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Autofluorescent gelatin nanoparticles as imaging probes to monitor matrix metalloproteinase metabolism of cancer cells Bo Cai,1 Lang Rao,1 Xinghu Ji,2 Lin-Lin Bu,3 Zhaobo He,1 Da Wan,1 Yi Yang,1 Wei Liu,1 Shishang Guo,1 Xing-Zhong Zhao1 1

Key Laboratory of Artificial Micro- and Nano-Structures of Ministry of Education, School of Physics and Technology, Wuhan University, Wuhan 430072, China 2 Key Laboratory of Analytical Chemistry for Biology and Medicine of Ministry of Education, College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China 3 The State Key Laboratory Breeding Base of Basic Science of Stomatology and Key Laboratory of Oral Biomedicine, Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan 430072, China Received 6 May 2016; revised 27 June 2016; accepted 30 June 2016 Published online 22 July 2016 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35823 Abstract: In this paper, autofluorescent gelatin nanoparticles were synthesized as matrix metalloproteinase (MMP) responsive probes for cancer cell imaging. A modified two-step desolvation method was employed to generate these nanoparticles whose size was controllable and had stable autofluorescence. As glutaraldehyde was introduced as the crosslinking agent, the generation of Schiff base (C@N) and double carbon bond (C@C) between glutaraldehyde and gelatin endowed these gelatin nanoparticles distinct autofluorescence. Considering MMPs were usually overexpressed on the surface of cancer cells and they had degradation ability

toward gelatin, we utilized these nanoparticles as imaging probes to responsively monitor the MMP metabolism of cancer cells according to the luminance change. As fluorescent probes, these nanoparticles had facile synthesis procedure and good biocompatibility, and provided a smart strategy to C 2016 Wiley Periodicals, Inc. J monitor cancer cell behaviors. V Biomed Mater Res Part A: 104A: 2854–2860, 2016.

Key Words: gelatin, nanoparticles, autofluorescence, matrix metalloproteinase, cell imaging

How to cite this article: Cai B, Rao L, Ji X, Bu L-L, He Z, Wan D, Yang Y, Liu W, Guo S, Zhao X-Z. 2016. Autofluorescent gelatin nanoparticles as imaging probes to monitor matrix metalloproteinase metabolism of cancer cells. J Biomed Mater Res Part A 2016:104A:2854–2860.

INTRODUCTION

Bioimaging provides us a wide range of choices to visualize and quantify multiple events in living cells dynamically.1,2 Among various bioimaging methods like (near) infrared and Raman spectroscopy, magnetic resonance imaging (MRI), radioimaging, CT imaging, positron emission tomography, and so forth, fluorescent imaging is the most widespread due to its sensitivity, selectivity and versatility.3 Recent advances in fluorescence imaging and probes enable us to image cellular activity in a real-time manner,4 especially about those metabolic activities in cancer cells (e.g., communications, invasiveness and metastasis), which greatly helps to reveal valuable insights about cancer occurrence, development and metastasis.5–8 Commonly-used fluorescent probes include fluorescently doped silicas and sol–gels, hydrophilic/hydrophobic polymers, quantum dots, carbon dots, upconversion nanoparticles, noble metal nanoparticles (e.g., gold or silver nanoparticles), and so on.3 However, when applying such probes for biological or biomedical imaging, the biocompatibility is always a big concern either

in vitro or vivo.9 The cytotoxicity of these nanomaterials or their byproducts causes cell dysfunctions and then leads to the failures of recording cell behaviors faithfully. A variety of coating methods are developed to increase the biocompatibility of these imaging nanoparticles, such as the PEGylation10 and the camouflage strategies using cellular membranes.11,12 Nevertheless, these coating procedures are always complicated to realize, thus hindering their popularization. So materials that facilitate the easy nanoparticle synthesis as well as maintain feasible biocompatibility attract more and more attention in bioimaging researches. Given abundant sources and excellent biocompatibility, natural extractives are more, and more preferred as the raw materials for designing nanomaterials toward biomedical applications.4 Their mimesis to the biological tissues and low antigenicity ensure the safety for in vitro/vivo applications. Also their various amounts of radicals enable versatile bioconjugation with functional agents (e.g., antibodies, luminous molecules and so on), which largely extends their functionalities.13–15 Among these extractives, gelatin, which

Additional Supporting Information may be found in the online version of this article. Correspondence to: X.-Z. Zhao; e-mail: [email protected]

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FIGURE 1. Schematic showing the MMP-responsive probing by using degradable autofluorescent GelNPs.

is the natural peptide/protein extractive from animal collagen, has been widely used as pharmaceutical carrier for drug delivery.16–19 Since the development of the facile twostep desolvation method for gelatin nanoparticle (GelNPs) synthesis,20 GelNPs have been widely used for cancer targeting and drug delivery.21–23 Herein, in order to utilize the great biocompatibility of gelatin and the facile synthesis procedure of GelNPs for cell imaging, we synthesized the autofluorescent GelNPs using a modified two-step desolvation method. Through introducing glutaraldehyde (GTA) as the crosslinking agent, the fluorescent bonds of Schiff base (C@N) and double carbon bonds (C@C) were generated among the amine groups of gelatin and aldehyde groups of GTA, thus endowing the GTA crosslinked GelNPs autofluorescence. This kind of photoluminescence showed robust photostability among the wavelength of 522 nm, demonstrating its potentials in cell imaging. As known, matrix metalloproteinases (MMPs), the enzymes which can regulate the tumor microenvironment, are usually overexpressed in cancer cells and can degrade the gelatin.24 This property of cancer cells enabled our autofluorescent GelNPs responsive to the MMP metabolism of the targeted cancer cells (Fig. 1). After labeling to cancer cells, those GelNPs showed the autofluorescence to display the cell morphology. And as the metabolism of MMPs went on, GelNPs were gradually degraded and thus the fluorescence intensity decreased as well. So according to the measurement of the fluorescence variation, we could effectively monitoring the MMP activity in cancer cells, which would provide us more insights about the cancer cell invasion and metastasis that were closely related to MMPs. It could be expected that our autofluorescent GelNPs would provide smart, facile and biocompatible cellular imaging probes to investigate the behaviors of cancer cells. MATERIALS AND METHODS

Chemicals and reagents Gelatin (Type A, 300 bloom) from porcine skin, glutaraldehyde (Grade I, 50%) (GTA), acetone, 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI), 3-(4,5-dimethylthiazol-2yl)22,5-diphenyltetrazolium bromide (MTT), hydrochloric

acid (HCl), paraformaldehyde (PFA) and acidified sodium dodecyl sulfate (SDS, 99%) were purchased from SigmaAldrich. Dulbecco’s modified eagle medium (DMEM, Hyclone, high glucose), PBS (Hyclone, 13, 0.0067M PO431), penicillin– streptomycin (10,000 U/mL, Gibco) and 0.25% trypsin–EDTA (Gibco, 13) were obtained from Thermo-Fisher Scientific. Fetal bovine serum (FBS) was obtained from Invitrogen. Human tissue inhibitor of metalloproteinases 2 (TIMP2) was purchased from Sino Biological. Deionized water (DI water) used for solutions was purified by a Millipore Direct-Q3 water purification system (Millipore, MA, USA). Instruments The morphology and size of GelNPs were characterized by field emission scanning electron microscope (FESEM, S4800, Hitachi, Japan) and dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern Instruments, UK), respectively. A UV–Vis spectrophotometer (UV-2550, Shimadzu, Japan) and a spectrofluorephotometer (RF-5301PC, Shimadzu, Japan) were employed for measuring UV–Vis absorption and fluorescence spectra. The Fourier transform infrared (FTIR) spectroscopy spectra was measured by a FTIR spectrometer (Nicolet 5700, Thermo-Fisher Scientific, USA). A fluorescent microscope (IX81, Olympus, Japan) was utilized for capturing bright field/fluorescent cellular images using image analysis software (Image Pro Plus). The morphology of GelNPs was characterized using a transmission electron microscope (TEM, JEM-2010 ES500W, Japan) operating at 200 keV. Confocal microscopy images were acquired by the A11 confocal laser microscope system (Nikon, Japan). Gelatin nanoparticle synthesis and purification Gelatin nanoparticles were synthesized according to a modification of the two-step desolvation method. 20 At first, 1.25 g gelatin powders were dissolved in 25 mL DI water at 508C. Then 25 mL acetone was added into the solution dropwise at 12 mL/min and stirred at 600 rpm during the heating. After the addition of acetone, the stirring was turned off. And exactly 1 min later, the supernatant containing the low molecular weight gelatin was decanted completely. The sediment of about 2 g hydrogel was redissolved at 508C using

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25 mL DI water again, and then half of the solution was removed. The pH of this solution was adjusted to 2.5. Subsequently, another 34 mL acetone was added dropwise at 1 mL/min, with stirring of 1000 rpm and temperature of 508C. When the acetone was totally added, the solution appeared cloudy due to the scattering of the synthesized gelatin nanoparticles. After then, the solution was removed from heating while kept stirring at 1000 rpm. Another 500 lL acetone containing different amounts of 50% glutaraldehyde solution (according to the need of the experiments) was added to the solution at 50 lL/min to crosslink the nanoparticles. Finally, the solution was stirring at 1000 rpm for about 16 h. The obtained nanoparticles were collected by centrifugation (10,000 rpm for 10 min) and washed three times with deionized water. Then these nanoparticles were lyophilized and kept at 48C for possible usage. Cell culture and cytotoxicity assay MCF-7 human breast cancer cells, CAL-27 tongue squamous cell carcinoma cell line and LO2 human normal liver cell line were cultured in DMEM with 10% FBS and 1% penicillin and streptomycin at 378C and 5% CO2 atmosphere in a cell incubator (Thermo Forma Series II, Thermo Scientific). In vitro cytotoxicity was determined through an established colorimetric MTT assay. For MTT assay, MCF-7 cells were seeded into 96-well cell culture plate at 1.0 3 104/well and then incubated for 24 h (378C, 5% CO2). After serum starvation for 24 h, MCF-7 cells were incubated with various concentrations of GelNPs (0, 0.1, 0.25, 0.5, 0.75, 1, and 1.5 mg/mL) or 0.1 mg/mL GelNPs with different weight ratio of GTA to gelatin (0, 0.005, 0.0125, 0.025, 0.05, 0.075, 0.1) in serum-free media for 48 h, respectively. For each well, 10 lL stock MTT (5 mg/mL) was added with further incubation at 378C for 4 h, acidified SDS was used to lyse the cells. The absorbance at 570 nm was tested by using the microplate reader (Bio-Rad 680, USA). All independent experiments were carried out three times at least. Based on these experiments, a linear relationship between cell number and optical density was achieved, and then it was capable to accurately quantify the changes in the rate of cell proliferation. Fluorescent cellular labeling MCF-7 cells were plated on to a 96-well plate at 1 3 105/well and incubated for 24 h before exposure to GelNPs and GelNPs/TIMP2. To see the fluorescent labeling of cells using GelNPs, the MCF-7 cells were first incubated in DMEM with 0.1 mg/mL GelNPs for 1 or 12 h, and then the cells were washed with PBS for three times and fixed by 4% PFA for 15 min. Another three times washes were implemented and 100 ng/mL DAPI was used to stain the cell nuclei. For the MMP responsiveness probing, the MCF-7 cells were firstly pretreated with DMEM with TIMP2 (0.1 mg/mL) to block MMPs. Then the cells were washed by PBS for three times and subsequently incubated in DMEM with 0.1 mg/mL GelNPs and 0.1 mg/mL TIMP2 for 12 h. After then, these cells were washed with PBS for three times and fixed with 4% PFA for 15 min. Another three times washes were implemented and 100 ng/mL DAPI was used to stain the cell nuclei.

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Confocal microscopy for DAPI and GelNP fluorescence At first, MCF-7, CAL-27 and LO2 cells were plated on to 12 glass-bottom dishes (15 mm, NEST Corporation) (4 dishes for every cell line) and incubated for 24 h. To monitor the MMP-responsive fluorescent labeling of MCF-7, CAL-27, and LO2 cells using GelNPs, all the three kinds of cell lines were first incubated in DMEM with 0.1 mg/mL GelNPs for 1, 4, 8, and 12 h, respectively. And then the cells were washed with PBS for three times and fixed by 4% PFA for 15 min. Another three times washes were implemented and 100 ng/mL DAPI was used to stain the cell nuclei. After washed again by PBS for three times, the cells were observed under a confocal microscope. For the MMP-inhibited probing, the CAL-27 cells were firstly pre-treated with DMEM with TIMP2 (0.1 mg/mL) to block MMPs. Then, the cells were washed by PBS for three times and subsequently incubated in DMEM with 0.1 mg/ mL GelNPs and 0.1 mg/mL TIMP2 for 1, 4, 8, and 12 h respectively. After then, these cells were washed with PBS for three times and fixed with 4% PFA for 15 min. Another three times washes were implemented and 100 ng/mL DAPI was used to stain the cell nuclei. RESULTS AND METHODS

Synthesis and characterization of GelNPs The synthesis of GelNPs was according to a modification of the two-step desolvation method.20 A typical SEM image of the synthesized GelNPs was shown in Figure 2(a), which displayed the fine sphere morphology of these nanoparticles. TEM images also indicated that GelNPs were spherical and monodispersed (Supporting Information Fig. S1). Figure 2(b) gave out the DLS hydrodynamic diameter distribution of the GelNPs synthesized under the same conditions as Figure 2(a). It could be seen that these GelNPs showed narrow size distribution among 200 nm. However, it was noteworthy that the hydrodynamic diameters of GelNPs measured by DLS (about 200 nm) was larger than what the SEM images showed (about 100 nm), which was due to the significant difference between the measurement principle of the two analytical methods used here. For DLS analysis, the particles were dispersed and measured in DI water, whereas SEM detection had to be performed with dried analytes under vacuum condition. Using the two-step desolvation method, the size of the synthesized GelNPs was regulated by a series of parameters, mainly the temperature, pH, speed of the acetone addition, and the amount of GTA for crosslinking.25 Considering the autofluorescence phenomenon was tightly related to the crosslinking agent GTA,26 we mainly investigated how the GTA amount added during the synthesis procedure influenced the diameter of those synthesized GelNPs. GTA was generally used to crosslink GelNPs to avoid possible nanoparticle disintegration, which was realized through the Schiff base generation between amidogen of gelatin and aldehyde group of GTA.27 The weight ratio of GTA to gelatin was employed as the parameter to investigate the relationship between the degree of crosslinking and the particle size. As shown in Figure 2(c), GelNP size was continuously

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FIGURE 2. (a) Typical SEM image of the synthesized GelNPs. (Inset) A SEM magnification of a GelNP. (b) Typical DLS analysis showing GelNP size distribution. (c) GelNP size regulation by the weight ratio of GTA to gelatin.

decreasing when the amount of GTA increased, which was due to the increasing degree of crosslinking among gelatin molecules inside the nanoparticles. However, the polydispersity index (PDI) varied a lot stochastically during the change of GTA dose. Mechanism of GelNP autofluorescence Indeed, the usage of GTA as the crosslinking agent not only stabilized the nanoparticles but also generated an autofluorescence signal. As reported by previous literatures, GTA would be condensed and dehydrated, and then generated unsaturated a–b double bonds (C@C).28,29 According to the reaction formula of gelatin and condensation product of GTA, two different kinds of double bonds could be found in the structure of GTA crosslinked GelNPs, i.e., C@C from condensation moiety of GTA, and C@N from the crosslinking reaction moiety between the amine groups in gelatin and aldehyde groups in the condensation product of GTA [Fig. 3(a)]. These two kinds of double bonds formed a conjugated planar structure in GelNPs, and it was known that C@N bonds were associated with n–p* transitions and that C@C bonds were related to p–p* transitions. The transition of electrons between these different orbitals generated the autofluorescence.30 Figure 3(b) showed the FTIR spectra which confirmed the presence of C@N and C@C bonds after GTA crosslinking. Compared to pure gelatin powders, two typical absorption peaks could be observed for GelNPs crosslinked by GTA. The peak with a strong intensity centered at 1643 cm21 was attributed to the imine bond

(C@N) and the peak at 1546 cm21 was associated with the C@C bond. The similar FTIR results of GTA crosslinked materials containing amino groups (such as gelatin and chitosan) were also reported in other literatures.27,28,31,32 We also investigated the fluorescence of different reagents used in the GelNP synthesis under excitation of 475 and 530 nm (usually used in fluorescent microscopy), as shown in Supporting Information Figure S2. It could be seen that only when gelatin (GEL) itself or GelNPs were crosslinked by GTA, C@N bonds were generated and then distinct autofluorescence was emitted, while acetone, gelatin, or GTA alone had no distinct fluorescence. For GTA only, although it could condense and generate C@C bonds, its autofluorescence could be negligible due to the low condensed degree. And the main source of the autofluorescence emitted by our synthesized GelNPs were still the C@N bonds of the Schiff base from the crosslinking of GTA and gelatin. Characterization of GelNP autofluorescence To further investigate the autofluorescence of the GTA crosslinked GelNPs, we implemented several optical characterizations. Figure 4(a) gave out the typical fluorescent excitation and emission spectra of these GelNPs. It could be seen that the emission peak occurred at 522 nm upon excitation at 491 nm. As the GelNP excitation/emission spectra contained quite a long range of wavelength, the autofluorescence of GelNPs varied its color for observation when excited under different excitation wavelength.


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FIGURE 3. (a) Schematic showing the mechanism of GelNP autofluorescence. (b) Typical FTIR spectra of gelatin and GTA crosslinked GelNPs. (Inset) A magnification of the GTA crosslinked GelNP FTIR spectra between wave number 1350 and 1750 cm21.

Also we investigated the photostability of the autofluorescence of the GelNPs [Fig. 4(b)]. These GelNPs were dispersed in DI water and stored in a transparent tube, which was exposed in the daylight ambient conditions directly during our experiment. Then we measured the fluorescent

intensity of such solution using a fluorescent microscope at different time point. It was noticeable that the fluorescent intensity of these GelNPs (emission at 520 and 590 nm) changed little for at least 1 week, which undoubtedly facilitated their possible use in biological applications and

FIGURE 4. (a) Typical fluorescence excitation and emission curves of the autofluorescent GelNPs. (b) Photostability of the GelNPs stored in ambient conditions at the emission of 520 and 590 nm. (c) UV–Vis spectra of GelNPs crosslinked with different ratio of GTA to gelatin from 0 to 0.1. (Inset) A magnification of GelNP UV–Vis spectra from wavelength 200 to 400 nm. (d) Fluorescent emission spectra of GelNPs crosslinked with different ratio of GTA to gelatin from 0 to 0.1.

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FIGURE 5. (a) MCF-7 cell viability in the presence of GelNPs with different concentrations. (b) MCF-7 cell viability in the presence of 0.1 mg/mL GelNPs crosslinked with different ratio of GTA to gelatin. (c) Probing MMP responsiveness using autofluorescent GelNPs for specific cancer cell imaging. The scale bars are 20 mm.

indicated that our GelNP probe needs no specific store requirements. And the fluorescent intensity of the 520 nm emission was nearly five times stronger than the 590 nm emission, which matched with the emission spectra in Figure 4(a) very well. As the autofluorescence was generated from the C@N and C@C bonds that related to GTA, it was obvious that the amount of GTA used for gelatin crosslinking counted importantly for the intensity of the autofluorescence. Figure 4(c) and (d) demonstrated how the weight ratio of GTA to gelatin in GelNP synthesis influenced the optical properties of GelNPs. When more GTA was added for more thorough crosslinking in GelNPs, more Schiff base bond would generate and induce stronger fluorescence. As shown in Figure 4(c), the gradual increase in the absorbance of GelNPs within 250–270 nm indicated that the increase of fluorescence intensity was caused by the formation of imine bonds, which was obviously induced by the higher weight ratio of GTA to gelatin.29 And the fluorescent emission spectra of

GelNPs crosslinked with different amount of GTA in Figure 4(d) demonstrated this conclusion more clearly, that was, when more GTA added during GelNP synthesis, the autofluorescence became brighter. GelNP cytotoxicity and MMP responsiveness probing In order to utilize GelNPs for cell biological assays, we investigated the cytotoxicity of these nanoparticles at first. As shown in Figure 5(a), the viability of the MCF-7 cells decreased slightly with the dose increase of GelNPs used. It could be seen that when the GelNP dose was as low as 0.1 mg/mL, the cell viability was almost the same compared to the control group. Although the GelNP concentration was quite low, these nanoparticles could still provide fluorescence that was strong enough for the metabolism observation. Considering the toxicity of GTA to cells, we also investigated the influence of the amount of GTA used in synthesis procedure on the cell viability. The results in Figure 5(b) indicated that, as GTA was consumed during the

FIGURE 6. The confocal microscopy showing continuous monitoring of the DAPI and GelNP fluorescence of MCF-7 cells without the TIMP2 treatment. The scale bars are 20 mm.


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crosslinking inside the GelNPs, the amount of GTA used for GelNP synthesis nearly did not harm the cells at all. Because MMPs were usually up-regulated in the cancer cells and gelatin could be degraded by these cell-secreting enzymes, we employed the synthesized autofluorescent GelNPs to study the MMP metabolism of cancer cells [Fig. 5(c)]. Firstly, GelNPs were incubated with MCF-7 cells. Then the nanoparticles could attach to the surface of these cancer cells to exhibit green fluorescence under the excitation of 491 nm (control group). As time went on, these cells secreted MMP enzymes and gradually degraded those GelNPs, causing the decrease of the autofluorescence intensity (control-12 h group). However, if the MMP inhibitor TIMP2 was introduced, GelNPs were kept well on the cell surface and made MCF-7 cells to still show green fluorescence (TIMP-12 h group). Similar results were also achieved using another kind of cancer cell line CAL-27 (tongue squamous cell carcinoma) (Supporting Information Fig. S3). Considering the fact that MMPs were up-regulated in almost every type of human cancer cells,24 we could monitor the MMP-responsive activity of cancer cells according to variation of the intensity of the GelNP autofluorescence. Also, we utilized confocal microscopy to continuously monitor the MMP metabolism of MCF-7 cells that were not treated with TIMP2. As shown in Figure 6, the green autofluorescence of GelNPs gradually decreased and finally disappeared as time went on, indicating the continuous degradation of GelNPs by the MMP metabolism of MCF-7 cells. However, for those cells that had normal MMP expression (e.g., human normal liver cells LO2),22 GelNPs were almost not degraded and thus their autofluorescence could be maintained during the coincubation with such cells (Supporting Information Fig. S4). CONCLUSION

In conclusion, we developed autofluorescent GelNPs as a kind of smart and biocompatible imaging probe for responsive monitoring the MMP metabolism of cancer cells. Through adjusting the amount of GTA as the crosslinker, we could effectively control the size and the intensity of autofluorescence of these nanoparticles. Due to the generation of Schiff base and double carbon bonds during the crosslinking procedure, the synthesized nanoparticles showed stable autofluorescence with the wavelength around 522 nm. Given the over-expression of MMPs in cancer cells and their ability of degrading gelatin, these autofluorescent GelNPs were employed as a kind of smart imaging probe which was responsive to the MMP metabolism. ACKNOWLEDGMENTS

The authors thank Mr. Junhua Xu, Mr. Wenqing Li, Mr. Xianyin Song, Mr. Zhuo Xing, Mr. Yunfeng Zuo from School of Physics and Technology, Wuhan University and Ms. Jingwen Lan from College of Chemistry and Molecular Sciences, Wuhan University for beneficial discussions and instrumental support. This project was funded by the National R&D Program for Major Research Instruments (No. 81527801), the National

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Science Fund for Talent Training in Basic Science (No. J1210061), the National Natural Science Foundation of China (Nos. 51272184, 81572860, and 61474084), and the Fundamental Research Funds for the Central Universities (No. 2015202020202). REFERENCES 1. Fernandez A, Vendrell M. Chem Soc Rev 2016;45:1182–1196. 2. Bartelmess J, Quinn SJ, Giordani S. Chem Soc Rev 2015;44:4672– 4698. 3. Wolfbeis OS. Chem Soc Rev 2015;44:4743 4. Peng HS, Chiu DT. Chem Soc Rev 2015;44:4699–4722. 5. Wang F, Zhu Y, Zhou L, Pan L, Cui Z, Fei Q, Luo S, Pan D, Huang Q, Wang R, Zhao C, Tian H, Fan C. Angew Chem Int Ed 2015;54: 7349–7353. 6. Rao W, Wang H, Han J, Zhao S, Dumbleton J, Agarwal P, Zhang W, Zhao G, Yu J, Zynger DL, Lu X, He X. ACS Nano 2015;9:5725– 5740. 7. Li C, Liang S, Zhang C, Liu Y, Yang M, Zhang J, Zhi X, Pan F, Cui D. Biomaterials 2015;54:177–187. 8. Zheng X, Tang H, Xie C, Zhang J, Wu W, Jiang X. Angew Chem Int Ed 2015;54:8094–8099. 9. Yang J, Zhang Y, Gautam S, Liu L, Dey J, Chen W, Mason RP, Serrano CA, Schug KA, Tang L. Proc Natl Acad Sci 2009;106: 10086–10091. 10. Knop K, Hoogenboom R, Fischer D, Schubert US. Angew Chem Int Ed 2010;49:6288–6308. 11. Rao L, Bu LL, Xu JH, Cai B, Yu GT, Yu X, He Z, Huang Q, Li A, Guo SS, Zhang WF, Liu W, Sun ZJ, Wang H, Wang TH, Zhao XZ. Small 2015;11:6225–6236. 12. Lang R, Jun-Hua X, Bo C, Huiqin L, Ming L, Yan J, Liang X, ShiShang G, Wei L, Xing-Zhong Z. Nanotechnology 2016;27:085106 13. Ferreira LS, Gerecht S, Fuller J, Shieh HF, Vunjak-Novakovic G, Langer R. Biomaterials 2007;28:2706 € ngst T, Hennink WE, Dhert 14. Malda J, Visser J, Melchels FP, Ju WJA, Groll J, Hutmacher DW. Adv Mater 2013;25:5011–5028. 15. Wegst UGK, Bai H, Saiz E, Tomsia AP, Ritchie RO. Nat Mater 2015;14:23–36. 16. Saraogi GK, Gupta P, Gupta UD, Jain NK, Agrawal GP. Int J Pharm 2010;385:143–149. 17. Ofokansi K, Winter G, Fricker G, Coester C. Eur J Pharm Biopharm 2010;76:1–9. 18. Tseng CL, Su WY, Yen KC, Yang KC, Lin FH. Biomaterials 2009;30: 3476–3485. 19. Won YW, Kim YH. J Control Release 2008;127:154 20. Coester KLHVBJKCJ. J Microencapsulat 2000;17:187–193. 21. Xu JH, Gao FP, Li LL, Ma HL, Fan YS, Liu W, Guo SS, Zhao XZ, Wang H. Microporous Mesoporous Mater 2013;182:165–172. 22. Xu JH, Gao FP, Liu XF, Zeng Q, Guo SS, Tang ZY, Zhao XZ, Wang H. Chem Commun 2013;49:4462–4464. 23. Wong C, Stylianopoulos T, Cui J, Martin J, Chauhan VP, Jiang W, Popovic´ Z, Jain RK, Bawendi MG, Fukumura D. Proc Natl Acad Sci 2011;108:2426–2431. 24. Egeblad M, Werb Z. Nat Rev Cancer 2002;2:161–174. 25. Azarmi S, Huang Y, Chen H, McQuarrie S, Abrams D, Roa W, Finlay WH, Miller GG, Lobenberg R. J Pharm Pharm Sci 2006;9: 124–132. 26. zur Nieden NI, Turgman CC, Lang X, Larsen JM, Granelli J, Hwang YJ, Lyubovitsky JG. ACS Appl Mater Interfaces 2015;7: 10599–10605. 27. Farris S, Song J, Huang Q. J Agric Food Chem 2010;58:998– 1003. 28. Hu H, Xin JH, Hu H, Chan A, He L. Carbohydr Polym 2013;91:305– 313. 29. Shin DH, Heo MB, Lim YT. Molecules (Basel, Switzerland) 2015; 20:4369–4382. 30. Wang K, Yuan X, Guo Z, Xu J, Chen Y. Carbohydr Polym 2014; 102:699–707. 31. Ding H, Yu SB, Wei JS, Xiong HM. ACS Nano 2016;10:484–491. 32. Wang K, Zhang X, Zhang X, Fan X, Huang Z, Chen Y, Wei Y. Polym Chem 2015;6:5891–5898.
