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 (LDLRs) is a prognostic indicator of metastatic potential⁷. Inspired by the LDL and its uptake by cancer cells, a new strategy has been called: Make the drug look like the cancer's food⁸. Thus, these new nanoparticle drug delivery designs^8,9,10 have been inspired by the core- and lipid-stabilized design of the natural LDLs¹¹ as a mechanism for delivering anticancer drugs to cancer cells. This passive targeting delivery system supports the encapsulating of, especially, hydrophobic drugs, which are usually given in oral dosage form but provide only a small amount of the drugs to the bloodstream, so limiting their expected efficacy¹². As with the stealth liposomes¹³, a polyethylene glycol (PEG) coating helps to reduce any immunologic response and extends the circulation in the bloodstream for optimum tumor uptake by the purported enhanced permeation and retention (EPR) effect^14,15. However, in addition to, in some instances, instability in the circulation and undesirable distribution in the system¹⁶, some obstacles remain unsolved, such as how and to what extent such nanoparticles are taken in by cells and what is their intracellular fate. It is here that this paper addresses the nanoparticle uptake of a particular hydrophobic anticancer drug falcarindiol, using confocal and epifluorescence imaging techniques.

The aim of the study is to fabricate lipid-coated nanoparticles of falcarindiol and to study their intracellular uptake in hMSCs. Thereby, potentially stabilizing its administration, overcoming the challenges associated with the delivery, and improving the bioavailability. Thus assessing a new delivery system for this anticancer drug. Previously, falcarindiol has been administrated orally via a high concentration purified falcarindiol as a food supplement¹⁷. However, there is need for a more structured approach to deliver this promising drug. Therefore, falcarindiol nanoparticles, with a phospholipid and cholesterol encapsulating monolayer with the purified drug constituting the core of the particle, were designed. The rapid injection method of solvent shifting, as recently developed by Needham et al.⁸, is used in this study to encapsulate the polyacetylene falcarindiol.

The method has previously been used for the fabrication of lipid nanoparticles to encapsulate diagnostic imaging agents^18,19, as well as test molecules (triolein)²⁷ and drugs (orlistat, niclosamide stearate)^8,27,28. It is a relatively simple technique when carried out with the right molecules. It forms nanosized particles, at the limit of their critical nucleation (~20 nm diameter), of highly insoluble hydrophobic solutes dissolved in a polar solvent. The solvent exchange is accomplished by a rapid injection of the organic solution into an excess of antisolvent (usually, an aqueous phase in a 1:9 organic:aqueous volume ratio)^20,21.

The compositional design of the nanoparticles give rise to multiple advantages. The DSPC:Chol components provide a very tight, almost impermeable, biocompatible, and biodegradable monolayer.The PEG provides a sterically stabilizing interface which acts as a shield from opsonization by the immune system, slowing any uptake by the reticuloendothelial system (liver and spleen) and protecting against the mononuclear phagocyte system, preventing their retention and degradation by the immune system, and hence, increasing their circulation half-time in blood²². This allows the particles to circulate until they extravasate at diseased sites, such as tumors, where the vascular system is leaky, allowing EPR-effect to give rise to passive accumulation of the particles. Additionally, the lipid coat allows one to have better control over the nanoparticles' size by kinetically trapping the core at its critical nucleus dimension^27,28. Lipids induce various surface properties (including peptide targeting, which was not yet available for this project), a pure drug core, and a low polydispersity^22,27,28. The method used for particle size analysis is DLS, a technique that allows researchers to measure the size of a large number of particles at the same time. However, this method can bias the measurements to bigger sizes, if the nanoparticles are not monodispersed²³. This issue is assessed with the lipid coat as well. More details of these fundamental designs and the quantification of all characteristics are given in other publications^27,28.

The drug encapsulated in the nanoparticles is falcarindiol, a dietary polyacetylene found in plants from the Apiaceae family. It is a secondary metabolite from the aliphatic C₁₇polyacetylenes type that has been found to display health-promoting effects, including anti-inflammatory activity, antibacterial effects, and cytotoxicity against a wide range of cancer cell lines. Its high reactivity is related to its ability to interact with different biomolecules, acting as a very reactive alkylating agent against mercapto and amino groups²⁴. Falcarindiol has previously been shown to reduce the number of neoplastic lesions in the colon^17,25, although the biological mechanisms are still unknown. However, it is thought that it interacts with biomolecules such as NF-κB, COX1, COX-2, and cytokines, inhibiting their tumor progression and cell proliferation processes, leading to arresting the cell cycle, endoplasmic reticulum (ER) stress, and apoptosis^17,26 in cancer cells. Falcarindiol is used in this study as an example anticancer drug due to its anticancer potential and mechanism are being studied currently, and because it shows promising anticancer effects. The cellular uptake of the nanoparticles is tested in hMSCs and imaged using epifluorescence and confocal microscopy. This cell type was chosen due to its large size, making them ideal for microscopy.

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.
   1. Insert the scintillation vial in the block heater; the sample concentrator delivers gas over the sample using stainless steel needles, concentrating the sample. Evaporate at room temperature; do not use heat.
4. Once dried, add the following components of the lipid coating into the above-mentioned scintillation vial: 16.3 µL of 31.64 mM DSPC chloroform stock solution, 3.4 µL of 17.82 mM DSPE PEG 2000 chloroform stock solution, 24 µL of 25 mM cholesterol chloroform stock solution, and 6 µL of 4 mM DiI chloroform stock solution. Clean the syringe with chloroform after adding each component to avoid cross contamination. 
   CAUTION: Immediately close the vials containing the lipids so that the solvent does not evaporate and, thereby, modify the concentration. Work in a fume hood.
   NOTE: The concentrations of chloroform stock solutions can vary, depending upon the chemical supplier or dilutions made in the lab.
5. Wrap the vial with aluminum foil to protect DiI from light. Leave the sample overnight in the desiccator to evaporate the chloroform.
6. Dissolve the desiccated sample in absolute ethanol to a final volume of 1.2 mL, which gives final concentrations of DSPC, DSPE PEG 2000, cholesterol, and DiI of 0.43 mM, 0.05 mM, 0.5 mM, and 0.02 mM, respectively. This solution represents the organic phase.
7. Take the 12 mL glass vial, fill it with 9 mL of purified water and, add the magnetic flea into the vial containing 9 mL of water. Keep the vial on the magnetic stirrer, stirring at 500 rpm (Figure 1).
8. Attach the 1 mL glass syringe to the dispensing system and clean it with chloroform to avoid any contamination. This by, slowly pulling the chloroform into the glass syringe and dispensing manually into a waste collector  at least 10 tiems.
   CAUTION: This must be done under a fume hood.
9. Prime the syringe with ethanol. Priming replaces the old solvent, as well as removes any air bubbles.
   CAUTION: This must be done under a fume hood.
10. Using the syringe, aspirate 1 mL of the organic phase.
11. Insert the syringe into the glass vial, up to the middle of the 9 mL watermark, and maintain it steady in the middle of the vial (as shown in Figure 1).
12. Inject the solution at the selected speed of injection (833 µL/s) by pressing the dispense button on the dispensing system (Figure 2). This generates 10 mL of 50 µM lipid-coated nanoparticles of falcarindiol in 10% ethanol-containing water.
    NOTE: This injection speed has been found to achieve the finest particles, obtaining a narrow particle size distribution. It is critical to make sure that the syringe is in the center, steady, and straight when dispensing the solution.
13. Immediately after the injection, remove the vial from the stirrer and transfer the sample to a 50 mL round-bottom flask (RBF).
14. Attach the RBF to the rotary evaporator and evaporate 1 mL of the organic solvent, using the rotary evaporator at room temperature. Avoid excess bubble formation.
    NOTE: This step will take ~5 min.
15. Transfer the nanoparticle suspension from the RBF to another 12 mL glass vial. Ensure that the volume is 9 mL. Split the sample in two 12 mL glass vials (put 4.5 mL in each).
16. Add 0.5 mL of ultrapure water to one of the vials and 0.5 mL of 10x phosphate-buffered saline (PBS) to the other vial. Take out 1 mL of each sample for the particle size measurement.

2. Particle size analysis using the DLS technique

NOTE: Size measurements were carried out by using a DLS analyzer which determines particle size distributions. It is equipped with a 100 mW laser that operates at a wavelength of 662.2 nm and with an avalanche photodiode detector placed at a 90° angle to the incident angle. The beam is scattered by the nanoparticles and detected by the photodetector.

1. Turn on the DLS instrument and set the desired temperature at 20 °C, until it stabilizes.
2. Set the instrument parameters as follows: data acquisition time = 4 s, number of acquisitions = 30, auto-attenuation function = On, and the auto-attenuation time limits = 0.
3. Fill the plastic cuvette with 1 mL of nanoparticle suspension and start the measurement.
4. Report the measured size depending on the solvent used (water or PBS).
   NOTE: The measurement in PBS is made to have an approximate idea of the size of the cells in the medium when treating the cells. The cell treatment will be done with the nanoparticles dissolved in water.
5. Repeat the measurements 24 h after the synthesis, to check for particle aggregation.

3. Cell treatment

1. Grow hMSCs in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, in a humified chamber at 37°C with 5% CO₂.
   CAUTION: Work in the sterile Laminar Flow Hood for steps 3.1, 3.2, and 3.3.
2. Seed approximately 50,000 cells to obtain a cell density of approximately 30% on previously absolute-EtOH-sterilized #1.5 coverslips placed in 6-well plates. Add MEM in order to have a final volume of 3 mL in each well. Incubate for 24 h under the same conditions as in step 3.1. Seed the cells 24 h before the treatment.
   NOTE: It is critical the cells are seeded at least 24 h before the nanoparticle treatment, to make sure that the cells are in an adequate confluence.
3. Without removing the medium, add 3 µL of the nanoparticle solution, for a final falcarindiol concentration of 5 µM. Incubate for 24 h in the same conditions as in step 3.1.
   NOTE: The nanoparticles' preparation was carried out on the day of the cell treatment, to avoid particle aggregation.
4. Subsequently, after 24 h of treatment, wash the cells 2x with PBS, fix them in 4% formaldehyde for 10 min at room temperature, and store them in PBS at 4°C for up to several months until imaged.
   CAUTION: This must be done under a fume hood.
   1. Alternatively, after fixation, a 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining can be performed. For this, after fixing the cells, permeabilize them with 0.1% Triton X-100 for 30 min, wash them 2x with PBS, and stain them with 250 µL of 300 nM DAPI for 5 minutes, protected from light.

4. Microscopy

1. Fluorescence microscopy
   1. Use a widefield fluorescence microscope equipped with an electron-multiplied CCD camera to acquire images. Use the 150x NA 1.45 oil objective and the GFP LP channel.
2. Confocal microscopy
   1. Acquire confocal microscopy images, using the 63x NA 1.4 oil objective, an Argon laser (514 nm) for DiI, and a two-photon laser (780 nm) for DAPI, to verify the uptake of the nanoparticles into the cells.

Representative 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, and so including the fluorescent dye, DiI, were of a similar size, namely 74.1 ± 6.7 nm; however, they were found to be relatively monodispersed and had a lower PDI of 0.182, which indicates a smaller distribution of particle sizes, since PDI describes the size distribution of the nanoparticle population. Generally, a PDI below 0.3 is desired when fabricating nanoparticles for pharmaceutical purposes.

Following the fabrication, the particle size was measured after 3 h and 24 h, times based on the delay required for the addition of nanoparticles to the cell culture. No aggregation was observed after 24 h, however  data is not shown in this manuscript as it will be reported in another study,  and it is recommended to test for particle stability after 24 h. After confirming the size stability of the lipid-coated nanoparticles, DiI-labeled, lipid-coated nanoparticles were fabricated by following the protocol and, eventually, used for the uptake study. For every study, a fresh nanoparticle sample was prepared. A schematic of the final falcarindiol nanoparticles’ structure is shown in Figure 3, and the particle’s size data after fabrication is shown in Table 1, as well as the measurement taken 3 h after fabrication.

As a first observation of the nanoparticles inside the cells, epifluorescence microscopy images were acquired after 24 h of treatment. The nanoparticles were visualized as white bright dots, and it could be hypothesized that nanoparticles were located inside the cells, surrounding the nucleus (Figure 4A).

To verify that falcarindiol nanoparticles had entered the cells, confocal microscopy was performed on hMSCs treated for 24 h. Confocal images confirmed that nanoparticles had entered the cells, and a large number of nanoparticles were scattered in the cytoplasm in every cell (Figure 4B-D). These results show that nanoparticles act as a stable drug delivery system for falcarindiol.

Figure 1: Nanoparticles preparation setup showing assembly for injection under stirring²⁷. The setup consists of the autopipette with a 1 mL glass syringe filled with 1 mL of the ethanolic solution containing the nanoparticles' components. The glass vial contains 9 mL of water and the magnetic flea is placed on the magnetic stirrer. Please click here to view a larger version of this figure.

Figure 2: Schematic of the mixing of solvents in the rapid injection method of solvent shifting²⁷. Panels show the injection of 1 mL of ethanolic phase containing nanoparticles' components at a speed of 833 µL/s into 9 mL of water while stirring at 500 rpm. The rapid injection with chaotic mixing of the ethanolic solution containing the nanoparticles' components (falcarindiol, DSPC, cholesterol, DSPE PEG 2000, DiI) into the antisolvent (water), leadd to the formation of the nanoparticles. The color is given by DiI. It can be observed how the ethanolic solution is mixed, rapidly increasing its concentration and the nanoparticles are formed. Please click here to view a larger version of this figure.

Figure 3: Schematic of the final falcarindiol nanoparticles' structure. Nanoparticle structure, including DSPC, DSPE PEG 2000, cholesterol, DiI, and falcarindiol. The different components are scaled according to their concentrations. Please click here to view a larger version of this figure.

Figure 4: Images of lipid-coated falcarindiol nanoparticles in human mesenchymal stem cells. (A) Epifluorescence microscopy image of hMSCs treated with falcarindiol nanoparticles for 24 h. The following panels show confocal microscopy images of the hMSCs treated with falcarindiol nanoparticles for 24 h: (B) DAPI stain of nuclei, (C) DiI nanoparticles, and (D) an overlay of both images, nuclei are shown in blue and the nanoparticles in red. The scale bars are 10 µm. Nanoparticles are visualized as white bright dots in the cell cytoplasm. The images show that a large number of nanoparticles have entered the cells after 24 h of incubation. Please click here to view a larger version of this figure.

Type of nanoparticles	Solvent	Nanoparticle size (nm)	Polydisperisty (nm)	Polydispersity Index (PDI)
Uncoated Falcarindiol	Water	83.9	± 23,9	0.571
	PBS	71	± 20,3	0.571
Lipid coated Falcarindiol	PBS	91.6	± 6,4	0.141
	PBS after 3h	93.5	± 5,7	0.122
Lipid coated Falcarindiol + DiI	PBS	74.1	± 6,7	0.182

Table 1: Different designs of the fabricated nanoparticles. Size and polydispersity index of the synthesized falcarindiol nanoparticles, depending on the solvent and nanoparticle type. Falcarindiol nanoparticles with and without a lipid coat were fabricated. Various concentrations of the coat components were tested. The particle aggregation was tested after 3 h.

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 obtained successfully. The possibility of induced polydispersity due to the involvement of turbulent mixing for the falcarindiol precipitation alone was controlled by the presence of coating lipids. The structure of these lipid-coated nanoparticles resembled the low-density lipoproteins with exception of the exclusion of the native 500,000 kDa ApoB100  and the additional presence of PEG-lipids for steric stability). This passively targeted drug delivery system allows the encapsulation of a broad range of especially hydrophobic drugs and diagnostic materials^18,19, reducing the immunologic response and increasing the accumulation in cancerous tissues^16,18. Furthermore, depending on the drug degradation reactions (e.g., hydrolysis, enzymolysis), the drug delivery system protects the drug from degradation during its circulation in vivo.

Therefore, falcarindiol nanoparticles, with a lipid-encapsulating monolayer containing a pure core of the purified drug, were designed and fabricated. The lipid monolayer coating consisted of DSPC, cholesterol, and DSPE-PEG 2000, with the fluorescent label DiI. The fabrication of the particles was carried out using the rapid injection method of solvent shifting, which consists of a rapid injection of an ethanolic solution containing the nanoparticles components into an excess of aqueous phase (1:9). The size of the nanoparticles was measured using DLS, and the uptake of the nanoparticles was examined in hMSCs and imaged using fluorescence and confocal microscopy.

Uncoated nanoparticles can also be obtained, with the sizes of 71 ± 20.3 nm. However, after following the protocol described above, nanoparticles of 74.1 nm ± 6.7 nm, with polydispersity values of 0.182, were fabricated. Thus, after modifying the nanoparticles by adding the lipid coat, the size and PDI of the nanoparticles was reduced, , making them more suitable for drug delivery.

It is important to be highly aware of the critical steps in the protocol, such as the importance of the syringe position when injecting the organic phase, the preparation of the nanoparticles on the same day of treatment to avoid aggregation, and the seeding of the cells the day before to ensure adequate confluence level. In fact, all the steps in the first part of the protocol can be considered critical as they either affect the size of the nanoparticles or the final concentration of the active compound and coating lipids. Considering ‘concentration’ as an important parameter, steps 1.2, 1.4, 1.6, 1.15, and 1.16 are critical. Considering ‘nanoparticle size’ as an important parameter, steps 1.7, 1.11, and 1.12 are critical.

Fluorescence and confocal microscopy showed that nanoparticles had entered the cells, and a large number of nanoparticles were scattered in the cytoplasm of every cell. These results suggest that nanoparticles designed in this study can function as a new, and stable drug delivery system for falcarindiol.

This technique provides a simple, fast, and reproducible approach to encapsulate different cancer drugs, and the limitations of the method are assessed with the lipid coat.

Materials

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