Supplementary Material for:

In vitro Biocompatibility Evaluations of Hyperbranched Polyglycerol Hybrid Nanostructureas a Candidate for Nanomedicine Applications

Ali Zarrabi[†,1], Mohammad Ali Shokrgozar2, Manouchehr Vossoughi3, Mehdi Farokhi4

1 Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan, Iran

2 National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran

3 Institute for Nanoscience and Nanotechnology, Sharif University of Technology, Tehran, Iran

4 Department of Tissue Engineering and Cell Therapy, School of Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran, Iran

Experimental

Materials, Cell Lines and Animals

β-CD, anhydrous methanol, dimethyl sulfoxide (DMSO), and acetone were purchased from Merck, Germany. Potassium methoxide solution (3.7 M in methanol) was purchased from Fluka, Germany, and used as received. Glycidol (96%) was purchased from Acros, Belgium, and purified by vaccum distillation and stored in a refrigerator (4 °C). For the in vitro biocompatibility tests, the polymer solutions of predefined concentrations were freshly prepared in isotonic saline solution and then neutralized and sterilized by filters (Whatman syringe filter, pore size: 200 nm) before introducing the media to the cell medium. MTT and NR reagent were purchased from Sigma, Germany. The L929 mouse fibroblast and breast cancer cells BT-20 were obtained from Pasteur Institute, National Cell Bank of Iran. Cell culture media, fetal bovine serum (FBS) and antibiotics were purchased from GIBCO, Germany. Blood was drawn from healthy unmedicated donors into anticoagulated glass tubes (Becton Dickinson, Franklin Lakes, NJ) containing 3.2% sodium citrate. Plasma was isolated from the blood by centrifugation and used immediately. Innovin® reagent and actin were purchased from Dade Behring, Germany. Single radial immuno-diffusion (SRID) immunoassay kit was purchased from Biogene, Mashhad, Iran. Formaldehyde solution 10%, melt paraffin and Eosin and Hematoxylin stains were purchased from Merck, Germany.The water used was double-distilled deionized. Other chemicals were analytical grade.

Characterization

Proton Nuclear Magnetic Resonance (1H NMR) spectra were recorded in D2O solution, on a Bruker DRX 400 (400 MHz, DMSO-d6, δ) spectrometer with the solvent proton signal for reference. Carbon Nuclear Magnetic Resonance (13C NMR, 100 MHz, CDCl3, δ) spectra were recorded on the same instrument using the solvent carbon signal as a reference. The molecular weight was determined by gel permeation chromatography (GPC). In brief, the synthesized hybrid nanostructures were dissolved in DMSO at a concentration of 5 mg ml-1 and then tested using a GF-710HQ column (Showa Denko Co., Ltd., Tokyo, Japan) with DI water as the eluent and pullulan as standard. The particle size and polydispersity were determined using Dynamic Light Scattering (DLS) (Zetasizer ZS, Malvern Instruments) using a 4mW He–Ne laser (633 nm wavelength) with a fixed detector angle of 173 °.

β-CD-g-PG hybrid nanostructure synthesis

The reaction was carried out in a glass reactor equipped with a mechanical stirrer under nitrogen atmosphere. In a typical synthesis, β-CD (0.5 g, 0.5 mmol) was first added to a methanol solution of potassium methoxide (3 ml, 0.78 g) and mixture was stirred for 1 h at room temperature for partial deprotonation of β-CD. Then methanol was evaporated using vacuum evaporator and glycidol (6.17 ml, 92 mmol) was added to the mixture gradually and drop wise at 100 ºC over2 h, choosing the initiator amount according to the monomer/initiator ratio. Mixture was stirred at this temperature for 12 h. Then it was cooled and dissolved in methanol and neutralized by filtration over cation-exchange resin. The product was twice precipitated from methanol into acetone as a viscous light yellow compound and dried using vacuum oven at 80 ºC for 6 h.

All steps of reactions were performed in a nitrogen atmosphere to avoid any oxygen interference with anionic species.

Results and discussion

β-CD-g-PG hybrid nanostructure synthesis

For initiating the polymerization of glycidol, β-CDwas deprotonated partially (10%) or completely using potassium methoxide and it was used to initiate the polymerization as multifunctional initiator(Scheme 1). According to literature, the use of a polyfunctional initiator results in considerable lowering of the polydispersity.6β-CD, as core initiator, has 21 hydroxyl group functionality on each core. Therefore, we expected to achieve a hybrid nanostructurewith the least polydispersity reported. Based on GPC results from several species of β-CD, our hybrid nanostructurehad polydispersity of mostly less than 1.3.

Synthesis of β-CD-g-PG hybrid nanostructures was based on anionic ring opening polymerization of glycidol under slow monomer addition conditions using hydroxyl functional groups of β-CD as initiators. The slow addition of monomer leads to well-defined growth of the supramolecule on the basis that monomer exclusively reacts with the growing multifunctional hyperbranched polymer.

Number and degree of polymerization (DP) of grafted PG branches to β-CD were strongly depended on the initiator (potassium methoxide) to hydroxyl groups of β-CD (P/OH) and monomer (glycidol) to initiator (G/P) ratios. In a constant G/P ratio, increasing the ratio of P/OH leads to an increase in the number of PG branches but a decrease in their DP, whereas in a constant P/OH ratio, increasing the G/P ratio leads to β-CD-g-PG hybrid nanostructures containing lower number of PG branches with higher DP. In this manuscript in a typical β-CD-g-PGx(y), x and y are P/OH and G/P ratios, respectively. For example β-CD-g-PG0.047(5) is synthesized using P/OH=1/21 and G/P=5, whereas β-CD-g-PG1(5) is synthesized using P/OH=1 and G/P=5. Comparing these two hybrid nanostructures, the number of conjugated PG branches to β-CD in the β-CD-g-PG1(5) is higher but the DP of conjugated PG branches is lower than that for β-CD-g-PG0.047(10). However increasing the ratio of glycidol to CD always result in hybrid nanostructures with higher molar masses.

Scheme 1. Synthesis of β-CD-g-PG hybrid nanostructures

Characterization

Nuclear magnetic resonance

Figure S1 a and b show the 1H NMR and 13C NMR spectra of β-CD-g-PG1(5) hybrid nanostructure. The 1H NMR spectra of β-CD-g-PG show the four methylene and one methane protons of PG as broad resonance band between 3.3 and 4.1 ppm and the hydroxyl protons (as well as water protons) give a signal at 4.8 ppm only in methanol. In the 1H NMR spectrum, signal at 5.26 ppm is assigned to anomeric proton of β-CD. The polyglycerol backbone consists of linear (L), dendritic (D) and terminal units (T). In the 13C NMR spectrum of β-CD-g-PG there are seven well-resolved peak regions between 60 to 90 ppm; (1) L13: –CH2OH carbon at 62.8 ppm, CH2 carbon at 71.3 ppm and -CHOH at 81 ppm. (2) L14: both CH2 carbons at 74 ppm, CHOH carbon at 70.8 ppm. (3) Terminal unit (T): CH2OH carbon at 65 ppm, CHOH carbon at 72.3 ppm, and the CH2 carbon at about 72.5 ppm; (4) dendritic unit (D): CH carbon at 80.2 ppm, one CH2 carbon at 73.0 ppm, and the other at about 72.5 overlapping with a CH2 carbon of a terminal unit. The C1, C2, C3, C4, C5 and C6 signals of β-CD are lied in the region between 59 to 101 ppm.

Figure S1. a) 1H NMR b) 13C NMR spectra of β-CD-g-PG1(5) hybrid nanostructure

Molecular weight measurements

GPC diagrams for another series of β-CD-g-PGx(y) hybrid nanostructures in which x varies but y is constant are shown in Figure S2. Here, the molecular weight of hybrid nanostructures increase is resulted from increasing the number of the grafted PG branches.

Figure S2. GPC diagrams for series of β-CD-g-PGx(y), x varies but y is constant

Size and size distribution measurements

Size of carriers in biological systems is a critical factor and it is limited to nanometer scale when going through the cell membrane. The size of synthesized hybrid nanostructures was investigated in water solution by DLS experiments. Due to the high functionality and three dimensionality of β-CD, the size of β-CD-g-PG hybrid nanostructures was small enough to be used in nanomedicine applications. According to DLS experiments, the size of synthesized hybrid nanostructures depends strictly on the DP of grafted PG branches, G/P molar ratio, and density of PG branches, P/OH molar ratio. For example, the size of β-CD-g-PG1(5), β-CD-g-PG1(10),and β-CD-g-PG1(20) hybrid nanostructures in water is 1, 2, and 5 nm, respectively (Figure S3, diagrams (a),(b),(d)). Clearly, there is a reverse relationship between the size of β-CD-g-PG hybrid nanostructures and the P/OH ratio. According to diagrams (b) and (c) in figure S3, size of β-CD-g-PG1(10) and β-CD-g-PG0.33(10) are 2 and 3 nm respectively. Increasing of the P/OH molar ratio leads to hybrid nanostructures with higher density but lower sizes.

Figure S3. DLS diagrams of a) β-CD-g-PG1(5), b) β-CD-g-PG1(10), c) β-CD-g-PG0.33(10) and d) β-CD-g-PG1(20) hybrid nanostructures in water

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[†,1]† Corresponding Author: