Electronic supplementary material
Hollow giant lipid vesicles prepared by coaxially electrospraying solutions of phospholipid and degradable polyelectrolyte
In Colloid and Polymer Science
Yuichi Funasaki, Eiko Tsuchiya, Tatsuo Maruyama*
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan
*Corresponding Author
Tel & Fax: +81-78-803-6070
E-mail:
Experimental
Materials
Calcein, fluorescein isothiocyanate (FITC), and fluorescein isothiocyanate–dextran (FITC-dextran, 70 kDa) were purchased from Sigma (St. Louis, MO). 1,3-Di-4-piperidylpropane, 1,4-bis(acryloyloxy)butane, dichloromethane, cystamine dihydrochloride, and triethylamine were purchased from Tokyo Chemical Industry Co., Ltd. L-a-lecithin (from soybean), containing 95.5 wt% phosphatidylcholine and 0.3 wt% triacylglycerol, was purchased from Calbiochem. Green fluorescent polystyrene microspheres (0.5 μm in a diameter) were purchased as a suspension from Duke Scientific. All other reagents were purchased from Wako Pure Chemical Industries.
Synthesis of poly(b-amino esters)
Poly(b-amino esters) was synthesized as described in previous studies [1]. 1,3-Di-4-piperidylpropane (4.93 g, 15 mmol) was dissolved in dichloromethane (10 mL). 1,4-Bis(acryloyloxy)butane (4.76 g, 4.49 mL, 24 mmol) was added to the solution via pipette. The solution was stirred using a magnetic stir-bar at 50 °C. After 24 h, the reaction solution was cooled to room temperature and dripped slowly into vigorously stirred n-hexane. The polymer was collected and dried under vacuum to yield poly(b-amino esters) (PBAE) as a white solid. The product was characterized by 1H NMR and elemental analysis. The molecular weight of PBAE was determined by GPC (Jasco LC2000 plus) equipped with an 80 × 300 mm column × 2 (Showa Denko K. K.) and a RI detector (Jasco RI2031 plus) at 40 °C. Chloroform/0.1 M piperidine was used as an elution solvent at a flow rate of 1.0 mL/min, and PMMA molecular weight standards were used for a standard curve. 1H NMR (300 MHz, CDCl3): δ (ppm) = 4.10 (t, 4H, -COO-CH2-), 2.98-2.39 (m, 12H, -N-CH2-), 1.95 (t, 4H, -CH2-COO-), 1.75-1.65 (m, 4H, -COO-CH2-C2H4), 1.65-1.54 (m, 6H, -C3H6-CH-), 1.37-1.24 (m, 2H, -CH-), 1.24-1.07 (m, 8H, -CH-CH2-).
Synthesis of FITC-NH2
Cystamine (1 g, 4.4 mmol), FITC (172.9 mg, 0.44 mol) and triethylamine (1.2 ml, 8.9 mmol) were dissolved in 15.2 mL of THF/water (1:1 volume ratio) mixture and stirred for 2 h at room temperature. After the solution was evaporated to half of its original volume, a 4 M HCl aqueous solution was added to adjust the resultant solution pH to 1−2. The precipitate obtained was centrifuged and washed with 1 mM HCl aqueous solution (10 mL) three times and freeze-dried overnight. The yield was 18%. The product was characterized by 1H NMR and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF-MS) (ultrafleXtreme-KB, Bruker Dultonics). 1H NMR (300 MHz, DMSO-d6): δ (ppm) = 6.69-6.54 (m, 9H, fluorescence), 3.16-2.94 (m, 8H, -CH2-). MALDI-TOF/MS ([M]+ calcd for C25H23O5N3S3, m/z = 541.7; found, 542.5).
Synthesis of FITC-PBAE
Acrylate end-functionalized PBAE (PBAEAcr) was synthesized according to the literature [2], starting from a reaction solution prepared as for PBAE polymerization. After stirring the reaction solution for 24 h, 1,4-butanediol diacrylate (496 mL, 490 mg, 2.47 mmol) was added and the solution was stirred at 50 °C for an additional 10 h. The resulting reaction solution was dripped slowly into vigorously stirred n-hexane, and the precipitate was dried under vacuum to yield PBAEAcr as a white solid. To synthesize FITC-PBAE, PBAEAcr (506.5 mg) was dissolved in anhydrous dichloromethane (6.5 mL) in a screw-capped vial. FITC-NH2 (20 mg) was dissolved in 1 ml of methanol, and added to the dissolved polymer via pipette. The reaction solution was stirred at 50 °C for 10 h. The product was precipitated by dripping the reaction solution into vigorously stirred n-hexane and dried under vacuum overnight. The crude polymer was then dissolved in 0.5 M HCl (4.2 mL) and precipitated into a vigorously stirred aqueous solution of 0.2 M NaOH (25 mL). The resulting suspension was centrifuged, and the resulting precipitate was washed three times with water. The final product was lyophilized to yield FITC-PBAE as an orange solid. The 1H NMR spectrum for FITC-PBAE was very similar to that of PBAE.
References
1. Lynn DM, Langer R (2000) Degradable poly(beta-amino esters): Synthesis, characterization, and self-assembly with plasmid DNA. J Am Chem Soc 122:10761-10768.
2. Bechler SL, Lynn DM (2011) Design and Synthesis of a Fluorescently End-Labeled Poly(beta-amino ester): Application to the Characterization of Degradable Polyelectrolyte Multilayers. J Polym Sci, Part A: Polym Chem 49:1572-1581.
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