Nucleic Acids Delivery Vehicles

Transcription

1 Supporting Information Controlled PEGylation Crowdedness for Polymeric Micelles to Pursue Ligand-specified Privilege as Nucleic Acids Delivery Vehicles Xiyi Chen, a,* Haifeng Gu, b Jinjun Yang, c Sudong Wu, d Jun Liu, e Xi Yang, f Qixian Chen e,* a School of Public Health, Dalian Medical University, No. 9 West Section Lvshun South Road, Dalian , China b College of Science, Dalian Ocean University, No. 52 Heishijiao Street, Dalian , China c School of Environmental Science and Safety Engineering, Tianjin University of Technology, Xiqing District, Tianjing , China d Ningbo Institute of Materials Technology and Engineering, China Academy of Sciences, Ningbo , China e Ningbo Hygeia Medical Technology Co., Ltd, No Lingyun Road, High-Tech Zone, Ningbo , China f Department of Neurosurgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai , China Corresponding Authors * Address correspondence to xychen@dmu.edu.cn (X. Chen) and qixian@mit.edu (Q. Chen). S-1

2 1. Materials and equipment α-methoxy-ω-amino-poly(ethylene glycol) (PEG) (M W 12,000) and acetal-peg-nh 2 was obtained from Nippon Oil and Fats Co., Ltd. (Tokyo, Japan). β-benzyl-l-aspartate N-carboxyanhydride (BLA-NCA) was obtained from Chuo Kaseihin Co., Inc. (Tokyo, Japan). Diethylenetriamine (DET), N,Ndimethylformamide (DMF), n-butylamine, dichloromethane, benzene, and trifluoroacetic acid were purchased from Wako Pure Chemical Industries, Ltd. 3,3'-Dithiodipropionic acid di(nhydroxysuccinimide ester) (NHS-SS-NHS) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). (3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was purchased from Fisher Scientific International, Inc. (Pittsburgh, PA). Alexa488 succinimidyl ester was a product of Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was purchased from Dainippon Sumitomo Parma Co., Ltd. (Osaka, Japan). Cell culture lysis buffer and luciferase Assay System Kit was purchased from Promega Co. (Madison, WI). Dulbecco s modified Eagle s medium (DMEM) was purchased from Sigma Aldrich (St. Louis, MO). For cellular uptake and intracellular distribution assay, mrna was labeled with Cy3 using a Label IT Nucleic Acid Labeling Kit from Mirus Bio Corporation (Madison, WI) according to the manufacturer s protocol. For aqueous phase SEC, LC-2000 system (JASCO, Tokyo, Japan) equipped with Superdex200 10/300 GL (GE Healthcare, Tokyo, Japan) and a UV detector was used. 2. Table S1 Chemical descriptions of a variety of polymers in the study. PEG (M w ) DP of PAsp(DET) DP of PAsp crgd PAsp PEG-PAsp(DET) 12 kda PEG-SS-PAsp(DET) 12 kda crgd-peg-pasp(det) 12 kda 38-98% 3. DTT-responsive cleavage of PEG-SS-PAsp(DET) The stock solution of PEG-SS-PAsp(DET) was dissolved in PBS (10 mm, ph 7.4) at a concentration of 1 mg/ml, which was subsequently resorted into two equal fractions, either supplemented with DTT- S-2

3 containing PBS solution or blank PBS solution to have the final DTT concentration of 50 mm or 0 mm and final polymer concentration of 0.5 mg/ml. The reaction solution was transferred for aqueous GPC measurement. 4. Preparation of polymeric micelles The stock solution of crgd-peg-pasp(det) together with PEG-SS-PAsp(DET) and PEG-PAsp(DET) was dissolved in PBS buffer (10 mm, ph 7.4) at a concentration of 10 mg/ml. Meanwhile, powder of PAsp was dissolved in PBS buffer (10 mm, ph 7.4) at a concentration of 10 mg/ml. Furthermore, aliquot of stock solution was mixed at an equal charge ratio under vortex for 30 s. The complexation solution was transferred at an ice bath for incubation for post treatment. 5. Crosslinking for polymeric micelles The prepared polymeric micelles in PBS buffer (ph 7.4) were supplemented with EDC solution. Note the molar ratio of EDC and carboxyl group of PAsp was 1:5 with the aim of coupling reaction between amine groups of PAsp(DET) and carboxyl groups of PAsp. The reaction was kept for 3 h at 4 C, followed by dialysis in PBS buffer (ph 7.4) for three times at 4 C to remove unreacted EDC. 6. DLS measurement The hydrodynamic diameter and polydispersity index (PDI) of polymeric micelles were measured by DLS using a Zetasizer Nanoseries instrument (Malvern Instruments Ltd., UK). The measurement was performed for three times at 25 C. The rate of decay in the photon correlation function was analyzed according to a cumulant method, and the corresponding diameter was calculated using the Stokes-Einstein equation. 7. ζ -potential measurement The ζ-potential of the polymeric micelles was measured by Nano ZS (ZEN3600, Malvern Instruments, Ltd., UK). The polymeric micelle solution, either with DTT (50 mm) treatment or no treatment was injected into folded capillary cells (Malvern Instruments, Ltd.). The ς-potential was determined from the laser-doppler electrophoresis using the Zetasizer nanoseries (Malvern Instruments Ltd., UK). From the obtained electrophoretic mobility, the ς-potential was calculated by using the Smoluchowski equation: ς = S-3

4 4πην/ε in which η is the electrophoretic mobility, ν is the viscosity of the solvent, and ε is the dielectric constant of the solvent. The results are expressed as the average of three experiments. 8. Cellular uptake of polymeric micelles Aiming for evaluation of cellular uptake activities of a class of polymeric micelles, crgd-peg- PAsp(DET) labeled by Alexa Fluor 488 NHS (NHS: N-Hydroxysuccinimide) was used to prepare polymeric micelles. Furthermore, U87 cells were seeded in 48-well plates at a density of 10,000 cells/well with 200 µl of DMEM containing 10% FBS and incubated 24 h (37 C, CO2 concentration 5%). After the culture medium was replaced with fresh medium, 20 µl polymeric micelle solutions were added to each well. The cells were then washed three times by PBS and 400 µl fresh medium was added. The cellular uptake efficiency was measured by a BD LSR II flow cytometer equipped with FACS-Diva software (BD Biosciences, Franklin Lakes, NJ). The obtained data were expressed as the mean fluorescence intensity from three independent samples (n = 4). 9. Protein adsorption by Isothermal Titration Calorimetry (ITC) The ITC investigations were carried out at 37 C, using a VP-ITC microcalorimeter (GE MicroCal Inc., USA). The protein adsorption reactions were performed by injecting 20 µm solution of BSA in 1 PBS into a 2 ml sample cuvette containing polymeric micelles at a concentration of 2 mg/ml in 1 PBS under a stirring speed of 300. A total of 50 injections were conducted with an interval of 240 s and a reference power of 10 µcal/s. Titration volumes of BSA were programmed as follows: an initial injection of 2 µl, followed by thirty four injections of 5 µl, and fourteen injections of 10 µl. Binding isotherms were plotted and analyzed using Origin Software (MicroCal Inc., USA), where the ITC measurements were fit into a one-site binding model. 10. Intracellular trafficking mrna was labeled with Cy5 using the Label IT Nucleic Acid Labeling Kit (Mirus, Madison, WI). U87 cells were seeded on a 35 mm glass base dish (Iwaki, Japan) at a density of 30,000 cells/well with 1 ml of DMEM containing 10% FBS and incubated 24 h (37 C, CO 2 concentration 5%). Both Cell Light GFP Endosome and Cell Light GFP Lysosome at dosage of 10 µl were added to label late-endosome/lysosome S-4

5 and incubated for 48 h staining. The cells were washed by 1 ml PBS and replaced with 1 ml fresh medium. A class of crgd-peg-pasp(det)/mrna polymeric solution (mrna concentration of 33.3 µg/ml, containing 4 µg Cy5-labeled mrna) with varying PEGylation degree was applied to the cells. After 24 h incubation, Hoechst solution (1 mg/ml, 5 µl) was added, followed by 5 min incubation for nuclei staining. The cells were washed twice with 1 ml PBS and replaced with 1 ml fresh medium. The CLSM observation was performed using LSM 780 (Carl Zeiss, Germany) with 40 objective (C- Apochromat, Carl Zeiss, Germany) at excitation wavelengths of 488 nm (Ar laser) and 633 nm (He-Ne laser) for Cell Light GFP and Cy5, respectively. 11. Cellular uptake of mrna polymeric micelles Aiming for evaluation of cellular uptake activity of a class of mrna polymeric micelles, mrna labeled by Cy3 was used to prepare mrna polymeric micelles. U87 cells were seeded in 48-well plates at a density of 10,000 cells/well with 200 µl of DMEM containing 10% FBS and incubated 24 h (37 C, CO2 concentration 5%). After the culture medium was replaced with fresh medium, polymeric micelles containing 0.5 µg Cy3-labeled mrna/well were added to each well. The cells were then washed three times by PBS and 400 µl fresh medium was added. The cellular uptake efficiency was measured by a BD LSR II flow cytometer equipped with FACS-Diva software (BD Biosciences, Franklin Lakes, NJ). The obtained data were expressed as the mean fluorescence intensity from three independent samples (n = 3). 12. Preparation of mrna polymeric micelles The stock solution of crgd-peg-pasp(det) together with PEG-SS-PAsp(DET) and PEG-PAsp(DET) was dissolved in PBS buffer (10 mm, ph 7.4) at a concentration of 10 mg/ml. Meanwhile, mrna (Luc) was dissolved in PBS buffer (10 mm, ph 7.4) at a concentration of 100 ng/µl. Polymeric micelles were prepared by mixing 1-unit volume of the PEG-PLys solution with 2-unit volume of mrna solution at an N/P ratio of 1 under vortex for 10 s. The N/P ratio is defined as the residual molar ratio of amine (N) groups of PEG-PLys to the phosphate (P) groups of mrna. The crosslinking for mrna polymeric micelles was conducted according to a similar procedure with the crosslinker of NHS-SS-NHS. Note that S-5

6 the molar ratio of the NHS moieties in NHS-SS-NHS and the Asp(DET) units was 1:5 with the aim of crosslinking of amine groups of PAsp(DET) segments. 13. Gene expression measurement U87 cells was seeded in 24-well plates at a density of 20,000 cells/well with 400 µl of DMEM containing 10% FBS and incubated 24 h (37 C, CO2 concentration 5%). After the culture medium was replaced with fresh medium, polymeric micelles containing 1 µg mrna (LUC)/well was added to each well. After 24 h of incubation, the medium was replaced with 400 µl of fresh medium, followed by another 24 h incubation. Luciferase expression was determined using a Luciferase assay system (Promega, Madison, WI) and a GloMaxTM 96 microplate luminometer (Promega, Madison, WI) following the manufacture s protocol (n = 4). The quantity of protein in the cell lysates was determined according to a MicroBCA Protein Assay Reagent Kit following the manufacture s protocol. On the other hand, polymeric micelles containing GFP mrna were utilized for direct observation of protein expression by following a same transfection protocol, where the GFP expression was captured by INCELL Analyzer (GE Healthcare UK Ltd., Buckinghamshire, England). Fig. S1 GFP expression by a class of polyplex micelles containing GFP mrna in U87 cells. S-6

7 14. Table S2 Physiological characterizations of a class of polymeric micelles with varying PEGylation degrees. PEGylation 10% 20% 40% 60% 80% 100% DLS size 51.4 ± ± ± ± ± ± 1.21 ζ potential 3.67 ± ± ± ± ± ± 0.06 In Table S2, a class of the polymeric micelles with varying PEGylation degrees appeared to possess a comparable DLS size (approximate 50 nm). This is reasonable because the pre-formulated polymeric micelles were subjected to crosslinking treatment for PAsp(DET)/DET complex core prior to DTT-based depegylation treatment. Basically, the manufactured polymeric micelles post DTT treatment still have distinct core-shell architecture, whose size should be contributed by the core size of PAsp(DET)/PAsp complex and the thickness of PEG. In this study, the core PAsp(DET)/PAsp was uniformly treated with crosslinking prior to DTT-based depegylation treatment, hence, we could anticipate a constant core size for polymeric micelles in spite of varying PEGylation degrees. Pertaining to the PEG thickness of PEG shell, the PEG in electrostatic-based polyplex micelle has validated to adopt either mushroom or squeezed conformation, S1 which elicits approximate PEG thickness. S1 Therefore, it should be reasonable that a comparable DLS for a class of polymeric micelles despite them with varying PEGylation degree. On the other hand, the zeta potential has been revealed to be not markedly affected by PEG density, S2 despite relatively more apparent zeta potential could be obtained for the polymeric micelles with lower PEGylation, which is also in good accordance to the present results. 15. GPC characterization of diverse polymers S-7

8 Fig. S2 GPC traces of diverse polymers. Red: PEG-PAsp(DET); Purple: crgd-peg-pasp(det); PEG-Grey: PEG- SS-PAsp(DET); and Green: homo-pasp. [S1] Tockary, T. A.; Osada, K.; Chen, Q.; Machitani, K.; Dirisala, A.; Uchida, S.; Nomoto, T.; Toh, K.; Matsumoto, Y.; Itaka, K.; Nitta, K.; Nagayama, K.; Kataoka, K. Tethered PEG Crowdedness Determining Shape and Blood Circulation Profile of Polyplex Micelle Gene Carriers. Macromolecules 2013, 46, [S2] Chen, Q.; Osada, Osada, K.; Ishii T.; Oba, M.; Uchida, S.; Tockary, T. A.; Endo, T.; Ge, Z.; Kinoh, H.; Kano, M. R.; Itaka K.; Kataoka, K. Homo-catiomer Integration into PEGylated Polyplex Micelle from Block-catiomer for Systemic Anti-angiogenic Gene Therapy for Fibrotic Pancreatic Tumors. Biomaterials 2012, 33, S-8