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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Cancer Research

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

Published: September 18th, 2018

DOI:

10.3791/58209

1Laboratory for Reproductive Health, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS), 2Guangdong Key Laboratory of Nanomedicine, CAS Key Lab for Health Informatics, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS), 3Department of Chemistry, Guangdong Medical University
* These authors contributed equally

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.

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.

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 per....

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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 solut.......

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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.......

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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.......

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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.

....

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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

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