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 polymerization bottle from step 1.1.</li>
	<li>Ventilate the reaction system in the polymerization bottle with nitrogen via three freeze-pump-thaw cycles.
	<ol>
		<li>In detail, freeze the solution in the polymerization bottle using a condensate trap, fix the polymerization bottle to the iron support, and vacuumize and inject nitrogen into the reaction mixture via a conduit tipped with a needle (#9 needle, inner diameter 0.65 mm, outer diameter 0.9 mm). Seal the polymerization bottle and thaw the solution at room temperature for 30 min.</li>
		<li>Repeat the freeze-pump-thaw cycles three times.</li>
	</ol>
	</li>
	<li>Put the polymerization bottle into a 70 &#176;C oil-bath and let the solution react for 24 h under the nitrogen atmosphere.</li>
	<li>Chill the polymerization bottle at 0 &#176;C and open the rubber stopper to terminate the polymerization process.</li>
	<li>Precool acetone in a -20 &#176;C fridge for 2 h and then mix it with the reaction solution of step 1.5 at 50:1 (v/v). After that, centrifuge at 8,200 x g for 10 min to remove acetone and collect the precipitate.</li>
	<li>To purify the synthesized PBNHEMA, dissolve the collected precipitate in 2 mL of pure water and then mix it with 100 mL of precooled acetone, at the ratio of 1:50 (v/v). Centrifuge the solution at 8,200 x g for 10 min and collect the precipitate. Repeat this process three times.</li>
	<li>Dry the produced PBNHEMA using a 50 &#176;C vacuum drier. Once dried, weigh the powder with a balance. Calculate the yield rate according to Equation 1. 
	<img alt="Equation 1" src="/files/ftp_upload/58527/58527eq01v2.jpg" style="opacity: 0.9;" />&#160;(1) 
	NOTE: In this experiment, the yield obtained was 77.2%.</li>
</ol>
2. Synthesis of BNHEMA-b-APMA polymer (BA)
<ol>
	<li>Dissolve 0.96 g of N-(3-aminopropyl)methacrylamide hydrochloride (APMA) and 0.93 g PBNHEMA in 5 mL of distilled water in a 10 mL beaker.</li>
	<li>Dissolve 0.01 g of 4,4&#8217;-azobis (4-cyanovaleric acid) (ACVA) in 0.5 mL of 1,4-dioxane and mix with the APMA-PBNHEMA solution from part 2.1.</li>
	<li>Transfer the mixture into a polymerization bottle and ventilate with dry nitrogen for 1 h.</li>
	<li>Put the polymerization bottle into a 70 &#176;C oil-bath and let it react for 24 h under the nitrogen atmosphere.</li>
	<li>Chill the polymerization bottle at 0 &#176;C and open the rubber stopper to terminate the polymerization process.</li>
	<li>Transfer the solution to the chilled acetone from step 1.6, and then centrifuge the solution at 8,200 x g for 10 min to precipitate the BA.</li>
	<li>Dissolve the BA in 2 mL of distilled water and precipitate the polymer in chilled acetone. Repeat three times.</li>
	<li>Dry the produced BA in a 50 &#176;C vacuum drier and weigh the obtained powder. Calculate the yield rate according to Equation 2. 
	<img alt="Equation 2" src="/files/ftp_upload/58527/58527eq02v2.jpg" style="opacity: 0.9;" /> 
	NOTE: In this experiment, the yield rate was calculated to be 82.0%.</li>
</ol>
3. Determine the mole percent of APMA in BA copolymer via the ninhydrin method
NOTE: Spectrophotometry is used to determine the contents of multicomponent amino acids. The principle is a color reaction of ninhydrin and amino acid where the absorbance is correlated with the amino acid content to a certain extent13,14.
<ol>
	<li>Dissolve 5 g of ninhydrin in 125 mL of boiling distilled water. Also, dissolve 5 g of Vitamin C in 250 mL of warm distilled water. Add the 250 mL of Vitamin C solution dropwise to the ninhydrin solution under magnetic stirring. Continue to stir for 15 min and then chill the reaction solution in a 4 &#176;C fridge.</li>
	<li>Take the solution out of the fridge and filter by suction using a Buchner funnel to obtain reduced ninhydrin. Collect the precipitate and preserve it in a phosphorus pentoxide dehydrator.</li>
	<li>Dissolve 85 mg of ninhydrin and 15 mg of reduced ninhydrin in 10 mL of ethylene glycol monomethyl ether to prepare the ninhydrin-coloring solution. 
	NOTE: Ninhydrin-coloring solution can react with &#945;-amino in the APMA and form a violet compound with a structure as described in a previous study15.</li>
	<li>Dilute 1 mL of 0, 1, 10, 100, 1,000 mg/mL APMA monomer solutions with 1 mL of acetate buffer (2 M, pH 5.4), and then add 1 mL of ninhydrin-coloring solution, respectively.</li>
	<li>Heat the mixtures for 15 min in a boiling water bath and then cool them using running water. Let the solutions sit for 5-10 min and dilute them with 3 mL of 60% ethyl alcohol and mix them thoroughly. Measure the absorbance at 570 nm using a spectrophotometer and draw the standard curve (Equation 3). 
	<img alt="Equation 3" src="/files/ftp_upload/58527/58527eq03v2.jpg" style="opacity: 0.9;" /> 
	NOTE: Equation 3 was derived from the linear fitting of the absorbance at 570 nm versus the APMA concentration.</li>
	<li>Dissolve 0.01 g of BA in 1 mL of distilled water; add 1 mL of acetate buffer (2 M, pH 5.4) and 1 mL of ninhydrin-coloring solution. Calculate the molar content of APMA according to the absorbance at 570 nm. 
	<img alt="Equation 4" src="/files/ftp_upload/58527/58527eq04v2.jpg" style="opacity: 0.9;" /> 
	<img alt="Equation 5" src="/files/ftp_upload/58527/58527eq05v2.jpg" style="opacity: 0.9;" />&#160;(5) 
	<img alt="Equation 6" src="/files/ftp_upload/58527/58527eq06v2.jpg" style="opacity: 0.9;" /> 
	NOTE: The calculation formulas are as follows (Equations 3-6).</li>
</ol>
4. Synthesis of methionine grafted BA polymer (mBA)
<ol>
	<li>Dissolve 8.9 mg of Fomc-Methionine in 5 mL of DMSO in a recovery flask.</li>
	<li>Add 6.92 mg of 1-ethyl-3-(3-dimethylaminopropyl ) carbodiimide hydrochloride (EDCl) and 4.86 mg of 1-hydroxybenzotriazole (HOBT) to the recovery flask and react at 0 &#176;C for 0.5 h.</li>
	<li>Dissolve 2.59 g of BA in 5 mL of DMSO solution and then add 50 &#956;L of trimethylamine; add this solution dropwise to the recovery flask (step 4.2) and let the solution react for 0.5 h at room temperature.</li>
	<li>Dialyze to remove DMSO and trimethylamine from the BA solution in step 4.3 using a dialysis bag (MWCO 10 kDa) in a 2 L beaker for 24 h; replace the deionized water every 6 h.</li>
	<li>Freeze-dry the obtained mBA and weigh to calculate the yield rate according to Equation 7. 
	<img alt="Equation 7" src="/files/ftp_upload/58527/58527eq07v2.jpg" style="opacity: 0.9;" /> 
	NOTE: In this case, the yield rate was determined to be 71%.</li>
	<li>Quantify the NH2 containing mBA by measuring the absorbance at 570 nm to calculate the amount of grafted methionine. Calculate the molar content of methionine according to Equation 8. 
	<img alt="Equation 8" src="/files/ftp_upload/58527/58527eq08v2.jpg" style="opacity: 0.9;" /></li>
</ol>
5. Synthesis of guanidinated and methionine conjugated BNHEMA-b-APMA polymer (mBG)
NOTE: Three different mBA1, mBA2 and mBA3 copolymer were synthesized. mBA3 copolymer is used as an example in the following steps.
<ol>
	<li>Dissolve mBA3 containing 60 &#956;mol of amino group in 5 mL of pure water. 
	NOTE: Amino group content was quantified using the ninhydrin method as described in step 3.5.</li>
	<li>Dissolve 40.6 mg (300 &#956;mol) of guanidinylation reagent 1-amidinopyrazole hydrochloride in mBA solutions.</li>
	<li>Adjust the pH to 9.0 with the saturated solution of sodium carbonate and let it stabilize for 24 h at room temperature.</li>
	<li>Dialyze the mBG product with deionized water using a dialysis bag in a beaker (MWCO 10 kDa, 2 L) and preserve it in the form of a freeze-dried powder. 
	The yield percentage was calculated to be 85% via Equation 8. 
	<img alt="Equation 9" src="/files/ftp_upload/58527/58527eq09v2.jpg" style="opacity: 0.9;" />&#160;(8)</li>
	<li>Dissolve mBG powder in D2O in the NMR tubes and characterize it using 1H nuclear magnetic resonance spectroscopy (1H NMR)16.</li>
</ol>
6. Preparation and characterization of mBG/pDNA polyplexes
<ol>
	<li>Dissolve 50 &#956;g of pDNA in 50 &#956;L of RNase/DNase-free water.</li>
	<li>Dissolve 1 mg of mBG copolymers in 1 mL of RNase/DNase-free water.</li>
	<li>Add the mBG copolymers solution directly into the pDNA solution according to different feeding ratios, that is, different N/P ratios (1:1, 4:1, 8:1, 16:1, and 32:1). 
	NOTE: N/P ratio is defined as the molar ratio of the guanidine group in the polymer and the phosphate group in pDNA, namely the molar ratio of the GPMA chain in the polymer and the mononucleotide in pDNA. The N/P ratio is calculated according to the molecular weights of amino nitrogen (N) in mBG and phosphate group (P) in pDNA .</li>
	<li>Mix the solutions with a vortex mixer and allow them to stand for 30 min at room temperature. After that, disperse the mixture in phosphate buffer solution (PBS, pH 7.4) and preserve the obtained mBG/pDNA polyplexes at 4 &#176;C for the follow-up experiments. 
	NOTE: The average particle size and Zeta potential of mBG and the complexes were detected using dynamic light scattering (DLS)17.</li>
	<li>Dilute 10 &#956;L of the mBG/pDNA polyplex solutions with 1 mL of PBS (pH 7.4) in the DLS and Zeta potential sample cells. 
	NOTE: Particle size and Zeta potential detection were performed three times and an average of the three values was taken.</li>
</ol>
7. Electrophoretic retardation experiment of mBG/pDNA polyplexes
NOTE: An electrophoretic retardation experiment was conducted to determine the minimum charge ratio.
<ol>
	<li>Take five groups of the mBG/pDNA polyplexes with different N/P ratios (1:1, 4:1, 8:1, 16:1, and 32:1) containing 50 &#956;g of pDNA .</li>
	<li>Add 6x loading buffer to the mBG/pDNA polyplex samples to a final concentration of 1x.</li>
	<li>Add the solutions to the 1.5% agarose gels and run the gel at 90 mV for 15 min, using pDNA as control .</li>
	<li>Take pictures of the gels using a gel imager.</li>
</ol>
8. Cytotoxicity of mBG/pDNA polyplexes
<ol>
	<li>Seed MCF-7 cells into the 96-well plates at a density of 104 cells per well. Then, culture the cells for 12 h using DMEM medium (10% FBS and 1% antibiotic) in a humidified 37 &#176;C incubator supplied with 5% CO2.</li>
	<li>Replace the culture medium with antibiotic-free DMEM culture media containing 10% fetal bovine serum (FBS) and mBG/pDNA polyplexes of different charge ratios (N/P 4, 8, 16, and 32, n=6) for 6 h, taking cells added with equal volumes of PBS solution as the control. Then, replace the culture medium with 150 &#956;L of fresh 1640 medium and further culture the cells for 24 h.</li>
	<li>Add 5 mg/mL 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (20 &#956;L/well) to the 96-well plates and further culture the cells for 4 h.</li>
	<li>Remove the solution and add 150 &#956;L of DMSO to each well and shake the 96-well plates 30 sec.</li>
	<li>Measure the optical density (OD) at 490 nm with a microplate reader to show the cell viability. Calculate the cell viability percentage according to Equation 9. 
	Cell viability % =&#160;<img alt="Equation 10" src="/files/ftp_upload/58527/58527eq10v2.jpg" style="opacity: 0.9;" />&#160;(9)</li>
</ol>
9. Transfection efficiency of mBG/ GFP-pDNA polyplexes
<ol>
	<li>Dissolve 50 &#956;g of pDNA containing the reporter gene green fluorescent protein (GFP) pDNA (GFP-pDNA) in 50 &#956;L of RNase/DNase-free water. Then dissolve 1 mg of mBG copolymers in 1 mL of RNase/DNase-free water. Mix pDNA and mBG solution at a charge ratio (N/P) of 1:1, 4:1, 8:1, 16:1, and 32:1 and incubate for 30 min at room temperature. Disperse the mBG/GFP-pDNA polyplexes solution using ultrasonic waves (30 s) and store at 4 &#176;C for the follow-up experiments. 
	NOTE: The N/P ratio is calculated according to the molecular weights of amino nitrogen (N) in mBG and phosphate group (P) in pDNA .</li>
	<li>Seed MCF-7 cells at a density of 2 &#215; 105 cells per well in a 6-well plate and culture them at 37 &#176;C and 5% CO2 in a humidified incubator for 12 h.</li>
	<li>Replace the culture medium with the fresh culture medium containing mBG/GFP-pDNA polyplexes of different N/P ratios (4, 8, 16, and 32) for 6 h.</li>
	<li>Replace the medium with 2 mL of fresh RPMI1640 medium and culture for 48 h.</li>
	<li>Collect the cells and detect the green fluorescence with a flow cytometer.</li>
</ol>

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The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in this article.

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>&amp;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&amp;rsquo;-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>&gt;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>&amp;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>&amp;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 ...

methionine-functionalized-biocompatible-block-copolymers-for-targeted

Research

JoVE Journal

Chemistry

5.7K Views.  Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research & Nanjing Medical University Affiliated Cancer Hospital. 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.

Video: Methionine Functionalized Biocompatible Block Copolymers for Targeted Plasmid DNA Delivery

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.

An example of a nano drug based on polymalic acid is presented towards the rational design of personalized medicine that is applicable to cancer. It describes the synthesis of a nano drug to treat Her2-positive human breast cancer in a nude mouse.

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this ...

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

Biochemistry

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Genetics

Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar concentration forming hydrophobic cores surrounded by hydrophilic shells in aqueous environments. The core of these micelles can be utilized to load hydrophobic, poorly water soluble drugs like docetaxel (DTX) and everolimus (EVR). Systematic characterization of the micelle structure and drug loading capabilities are important before in vitro and in vivo studies can be conducted. The goal of the protocol described herein is to provide the necessary characterization steps to achieve standardized micellar products. DTX and EVR have intrinsic solubilities of 1.9 and 9.6 &#181;g/ml respectively Preparation of these micelles can be achieved through solvent casting which increases the aqueous solubility of DTX and EVR to 1.86 and 1.85 mg/ml, respectively. Drug stability in micelles evaluated at room temperature over 48 hr indicates that 97% or more of the drugs are retained in solution. Micelle size was assessed using dynamic light scattering and indicated that the size of these micelles was below 50 nm and depended on the molecular weight of the polymer. Drug release from the micelles was assessed using dialysis under sink conditions at pH 7.4 at 37 oC over 48 hr. Curve fitting results indicate that drug release is driven by a first order process indicating that it is diffusion driven.

The goal of this protocol is to describe the preparation and characterization of physically entrapped, poorly water soluble drugs in micellar drug delivery systems composed of amphiphilic block copolymers.

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Bioengineering

Anionic Polymerization of an Amphiphilic Copolymer for Preparation of Block Copolymer Micelles Stabilized by &#960;-&#960; Stacking Interactions

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE). Characterization of the copolymer revealed a narrow molecular distribution (PDI &#60; 1.03) and confirmed the degree of polymerization of mPEG122-b-(PheGE)15. The critical micelle concentration of the copolymer was evaluated using an established fluorescence method with the aggregation behavior evaluated by dynamic light scattering and transmission electronic microscopy. The potential of the copolymer for use in drug delivery applications was evaluated in a preliminary manner including in vitro biocompatibility, loading and release of the hydrophobic anti-cancer drug doxorubicin (DOX). A stable micelle formulation of DOX was prepared with drug loading levels up to 14% (wt%), drug loading efficiencies &#62; 60% (w/w) and sustained release of drug over 4 days under physiologically relevant conditions (acidic and neutral pH, presence of albumin). The high drug loading level and sustained release is attributed to stabilizing &#960;-&#960; interactions between DOX and the core-forming block of the micelles.

The key steps of living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE) are described. The resulting block copolymer micelles (BCMs) were loaded with doxorubicin 14% (wt%) and sustained release of drug over 4 days under physiologically relevant conditions was obtained.

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl ...

Gene-therapy Inspired Polycation Coating for Protection of DNA Origami Nanostructures

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in physiological fluids induce denaturation and degradation of self-assembled DNA nanostructures. In non-viral gene delivery, enzymatic degradation of DNA is overcome by the encapsulation of the negatively charged DNA in a cationic shell. Herein, inspired by gene delivery advancements, a simple, one-step and robust methodology is presented for the stabilization of DNA origami nanostructures by coating them with chitosan and linear polyethyleneimine. The polycation coating efficiently protects DNA origami nanostructures in Mg-depleted and nuclease-rich media. This method also preserves the full addressability of enzyme- and aptamer-based functionalization of DNA nanostructures.

Here, a protocol for the protection of DNA origami nanostructures in Mg-depleted and nuclease-rich media using natural cationic polysaccharide chitosan and synthetic linear polyethyleneimine (LPEI) coatings is presented.

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in ...

Assembly and Characterization of Polyelectrolyte Complex Micelles

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful platform for exploring the physics of polyelectrolyte interactions and also offer a promising solution to the pressing problem of delivering therapeutic oligonucleotides in vivo. Developing predictive structure-property relationships for PCMs has proven difficult, in part due to the presence of strong kinetic traps during nanoparticle self-assembly. This article discusses criteria for choosing polymers for PCM construction and provides protocols based on salt annealing that enable assembly of repeatable, low-polydispersity nanoparticles. We also discuss PCM characterization using light scattering, small-angle X-ray scattering, and electron microscopy.

We provide protocols and representative data for designing, assembling, and characterizing polyelectrolyte complex micelles, core-shell nanoparticles formed by polyelectrolytes and hydrophilic charged-uncharged block copolymers.

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...

Production of Double-stranded DNA Ministrings

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ministrings possess a heightened safety profile and also efficiently deliver DNA cargo to targeted cells. Conventional DNA vectors carry undesirable prokaryotic sequences, including antibiotic resistance genes, CpG motifs, and bacterial origins of replication, which may lead to the stimulation of host immunological responses. The bioavailability of conventional DNA vectors is also compromised due to their larger molecular size. Their circular nature may also impart chromosomal integration, leading to insertional mutagenesis.
Bacterial sequences are excised from DNA minivectors, leaving only the gene of interest (GOI) and necessary eukaryotic expression elements. Our LCC DNA minivectors, or DNA ministrings, are devoid of immunogenic bacterial sequences; therefore improving their bioavailability and GOI expression. In the event of vector integration into the chromosome, the LCC DNA ministring will lethally disrupt the host chromosome, thereby removing the potentially dangerous mutant from the proliferating cell population. Consequently, DNA ministrings offer the benefits of &#39;minicircle&#39; DNA while eliminating the potential for undesirable vector integration events. In comparison to conventional plasmids and their isogenic circular covalently closed (CCC) counterparts, DNA ministrings demonstrate superior bioavailability, transfection efficiency, and cytoplasmic kinetics - they thus require lower amounts of cationic surfactants for effective transfection of target cells.
We have constructed a one-step inducible in vivo system for the production of DNA ministrings in Escherichia coli that is simple to use, rapid, and scalable.

The linear covalently closed (LCC) DNA minivector (DNA ministring) is a non-viral gene delivery vector offering high transfection efficiency and is relevant to any DNA delivery application. The production system is simple, rapid, and scalable in vivo. The following protocol provides visual step-by-step instructions for the production of DNA ministrings.

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ...