Name: methionine functionalized biocompatible block copolymers for targeted plasmid dna delivery
Uploaded: 2019-08-06T12:30:00
Description: Watch this Scientific Journal Video about Methionine Functionalized Biocompatible Block Copolymers for Targeted Plasmid DNA Delivery at JoVE.com

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).

1. Synthesis of BNHEMA polymer (PBNHEMA)
<ol>
	<li>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.</li>
	<li>Dissolve 0.03 g of 4-cyanopentanoic acid dithiobenzoate (CTP) and 0.02 g of 4,4&#8217;-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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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...

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 find a cationic polymer carrier with high efficiency, low toxicity, and good targeting ability5. Among all polymer carriers, block copolymers are one of the most widely used drug delivery systems. Block copolymers are intensively studied for their self-assembly property and abilities to form micelles, microspheres, and nanoparticles in drug delivery5. Block copolymers can be synthesized via living polymerization or click chemistry methods.
In 1956, Szwarc et al. raised the topic of living polymerization, defining it as a reaction without chain-breaking reactions6,7. Since then, multiple techniques had been developed to synthesize polymers using this method; thus, living polymerization is viewed as a milestone of polymer science8. Living polymerization can be classified into living anionic polymerization, living cationic polymerization, and reversible deactivation radical polymerization (RDRP)9. Living anionic/cationic polymerizations have a limited scope of application due to their strict reaction conditions10. Controlled/living radical polymerization (CRP) has mild reaction conditions, convenient disposition, and good yield and has thus been a major research focus in recent years11. In CRP, active propagation chains are reversibly passivated into dormant ones to reduce the concentration of free radicals and avoid the bimolecular reaction of propagating chain radicals. The addition polymerization can continue only if the inactive dormant propagating chains are reversibly animated into chain radicals. As one of the most promising forms of living radical polymerization, reversible addition-fragmentation chain transfer (RAFT) polymerization is a method applicable to yield block polymers with controlled molecular weight and structure, narrow molecular weight distribution, and carrying functional groups12. The key to successful RAFT polymerization is the effect of chain transfer agents, usually dithioesters, which possess very high chain transfer constant.
In this paper, a RAFT polymerization method was designed to prepare BNHEMA-b-APMA block polymer, taking 4,4&#8217;-azobis(4-cyanovaleric acid) (ACVA) as an initiating agent and 4-cyanopentanoic acid dithiobenzoate (CTP) as a chain transfer agent. RAFT polymerization was used twice to introduce BNHEMA into the cationic polymer carriers. Subsequently, the amine groups in the APMA chain were modified with methionine and the guanidinylation reagent 1-amidinopyrazole hydrochloride. Making the use of the positive charges of the guanidinylation reagent and methacrylamide polymer skeleton structure, the cellular uptake efficiency of the obtained block polymer carriers was improved.

<table>
<tbody>
<tr>
<td>1-hydroxybenzotriazole</td>
<td>Macklin Biochemical Co., Ltd,China</td>
<td>H810970</td>
<td>&ge;97.0%</td>
</tr>
<tr>
<td>1,4-dioxane</td>
<td>Sinopharm chemical reagent Co., Ltd, China</td>
<td>10008918</td>
<td>AR</td>
</tr>
<tr>
<td>1-amidinopyrazole Hydrochloride</td>
<td>Aladdin Co., Ltd., China</td>
<td>A107935</td>
<td>98%</td>
</tr>
<tr>
<td>1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride</td>
<td>Aladdin Co., Ltd., China</td>
<td>E106172</td>
<td>AR</td>
</tr>
<tr>
<td>4,4&#x2019;-azobis(4-cyanovaleric acid)</td>
<td>Aladdin Co., Ltd., China</td>
<td>A106307</td>
<td>Analytical reagent (AR)</td>
</tr>
<tr>
<td>4-cyano-4-(phenylcarbonothioylthio)pentanoic Acid</td>
<td>Aladdin Co., Ltd., China</td>
<td>C132316</td>
<td>&#x003E;97%(HPLC)</td>
</tr>
<tr>
<td>Acetate</td>
<td>Sinopharm chemical reagent Co., Ltd, China</td>
<td>81014818</td>
<td>AR</td>
</tr>
<tr>
<td>Acetone</td>
<td>Sinopharm chemical reagent Co., Ltd, China</td>
<td>10000418</td>
<td>AR</td>
</tr>
<tr>
<td>Agarose</td>
<td>Aladdin Co., Ltd., China</td>
<td>A118881</td>
<td>High resolution</td>
</tr>
<tr>
<td>Ascorbic acid</td>
<td>Aladdin Co., Ltd., China</td>
<td>A103533</td>
<td>AR</td>
</tr>
<tr>
<td>DMSO</td>
<td>Aladdin Co., Ltd., China</td>
<td>D103272</td>
<td>AR</td>
</tr>
<tr>
<td>Ethylene glycol</td>
<td>Aladdin Co., Ltd., China</td>
<td>E103319</td>
<td>AR</td>
</tr>
<tr>
<td>N-(3-aminopropyl)methacrylamide hydrochloride</td>
<td>Aladdin Co., Ltd., China</td>
<td>N129096</td>
<td>&ge;98.0%(HPLC)</td>
</tr>
<tr>
<td>N,N-bis(2-hydroxyethyl)methacrylamide</td>
<td>ZaiQi Bio-Tech Co.,Ltd, China</td>
<td>CF259748</td>
<td>&ge;98.0%(HPLC)</td>
</tr>
<tr>
<td>Ninhydrin</td>
<td>Aladdin Co., Ltd., China</td>
<td>N105629</td>
<td>AR</td>
</tr>
<tr>
<td>PBS buffer</td>
<td>Aladdin Co., Ltd., China</td>
<td>P196986</td>
<td>pH 7.4</td>
</tr>
<tr>
<td>Plasmid DNA</td>
<td>BIOGOT Co., Ltd, China</td>
<td>pDNA-EGFP</td>
<td>pDNA-EGFP</td>
</tr>
<tr>
<td>Plasmid DNA</td>
<td>BIOGOT Co., Ltd, China</td>
<td>Pdna</td>
<td>pDNA</td>
</tr>
<tr>
<td>Sodium carbonate decahydrate</td>
<td>Aladdin Co., Ltd., China</td>
<td>S112589</td>
<td>AR</td>
</tr>
<tr>
<td>Trimethylamine</td>
<td>Aladdin Co., Ltd., China</td>
<td>T103285</td>
<td>AR</td>
</tr>
</tbody>
</table>

methionine functionalized biocompatible block copolymers for targeted plasmid dna delivery

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&#8217;-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.

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...

Watch this Scientific Journal Video about Methionine Functionalized Biocompatible Block Copolymers for Targeted Plasmid DNA Delivery at JoVE.com

Methionine Functionalized Biocompatible Block Copolymers for Targeted Plasmid DNA Delivery

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 ...

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