Biomaterials 35 (2014) 8227e8235

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Dextran-based fluorescent nanoprobes for sentinel lymph node mapping Tingting Dai a, 1, Shuyan Zhou b, 1, Chuyang Yin c, 1, Shengli Li a, Weigang Cao a, Wei Liu a, Kang Sun b, Hongjing Dou b, *, Yilin Cao a, *, Guangdong Zhou a, * a Department of Plastic and Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Tissue Engineering, National Tissue Engineering Center of China, 639 Zhi Zao Ju Road, Shanghai 200011, PR China b The State Key Laboratory of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, PR China c Department of Breast Surgery, Obstetrics and Gynecology Hospital of Fudan University, Shanghai 200011, PR China

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Article history: Received 22 April 2014 Accepted 4 June 2014 Available online 21 June 2014

Biopsy of sentinel lymph node (SLN) has become a common practice to predict whether tumor metastasis has occurred, so proper SLN positioning tracers are highly required. Due to many drawbacks of SLN tracers currently used, developing ideal, biosafe SLN imaging agents is always an urgent issue. The current study designed a novel fluorescent nanoprobe for accurate SLN mapping. Dextran-based nanogel (DNG) was prepared through a highly efficient self-assembly assisted approach and serves as a multifunctional platform for conjugating wide spectra emitting fluorescent agents. The newly fabricated fluorescent DNG (FDNG) could be designed with optimum size and stable fluorescent intensity for specific SLN imaging. Furthermore, a long-term dynamic course in vivo (from 1 min to 72 h) revealed the satisfactory specificity, sensitivity, and stability for SLN mapping. Most importantly, both in vitro and in vivo evaluations indicated that FDNG had fine biosafety and biocompatibility with lymphatic endothelial cells. All these results supported that FDNG could be used as highly efficient molecular imaging probes for specific, sensitive, stable, non-invasive, and safe SLN mapping, which provides efficient and accurate location for SLN biopsy and thus predicts tumor metastasis as well as directs therapies. Besides, our recent studies further demonstrated that DNG could also serve as a specific and controllable drug carrier, indicating a potential application for specific therapies of various lymph-associated diseases. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Fluorescent dextran-based nanogels Sentinel lymph node Mapping Lymphatic endothelial cells Biosafety

1. Introduction Sentinel lymph nodes (SLNs) are the first lymph nodes (LNs) that are reached by cancer cells when primary tumor metastasis occurs via the lymphatic vessels (LVs) [1e3]. It is generally believed that if no metastasis is observed in the SLN, the risk of metastasis extending to the regional LNs will be low [4]. Therefore, the accurate localization of SLNs has been considered a very important step towards the biopsy of SLNs to determine whether tumor metastasis has occurred [5]. At present, SLNs are usually probed with methylene blue or with isotopes [6], but all these methods have drawbacks. For example, methylene blue displays non-selective in vivo

* Corresponding authors. E-mail addresses: [email protected] (H. Dou), [email protected] (Y. Cao), [email protected] (G. Zhou). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biomaterials.2014.06.012 0142-9612/© 2014 Elsevier Ltd. All rights reserved.

distribution and a large chance that the second or even third stand LNs may be mistaken for SLNs, which may result in incorrect conclusions about cancer metastasis states. Additionally, methylene blue guided methods need invasive surgery and thus the detection of SLNs largely depends on the operator's experience. Radionuclide lymphatic imaging involves complex preoperative preparation procedures and expensive equipment, and the radioactivity also limits the application of isotopic probes. The demand for specific, biosafe, and stable nanoprobes that can provide accurate and longlasting mapping of SLNs has become an overwhelming issue in biomedical field. The rapid development of molecular imaging methods offers great possibilities for highly efficient and non-invasive imaging of SLNs [5,7e12]. In various strategies employed for SLN detection [8,13e16], fluorescent imaging has proven to be an efficient approach to address this challenge because of its high sensitivity and radiation-free nature [17]. Among various fluorescent agents, the inorganic nanocrystals [18] including quantum dots (QDs)

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[19e22] and fluorescent nanodiamonds [23] have been reported as probes for SLN mapping. Nevertheless, the potential toxicity of QDs and the complicated fabrication procedure of nanodiamonds greatly compromise their usage for in vivo imaging. Meanwhile, as a class of nanocarriers possessing three-dimensional hydrophilic polymer networks [24,25], nanogels have been favored in many biomedical applications, including drug delivery and cell imaging, etc. [26e31]. Noh and his co-workers synthesized a near-infrared (NIR) fluorescent probe that consisted of pullulan-cholesterol nanogels and IRDye800 payload [32]. They found that these nanogels, with a hydrodynamic diameter of 30e50 nm, could accumulate selectively in the LNs and thus were very efficient for specific SLN mapping. Although some existing reports have preliminarily described the potential applications of polymeric fluorescent nanogels for SLN mapping [31e33], several important issues still need to be addressed to predict practical clinical feasibility of fluorescent nanogels for specific and safe SLN mapping: 1) The feasibility of fluorescent nanogel fabrication based on biomacromolecule with proper diameters and stable fluorescence properties; 2) The longterm in vivo dynamic course of the fluorescent nanogels for SLN mapping; 3) The biosafety of these fluorescent nanogels; and 4) The possible impact of fluorescent nanogels on the characteristic and function of lymphatic endothelial cells (LECs), a dominant cell type that lines the inner surface of LVs. To address the above issues, in the current study, we designed a dextran-poly (acrylic acid) nanogel (DNG) through a highly efficient self-assembly assisted (SAA) approach referred to our previous study [34], and then conjugated it with amine-bearing fluorochromes with broad-spectrum fluorescence to produce various fluorescent nanogels (FDNGs). The characteristic of a representative green light emitting FDNG as a probe for SLN imaging was investigated in detail. Cytotoxicity, biocompatibility, and FDNG's influence on cell function were further evaluated to predict the biosafety in cell level. Most importantly, FDNG was introduced for SLN mapping in a BALB/c mouse model and an in vivo long-term dynamic course was further investigated to explore the feasibility for accurate SLN location. Besides, FDNG's influence on body weight, behavior, blood biochemistry, and important organs was also evaluated to predict the biosafety for future clinical application. 2. Materials and methods 2.1. Materials Dextran-poly(acrylic acid) nanogel (DNG) was fabricated via an SAA method developed by our group, as described in previous reports [34,35]. 5Aminofluorescein (5-AF) was purchased from Sigma Aldrich (St. Louis, U.S.A.), 7amino-4-methyl coumarin (AMC) was purchased from Aladdin (Shanghai, China), near-infrared light emitting CdSeTe/CdS/ZnS core/shell/shell quantum dots (QDs) were fabricated in our laboratory as described in previous works [36,37] and further modified with glutathione (GSH) [38]. 1,10 -dioctadecyl-3,3,3,30 -tetramethyl-indocarbocyanine-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) was purchased from Molecular Probes (Oregon, U.S.A.), and matrigel was from BD Biosciences (New Jersey, U.S.A.). Microvascular Endothelial Cell Growth Medium-2 (EGM2-MV) was purchased from Lonza (Basel, Switzerland). 2.2. Preparation and characterization of FDNGs FDNGs were fabricated through conjugation of DNG with three different aminebearing fluorescent agents, 5-AF, AMC, or GSH-modified CdSeTe/CdS/ZnS core/shell/ shell QDs respectively, under 1- (3-dimethylaminopropyl) -3- ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) catalysis to produce green, blue, or deep-red fluorescence-emitting nanogels. The fluorescence intensities exhibited by FDNGs were measured using a fluorescence spectrophotometer (RF-5301PC, Shimadzu, Japan), with excitation wavelengths of 350 nm, 492 nm, and 450 nm for FDNG(AMC), FDNG(5-AF), and FDNG(QDs) samples respectively. As FDNG(5-AF) showed strong green fluorescence and green light was readily detected by both human eyes and instruments [39], FDNG(5-AF) was used for future studies. The diameters and zeta potentials of DNG and FDNG(5-AF) were characterized using Zetasizer Nano ZS90 (Malvern, U.K.). TEM images were recorded using a JEM-2100 (JEOL, Japan) TEM system, and the samples were negatively stained with

phosphotungstic acid. 1H NMR spectra of DNG and FDNG(5-AF) samples (dissolved in D2O, adjusted by 40 wt% NaOD) were measured using an Avance III 400 MHz NMR spectrometer (Bruker, Switzerland). The fluorescence stability of FDNG(5-AF) sample was tested during a storage period of over 30 days at 37  C. 2.3. In vivo lymphatic node mapping Female BALB/c mice aged 5e6 weeks (acquired from the Animal Institute of Chinese Academy of Science, Shanghai, China) were maintained under specific pathogen free conditions. All experiments employing mice were performed in accordance with the Chinese NIH guidelines for the care and use of laboratory research animals. For in vivo experiments, according to Young-Woock Noh's methods [32], the FDNG(5-AF) nanoprobe (20 mg of samples in 20 mL of water) was intradermally injected into the front paw of a mouse and imaged for in vivo migration of FDNG(5AF) nanoprobe using a fluorescent optical imaging system. The SLN and LVs of the mice injected with FDNG(5-AF) nanoprobe were photoed at 0, 1 min, 5 min, 30 min, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 60 h and 72 h. Methylene blue dye at a concentration of 1% was then reinjected at the same site to further confirm the position of SLN. After skin removal, photographs of SLN were obtained using a Canon digital camera. 2.4. In situ histofluorescence The in situ distribution of FDNG(5-AF) was analyzed by dissecting the axillary SLN after 12 h of injection. According to Noh's methods [33], the lymph node was embedded, frozen, cut to sections, and transferred to glass slides. The slides were stained with rabbit anti-mouse Lymphatic Vessel Endothelial Hyaluronic Acid Receptor 1 (LYVE-1) (Abcam, U.S.A.) to label lymphatic endothelial cells (LECs) as described in our previous study [40]. 2.5. In vitro co-incubation of LECs and FDNG(5-AF) To further analyze the possible mechanism of FDNG(5-AF) on SLN mapping at cell level, in vitro experiments were performed by incubating FDNG(5-AF) with LECs, a dominant cell type that lines the inner surface of LVs in lymph node. The isolation, culture, and identification of LECs were described in our previous study [40]. FDNG(5-AF) was added into EGM-2-MV to a final concentration of 100 mg/mL, and co-incubated with 0.5  106 LECs for 24 h. The resultant LECs were abbreviated as FDNG-LECs. Fluorescence images were captured under a fluorescence microscope (IX-70, Olympus, Japan) after washing and fixing. The positive rate of FDNG(5-AF) uptake was analyzed via a flow cytometer (Beckman Coulter, U.S.A.). For TEM observation, samples were prepared according to previous reports [41] and observed with TEM (Quanta 200, FEI Hillsboro, U.S.A.). 2.6. In vitro cytotoxicity and proliferation assays Cytotoxicity of FDNG(5-AF) on LECs, kidney NRK cells, and liver BRL-3A cells were evaluated by MTT method (NRK and BRL-3A cell were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai, China). Cells were plated at a density of 6  103 cells/well in 96-well plates and incubated with FDNG(5-AF) at different concentrations for 24 h or 48 h followed by MTT assays. For cell proliferation evaluation, LECs or FDNG-LECs were seeded into 96-well plates at a density of 2  103 cells/well, and cell proliferation rates were detected daily for eight days by MTT method. Data expressed as the mean ± the standard deviation was used to prepare the growth curves. 2.7. Cell functional evaluation FDNG-LECs in EGM-2-MV were seeded onto matrigel-coated 24-well plate at a density of 2  104 cells/well and then incubated for at least 4 h at 37  C. The formation of microtube-like structure was analyzed using a Nikon TS-100 microscope and images were captured with a Nikon digital camera (DXM 1200). Uptake of DiIAc-LDL by FDNG-LECs was analyzed via fluorescence microscopy using a previously described method [42]. 2.8. In vivo biosafety evaluation BALB/c mice were randomly injected with 20 mL saline (control group) or 20 mL FDNG(5-AF) (1 mg/mL). At predetermined time points, mice were weighed and assessed for behavior changes. Using a standard blood collection technique from the heart, blood was drawn for biochemistry examination. For blood analysis, 1 mL of blood was collected from mice and separated by centrifugation into cellular and plasma fractions. Upon the completion of the last time point, mice were sacrificed by isoflurane anesthetic. Major organs in both FDNG(5-AF) treated mice and control mice were harvested, fixed in 4% neutral buffered formalin, embedded routinely into paraffin, stained with hematoxylin and eosin (H&E), and the histopathological lesions were examined and evaluated according to previously reported methods [43]. 2.9. Doxorubicin (DOX) loading DOX was conjugated onto DNG via hydrazone bond as described in previous report with minor modifications [44]. The hydrodynamic diameter () of the resultant DNG(DOX) was acquired from Zetasizer Nano ZS90 (Malvern, U.K.), while

T. Dai et al. / Biomaterials 35 (2014) 8227e8235 its morphology was observed under TEM. DNG(DOX) was incubated with human cervical cancer (HeLa) cells (purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai, China) for 72 h and the relevant fluorescence images were taken under fluorescence microscope (IX-70, Olympus, Japan). 2.10. VEGF-C loading Vascular endothelial growth factor C (VEGF-C, the key growth factor to promote regeneration of LVs) was loaded into DNG through Van der Waals forces and hydrophobic interactions. 25 mg VEGF-C (dissolved in 4.5 mL water) was mixed with 0.6 mL DNG (1 mg/mL) under gentle stirring at room temperature for 12 h. The mixture was centrifuged at 14,000 rpm for 45 min, and the UVevisible absorbance spectrum of the supernatant was measured by UV-2550 UVeVis spectrophotometer (Shimadzu, Japan). The loading content (LC) and encapsulation efficiency (EE) of VEGF-C were calculated according to the calibration curve of VEGF-C [45].

3. Results and discussion 3.1. Synthesis of FDNGs FDNGs were prepared through the conjugation of carboxylbearing DNG with amine-containing fluorochromes through amide condensation reactions, as shown in Fig. 1. Firstly, DNG was prepared through a facile and environmental-friendly SAA fabrication method [34]. This assembly was driven by hydrogen bond interactions between the hydroxyl groups of dextran and the carboxyl groups of poly (acrylic acid) (PAA) chains. The abundance of carboxyl groups attached to DNG provided the opportunity for further modification with various amine-bearing fluorescent agents. Examples of the amine-bearing fluorescent agents that had been employed in the current study included blue 7-amino-4methyl coumarin (AMC, Em ¼ 415 nm), green 5aminofluorescein (5-AF, Em ¼ 516 nm), or NIR glutathione (GSH) e modified CdSeTe@CdS@ZnS QDs (Em ¼ 688 nm). These modifications thus yielded FDNGs with various fluorescent properties

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and controllable particle sizes (Fig. S1). Moreover, the newly fabricated fluorescent nanogel had displayed potential application in stem cell imaging according to our recent study [35]. Therefore, the successful fabrication of FDNG(AMC), FDNG(5-AF), and FDNG(QDs) implied that DNG might serve as a multi-functional platform for various fluorescent imaging applications including SLN mapping. 3.2. Characterization of FDNG(5-AF) Since green light is readily detected by both human eyes and instruments [39], and FDNG(5-AF) showed the highest fluorescence intensity among the fabricated FDNGs (Fig. S1, DeF), subsequent experiments were mainly carried out with FDNG(5-AF) as a research model. The characteristic of FDNG(5-AF) was firstly investigated to predict the feasibility as a nanoprobe for specific SLN imaging. It is known that the specificity of SLN imaging mainly depends on the particle size of imaging tracers. Although it is still a controversial issue about the optimal particle size for specific SLN imaging, the generally accepted particle size for specific uptake by LVs is 30e300 nm [32,46]. According to the current results, transmission electron microscopy (TEM) observation revealed that both DNG and FDNG(5-AF) exhibited a regular spherical morphology (Fig. 2A and B). Dynamic light scattering (DLS) measurements indicated that the DNG and FDNG(5-AF) had a hydrodynamic diameter of ~113 nm and ~180 nm respectively (Fig. 2C), a very proper particle size for specific SLN mapping. It was worth noting that the diameters shown in TEM images were much smaller than those determined by DLS, which was caused by the shrinkage of the nanogels during the water evaporation process for the preparation of TEM samples [47,48]. The successful fabrication of FDNG(5-AF) was also verified via 1H NMR spectroscopy, as shown in Fig. 2E. The

Fig. 1. Facile fabrication of DNG and its modification with various fluorescent agents and spectra analysis. The inset images in the fluorescence spectra show the photos of various FDNG solutions under sunlight and UV light. Under UV light excitation, FDNG(AMC), FDNG(5-AF), and FDNG(QDs) emit blue, green, and deep-red fluorescence respectively. (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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Fig. 2. Characterization of DNG and FDNG(5-AF). (A, B) TEM images of DNG and FDNG(5-AF) (negatively stained by phosphotungstic acid); (C) Size distributions of DNG and FDNG(5-AF) measured by DLS; (D) Fluorescence stability of FDNG(5-AF) at 37  C with a period over 30 d; (E) 1H NMR spectra of dextran, DNG, and FDNG(5-AF).

signals observed in DNG around 0.8e2.2 ppm corresponded to the protons of the PAA chains. Meanwhile, the new peaks appeared in FDNG(5-AF) around 5.5e7.0 ppm corresponded to the phenyl rings of 5-AF. At last, the stability of FDNG(5-AF) was also evaluated to

predict its in vivo efficacy. As shown in Fig. 2D, FDNG(5-AF) showed good fluorescence stability when it was stored at 37  C. For example, FDNG(5-AF) retained over 90% of its original fluorescence intensity at 37  C for 7 days and over 60% for 30 days. All these

Fig. 3. Images of forelimb axillary SLN and LVs after injection of FDNG(5-AF). Fluorescent (A) and optical (B) images of FDNG(5-AF) selectively entering LVs and SLN; Fluorescent (C) and optical (D) images of FDNG(5-AF) and methylene blue co-injected mice after skin removal: FDNG(5-AF) labeled SLN shows clear borderline with surrounding tissue, indicating the specificity of FDNG(5-AF) for SLN labeling; Fluorescent (E) and optical (F) images of dissected SLN and adjacent fat tissues: SLN shows strong fluorescent profile while adjacent fat tissue shows negative fluorescent signals; (G) Immunohistofluorescence staining of dissected SLN from FDNG(5-AF) treated mouse after 12 h of injection. (H) The partially enlarged image of (G). Green: FDNG(5-AF), Red: LECs (stained by lymphatic vessel endothelial hyaluronan receptor (LYVE-1)), Blue: nucleus (stained by 40 ,6- diamidino-2phenylindole (DAPI)). (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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results indicated that FDNG(5-AF) could be successfully fabricated with proper particle size and superior stability for specific SLN mapping. 3.3. In vivo SLN mapping Although the particle size and fluorescent property of FDNG(5AF) seemed to be suitable for SLN mapping, the practical efficacy of in vivo SLN imaging is the golden standard to determine whether it can be used for future clinical study. To address this issue, FDNG(5AF) (20 mg of samples in 20 mL of water) was intradermally injected into the forepaw of a mouse and its in vivo migration was recorded using a fluorescent optical imaging system. The results revealed that FDNG(5-AF) could rapidly, selectively diffuse into LVs and SLN as early as less than 1 min after injection (Fig. 3A, B and Fig. 4A, B). Methylene blue was also injected at the same site as FDNG(5-AF), and the green fluorescence overlapped with the blue dye (Fig. 3C and D), confirming the accurate location of SLN with this fluorescent nanoprobe. Moreover, SLN showed strong fluorescent profile while adjacent fat tissue showed negative fluorescence labeling (Fig. 3E and F), which indicated high specificity of FDNG(5-AF) for

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SLN imaging and thus guaranteed the accuracy of SLN biopsy. Besides, immunofluorescence staining of the dissected SLN further revealed that abundant FDNG gathered in the LVs of SLN, providing direct evidence for specific uptake of FDNG(5-AF) by SLN (Fig. 3G and H). Taken together, all these results implied that FDNG(5-AF) could be used as an efficient molecular imaging probe for accurate, sensitive, and specific SLN mapping. Besides sensitivity and specificity, the stability of FDNG(5-AF) fluorescence intensity in SLN is another key parameter that directly influences practical clinical efficacy for SLN mapping. To clarify this issue, a long-term in vivo dynamic course was investigated in the current study to evaluate the stability of FDNG(5-AF) for SLN mapping. As shown in Fig. 4, the fluorescence signals of SLN were strong enough to be captured and recognized during the first 60 h after FDNG(5-AF) injection. Especially, very strong fluorescent signals in SLN could be specifically detected within 12 h after injection, which provided long enough time for surgical procedure of SLN biopsy. The long retention time of FDNG(5-AF) in SLN might be related to its large particle size, which has a strong impact on the migration time during SLN mapping [32,49], and the depth of anesthesia, which can change hemodynamics and thus influence

Fig. 4. Dynamic imaging of axillary SLN and forelimb LVs after injection of FDNG(5-AF). Images (AeC) are taken under both white light and blue light. Images (DeL) are taken under blue light. The scale bars indicate 1 mm.

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lymphatic flow [50]. Another possible reason was that part of FDNG(5-AF) particles might have been phagocytized by LECs or phagocytic cells and thus retained for a long time in SLN. The coexpression of LEC specific marker and FDNG(5-AF) fluorescence (Fig. 3H, yellow staining area) provided direct evidence for this speculation, which was further supported by in vitro study that FDNG(5-AF) could be efficiently taken up by LECs after coincubation of 24 h with a high positive rate (Fig. 5C and D). All these factors ensured a long retention time of FDNG(5-AF) in SLN and thus enhanced the stability of SLN mapping.

3.4. In vitro and in vitro biosafety evaluation Despite the above advantages, biosafety remains to be a great concern to determine whether FDNG(5-AF) can be used for future clinical study [43,51]. To address this issue, cytotoxicity, biocompatibility, and FDNG's influence on cell function were firstly evaluated in vitro to predict its biosafety at cell level. The results suggested that FDNG(5-AF) had a very low cytotoxicity to multiple cells (Fig. 5A and Fig. S2) and had no visible effects on the proliferation (Fig. 5B), specific morphology (Fig. 5E), and phenotype of

Fig. 5. The biocompatibility of FDNG(5-AF) with LECs. (A) Cell viability of LECs after co-incubation with FDNG(5-AF) for 24 h and 48 h; (B) Cell growth curves of normal and FDNG(5AF) treated LECs (FDNG-LECs) during a period of 8 days, no obvious difference is observed; (C) Fluorescence images of FDNG-LECs after 24 h's co-incubation; (D) Flow cytometry of FDNG-LECs, the positive labeling rate is about 99.4%; (E) Optical images of FDNG-LECs, no morphology change is found; (F) TEM image of FDNG-LECs, FDNG(5-AF) is mainly distributed within lysosomes; (GeL) Cell function studies: FDNG-LECs retain the specific functions of DiI-Ac-LDL uptake (GeJ) and microtube-like formation (K, L). Green: FDNG(5AF), Red: DiI-Ac-LDL, Blue: nucleus (stained by DAPI). (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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LECs (Fig. S3 and S4). Furthermore, after incubation with FDNG(5AF), LECs retained the ability to take up 1,10 -dioctadecyl-3,3,3,30 tetramethyl-indocarbocyanine -labeled acetylated low-density lipoprotein (DiI-Ac-LDL) and to form three dimensional microtubelike structure in matrigel, indicating a fine function of LECs (Fig. 5GeL). It was worth noting that after phagocytized by LECs, FDNG(5-AF) mainly distributed in lysosomes (Fig. 5F), and then disappeared after several days (data not shown), implying that FDNG(5-AF) might be degraded by lysosomes and thus had a very low possibility of accumulation in cells. All these in vitro data demonstrated that FDNG(5-AF) had good biocompatibility and biosafety at the cell level. In vivo investigations were further performed to clarify the practical biosafety. The intradermal injection of FDNG(5-AF) had no significant influences on animal body weight compared with the control group (Fig. 6A) and no abnormal clinical signs and behaviors were detected in the treated group. The histological examination further showed that the liver, kidney, spleen, and heart of FDNG(5-AF) treated animals had no appreciable pathological lesion after one month of injection (Fig. 6B). To quantitatively evaluate the acute and long-term toxicity of FDNG(5-AF), the blood biochemistry indices including total protein (TP), albumin (ALB), globulin

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(GLOB), albumin e globulin ratio (A/G), alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine (CREA) were measured. Among the above indices, ALT and AST are closely related to the liver function of animals, while CREA and BUN to the kidney function [43]. No significant differences were observed in all the indices of treated mice within an observation period of one month except a transient increase of ALT and AST at 24 h, indicating that FDNG(5-AF) might be metabolized by liver and thus result in a transient and slight liver damage (Fig. 6C). All the in vivo results suggested that FDNG(5-AF) had good biosafety for SLN mapping. 3.5. Potential application of DNG as a drug carrier Apart from loading fluorescent agents for SLN mapping, our recent studies demonstrated that DNG could also be employed as a candidate drug carrier for the treatment of various lymphassociated diseases. For example, antitumor drugs, such as doxorubicin (DOX), could be successfully conjugated onto DNG. The resultant DNG(DOX) had proper particle size and readily entered into tumor cells (Fig. S5), which might help to promote the drugs to selectively diffuse into LVs surrounding tumor and thus efficiently

Fig. 6. In vivo biosafety evaluation. (A) Effects of FDNG(5-AF) on body weight of mice. Body weight records show similar increasing trend in both FDNG(5-AF) treated mice and normal control; (B) Histological assessment of liver, kidney, spleen, and heart in the treated mice after 1 month of FDNG(5-AF) injection. No appreciable pathological changes are found in liver, kidney, spleen, and heart of the treated mice. Scale bars indicate 100 mm; (C) Influence of FDNG(5-AF) on blood biochemistry indices of the treated mice at 24 h, 48 h, 1 week, and 1 month. Blood biochemistry analysis shows that no significant changes are observed between FDNG(5-AF) treated mice and control mice in all kinds of indices at all time points except a transient increase of ALT and AST at 24 h. TP (Total protein), ALB (Albumin), GLOB (Globulin), A/G (Albumin e globulin ratio), ALT (Alanine aminotransferase), AST (Aspartate aminotransferase), BUN (Blood urea nitrogen), CREA (Creatinine).

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inhibit lymphatic metastasis of malignant tumor. As another example, DNG could also load growth factors, such as vascular endothelial growth factor C (VEGF-C, the key growth factor to promote regeneration of LVs) (Fig. S6), which provides the possibility for targeted therapy of lymphedema. More detailed results will be included in another paper which will be finished in the near future. 4. Conclusions In summary, the current study testified that dextran-based nanogel could serve as a multi-functional platform for conjugating wide spectra emitting fluorescent agents, and these newly fabricated FDNGs could be designed with optimum size for specific SLN imaging. Furthermore, a long-term dynamic course in vivo revealed the satisfactory stability for SLN mapping, which might attribute to proper particle size and the uptake of FDNG by LECs. In addition, both in vitro and in vivo evaluation results indicated that FDNG had fine biocompatibility and biosafety. All these current results supported that FDNG could be used as highly efficient molecular imaging probes for specific, sensitive, stable, non-invasive, and safe SLN mapping, which provides efficient and accurate location for SLN biopsy and thus predicts tumor metastasis as well as directs operative procedures. Besides, our recent studies further demonstrated that DNG could also serve as a specific and efficient drug carrier, indicating a potential application for specific therapies of various lymph-associated diseases. Acknowledgments This research was supported by Hi-Tech Research and Development Program of China (2012AA020507), National Natural Science Foundation of China (81371703, 21374061, 31271046, 21174082, 81371700, and 81372080). The authors appreciate the technical supports and other helps from Demin Ying, Lijuan Zong, Juanjuan Wu, Jinjun Chen, Xiuzhen Li, and Wanyao Xia in the laboratory and National Tissue Engineering Center of China. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.06.012. References [1] Kim K-R, Lee Y-D, Lee T, Kim B-S, Kim S, Ahn D-R. Sentinel lymph node imaging by a fluorescently labeled DNA tetrahedron. Biomaterials 2013;34: 5226e35. [2] Giuliano AE, Kirgan DM, Guenther JM, Morton DL. Lymphatic mapping and sentinel lymphadenectomy for breast cancer. Ann Surg 1994;220:391e8. discussion 8e401. [3] Skobe M, Hawighorst T, Jackson DG, Prevo R, Janes L, Velasco P, et al. Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat Med 2001;7:192e8. [4] Thompson JF, McCarthy WH, Bosch CM, O'Brien CJ, Quinn MJ, Paramaesvaran S, et al. Sentinel lymph node status as an indicator of the presence of metastatic melanoma in regional lymph nodes. Melanoma Res 1995;5:255e60. [5] Huang X, Zhang F, Lee S, Swierczewska M, Kiesewetter DO, Lang L, et al. Longterm multimodal imaging of tumor draining sentinel lymph nodes using mesoporous silica-based nanoprobes. Biomaterials 2012;33:4370e8. [6] Tanaka E, Choi H, Fujii H, Bawendi M, Frangioni J. Image-guided oncologic surgery using invisible light: completed pre-clinical development for sentinel lymph node mapping. Ann Surg Oncol 2006;13:1671e81. [7] Weissleder R. Molecular imaging: exploring the next frontier1. Radiology 1999;212:609e14. [8] Zhang C, Liu T, Su Y, Luo S, Zhu Y, Tan X, et al. A near-infrared fluorescent heptamethine indocyanine dye with preferential tumor accumulation for in vivo imaging. Biomaterials 2010;31:6612e7.

[9] Tang J, Kong B, Wu H, Xu M, Wang Y, Wang Y, et al. Carbon nanodots featuring efficient FRET for real-time monitoring of drug delivery and two-photon imaging. Adv Mater 2013;25:6569e74. [10] Pourtau L, Oliveira H, Thevenot J, Wan Y, Brisson AR, Sandre O, et al. Antibody-functionalized magnetic polymersomes: in vivo targeting and imaging of bone metastases using high resolution MRI. Adv Healthc Mater 2013;2: 1420e4. [11] Pan D, Cai X, Kim B, Stacy AJ, Wang LV, Lanza GM. Rapid synthesis of near infrared polymeric micelles for real-time sentinel lymph node imaging. Adv Healthc Mater 2012;1:582e9. [12] Pan D, Cai X, Yalaz C, Senpan A, Omanakuttan K, Wickline SA, et al. Photoacoustic sentinel lymph node imaging with self-assembled copper neodecanoate nanoparticles. ACS Nano 2012;6:1260e7. [13] Rockall AG, Sohaib SA, Harisinghani MG, Babar SA, Singh N, Jeyarajah AR, et al. Diagnostic performance of nanoparticle-enhanced magnetic resonance imaging in the diagnosis of lymph node metastases in patients with endometrial and cervical cancer. J Clin Oncol 2005;23:2813e21. [14] Wunderbaldinger P. Optical imaging of lymph nodes. Eur J Radiol 2006;58: 390e3. [15] Erpelding TN, Kim C, Pramanik M, Jankovic L, Maslov K, Guo Z, et al. Sentinel lymph nodes in the rat: noninvasive photoacoustic and US imaging with a clinical US system1. Radiology 2010;256:102e10. [16] Zhang C, Wang S, Xiao J, Tan X, Zhu Y, Su Y, et al. Sentinel lymph node mapping by a near-infrared fluorescent heptamethine dye. Biomaterials 2010;31:1911e7. [17] Akers WJ, Kim C, Berezin M, Guo K, Fuhrhop R, Lanza GM, et al. Noninvasive photoacoustic and fluorescence sentinel lymph node identification using dyeloaded perfluorocarbon nanoparticles. ACS Nano 2010;5:173e82. [18] Erogbogbo F, Yong K-T, Roy I, Hu R, Law W-C, Zhao W, et al. In vivo targeted cancer imaging, sentinel lymph node mapping and multi-channel imaging with biocompatible silicon nanocrystals. ACS Nano 2010;5:413e23. [19] Pons T, Pic E, Lequeux N, Cassette E, Bezdetnaya L, Guillemin F, et al. Cadmium-free CuInS2/ZnS quantum dots for sentinel lymph node imaging with reduced toxicity. ACS Nano 2010;4:2531e8. [20] Kim S, Lim YT, Soltesz EG, De Grand AM, Lee J, Nakayama A, et al. Nearinfrared fluorescent type II quantum dots for sentinel lymph node mapping. Nat Biotechnol 2004;22:93e7. [21] Ballou B, Ernst LA, Andreko S, Harper T, Fitzpatrick JAJ, Waggoner AS, et al. Sentinel lymph node imaging using quantum dots in mouse tumor models. Bioconjugate Chem 2007;18:389e96. [22] Pic E, Pons T, Bezdetnaya L, Leroux A, Guillemin F, Dubertret B, et al. Fluorescence imaging and whole-body biodistribution of near-infrared-emitting quantum dots after subcutaneous injection for regional lymph node mapping in mice. Mol Imaging Biol 2010;12:394e405. [23] Vaijayanthimala V, Cheng P-Y, Yeh S-H, Liu K-K, Hsiao C-H, Chao J-I, et al. The long-term stability and biocompatibility of fluorescent nanodiamond as an in vivo contrast agent. Biomaterials 2012;33:7794e802. [24] Kabanov AV, Vinogradov SV. Nanogels as pharmaceutical carriers: finite networks of infinite capabilities. Angew Chem Int Ed 2009;48:5418e29. [25] Oh JK, Drumright R, Siegwart DJ, Matyjaszewski K. The development of microgels/nanogels for drug delivery applications. Prog Polym Sci 2008;33: 448e77. [26] Shah PP, Desai PR, Patel AR, Singh MS. Skin permeating nanogel for the cutaneous co-delivery of two anti-inflammatory drugs. Biomaterials 2012;33: 1607e17. [27] Wu W, Aiello M, Zhou T, Berliner A, Banerjee P, Zhou S. In-situ immobilization of quantum dots in polysaccharide-based nanogels for integration of optical pH-sensing, tumor cell imaging, and drug delivery. Biomaterials 2010;31: 3023e31. [28] Wu W, Shen J, Banerjee P, Zhou S. Coreeshell hybrid nanogels for integration of optical temperature-sensing, targeted tumor cell imaging, and combined chemo-photothermal treatment. Biomaterials 2010;31:7555e66. [29] Hong-shang P, Stolwijk JA, Li-Ning S, Wegener J, Wolfbeis OS. A nanogel for ratiometric fluorescent sensing of intracellular pH values. Angew Chem Int Ed 2010;49:4246e9. [30] Wu W, Shen J, Banerjee P, Zhou S. A multifuntional nanoplatform based on responsive fluorescent plasmonic ZnO-Au@PEG hybrid nanogels. Adv Healthc Mater 2011;21:2830e9. [31] Mok H, Jeong H, Kim S-J, Chung BH. Indocyanine green encapsulated nanogels for hyaluronidase activatable and selective near infrared imaging of tumors and lymph nodes. Chem Commun 2012;48:8628e30. [32] Noh Y-W, Kong S-H, Choi D-Y, Park HS, Yang H-K, Lee H-J, et al. Near-infrared emitting polymer nanogels for efficient sentinel lymph node mapping. ACS Nano 2012;6:7820e31. [33] Noh Y-W, Park HS, Sung M-H, Lim YT. Enhancement of the photostability and retention time of indocyanine green in sentinel lymph node mapping by anionic polyelectrolytes. Biomaterials 2011;32:6551e7. [34] Tang M, Dou H, Sun K. One-step synthesis of dextran-based stable nanoparticles assisted by self-assembly. Polymer 2006;47:728e34. [35] Zhou S, Dou H, Zhang Z, Sun K, Jin Y, Dai T, et al. Fluorescent dextran-based nanogels: efficient imaging nanoprobes for adipose-derived stem cells. Polym Chem 2013;4:4103e12. [36] Wang X, Wang G, Li W, Zhao B, Xing B, Leng Y, et al. NIR-emitting quantum dot-encoded microbeads through membrane emulsification for multiplexed immunoassays. Small 2013;9:3327e35.

T. Dai et al. / Biomaterials 35 (2014) 8227e8235 [37] Xing B, Li W, Wang X, Dou H, Wang L, Sun K, et al. Highly-fluorescent alloyed quantum dots of CdSe1-xTex synthesized in paraffin liquid: gradient structure and promising bio-application. J Mater Chem 2010;20:5664e74. [38] Wei Y, Yang J, Ying JY. Reversible phase transfer of quantum dots and metal nanoparticles. Chem Commun 2010;46:3179e81. [39] Nathans J, Thomas D, Hogness DS. Molecular genetics of human color vision: the genes encoding blue, green, and red pigments. Science (New York, NY) 1986;232:193e202. [40] Tt Dai, Zh Jiang, Li Sl, Zhou Gd, Kretlow JD, Wg Cao, et al. Reconstruction of lymph vessel by lymphatic endothelial cells combined with polyglycolic acid scaffolds: a pilot study. J Biotechnol 2010;150:182e9. [41] Peckys DB, de Jonge N. Visualizing gold nanoparticle uptake in live cells with liquid scanning transmission electron microscopy. Nano Lett 2011;11: 1733e8. [42] Craig LE, Spelman JP, Strandberg JD, Zink MC. Endothelial cells from diverse tissues exhibit differences in growth and morphology. Microvasc Res 1998;55:65e76. [43] Zhang X-D, Wu D, Shen X, Liu P-X, Fan F-Y, Fan S-J. In vivo renal clearance, biodistribution, toxicity of gold nanoclusters. Biomaterials 2012;33:4628e38. [44] Xiong W, Gao X, Zhao Y, Xu H, Yang X. The dual temperature/pH-sensitive multiphase behavior of poly(N-isopropylacrylamide-co-acrylic acid)

[45]

[46] [47]

[48]

[49]

[50] [51]

8235

microgels for potential application in in situ gelling system. Colloid Surf B 2011;84:103e10. Xu Q, Liu Y, Su S, Li W, Chen C, Wu Y. Anti-tumor activity of paclitaxel through dual-targeting carrier of cyclic RGD and transferrin conjugated hyperbranched copolymer nanoparticles. Biomaterials 2012;33:1627e39. Thompson JF, Morton DL, Kroon BB. Textb melanoma. Lond: Martin Dunitz; 2004. Dou H, Jiang M, Peng H, Chen D, Hong Y. PH-dependent self-assembly: micellization and micelleehollow-sphere transition of cellulose-based copolymers. Angew Chem Int Ed 2003;42:1516e9. Dou H, Tang M, Sun K. A facile one-pot synthesis to dextran-based nanoparticles with carboxy functional groups. Macromol Chem Phys 2005;206: 2177e81. Reddy ST, van der Vlies AJ, Simeoni E, Angeli V, Randolph GJ, O'Neil CP, et al. Exploiting lymphatic transport and complement activation in nanoparticle vaccines. Nat Biotechnol 2007;25:1159e64. Rao RR, Bequette BW, Roy RJ. Simultaneous regulation of hemodynamic and anesthetic states: a simulation study. Ann Biomed Eng 2000;28:71e84. Chen Y, Chen H, Shi J. In vivo bio-safety evaluations and diagnostic/therapeutic applications of chemically designed mesoporous silica nanoparticles. Adv Mater 2013;25:3144e76.