Chemicals, peptides and cell lines
Boron clusters (as sodium salts) were from Katchem, streptomycin sulfate and kanamycin A monosulfate both from Sigma, MMAF from Carbosynth and dBET1 from Cayman Chemicals. Peptides (WR7 and WK7) were custom-made by Biosyntan in >98% purity as confirmed by HPLC and MS. TAMRA-R8 was synthesized by solid-phase peptide synthesis, as reported51. HeLa, HEK293, ARPE-19 and A549 cells were obtained from ATCC, and GT1-7 cells were obtained from Millipore. HeLa, HEK293, GT1-7 and A549 cells were maintained in DMEM, and ARPE-19 in DMEM/F-12, in all cases supplemented with 10% FBS and 1% penicillin–streptomycin–glutamine mix, at 37 °C, 5% CO2 and 95% humidity.
A thin lipid film was prepared by evaporating a lipid solution with a stream of nitrogen and then dried in vacuo overnight. For the zwitterionic vesicles, 25 mg EYPC in 1 ml of CHCl3 was used, and for the anionic vesicles, DMPE/DPPG/CHOL (4.4/10.4/2.6 mg, 1/2/1 molar ratio) in a 1:1 mixture of CHCl3 and MeOH (1 ml) was used. To prepare the lipid⊃HPTS/DPX vesicles (where ⊃ indicates encapsulation), the dry film was rehydrated (for 30 min at ambient temperature for EYPC, and for 60 min at 55 °C for DMPE/DPPG/CHOL) with 1 ml buffer (5 mM HPTS, 16.5 mM DPX, 10 mM Tris, 72 mM NaCl, pH 7.4) and subjected to 10 freeze–thaw cycles and extrusions (15 times) through a polycarbonate membrane (pore size 100 nm). Extravesicular components were eluted by size exclusion chromatography (NAP-25 column Sephadex G-25 DNA grade) with 10 mM Tris, 107 mM NaCl, pH 7.4 (ambient temperature for EYPC, 65 °C for DMPE/DPPG/CHOL). The lipid⊃CF vesicles were prepared analogously, except for the types of rehydration/elution buffers, which were 50 mM CF, 10 mM HEPES, pH 7.5/10 mM HEPES, 107 mM NaCl, pH 7.5 for EYPC⊃CF and 100 mM CF, 10 mM Tris, pH 7.4/10 mM Tris, 140 mM NaCl, pH 7.4 for DMPE/DPPG/CHOL⊃CF.
Transport experiments in HPTS/DPX vesicles
EYPC vesicle stock solutions (5–8 µl) were diluted with buffer (10 mM Tris, 107 mM NaCl, pH 7.4) in a disposable plastic cuvette and gently stirred (total volume 2,000 µl, final lipid concentration 13 µM). HPTS fluorescence was monitored at wavelength λem = 511 nm (λex = 413 nm) as a function of time after addition of boron clusters at 50 s, analyte at 100 s and Triton X-100 (24 µl, 1.2% wt/vol) at 600 s, the latter to lyse the vesicles, for calibration. Fluorescence intensities were normalized to fractional emission as I(t) = (It − I0)/(I∞ − I0), where I0 = It before cluster addition and I∞ = It after lysis. For Hill analysis, It before lysis was defined as transport activity, Y, and plotted against cluster (or analyte) concentration, c, and fitted to the Hill equation Y = Y0 + (Ymax − Y0)/(1 + (EC50/c)n), to give the activity in the absence of cluster, Y0, the maximal activity, Ymax, the concentration needed to achieve 50% of maximal activity, EC50, and the Hill coefficient, n.
In the activator measurements, in which different activators were tested with the same cargo, the activator efficiency (Ea) is determined from their ability to activate the transport of an impermeable cargo molecule and is characterized by Ymax, its maximal activity, and EC50, the effective activator concentration. A potent activator reaches high Ymax at low EC50. To reflect both factors, the activator efficiency is defined as Ea = Ymax × (pEC50/fa), where pEC50 is the negative logarithm of EC50. To enable comparison with literature studies, which aimed for a scale of Ea values from 0 to 10 (ref. 18), the scaling factor fa was set to 20.6.
In the transport measurements, in which different types of cargo were tested with the same activator, the transport efficiency (Et) reports on the sensitivity of the cargo for being transported and is described by Ymax, the maximal activity, and EC50, the effective cargo concentration. An easily accessible cargo reaches high Ymax at low EC50. The transport efficiency is defined as a composition of both parameters according to Et = Ymax × (pEC50/ft), where pEC50 is the negative logarithm of EC50. The scaling factor ft was deliberately set to 19.8 such that the Et value of the reference compound, WR7, equals 10.0, also in an effort to set up a scale from 0 to 10.
Leakage experiments in CF vesicles
For leakage experiments with the lipid⊃CF vesicles, stock solutions (6 µl) were diluted with the respective buffer in a disposable plastic cuvette and gently stirred (total volume 2,000 µl, final lipid concentration 13 µM). CF fluorescence was monitored at λem = 517 nm (λex = 492 nm) as a function of time after addition of the respective activating or disrupting agent (cluster, WR7 or pyrenebutyrate) at 50 s, and Triton X-100 (24 µl 1.2% (wt/vol)) at 600 s, the latter to lyse the vesicles, for calibration. Fluorescence intensities were normalized to fractional emission intensity as I(t) = (It − I0)/(I∞ − I0), where I0 = It before disrupting agent addition and I∞ = It after Triton X-100 lysis. For Hill analysis of the data for the DMPE/DPPG/CHOL⊃CF vesicles, It before Triton X-100 lysis was defined as membrane-disrupting activity, Y, and plotted against disrupting agent concentration, c, and fitted to the Hill equation Y = Y0 + (Ymax − Y0)/(1 + (EC50/c)n), to give Y0, Ymax, EC50 and n.
U-tube transport experiments
The U-tubes were home-made, similarly to those of Rebek and co-workers52 and Matile and co-workers19, and consisted of a small beaker with a central glass barrier separating the two aqueous phases, namely cis (sampling phase) and trans (receiving phase), but enabling the placement of an interfacing chloroform layer below the cis and trans phases. A 3 ml portion of CHCl3 was located in the U-tube and 1 ml of the cis and trans phases were added. The organic phase was stirred at 700 r.p.m. at room temperature. Aliquots (20 μl) from the aqueous trans phase were taken at different times, diluted to 450 μl with buffer (10 mM Tris, 107 mM NaCl, pH 7.4) and measured by fluorescence.
Isothermal titration calorimetry
All experiments were performed in a VP-ITC MicroCalorimeter from MicroCal, at atmospheric pressure and 25 °C. Solutions were degassed and thermostated before the titration experiments in a ThermoVac accessory. A constant volume of B12Br122− (10 µl per injection) was injected into the peptide solution (WR7 or WK7) in water to determine the apparent binding affinity of B12Br122− with the peptides. Dilution heats were determined by titration of B12Br122− into water and subtracted from the reaction heat. The neat reaction heat was fitted with Origin v.7.0 and v.8.0 software by using a one-set-of-sites model to obtain the complex stability constant (Ka) and molar reaction enthalpy (ΔHº). The free energy (ΔGº) and entropy changes (ΔSº) were obtained according to the relation ΔGº = −RTlnKa = ΔHº − TΔSº.
Dynamic light scattering
DLS experiments were carried out on a Malvern Instruments DTS Nano 2000 Zeta-Sizer. Note that DLS measurements of the combinations of the B12Br122− clusters with the different cargos did not show any detectable signal of particles of DLS-measurable size.
Cell culture and confocal imaging
For confocal microscopy studies, HeLa cells were seeded the day before on a µ-Slide 8 well (ibidi) at a density of 30,000 cells per well. The clusters and/or peptides were diluted in HKR buffer (5 mM HEPES, 137 mM NaCl, 2.68 mM KCl, 2.05 MgCl2, 1.8 CaCl2, pH 7.4) and added to the cells previously washed with HKR. HeLa cells were incubated with TAMRA-R8 (1 µM) and dodecaborate clusters in HKR buffer for 1 h at 37 °C, 5% CO2, washed with DMEM without phenol red and immediately imaged using Fusion software (Andor) with a Dragonfly spinning disc confocal microscope mounted on a Nikon Eclipse Ti-E and equipped with an Andor Zyla 4.2 PLUS sCMOS digital camera. For the phalloidin delivery studies, HeLa, GT1-7, ARPE-19 and A549 cells were incubated with phalloidin-TRITC and the boron cluster for 3 h and subsequently the nuclei were stained with 1 µM Hoechst 33342 for 20 min right before imaging. Images were processed with FIJI v. 2.1.0/1.53e (ref. 53).
Cell viability assay
For MTT assays in the presence of the clusters and TAMRA-R8, HeLa cells were seeded the day before in 96-well plates at 10,000 cells per well. Cells were incubated with the clusters dissolved in DMEM, in the presence or absence of 1 µM TAMRA-R8 for 1 h. The incubation mixtures were replaced with DMEM + 10% FBS + 0.5 mg ml–1 MTT. For the viability assays in the presence of B12Br122− or R8, HeLa, GT1-7, ARPE-19 and A549 cells were seeded the day before in 96-well plates at 6,000 cells per well. Cells were incubated with B12Br122− or R8 dissolved in HKR buffer for 3 h and, thereafter, incubated for 24 h with complete medium before incubating with complete medium and 0.5 mg ml−1 MTT. For viability studies in the presence of MMAF, HeLa cells were incubated with MMAF and B12Br122− diluted in DMEM (without serum or antibiotics) for 3 h. Cells were washed with 0.1 mg ml−1 heparin and further incubated for 21 h in complete medium and 2 h in complete medium containing 0.5 mg ml−1 MTT. For all types of assays, after 2 h of incubation, the medium was carefully removed, and formazan crystals dissolved by addition of DMSO. The absorbance at 570 nm was measured with a plate reader (Tecan Infinite F200Pro) and the data normalized to the value of untreated cells (100% viability). Data were analysed with R (v. 4.0.3)54.
Kanamycin A delivery in E. coli
A preculture of E. coli Top10 cells was incubated overnight in LB medium with 50 µg ml−1 streptomycin sulfate. The following day, 103–104 colony forming units per ml were grown in Costar cell culture 96-well plates in the presence of different concentrations of kanamycin A monosulfate (0, 2.5, 3 or 3.5 µg ml−1) and B12Br122− (0, 500, 750 or 1,000 µM) in LB medium without streptomycin at 37 °C in a shaking incubator. After 18 h, the optical density at 570 nm, as an indicator of bacterial growth, was measured with a Tecan Infinite F200Pro microplate reader. Data were normalized for each concentration of B12Br122− relative to the control condition without antibiotic.
CRBN target engagement assay
This assay was performed according to the protocol by the manufacturer (Promega), with the required adaptation for carrier addition. HEK293 cells were co-transfected with the plasmids for NanoLuc-CRBN and DDB1 expression using Lipofectamine 2000. Cells were trypsinized, resuspended in Opti-MEM I at 200,000 cells per ml, and 34 μl dispensed on a white, non-binding surface plate (Corning). dBET1 serial dilutions were prepared in DMSO at 1,000× concentration, and further diluted in Opti-MEM I to 20×. B12Br122− was diluted to 20× in Opti-MEM I and mixed 1:1 with the dBET1 solutions. A 2 μl portion of NanoBRET target tracer CRBN reagent (final concentration, 0.5 μM) and 4 μl of the dBET1/B12Br122− mixtures were added to the cells, which were incubated for 2 h at 37 °C. Complete substrate-plus-inhibitor solution was prepared and bioluminescence resonance energy transfer (BRET) was measured with a Tecan Infinite 200Pro plate reader (filters Blue2 and Red; integration time of 1 s). Background correction was carried out by subtracting the signal of a sample without tracer. Values of each B12Br122− concentration series were normalized to the BRET readout of the controls without dBET1. Data were analysed with R (v.4.0.3)54.
Cytosolic TAMRA-R8 concentration
Cytosolic extracts were obtained according to a previously described protocol55 by incubation with digitonin, a steroidal saponin that preferentially permeabilizes cholesterol-rich membranes, such as the plasma membrane, with minor effects on intracellular membranes. Briefly, HeLa cells were seeded at 260,000 cells per well in six-well plates, washed the next day twice with HKR, incubated with 1 µM TAMRA-R8 (the L enantiomer) in the presence or absence of 10 µM B12Br122− for 1 h, washed twice with HKR, three times with 2 mg ml−1 heparin in HKR and once with ice-cold PBS containing calcium and magnesium. Cells were incubated on ice with 600 µl of 35 µg ml−1 digitonin in PBS Ca/Mg for 10 min, the supernatant with the cytosolic fraction collected and cells washed with 200 µl of PBS Ca/Mg, combining this supernatant with the previous extract. The non-cytosolic fraction was collected by incubation of the cells with 800 µl of 1% Triton X-100 in PBS. TAMRA fluorescence of the extracts was determined in a plate reader (Tecan Infinite 200Pro, λex = 555 nm, λem = 585 nm) and concentrations were calculated by using a calibration curve with serial dilutions of TAMRA-R8. For the complementary HPLC analysis, phosphate buffer was replaced by TBS (20 mM Tris–HCl, pH 7.2, 150 mM NaCl, 0.5 mM CaCl2, 0.5 mM MgCl2) and digitonin extraction was performed as indicated above. An aliquot of these extracts was used for β-hexosaminidase activity determination. Cytosolic extracts were lyophilized and resuspended in 1:10 volumes of H2O:CH3CN 1:1 with 1% TFA, and analysed by HPLC (RP-HPLC Agilent Luna 5U C18 100 Å, H2O (0.1% TFA)/CH3CN (0.1% TFA) 100:0 (0→5 min); 100:0→5:95 (5→20 min)) by monitoring the 555-nm absorbance of the TAMRA chromophore.
The quality of fractionation was assessed by lysosomal β-hexosaminidase activity, using 4-nitrophenyl 2-acetamido-2-deoxy-β-d-glucopyranoside as substrate. Briefly, 20 µl of extract was incubated with 80 µl of 7.5 mM substrate in 100 mM citrate buffer, pH 4.7, for 40 min at 37 °C, and the reaction was stopped by addition of 200 µl of 0.2 M Tris solution. Absorbance at 405 nm was measured in a plate reader. As blank, wells containing only the substrate were used. The enzymatic activities were found to be 3.2 ± 2.0% in the presence of the peptide and 5.4 ± 1.0% in the presence of peptide and cluster, confirming a high purity of the cytosolic fractions.
HeLa cells, seeded at 260,000 cells per well in six-well plates the day before, were washed with HKR and incubated for 3 h with 2.5 ml per well of 50 µM of each boron cluster diluted in HKR. Cells were washed with HKR containing 0.1 mg ml−1 heparin, twice with HKR and subsequently lysed with concentrated nitric acid (69% HNO3). Cells from nine wells were pooled for each sample. Lysates were diluted before analysis by ICP-MS in an Agilent 7700x equipped with a MicroMist glass low-flow nebulizer, a double-pass spray chamber with a Peltier system (2 °C) and a quartz torch. A calibration curve for the element boron (B) between 10 and 1,000 μg l−1 was prepared with the element germanium (Ge) as internal standard. The ICP-MS instrument parameters were as follows: RF power, 1,550 W; sample depth, 8 mm; carrier gas flow, 1.1 l min−1; nebulizer pump speed, 0.1 r.p.s.; S/C temperature, 2 °C. Other parameters were set as follows: extract 1, 0; extract 2, −175; omega bias, −100; omega lens, 12.6; cell entrance, −40; cell exit, −60; deflect, 0.4; plate bias, −60; QP bias, −15; OctP RF, 180; OctP bias, −18; He gas, 3.6; discriminator, 4.5 mV; analogue HV, 1,730 V; pulse HV, 954 V.
HeLa cells were seeded at 10,000 cells per well in 96-well plates. The next day, they were incubated for 1 h with the indicated compounds diluted in HKR. Cells were subsequently washed for 5 min with HKR containing 0.1 mg ml−1 heparin, washed again with HKR and trypsinized. Trypsin was neutralized with PBS containing 2% FBS and 5 mM EDTA. TAMRA fluorescence was excited with a green laser (532 nm) and measured on a Guava easyCyte BG HT collecting the emission at 620/52 nm (Orange-G channel) and using InCyte v.3.2 (GuavaSoft, Millipore). Data were analysed with R (v.4.0.3)54 and the packages CytoExploreR (v.1.0.8)56 and ggcyto (v.1.18.0)57 Cells with typical FSC and SSC parameters were selected and the median fluorescence intensity calculated for each sample. Each condition was measured in triplicate.
Synthesis and characterization of TAMRA-D-R8
TAMRA-D-R8 was synthesized via manual Fmoc solid-phase peptide synthesis, using Fmoc-Rink amide resin (loading, 0.19 mmol g−1), as previously described51. TAMRA-D-R8 was obtained after RP-HPLC purification with an overall yield of 17% (15 mg) in 99% purity. It was characterized on an RP-HPLC Agilent SB-C18 column, H2O (0.1% TFA)/CH3CN (0.1% TFA) 95:5→5:95 (0→12 min)]. Rt, 5.96 min. MS (ESI): 1,124.7 (9, [M+2H+4TFA]2+), 1,067.9 (17, [M+2H+3TFA]2+), 1,011.0 (14, [M+2H+2TFA]2+), 712.4 (37, [M+3H+3TFA]3+), 674.2 (100, [M+3H+2TFA]3+), 636.3 (95, [M+3H+TFA]3+), 598.2 (36, [M+3H]3+), 534.5 (24, [M+4H+3TFA]4+), 506.0 (36, [M+4H+2TFA]4+), 477.5 (48, [M+4H+TFA]4+), 449.1 (62, [M+4H]4+).
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