Native Zinc Catalyzes Selective and Traceless Release of Small Molecules in β-Cells

The loss of insulin-producing β-cells is the central pathological event in type 1 and 2 diabetes, which has led to efforts to identify molecules to promote β-cell proliferation, protection, and imaging. However, the lack of β-cell specificity of these molecules jeopardizes their therapeutic potential. A general platform for selective release of small-molecule cargoes in β-cells over other islet cells ex vivo or other cell-types in an organismal context will be immensely valuable in advancing diabetes research and therapeutic development. Here, we leverage the unusually high Zn(II) concentration in β-cells to develop a Zn(II)-based prodrug system to selectively and tracelessly deliver bioactive small molecules and fluorophores to β-cells. The Zn(II)-targeting mechanism enriches the inactive cargo in β-cells as compared to other pancreatic cells; importantly, Zn(II)-mediated hydrolysis triggers cargo activation. This prodrug system, with modular components that allow for fine-tuning selectivity, should enable the safer and more effective targeting of β-cells.

All reagents were purchased and used as received from commercial sources without further purification.
Reactions were performed in round-bottom flasks stirred with Teflon®-coated magnetic stir bars. Moisture and air-sensitive reactions were performed under a dry nitrogen/argon atmosphere. Moisture and airsensitive liquids or solutions were transferred via nitrogen-flushed syringes. As necessary, organic solvents were degassed by bubbling nitrogen/argon through the liquid. The reaction progress was monitored by thin-layer chromatography (TLC) and ultra-performance liquid chromatography mass spectrometry (UPLC-MS). Flash column chromatography was performed using silica gel (60 Å mesh, 20-40 µm) on a Teledyne Isco CombiFlash Rf system. Analytical TLC was performed using Merck Silica gel 60 F254 pre-coated plates (0.25 mm); illumination at 254 nm allowed the visualization of UV-active material, and a phosphomolybdic acid (PMA) stain was used to visualize UV-inactive material. UPLC-MS was performed on a Waters ACQUITY UPLC I-Class PLUS System with an ACQUITY SQ Detector 2. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 400 Spectrometer ( 1 H NMR, 400 MHz; 13 C, 101 MHz) at the Broad Institute of MIT and Harvard. 1 H and 13 C chemical shifts are indicated in parts per million (ppm) relative to SiMe 4 (δ = 0.00 ppm) and internally referenced to residual solvent signals. NMR solvents were purchased from Cambridge Isotope Laboratories, Inc., and NMR data were obtained in CDCl 3 or DMSO. Data for 1 H NMR are reported as follows: chemical shift value in ppm, multiplicity (s = singlet, d = doublet, t = triplet, dd = doublet of doublets, and m = multiplet), integration value, and coupling constant value in Hz. Tandem liquid chromatography-mass spectrometry (LCMS) was performed on a Waters 2795 separations module with a 3100 mass detector. High-resolution mass spectra were recorded on a JEOL AccuTOF LC-Plus 46 DART system at the department of chemistry instrumentation facility at the Massachusetts Institute of Technology and a Thermo Q Exactive Plus mass spectrometer system equipped with an HESI-II electrospray ionization source at Harvard Center for Mass Spectrometry at the Harvard FAS Division of Science Core Facility.
Compounds 2 and 3 were made the same way as compound 1.
The linker was prepared as described by Gillies et al., and the spectroscopy data matched literature values (J. Am. Chem. Soc. 2009, 131, 18327-18334).

Beta-cell specific fluorophore release
In a 96-well plate, INS-1E (30,000 cells), HEK293T (12,000 cells), alpha TC1 clone 6 (30,000 cells), and PANC-1 (20,000 cells) were plated per well and cultured for 24 h at 37 °C with 5% CO 2 . Compounds (DA-ZP1, DA-FC, ZnPD1, ZnPD4, ZnPD5, and ZnPD1ctrl) at the indicated concentration in the corresponding cell culture medium containing 2× DAPI were incubated for 1 h inside the incubator. The medium containing compound was removed and washed with 3× 100 µL of fresh medium, and finally, another 100 µL of fresh, dye-free medium was added. The cells were immediately imaged using a high-content fluorescence microscope (Molecular Devices or Operetta Phenix) in both the DAPI and FITC channels at 20X magnification. Images were analyzed using the MetaXpress or Harmony software and data were plotted using GraphPad Prism 6.

Beta-cell specific staining of human islets
Approximately 30,000 freshly dissociated human islet cells per well were plated in a 96-well format and incubated for 24 h at 37 °C with 5% CO 2 . Cells were incubated with DA-ZP1 (5 µM) at the indicated S10 concentration in the human islet cell culture medium for 1 h in the incubator. Cells were then washed with 2× 100 µL of fresh medium before fixing and permeabilizing with cold methanol for 1-2 min following a literature protocol (Jamur et al., 2010). Cells were then immunostained with rat anti-C-Peptide antibody (Cell Signaling) overnight at 4 °C and subsequently stained with Alexa Fluor 647 anti-rat (ThermoFisher) and 1× DAPI (ThermoFisher) for 1 h. The plates were imaged using the Phenix high-content confocal microscope (Perkin Elmer) with DAPI as the nuclear counterstain.

DA-ZP1-or ZnPD4-assisted β-cell sorting
Upon receipt, islets were centrifuged at 1000 rpm for 1 min and resuspended in supplemented CMRL 1066 medium. Islets were then transferred to 10 cm culture dishes and cultured overnight to 24 h. Healthy islets were handpicked and washed with DPBS. For DA-ZP1 or ZnPD4 staining, islets were dissociated into single cells by TrypLE. Briefly, 1 mL TrypLE was added to human islet pellet and incubated at 37 °C for 12 min by mixing the tube every 3-4 min. At the end of incubation, TrypLE was neutralized by adding 9 mL DMEM HG containing 10% FBS. The cell suspension was filtered using a 30 m filter to remove any aggregates, counted on a hemocytometer, and pelleted by spinning cells at 1200 rpm for 5 min. Cells were washed with CMRL 1066 final wash/culture medium and resuspended in the final wash/culture medium with DA-ZP1 or ZnPD4 (160 nM) for 30 min at 37 °C. At the end of the incubation period, cells were washed and the cell pellet was resuspended in the final wash/culture medium. DA-ZP1 or ZnPD4 positive and negative cells were sorted by FACSAria cell sorter (BD Biosciences, Joslin Flow Cytometry Core).
Sorted cells were spun at 1200 rpm for 5 min and washed with DPBS, then fixed in 4% PFA for 15 min at room temperature. Cells were washed and embedded in agarose and paraffin, sectioned and used for immunostaining as previously described by El Ouaamari et al., (Cell Metab. 2016, 23, 194-205). Analysis of flow cytometry data was completed using FlowJo 10.4.2 (FlowJo LLC, Ashland, OR). The gating strategy is shown in Figures S3 and S7. The donor demographic information is summarized in Table S1.

Kinetics of DA-ZP1 and ZnPD4 in cells
The cells were plated in black-walled, clear-bottom 96-well plates coated with poly-D-lysine (PDL, Sigma Aldrich) for 1 h and washed with 200 µL of PBS/well. INS-1E and TC-1.6 cells were plated at 30,000 S11 cells/well, and PANC-1 and HEK293T cells were plated at 11,250 cells/well. After plating, the cells were incubated overnight at 37 °C. DAZP1 or ZnPD4 solutions were prepared in microcentrifuge tubes using the respective, dye-free cell mediums: RPMI 1640, DMEM + GlutaMAX, or DMEM (low glucose). The medium in the 96-well plates was removed by gently inverting the plates upside down. Any medium remaining in the wells was not removed. DAZP1 or ZnPD4 solutions (10 M-10 nM) in dye-free medium were added to the wells (100 L/well). After the solutions were added, the plates were incubated for 1-25 h at 37 °C, after which time the plates were gently inverted to remove the medium. Any residual medium was removed with a multichannel pipette. The wells were then gently washed twice with reconstituted dyefree cell medium (100 L/well). DAPI was then added to the wells in dye-free cell medium (100 L/well), and the plates were incubated at 37 °C for 30 min. DAPI was removed by gently inverting the plates, and the residual medium was removed with a multichannel pipette. The wells were washed with 50 L/well of dye-free INS-1E/PANC-1 medium. Finally, 100 L/well of dye-free cell medium was added to wells, and the plates were sealed for live-cell imaging.

Cargo release detection by LC-MS
Samples of 1 × 10 6 cells were treated with compound (5 µM) in a 6-well plate for the indicated duration before collecting by treatment with trypsin. Cells were re-suspended in 1 mL PBS and pelleted down at 4000×g for 2 min. The supernatant was removed carefully keeping the residual PBS along with the cell pellet. Cells were briefly vortexed and 10 µL DMSO was added before vortexing vigorously for 15 s. The cell suspension was flash-frozen in liquid nitrogen followed by thawing and vortexing for 15 s. This process was repeated twice. A 50-µL aliquot of acetonitrile containing 2 µM spautin as an internal standard was added and the mixture was vortexed for 30 s. The suspension was centrifuged at 10000×g for 1 min and then the supernatant was collected for mass spectrometric analysis.

Fluorescence spectroscopic studies on ZnPDs
The compound in PBS was taken in a 96-well microplate (250 µL, 10 µM per well, Nunc 96 Well Plates) and incubated with different concentrations of ZnSO 4 (0-1 mM). Reaction kinetics were monitored by measuring the fluorescence of the activated fluorescein dye (λ ex = 490 nm, λ em = 522 nm) over 20 h.

Stability of ZnPD5 in cell culture media
Samples of the compound in INS-1E and human islet media (250 µL, 10 µM per well) alone or in the presence of varying concentrations of ZnSO 4 (125-1000 µM) were incubated and the stability was monitored by measuring the fluorescence signal (λ ex = 490 nm, λ em = 522 nm) for over 12 h.

Cellular localization
Localization of DA-ZP1 release was performed following a reported cell-painting method (Nat. Protoc. 2016, 11, 1757. In a typical experimental procedure, INS-1E cells were treated with 5 µM DA-ZP1 for 1 h in a 96-well plate. The media was removed and 60 µL/well MitoTracker (Invitrogen, cat. no. M22426) solution containing 2× DAPI was added and incubated for 30 min in the dark. To the same well, 20 µL 16% PFA was added and incubated for an additional 20 min in dark. Cells were then washed twice with 100 µL HBSS and then processed for imaging. The cells were imaged by confocal microscopy using DAPI, FITC, and deep red (647 nm) channels.

Effect of the metal chelator
INS-1E cells were first incubated in media containing either DMSO or 25 µM TPEN for 30 min followed incubating with DA-ZP1 (5 µM) or treated with 4% PFA for fixation. DA-ZP1 treated live cells were processed for imaging after 30 min of incubation. The fixed cells were incubated with ZnPD5 (5 µM) in PBS containing 1× DAPI for 3 h. Cells were then imaged under a fluorescence microscope using DAPI and FITC channels.

Intact islet staining
Intact islet cells were pipetted up and down 5-7 times in culture media using 1 mL tips and then portioned (60-80 islets per condition) into 1 mL microtubes. Islets were incubated with ZnPD5 at 5 µM for 3 h inside the incubator with occasional mixing. Cells were then spun down (200 RCF, 2 min) and the supernatant was carefully removed. 300 µL of TrypLE was added to each tube and incubated at 37 °C for 12 min with occasional mixing. Culture media (700 µL) was added to each tube and pipetted up and down 7-10 times using a 1 mL pipet. Cells were then spun down at 200 RCF for 5 min and the supernatant was carefully removed. Cells were then resuspended in 300 µL media and transferred to a pre-coated 96-well plate in two replicates. Cells were allowed to adhere by culturing for 12 h and then fixed with 4% PFA for 20 min and subsequently permeabilized with 0.2% Triton-X for 20 min. Cells were then processed for primary and secondary antibody staining following the protocol as discussed earlier. Cells were imaged by using fluorescence microscopy under DAPI, FITC, and 647 nm channels.

Immunostaining
Sections of agar and paraffin-embedded cells were stained as previously described (J. Diabetes Res. 2015, 2015. Primary antibodies to all antigens and appropriate secondary antibodies with their working dilutions are listed in Table S2. Digital images were taken with an AXIO Imager A2 upright microscope equipped with an X-Cite series 120Q light source, Axiocam 512 color camera, and Zen 2.3 lite software. Images were overlaid and hormone-positive cells counted using ImageJ software. ≥ 5,000 cells were counted per group (n=3 replicate slides).

Human Islet proliferation
Human islets were dissociated into a single cell suspension using TrypLE and plated at 10,000 islet cells per well in coated 384-well plates. Islet cells were cultured in FBS-supplemented CMRL 1066 medium with 10 µM 5-ethynyl-2'-deoxyuridine (EdU) and incubated at 37 °C with 5% CO 2 . Every 2 days, cells were treated with DMSO and ZnPD5 in duplicate at doses ranging from 312.5 nM to 2.5 µM. After 6 days, cells were fixed using 3.7% PFA, permeabilized using 0.2% Triton X-100, and copper-catalyzed click chemistry S14 was performed to visualize proliferating cells using Alexa Fluor 488 azide (ThermoFisher). Cells were subsequently immunostained with rat anti-C-peptide antibody and goat anti-rat Alexa Fluor 647 (ThermoFisher) to mark β-cells along with DAPI as a nuclear counterstain. For each well, 16 fields were acquired using the Opera Phenix™ (PerkinElmer) and image analysis was performed using Harmony® Software (PerkinElmer) to calculate β-cell proliferation.