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...

<ol>
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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 ...

Cationic polymer drug carriers have the advantages of good stability, low immunogenicity, and facile preparation and modification. Here, we offer a rapid and simple method for its synthesis. Reversible addition-fragmentation chain transfer polymerization method is a method applicable to yield block polymers with controlled molecular weight and structure and carrying functional groups.These drug carriers can simultaneously carry different drugs to achieve the collaborate treatment of cancer, inflammation, and other diseases. IFT polymerization is a classic method, and this protocol gives an example of its application in gene therapy. The key to the success for synthesis of this polymer is to precisely control the reaction temperature.It is more advisable to use an oil bath than a water bath. To begin this procedure, obtain a round-bottom flask with a rubber stopper and a magnetic stirrer to be used as the polymerization bottle. Dissolve 1.87 grams of bis-hydroxy HEMA in one milliliter of distilled water in the polymerization bottle.In a five-milliliter beaker, dissolve 0.03 grams of CTP and 0.02 grams of ACVA in 0.5 milliliters of 1, 4-dioxane. Add this mixture to the polymerization bottle. Then, use a condensate trap to freeze the solution in the polymerization bottle.Fix the bottle to the iron support, and use a conduit tripped with a needle to vacuumize and inject nitrogen into the reaction mixture. Then, seal the polymerization bottle, and thaw the solution at room temperature for 30 minutes. Repeat this freeze-pump-thaw cycle three times.After this, place the bottle into an oil bath at 70 degrees Celsius and let the solution react for 24 hours under the nitrogen atmosphere. The next day, chill the polymerization bottle at zero degrees Celsius and open the rubber stopper to terminate the polymerization reaction. Next, precool acetone in a refrigerator at negative 20 degrees Celsius for two hours, and mix it with the reaction solution in the polymerization bottle at a ratio of 50 to one.Centrifuge this mixture at 8, 200 times g for 10 minutes to remove the acetone and collect the precipitate. To purify the synthesized poly bis-hydroxy HEMA, dissolve the collected precipitate in two milliliters of pure water. Then, mix it with 100 milliliters of precooled acetone at a ratio of one to 50.Centrifuge the solution at 8, 200 times g for 10 minutes to collect the precipitate. Repeat this purification process of dissolving, mixing, and centrifuging the precipitate three times. First, dissolve 0.96 grams of APMA and 0.93 grams of the purified poly bis-hydroxy HEMA in five milliliters of distilled water in a 10-milliliter beaker.Dissolve 0.01 grams of ACVA in 0.5 milliliters of 1, 4-dioxane, and add this to the APMA and poly bis-hydroxy HEMA solution. Transfer this mixture to a polymerization bottle, and ventilate with dry nitrogen for one hour. Then, put the polymerization bottle into an oil bath at 70 degrees Celsius, and let it react for 24 hours under the nitrogen atmosphere.The next day, chill the polymerization bottle at zero degrees Celsius and open the rubber stopper to terminate the polymerization process solution. To begin, seed MCF-7 cells into the wells of a six-well plate at a density of 20, 000 cells per well. Culture the cells in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide for 12 hours.After this, replace the culture medium with the fresh culture medium containing cationic polymer and pDNA polyplex polyplexes of different ratio of nitrogen-to-phosphorus ratios. Culture the cells for six hours, and then replace the medium with two milliliters of fresh RPMI 1640 medium. Continue culturing for 48 hours.Then, collect the cells, and use a flow cytometer to detect the green fluorescence. In this study, methionine-functionalized biocompatible block copolymers are prepared via the reversible addition-fragmentation chain transfer method, and the plasmid DNA complexing ability of the obtained mBG and their transfection efficiency is investigated. The average particle size and zeta potential of mBG/pDNA polyplexes are 124 nanometers and plus 15.7 millivolts, respectively, at an N-to-P ratio of 16.Electrophoretic retardation reveals that pDNA with an N-to-P ratio of zero can move without restraint in the agarose gel and is observed as a stripe in the gel imager. When pDNA is complexed with mBG, the movement of pDNA is retarded, and subsequently, the brightness of the band is reduced. This shows that mBG can completely complex the pDNA when the N to P is higher than four.The cytotoxicity of the mBG/pDNA polyplexes is then measured using the standard MTT assay These results show that mBG/pDNA polyplexes have cytotoxicity than PEI/pDNA polyplexes at the N-to-P ratios of four, eight, 16, and 32. As can also be seen, the cytotoxicity of the mBG/pDNA polyplexes increases with increased N-to-P ratio. This increased cytotoxicity is a result of the positively charged GPMA component.The N-to-P ratio used in the pDNA transfection experiment is selected according to the cytotoxicity results. The transfection abilities of mBG1, mBG2, and mBG3 are compared by measuring the GFP fluorescence intensity, and the results showed that mBG3 is the optimal gene carrier. It is important to exhaust air from the silt round-bottom flask and maintain the reaction temperature.The obtained polymer drug carriers can be applied to the co-delivery of chemotherapy drugs and gene drugs to synergistically treat drug-resistant cancer. The molecular mechanism of combination of gene drugs and chemotherapeutic drugs can be further explored for the treatment of drug-resistant tumors. Acetone is rated as low toxic for its acute toxicity.Keys, inhalation or contact with eyes or skin should be avoided. Avoid to the risk of scalding during the heating procedure.

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