Name: uptake of new lipid-coated nanoparticles containing falcarindiol by human mesenchymal stem cells
Uploaded: 2019-02-09T14:00:00.000+00:00
Duration: 9 min 34 s
Description: Watch this Scientific Journal Video about Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells at JoVE.com

The authors thank Dr. Moustapha Kassem (Odense University Hospital, Denmark) for the human mesenchymal stem cells. The authors thank the Danish Medical Bioimaging Center for access to their microscopes. The authors thank the Carlsberg and Villum foundations for financial support (to E.A.C.). The authors acknowledge the financial support provided by the Niels Bohr Professorship award from the Danish National Research Foundation.

1. Nanoparticle synthesis by rapid solvent shifting technique
<ol>
	<li>Set up the following for the nanoparticles&#39; 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.</li>
	<li>Dispense 2.4 mL of 250 &#181;M falcarindiol stock dissolved in 70% EtOH water mixture in a scintillation vial.</li>
	<li>Evaporate the liquid fraction, using the sample concentrator for approximately 4 h, to obtain dry falcarindiol.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Once dried, add the following components of the lipid coating into the above-mentioned scintillation vial: 16.3 &#181;L of 31.64 mM DSPC chloroform stock solution, 3.4 &#181;L of 17.82 mM DSPE PEG 2000 chloroform stock solution, 24 &#181;L of 25 mM cholesterol chloroform stock solution, and 6 &#181;L of 4 mM DiI chloroform stock solution.&#160;Clean the syringe with chloroform after adding each component to avoid cross contamination.&#160; 
	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.</li>
	<li>Wrap the vial with aluminum foil to protect DiI from light.&#160;Leave the sample overnight in the desiccator to evaporate the chloroform.</li>
	<li>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.</li>
	<li>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.&#160;Keep the vial on the magnetic stirrer, stirring at 500 rpm (Figure 1).</li>
	<li>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&#160; at least 10 tiems. 
	CAUTION: This must be done under a fume hood.</li>
	<li>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.</li>
	<li>Using the syringe, aspirate 1 mL of the organic phase.</li>
	<li>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).</li>
	<li>Inject the solution at the selected speed of injection (833 &#181;L/s) by pressing the dispense button on the dispensing system (Figure 2). This generates 10 mL of 50 &#181;M 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.</li>
	<li>Immediately after the injection, remove the vial from the stirrer and transfer the sample to a 50 mL round-bottom flask (RBF).</li>
	<li>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.</li>
	<li>Transfer the nanoparticle suspension from the RBF to another 12 mL glass vial. Ensure that the volume is 9 mL.&#160;Split the sample in two 12 mL glass vials (put 4.5 mL in each).</li>
	<li>Add 0.5 mL of ultrapure water to one of the vials and 0.5 mL of 10x phosphate-buffered saline (PBS) to&#160;the other vial. Take out 1 mL of each sample for the particle size measurement.</li>
</ol>
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&#176; angle to the incident angle. The beam is scattered by the nanoparticles and detected by the photodetector.
<ol>
	<li>Turn on the DLS instrument and set the desired temperature at 20 &#176;C, until it stabilizes.</li>
	<li>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.</li>
	<li>Fill the plastic cuvette with 1 mL of nanoparticle suspension and start the measurement.</li>
	<li>Report the measured size depending on the&#160;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.</li>
	<li>Repeat the measurements 24 h after the synthesis, to check for particle aggregation.</li>
</ol>
3. Cell treatment
<ol>
	<li>Grow hMSCs in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, in a humified chamber at 37&#176;C with 5% CO2. 
	CAUTION: Work in the sterile Laminar Flow Hood for steps 3.1, 3.2, and 3.3.</li>
	<li>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.</li>
	<li>Without removing the medium, add 3 &#181;L of the nanoparticle solution, for a final falcarindiol concentration of 5 &#181;M. Incubate for 24 h in the same conditions as in step 3.1. 
	NOTE: The nanoparticles&#39; preparation was carried out on the day of the cell treatment, to avoid particle aggregation.</li>
	<li>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&#176;C for up to several months until imaged. 
	CAUTION: This must be done under a fume hood.
	<ol>
		<li>Alternatively, after fixation, a 4&#8242;,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 &#181;L of 300 nM DAPI for 5 minutes, protected from light.</li>
	</ol>
	</li>
</ol>
4. Microscopy
<ol>
	<li>Fluorescence microscopy
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Confocal microscopy
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 followed27,28 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&#160; 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 materials18,19, reducing the immunologic response and increasing the accumulation in cancerous tissues16,18. 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 &#177; 20.3 nm. However, after following the protocol described above, nanoparticles of 74.1 nm &#177; 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 &#8216;concentration&#8217; as an important parameter, steps 1.2, 1.4, 1.6, 1.15, and 1.16 are critical. Considering &#8216;nanoparticle size&#8217; 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.

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&#39;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&#39;s food8. Thus, these new nanoparticle drug delivery designs8,9,10&#160;have been inspired by the core- and lipid-stabilized design of the natural LDLs11&#160;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) effect14,15. 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.&#160;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&#160;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 agents18,19, 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&#160;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&#160;allows one to have better control over the nanoparticles&#39; size by kinetically trapping the core at its critical nucleus dimension27,28. Lipids induce various surface properties (including peptide targeting, which was not yet available for this project), a pure drug core, and a low polydispersity22,27,28. 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 publications27,28.
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 colon17,25, although the biological mechanisms are still unknown. However, it is thought that it interacts with biomolecules such as NF-&#954;B, COX1, COX-2, and cytokines, inhibiting their tumor progression and cell proliferation processes, leading to arresting the cell cycle, endoplasmic reticulum (ER) stress, and apoptosis17,26 in cancer cells. Falcarindiol is used in this study as an example anticancer drug due to&#160;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.

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

uptake of new lipid-coated nanoparticles containing falcarindiol by human mesenchymal stem cells

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&#160;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 &#177; 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.

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 &#177; 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 &#177; 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&#160; data is not shown in this manuscript as it will be reported in another study,&#160; 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&#8217; structure is shown in Figure 3, and the particle&#8217;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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59094/59094fig1.jpg" /> 
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&#39; components. The glass vial contains 9 mL of water and the magnetic flea is placed on the magnetic stirrer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59094/59094fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59094/59094fig2.jpg" /> 
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&#39; components at a speed of 833 &#181;L/s into 9 mL of water while stirring at 500 rpm. The rapid injection with chaotic mixing of the ethanolic solution containing the nanoparticles&#39; 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&#160;the nanoparticles are formed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59094/59094fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59094/59094fig3.jpg" /> 
Figure 3: Schematic of the final falcarindiol nanoparticles&#39; structure. Nanoparticle structure, including DSPC, DSPE PEG 2000, cholesterol, DiI, and falcarindiol. The different components are scaled according to their concentrations. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59094/59094fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59094/59094fig4.jpg" /> 
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 &#181;m. 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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59094/59094fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Type of nanoparticles</td>
			<td>Solvent</td>
			<td>Nanoparticle size (nm)</td>
			<td>Polydisperisty (nm)</td>
			<td>Polydispersity Index (PDI)</td>
		</tr>
		<tr>
			<td>Uncoated Falcarindiol</td>
			<td>Water</td>
			<td>83.9</td>
			<td>&#177; 23,9</td>
			<td>0.571</td>
		</tr>
		<tr>
			<td></td>
			<td>PBS</td>
			<td>71</td>
			<td>&#177; 20,3</td>
			<td>0.571</td>
		</tr>
		<tr>
			<td>Lipid coated Falcarindiol</td>
			<td>PBS</td>
			<td>91.6</td>
			<td>&#177; 6,4</td>
			<td>0.141</td>
		</tr>
		<tr>
			<td></td>
			<td>PBS after 3h</td>
			<td>93.5</td>
			<td>&#177; 5,7</td>
			<td>0.122</td>
		</tr>
		<tr>
			<td>Lipid coated Falcarindiol + DiI</td>
			<td>PBS</td>
			<td>74.1</td>
			<td>&#177; 6,7</td>
			<td>0.182</td>
		</tr>
	</tbody>
</table>
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.

Watch this Scientific Journal Video about Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells at JoVE.com

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

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;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.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

uptake-new-lipid-coated-nanoparticles-containing-falcarindiol-human

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.9K Views.  University of Southern Denmark. De fabricage van lipide-gecoate nanodeeltjes met de snelle injectiemethode biedt een stabiel systeem voor de levering van geneesmiddelen voor kankertherapie. Deze techniek is een eenvoudige, snelle, schaalbare en reproduceerbare techniek voor nanodeeltjesfabricage die hydrofobe geneesmiddelen inkapselt. Deze techniek is toepasbaar voor het fabriceren van diagnostische en therapeutische nanodeeltjes uit hydrofobe materialen die effectief kunnen worden gebruikt in verschillende aandoeningen.

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

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes.
A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages 1. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo.
Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles 2-4. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images 5. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM-1 sec-1 strength4. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure 6. Our group has applied this agent in an &ldquo;off label&rdquo; use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.

We describe a technique for labeling and tracking stem cells with FDA-approved, superparamagnetic iron oxide (SPIO), ferumoxytol (Feraheme). This cellular imaging technique that utilizes magnetic resonance (MR) imaging for visualization, is readily accessible for long-term monitoring and diagnosis of successful or unsuccessful stem cell engraftments in patients.

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

Medicine

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Genetics

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the efficacy of cell persistence and biodistribution of haMSCs in animal models. We demonstrated a method to label the cell membrane of haMSCs with lipophilic fluorescent dye. Subsequently, intra-articular injection of the labeled cells in rats with surgically induced KOA was monitored dynamically by an in vivo imaging system. We employed the lipophilic carbocyanines DiD (DilC18 (5)), a far-red fluorescent Dil (dialkylcarbocyanines) analog, which utilized a red laser to avoid excitation of the natural green autofluorescence from surrounding tissues. Furthermore, the red-shifted emission spectra of DiD allowed deep-tissue imaging in live animals and the labeling procedure caused no cytotoxic effects or functional damage to haMSCs. This approach has been shown to be an efficient tracking method for haMSCs in a rat KOA model. The application of this method could also be used to determine the optimal administration route and dosage of MSCs from other sources in pre-clinical studies.

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

This protocol describes an efficient way to monitor the cell persistence and biodistribution of human adipose-derived mesenchymal stem cells (haMSCs) by far-red fluorescence labeling in a rat knee osteoarthritis (KOA) model via intra-articular (IA) injection.

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

Developmental Biology

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 ...

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2.
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3.
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4.
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.

Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

Biology

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

The immunomodulatory properties of human mesenchymal stem cells (MSC) appear increasingly relevant for clinical application. Using a co-culture system of MSCs and peripheral blood leukocytes pre-stained with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), we describe the in vitro assessment of MSC immunomodulation on effector leukocyte proliferation and specific subpopulations.

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

Immunology and Infection

Ultracentrifuge-Based Isolation of Extracellular Vesicles from Human ADSCs

Purification and Characterization of Extracellular Vesicles from Human Adipose-Derived Mesenchymal Stem Cells

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research suggests that their regenerative function may be attributed to cell-cell contact and cell paracrine effects. The paracrine effect is an important way for cells to interact and transfer information over short distances, in which extracellular vesicles (EVs) play a functional role as carriers. There is significant potential for ADSC EVs in regenerative medicine. Multiple studies have reported on the effectiveness of these methods. Various methods for extracting and isolating EVs are currently described based on principles such as centrifugation, precipitation, molecular size, affinity, and microfluidics. Ultracentrifugation is regarded as the gold standard for isolating EVs. Nevertheless, a meticulous protocol to highlight precautions during ultracentrifugation is still absent. This study presents the methodology and crucial steps involved in ADSC culture, supernatant collection, and EV ultracentrifugation. However, even though ultracentrifugation is cost-effective and requires no further treatment, there are still some inevitable drawbacks, such as a low recovery rate and EV aggregation.

Author Spotlight: Standardizing and Improving the Extraction and Purification of Extracellular Vesicles from Human ADSCs

This protocol describes all procedures, from culturing human adipose-derived mesenchymal stem cells (ADSCs) and collecting supernatant to extracting extracellular vesicles (EVs) using ultracentrifugation.

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research ...

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs&#39; size was 30-200 nm, with a particle concentration of 7.75 &#215; 1010 particles/mL and a protein concentration of 80 &#181;g/mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).

This protocol demonstrates how to isolate human umbilical cord-derived mesenchymal stem cells&#39; small extracellular vesicles (hUC-MSC-sEVs) with a simple lab-scale benchtop setting. The size distribution, protein concentration, sEVs markers, and morphology of isolated hUC-MSC-sEVs are characterized by nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively.

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this ...

Functional Analysis of BAMBIhighMFGE8high UC-MSC Subpopulations

Isolation of Human BAMBIhighMFGE8high Umbilical Cord-Derived Mesenchymal Stromal Cells

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various diseases. Human UC-MSCs are a heterogeneous population consisting of three main subpopulations with different cell shapes, proliferation rates, differentiation abilities, and immune regulatory functions. Previously, BAMBIhighMFGE8high UC-MSCs, the first subgroup successfully isolated from UC-MSCs were found to fail to alleviate lupus nephritis. Hence, the function and underlying mechanism of this subgroup in MSC therapy for diseases remains unknown. It is necessary to isolate and further investigate BAMBIhighMFGE8high UC-MSCs in terms of their phenotype, metabolism, and function to completely understand the nature of this MSC subgroup. In this protocol, we describe a detailed method for isolating the BAMBIhighMFGE8high subpopulation from human UC-MSCs. The subpopulation of UC-MSCs is labeled with two surface markers, BAMBI and MFGE8, by flow cytometry sorting. The isolated cells are cultured and verified by flow cytometry analysis. The specific genes expressed in the BAMBIhighMFGE8high UC-MSCs are identified by RT-qPCR. This protocol results in highly efficient and pure cell sorting and describes the marker profiles of the BAMBIhighMFGE8high UC-MSCs.

The isolation of BAMBIhighMFGE8high MSCs, one of the three main subgroups constituting heterogeneous human UC-MSCs, is helpful for fully understanding the characteristics and functions of this subtype for its future application to improve clinical efficacy in specific diseases. Here, we present a method for sorting BAMBIhighMFGE8high UC-MSCs.

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various ...

Fluorescence-Activated Single-Cell Sorting of Human Mesenchymal Stem Cells

Single-Cell Sorting of Immunophenotyped Mesenchymal Stem Cells from Human Exfoliated Deciduous Teeth

The mesenchymal stem cells (MSCs) of an organism possess an extraordinary capacity to differentiate into multiple lineages of adult cells in the body and are known for their immunomodulatory and anti-inflammatory properties. The use of these stem cells is a boon to the field of regenerative biology, but at the same time, a bane to regenerative medicine and therapeutics owing to the multiple cellular ambiguities associated with them. These ambiguities may arise from the diversity in the source of these stem cells and from their in vitro growth conditions, both of which reflect upon their functional heterogeneity.
This warrants methodologies to provide purified, homogeneous populations of MSCs&#160;for therapeutic applications. Advances in the field of flow cytometry have enabled the detection of single-cell populations using a multiparametric approach. This protocol outlines a way to identify and purify&#160;stem cells from&#160;human exfoliated deciduous teeth (SHEDs) through fluorescence-assisted single-cell sorting. Simultaneous expression of surface markers, namely, CD90-fluorescein isothiocyanate (FITC), CD73-peridinin-chlorophyll-protein (PerCP-Cy5.5), CD105-allophycocyanin (APC), and CD44-V450, identified the &#34;bright,&#34; positive-expressors of MSCs&#160;using multiparametric&#160;flow cytometry. However, a significant drop was observed in percentages of quadruple expressors of these positive markers from passage 7 onwards to the later passages.
The immunophenotyped subpopulations were sorted using the single-cell sort mode where only two positive and one negative marker constituted the inclusion criteria. This methodology ensured the cell viability of the sorted populations and maintained cell proliferation post sorting. The downstream application for such sorting can be used to evaluate lineage-specific differentiation for the gated subpopulations. This approach can be applied to other single-cell systems to improve isolation conditions and for acquiring multiple cell surface marker information.

Author Spotlight: Importance of Single Cell Sorting in Isolating Purified Populations of Mesenchymal Stem Cells 

This protocol describes the use of fluorescence-activated cell sorting of human mesenchymal stem cells using the single-cell sorting method. Specifically, the use of single-cell sorting can achieve 99% purity of the immunophenotyped cells from a heterogeneous population when combined with a multiparametric flow cytometry-based approach.

The mesenchymal stem cells (MSCs) of an organism possess an extraordinary capacity to differentiate into multiple lineages of adult cells in the body and ...

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

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