Supplementary Information. Overcoming Endosomal Barrier by Amphotericin B-loaded Dual ph- Responsive PDMA-b-PDPA Micelleplexes for sirna Delivery

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1 Supplementary Information Overcoming Endosomal Barrier by Amphotericin B-loaded Dual ph- Responsive PDMA-b-PDPA Micelleplexes for sirna Delivery Haijun Yu, Yonglong Zou,, Yiguang Wang, Xiaonan Huang, Gang Huang, Baran D. Sumer ξ, David A. Boothman,, Jinming Gao,* Department of Pharmacology, Department of Radiation Oncology, ξ Department of Otolaryngology, Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA Materials and Methods for Co-polymer Syntheses Materials. Dimethyl amino ethyl methacrylate (DMA) and styrene were ordered from Sigma (River Edge, NJ, USA) and purified by vacuum distillation over calcium hydride. Methoxypolyethylene glycol 5000 (PEG 114 -OH), CuBr (99.99%), Ethyl 2-bromoisobutyrate (EBiB, g mol -1 ), 2,2'-bipyridine (bpy, g mol -1 ), copper bromide (CuBr, g mol -1 ), diethyl pyrocarbonate (DEPC, g mol -1 ) DMF, isopropyl alcohol (i-pa), and N,N,N,N,N -Pentamethyldiethylenetriamine (PMDETA, g mol -1 ) were ordered from Sigma-Aldrich (River Edge, NJ, USA) and used without further purification. 2- (Diisopropylamino) ethyl methacrylate (DPA) was purchased from Scientific Polymer Products Inc. (Ontario, NY, USA) and purified by vacuum distillation over calcium hydride. Dialysis tubing (MWCO 3500 Da) and syringe filter (0.45 µm, Whatman) were ordered from Fisher Scientific. Inc. (IL, USA). Tetramethylrhodamine isothiocyanate Dextran (TMR-dextran, average mol wt 65,000-76,000) was order from Sigma. Amphotericin B (AmB) and Bafilomycin 1

2 A1 (Baf-A1) were both ordered from Sigma and dissolved in DMSO at concentration of 0.1 mmol L -1 as stock solutions. Synthesis of PEG-Br macroinitiator. PEG 114 -Br macroinitiator was prepared according to the literature method. 1 Typically, PEG 114 -OH (10.0 g, mol) was dissolved in 250 ml of anhydrous dichloromethane (DCM) followed by addition of anhydrous triethylamine (2.75 ml, mol). 2-bromoisobutyryl bromide (2.50 ml, mol) in anhydrous DCM was added dropwise into the mpeg DCM solution over 2 h in ice bath. The reaction was continued overnight at room temperature. The crude product was purified by diethyl ether precipitation and ethanol recrystallization. Yield: ~80%. 1 H NMR (500 MHz, CDCl 3 ): δ 1.93 (single, 6H, BrC(CH 3 ) 2 ), 3.38 (single, 3H, -OCH 3 ). Syntheses of PEG-b-PDPA and PEG-b-PDMA diblock copolymers via ATRP. 2 Macroinitiator PEG 114 -Br (0.50 g, mmol), CuBr (14.3 mg, mmol), PMDETA (43.3 mg, mmol) and DPA (2.13 g 10.0 mmol) were charged into a schlenk flask. One milliliter of DMF/i-propanol mixture (v/v 1:1) was added as the solvent. The reactant mixture was degassed by three freezing-thawing cycles. The polymerization was carried out at 40 C for 24 h. The reaction mixture was diluted by THF and passed through a neutral alumina column. The product was purified by dialyzing against Milli-Q water and dried by lyophilizing (see Fig. SI 1A for chemical structure). 1 H NMR (500 MHz, CDCl 3 ): δ 0.8~1.1 (broad, 15 H x 42, -CH(CH 3 ) 2, - CCH 3 ), 1.6~2.1(broad, 2 H x 42, -CCH 2 -), 2.62 (broad, 2 H x 42, -CH(CH 3 ) 2 ), 2.98 (broad, 2 H x 42, -CH 2 CH 2 N-), 3.64 (broad, 4 H x 113, -O(CH 2 ) 2 -), 3.75~4.0 (broad, 2 H x 42, -NCH 2 CH 2 O-). To synthesize PEG-b-PDMA copolymers, PEG 114 -Br (0.50 g, mmol), CuBr (14.3 mg, mmol), bpy (43.3 mg, mmol) and DMA (2.13 g 10.0 mmol) were charged into a schlenk flask with 1.0 ml of methanol addition. The polymerization was carried out at room 2

3 temperature for 12 h. The reaction mixture was diluted by THF and passed through a neutral alumina column. The product was purified by dialyzing against Milli-Q water and dried by lyophilizing (Fig. SI 1B). 1 H NMR (500 MHz, CDCl 3 ): δ 0.8~1.1 (broad, 3 H x 50, -CCH 3 ), 1.6~2.1(broad, 2 H x 50, -CCH 2 -), 2.31 (single, 6 H x 50, -N(CH 3 ) 2 ), 3.64 (broad, 4 H x 113, - O(CH 2 ) 2 -), 4.05~4.15 (broad, 2 H x 50, -NCH 2 CH 2 O-). Synthesis of PDMA-b-PDPA diblock copolymers via ATRP. Typically, DMA (1.60 g, 10.2 mmol), bpy (62.0 mg, mmol) and EBiB (77.0 mg, mmol) were added into a schlenk flask with 2.0 ml of anhydrous methanol addition. The mixture was degassed by three freezing-thawing cycles under vacuum condition. After charged by CuBr (28.6 mg, 0.20 mmol), the polymerization was initiated and continued for 4.0 h at room temperature. Afterwards, the flask was injected with DPA monomer (2.50 g, 11.8 mmol in 2.0 ml of methanol). The DPA methanol solution was degassed by repeated freezing-thawing treatment under vacuum condition. The polymerization was continued for 24 h at room temperature and quenched by exposing to air oxygen. The reaction mixture was diluted by THF and passed through an activated neutral alumina column. The collected solution was condensed and dialyzed against Milli-Q water. Purified product was obtained by lyophilizing (Fig. SI 1C). 1 H NMR (500 MHz, CDCl 3 ): δ 0.8~1.1 (broad, 15 H x H x 50, -CH(CH 3 ) 2, -CCH 3 ), 1.6~2.1 (broad, 2 H x H x 50, -CCH 2 -), 2.31 (single, 6 H x 50, -N(CH 3 ) 2 ), 2.55~2.75 (broad, 2 H x H x 50, -CH(CH 3 ) 2, - NCH 2 CH 2 O-), 3.0 (broad, 2 H x 65, -NCH 2 CH 2 O-, 3.75~4.0 (broad, 2H x 65, -NCH 2 CH 2 O-), δ 4.05~4.15 (broad, 2H x 50, -NCH 2 CH 2 O-). Synthesis PSt-b-PDMA diblock copolymer via ATRP. 3 Firstly, macroinitiator PSt-Br was prepared by bulk polymerization with CuBr/PMDETA as the catalyst and EBiB as the initiator with the following recipe: Styrene (4.45 g, 42.8 mmol), CuBr (128 mg, 0.89 mmol), 3

4 PMDETA (154 mg, 0.89 mmol), and EBiB (164.7 mg, 0.89 mmol), After 4 h reaction, the conversion ratio reached 90%, and the reaction mixture was diluted with THF and passed over an alumina column with THF as the eluent. The polymer was precipitated in methanol and dried in vacuo for 48 h. The Mn and PDI values were 5000 and 1.22 as determined by GPC measurement, respectively. 1 H NMR (500 MHz, CDCl 3 ): δ 0.8~2.1 (broad, 3 H x n, -CHCH 2 -), 6.0~7.2 (broad, 5 H x n, -C 6 H 5 ). Thereafter, PSt-Br macroinitiator (0.50 g, 0.10 mmol) was mixed with DMA (4.5 g, 5.0 mmol), benzene (4.2 ml), EtOH (0.8 ml), CuCl (20 mg, 0.18 mmol), and bpy (60 mg, 0.36 mmol) to conduct the polymerization. Twenty four hours later, the reaction mixture was diluted with THF and passed through an alumina column with THF as the eluent. The collected solution was concentrated, dialyzed against Milli-Q water, and dried by lyophilizing (Fig. SI 1D). 1 H NMR (500 MHz, CDCl 3 ): δ 0.8~2.1 (broad, 3 H x H x 35, -CH(CH 3 ) 2, -CCH 3 ), 2.31 (single, 6 H x 35, -N(CH 3 ) 2 ), 2.55~2.75 (broad, 2 H x 35 -NCH 2 CH 2 O-), 4.05~4.15 (broad, 2H x 35, -NCH 2 CH 2 O-). Measurement of critical micelle concentrations (CMC). The CMC of the prepared micelles was determined by fluorescence measurement. 4 The micelle stock solution was freshly prepared by solvent evaporation method and diluted into a set of solutions with different copolymer concentrations. 5 Pyrene solution in THF (0.1 mg L -1 ) was added into each micelle solution with final pyrene concentration of mol L -1. Steady-state fluorescence spectra were obtained with a Hitachi fluorescence spectrometer (FL-7000, Tokyo, Japan). For the fluorescence measurements, 3.0 ml of each sample was placed in a 1.0 cm square quartz cell. The excitation wavelength was set at 339 nm for fluorescence emission spectra determination, and the detection wavelength was set at 390 nm for excitation spectra examination, respectively. 4

5 The slit width was set at 2.0 nm for both excitation and emission fluorescence spectra measurements. ph titration of different copolymers. The diblock copolymers were firstly dissolved in 0.1 N HCl to prepare stock solution at a final concentration of 10 mg ml -1, respectively. ph titration was carried out by adding small volumes (80 µl increments) of 0.1 N NaOH solution under stirring. The ph increase in the range of 3 to 10 was monitored as a function of total added volume of NaOH (V NaOH ). The ph values were measured using a Mettler Toledo ph meter with a microelectrode. Figure 2b shows the representative titration curves for the copolymers with different block components. Fluorescence characterization. The fluorescence emission spectra of TMR-labeled PDMA-b-PDPA micelles were obtained on a Hitachi fluorometer (F-7500 model). For each copolymer, the sample was initially prepared in Milli-Q water at the concentration of 5 mg/ml. Then the stock solution was diluted in 0.2 M citric-phosphate buffers with different ph values. The terminal polymer concentration was controlled at 0.1 mg ml -1. The samples were excited at 545 nm, and the emission spectra were collected from 560 to 680 nm. The emission and excitation slits were both 5 nm. The emission intensity at 580 nm was used to demonstrate the ph-responsive property of the micelle as shown in the inner graph of Figure SI 4. AmB-loading ratio and encapsulation efficiency. The AmB-loaded PDMA-b-PDPA micelles were freshly prepared by the solvent evaporation method. The trace THF was removed by twice ultra-centrifugation (ultra-centrifugal unit, MWCO 100 kda). The concentrated micelle solution was diluted into 3.0 mg ml -1 by sterile Milli-Q water and stored at 4 C. To determine the AmB loading percentage and encapsulation efficiency, the AmB-loaded micelle solution (5.0 mg ml -1 ) was acidified to ph 6.0 with 1.0 M HCl solution and diluted with large excess of 5

6 ethanol. The AmB concentration was determined by UV-Vis spectra (Abs 384 nm ). The UV-Vis absorption of a set of AmB ethanol solution with defined concentration was measured as standard curve. To elucidate the polymer influence on the UV-Vis absorption, a certain amount of 5.0 mg ml -1 copolymer solution was added into each AmB standard solution before the measurement. The determined AmB-loading ratio and encapsulation efficiency were included in Table SI 2. Flow cytometry. Cells treated with TMR-labeled micelleplexes were analyzed on a Beckman-Coulter flow cytometer equipped with a 488 nm Argon laser. Fluorescence of TMR and Alexa was detected at 575 nm (FL2) and 643 nm (FL4), respectively. Background fluorescence and autofluorescence were determined using mock-treated cells. Approximately 10,000 events were acquired per sample. Data analysis was carried out using the FCS Express3 software (De Novo Software, CA, USA), and the results were shown in Figure SI 5. REFERENCES 1. Pioge, S.; Fontaine, L.; Gaillard, C.; Nicol, E.; Pascual, S. Self-Assembling Properties of Well-Defined Poly(ethylene oxide)-b-poly(ethyl acrylate) Diblock Copolymers. Macromolecules 2009, 42, Du, J. Z.; Armes, S. P. ph-responsive Vesicles Based on A Hydrolytically Self-Cross- Linkable Copolymer. J. Am. Chem. Soc. 2005, 127, Jewrajka, S. K.; Chatterjee, U. Block Copolymer Mediated Synthesis of Amphiphilic Gold Nanoparticles in Water and An Aqueous Tetrahydrofuran Medium: An Approach for the Preparation of Polymer-Gold Nanocomposites. J. Polym. Sci. Part A: Polym. Chem. 2006, 44, Dai, Z. L.; Piao, L. H.; Zhang, X. F.; Deng, M. X.; Chen, X. S.; Jing, X. B. Probing the Micellization of Diblock and Triblock Copolymers of Poly(L-lactide) and Poly(ethylene glycol) in Aqueous and NaCl Salt Solutions. Colloid Polym. Sci. 2004, 282, Blanco, E.; Bey, E. A.; Dong, Y.; Weinberg, B. D.; Sutton, D. M.; Boothman, D. A.; Gao, J. M. Beta-Lapachone-Containing PEG-PLA Polymer Micelles as Novel Nanotherapeutics Against NQO1-Overexpressing Tumor Cells. J. Control. Release 2007, 122,

7 Table SI 1. Characterization of molecular weights and CMCs for different diblock copolymers. Sample Composition a Mn a (*10 3 ) Mw c (*10 3 ) Mn c (*10 3 ) Mw/Mn c PDI CMC d (10-4 mg ml -1 ) 1 PDMA 50 -b-pdpa PEG 114 -b-pdma PEG 114 -b-pdpa PSt 50 -b-pdma a Copolymer composition and number-averaged molecular weight were determined by 1 H NMR. b Weight-averaged molecular weights (Mw) and molecular weight polydispersity (PDI, Mw/Mn) were determined by GPC measurement using THF as an eluent. c Critical micelle concentration (CMC) of the micelles was determined by the pyrene method. 7

8 Table SI 2. UV-Vis spectra determined AmB-loading percentages and encapsulation efficiency Feeding ratio (wt %) Loading ratio (wt %) Encapsulation efficiency (%) Table SI 3. Particle size and zeta-potential of AmB-loaded PDMA-b-PDPA micelles. AmB loading (wt %) Particle size (d, nm) Zeta potential (mv) (±2.0) 31.3 (±2.2) (±0.40) 30.4 (±1.4) (±1.1) 28.7 (±1.1) (±1.1) 30.2 (±0.90) 8

9 Figure SI 1. Scheme for the syntheses of (A) PEG-b-PDPA, (B) PEG-b-PDMA, (C) PDMA-b- PDPA, and (D) PSt-b-PDMA copolymers. All diblock copolymers used in this study were synthesized by the ATRP method. 9

10 A polymer/sirna (w/w) Particle Size (d, nm) B AmB-loading (wt%) Polymer/siRNA (w/w) Zeta Potentials (mv) AmB-loading (wt%) Figure SI 2. Characterization of AmB-loaded PDMA-b-PDPA/siRNA micelleplexes. (A) Particle size, and (B) zeta-potential of AmB-loaded PDMA-b-PDPA/siRNA micelleplexes at different polymer/sirna ratios and AmB loading densities. 10

11 Fluoresence Intensity ph Relative FI ph Value Wavelength (nm) Figure SI 3. Fluorescence emission spectra of TMR-labeled PDMA-b-PDPA micelles at different phs. The micelles displayed increased fluorescence intensity (Em 580nm) in response to ph decrease (insert graph). 11

12 Figure SI 4. CLSM examination for intracellular dissociation of TMR-labeled PDMA-b-PDPA micelleplexes (scale bar = 10 µm). (A) Intracellular dissociation of the PDMA-b-PDPA micelleplexes in A549-Luc cells with Rab5a-GFP-labeled early endosomes (top panel), or Lamp1-GFP-labeled late endosomes/lysosomes (middle panel). (B) Baf-A1 inhibited the intracellular dissociation of PDMA-b-PDPA micelleplexes in A549-Luc cells with Lamp1-GFPlabeled late endosomes/lysosomes (bottom panel). The cells were pre-treated with Baf-A1 1 h before addition of micelleplexes. 12

13 C Positive Cell Percentage (%) TMR-nanoparticle Alexa-siRNA Baf-A1 (-) Baf-A1 (+) Figure SI 5. Flow cytometry histogram of (A) TMR or (B) Alexa positive A549-Luc cells with Baf-A1 (Baf-A1 (+)) or without Baf-A1 (Baf-A1 (-)) pre-treatment. (C) TMR or Alexa positive cell percentages in A549-Luc cell population with or without Baf-A1 pre-treatment as determined by flow cytometry analysis. The cells were pre-treated by Baf-A1 1 h before micelleplex addition, and incubated with the micelleplexes for 4 h before flow cytometry analysis. 13

14 Figure SI 6. CLSM images of A549-Luc cells after incubation with AmB-loaded micelleplexes. The images were taken (A) 6 h, or (B) 24 h post-micelleplex-incubation (scale bar = 20 µm and applied for all images). 14

15 100 A Polymer/siRNA (w/w) Rel Luc Knockdown (%) B Free AmB (wt%) Polymer/siRNA (w/w) Rel Cell Viability (%) Free AmB (wt%) Figure SI 7. Luc knockdown activity and cytotoxicity of the mixed solution of free AmB and PDMA-b-PDPA micelleplexes. The Rel Luc knockdown was determined by normalizing the Luc activity of sirna-luc treated cells by that of the sirna-scr treated cells. The relative cell viability was determined by MTT assay and normalized by the viability of untreated cell control. 15