huge potential in biomedical applications, including wounding dressings, tissue engineering, and biological stents, however, further modification of the silk is usually needed. In addition, these approaches inevitably require the use of toxic chemical solvents and complex multistep procedures.13 Therefore, the development of an easy and green in situ modification route for the production of the functionalized silk fibers by directly feeding specific diets to silkworms would be meaningful for further applications.

To date, various materials, including gold nanoclusters,14 genes15,16 and lanthanide (Ln)-doped upconversion nanoparticles (UCNPs)17 have been incorporated into silk. The gold nanoclusters have demonstrated excellent optical properties, however the limited penetration depth remarkably impedes their widespread use in bioimaging applications.14 Genetic alteration is environmentally friendly and retains the pristine properties of the silk, however, complicated procedures are required, and the biosafety of silkworms should be carefully considered.15,16 Although UCNPs nanomaterials are considered as promising optical probes for fluorescence imaging owing to their high biocompatibility and the reduced photo-damage to biological samples, the spatial resolution and signal-to-noise (S/N) ratio are still relatively attenuated in the near infrared (NIR)-I (750–900 nm) regions.17–23

Compared to traditional optical imaging in the visible and NIR-I regions, a newly developed fluorescence imaging technique using a photon in the NIR-II region (1000–1700 nm) has emerged as the next-generation in vivo optical imaging method owing to its remarkably decreased scattering losses and significant improvement in the imaging quality and spatial resolution.18,19,24–26 Recently, lanthanide-based nanoprobes with NIR-II emission have been developed as promising agents for in vivo vascular imaging, the detection of tiny tumors, and imaging-guided tumor dissection.27–30 Therefore, it is significantly important to design NIR-II emissive silk fibers by incorporating lanthanide-based nanomaterials into silk, which still remains unexplored.

Herein, a general feeding method for designing NIR-II emissive silk-based materials was explored. The obtained hybrid silks incorporated with rare-earth nanoparticles present a bright NIR-II emission beyond 1500 nm. The NIR-II emitting silk-based materials were successfully used for highly sensitive in vivo optical bioimaging and presented a high biocompatibility. More importantly, in situ monitoring of the in vivo state of the implanted silk-based stent models was also achieved.

Experimental

Chemicals and materials

GdCl3·6H2O (99.99%), YCl3·6H2O (99.99%), YbCl3·6H2O (99.99%), and ErCl3·6H2O (99.9%) were purchased from QingDa elaborate Chemical Reagent Co. Ltd (Shandong). NaOH (98%), NH4F (98%), 1-octadecene (90%), oleic acid (90%), absolute ethanol, CO-520, tetraethyl orthosilicate (TEOS), and other reagents were purchased from Sinopharm Chemical Reagent Co., China. In this work, Bombyx mori larval silkworms were reared by an artificial diet. The normal mulberry leaves were obtained from the supermarket.

Synthesis of NaYF4 nanocrystals

NaYF4 nanocrystals were synthesized using a high-temperature co-precipitation method31 as follows: YbCl3, YCl3, ErCl3, and GdCl3 (20[thin space (1/6-em)]:[thin space (1/6-em)]38:2[thin space (1/6-em)]:[thin space (1/6-em)]40) with a total amount of 1 mmol of lanthanide ions were added to a 100 mL three neck flask containing 12 mL oleic acid and 30 mL 1-octadecene. The mixed solution was then heated at 160 °C for 1 h to evaporate off the water under argon protection. Subsequently, the solution was cooled down to 90 °C. 4 mmol NH4F and 2.5 mmol NaOH were dissolved in 8/20 mL of methanol, respectively, and slowly added into the reaction flask over 20 min. The solution was stirred for another 1 h at room temperature, and then the mixed solution was maintained at 60 °C and stirred for 1 h. After removing methanol, the solution was heated to 305 °C and maintained at that temperature for 25 min under argon gas protection, and then cooled down to room temperature. The as-prepared products were collected by centrifugation, washed with ethanol three times and finally dispersed in 10 mL cyclohexane.

Synthesis of NaYF4@SiO2 nanoparticles

In a typical synthesis,17 2 mL of NaYF4:Gd3+/Yb3+/Er3+ nanocrystals (0.1 mmol mL−1) were mixed with 0.1 mL of CO-520 in 40 mL of cyclohexane and stirred for 10 min. Then, 0.4 mL of CO-520 and 0.8 mL of concentrated ammonia were added and sonicated for 30 min. After that, 0.6 mL of TEOS was added, and the mixture was stirred for 48 h at room temperature. The rotational speed of the magnetic rotor was 650 rpm. The nanoparticles were precipitated with acetone and washed three times with a 1[thin space (1/6-em)]:[thin space (1/6-em)]1 volume of ethanol/water solution and finally dispersed in 10 mL water for further use.

Feeding of silkworms

The obtained NaYF4@SiO2 solutions (4 mg mL−1) were uniformly sprayed onto fresh mulberry leaves using an airbrush, which were then directly used as a diet for the silkworms. A total of 40 Bombyx mori silkworms in third instars were divided into two groups and reared under the conditions of a temperature of 25 °C, a relative humidity of 20% and feeding of the diets.

Characterization

X-ray diffraction (XRD) measurements were performed using a Rigaku D/max 2500 X-ray diffractometer with Cu-Kα radiation (λ = 0.15406 nm) at 40 kV and 250 mA. The shape and structure of the as-prepared NaYF4 and NaYF4@SiO2 were characterized using transmission electron microscopy (TEM, FEI Tecnai F20). The shapes of the silks were characterized using scanning electron microscopy (SEM, S-4800, Hitachi Ltd, Japan). The upconversion (UC) emission spectrum was recorded by a Zolix Analytical Instrument (fluoroSENS 9000 A) under the excitation of a 980 nm laser. The down-shifting NIR-II emission spectrum was detected using a NIR spectrometer (NIRQuest512, Ocean Optics) in the 900–1700 nm spectral region under a 980 nm laser excitation. The element composition in silk was determined using inductively coupled plasma mass spectrometry (ICP-MS, iCAP RQ, Germany). The surface ligands of the silk were detected by Fourier transform infrared spectrometer (FTIR, PerkinElmer Frontier Mid-IR FTIR spectrometer). The strain-stress data were measured using silk fibers and a SHIMADZUAG-IS tensile tester. The dynamic light scattering (DLS) and zeta potential of the nanocrystals were recorded on the Zetasizer Nano ZS system.

Preparation of the phantom implanted biological stent models

The NIR-II emitting NaYF4-silk was soaked in alcohol at a concentration of 99% (w/v) with 6 h for sterilization before implantation. Then, the NaYF4-silk was cut into square and “H”,“N”,“U” shapes as biological stent models. The as-prepared models were baked in a 60 °C oven. The operative area of the mouse abdomen skin was cleaned with alcohol, and the implanted biological stent models were implanted on the shaved abdomen of each mouse at depth of 0.8 mm.

Cell viability assay

The cytotoxicity of the as-prepared samples was studied by using a 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) experiment. The 4T1 cells were cultured in 96-well plates. After 24 h of incubation, the medium was removed and a series of concentrations of NaYF4@SiO2 solutions were added into the well and further cultured for another 24 h. After that, the cell viability was evaluated using a MTT assay.

Histological tests

In order to assess the in vivo toxicity of the NaYF4@SiO2 nanoparticles, a histological test was performed. The Kunming mice were intravenously injected with NaYF4@SiO2 nanoparticles dispersed in water at a total dose of 150 μL (4 mg mL−1) for 15 and 30 d as the test group. Untreated mice were used as the control group. Then, the heart, liver, spleen, lung, and kidney were collected from the test and control groups. These organs were sliced and stained with hematoxylin and eosin (H&E) to monitor the histological changes.

In vivo optical imaging

In vivo optical imaging of the NaYF4-silk and the implanted biological stent models in the UC region was performed by using a multi-modal imaging system (Bruker In Vivo FX Pro) excited by a 980 nm laser at a power density of 100 mW cm−2 and an exposure time of 30 s. The NIR-II optical imaging of silkworms, NaYF4-silk, and the implanted biological stent models was performed using the home-made NIR-II imaging system32 under excitation by a 980 nm laser with a power density of 100 mW cm−2 and an exposure time of 2 s. All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Hunan Normal University, and the experiments were approved by the Animal Ethics Committee of Hunan Province.

Results and discussion

The general strategy for designing NIR-II emitting silk-based materials by the feeding of rare earth nanoparticles is demonstrated in Scheme 1. Then, the NIR-II emissive properties and structure of the silk hybrid materials were studied. As demonstrated in Fig. 1a and Fig. S1 (ESI), the NaYF4 nanocrystals present a uniform size with a high monodispersity. For further biological applications, the surface coating of SiO2 (Fig. 1b and c) can provide the core nanoparticles with protection against the external environment and improve the biocompatibility of NaYF4 nanomaterials.17,33 As demonstrated in Fig. 1 and Fig. S2 (ESI), the NaYF4 nanocrystals present a good monodispersity with an average size of about 10 ± 0.9 nm. After coating SiO2, the average size of the NaYF4@SiO2 is increased to 17 ± 2 nm. The DLS measurement (Fig. S3a, ESI) was also performed to evaluate the degree of aggregation of the samples. The mean hydrodynamic diameter of the NaYF4@SiO2 nanocrystals was estimated to be 36 nm with a standard deviation of 1.97 nm. Furthermore, the surface of the NaYF4@SiO2 nanocrystals indicates the negatively charged nature in the water and the zeta potential of the nanocrystals is about −30 mV (Fig. S3b, ESI). The characteristic emission peaks (Fig. 1d) located at 522/541, 650, and 1525 nm of both the NaYF4@SiO2 nanocrystals embedded silk (NaYF4-silk) hybrid and the pure NaYF4@SiO2 nanocrystals were detected under the excitation of a 980 nm laser, corresponding to the 2H11/2/4S3/24I15/2, 4F9/24I15/2 and 4I13/24I15/2 electronic transitions of Er3+, respectively (Fig. 1f).34 Moreover, the photo-stability curve (Fig. S4, ESI) reveals that the designed rare earth-based nanoparticles present a superior photo-stability in water with a photo-bleaching degree of 0.72% in 40 min, which is significantly lower than the previously reported small molecular probe.24 To further reveal the NIR-II emissive properties, in vitro phantom imaging of the pure silk and NaYF4-silk hybrids was performed. As shown in Fig. 1e, there are no optical signals observed in the pure silk. In contrast, obvious UC and NIR-II fluorescence signals were detected in the NaYF4-silk hybrids, validating the successful construction of the UC/NIR-II emissive silk. Additionally, the NaYF4-silk hybrids present a high S/N of 21, implying its high potential for in vivo NIR-II optical imaging, owing to the low photon scattering losses.28 Moreover, the in vitro NIR-II phantom bioimaging (Fig. S5a, ESI) revealed that the designed NaYF4-silk hybrid presented a superior photo-stability over 20 d. The in vivo stability of the implanted silk-based stent model was also monitored for 20 d. As demonstrated in Fig. S5b (ESI), the NIR-II signal intensity was gradually decreased, indicating the partial biodegradation of the silk hybrid.35 To further reveal the incorporation of the rare earth nanoparticles in the silk matrix, an XRD test was performed. As demonstrated in Fig. 1g, two characteristic broad peaks located at 2θ = 20.66° and 28.74° are observed in the pure silk and NaYF4-silk hybrids, which are assigned to the crystalline diffraction of silk II and silk I, respectively.36–38 Moreover, compared to the pure silk, some additional diffraction peaks located at 2θ = 61°, 62°, 71°, 72°, and 78° were detected in the NaYF4-silk hybrids, which are attributed to the (112), (220), (311), (212) and (401) crystal planes of the hexagonal phase NaYF4 (JCPDS File no. 16-0334), respectively. It should be noted that the pure silk presents a large amorphous crystal background from 15° to 55°, resulting in diffraction peaks for NaYF4 in low diffraction angle being missed, which is consistent with the XRD results obtained for the silk incorporated with other nanoparticles.39 The XRD results unambiguously reveal that the NaYF4@SiO2 nanoparticles are successfully incorporated into the silk matrix, leading to the additional downshifting emission properties of the silk hybrids.
image file: c9tb02685a-s1.tif
Scheme 1 A schematic illustration showing the design of NIR-II emissive silk using a general strategy of feeding rare earth-based NaYF4@SiO2 nanocrystals to silkworms for in vivo NIR-II bioimaging.

image file: c9tb02685a-f1.tif
Fig. 1 Typical TEM images of (a) pure NaYF4:Gd/Yb/Er and (b) NaYF4@SiO2, and (c) a scanning TEM (STEM) image of NaYF4@SiO2. (d) UC and down-shifting NIR-II emission spectra of pure NaYF4@SiO2 and NaYF4-silk hybrids under the excitation of a 980 nm laser. (e) In vitro phantom imaging of the pure silk and NaYF4-silk hybrid in the UC and NIR-II regions. (f) A simplified energy level diagram for the UC and down-shifting NIR-II emission. (g) XRD patterns of pure silk, NaYF4@SiO2, and the NaYF4-silk hybrid.

To achieve the NIR-II emissive silk, the silkworms were fed with mulberry leaves, which were pre-sprayed with NaYF4@SiO2 water solutions (Fig. 2a). A total of 40 silkworms were divided into two groups by feeding with pure mulberry leaves (control group) and NaYF4@SiO2 stained mulberry leaves (NIR-II emissive silk). No differences were observed between the silkworms fed with NaYF4@SiO2 and the control group until they produced cocoons, indicating that diets containing the small proportion of NaYF4@SiO2 used in this study were safe for raising silkworms. Compared to the control group (Fig. 2c), the silkworms fed with NaYF4@SiO2 stained mulberry leaves (Fig. 2d–g) present a bright NIR-II fluorescent signal in the body under 980 nm laser excitation. These results indicate that the ingested nanomaterials were successfully incorporated into the silkworm. Meanwhile, NIR-II imaging (Fig. S6, ESI) of the feces collected from silkworms after feeding with NaYF4@SiO2 was performed and obvious fluorescence signals were observed. This result revealed that some of ingested nanoparticles were excreted from silkworms in the feces. In addition, in vivo NIR-II imaging of mice injected with NaYF4@SiO2 was also performed. As shown in Fig. S7 (ESI), a significant NIR-II signal from the liver and spleen region can be detected after 30 min, and the main excretion mechanism of NaYF4@SiO2 may be the hepatic excretion route. These findings indicate that the NIR-II emitting NaYF4@SiO2 nanocrystals can also be applied as optical nanoprobes for in vivo optical bioimaging.


image file: c9tb02685a-f2.tif
Fig. 2 (a) A schematic diagram showing the feeding of silkworms with mulberry leaves pre-sprayed with NaYF4@SiO2 water solution. (b) A digital photograph of a silkworm. (c) In vivo NIR-II optical imaging of a silkworm (control group). (d)–(g) In vivo NIR-II optical imaging of a silkworm fed with mulberry leaves pre-sprayed with NaYF4@SiO2 water solution.

To further reveal the NIR-II emission from the cocoon, in vitro phantom optical imaging of the cocoon (Fig. 3a–c) was performed in the NIR-II region with a field of view (FOV) of 26 × 21 mm. As demonstrated, the silks presented an obvious NIR-II signal, further validating the successful incorporation of the NaYF4 nanoparticles into silk. A region of silk was chosen for evaluating the high spatial resolution imaging (Fig. 3d and e). As illustrated, all of the small silks were distinctly identified. Then, cross-sectional analyses (Fig. 3f and g) across the colour lines in Fig. 3e were performed and fitted using Gaussian functions to determine the widths. The silk widths were determined to be 42 and 67 μm, which were consistent with the SEM observation (Fig. 3h and i). The silks containing NaYF4 were further processed into a phantom silk thread (Fig. 3j). A remarkable NIR-II luminescence (Fig. 3k and l) can be observed, verifying the possibility of real-time visualization of the state of the implanted silk thread. To further reveal the incorporation of nanoparticles in silk, concentration-dependent NIR-II emission (Fig. S8, ESI) was first evaluated, indicating an excellent linear correlation between the concentration and the NIR-II fluorescence intensity. From Fig. 3c and e, the cocoon also presented a relatively uniform NIR-II optical signal with small bright spots. Based on this, we speculate that the distribution of nanoparticles in silk is relatively uniform with small aggregations. To assess the amount of incorporation of the nanoparticles in silk, an ICP-MS test was performed, demonstrating the presence of Gd, Y, Yb, Er and Si elements (Tables S1 and S2, ESI) in the NaYF4-silk hybrids after feeding nanoparticles. These findings further verify that the NaYF4 nanoparticles are successfully incorporated into silk and the NIR-II emission in the silk hybrids originates from the embedded nanoparticles in the silk. In addition, an FTIR study was performed to reveal the possible surface ligands of silk. As shown in Fig. S9 (ESI), no significant differences were observed between pure silk and the NaYF4-silk hybrids, suggesting that there were no strong covalent interactions40 between the silk fibroin and the NaYF4@SiO2 nanocrystals. It should be noted that both the pure silk and NaYF4-silk present a peak located at about 1407 cm−1 related to the vibrations of the CH group, implying that the embedded NaYF4 nanoparticles in silk do not significantly change the inter-structure of the silk protein.41 We have further studied the mechanical properties of the silk. As demonstrated in Table S3, the NaYF4-silk presents a similar tensile strength to pure silk, demonstrating the limited influence on the mechanical properties.


image file: c9tb02685a-f3.tif
Fig. 3 (a) A schematic illustration of the in vitro phantom imaging of hybrid silk under the excitation of a 980 nm laser. (b) A digital photograph of a silk cocoon. (c) NIR-II fluorescence imaging of the silk cocoon. (d) A bright field image of the NaYF4-silk hybrids. (e) NIR-II fluorescence imaging of silk with a FOV of 26 × 21 mm. (f) and (g) The corresponding cross-sectional fluorescence intensity profiles (black) and Gaussian fit (red, purple) along the red and purple lines in (e). (h) An SEM image. (i) A high-magnification SEM image. (j) A typical photograph of the explored silk thread. (k) In vitro phantom imaging of the silk thread under 980 nm laser excitation. (l) High magnification NIR-II fluorescence imaging of the silk thread.

Owing to the excellent in vitro phantom imaging, in vivo NIR-II imaging guided real-time visualization (Fig. 4a) of the implanted biological stent model made from the hybrid silks was performed under the irradiation of a 980 nm laser with a power density of 100 mW cm−2. Before the in vivo test, the preliminary toxicity and inflammatory responses were evaluated by directly exposing the mouse skin to the hybrid silks for 96 h (Fig. 4b), indicating no allergic or inflammatory response occurred. We have further studied the in vitro and in vivo toxicity of the rare earth-based nanoparticles. As shown in Fig. S10 and S11 (ESI), the results demonstrated that the rare earth-based nanoparticles presented a low cytotoxicity and high biocompatibility. Then, the in vivo UC and NIR-II imaging (Fig. 4c) of the implanted biological stent model with a square shape were performed. The maximum S/N in the NIR-II imaging (Fig. 4d) was evaluated to be approximately 10-fold larger than that of the UC imaging, verifying the superior sensitive bioimaging in NIR-II imaging beyond 1500 nm compared to UC imaging. Encouraged by the high-performance NIR-II emission of the NaYF4-silk hybrids, the other preliminary implanted biological stent models (“H”,“N”,“U”, Fig. 4e) made from the hybrid silks were further used for in situ visualization. As demonstrated, all of these letters present a bright NIR-II fluorescence signal. After subcutaneous implantation of the “H”,“N”,“U” stent in the mouse abdomen (Fig. 4f), clear “H”,“N”,“U” shapes (Fig. 4g) can be obviously observed in the NIR-II imaging, further validating the feasibility of in situ deep-tissue monitoring of the state of the implanted biological stent. Therefore, the designed NIR-II emissive silk hybrids can be used as promising biological stent materials for highly sensitive visualization of the in vivo state of the implanted devices.


image file: c9tb02685a-f4.tif
Fig. 4 (a) A schematic illustration of the in vivo NIR-II imaging of the implanted biological stent model made from the hybrid silks. (b) Allergy test of a mouse by directly exposing the abdomen skin to the hybrid silks for 96 h. (c) In vivo UC/NIR-II imaging of the subcutaneously implanted stent model with a square shape made from the hybrid silk under 980 nm laser excitation (inset: a digital photograph of the implanted stent model). (d) The corresponding fluorescence intensity profiles across the purple and blue lines in (c). (e) A digital photograph and in vitro phantom NIR-II imaging of other phantom biological stents with “H”, “N”, and “U” shapes. (f) and (g) In vivo NIR-II imaging after subcutaneously implanting the models in the mouse abdomen under the excitation of a 980 nm laser.

Conclusions

In summary, optically functionalized silks with NIR-II emission were explored using a general method of feeding silkworms with rare earth-based nanoparticles. The silks incorporated with the rare earth nanoparticles presented excellent NIR-II emission beyond 1500 nm and were successfully used for in vivo highly sensitive NIR-II bioimaging with good biocompatibility. More importantly, the designed NIR-II emissive silks can be used for in situ monitoring of the in vivo states of the implanted biological stents.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 21671064), the Science and Technology Planning Project of Hunan Province (No. 2017RS3031), the Scientific Research Fund of Hunan Provincial Education Department (19A329), the Natural Science Foundation of Hunan Province, China (No. 2019JJ10002), and the Hunan Provincial Innovation Foundation For Postgraduates (CX2018B238).

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Footnotes

Electronic supplementary information (ESI) available. See DOI: 10.1039/c9tb02685a
These authors contributed equally to this paper.

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