Name: synthesis of aptamer-pei-g-peg modified gold nanoparticles loaded with doxorubicin for targeted drug delivery
Uploaded: 2020-06-23T13:30:00
Description: Watch this Scientific Journal Video about Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery at JoVE.com

This research was funded by the National Natural Science Foundation of China (31700840); the Key Scientific Research Project of Henan Province (18B430013, 18A150049). This research was supported by the Nanhu Scholars Program for Young Scholars of XYNU. The authors would like to thank bachelor student Zebo Qu from the College of Life Sciences in XYNU for his helpful works. The authors would like to acknowledge the Analysis &#38; Testing Center of XYNU for the use of their equipment.

CAUTION: Make sure to consult all relevant material safety data sheets (MSDS) before using all chemicals. Several of the chemicals used for preparing copolymer and nanoparticles are acutely toxic. Nanoparticles also have potential hazards. Make sure to use all appropriate safety practices and personal protective equipment, including gloves, lab coat, hoods, full-length pants, and close-toed shoes.
1. Synthesis of double-carboxyl polyethylene glycol (CT-PEG)33
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanoparticles+in+delivery+applications.">Gold nanoparticles in delivery applications.</a> Advanced Drug Delivery Reviews. 60 (11), 1307-1315 (2008).</li><li>Vandghanooni, S., Eskandani, M., Barar, J., Omidi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antisense+LNA-loaded+nanoparticles+of+star-shaped+glucose-core+PCL-PEG+copolymer+for+enhanced+inhibition+of+oncomiR-214+and+nucleolin-mediated+therapy+of+cisplatin-resistant+ovarian+cancer+cells.">Antisense LNA-loaded nanoparticles of star-shaped glucose-core PCL-PEG copolymer for enhanced inhibition of oncomiR-214 and nucleolin-mediated therapy of cisplatin-resistant ovarian cancer cells.</a> International Journal of Pharmaceutics. 573, 118729 (2020).</li><li>Andghanooni, S., Eskandani, M., Barar, J., Omidi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AS1411+aptamer-decorated+cisplatin-loaded+poly(lactic-co-glycolic+acid)+nanoparticles+for+targeted+therapy+of+miR-21-inhibited+ovarian+cancer+cells.">AS1411 aptamer-decorated cisplatin-loaded poly(lactic-co-glycolic acid) nanoparticles for targeted therapy of miR-21-inhibited ovarian cancer cells.</a> Nanomedicine. 13 (21), 2729-2758 (2018).</li><li>Palmieri, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+anti-nucleolin+recombinant+immunoagent+for+cancer+therapy.">Human anti-nucleolin recombinant immunoagent for cancer therapy.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (30), 9418-9423 (2015).</li><li>Pichiorri, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+NCL+targeting+affects+breast+cancer+aggressiveness+through+miRNA+regulation.">In vivo NCL targeting affects breast cancer aggressiveness through miRNA regulation.</a> Journal of Experimental Medicine. 210 (5), 951-968 (2013).</li><li>Hou, S., McCauley, L. 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X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+erosion+properties+of+PEG-containing+polyanhydrides.">Synthesis and erosion properties of PEG-containing polyanhydrides.</a> Macromolecular Bioscience. 7 (5), 620-628 (2007).</li><li>Nie, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+Vaginal+Hydrogels+as+a+Multi-Drug+Carrier+for+Contraception.">Injectable Vaginal Hydrogels as a Multi-Drug Carrier for Contraception.</a> Applied Sciences. 9 (8), 1638 (2019).</li><li>Zou, P., Suo, J., Nie, L., Feng, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-responsive+biodegradable+star-shaped+block+copolymers+for+vaginal+gels.">Temperature-responsive biodegradable star-shaped block copolymers for vaginal gels.</a> Journal of Materials Chemistry. 22 (13), 6316-6326 (2012).</li><li>Etrych, T., &#352;ubr, V., Laga, R., &#344;&#237;hov&#225;, B., Ulbrich, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+conjugates+of+doxorubicin+bound+through+an+amide+and+hydrazone+bond:+Impact+of+the+carrier+structure+onto+synergistic+action+in+the+treatment+of+solid+tumours.">Polymer conjugates of doxorubicin bound through an amide and hydrazone bond: Impact of the carrier structure onto synergistic action in the treatment of solid tumours.</a> European Journal of Pharmaceutical Sciences. 58, 1-12 (2014).</li><li>Safari, F., Tamaddon, A. 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The 1H NMR spectrum (Figure 2) confirm the successful synthesis of CT-PEG copolymer and PEI-g-PEG copolymer. The molecular weights of PEG and PEI were 1,000 and 1,200, respectively. Additionally, the EDC/NHS catalytic system was used to synthesize PEI-g-PEG copolymer via amide reactions. It should be noted that if the molecular weights of PEG and PEI changed for synthesizing PEI-g-PEG copolymer, then the reaction time and catalytic system needs to be reevaluated. Also, the reactio...

Being the major public health problem worldwide, cancer is widely characterized as having a low cure rate, high recurrence rate, and high mortality rate1,2. Current conventional anti-cancer methods include surgery, chemotherapy, and radiotherapy3, among which chemotherapy is the primary treatment for cancer patients in the clinic4. Clinical used anticancer drugs mainly include paclitaxel (PTX)5 and doxorubicin (DOX)6,7. DOX, an antineoplastic drug, has been broadly applied in clinical chemotherapy, due to the advantages of cancer cytotoxicity and inhibition of cancer cell proliferation8,9. However, DOX causes cardiotoxicity10,11, and the short half-life of DOX restricts its application in the clinic12. Therefore, degradable drug carriers are needed to load DOX and subequently release in a controlled fashion to a targeted area.
Nanoparticles have been widely used in targeted drug delivery systems and have several advantages in cancer treatment (i.e., sizeable surface-to-volume ratio, small size, ability to encapsulate various drugs, and tunable surface chemistry, etc.)13,14,15. In particular, gold nanoparticles (AuNPs) have been widely used in biological and biomedical applications, such as photothermal cancer therapy16,17. The unique properties of AuNPs, such as facile synthesis and general surface functionalization, have excellent prospects in the clinical field of cancer therapy18. Also, AuNPs have been used to identify drug delivery strategies, diagnose tumors, and overcome resistance in many studies19,20.
Notwithstanding, AuNPs need to be further tailored to overcome drug resistance via high local release at tumor lesions through enhanced permeation and retention (EPR), such as the targeting and accessibility properties. Polymer functionalized AuNPs have exhibited unique advantages, such as improved water solubility of hydrophobic anticancer drugs and prolonged circulation time21,22. Various biocompatible polymers have been used for AuNP coatings, such as polyethylene glycol (PEG), polyethyleneimine (PEI), hyaluronic acid, heparin, and xanthan gum. Then the stability, as well as the payload, of AuNPs is improved well23. Specifically, PEI is a highly branched polymer that is composed of many repeating units of primary, secondary, and tertiary amines24. PEI has excellent solubility, low viscosity, and a high degree of functionality, which is suitable for coating on AuNPs.
On the other hand, anti-cancer drugs need to be delivered to cancer cells directly with improved loading efficiency, and with lower toxicity for treating primary and advanced metastatic tumors25. Targeted ligands have great potential for anti-cancer drug targeted delivery systems26. Its selectivity for target molecule binding confers anti-cancer drug targeting specificity and increases drug enrichment in diseased tissues27. More ligands include antibodies, polypeptides, and small molecules. Compared to other ligands, nucleic acid aptamers can be synthesized in vitro and are easy to modify. AS1411 is an unmodified 26 bp phosphodiester oligonucleotide that forms a stable dimeric G-tetramer structure to specifically bind to an overexpressed target nuclear protein receptor on cancer cells28,29,30. AS1411 inhibits the proliferation of many cancer cells but does not affect the growth of healthy cells31,32. As a result, AS1411 has been used to fabricate an ideal targeted drug delivery system.
In this study, a PEI-g-PEG copolymer is synthesized via an amide reaction, then PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are fabricated. Additionally, DOX and AS1411 are sequentially linked to the prepared PEI-g-PEG@AuNPs, as shown in Figure 1. This detailed protocol is intended to help researchers avoid many of the common pitfalls associated with the fabrication of new PEI-g-PEG@AuNPs loaded with DOX and AS1411.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-Dimethylaminopyridine</td>
			<td>Macklin</td>
			<td>D807273</td>
			<td></td>
		</tr>
		<tr>
			<td>A549 cell</td>
			<td></td>
			<td>ATCC CCL-185TM</td>
			<td></td>
		</tr>
		<tr>
			<td>AS1411</td>
			<td>BBI Life Sciences Corporation</td>
			<td></td>
			<td>5&#39;-d (TTTGGTGGTGGTGGTTGTGGTGGTGGTGG) FL-AS1411 (fluorophore-labeled AS1411)</td>
		</tr>
		<tr>
			<td>Anhydrous Tetrahydrofuran (THF)</td>
			<td>SinoPharm Chemical Reagent Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counting kit-8 (CCK-8)</td>
			<td>Sigma Aldrich</td>
			<td>96992-500TESTS-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Traditional Chinese medicine</td>
			<td>80047318</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether (Et2O)</td>
			<td>SinoPharm Chemical Reagent Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Macklin</td>
			<td>D806645</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium (DMEM)</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Doxorubicin hydrochloride</td>
			<td>Rhawn</td>
			<td>R017518</td>
			<td></td>
		</tr>
		<tr>
			<td>Ether absolute</td>
			<td>Traditional Chinese medicine</td>
			<td>80059618</td>
			<td></td>
		</tr>
		<tr>
			<td>Field Emission Transmission Electron Microscope</td>
			<td>FEI Company</td>
			<td>Tecnai G2 F 20</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold(III) chloride trihydrate</td>
			<td>Rhawn</td>
			<td>R016035</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Particle-size Instrument</td>
			<td>Malvern Instruments Ltd</td>
			<td>ZetasizerNanoZS/Masterszer3000E</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Reader</td>
			<td>Molecular Devices</td>
			<td>SpectraMax 190</td>
			<td></td>
		</tr>
		<tr>
			<td>N-(3-Dimethylaminopropyl)-N&#39;-ethylcarbodiimide hydrochloride</td>
			<td>Macklin</td>
			<td>N808856</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Hydroxysuccinimide</td>
			<td>Macklin</td>
			<td>H6231</td>
			<td></td>
		</tr>
		<tr>
			<td>NMR software</td>
			<td>Delta 5.2.1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclear Magnetic Resonance Spectrometer</td>
			<td>JEOL</td>
			<td>JNM-ECZ600R/S3</td>
			<td></td>
		</tr>
		<tr>
			<td>Origin 8.5</td>
			<td>OriginLab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Sigma Aldrich</td>
			<td>V900929-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Sigma Aldrich</td>
			<td>P4417-100TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(ethylene glycol)</td>
			<td>Sigma Aldrich</td>
			<td>81188</td>
			<td>BioUltra, average Mn ~ 1000</td>
		</tr>
		<tr>
			<td>Poly (ethyleneimine) solution</td>
			<td>Sigma Aldrich</td>
			<td>482595</td>
			<td>average Mn ~ 1200, 50 wt.% in H2O</td>
		</tr>
		<tr>
			<td>Sodium borohydride, powder</td>
			<td>Acros</td>
			<td>C18930</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Sigma Aldrich</td>
			<td>85886-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Succinic anhydride</td>
			<td>Traditional Chinese medicine</td>
			<td>30171826</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrahydrofuran</td>
			<td>Traditional Chinese medicine</td>
			<td>40058161</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Traditional Chinese medicine</td>
			<td>80134318</td>
			<td></td>
		</tr>
		<tr>
			<td>UV/VIS/NIR Spectrometer</td>
			<td>Lambda950</td>
			<td>Lambda950</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray Photoelectron Spectrometer</td>
			<td>Thermo&#8194;Fisher&#8194;Scientific</td>
			<td>K-ALPHA 0.5EV</td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis of aptamer-pei-g-peg modified gold nanoparticles loaded with doxorubicin for targeted drug delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

1H NMR spectroscopy was used to confirm the successful synthesis of CT-PEG polymer and PEI-g-PEG copolymers (Figure 2). Figure 2a shows that the methylene proton signal at &#948; = 3.61 ppm and carboxyl proton signal at &#948; = 2.57 ppm confirm the successful synthesis of CT-PEG polymers. Figure 2b shows that the methylene proton signal of PEG at &#948; = 2.6 ppm and proton signal of PEI at <em...

Watch this Scientific Journal Video about Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery at JoVE.com

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...

Because of the drug resistance and toxicity, the use of doxorubicin is limit in the clinic. This protocol provides a degradable carrier for keeping the release of this therapeutic agent. The advantage of this method is that the synthesized carrier can be used to deliver the drug directly to cancer cells, inducing cell death in the cancer cells only.For CT-PEG synthesis, first add 1.46 grams of succinic anhydride and 209 milligrams of 4-dimethylaminopyridine to a 100 milliliter round bottom flask. Next, add 15 milliliters of anhydrous tetrahydrofuran to the flask and fit a glass stopper into the flask, before incubating the reaction for 30 minutes at zero degrees Celsius. During the incubation, add 4.208 grams of PEG, 1.8 milliliters of triethylamine and 15 milliliters of anhydrous tetrahydrofuran to a new flask and close the flask with a glass stopper.At the end of the incubation, use a syringe under nitrogen atmosphere to slowly transfer the second solution to the first flask. Stir the new solution for two hours at zero degrees Celsius, before continuing the reaction at room temperature overnight. The next morning, use a rotary evaporator at 40 degrees Celsius and 0.1 megapascals to concentrate the reaction solution and to remove the tetrahydrofuran solvent.After an hour, dissolve the reaction solution in 15 milliliters of 1.325 gram per milliliter of dichloromethane at room temperature, before adding 15 milliliters of cold diethyl ether to obtain the PEG diacid precipitation product. Then remove the solvent via filter paper and dry the precipitate under vacuum for 48 hours at room temperature. For PEI-g-PEG copolymer synthesis, add 305.47 milligrams of the CT-PEG precipitate and five milliliters of DMSO to a new flask and stir the reaction at room temperature until the CT-PEG is fully dissolved.Next, dissolve 49.46 milligrams of EDC in five milliliters of DMSO and add the solution to the flask. Stir the reaction for 30 minutes at room temperature, before dissolving 29.69 milligrams of N-Hydroxysuccinimide and five milliliters of DMSO and adding the resulting solution to the flask. Continue to stir the reaction for three hours at room temperature.Near the end of the incubation, dissolve 28.6 microliters of PEI into 10 milliliters of DMSO and add the solution drop wise to the flask. Stir the reaction at room temperature for at least three days. At the end of the incubation, transfer the reacted solution to a dialysis bag with a 1000 molecular weight cutoff and place the bag into a one liter beaker containing 500 milliliters of ultra pure water as the dialysate.At the end of the dialysis, transfer the solution to a dialysis bag with a 10, 000 molecular weight cutoff and placed the bag into a one liter beaker with 500 milliliters of ultra pure water as the dialysate. At the end of the second dialysis, use a rotary evaporator at 40 degrees Celsius and 0.1 megapascals to concentrate the solution and freeze dry the sample to obtain the PEI-g-PEG powder. For the coating of gold nanoparticles with the PEI-g-PEG product, dissolve five milligrams of the prepared PEI-g-PEG in five milliliters of ultra pure water in a new flask and fit the flask with a glass stopper.Add 100 milliliters of 0.3 millimolar auric chloride solution to the flask and stir the solution for three hours at room temperature. The colors should change immediately from light yellow to dark yellow. At the end of the incubation, add one milliliter of one milligram per milliliter of sodium borohydride solution to the flask.The color should change immediately from dark yellow to golden yellow, and stir the solution for an additional three hours at room temperature. Then dialyze the reaction product with a 1000 molecular weight cutoff dialysis bag for three days as demonstrated to obtain the PEI-g-PEG coated gold nanoparticle solution. For grafting of doxorubicin to the PEI-g-PEG coated gold nanoparticles.Add one milliliter of 2.2 milligram per milliliter doxorubicin solution and 20 milliliters of the PEI-g-PEG coated gold nanoparticle solution to a new flask and fit the flask with a glass stopper. Next, dissolve 0.727 milligrams of EDC in one milliliter of ultra pure water and add the EDC solution to the flask. Dissolve 0.437 milligrams of N-Hydroxysuccinimide in one milliliter of ultra pure water, and add the N-Hydroxysuccinimide solution to the flask for a one hour incubation with stirring at room temperature.At the end of the incubation, dialyze the reaction product in a 1000 molecular weight cutoff dialysis bag for three days as demonstrated to obtain the doxorubicin g-PEI-g-PEG coated gold nanoparticle solution. For grafting of the AS1411 aptamer to the DOX-g-PEI-g-PEG. Add 20 milliliters of the doxorubicin DOX-g-PEI-g-PEG coated gold nanoparticle solution to an approximately four optical density concentration of AS1411.Dissolve 28.76 milligrams of EDC in one milliliter of ultra pure water and add the EDC solution to the flask. Dissolve 17.27 milligrams of N-Hydroxysuccinimide in one milliliter of ultra pure water and add the N-Hydroxysuccinimide solution to the flask for a one hour incubation with stirring at room temperature. Then dialyze the reaction product in a 1000 molecular weight cutoff dialysis bag for three days as demonstrated to obtain AS1411-g-Doxorubicin-g-PEI-g-PEG coated gold nanoparticles.Proton NMR spectroscopy can be used to confirm the successful synthesis of CT-PEG polymer and PIE-g-PEG copolymers. Ultra violet spectroscopy can be conducted to determine the successful functionalization and gradual loading of prepared copolymer on gold nanoparticles. X-ray photo electron spectroscopy can be used to investigate the chemical bond of co-polymer on gold nanoparticles.Dynamic light scattering can be performed to determine the size distribution of the prepared nanoparticles. In this analysis, transmission electron microscopy revealed non-clustered nanoparticles with a uniform morphology. Cell viability analysis revealed a decrease in A549 cell numbers over time and in response to increasing concentrations of nanoparticle.Compare to the free doxorubicin group, the cell numbers increased, however, indicating a reduced toxicity. Further, analysis of the doxorubicin release profile reveals that the sustained release of doxorubicin from functionalized nanoparticles caused a decrease in A549 cells. The PEI-g-PEG copolymer preparation and AS1411 grafting steps are important for success for implementation of the procedure.The same drug carrier can be achieved by grafting AS1411 before grafting drugs, but the drug loading efficiency will be decreased.

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