Tuning the cationic interface of simple polydiacetylene micelles to improve siRNA delivery at the cellular level

Polydiacetylene micelles were assembled from four different cationic amphiphiles and photopolymerized to reinforce their architecture. The produced micelles were systematically investigated, in interaction with siRNAs, for intracellular delivery of the silencing nucleic acids. The performances of the carrier systems were rationalized based on the cell penetrating properties of the micelles and the nature of their cationic complexing group, responsible for efficient siRNA binding and further endosomal escape.


1-General
Unless otherwise specified, chemicals were purchased from Sigma-Aldrich and used without further purification. CH2Cl2 was distilled from calcium hydride before use. 1 H and 13 C NMR spectra were recorded on a Bruker Avance DPX spectrometer operating at 400 and 100 MHz, respectively. Chemical shifts (δ) are given in ppm relative to the NMR solvent residual peak and coupling constants (J) in hertz. Mass spectra were recorded using a MarinerTM ESI-TOF spectrometer. Wavenumbers are given in cm -1 at their maximum intensity. Dynamic light scattering (DLS) measurements were carried out using a Vasco Flex instrument by Cordouan Technologies equipped with a laser diode (λ = 450 nm). Zeta potential measurements were performed on a Wallis instrument from Cordouan Technologies equipped with a laser diode (λ = 635 nm). For ultrasonic mixing, an ultrasonic probe (Branson Sonifier 450, Output 4, Duty cycle 30%) was used. Photo-polymerization experiments were carried out using a low pressure 40 W mercury UV lamp (Heraeus) at 254 nm.

3-Characterization Critical Micelle Concentration (CMC) measurements.
CMC values were measured using the pyrene inclusion method. This method takes advantage of the environment specific fluorescence of the pyrene probe, as a detection of organized lipidic environment. A set of dilutions were prepared from 10 mM stock solutions of micelles ranging from 2 mM to 1 µM. 1 µL of 1 mM DMSO solution of pyrene was added to 1 mL of each sample and stirred vigorously for 2 h before fluorescence measurement. Fluorescence spectra were recorded at 339 nm UV excitation wavelength at 5 nm band pass. The relative intensities at 373 nm and 384 nm were recorded. The ratios of the relative fluorescence intensities I373nm/I384nm were plotted against log of mM concentrations. CMC is deduced from the inflexion point.

Assembly and dialysis of the micelles.
Amphiphile (10 mg) was first protonated in 1 mL of CH2Cl2/CH3OH 80:20 and 1 µL of HCl (37%). The solvent was evaporated under reduced pressure and a white solid was formed. The solid was then solubilized in 1 mL of 10 mM HCl and sonicated with an ultrasonic probe for 30 min. The solution was then subjected to UV irradiation at 254 nm for 5 h to yield a pale yellow solution of photo-polymerized product. Deionized water was added to replace the volume that was lost by evaporation during the photo-polymerization process. The dialyses were performed in 3 000 MWCO dialysis membranes (Thermo Fisher) against a 1000 times larger volume of slightly acidic water (0.1% v/v of HCl 37%) over 7 days.

Size measurements by Dynamic Light Scattering (DLS).
Six acquisitions (60 s each) were performed on the colloid samples. Mean hydrodynamic diameter values of 6.6, 7.6, 9.2, and 7.6 nm were recorded for pDA-AM 1, pDA-AM 2, pDA-AM 3, and pDA-AM 4 micelles, respectively.

Zeta potential.
Samples of the micelle colloids (10 mg mL −1 , 1 mL) were introduced in a dedicated cuvette equipped with an electrode and measurements were performed under 7.2 mV electric field. Zeta potential values of 29, 17, 15, and 30 mV were recorded for pDA-AM 1, pDA-AM 2, pDA-AM 3, and pDA-AM 4 micelles, respectively.

4-Biology siRNA transfection and proliferation/survival assay.
The human cervical adenocarcinoma Hela cell line (ATCC) was routinely grown in DMEM (Sigma) supplemented with 10% [v/v] Fetal Bovine Serum (PAA), Penicillin (100 UI mL −1 , Sigma) and Streptomycin (100 µg mL −1 , Sigma). For transfection purpose, siRNAs diluted in OPTIMEM (Gibco) were complexed with either our micelles at various N/P ratios, or with Lipofectamine RNAiMAX (Life Technologies), in collagen-coated (Rat tail collagen, Sigma), clear bottom, blackwalled 384-well culture plates (Greiner μClear plates, Cat# 781091). After 20 min of complexation, Hela cells were seeded on top of the complexes (1000 cells/well; final [siRNA] = 20 nM), and incubated for three days at 37 °C and 5% CO2 in a humidified incubator (Forma Stericycle, Thermo). Plates were then fixed overnight with para-formaldehyde (4% [w/v] in PBS, Sigma), and nucleic acids were stained with Hoechst 33342 (2 μg mL −1 , Sigma). After a PBS wash, plates were imaged on a High Content Imaging device (Operetta HCS epifluorescence microscope, Perkin Elmer). Three fields per well were acquired at 10× magnification in the blue channel (λex = 380 ± 20 nm; λem = 445 ± 35 nm). An automated algorithm was developed under Harmony 3.0 (Perkin Elmer) as described before. 17 Briefly, the algorithm segments nuclear Regions of Interest (ROI) based on the DNA-bound Hoechst fluorescence, then quantifies the total cell amount per well. All results were normalized to the untreated wells, and expressed as averaged results ± SD of four wells per condition. A pool of cytotoxic siRNAs (Allstars maximal death control, Qiagen) and a scrambled siRNA (UNR, target sequence: AAGCCGGTATGCCGGTTAAGT, Qiagen) were used as positive and negative phenotypic controls of transfection efficiencies, respectively.

Gel retardation assay.
siRNA/micelle complexes were prepared by mixing 20 pmol of scrambled (UNR) siRNA diluted in OptiMEM with varying amounts of micelles to achieve specific N/P ratios, ranging from 1 to 100. After 20 min of incubation, DNA loading buffer (Thermo Fisher) was added to the resulting complexes, and samples were loaded into a 2% (w/v) agarose gel containing 1 mM EDTA and 40 mM Tris acetate buffer pH 8.0 (TAE, Gibco). Sample migration was performed at 70 V for 80 min, and after electrophoresis, the gel was stained during 30 min with TAE buffer containing 0.5 µg mL −1 ethidium bromide (Sigma-aldrich). After two subsequent washes in TAE buffer, the siRNA@micelle complexes were detected with an UV Transilluminator (Vilber Lourmat) at 254 nm. 1 H-NMR 13 C-NMR S13 6-Cytotoxicity studies of micelles Figure S1: Cell proliferation/survival assay of Hela cells exposed to increasing concentrations of pDA-AM 1, pDA-AM 2, pDA-AM 3, and pDA-AM 4 micelles. 72 h post exposure to the micelles, cells were fixed, nuclei were stained with Hoechst 33342, and the cell amounts into culture wells were assessed by High Content imaging. Results are expressed as the average relative cell amount in treated wells normalized to untreated wells.