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Dendron-Grafted Polylysine-Based Dual-Modal Nanoprobe for Ultra-Early Diagnosis of Pancreatic Precancerosis via Targeting a Urokinase-Type Plasminogen Activator Receptor Hui Li, Ping Wang, Wenyu Gong, Qi Wang, Jia Zhou, Wei-Hong Zhu,* and Yingsheng Cheng* become the second most common cause of cancer-related death by 2030.[1] PDAC is believed to be developed from histologically identifiable intraductal lesions known as pancreatic intraepithelial neoplasias (PanINs) that undergo a series of architectural, cytologic, and genetic changes.[2] Despite research efforts over the past 50 years, the conventional treatment approaches such as surgery, radiation, chemotherapy, or combinations of these treatments have little efficacy on development of aggressive neoplasm. Because of the modest benefits from radio therapy and chemotherapy and limited crowd of surgery (15–20% patients), the early detection of PDAC (PanIN-III and early-stage cancers) provides opportunities for early intervention, thus offering the best hope for longer survival time.[3] Unfortunately, due to the lack of specific symptoms and limitations in diagnostics, small PanIN lesions and early-stage cancers are frequently eluded from detection in the clinic. Accordingly, a powerful sensitive tool for detection of precancerous lesions and early-stage cancers is urgently needed. Nanoparticles are extensively studied for their unique size-dependent properties[4] and their potential applications as nanoprobes in biomedical imaging.[5–10] Recently, various multimodal imaging nanoprobes have been fabricated by combining
Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer death. Early detection of precancerous pancreatic intraepithelial neoplasia (PanIN) tissues is an urgent challenge to improve the PDAC prognosis. Here, a urokinase-type plasminogen activator receptor (uPAR)-targeted magnetic resonance (MR)/near-infrared fluorescence (NIRF) dual-modal nanoprobe dendron-grafted polylysine (DGL)-U11 for ultra-early detection of pancreatic precancerosis is reported. Because of its good biocompatibility and biodegradability, globular architecture, and well-defined reactive groups, the DGL is chosen as the platform to load with a pancreatic tumor-targeting peptide U11, a magnetic resonance contrast agent Gd3+-diethylene triamine pentaacetic acid, and a near-infrared fluorescent cyanine dye Cy5.5. The nanoprobe DGL-U11 has several preferable characteristics, such as active peptide targeting to activator receptor, good biocompatibility, dual-modal imaging diagnosis, and well controlled diameter in a range of 15–25 nm. Upon incorporation of the active U11 peptide target to the overexpressed activator receptor uPAR, the targeted nanoprobe DGL-U11 can increase to the earlier PanIN-II stage through in vivo NIRF imaging. Labeled with both MR and NIRF bioimaging reporters, the uPAR-targeted dual-modal nanoprobe is very effective in the targeted imaging of precancerous PanINs and PDAC lesions with high sensitivity and spatial resolution, providing a promising platform to the ultra-early detection of PDAC.
1. Introduction Pancreatic ductal adenocarcinoma (PDAC), with 5-year survival rate of 6% and an increasing incidence, is on track to Dr. H. Li, J. Zhou, Prof. Y. Cheng Department of Radiology Shanghai Jiao Tong University Affiliated Sixth People’s Hospital No. 600 Yi Shan Road, Shanghai 200233, P. R. China E-mail:
[email protected] Dr. P. Wang Molecular Imaging Laboratory MGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging Department of Radiology Massachusetts General Hospital Harvard Medical School Boston, MA 02129, USA
W. Gong Department of CT the First People’s Hospital of Yancheng City Jiangsu 224005, China Dr. Q. Wang, Prof. W.-H. Zhu Key Laboratory for Advanced Materials and Institute of Fine Chemicals Shanghai Key Laboratory of Functional Materials Chemistry School of Chemistry and Molecular Engineering East China University of Science and Technology Shanghai 200237, P. R. China E-mail:
[email protected]
The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adhm.201700912.
DOI: 10.1002/adhm.201700912
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different functional nanoparticles for the sensitive and specific detection of unique cellular processes[11–15] or even for the treatment of diseases directly.[16–21] Developing multifunctional nanoparticles with multimodal imaging and tumor-targeting capabilities may open a window to the early detectability of pancreatic precancerosis.[22,23] Although considerable efforts have been devoted to imaging relatively large and readily apparent pancreatic tumors,[24–26] studies on the early detection of PanIN lesions are lacking due to the negative target legend expression in precancerous lesions cells. Serine protease urokinase-type plasminogen activator (uPA) and its receptor (uPAR) have been known to involve in the invasion and metastasis of many solid tumors.[27] Specifically, uPAR is found to be highly expressed in pancreatic cancer tissues.[28] Hildenbrand et al. have further found that uPAR overexpresses in high-grade PanIN lesions.[29] That is, both high-grade precursor lesions (PanIN-III) and the substantial proportion of PanIN-II lesions are overexpressed with uPAR, while PanIN-I lesions and nonneoplastic tissues are usually negative. Therefore, uPAR is a promising target to modify the diagnosis nanoparticles for the early detection of pancreatic precancerosis. Noninvasive diagnostic methods are especially advantageous for identifying precancerous lesions in high-risk populations. Magnetic resonance (MR) imaging has been currently developed as one of the best methodologies for assessing the anatomy and function of tissues in clinical medicine since it offers excellent temporal and spatial resolution,[30] acquires images rapidly in vivo, and enables long effective imaging window.[31–35] However, MR imaging suffers from limited sensitivity and incongruence
between the tumor area outlined in the preoperative MRI and the actual tumor area during surgery.[36,37] Among optical imaging technologies, near-infrared fluorescence (NIRF) imaging within a wavelength range of 650–1000 nm has received considerable attention due to its high sensitivity, rapid real-time imaging, and relatively low autofluorescence from organisms and tissues,[38–40] making it especially suitable for endoscopic imaging and imaging guided surgery. However, NIRF imaging suffers from limited penetration depth.[41–45] Therefore, dual-modal MR/NIRF imaging system is expected to offer a promising approach to compensate the inherent shortcomings of each single-modal system, especially for the sake of detecting early PanINs with enhanced spatial resolution and high sensitivity. In this work, uPAR-targeted dual-modal MR/NIRF bioimaging nanoprobe DGL-U11 (Figure 1) is developed to facilitate the early detection of pancreatic precancerosis. Dendron-grafted poly-l-lysine (DGL) has emerged in biomedical research as a versatile platform to nanomaterials with desirable properties, such as drug delivery and molecular bioimaging. The adaptability of DGL is delineated by good biocompatibility, biodegradability, well-defined structures, distinct sizes, and a potentially large number of accessible reactive functional groups on its periphery.[46,47] With this in mind, the third generation dendron of DGL is utilized as the nanoprobe platform. Derived from the binding domain of the natural ligand to the uPAR receptor,[48] U11 peptide is linked with polyethylene glycol (PEG) to the nanoprobe for improving biocompatibility. The nanoprobe is labeled with a NIR fluorophore Cy5.5 for achieving fluorescence imaging, which can avoid the tissue absorption and
Figure 1. Synthetic route to uPAR-targeted dual-modal nanoprobe. A) A control nanoprobe DGL-PEG without the targeting legend U11 for insight into the active targeting capacity. B) uPAR-targeted dual-modal nanoprobe DGL-U11 with three steps: PEG was conjugated with pancreatic tumor-targeting peptide U11, then U11 peptide modified PEG layer was covalently conjugated to the DGL nanospheres modified with NIR fluorescent cyanine dye Cy5.5, and finally coated with the MR contrast agent Gd3+-DTPA to yield the uPAR-targeted dual-modal nanoprobe DGL-U11.
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autofluorescence in the region of 650–900 nm. The nanoprobe is further functionalized with a high thermodynamic stable and kinetic inert MR contrast agent, Gd3+-diethylene triamine pentaacetic acid (Gd3+-DTPA). In vitro and in vivo results demon strate that the uPAR-targeted dual modal nanoprobe DGL-U11 bestows the preferable biocompatibility, superior active targeting ability, and excellent MR and NIRF imaging capabilities. As demonstrated, the dual-modal nanoprobe DGL-U11 is a promising tool for the ultra-early detection of pancreatic precancerosis.
2. Results and Discussion 2.1. uPAR-Targeted Nanoprobe DGL-U11 for MR/NIRF Dual-Modal Imaging As specific dendron, DGL has emerged in biomedical research as a versatile platform to nanomaterials with desirable properties, such as drug delivery and molecular bioimaging. Here we exploited the third generation dendron G3 of DGL (DGL-G3) due to its good biocompatibility, biodegradability, well-defined structures, distinct sizes, and a potentially large number of accessible reactive functional groups on its periphery.[46,47] Dendrigraft DGL G3 contains 123 lysine groups on DGL-G3 surface, which is convenient for chemical modification. The PEG modified dendrimer (DGL-PEG) with a hydrodynamic diameter of 6–7 nm was measured with a circulation lifetime of 1.5 h in mouse. Also, this conjugate is mainly distributed in liver and kidney, but excreted mainly through the kidney after the intravenous injection. Figure 1 shows the synthetic route for both the targeted nanoprobe DGL-U11 and control DGL-PEG. Here the control nanoprobe DGL-PEG without the targeting legend U11 was prepared for further insight into the active targeting capacity of dual-modal nanoprobe. A dendritic strategy with 3rd generation is utilized for creating branch points that allow conjugation of pancreatic tumor-targeting peptide U11, NIR fluorescent cyanine dye Cy5.5, and the MR contrast agents Gd3+-DTPA. Briefly, treating N-Hydroxysuccinimide (NHS) esters of Cy5.5 with 3rd generation DGL (containing 123 primary amino groups, Mw: 22 kDa) in 0.1 m 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 8.3) yielded compound 1. The mono-activated PEG2k-NHS ester was reacted with compound 1, and further treated with DTPA-dianhydride and GdCl3 to yield the control nanoprobe DGL-PEG. Following the similar way, the bis-activated PEG moiety Mal-PEG2K-NHS reacted with U11 peptide to produce Mal-PEG2K-U11, which was further treated with compound 1, DTPA-dianhydride and GdCl3 to obtain the targeted nanoprobe DGL-U11 (Figure 1). The physical parameters of nanoprobes DGL-PEG and DGLU11 are listed in Table 1. As shown in Figure 2, the hydrodynamic diameter of the targeted nanoprobe DGL-U11 was 22.98 nm, which was similar to that of control nanoprobe DGLPEG (19.97 nm). The polydispersity index (PDI) of all nanoprobes remained below 0.3, that is, 0.150 for DGL-U11 and 0.217 for DGL-PEG. The nanoprobe size is sufficiently small, in the range of 15–25 nm in diameter, to traverse the vasculatures in the pancreatic lesions and sufficiently large to achieve enhanced permeability and retention effects. Both DGL-U11 and DGL-PEG
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Table 1. Size, zeta potential, and molecular composition of uPAR targeted dual-modal nanoprobe DGL-U11 and control nanoprobe DGL-PEG. d [nm]a)
PDIa)
ζa)
Molar ratiob)
DGL-PEG
19.97
0.217
+6.36 mV
1/1.2/10.4/N/89/24
DGL-U11
22.98
0.150
+6.30 mV
1/1.2/10.1/9.5/84/25
Nanoprobe
(d), polydispersity index (PDI), and zeta potential (ζ) measured by dynamic light scattering (DLS); b)Molar ratio of DGL/Cy5.5/PEG/Peptide/DTPA/ Gd in the nanoprobes. a)Diameter
showed positive charges in physiological pH. The average zeta potentials of DGL-PEG and DGL-U11 were measured to be +6.36 and +6.30 mV. As indicated by 1H NMR spectroscopy, the molar ratio of U11/PEG/DTPA/DGL was 9.5/10.1/84/1 in DGL-U11 and 0/10.4/89/1 in DGL-PEG (Figures S1 and S2, Supporting Information). Based on the standard absorption working curve, the average 1.2 Cy5.5 fluorophores were labeled on each nanoprobe, making them sufficiently sensitive to be visualized through optical imaging. Longitudinal relaxivity r1p values were determined according to the equation r1p = (1/Tsample − 1/TPBS)/ [Gd3+]. The longitudinal relaxivities r1p of DGL-PEG and DGL-U11 were measured as 6.3 and 6.2 × 10−3 m−1 s−1/Gd3+, respectively, which were sufficiently sensitive for MR imaging.
2.2. Negligible Cytotoxicity and High Cellular Uptake of DGL-U11 to uPAR-Overexpressed Pancreatic Cancer Cells Low cytotoxicity is a key criterion for biomaterials used in medical applications. The main components such as DGL and PEG have been reported to be biodegradable and exhibit no significant cytotoxicity. Here, the cytotoxicity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at the range of 1 × 10−9 –200 × 10−6 m for three times. The experimental median toxic concentration (TC50) values against PANC1 cells were calculated to be 56 × 10−6 m for DGL-U11 (Figure 3A) and 58 × 10−6 m for DGL-PEG (Figure 3B), around 28 and 29 fold of the working concentration for diagnostic applications in tumor cells, suggestive of the low cytotoxicity with nanoprobes DGL-U11 and DGL-PEG. Here dendrigraft DGL shows lower cytotoxicity compared to currently widely used polyamidoamine.[49,50] The cytotoxicities of the DGL depend on their surface charge and terminal modifications. The positively charged DGL could nonspecifically bind to the negatively charged biological membranes. In this work, averagely ten PEG chains were modified on the DGL surface to shield the positive charge and improve the biocompatibility. Confocal laser microscopy imaging was performed to study the active targeting behavior of nanoprobes to uPAR, which is highly overexpressed in pancreatic lesions cells. As shown in Figure 3C, the fluorescence intensity of PANC1 cells incubated with the targeted nanoprobe DGL-U11 for 2 h at 37 °C was significantly higher than that of cells treated with the control nano probe DGL-PEG. The mean values of fluorescence intensities in PANC1 cells were 5.6 and 2.8 for DGL-U11 and DGL-PEG, respectively (Figure 3D), indicative of 2.0-fold higher uptake of the targeted nanoprobe DGL-U11 than control nanoprobe DGL-PEG in uPAR overexpressed cells. In addition, when incubated with DGL-U11 solution (2 × 10−6 m) for 2 h at 37 °C,
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Figure 2. Characterization of nanoprobes. A) Hydrodynamic size of DGL-U11 and DGL-PEG, well controlled in a diameter range of 15–25 nm. B) Zeta potential of DGL-U11 and DGL-PEG.
the fluorescent signal was clearly visible in the PANC1 cells. However, when the cells were pre-incubated with a solution containing excess U11 before incubation with DGL-U11, the fluorescent signal almost disappeared, suggesting that the uptake of DGL-U11 nanoprobes was markedly accelerated with incorporation of U11 peptide unit. Additionally, the fluorescent signal was undetectable in the T47D cells with no overexpression of uPAR[29] under treatment of DGL-U11 solution (2 × 10−6 m) for 2 h at 37 °C. These results imply that incorporation of U11
peptide in DGL-U11 is very effective for actively targeted imaging of high uPAR overexpressed PanINs and PDAC lesions.
2.3. Detection of Pancreatic Lesions as Early as PanIN-II Stage using NIRF Imaging, and PanIN-III Stage Using MR Imaging During the pancreatic carcinogenesis of the dimethylolbutanoic acid [2,2-bis(hydroxymethyl)butyric acid] (DMBA)-induced
Figure 3. Negligible cytotoxicity and high actively targeted cellular uptake of dual-modal nanoprobe DGL-U11 to uPAR overexpressed pancreatic cancer PANC-1 cells. A) Cytotoxicity of targeted nanoprobe DGL-U11 in PANC-1 cells and B) cytotoxicity of control nanoprobe DGL-PEG in PANC-1 cells, showing the negligible cytotoxicity of DGL-U11 with high cell viability and high experimental median toxic concentration (TC50) values. C) Microscopic fluorescence images of the uPAR overexpressed cells PANC1 or no uPAR overexpressed T47D cells under treatment of 2.0 × 10−6 m nanoprobe for 2 h at 37 °C. D) Relative cellular fluorescence intensity under nanoprobe treatment (n = 4). Note: Compared with DGL-PEG, the high uptake rate of DGL-U11 in uPAR overexpressed cells PANC1 indicates the active targeting ability of DGL-U11. All data are presented as the mean ± SD, *P < 0.05, Mann–Whitney U-test.
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Figure 4. In vivo MR imaging of SD rats at PanIN-I, PanIN-II, PanIN-III, and PDAC lesions at 24 h after intravenous injection with the uPAR targeted dual-modal nanoprobe DGL-U11 or control nanoprobe DGL-PEG. Note: Arrow indicates the lesion site, and using DGL-U11 probe could visualize the PanIN-III and PDAC lesions directly.
rat model, the dynamic in vivo MR imaging was performed at various stages (30, 60, 90, and 120 d after DMBA implantation, corresponding to PanIN-I, PanIN-II, PanIN-III, and PDAC stages, respectively). In vivo MR imaging was performed in the DMBA-induced rat model at 24 h after intravenous injection of the nanoprobes. The pancreatic lesions were located through T2-weighted imaging (T2WI) by highlighting the area with a high water concentration. T1-weighted imaging (T1WI) showed an enhanced MR signal in the pancreatic lesions after injection with DGL-U11. The results showed that the T1WI signal enhancement in the pancreatic lesions gradually increased with the increasing delay before DMBA implantation. As shown in Figure 4, at the PanIN-I and PanIN-II stages, the contrast between the surrounding normal pancreatic tissues and pancreatic lesions was negligible after 24 h injection administration of DGL-U11. The negative results can be attributed to be either absent or relatively low expression of uPAR during these two stages. However, the PanIN-III lesion was visualized with an obvious MR signal enhancement at 24 h after injection with DGL-U11. The PDAC lesions displayed a higher T1WI signal than the PanIN-III lesions. The T/B ratio was also significantly higher in the PDAC lesions than in the PanIN-III lesions. While the T1WI signals at the PanIN-III and PDAC stages were clearly enhanced under treatment with the targeted nanoprobe DGL-U11, the signal in the lesions was almost unchanged for all of the stages during the pancreatic carcinogenesis upon treatment of control nanoprobe DGL-PEG. These results demonstrate the feasibility of the targeted nanoprobe DGL-U11 for the early detection of PanIN-III and PDAC lesions. Simultaneously, in vivo NIRF imaging was performed in a DMBA-induced rat model at 24 h after intravenous injection with the nanoprobes using confocal laser endomicroscopy. As shown in Figure 5, only trace fluorescent signals were observed at the PanIN-I stage, and stronger fluorescent signals were found at the PanIN-II stage, indicative of the increased distribution of nanoprobe DGL-U11 in the pancreatic lesions. Moreover, the fluorescent signal was significantly enhanced at
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the PanIN-III stage, and the strongest at the PDAC stage. As expected, the actively targeted nanoprobe DGL-U11 resulted in stronger fluorescent signals in the pancreatic lesions than the control nanoprobe DGL-PEG. Actually, in the rats injected with the DGL-PEG, the fluorescent signal in the pancreatic lesions was almost invisible at the PanIN-I stage. Although a higher signal was also found in the PanIN-II, PanIN-III, and PDAC lesions, all the levels were significantly lower than those observed in the rats injected with DGL-U11. Next, the nanoprobe distribution in the pancreatic lesions was also displayed using ex vivo NIRF imaging. As shown in Figure 6A, the fluorescent signals increased along with the progress of the carcinogenesis, which were consistent with the results of the in vivo confocal laser endomicroscopy imaging. The enhanced signal could be attributed to the relatively high overexpression level of uPAR in the pancreatic lesions at these stages since a high overexpression level of uPAR may facilitate the uptake of the actively targeted nanoprobe DGL-U11. As confirmed in our previously published work, pancreatic lesions were develop into the PanIN-I, PanIN-II, PanIN-III, and PDAC stages at 30, 60, 90, and 120 d, respectively, in the pancreatic carcinogenesis model. To further verify the progression of the pancreatic lesions measured in the ex vivo optical imaging studies, a histopathological examination was conducted. As shown in Figure 6B, PanIN-1 shows flat epithelial lining with tall columnar cells with basally located nuclei and abundant supranuclear mucin. PanIN-2 demonstrates fullthickness pseudostratification of nuclei with moderate cytologic abnormalities. PanIN-3 is characterized by complete loss of polarity, budding of cellular tufts into the duct lumen, and significant nuclear pleomorphism. PDAC shows significant architecture and cytological abnormalities followed by basement membrane invasion. The pathological changes could also be clearly observed as the disease progressed, which is consistent with the histological features of different stages of human pancreatic carcinogenesis. To verify the active role of
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Figure 5. In vivo confocal laser endomicroscopy imaging of SD rats at PanIN-I, PanIN-II, PanIN-III, and PDAC lesions at 24 h after intravenous injection with the uPAR targeted dual-modal nanoprobe DGL-U11 or control nanoprobe DGL-PEG. Note: The fluorescence intensity in nanoprobe DGL-U11 group is brighter than that in control nanoprobe DGL-PEG, indicative of the efficient active targeting of DGL-U11 to the receptor uPAR. The fluorescence intensities in the PanIN-II, PanIN-III, and PDAC lesions are clearly visible, and an increasing trend along with the progressive grade is indicative that DGL-U11 probe could be used for ultra-early detection of the pancreatic lesions as early as PanIN-II stage using NIRF imaging.
uPAR in mediating nanoprobe delivery, the immunohistochemistry study was conducted. Images of the pancreatic lesion sections demonstrated the remarkably increased expression level of uPAR along with the progress of carcinogenesis (Figure 6C).
2.4. Negligible Systemic Toxicity of Nanoprobe DGL-U11 Evaluated by In Vivo Safety Studies We carefully studied the systemic toxicity of the targeted nano probe DGL-U11. SD rats injected with saline and DGL-U11
Figure 6. NIRF imaging of the pancreatic carcinogenesis lesions with uPAR targeted nanoprobe DGL-U11. A) Ex vivo NIRF imaging of the pancreatic lesions at 24 h after intravenous injection of nanoprobe DGL-U11 or DGL-PEG (n = 4). The fluorescence in tumor of DGL-11 decreased in the order PDAC > PanIN-III > PanIN-II > PanIN-I, and the fluorescence intensity in PanIN-II, PanIN-III, and PDAC lesions are clearly visible, indicating that DGLU11 probe could be used for detecting PanIN-II, PanIN-III, and PDAC lesions. B) H&E images of pancreatic lesions verified the progressive grade of the pancreatic tissue detected in the ex vivo optical imaging studies (n = 4). C) Immunohistochemistry images of the uPAR overexpression in pancreatic lesions along with the progress of carcinogenesis (n = 4).
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Figure 7. Histological analysis of the major organs (heart, liver, spleen, lung, kidney, and brain) from healthy rats treated with saline or rats treated with the targeted nanoprobe DGL-U11 for 1 or 7 d, indicating that uPAR-targeted nanoprobe DGL-U11 is negligible cytotoxicity, preferably safe for biomedical applications (n = 4).
(25 mg kg−1) were investigated at 1 and 7 d. Compared with the saline group, no death or serious body weight loss was observed in either test group during the study period following one intravenous injection of 25 mg kg−1 DGL-U11 per day. To evaluate the acute toxicity of tissues caused by DGL-U11, sections of the heart, liver, spleen, lung, kidney and brain were collected at 24 h after the last administration of the nanoprobes, and then stained with H&E. As shown in Figure 7, compared to the control group, the cellular structure apparently did not change, and no necrosis, congestion or hydropic degeneration was observed in the tissue sections, indicating that no visible lesions were induced in these tissues upon DGL-U11 treatment. This outcome is consistent with the results based on the in vitro cytotoxicity studies. Thus, the targeted nanoprobe DGLU11 is expected to be safe for biomedical applications, especially enhancing the early detection and surgery incision with the endoscopic technique.
3. Conclusion We have successfully developed uPAR-targeted MR/NIRF dual-modal imaging nanoprobe DGL-U11 for ultra-early detection of pancreatic precancerosis based on a dendritic functionalization strategy. The nanoprobe was developed by coating dendron-grafted polylysine DGL with a pancreatic tumor-targeting peptide U11, a MR contrast agent Gd3+-DTPA as well as a NIR fluorescent cyanine dye Cy5.5. Both uPAR-targeted nanoprobe DGL-U11 and control nanoprobe DGL-PEG can be well controlled in a diameter range of 15–25 nm, bestowing high biocompatibility for negligible cytotoxicity and histological analysis, and exhibiting the high active targeting to the
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overexpressed receptor uPAR. With the active targeting dualmodal bioimaging, pancreatic lesions can be detected as the PanIN-III stage through in vivo magnetic resonance imaging, and improved to the earlier PanIN-II stage through in vivo NIR fluorescent imaging. As a whole, the uPAR-targeted dualmodal nanoprobe DGL-U11 provides a promising strategy for ultra-early diagnostic of pancreatic precancerosis in the clinical prognosis.
4. Experimental Section Materials: All organic solvents were analytical grade and purchased from Aladdin Reagent (Shanghai, China) unless otherwise specified. The third generation DGL (containing 123 primary amino groups, Mw: 22 kDa) was purchased from COLCOM (Montpellier, France). PEG2Khydroxysuccinimide (PEG2K-NHS) and maleimide derivative (MalPEG2k-NHS) were purchased from JenKem Technology (Beijing, China). Cy5.5-NHS (IR783-NHS) was purchased from Sigma-Aldrich (Saint Louis, USA). The uPAR-targeted peptide U11 was synthesized by China Peptides Co., Ltd. (Shanghai, China). Fetal bovine serum, Dulbecco’s modified Eagle’s medium, trypsin, penicillin, and streptomycin were purchased from Life Technologies Inc. (Carlsbad, USA). The nanoprobes were synthesized according to previously published methods.[51,52] The conjugation quantity of PEG, DTPA, and U11 was controlled by regulating the proportions of the raw materials. The molar ratio of U11/PEG/DTPA/DGL was determined by 1H-NMR spectroscopy. The Cy5.5 labeling degrees were determined by measuring the absorbance of Cy5.5. The Gd3+ labeling degrees were determined by inductively coupled plasma atomic emission spectrometry. DMBA was purchased from Sigma Pharmaceuticals (South Croydon, Victoria, Australia). Synthesis of Control Nanoprobe DGL-PEG: Cy5.5-NHS ester (IR783-NHS) dissolved in anhydrous DMF was added dropwise to DGL in a 0.1 m HEPES solution (pH 8.3). After stirring at room temperature for 1 h, the fluorophore-conjugated compound 1 was obtained and
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purified by centrifuge with a centrifugal filter (10 000 MW cut-off) at a speed of 4000 rpm. PEG2K-NHS ester in a 0.1 m HEPES solution (pH 8.3) was added to a solution of compound 1, and the mixture was stirred at room temperature for 1 h. The product was purified with a centrifugal filter (10 000 MW cut-off) to obtain compound 2. DTPA dianhydride was added to a solution of compound 2 in 0.5 m HEPES buffer (pH 8.3), and the pH of the solution was monitored and adjusted to reach approximately 8.3 by adding 1.0 m NaOH solution. The mixture was stirred at room temperature for 3 h and purified by centrifugal filtration (10 000 MW cut-off) to produce compound 3. GdCl3 was added to a solution of compound 3 in deionized water (pH 7.4). The suspension was stirred at room temperature overnight, and was purified with a centrifugal filter (10 000 MW cut-off) to yield the control nanoprobe DGL-PEG. Synthesis of Targeted Nanoprobe DGL-U11: The U11 peptide and Mal-PEG2K-NHS ester were added to anhydrous DMF. The mixture was stirred at room temperature for 2 h and added to a solution of compound 1 in 0.1 m HEPES buffer (pH 8.3). This mixture was stirred at room temperature overnight, and purified with a centrifugal filter (10 000 MW cut-off) to yield compound 4. DTPA dianhydride was added to a solution of compound 4 in 0.5 m HEPES buffer (pH 8.3), and the pH of the solution was monitored and adjusted to approximately 8.3 by adding 1.0 m NaOH solution. The mixture was stirred at room temperature for 3 h and purified by centrifugal filtration (10 000 MW cut-off) to yield compound 5. GdCl3 was added to a solution of compound 5 in deionized water (pH 7.4). The suspension was allowed to stir at room temperature overnight, and purified with a centrifugal filter (10 000 MW cut-off) to obtain the targeted nanoprobe DGL-U11. Characterization of Nanoprobes: The molar ratios between DGL, PEG, U11, and DTPA in the nanoprobes were quantified by 1H NMR (Varian Mercury 400 spectrometer, USA). The absorbance of Cy5.5 in the nanoprobes was measured on a UV-2401PC absorption spectrometer (SHIMADZU, Japan). The size distribution and zeta potentials of nanoprobes were determined by a Malvern Zetasizer dynamic light scattering instrument (Malvern Instruments Inc., USA). The gadolinium ion concentrations were determined with a Hitachi P-4010 inductively coupled plasma atomic emission spectroscopy system (Tokyo, Japan). Development of Pancreatic Adenocarcinoma Progression Model: Animal procedures were performed according to the guide recommendations for the care and use of laboratory animals published by the National Institutes of Health. A pancreatic ductal adenocarcinoma progression model was established in rats according to a previously reported method.[53–55] In brief, SD rats weighing 200 g were anesthetized with a mixture of ketamine (100 mg kg−1) and xylazine (10 mg kg−1) via intraperitoneal injection. A median incision was made in the rat abdomen to expose the pancreas. A pocket was developed at the pancreas tail. DMBA (Sigma Pharmaceuticals, South Croydon, Victoria, Australia) at a dose of 5 mg/100 g was implanted, and secured with a 6.0 prolene purse string suture. Then, the abdominal cavity was sutured. The histological analysis in our previously published study confirmed that the pancreatic lesions would develop into PanIN-I, PanIN-II, PanIN-III, and PDAC stages at 30, 60, 90 and 120 d, respectively, after establishing the pancreatic carcinogenesis model. In Vivo MR Imaging Studies: All MR imaging experiments were conducted on a Bruker Pharmascan 7.0 T micro-MRI scanner (Bruker, Germany). The SD rats were used for in vivo imaging 1, 2, 3, and 4 months after DMBA implantation, which corresponded to a normal pancreas, PanIN-I, PanIN-II, PanIN-III, and PDAC stages of pancreatic carcinogenesis, respectively. The nanoprobes were injected with a T1 value of 60 ms via caudal veins. At select time points, animals were anesthetized with a mixture of ketamine (100 mg kg−1) and xylazine (10 mg kg−1) and positioned prostrate within the magnet bore. Their respiration rate was monitored by a Bruker Physiogard system with 30–40 breaths min−1. In vivo T2WI and T1WI images of the rat abdomens were sequentially acquired: T2WI [repetition time (TR)/ echo time (TE) = 3800/95 ms, acquisition matrix = 256 × 256; field of view = 40 × 40 mm; number of slices = 12; slice thickness = 2 mm,
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rare factor = 8, flip angle = 150°]. T1WI [TR/TE = 600/11 ms; acquisition matrix = 256 × 256; field of view = 40 × 40 mm; number of slices = 12, slice thickness = 2 mm, flip angle = 80°]. Optical Imaging Studies: In vivo NIRF images of rat PanIN and PDAC tissues were obtained using confocal laser endomicroscopy (Cellvizio Image Cell, Mauna Kea Technology Inc., France). After 24 h administration of nanoprobes, the left upper rat abdominal wall was disinfected, and a small incision was made. The handheld endomicroscopy probe, which scans and records the signals of the tumor, was inserted. The scanning parameters included an excitation wavelength of 670 nm and an acquisition bandwidth of 680–700 nm. Subsequently, the rats were sacrificed and perfused transcardially with phosphate buffer saline (PBS) followed by 4% paraformaldehyde. The PanINs and PDAC tissues were excised carefully, and ex vivo NIRF images were acquired using a Maestro Imaging System (CRi Inc., Woburn, MA, USA) with a bandpass excitation filter from 650 to 670 nm and a longpass emission filter over 680 nm. The regions of interest were defined and quantified using the instrument software (Maestro software, CRi). Histological H&E Staining: After ex vivo optical imaging studies, the isolated rat PanINs and PDAC tissues were fixed in 4% paraformaldehyde for 12 h, dehydrated in ascending grades of ethanol and embedded in paraffin. The rat PanINs and PDAC tissues were sectioned with a thickness of 5.0 µm, stained with H&E and visualized under an optical microscope (Olympus BX51, Japan). Statistical Analysis: All statistical analyses were performed using SPSS version 21.0 (SPSS Inc., Chicago, IL, USA). Data were reported as the mean ± SD. The experimental data were statistically analyzed by using the Mann–Whitney U-test. P values were considered statistically significant when less than 0.05.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements This work was supported by the Natural Science Foundation of Shanghai (Nos. 15ZR1431800 and 13ZR1459400), the Interdisciplinary Program of Shanghai Jiao Tong University (No. YG2013MS48), and the Program of Shanghai Science Municipal for Basic Focus (No. 10JC1412900).
Conflict of Interest The authors declare no conflict of interest.
Keywords activator receptors, dendron-loaded nanoprobes, magnetic resonance imaging, NIR fluorescence bioimaging, pancreatic cancer Received: July 31, 2017 Revised: September 17, 2017 Published online:
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