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 solution by dissolving 20 mg of soybean lecithin in 20 mL of the aqueous solution of 4% ethanol. Store the soybean lecithin stock solution at 4 &#176;C.</li>
	<li>Prepare a 25 mg/mL DSPE-PEG (2000)-COOH stock solution by dissolving 100 mg of DSPE-PEG-COOH in 4 mL of the aqueous solution of 4% ethanol. Store the stock solution at -20 &#176;C.</li>
	<li>Prepare a 10 mg/mL DOX stock solution by dissolving 50 mg of DOX in 5 mL of ultrapure water. Store the DOX stock solution at 4 &#176;C in the dark.</li>
	<li>Prepare a 2 mg/mL PLGA stock solution by dissolving 20 mg of PLGA in 10 mL of acetonitrile. Store the PLGA stock solution at 4 &#176;C. 
	CAUTION: Acetonitrile is flammable and toxic. Operate with care in a fume hood, and wear appropriate personal equipment, such as lab coats, safety glasses and latex gloves.</li>
	<li>Prepare a 0.1 M 2-(morpholino) ethanesulfonic acid (MES, pH 6.0) stock solution by dissolving 2.17 g of MES in 100 mL of ultrapure water. Store the stock solution at 4 &#176;C.</li>
</ol>
2. Synthesis of DNPs
NOTE: To avoid DOX photochemical degradation, all operations were performed in the dark. 
Nanoparticles were synthesized by a previously reported single-step sonication method11,13,14.
<ol>
	<li>Add 3 mL of the aqueous solution of 4% ethanol to a sterile 10 mL centrifuge tube. Then, add 90 &#181;g of the soybean lecithin stock solution, 210 &#181;g of the DSPE-PEG-COOH stock solution and 750 &#181;g of the DOX stock solution to 3 mL of the aqueous solution of 4% ethanol.</li>
	<li>Place the centrifuge tube into an ice bath, and place the ice bath on an ultrasonic processor.</li>
	<li>With a 1 mL syringe, pipette 2 mg of the PLGA stock solution dropwise (1 drop/4-6 s) into the centrifuge tube. Meanwhile, sonicate the tube using an ultrasonic processor at a frequency of 20 kHz and an output amplitude of 30% for 5 min to synthesize the DNPs. 
	NOTE: To synthesize uniform particles with small sizes, the speed at which the PLGA solution is dripped into the tube needs to be slow, and bubble generation should be avoided.</li>
	<li>Purify the DNPs by washing the above solution in 0.1 M MES buffer (pH 6.0) 3 times using a centrifuge filter (MWCO, 10 kDa). Centrifuge at 4 &#176;C and 1000 &#215; g for 3 min each time. Finally, approximately 1 mL of MES-solved nanoparticles should remain. 
	NOTE: This is an acceptable stopping point in the procedure. If not used to conjugate peptides, the nanoparticles can be purified by PBS buffer (pH 7.4), and the purified DNPs can be stored at 4&#176;C in the dark.</li>
</ol>
3. Conjugation of Peptides to DNPs
<ol>
	<li>Ester activation
	<ol>
		<li>Add 0.4 mg of EDC (final concentration 2 mM) to 1 mL of DNPs.</li>
		<li>Add 0.24 mg of NHS to the reaction (final concentration 2 mM). 
		NOTE: For easy addition of the correct quantity of EDC and NHS, a stock solution may be prepared if the reagents are dissolved and used immediately.</li>
		<li>Mix the reaction components well, and place the reaction on a shaker; allow reaction for 30 min-1 h at room temperature in the dark.</li>
	</ol>
	</li>
	<li>Amine reaction
	<ol>
		<li>Increase the buffer pH to 7.2-7.5 using PBS (20&#215;, pH 7.4).</li>
		<li>Add 0.5 mg of the plCSA-targeting peptide (plCSA-BP, EDVKDINFDTKEKFLAGCLIVSFHEGKC) to the reaction solution. 
		NOTE: Before addition, dissolve the peptides in 20% acetonitrile. If peptides are not dissolvable, sonication in a bath sonicator may helpful.</li>
		<li>Mix the solution well, and then place on a shaker; allow the reaction to proceed at 4 &#176;C overnight in the dark.</li>
		<li>Place conjugate solution into dialysis bags (MWCO, 3,500 Da) to dialyze and purify the plCSA-DNPs using PBS (pH 7.4) buffer at room temperature for 24 h in the dark. 
		NOTE: Alternatively, purification could be performed as in step 2.4 to obtain plCSA-DNPs.</li>
		<li>For cell culture applications, filter the reconstituted solution through a 0.22 &#181;m sterile syringe filter to remove potential precipitates.</li>
	</ol>
	</li>
</ol>
4. Characterization of plCSA-Targeted Lipid-Polymer Nanoparticles
<ol>
	<li>Measurement of the hydrodynamic nanoparticle size using dynamic light scattering (DLS)
	<ol>
		<li>Dilute nanoparticles with ultrapure water (50-fold dilution). Load 500 &#181;L of the sample into a cuvette according to the instructions of the DLS or zeta potential instrument. 
		NOTE: Zeta potential and DLS cuvettes can differ based on the instrument&#39;s specifications.</li>
		<li>After the measurement is completed, record the particle diameter, polydispersity index (PDI) and zeta potential. Average the results obtained from 4 repeated readings, and calculate the standard deviation.</li>
	</ol>
	</li>
	<li>Transmission electron microscopy (TEM) 
	NOTE: The morphology of the nanoparticles was observed by TEM with the negative stain method.24
	<ol>
		<li>Dilute nanoparticles with ultrapure water (400-fold dilution). Add 20 &#181;L of the sample onto a TEM grid, and allow to sit for 5 min.</li>
		<li>Add 100 &#181;L of 2% (w/v) phosphotungstic acid and allow to sit for 2 min. Wick away the droplet.</li>
		<li>Dry the TEM grid at room temperature.</li>
		<li>Set the TEM acceleration voltage at 80 kV, and magnify the image to 100,000&#215; to visualize the nanoparticles.</li>
	</ol>
	</li>
	<li>Determination of the encapsulation efficiency (EE) and LE
	<ol>
		<li>Standard curve generation. Dissolve DOX in ultrapure water to prepare DOX solutions of five different concentrations: 1 &#181;g/mL, 5 &#181;g/mL, 10 &#181;g/mL, 50 &#181;g/mL, and 100 &#181;g/mL. Measure the absorption of the DOX solutions at 480 nm with a UV-VIS spectrometer. Generate a standard curve based on the DOX concentrations.</li>
		<li>Dilute 25 &#181;L of nanoparticles with 500 &#181;L of ultrapure water. Measure the absorption at 480 nm with a UV-VIS spectrometer. Calculate the drug concentrations by the standard curve.</li>
		<li>Calculate the LE using the following equation: 
		LE=((amount of drugs in nanoparticles)/(total weight of materials))&#215;100%.</li>
		<li>Calculate the EE using the following equation: 
		EE=((amount of drugs in nanoparticles)/(amount of added drug))&#215;100%.</li>
	</ol>
	</li>
	<li>Measurement of the conjugation efficiency using the bicinchoninic acid (BCA) assay25,26,27
	<ol>
		<li>Pipette 25 &#181;L of standard peptide solutions (Table 1) or plCSA-DNPs into microplate wells in duplicate. Add 200 &#181;L of the working reagent to each well, and mix the plate well on a plate shaker for 30 s.</li>
		<li>Cover the plate, and incubate at 37 &#176;C for 30 min.</li>
		<li>Measure the absorbance at 562 nm on a plate reader. Use the generated standard curve to calculate the plCSA concentrations of plCSA-DNPs.</li>
		<li>Calculate the conjugation efficiency using the following equation: 
		Conjugation efficiency=((amount of peptide in nanoparticles)/(amount of added peptide))&#215;100%.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vial</td>
			<td>Volume of diluent (&#956;L)</td>
			<td>Volume and source of peptide (&#956;L)</td>
			<td>Final peptide concentration (&#956;g/mL)</td>
		</tr>
		<tr>
			<td>A</td>
			<td>0</td>
			<td>300 of Stock</td>
			<td>1000</td>
		</tr>
		<tr>
			<td>B</td>
			<td>250</td>
			<td>250 of vial A dilution</td>
			<td>500</td>
		</tr>
		<tr>
			<td>C</td>
			<td>250</td>
			<td>250 of vial B dilution</td>
			<td>250</td>
		</tr>
		<tr>
			<td>D</td>
			<td>250</td>
			<td>250 of vial C dilution</td>
			<td>125</td>
		</tr>
		<tr>
			<td>E</td>
			<td>300</td>
			<td>200 of vial D dilution</td>
			<td>50</td>
		</tr>
		<tr>
			<td>F</td>
			<td>250</td>
			<td>250 of vial E dilution</td>
			<td>25</td>
		</tr>
		<tr>
			<td>G</td>
			<td>400</td>
			<td>100 of vial F dilution</td>
			<td>5</td>
		</tr>
		<tr>
			<td>H</td>
			<td>500</td>
			<td>0</td>
			<td>0</td>
		</tr>
	</tbody>
</table>
Table 1. Preparation of Standard peptides
5. Fluorescence Microscopy Assessment of plCSA-Targeted Nanoparticle Uptake in Choriocarcinoma (JEG3) Cells
<ol>
	<li>Seed cells onto sterile 12-well plates at 1.0&#215;104 cells/well with complete DMEM/F12 (cDMEM/F12, containing 1% penicillin/streptomycin and 10% fetal bovine serum (FBS)). Allow the cells to grow to 60% confluence at 37 &#176;C and 5% CO2 under humid condition.</li>
	<li>Remove the media, and add 1 mL of cold fresh media with a low serum content (5% FBS) and DNPs or plCSA-DNPs (5 &#181;g of DOX equivalent).</li>
	<li>Incubate the mixture of cells and nanoparticles at 4 &#176;C for 1 h.</li>
	<li>After incubation, remove the media, and wash the cells three times with PBS. Then, add 1 mL of fresh cDMEM/F12, and incubate the cells at 37 &#176;C for 30 min.</li>
	<li>Remove the cDMEM/F12, and wash the cells three times with PBS.</li>
	<li>Add 2 mL of cold 4% paraformaldehyde (PFA), and incubate at room temperature for 15 min to fix the cells.</li>
	<li>Remove the PFA. Wash the cells with 2 mL of PBS once. Add 1 mL of PBS containing DAPI (1 &#181;g/mL) for nuclei staining, and incubate at room temperature for 10 min.</li>
	<li>Aspirate the PBS, and wash the cells three times with PBS.</li>
	<li>Add mounting medium, and image the fluorescence with a fluorescence microscopy, using green and blue channels to visualize the DOX and nuclei, respectively.</li>
</ol>

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X.F. and B.Z. are the inventors on the patent PCT/CN2017/108646 and 201710906587.6 submitted by SIAT that covers a plCSA-targeted nanoparticle synthesis method and application. No potential conflicts of interest were disclosed by the other authors.

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 &amp;mu;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&lt;br/&gt; 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&amp;trade; 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 ...

synthesis-characterization-placental-chondroitin-sulfate-plcsa

Research

JoVE Journal

Cancer Research

8.7K Views.  Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS). Deze methode kan helpen bij het beantwoorden van belangrijke vragen in het gebied van de levering van geneesmiddelen, zoals gerichte levering van therapieën aan menselijke kankers en de placenta cystoblast. Het belangrijkste voordeel van deze techniek is dat het een eenvoudige en reproduceerbare methode is om peptiden geconjugeerd in andere deeltjes te synthetiseren. Om te beginnen, voeg drie millimeter van vier procent ethanol aan een steriele 10 milliliter centrifuge buis.Voeg vervo...

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

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

Bioengineering

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

Chemistry

Intra-lymph Node Injection of Biodegradable Polymer Particles

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g.&#160;size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.

Lymph nodes are the immunological tissues that orchestrate immune response and are a critical target for vaccines. Biomaterials have been employed to better target lymph nodes and to control delivery of antigens or adjuvants. This paper describes a technique combining these ideas to inject biocompatible polymer particles into lymph nodes.

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these ...

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices&#39; defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes.

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

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

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