Name: synthesis and characterization of placental chondroitin sulfate a (plcsa)-targeting lipid-polymer nanoparticles
Uploaded: 2018-09-18T14:00:00
Description: Watch this Scientific Journal Video about Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles at JoVE.com

This work was supported by grants from the National Key Research and Development Program of China (2016YFC1000402), the National Natural Sciences Foundation (81571445 and 81771617) and the Natural Science Foundation of Guangdong Province (2016A030313178) to X.F. and the Shenzhen Basic Research Fund (JCYJ20170413165233512) to X.F.

1. Preparation of Stock Solutions
<ol>
	<li>Prepare an aqueous solution of 4% ethanol by diluting 4 mL of absolute ethanol with 100 mL of ultrapure water. Store the solution at 4 &#176;C. 
	NOTE: Ultrapure water is defined as water without contaminants such as bacteria, particulates, ions, or nucleases. Ultrapure water was obtained from a water purification system with a target resistivity of up to 18.2 m&#937;&#183;cm, which means low anionic contamination.</li>
	<li>Prepare a 1 mg/mL soybean lecithin stock solut...

<ol>
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H., Aryal, S., Hu, C. -. M. J., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quick+synthesis+of+lipid&#8722;+polymer+hybrid+nanoparticles+with+low+polydispersity+using+a+single-step+sonication+method.">Quick synthesis of lipid&#8722; polymer hybrid nanoparticles with low polydispersity using a single-step sonication method.</a> Langmuir. 26 (22), 16958-16962 (2010).</li><li>Gu, 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=Folate-modified,+indocyanine+green-loaded+lipid-polymer+hybrid+nanoparticles+for+targeted+delivery+of+cisplatin.">Folate-modified, indocyanine green-loaded lipid-polymer hybrid nanoparticles for targeted delivery of cisplatin.</a> Journal of Biomaterials Science, Polymer Edition. 28 (7), 690-702 (2017).</li><li>Mandal, B., Mittal, N. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-hydroxysulfosuccinimide+active+esters:+bis(N-hydroxysulfosuccinimide)+esters+of+two+dicarboxylic+acids+are+hydrophilic,+membrane-impermeant,+protein+cross-linkers.">N-hydroxysulfosuccinimide active esters: bis(N-hydroxysulfosuccinimide) esters of two dicarboxylic acids are hydrophilic, membrane-impermeant, protein cross-linkers.</a> Biochemistry. 21 (17), 3950-3955 (1982).</li><li>Valencia, P. 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This protocol provides an efficient and reproducible method for synthesizing plCSA-BP-conjugated lipid-polymer nanoparticles. The single-step sonication method to prepare lipid-polymer nanoparticles is fast, reproducible and different from typical nanoprecipitation methods that involve heating, vortexing, or evaporation. Hence, the developed method significantly reduces the synthesis time. In addition, the EDC/NHS bioconjugate used in this protocol is a commonly used and convenient technique to conjugate peptides and ant...

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Hence, selective tumor targeting is the key to exploring successful therapeutic methods. Nanoparticles offer a promising opportunity for cancer therapy, and molecular assemblies with different functional groups will enhance drug efficacy and reduce associated side effects1,2. Moreover, nanoparticle systems mainly utilize passive and active targeting to reach target tumors3.
Passive targeting exploits the innate characteristics of nanoparticles and enhanced permeability and retention (EPR) effects to reach tumor cells. Cationic liposomes have been successfully used to deliver various anticancer drugs to tumors in clinical applications4,5,6. Despite the potential effective cancer therapeutic effect, a low drug concentration in the tumor region and an inability to distinguish tumor cells from normal tissues are two major limitations of passive-targeting nanoparticles7.
Active targeting strategies take advantage of antigen-antibody, ligand-receptor and other molecular recognition interactions to specifically deliver drugs to tumors8. The glycosaminoglycan placental chondroitin sulfate A (plCSA) is broadly expressed on most cancer cells and placental trophoblasts. Moreover, the malarial protein VAR2CSA can specifically bind to plCSA9,10. Hence, VAR2CSA can be a tool for targeting human cancer cells. However, when VAR2CSA is conjugated to nanoparticles, the full-length protein may limit the penetration of nanoparticles into tumor cells. Recently, we discovered a plCSA binding peptide (plCSA-BP), derived from the malarial protein VAR2CSA. plCSA-BP-conjugated lipid-polymer nanoparticles rapidly bonded to choriocarcinoma cells and significantly increased doxorubicin (DOX) anticancer activity in vivo11; these particles also specifically bonded to placental trophoblasts and could serve as a tool for the targeted delivery of drugs to the placenta12.
Lipid-polymer nanoparticles consist of a lipid monolayer shell and a hydrophobic polymeric core and represent a new carrier for drug delivery. These nanoparticles combine the advantages of liposomes and polymeric nanocarriers, such as controllable nanoparticle size, high biocompatibility, sustained drug release, high drug loading efficiency (LE), and excellent stability13. In this work, we used a single-step sonication method to synthesize lipid-polymer nanoparticles. This method is fast, convenient and suitable for scale-up and has been widely used to prepare lipid-polymer nanoparticles by our group11,14 and others15,16,17,18.
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is a popular carbodiimide used as a crosslinking agent for conjugating biomolecules containing amines and carboxylates19. In addition to EDC, N-hydroxysulfosuccinimide (NHS) is the most common conjugation reagent in surface and nanoparticle conjugation reactions20,21. NHS can reduce the number of side reactions and enhance the stability and yield of ester intermediates22,23.
Here, we describe a protocol for synthesizing plCSA-targeted lipid-polymer nanoparticles. First, the single-step sonication synthesis of DOX-loaded lipid-polymer nanoparticles (DNPs) is described. Then, an EDC/NHS bioconjugate technique for generating plCSA-BP-conjugated lipid-polymer nanoparticles is introduced. This bioconjugate technique can also be used to conjugate other antibodies and peptides to nanoparticles. Finally, we describe the physicochemical properties and in vitro assay used to characterize the plCSA-targeted lipid-polymer nanoparticles. We believe that these plCSA-targeted lipid-polymer nanoparticles could constitute an effective system for the targeted delivery of drugs to most human cancers and the targeted delivery of payloads to the placenta to treat placental disorders.

<table>
	<tbody>
		<tr>
			<td>plCSA peptide</td>
			<td>Shanghai GL Biochem</td>
			<td>573518</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Sinopharm Chemical</td>
			<td>10009218</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Soybean lecithin</td>
			<td>Avanti Polar Lipids</td>
			<td>441601</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>DSPE-PEG-COOH</td>
			<td>Avanti Polar Lipids</td>
			<td>880125</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Doxorubicin</td>
			<td>JKChemical</td>
			<td>113424</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Shanghai Lingfeng</td>
			<td>1008621</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>PLGA</td>
			<td>Sigma-Aldrich</td>
			<td>719897</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Ultrasonic processor</td>
			<td>Sonics</td>
			<td>VCX130</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Centrifuge filter (MWCO 10 kDa)</td>
			<td>Millipore</td>
			<td>UFC801024</td>
			<td>for nanoparticles purification</td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Sigma</td>
			<td>3-18KS</td>
			<td>for nanoparticles purification</td>
		</tr>
		<tr>
			<td>2-[morpholino]ethanesulfonic acid(MES)</td>
			<td>Sigma-Aldrich</td>
			<td>M3671</td>
			<td>for peptide conjugation</td>
		</tr>
		<tr>
			<td>1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)</td>
			<td>Sigma-Aldrich</td>
			<td>3450</td>
			<td>for peptide conjugation</td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide (NHS)</td>
			<td>Sigma-Aldrich</td>
			<td>56480</td>
			<td>for peptide conjugation</td>
		</tr>
		<tr>
			<td>Dialysis bags</td>
			<td>Spectrum</td>
			<td>132592T</td>
			<td>for nanoparticles purification</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Hyclone</td>
			<td>SH30028.01</td>
			<td>for cell culture</td>
		</tr>
		<tr>
			<td>10 mL centrifuge tubes, polypropylene</td>
			<td>Aladdin</td>
			<td>S-025</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes, polypropylene</td>
			<td>Corning</td>
			<td>430791</td>
			<td>for various applications</td>
		</tr>
		<tr>
			<td>0.22 &#956;m sterile syringe filter</td>
			<td>Millipore</td>
			<td>SLGV033RB</td>
			<td>for nanoparticles purification</td>
		</tr>
		<tr>
			<td>1 ml syringe, polypropylene</td>
			<td>BD</td>
			<td>328421</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Malvern Zetasizer</td>
			<td>Malvern</td>
			<td>Nano ZS</td>
			<td>for particle size analyer</td>
		</tr>
		<tr>
			<td>Phosphotungstic acid</td>
			<td></td>
			<td></td>
			<td>for TEM</td>
		</tr>
		<tr>
			<td>TEM grid</td>
			<td>EMCN</td>
			<td>BZ10024a</td>
			<td>for TEM</td>
		</tr>
		<tr>
			<td>UV-VIS spectrometer</td>
			<td>Leagene</td>
			<td>DZ0035</td>
			<td>for TEM</td>
		</tr>
		<tr>
			<td>Transmission 
			electron microscope</td>
			<td>JEOL</td>
			<td>JEM-100CXII</td>
			<td>for particle size analyer</td>
		</tr>
		<tr>
			<td>BCA reagent A</td>
			<td>Thermo Fisher Scientific</td>
			<td>23228</td>
			<td>for BCA assay</td>
		</tr>
		<tr>
			<td>BCA reagent B</td>
			<td>Thermo Fisher Scientific</td>
			<td>23224</td>
			<td>for BCA assay</td>
		</tr>
		<tr>
			<td>96-Well Plates</td>
			<td>Corning</td>
			<td>3599</td>
			<td>for BCA assay</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Thermo Fisher Scientific</td>
			<td>Multiskan&#8482; GO</td>
			<td>for BCA assay</td>
		</tr>
		<tr>
			<td>12-well plates</td>
			<td>Corning</td>
			<td>3513</td>
			<td>for cell culture</td>
		</tr>
		<tr>
			<td>JEG3 cell</td>
			<td>Cell Bank of the Chinese Academy of Sciences</td>
			<td>TCHu195</td>
			<td>Human placenta</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Hyclone</td>
			<td>SH30272.01</td>
			<td>phenol red-free</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>GIBCO</td>
			<td>10100</td>
			<td>for cell culture</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>GIBCO</td>
			<td>15070063</td>
			<td>for cell culture</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>OLYMPUS</td>
			<td>CKK53</td>
			<td>for celluar uptake</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Shanghai Lingfeng</td>
			<td>1372021</td>
			<td>for celluar uptake</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sangon Biotech</td>
			<td>A606584</td>
			<td>for celluar uptake</td>
		</tr>
		<tr>
			<td>Mounting medium</td>
			<td>Life</td>
			<td>P36961</td>
			<td>for celluar uptake</td>
		</tr>
	</tbody>
</table>

synthesis and characterization of placental chondroitin sulfate a (plcsa)-targeting lipid-polymer nanoparticles

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising approach to cancer therapy. The glycosaminoglycan placental chondroitin sulfate A (plCSA) is expressed on a wide range of cancer cells and placental trophoblasts, and malarial protein VAR2CSA can specifically bind to plCSA. A reported placental chondroitin sulfate A binding peptide (plCSA-BP), derived from malarial protein VAR2CSA, can also specifically bind to plCSA on cancer cells and placental trophoblasts. Hence, plCSA-BP-conjugated nanoparticles could be used as a tool for targeted drug delivery to human cancers and placental trophoblasts. In this protocol, we describe a method to synthesize plCSA-BP-conjugated lipid-polymer nanoparticles loaded with doxorubicin (plCSA-DNPs); the method consists of a single sonication step and bioconjugate techniques. In addition, several methods for characterizing plCSA-DNPs, including determining their physicochemical properties and cellular uptake by placental choriocarcinoma (JEG3) cells, are described.

In this protocol, PLGA, DSPE-PEG-COOH and soybean lecithin are a representative polymer, lipid-PEG-COOH conjugate and lipid, respectively. The synthesis of plCSA-targeted lipid-polymer nanoparticles via a single-step sonication method and an EDC/NHS technique is illustrated in Figure 1. First, under sonication conditions, soybean lecithin, PLGA and DSPE-PEG-COOH self-assemble to form core-shell structured DNPs. The core consists of PLGA and encapsula...

Watch this Scientific Journal Video about Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles at JoVE.com

Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles

Here, we present a protocol for the synthesis of placental chondroitin sulfate A binding peptide (plCSA-BP)-conjugated lipid-polymer nanoparticles via single-step sonication and bioconjugate techniques. These particles constitute a novel tool for the targeted delivery of therapeutics to most human tumors and placental trophoblasts to treat cancers and placental disorders.

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising ...

This method can help answer key questions in the drug delivery field, such as targeted delivery of therapeutics to human cancers and the placenta cystoblast. The main advantage of this technique is that it is a simple and reproducible method to synthesize peptides conjugated in other particles. To begin, add three millimeters of four percent ethanol to a sterile 10 milliliter centrifuge tube.Then, add 90 micrograms of soybean lecithin stock solution, 210 micrograms of DSPE-PEG-carboxylic acid stock solution, and 750 micrograms of DOX stock solution. Use a one milliliter syringe to pipette two milligrams of PLGA stock solution. Place the centrifuge tube in an ice bath on an ultrasonic processor and sonicate for two minutes.Then, add PLGA stock solution drop-wise into the centrifuge tube. To purify the DNPs, use a centrifuge filter to wash the solution in 0.1 molar MES buffer. Then, centrifuge the solution at 1000 G for three minutes at four degrees Celsius.To begin ester activation, add 0.4 milligrams of EDC to one milliliter of DNPs. Then, add 0.24 milligrams of NHS to the reaction. Then, allow the reaction to occur for up to one hour at room temperature in the dark.After this, mix the reaction components thoroughly and place the reaction on a shaker. To start the amine reaction, use PBS to increase the buffer pH from 7.2 to 7.5. Then, add 0.5 milligrams of pICSA targeting peptide to the reaction solution.Mix the solution well and place it on a shaker. Then, allow the reaction to proceed at four degrees Celsius overnight in the dark. The next day, fill dialysis bags with the conjugate solution.Purify the pICSA DNPs with PBS buffer at room temperature for 24 hours in the dark. After culturing cells according to the text protocol, remove the media and add one milliliter of fresh, cold media with five percent FBS and pICSA DNPs or DNPs. Then, incubate the cell and nano-particle mixture for one hour at four degrees Celsius.After this, remove the media and wash the cells three times with PBS. Then, add one milliliter of fresh media and incubate the cells for 30 minutes at 37 degrees Celsius. After this, remove the media and wash the cells three times with PBS.Then, add two milliliters of cold, four percent PFA and incubate the mixture at room temperature for 10 minutes. Remove the PFA and wash the cells with two milliliters of PBS. Then, add one milliliter of PBS with DAPI for nuclei staining.Allow the mixture to incubate at room temperature for 10 minutes. Finally, add mounting medium and image the fluorescence with a fluorescence microscope. Using this protocol, DNPs were successfully prepared and conjugated to pICSA BP.After conjugation, the diameter of the primary DNPs increased to approximately 109 nanometers.The TEM images of the DNPs and the pICSA DNPs showed that the particles were well dispersed and generally demonstrated spherical morphology. The zeta potential of the nano-particles was negative 20.1 and 29.9 millivolts, indicating that the nano-particles were highly stable. The JEG3 cellular uptake assay indicated the pICSA DNPs bound to the JEG3 cells within 30 minutes.Therefore, the pICSA BP was efficiently conjugated to the surface. While attempting this procedure, it's important to remember to sonicate the mixture for one to two minutes before adding PLGA. Following this procedure, our methods like HPLC assay can be performed in order to determine the conjugation efficiency more accurately.After its development, this technique paved the way for researchers in the field of reproductive biology to explore the possibility of placenta-targeting treatment in pregnancy complications.

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