Data on peptidyl platform-based anticancer drug synthesis and triton-x-based micellar clusters (MCs) self-assembly peculiarities for enhanced solubilization, encapsulation of hydrophobic compounds and their interaction with HeLa cells

The data presented here refer to a research article entitled “Self-Assembled Micellar Clusters Based on Triton-X-family Surfactants for Enhanced Solubilization, Encapsulation, Proteins Permeability Control, and Anticancer Drug Delivery” Solomonov et al., 2019. The present article provides the General Procedure for clusterization of Triton-X-based micelles and the effect of (i) metal ion, surfactant, and chelator concentration on the developed clusters formation, (ii) surfactant-chelator relation change, (iii) metal ion-micelles concertation ratio variation, (iv) metal ion replacement, (v) solvent replacement, (vi) kinetics of clusters formation, (vii) hydrophobic fluorescent dye (Coumarin 6) solubilization in aqueous MCs media, (viii) novel anticancer peptidyl drug synthesis and characterization and (ix) the viability of HeLa cells with and without the presence of drug-free Triton-X-based family MCs. These data provide additional insights useful for understanding all aspects of the micellar clusters formation, optimization, and control.


Specifications
Value of the data The data provide new insights into the micellar clusters synthesis based on Triton X-100 and X-114 surfactants, which can be of great value for researchers studying emulsions, surfactants, and self-assembly The data describes several routes to micellar clusters self-assembly with a different optical and chemical composition by varying the preparation conditions that will be helpful for optimizing experimental conditions for future effective hydrophobic compounds encapsulation The data describes the synthesis and characterization of newly developed anti-cancer PTR-58-CLB-CAMP peptide drug and demonstrates the viability of HeLa cells with and without the presence of drug-free Triton-X based micellar clusters, that may be relevant for future understanding differences in the interaction of other cell lines with the clusters This data could be relevant for researchers specializing in the fields of interfacial chemistry and self-assembly and may be used in future for the development of novel anticancer drugs, drug carriers for targeted drug delivery or enhanced solubilization of hydrophobic compounds

Data
This article includes the General Procedure to synthesize micellar clusters (MCs) based on the Triton-X family surfactants (TX-100 and TX-114). Application of the General Procedure and the effect of numerous alterations in the solution composition to prepare various MCs are presented in Figs. 1e3 and Table 1. Raw images measured by optical microscopy of the varying effect of metal ion, surfactant, and chelator concentration are shown in Figs. 4e16, the effect of surfactant-chelator relation change is shown in Table 2 and Fig. 17, while metal ion-micelle concertation ratio variation is presented in Table 3 and Figs. 18e19. The effect of metal ions replacement by co-chelator on the MCs formation is demonstrated in Figs. 20e23 and the effect of solvent and chelator replacement is shown in Fig. 24 and Table 4. Kinetics of the MCs formation is presented in Figs. 25e27. The solubilization of fluorescent dye such as Coumarin 6 in the MCs is shown in Fig. 28. The scheme of PTR-58-CLB-CAMP synthesis (Scheme 1), HPLC and LCMS chromatograms of the drug are presented in Fig. 29 and Tables 5e6. Finally, the  viability of HeLa cells with and without the presence of drug-free Triton-X-based family MCs is shown in Fig. 30. A summary table describing the effect of TX-100 and TX-114 applying in the micellar clustering and the most useful parameters of the MCs as well as additional data of the cells viability when treating with MCs are presented in the supplementary file (Scheme S1).

Application of the General Procedure for clusterization of triton micelles
To prove that the TX-100 and TX-114 clusters formation mechanism is specific and requires simultaneous presence all of four components in the mixture, according to the General Procedure, we have attempted to remove one or two of the components of the mixture. Therefore, the variations in the preparation procedure may be presented schematically as follows for each scheme in Table 1

Varying of metal ion, surfactant, and chelator concentration
In the general procedure, initial concentrations of all reagents were varied, while the concentration of NaCl was constant. The data are presented on Figs. 4e16.    Table 2 and Fig. 17.

Effect of metal ion replacement
Fe 2þ salt in the General Procedure was replaced by the salts, containing appropriate metal ion (NiCl 2 , MnCl 2 , CuCl 2 , ZnCl 2 , MgCl 2 , CaCl 2 ) or mixture. Initial salt concentration and concentrations of           other components remained the same as in the case of the General Procedure. The data are presented in Figs. 20e23.

Effect of solvent replacement
The data are presented in Fig. 24 and Table 4.

Kinetics of clusters formation
The data are presented in Figs. 25e27.

Fluorescent dye solubilization
A saturated dye solution of Coumarin 6 was prepared by dissolving it in MeOH. The prepared solution is vortexed vigorously for 5 min followed by centrifugation for 1 min at 5000 rpm and removing of undissolved substance. The concentration of the dye in the saturated solution was about 15 mM.     (Fig. S1).
For encapsulation of the dye, several routes are possible. The first one is to add 1 ml of desired dye to 10 ml drop, containing freshly prepared micellar clusters, obtained by General Procedure, based on Ni-BPhen complex. The second is to add 5 mle50 ml of freshly prepared micelle-chelator complex, to take 5 ml of the solution and to mix it with 5 ml of the salts solution (solution 2), as in the case of the General Procedure. The third route is to add 1 ml of desired dye to 5 or 10 ml of pure surfactant or 1 mle5 ml (or 10 ml, with NaCl) of micelle-chelator aggregate ('Support compound Scheme'), followed by incubation of the drops at 18e20 C over a reservoir sealed with silicon grease (24 well tissue culture plate VDX (Hampton Research) or Corning Inc.) containing 0.5 ml 200 mM NaCl or H 2 O. The data are presented in Fig. 28.
Summary of the most useful parameters for Triton-X-based MCs synthesis and followed encapsulation of the peptide anticancer drugs and hydrophobic compounds is presented in the supporting file (Table S1).

Anticancer drug synthesis
The data are presented in Scheme 1, Fig. 29 and Tables 5 and 6.

The viability of HeLa cells with and without the presence of drug-free triton-x-based family MCs
The data are presented in Fig. 30 and in Fig. S1 (supporting information).

The General Procedure of MCs droplets synthesis
The General Procedure of the Triton-X based MCs formation procedures is described in [1] and is based on the works [2e7].

Anticancer peptide drug conjugates synthesis
Camptothecin (CAMP), chlorambucil (CLB), all protected amino acids, resin, and coupling reagents were purchased from Tzamal D-Chem Laboratories Ltd. Petah-Tikva, Israel. All the solvents were purchased from Bio-Lab Ltd. Jerusalem, Israel or Gas Technologies Ltd., Kfar-Saba, Israel.
Loading of amino acid Fmoc Lys (Dde)OH. To resin with the provides described sequence (0.300 mg, 0.168 mmol loading) in a jacketed fritted peptide vessel was added a solution of protected amino acid Fmoc-Lys-(Dde)-OH (0.268 mg, 0.504 mmol) in NMP (3.5 ml), and after addition of DIPEA (0.165 ml, 1.01 mmol) the mixture was shaken for 1.5 h. After that, usual washings with NMP (5 times, 7 ml, 2 min each time) were applied to afford resin for ready for the next step.
Loading of CLB and CAMP. After post coupling wash and Fmoc-deprotection CLB (156 mg, 0.504 mmol), DIPEA (0.165 ml, 1.01 mmol) and coupling reagent PyBop (262 mg, 0.504 mmol) were preactivated in NMP (3.5 ml each) for 2 min at rt in usual manner and added to the peptidyl resin and shaken for 2 h). Completion of the reaction was monitored by ninhydrin test (Kaiser test, yellow). DDE group was removed by treatment with 2% hydrazine in DMF (2 Â 3min, 3.5 mL each) and subsequent usual washings with NMP (5 times, 7 ml, 2 min each time), obtaining deprotected peptidyl resin ready for the next step CAMPeCO 2 C 6 H 4 ep-(NO 2 ) (0.258 mg, 0.504 mmol) were dissolved in DMF (3.5 ml) and DIPEA (0.165 ml, 1.01 mmol), and then the pre-activated compound was added to the resin for coupling and shaken for 2h at rt. Then the resin was washed with NMP (5 times, 7 ml, 2 min each time). After the usual work up washing with (3 Â DCM, 5 ml each) the resin dried under the nitrogen and transferred to a vial for cleavage.
General Procedure for cleavage of loaded peptidyl platforms from Cl-Trt (2-Chlorotritylchloride) resin. A cold cleavage solution TFA (Trifluoroacetic acid)/triisopropylsilane/H 2 O 95:2.5:2.5, 5 ml) was added to the dried resin in the cleavage vessel. After shaking for 2 h, the solution was collected, and the resin washed with cold TFA (2 Â 1 ml each). After combining the TFA solutions, the solvent was evaporated under an N 2 stream and then precipitated by diethyl ether, purified by preparative HPLC on RP-18 (reverse phase-18). After purification, the collected fraction with the desired product was lyophilized to give PTR-58-CAMP-CLB. Analytical data: yield (87%), Purity (HPLC, 81%), LCMS m/z calcd for C 106 H 133 Cl 2 N 21 O 18 S 2 (Ms2H þ ) 2123.90, found (Ms/2) 1061.4. Labelling of the compound with the fluorescent dye have been done using the BODIPY-FL.

Fluorescence spectroscopy
The fluorescence spectra recording procedure is described in [1].

Optical microscopy
Images obtaining procedure is described in [1].

Staining of the cells for detection of changes in morphology
The procedure of cells preparation is described in [1].

High-performance liquid chromatography (HPLC)
All HPLC purifications were done via reverse phase on ECOM semi-preparative system with TOPAZ dual UV detection at 254 nm and 230 nm. Phenomenex Gemini ® 10 mm C18 110 Å, LC 250 Â 21.2 mm column was utilized. The column was kept at room temperature. Peaks were detected at 220 nm and 280 nm. Analytical RP-HPLC was performed on an UltiMate 3000 system (Dionex) using a Vydac C18 column (250 Â 4.6 mm) with silica (300 Å pore size) as a stationary phase. Linear gradient elution with eluent A (0.1% TFA in water) and eluent B (Acetonitrile) was used at a flow rate of 1 mL/min. Peaks were detected at 254 nm.

Liquid chromatography mass spectrometry (LCMS)
Electron spray ionization mass spectra (ESI-MS) were obtained using an Autoflex III smart-beam (MALDI, Bruker), Q-TOF micro (Waters) or LCQ FleetTM ion trap mass spectrometer (Finnigan/ Thermo). HPLC/LC-MS analyses were made using Agilent infinity 1260 connected to Agilent quadruple LC-MS 6120 series equipped with ZORBAX SB-C18, 2.1 Â 50 mm, 1.8 mm HPLC column. In all cases, the eluent solvents were A (0.1% Formic acid in H 2 O) and B (100% CH 3 CN). The UV detection was at 254 nm. The column temperature was kept at 50 C. The flow rate was 0.4 ml/min. The MS fragmentor was tuned on 30 V or 70 V in positive or negative mode.