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Summary

Abstract

Introduction

Protocol

Representative Results

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Materials

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Chemistry

Methionine Functionalized Biocompatible Block Copolymers for Targeted Plasmid DNA Delivery

Published: August 6th, 2019

DOI:

10.3791/58527

1Research Center of Clinical Oncology, Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research & Nanjing Medical University Affiliated Cancer Hospital, 2Department of General Surgery, The First Affiliated Hospital with Nanjing Medical University, 3School of Clinical Medicine, Xuzhou Medical University
* These authors contributed equally

This work presents the preparation of methionine functionalized biocompatible block copolymers (mBG) via the reversible addition-fragmentation chain transfer (RAFT) method. The plasmid DNA complexing ability of the obtained mBG and their transfection efficiency were also investigated. The RAFT method is very beneficial for polymerizing monomers containing special functional groups.

Reversible addition-fragmentation chain transfer (RAFT) polymerization integrates the advantages of radical polymerization and living polymerization. This work presents the preparation of methionine functionalized biocompatible block copolymers via RAFT polymerization. Firstly, N,N-bis(2-hydroxyethyl)methacrylamide-b-N-(3-aminopropyl)methacrylamide (BNHEMA-b-APMA, BA) was synthesized via RAFT polymerization using 4,4’-azobis(4-cyanovaleric acid) (ACVA) as an initiating agent and 4-cyanopentanoic acid dithiobenzoate (CTP) as the chain transfer agent. Subsequently, N,N-bis(2-hydroxyethyl)methacrylamide-b-N-(3-guanidinopropyl)methacrylamide (methionine grafted BNHEMA-b-GPMA, mBG) was prepared by modifying amine groups in APMA with methionine and guanidine groups. Three kinds of block polymers, mBG1, mBG2, and mBG3, were synthesized for comparison. A ninhydrin reaction was used to quantify the APMA content; mBG1, mBG2, and mBG3 had 21%, 37%, and 52% of APMA, respectively. Gel permeation chromatography (GPC) results showed that BA copolymers possess molecular weights of 16,200 (BA1), 20,900(BA2), and 27,200(BA3) g/mol. The plasmid DNA (pDNA) complexing ability of the obtained block copolymer gene carriers was also investigated. The charge ratios (N/P) were 8, 16, and 4 when pDNA was complexed completely with mBG1, mBG2, mBG3, respectively. When the N/P ratio of mBG/pDNA polyplexes was higher than 1, the Zeta potential of mBG was positive. At an N/P ratio between 16 and 32, the average particle size of mBG/pDNA polyplexes was between 100-200 nm. Overall, this work illustrates a simple and convenient protocol for the block copolymer carrier synthesis.

In recent years, gene therapy has emerged for the therapeutic delivery of nucleic acids as drugs to treat all kinds of diseases1. The development of gene drugs including plasmid DNA (pDNA) and small interfering RNA (siRNA) relies on the stability and efficiency of the drug delivery system (DDS)2. Among all DDS, cationic polymer carriers have the advantages of good stability, low immunogenicity, and facile preparation and modification, which give cationic polymer carriers broad application prospects3,4. For practical applications in biomedicine, researchers must f....

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1. Synthesis of BNHEMA polymer (PBNHEMA)

  1. Dissolve 1.87 g of N, N-bis(2-hydroxyethyl)methacrylamide (BNHEMA) in 1 mL of distilled water in a polymerization bottle.
    NOTE: The polymerization bottle is a round-bottom flask with a rubber stopper and a magnetic stirrer.
  2. Dissolve 0.03 g of 4-cyanopentanoic acid dithiobenzoate (CTP) and 0.02 g of 4,4’-azobis (4-cyanovalericacid ) (ACVA) in 0.5 mL of 1,4-dioxane in a 5 mL beaker. Then, add the CTP and ACVA solution to the polym.......

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BNHEMA was fed according to the objective degree of polymerization shown in Table 1; the synthesis procedure of mBG is shown in Figure 1. Firstly, BNHEMA homopolymer was prepared via the reversible addition-fragmentation chain transfer (RAFT) method in the water-dioxane system, using 4-cyanopentanoic acid dithiobenzoate as a chain transfer agent. Secondly, PBNHEMA was used as a chain transfer agent to prepare BNHEMA-b-APMA block polymer. APMA monomer was fed accordi.......

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This study introduced a series of BNHEMA-b-APMA block polymer cationic gene carriers. These block polymers were synthesized via the reversible addition-fragmentation chain transfer (RAFT) method. The hydrophilic segment BNHEMA was introduced to improve solubility. Methionine and guanidine groups were modified to improve the target ability and transfection efficiency5. The APMA chain content increased and guanidinylation in mBG copolymer reduced the particle size of mBG/pDNA polyplexes. The particl.......

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This research was supported by the National Key Research and Development Program of China (No. 2016YFC0905900), National Natural Science Foundation of China (Nos. 81801827, 81872365), Basic Research Program of Jiangsu Province (Natural Science Foundation, No. BK20181086), and Jiangsu Cancer Hospital Scientific Research Fund (No. ZK201605).

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Name Company Catalog Number Comments
1-hydroxybenzotriazole Macklin Biochemical Co., Ltd,China H810970 ≥97.0%
1,4-dioxane Sinopharm chemical reagent Co., Ltd, China 10008918 AR
1-amidinopyrazole Hydrochloride Aladdin Co., Ltd., China A107935 98%
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride Aladdin Co., Ltd., China E106172 AR
4,4’-azobis(4-cyanovaleric acid) Aladdin Co., Ltd., China A106307 Analytical reagent (AR)
4-cyano-4-(phenylcarbonothioylthio)pentanoic Acid Aladdin Co., Ltd., China C132316 >97%(HPLC)
Acetate Sinopharm chemical reagent Co., Ltd, China 81014818 AR
Acetone Sinopharm chemical reagent Co., Ltd, China 10000418 AR
Agarose Aladdin Co., Ltd., China A118881 High resolution
Ascorbic acid Aladdin Co., Ltd., China A103533 AR
DMSO Aladdin Co., Ltd., China D103272 AR
Ethylene glycol Aladdin Co., Ltd., China E103319 AR
N-(3-aminopropyl)methacrylamide hydrochloride Aladdin Co., Ltd., China N129096 ≥98.0%(HPLC)
N,N-bis(2-hydroxyethyl)methacrylamide ZaiQi Bio-Tech Co.,Ltd, China CF259748 ≥98.0%(HPLC)
Ninhydrin Aladdin Co., Ltd., China N105629 AR
PBS buffer Aladdin Co., Ltd., China P196986 pH 7.4
Plasmid DNA BIOGOT Co., Ltd, China pDNA-EGFP pDNA-EGFP
Plasmid DNA BIOGOT Co., Ltd, China Pdna pDNA
Sodium carbonate decahydrate Aladdin Co., Ltd., China S112589 AR
Trimethylamine Aladdin Co., Ltd., China T103285 AR

  1. Flotte, T. R. Gene and Cell Therapy in 2018: A Look Ahead. Human Gene Therapy. 29, 1-1 (2018).
  2. Huang, W., et al. Nanomedicine-based combination anticancer therapy between nucleic acids and small-molecular drugs. Advanced Drug Delivery Reviews. 115, 82-97 (2017).
  3. Wu, Y., et al. Reversing of multidrug resistance breast cancer by co-delivery of P-gp siRNA and doxorubicin via folic acid-modified core-shell nanomicelles. Colloids & Surfaces B Biointerfaces. 138, 60-69 (2016).
  4. Quader, S., Kataoka, K. Nanomaterial-Enabled Cancer Therapy. Molecular Therapy. 25, 1501-1513 (2017).
  5. Wu, Y., et al. Multivalent methionine-functionalized biocompatible block copolymers for targeted siRNA delivery and subsequent reversal effect on adriamycin resistance in human breast cancer cell line MCF-7/ADR. Journal of Gene Medicine. 19, e2969 (2017).
  6. Szwarc, M. ‘Living’ Polymers. Nature. 178, 168-169 (1956).
  7. Szwarc, M., Rembaum, A. Polymerization of methyl methacrylate initiated by an electron transfer to the monomer. Journal of Polymer Science. 22 (100), 189-191 (1956).
  8. Mukhopadhyay, R. D., Ajayaghosh, A. Living supramolecular polymerization. Science. 349, 241 (2015).
  9. Ozkose, U. U., Altinkok, C., Yilmaz, O., Alpturk, O., Tasdelen, M. A. In-situ preparation of poly(2-ethyl-2-oxazoline)/clay nanocomposites via living cationic ring-opening polymerization. European Polymer Journal. 88, 586-593 (2017).
  10. Wu, W., Wang, W., Li, J. Star polymers: Advances in biomedical applications. Progress in Polymer Science. 46, 55-85 (2015).
  11. Boyer, C., et al. Copper-Mediated Living Radical Polymerization (Atom Transfer Radical Polymerization and Copper(0) Mediated Polymerization): From Fundamentals to Bioapplications. Chemical Reviews. 116, 1803-1949 (2016).
  12. Keddie, D. J. A guide to the synthesis of block copolymers using reversible-addition fragmentation chain transfer (RAFT) polymerization. Chemical Society Reviews. 43, 496-505 (2014).
  13. Wu, Y., et al. Guanidinylated 3-gluconamidopropyl methacrylamide-s-3-aminopropyl methacrylamide copolymer as siRNA carriers for inhibiting human telomerase reverse transcriptase expression. Drug Delivery. 20, 296-305 (2013).
  14. Qin, Z., Liu, W., Guo, L., Li, X. Studies on Guanidinated N-3-Aminopropyl Methacrylamide-N-2-Hydroxypropyl Methacrylamide Co-polymers as Gene Delivery Carrier. Journal of Biomaterials Science, Polymer Edition. 23, 1-4 (2012).
  15. Friedman, M. Applications of the Ninhydrin Reaction for Analysis of Amino Acids, Peptides, and Proteins to Agricultural and Biomedical Sciences. Journal of Agricultural and Food Chemistry. 52, 385-406 (2004).
  16. Habuchi, S., Yamamoto, T., Tezuka, Y. Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level. Journal of Visualized Experiments. (115), 1-9 (2016).
  17. Rao, D. A., Nguyen, D. X., Mishra, G. P., Doddapaneni, B. S., Alani, A. W. Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles. Journal of Visualized Experiments. 102, 1-5 (2015).

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