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  • br Materials and methods br All solvents and reagents

    2020-08-30


    2. Materials and methods
    All solvents and reagents were purchased from Sigma-Aldrich and used as received unless otherwise noted. Peptide synthesis reagents were purchased from AnaSpec (Fremont, CA).
    2.1. Peptide synthesis
    H8R8 was synthesized by conventional solid-phase microwave as-sisted peptide synthesis techniques (CEM Liberty 1) where double his-tidine and arginine couplings were employed. To make amide peptides, rink amide ProTide Resins were used (CEM Corp, NC, USA). Standard microwave-assisted Fmoc deprotection was used [17]. Peptides were prepared at 0.25 mmol scale using HOBt/DIC/Oxyma for activation. Fmoc-Arg(Pbf)-OH was purchased from Ark Pharm (Illinois, USA) and Fmoc-His(Trt)-OH were from EMD Millipore (Massachusetts, USA). 
    2.2. Synthesis of vitamin E-oxy-butyric acid
    Vitamin E was modified as follows: in a 250-mL round-bottom flask, 417.69 mg (1.46 eq) of NaH were dissolved in 50 mL of THF and stirred on ice under argon for 10 min. 5.12 g (1 eq) of Vitamin E (α-tocopherol) were dissolved in 50 mL of THF and added to the NaH solution, before stirring on ice for 15 min under argon. 2.492 mL (1.46 eq) of bro-moethyl butyrate were added to the solution before stirring under argon first on ice for 20 min and then at room temperature overnight. The solution was transferred to a large beaker and diluted with 300 mL of CH2Cl2. Liquid-liquid extraction with excess DI water was used to purify the Vitamin E-oxy-ethylbutyrate intermediate (ESI-MS+: C35H60O4 expected m/z 544.9, found m/z 545.5 corresponding to M + H). 1 g of Vitamin E-oxy-ethylbutyrate was hydrolyzed in a mix of 10% w/v KOH (5 mL) and THF (10 mL) overnight. The solution was then quenched with 10 mL DI water, adjusted to pH 3 with con-centrated HCl (5–10 drops), and extracted 3 times with CH2Cl2. The organic phase was washed 2 times with saturated NaCl solution, dried on MgSO4 and evaporated under vacuum to give the final product. The crude was then purified on a silica column, using chloroform as eluting solvent. Impurities were washed off in chloroform, and the final pro-duct was eluted in chloroform:methanol (9:1 v/v). The organic solvent was removed by rotary evaporation and dried in an oven. (ESI-MS: C33H56O4 expected m/z 516.8, found 515.4 corresponding to M-H).
    2.3. N-terminal peptide acylation
    To modify the N-terminus of the peptide (0.125 mmol), 4.9 eq. of HCTU were used to activate 5.0 eq. of 2NBDG (stearic acid or vitamin e succinate) in DMF for 15 min at room temperature. The activated acid was then added to the peptides on resin, while adding 1 eq. of N,N-diisopropylethylamine (DIPEA). The reaction was allowed to proceed for 24 h before the resin was washed 2 times with DMF, then 2 times with DCM. Conjugation was evaluated with the 2,4,6-Trinitrobenzenesulfonic acid (TNBS) test [18].
    2.4. Fluorescein labeling of peptide
    To synthesize the fluorescein-modified peptide, Fmoc-Lys(alloc)-OH (EMD Millipore, Massachusetts, USA) was used and inserted between H8 and R8 as NH2-H8K(Alloc)-R8-Resin. VES and stearic acid were se-parately conjugated to the N-terminus using HCTU as above. Then the alloc protecting group was deprotected 3-times, 20 min each, using 0.1 eq. of tetrakis(triphenylphosphine) palladium(0) (Pd(PPh3)), and 10 eq. of borane dimethylamine complex (Me2NH·BH3). Finally, 5(6)-Carboxyfluorescein was activated using HCTU and conjugated to the
    peptide as stated above. Fmoc-NH2-H8K(Alloc)-R8 -Resin was used to prepare the fluorescein modified H8R8 where alloc was deprotected as above, and fluorescein was added to the free amine on lysine as above. Fmoc was then deprotected using 20% piperidine in DMF.
    2.5. Cleavage and purification
    Final deprotection and cleavage off of the resin was completed using a cleavage cocktail composed of trifluoroacetic acid:water:-triisopropylsilane (TFA:H2O:TIS, 95:2.5:2.5 v/v/v) for 5 h. The peptide was then precipitated in cold ether, centrifuged and washed with cold ether and allowed to dry overnight. The precipitate was then dissolved in TFA, diluted in acetonitrile:water (ACN:H2O:TFA, 90:9.9:0.1 v/v/v), and purified through a C18 reverse phase column (Silicycle, Quebec, Canada) using a gradient of acetonitrile from 10 to 100%. Peptide mass was verified through electrospray ionization (EI) or matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectro-metry using the Agilent 6538 Q-TOF mass spectrometer. Stock solutions of peptides were prepared in DMSO.
    2.6. Nanoparticle characterization and critical micelle concentration measurements
    Str-H8R8 and VES-H8R8 were dissolved in DMSO at a concentration of 250 mg/mL, and diluted in PBS (pH 7.4), 1.5 μM citric acid in PBS (pH 6.3), or 3 μM citric acid in PBS (pH 5.3). The peptides were passed through a 0.2 μm polyethylsulfone (PES) filter and nanoparticle dia-meters 2NBDG were measured using a Malvern Zetasizer Nano ZS (4 mW, 633 nm laser) at a concentration of 2.5 mg/mL. Critical micelle con-centration (CMC) was calculated based on the scattering intensity measured by dynamic light scattering as previously described [19]. The scattering intensity was measured using a DynaPro Plate Reader II (Wyatt Technologies) configured with a 60 mW, 830 nm laser and a detector angle of 158°. Str-H8R8 and VES-H8R8 were added to clear bottom 96-well plates at a peptide concentration of 16.6 mg/mL in PBS (pH 7.4), and serially diluted with measurements of three acquisitions per sample. Curve fitting algorithms were used to determine the CMC of the nanoparticles.