Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Summary

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Abstract

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 ± 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

Introduction

Lipid-coated nanoparticles are seeing an increased interest regarding their function as drug delivery systems for cancer therapy¹. Cancers have an altered lipid-metabolic reprogramming² and an increased need for cholesterol to proliferate³. They overexpress LDLs¹ and take in more LDLs than normal cells, to the extent that a cancer patient's LDL count can even go down⁴. LDL uptake promotes aggressive phenotypes⁵ resulting in proliferation and invasion in breast cancer⁶. An abundance of LDL receptors ....

Protocol

1. Nanoparticle synthesis by rapid solvent shifting technique

1. Set up the following for the nanoparticles' preparation: a block heater/sample concentrator, a desiccator, a digital dispensing system with a 1 mL glass syringe, a 12 mL glass vial, a magnetic stirrer, a magnetic flea (15 mm x 4.5 mm, in a cylindrical shape with polytetrafluoroethylene [PTFE] coating) inside the glass vial, and a rotatory evaporator.
2. Dispense 2.4 mL of 250 µM falcarindiol stock dissolved in 70% EtOH water mixture in a scintillation vial.
3. Evaporate the liquid fraction, using the sample concentrator for approximately 4 h, to obtain dry falcarindiol. <....

Results

Two different types of nanoparticles were fabricated, namely pure falcarindiol nanoparticles and lipid-coated falcarindiol nanoparticles. Various concentrations of lipids and cholesterol were tested. As shown in Table 1, uncoated nanoparticles formed in water and measured in PBS had a diameter of 71 ± 20.3 nm with a polydispersity index (PDI) of 0.571. Those parameters were measured on a DLS analyzer. The lipid-coated nanoparticles of falcarindiol used in the experiments,.......

Discussion

A detailed protocol for fabricating lipid-coated nanoparticles for drug delivery with the simple, fast, reproducible, and scalable rapid injection method of solvent shifting was followed^27,28 and is presented in this paper, as applied to falcarindiol. By controlling the speed of the injection of the organic phase into aqueous phase and by using coating lipids at appropriate concentrations to coat the falcarindiol core, particle in the sub-100 nm range could be ob.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
12 mL Screw Neck Vial (Clear glass, 15-425 thread, 66 X 18.5 mm)	Microlab Aarhus A/S	ML 33154LP
6 well plates	Greiner Bio One International GmbH	657160
Absolute Ethanol	EMD Millipore (VWR)	EM8.18760.1000
Chloroform	Rathburn Chemicals Ltd.	RH1009
Cholesterol	Avanti Polar Lipids, Inc.	700000P
Confocal Microscope	Zeiss LSM510
Cover Slips thickness #1.5	Paul Marienfeld GmbH & Co	117650
Desiccator	Self-build
DiI	Invitrogen	D282
DLS	Beckman Coulter	DelsaMAXpro 3167-DMP
DSPC (Chloroform stock)	Avanti Polar Lipids, Inc.	850365C
DSPE PEG 2000 (Chloroform stock)	Avanti Polar Lipids, Inc.	880120C
eVol XR	SGE analytical science, Trajan Scientific Australia Pty Ltd.	2910200
Fetal Bovine serum	Gibco	10270-106
Fluorescence Miccroscope	Olymous IX81		With Manual TIRF and Andor iXon EMCCD
Incubator	Panasonic	MCO-18AC
Magnetic flea	VWR Chemicals	15 x 4.5 mm	Cylindrical shape with PTFE coating
Magnetic stirrer	IKA	RT-10
Minimum Essential Media	Gibco	32561-029
PBS tablets for cell culture	VWR Chemicals	97062-732
Pen/strep	VWR Chemicals	97063-708
Phosphate Buffer Saline (PBS, pH 7.4)	Thermo Fisher	10010031
Rotary Evaporator	Rotavapor, Büchi Labortechnik AG	R-210
Sample concentrator	Stuart, Cole-Parmer Instrument Company, LLC	SBHCONC/1