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

Summary

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.

Abstract

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.

Introduction

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 effects^1,2. 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 applications^4,5,6. 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 plCSA^9,10. 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 group^11,14 and others^15,16,17,18.

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 reactions^20,21. NHS can reduce the number of side reactions and enhance the stability and yield of ester intermediates^22,23.

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.

Protocol

1. Preparation of Stock Solutions

1. 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 °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 mΩ·cm, which means low anionic contamination.
2. 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 °C.
3. 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 °C.
4. 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 °C in the dark.
5. 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 °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.
6. 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 °C.

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 method^11,13,14.

1. Add 3 mL of the aqueous solution of 4% ethanol to a sterile 10 mL centrifuge tube. Then, add 90 µg of the soybean lecithin stock solution, 210 µg of the DSPE-PEG-COOH stock solution and 750 µg of the DOX stock solution to 3 mL of the aqueous solution of 4% ethanol.
2. Place the centrifuge tube into an ice bath, and place the ice bath on an ultrasonic processor.
3. 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.
4. 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 °C and 1000 × 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°C in the dark.

3. Conjugation of Peptides to DNPs

1. Ester activation
   1. Add 0.4 mg of EDC (final concentration 2 mM) to 1 mL of DNPs.
   2. 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.
   3. 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.
2. Amine reaction
   1. Increase the buffer pH to 7.2-7.5 using PBS (20×, pH 7.4).
   2. 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.
   3. Mix the solution well, and then place on a shaker; allow the reaction to proceed at 4 °C overnight in the dark.
   4. 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.
   5. For cell culture applications, filter the reconstituted solution through a 0.22 µm sterile syringe filter to remove potential precipitates.

4. Characterization of plCSA-Targeted Lipid-Polymer Nanoparticles

1. Measurement of the hydrodynamic nanoparticle size using dynamic light scattering (DLS)
   1. Dilute nanoparticles with ultrapure water (50-fold dilution). Load 500 µL 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's specifications.
   2. 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.
2. Transmission electron microscopy (TEM)
   NOTE: The morphology of the nanoparticles was observed by TEM with the negative stain method.²⁴
   1. Dilute nanoparticles with ultrapure water (400-fold dilution). Add 20 µL of the sample onto a TEM grid, and allow to sit for 5 min.
   2. Add 100 µL of 2% (w/v) phosphotungstic acid and allow to sit for 2 min. Wick away the droplet.
   3. Dry the TEM grid at room temperature.
   4. Set the TEM acceleration voltage at 80 kV, and magnify the image to 100,000× to visualize the nanoparticles.
3. Determination of the encapsulation efficiency (EE) and LE
   1. Standard curve generation. Dissolve DOX in ultrapure water to prepare DOX solutions of five different concentrations: 1 µg/mL, 5 µg/mL, 10 µg/mL, 50 µg/mL, and 100 µg/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.
   2. Dilute 25 µL of nanoparticles with 500 µL of ultrapure water. Measure the absorption at 480 nm with a UV-VIS spectrometer. Calculate the drug concentrations by the standard curve.
   3. Calculate the LE using the following equation:
      LE=((amount of drugs in nanoparticles)/(total weight of materials))×100%.
   4. Calculate the EE using the following equation:
      EE=((amount of drugs in nanoparticles)/(amount of added drug))×100%.
4. Measurement of the conjugation efficiency using the bicinchoninic acid (BCA) assay^25,26,27
   1. Pipette 25 µL of standard peptide solutions (Table 1) or plCSA-DNPs into microplate wells in duplicate. Add 200 µL of the working reagent to each well, and mix the plate well on a plate shaker for 30 s.
   2. Cover the plate, and incubate at 37 °C for 30 min.
   3. Measure the absorbance at 562 nm on a plate reader. Use the generated standard curve to calculate the plCSA concentrations of plCSA-DNPs.
   4. Calculate the conjugation efficiency using the following equation:
      Conjugation efficiency=((amount of peptide in nanoparticles)/(amount of added peptide))×100%.

Vial	Volume of diluent (μL)	Volume and source of peptide (μL)	Final peptide concentration (μg/mL)
A	0	300 of Stock	1000
B	250	250 of vial A dilution	500
C	250	250 of vial B dilution	250
D	250	250 of vial C dilution	125
E	300	200 of vial D dilution	50
F	250	250 of vial E dilution	25
G	400	100 of vial F dilution	5
H	500	0	0

Table 1. Preparation of Standard peptides

5. Fluorescence Microscopy Assessment of plCSA-Targeted Nanoparticle Uptake in Choriocarcinoma (JEG3) Cells

1. Seed cells onto sterile 12-well plates at 1.0×10⁴ 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 °C and 5% CO₂ under humid condition.
2. Remove the media, and add 1 mL of cold fresh media with a low serum content (5% FBS) and DNPs or plCSA-DNPs (5 µg of DOX equivalent).
3. Incubate the mixture of cells and nanoparticles at 4 °C for 1 h.
4. 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 °C for 30 min.
5. Remove the cDMEM/F12, and wash the cells three times with PBS.
6. Add 2 mL of cold 4% paraformaldehyde (PFA), and incubate at room temperature for 15 min to fix the cells.
7. Remove the PFA. Wash the cells with 2 mL of PBS once. Add 1 mL of PBS containing DAPI (1 µg/mL) for nuclei staining, and incubate at room temperature for 10 min.
8. Aspirate the PBS, and wash the cells three times with PBS.
9. Add mounting medium, and image the fluorescence with a fluorescence microscopy, using green and blue channels to visualize the DOX and nuclei, respectively.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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