Prediction of Drug-Induced Nephrotoxicity with a Hydroxyl Radical and

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Prediction of Drug-Induced Nephrotoxicity with a Hydroxyl Radical and Caspase Light-Up Dual-Signal Nanoprobe Yuling Tong, Xitong Huang, Mi Lu, Bo-Yang Yu, and Jiangwei Tian Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05454 • Publication Date (Web): 14 Feb 2018 Downloaded from http://pubs.acs.org on February 15, 2018

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Analytical Chemistry

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Prediction of Drug-Induced Nephrotoxicity with a

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Hydroxyl Radical and Caspase Light-Up Dual-

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Signal Nanoprobe

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Yuling Tong, Xitong Huang, Mi Lu, Bo-Yang Yu,* and Jiangwei Tian*

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State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of TCM Evaluation and

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Translational Research, School of Traditional Chinese Pharmacy, China Pharmaceutical

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University, Nanjing 211198, P.R. China

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[email protected]

Corresponding authors, Bo-Yang Yu, Email: [email protected]; Jiangwei Tian, Email:

ACS Paragon Plus Environment

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

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The development of well-designed nanoprobes for specific imaging of multiple biomarkers in

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renal cells will afford beneficial information related to the transmutation process of drug-induced

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kidney injury (DIKI). However, the most reported nanoprobes for DIKI detection were

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dependent on single-signal output and lack of kidney targeting. In this work, we reported a renal

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cell targeting and dual-signal nanoprobe by encapsulating Brite 670 and Dabcyl-KFFFDEVDK-

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FAM into low molecular weight chitosan nanoparticle. Confocal fluorescence imaging results

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demonstrated that the nanoprobe could visualize the upregulation of hydroxyl radical in early

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stage and the activation of caspase-3 in late stage of DIKI both at renal cell and tissue level. In a

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mice DIKI model, the positive time of 8 h using the nanoprobe imaging was superior to that of

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72 h for serum creatinine or blood urea nitrogen, 16 h for cystatin-C, and 24 h for kidney injury

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molecule-1 with conventional methods. These results demonstrated that the nanoprobe may be a

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promising tool for effective early prediction and discriminative imaging of DIKI.

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Keywords: drug-induced kidney injury, dual-signal nanoprobe, renal targeting, fluorescence

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imaging, visualization


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The kidney is an important organ of excretion in the body that is naturally exposed to a greater

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proportion of circulating drugs or their metabolic products1. Thus, the kidney is more susceptible

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to be damaged by drugs, which ultimately results in drug-induced kidney injury (DIKI)2,3. DIKI

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remains a long-term concern in clinical setting as well as pharmaceutical research and has

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attracted increasing attention as a public health issue4. The DIKI progressing at early stage is

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reversible and the function of kidney can be recovered if DIKI is discovered and treated in a

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timely manner. Otherwise, irreversible damage to kidney will occur with the DIKI progressing to

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the late stage5. Therefore, discriminative detection of DIKI at early and late stages holds great

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promise for predicting the drug nephrotoxicity, adjusting the dosage regimen, and improving the

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success rate of new drug research. At present, serum creatinine (sCrea) and blood urea nitrogen

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(BUN) have been used mainly to monitor kidney damage pre-clinically and clinically6, but they

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are both late biomarkers7 that fail to reveal kidney damage until 65-75% of the normal kidney

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function has already been lost8. Although the detection sensitivity of several early biomarkers

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including kidney injury molecule-1 (KIM-1)9, clusterin10, cystatin-C (Cys-C)11, neutrophil

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gelatinase-associated lipocalin (NGAL)12, and heme oxygenase 1 (HO-1)13 is better than that of

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the current sCrea and BUN, the specificity and accuracy are poorer because the detection results

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of these biomarkers can be affected by other diseases14. Renal biopsy is well-deserved, selective,

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and accurate, unfortunately, the operation process is traumatic, complicated, and high-risk15.

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More importantly, these methods are not suitable for monitoring the process of DIKI from early

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to late stage. Therefore, developing a new strategy for noninvasively distinguishing DIKI at

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different stages is highly desired.

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There is clear evidence that the reactive metabolites formed from drugs during initial period

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are responsible for most cases of DIKI, which are usually accompanied by the generation of


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reactive oxygen species (ROS)16. ROS overproduction often results in intracellular oxidative

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stress and may be an early event in the process of DIKI. As the progressing of oxidative stress,

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caspase-3 as a downstream protease of apoptosis will be activated ultimately17, leading to

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irreversible renal cell death. Cisplatin (cis-diammineplatinum dichloride, CDDP) is one of the

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most widely used chemotherapeutic agents for cancer treatment due to its strong anticancer

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effects18,19. However, CDDP-induced nephrotoxicity limits the use of CDDP and related

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platinum-based therapeutics20,21. CDDP can selectively accumulate in renal tubular epithelial cell

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depending on organic cation transporter 2 (OCT2)22 and induce renal cell apoptosis by initial

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ROS especially hydroxyl radical (•OH)23 overgeneration and eventual caspase-3 activation24.

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Hence, the development of intracellular •OH and caspase-3 dual-activatable noninvasive

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detection technology will be an efficient way to evaluate and predict DIKI.

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Fluorescence imaging assay provides new opportunities to achieve this goal due to its

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noninvasive characteristics, high sensitivity and selectivity, intuitive visualization, and

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satisfactory temporal and spatial resolution25. In this regard, a newly designed dual-signal

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nanoprobe with renal cell targeting has been developed for dynamically monitoring the

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transmutation process from early to late stage via imaging of •OH and caspase-3 during DIKI.

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For renal cell targeting, low molecular weight chitosan (LMWC)26, a copolymer of D-

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glucosamine and N-acetylglucosamine derived from chitin, is chosen as the nanocarrier due to its

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excellent biocompatibility and biodegradability27. Notably, LMWC can specially bind to megalin

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receptor28,29 which is highly expressed on renal tubular epithelial cells. Furthermore, Brite 670 as

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a •OH fluorescence probe and Dabcyl-KFFFDEVDK-FAM (D-DEVD-F) as a caspase-3

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activatable fluorescent peptide30,31 are encapsulated into LMWC to construct the nanoprobe

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(Brite/DEVD@LMWC NP, Scheme 1). The nanoprobe is stable in physiological condition and


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can be selectively uptaken into the lysosome via megalin receptor-mediated endocytosis, in

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which the acidic pH environment induces the dissociation of nanocarrier and the subsequent

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release of Brite 670 and D-DEVD-F. At the early stage of DIKI, the increased •OH reacts with

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Brite 670 to emit strong red fluorescence. With the progressing to late stage, the green

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fluorescence of D-DEVD-F is generated upon reaction with caspase-3 owing to the

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disappearance of fluorescence resonance energy transfer (FRET) effect. As far as we know, this

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is the first time that a dual-signal nanoprobe has been used for discriminative imaging of DIKI at

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different stages. The proposed approach can provide a new strategy for early warning of DIKI

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and predicting the drug nephrotoxicity.

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Scheme 1. Schematic illustration about structure and function of Brite/DEVD@LMWC NP for

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hydroxyl radical and caspase-3 light-up discriminative imaging of drug-induced kidney cell

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injury stages.


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EXPERIMENTAL SECTION

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Synthesis of Brite/DEVD@LMWC NP. 0.5 mL dimethyl sulfoxide (DMSO) containing 4 mg

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Brite 670 and 2.5 mg D-DEVD-F was added to 8.5 mL LMWC solution (20 mg of LMWC) to

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obtain a mixture solution. 1 mL 0.05 wt% TPP solution was added dropwise into the mixture

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solution while stirring at 1000 rpm. The conjugation reaction was maintained for 1 h, and the

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product was then purified by successive dialysis (molar weight cut-off, MWCO 10000) against

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deionized water for 48 h. The final product was freeze-dried and kept in desiccators until use.

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Renal Injury Detection in Vitro. To detection of •OH production, HK-2 cells were seeded into

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96-well plates at a seeding density of 1 × 103 cells per well in 200 µL complete medium and

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incubated at 37 °C for 24 h. After rinsing with PBS, HK-2 cells were incubated with 200 µL

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culture media containing serial concentrations (0, 5, 10, 20, 40, 60, 80, 100 µM) of CDDP or

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CTN for 4 h at 37 °C. After incubation, the cells were treated with 10 µM H2DCFDA for 30 min

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and then the fluorescence intensity was measured using a spectrofluorometer at λex/λem =492/524

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nm.

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Confocal fluorescence imaging was used to monitor the process of drug-induced HK-2 injury,

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HK-2 cells were seeded into 35-mm confocal dishes (Glass Bottom Dish) at a density of 1 × 104

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per dish. The cells were pre-incubated with 40 µg/mL Brite/DEVD@LMWC NP for 4 h at 37

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°C, and then treated with different concentrations (0, 5, 10, 20, 40, 60, 80, 100 µM ) of CDDP

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(CTN) for 4 h or 16 h. After incubation, the cells were stained with 1.0 µM Hoechst 33342 for 25

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min, rinsed three times with PBS to perform fluorescence imaging with a CLSM at stationary

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parameters including the laser intensity, exposure time, and objective lens. Hoechst 33342 was

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excited at 405 nm with a violet laser diode and the emission was collected from 420 to 500 nm.


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FAM was excited at 488 nm with a helium-neon laser and the emission was collected from 500

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to 650 nm. Brite 670 was excited at 639 nm with an argon ion laser and the emission was

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collected from 650 to 800 nm. All images were digitized and analyzed by a ZEN imaging

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software.

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To monitor the change of CDDP-induced injury to HK-2 cells over time, HK-2 cells were pre-

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incubated for 40 µg/mL Brite/DEVD@LMWC NP for 4 h at 37 °C, and then treated with 50 µM

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CDDP for 0, 2, 4, 8, 16, 24 h at 37 °C. Furthermore, Brite/DEVD@LMWC NP-loaded HK-2

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cells were pretreated with 5 µM CUR or 10 µM RES for 2 h and then incubated with 50 µM

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CDDP for 16 h at 37 °C to implement confocal fluorescence imaging.

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Flow cytometric assay was designed for further detecting CDDP-induced HK-2 cell injury, the

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cells were pre-incubated with Brite/DEVD@LMWC NP for 4 h, and then treated with different

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concentrations (0, 5, 10, 20, 40, 60, 80, 100 µM) of CDDP for 4 h or 16 h, or incubated with 50

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µM CDDP for 0, 2, 4, 8, 16, and 24 h at 37 °C. The different treatment groups of HK-2 cells

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were also trypsinized, harvested, rinsed with PBS, and resuspended, and the different

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fluorescence of Brite 670 and FAM was measured using MACSQuant Analyzer 10. All

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experiments detected at least 10,000 cells and the data were analyzed with FCS Express V3.

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Renal Injury Detection in Tissue. Renal function was assessed by measuring sCrea, BUN, Cys-

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C, and KIM-1 levels. Blood samples were collected and centrifuged at 3000 rpm for 15 min at 4

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°C to separate serum. The levels of sCrea, BUN, Cys-C, and KIM-1 were measured through

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relevant assay kits. The kidneys were fixed with 4% formalin solution, and then dehydrated,

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embedded, frozensliced into pieces of 10 µm thick for confocal fluorescence imaging after

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staining with Hoechst 33342. Hoechst 33342 was excited at 405 nm with a violet laser diode and

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the emission was collected from 420 to 500 nm. FAM was excited at 488 nm with an argon ion


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laser and the emission was collected from 500 to 650 nm. Brite 670 was excited at 639 nm with a

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helium-neon laser and the emission was collected from 650 to 800 nm. All images were digitized

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and analyzed by a ZEN imaging software.

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RESULTS AND DISCUSSION

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Characterization of Brite/DEVD@LMWC NP.

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The Brite/DEVD@LMWC NP was prepared using an ionic crosslinking method32. TEM

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revealed that the nanoprobe was well-dispersed single spherical structure with size on the order

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of 40-60 nm (Figure 1A). The DLS measurements indicated the nanoprobe had an average

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hydrodynamic diameter of 72.3 ± 3.3 nm (Figure 1B) which was larger than TEM analysis due to

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the presence of hydration layers. In addition, FITC labeled LMWC nanoparticle (FITC-LMWC

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NP) was also synthesized to evaluate the renal cell targeting of LMWC-based nanocarrier. The

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absorption spectra of FITC-LMWC NP showed a characteristic peak of FITC-LMWC at 490 nm

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compared to LMWC and LMWC NP (Figure S1A). A significant fluorescence peak at 518 nm

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was observed in the fluorescence spectra (Figure S1B), and the fluorescence intensity at 518 nm

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of FITC-LMWC NP showed good linearity over the concentration range from 0 to 10 µg/mL

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with the linear correlation coefficient of 0.99979 (Figure S1C), which further indicated the

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successful conjugation of FITC to LMWC NP. The content of FITC conjugated on FITC-LMWC

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NP was determined with a standard curve method to be 68.74 ± 0.15 µg/mg (Figure S2). The

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zeta potentials of LMWC NP, FITC-LMWC NP, and Brite/DEVD@LMWC NP were obtained

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to be +35.2 mV, +34.5 mV, and +31.2 mV (Table S1), suggesting good stability of these

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colloidal particles33 and transportation in glomerular filtration34 of these nanoparticles.


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Figure 1. Characterization and properties of Brite/DEVD@LMWC NP. (A) TEM image of

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Brite/DEVD@LMWC NP. Scale bar: 50 nm. (B) Size distribution of Brite/DEVD@LMWC NP

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by DLS. In vitro release profiles of (C) Brite 670 and (D) D-DEVD-F from

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Brite/DEVD@LMWC NP at different pHs.

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Encapsulation and Release Profiles of Brite/DEVD@LMWC NP.

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To calculate the encapsulation efficiency (EE) and loading content (LC) of the nanoprobe, the

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fluorescence standard curves of Brite 670 (Figure S3A) and D-DEVD-F (Figure S3B) were

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obtained. The EE of Brite 670 and D-DEVD-F was measured to be 87.00 ± 0.04% and 79.66 ±

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0.81%, respectively (Table S2). The LC of Brite 670 and D-DEVD-F was measured to be 13.66

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± 0.01% and 7.82 ± 0.08%, respectively. In vitro release behaviors of the nanoprobe at different

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pHs were investigated by a dialysis method35. At pH 7.4 and 6.8, the nanoprobe was stable over

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48 h incubation (Figure 1C and D). On the contrary, at acidic environment of pH 5.0, the release

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speed was apparently accelerated. After 4 h incubation, the accumulative release rate of Brite

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670 and D-DEVD-F could reach at 78.86 ± 0.69% and 70.74 ± 1.41%, respectively, which

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ensured an effective probes delivery and release into the acidic lysosomes of kidney cells.


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Fluorescent Response of Brite/DEVD@LMWC NP.

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Fluorescence assays were performed to test the validity of Brite 670 and D-DEVD-F for the

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response to •OH and caspase-3 activity. Upon reaction with •OH, the fluorescence of Brite 670

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recovered gradually along with •OH concentration and showed a fluorescent peak at 663 nm

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under 650 nm excitation (Figure S4A). Furthermore, the fluorescence response to •OH was

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selective, other ROS including OCl ― , H2O2, TBHP, •OtBu did not induce the fluorescence

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recovery of Brite 670 (Figure S5). In the absence of caspase-3, the fluorescence emission of D-

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DEVD-F was very weak due to the energy transfer from FAM to Dabcyl. After caspase-3 was

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added into D-DEVD-F solution, a significant fluorescence peak at 514 nm was observed (Figure

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S4B), suggesting the cleavage of D-DEVD-F and the release of FAM. These results

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demonstrated the fluorescent response feasibility of Brite 670 and D-DEVD-F to •OH and

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caspase-3.

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The Brite/DEVD@LMWC NP was reacted with •OH or caspase-3 at different pHs. There was

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no apparent fluorescence of the nanoprobe whether at pH 7.4 or pH 5.0. After added the •OH or

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caspase-3 into the nanoprobe solution at pH 7.4, the fluorescence of Brite 670 or FAM did not

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recovery because the LMWC nanocarrier prevented the interaction between the inside probe and

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outside substrate. On the contrary, after changing the solution pH to 5.0, the addition of •OH or

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caspase-3 led to remarkable fluorescence enhancement of Brite 670 (Figure S4C) or FAM

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(Figure S4D), indicating that acid pH ruptured the LMWC nanocarrier to release the inside

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probes. These results demonstrated the successful encapsulation of Brite 670 and D-DEVD-F

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into the LMWC NP and the pH-dependent release of the nanoprobe.

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Renal Cell Uptake Assay of Brite/DEVD@LMWC NP.


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To examine the cell specificity and the major route of cellular uptake of the nanocarrier, human

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proximal renal tubular epithelial HK-2 cells, mouse fibroblast L929 cells, rat heart muscle H9c2

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cells, human umbilical vein endothelial HUVEC cells, and human normal liver L-02 cells were

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incubated with FITC-LMWC NP. Confocal fluorescence images showed that the green

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fluorescence of FITC-LMWC NP-incubated HK-2 cells increased gradually along with

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increasing concentrations (Figure S6A) and incubation times (Figure S6B), while the

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fluorescence of FITC-LMWC NP was scarcely observed in L929 cells (Figure S7), H9c2 cells,

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HUVEC cells, and L-02 cells (Figure S8), indicating that FITC-LMWC NP was specially

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accumulated in HK-2 cells. Furthermore, the FITC-LMWC NP-incubated HK-2 cells were co-

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stained with a lysosome probe, LysoTracker Deep Red, and a nucleus dye, Hoechst 33342 to

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study the subcellular distribution of FITC-LMWC NP in cells. The green fluorescence of FITC

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was overlapped well with the red LysoTracker fluorescence (Supplementary Video), judging by

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yellow fluorescence, indicating that the localization of FITC-LMWC NP was in the lysosomal

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compartment. Flow cytometric assays were also carried out, which revealed that the fluorescence

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intensity increase of FITC-LMWC NP in HK-2 cells was concentration (Figure 2A) and time

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(Figure 2C) dependent and much greater than that in L929 cells (Figure 2B and D). Furthermore,

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quantitative analysis of intracellular uptake was performed and expressed as the amount of

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FITC-LMWC NP associated with a unit weight of cellular protein (Figure S9). The cellular

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uptake of FITC-LMWC NP into HK-2 cells was significantly much higher than that of L929

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cells either at different concentrations (Figure S10A) or for different incubation times (Figure

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S10B). These results proved the specific targeting capability of FITC-LMWC NP for HK-2 cells.

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Furthermore, the mechanism of the enhanced cellular uptake of FITC-LMWC NP into HK-2

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cells was investigated. In the presence of ethylene diamine tetraacetic acid (EDTA), free LMWC,


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or glucosamine as the inhibitors of megalin recptor36, the uptake of FITC-LMWC NP was

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obviously inhibited (Figure S10C), suggesting that LMWC nanocarrier could be internalized into

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HK-2 cells through a megalin receptor-mediated endocytosis pathway.

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Figure 2. Flow cytometric assays of (A) HK-2 cells and (B) L929 cells incubated with 0 µg/mL

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(black), 2 µg/mL (red), 5 µg/mL (blue), 10 µg/mL (purple), 20 µg/mL (green) FITC-LMWC NP

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for 4 h, respectively. Flow cytometric assays of (C) HK-2 cells and (D) L929 cells incubated

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with 10 µg/mL FITC-LMWC NP for 0 h (black), 0.5 h (red), 1 h (blue), 2 h (purple), 4 h (green),

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and 8 h (yellow), respectively.

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Biocompatibility and Security Evaluation of Brite/DEVD@LMWC NP.

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To investigate the biocompatibility and security of Brite/DEVD@LMWC NP, the cytotoxicities

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of LMWC NP, FITC-LMWC NP, and Brite/DEVD@LMWC NP to HK-2 and L929 cell lines

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were evaluated using methyl thiazolyl tetrazolium (MTT) assays. After incubation for 24 h, the

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cellular viabilities of HK-2 (Figure S11A) and L929 cells (Figure S11B) could maintained more

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than 90% and only a slight decrease of cell viability was observed when the concentration was

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higher than 2.0 mg/mL, which confirmed that the nanoparticles had superior biocompatibility

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with both HK-2 and L929 cells.


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Kidney Cell Injury Imaging and Detection with Brite/DEVD@LMWC NP.

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CDDP-induced •OH production to HK-2 cells were measured using a commercial ROS probe

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dichlorodihydrofluorescein diacetate (H2DCFDA). After HK-2 cells were treated with different

4

concentrations of CDDP for 4 h, the fluorescence intensity in HK-2 cells increased gradually

5

with CDDP concentration (Figure S12A), and the cytotoxicity of CDDP to HK-2 cells was

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significant when the concentration reached at 40 µM using MTT assay (Figure S12B). To

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investigate the feasibility of Brite/DEVD@LMWC NP for imaging of CDDP-induced HK-2 cell

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injury, confocal fluorescence imaging was performed. 40 µg/mL Brite/DEVD@LMWC NP-

9

loaded HK-2 cells were first treated with different concentrations of CDDP for 4 h. The confocal

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fluorescence images showed that the red fluorescence of Brite 670 in HK-2 cells increased along

11

with increasing incubation concentrations of CDDP (Figure S13), indicating the increasing •OH

12

production during the CDDP treatment at a very early time. On the contrary, the green

13

fluorescence of FAM was rarely seen at this time point, which could be ascribed to the inactivity

14

of caspase-3 at 4 h post treatment of CDDP in the process of apoptosis37. Moving on,

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Brite/DEVD@LMWC NP-loaded HK-2 cells were treated with different concentrations of

16

CDDP for 16 h. The confocal fluorescence images in HK-2 cells showed obvious red

17

fluorescence of Brite 670 and increasing green fluorescence of FAM along with increasing

18

incubation concentrations of CDDP (Figure 3), suggesting abundant caspase-3 was activated to

19

cleave the D-DEVD-F and then release FAM. Furthermore, for the HK-2 cells treated with 50

20

µM CDDP, the red fluorescence of Brite 670 and the green fluorescence of FAM started to

21

appearance at the 2 h and 8 h post administration (Figure S14), respectively. Meanwhile,

22

confocal fluorescence imaging in the same field of Brite/DEVD@LMWC NP-loaded HK-2 cells

23

clearly revealed the fluorescent and morphological change in the process of CDDP-induced


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apoptosis (Figure S15). At 2 h incubation, bright red fluorescence was observed although the cell

2

morphology was intact, suggesting the •OH production at the early stage. With progressing to 8

3

h, the cells became shrinking and round, which indicated the late stage of apoptosis. At this time

4

point, the green fluorescence of FAM was apparent. These results manifested that the nanoprobe

5

could be used to detect early renal cell injury and discriminative imaging of CDDP-induced HK-

6

2 cell injury at different stages.

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Figure 3. Confocal fluorescence imaging of 40 µg/mL Brite/DEVD@LMWC NP-loaded HK-2

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cells incubated with different concentrations of CDDP for 16 h. Scale bar: 10 µm.

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Flow cytometric assays were further carried out for discriminative detection of CDDP-induced

11

HK-2 cell injury at different stages with dual-signal Brite/DEVD@LMWC NP. The Brite

12

670+/FAM+ region was representative for the cells with •OH production and caspase-3

13

activation. At 4 h incubation with different concentrations of CDDP, the cells were almost in the

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Brite 670+/FAM- region (Figure S16). The ratio of cells with 50 µM CDDP incubation in the

15

Brite 670+/FAM- region increased from 0.25% at 0 h to be 64.57% at 4 h (Figure 4), indicating

16

•OH production at the early stage of apoptosis. At 16 h incubation, cells in the Brite 670+/FAM+


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Page 16 of 26

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region became apparent when the concentration beyond 40 µM (Figure S16) and the ratio of cells

2

in the Brite 670+/FAM+ was reached up to 54.59% with 50 µM CDDP (Figure 4), which was

3

attributed to caspase-3 activation at the late stage. These results further demonstrated the dual-

4

signal advantage for discriminative detection of CDDP-induced renal cell injury at different

5

stages.

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Figure 4. Flow cytometric assays of 40 µg/mL Brite/DEVD@LMWC NP-loaded HK-2 cells

8

treated with 50 µΜ CDDP for different times.

9

Subsequently, the dual-signal Brite/DEVD@LMWC NP was used to assess the protective

10

effect of drugs on the CDDP-induced renal injury. Curcumin (CUR)38 and resveratrol (RES)39 as

11

active ingredients for protecting the renal injury by adjusting oxidation-reduction stress were

12

added before CDDP incubation. As seen in Figure S17, the pretreatment with CUR or RES in

13

HK-2 cells could remarkably reduce the fluorescence of Brite 670 and FAM compared to single

14

CDDP treatment. In the meantime, the cellular viability was significantly improved by CUR or

15

RES for the CDDP treated HK-2 cells (Figure S18). It demonstrated that the nanoprobe could be

16

employed to visualize the protective effect of CUR and RES on CDDP-induced renal injury.

17

Brite/DEVD@LMWC NP was further expanded to monitor citrinin (CTN)-induced HK-2

18

cells injury. CTN was another compound which could typically damage the renal cells via •OH

19

production and caspase-3 activation40. ROS generation as reflected by the relative fluorescence


16

Page 17 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

intensity of H2DCFDA was distinct in the CTN-incubated HK-2 cells (Figure S19A), and the

2

oxidative stress led to a CTN concentration-dependent cell death determined by MTT assay

3

(Figure S19B). The different concentrations of CTN induced HK-2 cell injury were evaluated

4

with dual-signal Brite/DEVD@LMWC NP by confocal fluorescence imaging. After 4 h

5

incubation, the red fluorescence of Brite 670 was light-up when the CTN concentration was

6

greater than 20 µM (Figure S20A), indicating the early stage of apoptosis. After 16 h incubation,

7

both red fluorescence of Brite 670 and green fluorescence of FAM was bright (Figure S20B) due

8

to the •OH production and caspase-3 activation in the late stage. The result demonstrated the

9

generality of Brite/DEVD@LMWC NP for early and discriminative imaging of drug-induced

10

renal cell injury.

11

Kidney Tissue Injury Detection with Brite/DEVD@LMWC NP.

12

Mice in group were given a single intraperitoneal injection of CDDP with a dose of 20 mg/kg41,42

13

to obtain the animal model of CDDP-induced kidney injury. After receiving CDDP treatment,

14

the mice in model group showed varying degree of toxic signs, including lethargy, depression,

15

inappetence, dullness, weakness, change in behavior, disinclination to move, and loss of hair. In

16

addition, body weight of the mice in model group decreased and was obviously lower as

17

compared to the control group mice (Figure S21). The conventional sCrea and BUN as clinical

18

examination biomarkers was used to assess the time-dependent CDDP-induced kidney injury.

19

After 72 h CDDP administration, the content of sCrea (Figure S22A) and BUN (Figure S22B)

20

increased significantly, indicative of a severe loss of renal function. However, these two

21

biomarkers were not suitable for early-stage DIKI detection because the content change was not

22

obvious during the 48 h CDDP treatment. Cys-C and KIM-1 were used as early biomarkers43 to


17


Page 18 of 26

1

detect DIKI. The remarkable content change of serum Cys-C (Figure S22C) and KIM-1 (Figure

2

S22D) could be observed at 16 h and 24 h CDDP administration, respectively.

3

Compared with conventional methods, the dual-signal Brite/DEVD@LMWC NP was used to

4

detect DIKI. Mice intravenously injected with Brite/DEVD@LMWC NP were sacrificed and the

5

major organs were obtained to implement histopathological examination by hematoxylin-eosin

6

(HE) staining. No obvious pathological abnormality was observed in major organs including

7

heart, liver, spleen, lung, kidney, large intestine, and small intestine for the nanoprobe treated

8

mice compared with the untreated mice as control (Figure S23), which strongly indicated the

9

biocompatibility and biosafety of the nanoprobe in vivo. Mice were intraperitoneally injected

10

with CDDP or normal saline and then intravenously injected with Brite/DEVD@LMWC NP.

11

The kidney tissues were obtained at different time points of CDDP treatment to perform confocal

12

fluorescence imaging. For the normal saline-treated mice, no Brite 670 or FAM fluorescence was

13

observed (Figure S24), revealing no injury or nephrotoxicity with kidney tissues of mice in the

14

control group. For the model group of mice treated with CDDP and Brite/DEVD@LMWC NP,

15

the red fluorescence of Brite 670 appeared in the kidney slice after 8 h CDDP administration and

16

could be sustained afterwards (Figure 5) which manifested •OH production and alarmed us the

17

early damage to kidney. It was noteworthy that the positive time of 8 h using the nanoprobe

18

imaging was superior to that of 72 h for sCrea or BUN, 16 h for Cys-C, and 24 h for KIM-1

19

based conventional detection methods, demonstrating the advantage of nanoprobe for early

20

detection of DIKI. At 16 h CDDP administration, the green fluorescence of FAM in the kidney

21

slice appeared and increased gradually afterwards (Figure 5), suggesting the caspase-3 activation

22

and late-stage renal cell apoptosis. For the mice treated with free Brite 670 and D-DEVD-F, no

23

fluorescence was observed in the kidney tissue slices (Figure S25), illustrating the essential role


18

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1

of LMWC nanocarrier in the targeted delivery of Brite 670 and D-DEVD-F for DIKI imaging.

2

Based on the above results, the dual-signal nanoprobe could be used for early detection and

3

discriminative imaging of DIKI stages in the kidney tissue. Although the fluorescence was only

4

detected at the kidney tissue level due to the visible excitation and emission wavelength of Brite

5

670 and FAM (400-700 nm), this work should substantially broaden the prospect to further

6

develop a bioluminescent44, near-infrared (NIR) I (700-1000 nm)45, or NIR II (1000-1700 nm)46

7

nanoprobe. The bioluminescence or NIR fluorescence-based imaging could effectively reduce

8

the autofluorescence of body and enhance the penetrability of light, which will be powerful for in

9

vivo imaging DIKI and enrich preclinical and clinical investigations.

10 11

Figure 5. Confocal fluorescence images of kidney tissue slices for the mice injected with 20

12

mg/kg CDDP and 20 mg/kg Brite/DEVD@LMWC NP. The slices were stained with Hoechst

13

33342 to indicate nucleus. Scale bar: 50 µm.


19


Page 20 of 26

1

CONCLUSION

2

In summary, a dual-signal nanoprobe named as Brite/DEVD@LMWC NP has been successfully

3

developed by encapsulating Brite 670 and D-DEVD-F into LMWC with an ionic crosslinking

4

method. The presence of LMWC enables the nanoprobe with remarkable targeting and

5

outstanding biocompatibility. The nanoprobe can be effectively internalized into renal cells via a

6

megalin receptor-mediated endocytosis pathway and subsequently release Brite 670 and D-

7

DEVD-F. The released Brite 670 can respond to drug-induced •OH production to emit red

8

fluorescence with good sensitivity and specificity for early detection of DIKI. The positive time

9

is superior to that of conventional methods. The green fluorescence of D-DEVD-F can be light-

10

up by caspase-3 to reflect the late stage of DIKI. To the best of our knowledge, this is the first

11

time that a dual-signal nanoprobe has been used for early detection and discriminative imaging

12

of DIKI at different stages. Our further studies will focus on developing bioluminescent or NIR

13

nanoprobe to reduce the autofluorescence of body and enhance the penetrability of light for in

14

vivo imaging of DIKI, which will provide a powerful technology to predict DIKI and help us

15

prevent and treat drug-induced side effects timely.

16

ASSOCIATED CONTENT

17

Supporting Information

18

The Supporting Information is available free of charge on the ACS Publications website at DOI:

19

Supplementary experiments, tables, figures, and references (PDF)

20

Supplementary video for 18 s (AVI)


20

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1

AUTHOR INFORMATION

2

Corresponding Authors

3

E-mail: [email protected].

4

E-mail: [email protected].

5

ORCID

6

Bo-Yang Yu: 0000-0002-1401-9250

7

Jiangwei Tian: 0000-0003-1018-4694

8

Notes

9

The authors declare no competing financial interest.

10

ACKNOWLEDGMENTS

11

This research was supported by National Natural Science Foundation of China (21775166,

12

21505161), Fundamental Research Funds for the Central Universities (2632017ZD10), Natural

13

Science Foundation of Jiangsu Province (BK20150701) and Six Talent Peaks Project in Jiangsu

14

Province. The authors acknowledge the Cellular and Molecular Biology Center of China

15

Pharmaceutical University for assistance with confocal fluorescence imaging and we are grateful

16

to Xiao-Nan Ma for her technical help.


21


Page 22 of 26

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Prediction of Drug-Induced Nephrotoxicity with a Hydroxyl Radical and

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