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...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-Density+Lipoprotein+as+a+Vehicle+for+Targeting+Antitumor+Compounds+to+Cancer+Cells.">Low-Density Lipoprotein as a Vehicle for Targeting Antitumor Compounds to Cancer Cells.</a> Bioconjugate Chemistry. 5 (2), 105-113 (1994).</li><li>Beloribi-Djefaflia, S., Vasseur, S., Guillaumond, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+metabolic+reprogramming+in+cancer+cells.">Lipid metabolic reprogramming in cancer cells.</a> Oncogenesis. 5 (1), 189 (2016).</li><li>Xin, Y., Yin, M., Zhao, L., Meng, F., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+on+nanoparticle-based+drug+delivery+systems+for+cancer+therapy.">Recent progress on nanoparticle-based drug delivery systems for cancer therapy.</a> Cancer Biology &amp; Medicine. 14 (3), 228 (2017).</li><li>Merriel, S. W. D., Carroll, R., Hamilton, F., Hamilton, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+between+unexplained+hypoalbuminaemia+and+new+cancer+diagnoses+in+UK+primary+care+patients.">Association between unexplained hypoalbuminaemia and new cancer diagnoses in UK primary care patients.</a> Family Practice. 33 (5), 449-452 (2016).</li><li>Yue, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesteryl+ester+accumulation+induced+by+PTEN+loss+and+PI3K/AKT+activation+underlies+human+prostate+cancer+aggressiveness.">Cholesteryl ester accumulation induced by PTEN loss and PI3K/AKT activation underlies human prostate cancer aggressiveness.</a> Cell Metabolism. 19 (3), 393-406 (2014).</li><li>dos Santos, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LDL-cholesterol+signaling+induces+breast+cancer+proliferation+and+invasion.">LDL-cholesterol signaling induces breast cancer proliferation and invasion.</a> Lipids in Health and Disease. 13 (16), (2014).</li><li>Gallagher, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+tumor+LDLR+expression+accelerates+LDL+cholesterol-mediated+breast+cancer+growth+in+mouse+models+of+hyperlipidemia+HHS+Public+Access.">Elevated tumor LDLR expression accelerates LDL cholesterol-mediated breast cancer growth in mouse models of hyperlipidemia HHS Public Access.</a> Oncogene. 36 (46), 6462-6471 (2017).</li><li>Needham, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bottom+up+design+of+nanoparticles+for+anti-cancer+diapeutics:+"put+the+drug+in+the+cancer's+food".">Bottom up design of nanoparticles for anti-cancer diapeutics: "put the drug in the cancer's food".</a> Journal of Drug Targeting. 24 (9), 836-856 (2016).</li><li>Lacko, A. G., Mconnathy, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+cancer+chemotherapy+using+synthetic+nanoparticles.">Targeted cancer chemotherapy using synthetic nanoparticles.</a> United States Patent Application Publication. , (2009).</li><li>Nikanjam, M., Gibbs, A. R., Hunt, C. A., Budinger, T. F., Forte, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+nano-LDL+with+paclitaxel+oleate+as+a+targeted+drug+delivery+vehicle+for+glioblastoma+multiforme.">Synthetic nano-LDL with paclitaxel oleate as a targeted drug delivery vehicle for glioblastoma multiforme.</a> Journal of Controlled Release. 124 (3), 163-171 (2007).</li><li>Teerlink, T., Scheffer, P. G., Bakker, S. J. L., Heine, R. 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M., Hansen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics+of+stealth+versus+conventional+liposomes:+effect+of+dose.">Pharmacokinetics of stealth versus conventional liposomes: effect of dose.</a> Biochimica et Biophysica Acta (BBA) - Biomembranes. 1068 (2), 133-141 (1991).</li><li>Maeda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Enhanced+Permeability+and+Retention+(EPR)+Effect+in+Tumor+Vasculature:+The+Key+Role+of+Tumor-Selective+Macromolecular+Drug+Targeting.">The Enhanced Permeability and Retention (EPR) Effect in Tumor Vasculature: The Key Role of Tumor-Selective Macromolecular Drug Targeting.</a> Advances in Enzyme Regulation. 41 (1), 189-207 (2001).</li><li>Wong, A. D., Ye, M., Ulmschneider, M. B., Searson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+of+the+Enhanced+Permeation+and+Retention+(EPR)+Effect.">Quantitative Analysis of the Enhanced Permeation and Retention (EPR) Effect.</a> PLoS ONE. 10 (5), 0123461 (2015).</li><li>Khodabandehloo, H., Zahednasab, H., Hafez, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanocarriers+Usage+for+Drug+Delivery+in+Cancer+Therapy.">Nanocarriers Usage for Drug Delivery in Cancer Therapy.</a> Iranian Journal of Psychiatry and Behavioral Sciences. 9 (2), (2016).</li><li>Kobaek-Larsen, M., El-Houri, R. B., Christensen, L. P., Al-Najami, I., Frett&#233;, X., Baatrup, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+polyacetylenes,+falcarinol+and+falcarindiol,+isolated+from+carrots+prevents+the+formation+of+neoplastic+lesions+in+the+colon+of+azoxymethane-induced+rats.">Dietary polyacetylenes, falcarinol and falcarindiol, isolated from carrots prevents the formation of neoplastic lesions in the colon of azoxymethane-induced rats.</a> Food &amp; Function. 8, 964-974 (2017).</li><li>Hervella, P., Parra, E., Needham, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+and+retention+of+chelated-copper+inside+hydrophobic+nanoparticles:+Liquid+cored+nanoparticles+show+better+retention+than+a+solid+core+formulation.">Encapsulation and retention of chelated-copper inside hydrophobic nanoparticles: Liquid cored nanoparticles show better retention than a solid core formulation.</a> European Journal of Pharmaceutics and Biopharmaceutics. 102, 64-76 (2016).</li><li>Hervella, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chelation,+formulation,+encapsulation,+retention,+and+in+vivo+biodistribution+of+hydrophobic+nanoparticles+labelled+with+57Co-porphyrin:+Octyl+Amine+ensures+stable+chelation+of+cobalt+in+Liquid+Nanoparticles+that+accumulate+in+tumors.">Chelation, formulation, encapsulation, retention, and in vivo biodistribution of hydrophobic nanoparticles labelled with 57Co-porphyrin: Octyl Amine ensures stable chelation of cobalt in Liquid Nanoparticles that accumulate in tumors.</a> Journal of Controlled Release. , (2018).</li><li>Zhigaltsev, I. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bottom-up+design+and+synthesis+of+limit+size+lipid+nanoparticle+systems+with+aqueous+and+triglyceride+cores+using+millisecond+microfluidic+mixing.">Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing.</a> Langmuir. 28 (7), 3633-3640 (2012).</li><li>Aubry, J., Ganachaud, F., Cohen Addad, J. -. 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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 ob...

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 border="0" cellpadding="0" cellspacing="0" style="width:965px;" width="966">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">12 mL Screw Neck Vial (Clear glass, 15-425 thread, 66 X 18.5 mm)</td>
			<td style="width:384px;">Microlab Aarhus A/S</td>
			<td style="width:160px;">ML 33154LP</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">6 well plates</td>
			<td>Greiner Bio One International GmbH</td>
			<td style="width:160px;">657160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Absolute Ethanol</td>
			<td>EMD Millipore (VWR)</td>
			<td>EM8.18760.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Chloroform</td>
			<td>Rathburn Chemicals Ltd.</td>
			<td>RH1009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Cholesterol</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>700000P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Confocal Microscope</td>
			<td style="width:384px;">Zeiss LSM510</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Cover Slips thickness #1.5</td>
			<td>Paul Marienfeld GmbH &#38; Co</td>
			<td>117650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Desiccator</td>
			<td style="width:384px;">Self-build</td>
			<td style="width:160px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DiI</td>
			<td>Invitrogen</td>
			<td style="width:160px;">D282</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">DLS</td>
			<td style="width:384px;">Beckman Coulter</td>
			<td style="width:160px;">DelsaMAXpro 3167-DMP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DSPC (Chloroform stock)</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>850365C&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DSPE PEG 2000 (Chloroform stock)</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>880120C</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">eVol XR</td>
			<td>SGE analytical science, Trajan Scientific Australia Pty Ltd.</td>
			<td>2910200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Fetal Bovine serum</td>
			<td>Gibco</td>
			<td style="width:160px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Fluorescence Miccroscope</td>
			<td style="width:384px;">Olymous IX81</td>
			<td style="width:160px;"></td>
			<td>With Manual TIRF and Andor iXon EMCCD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Incubator</td>
			<td style="width:384px;">Panasonic&#160;</td>
			<td style="width:160px;">MCO-18AC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Magnetic flea</td>
			<td>VWR Chemicals</td>
			<td>15 x 4.5 mm</td>
			<td>Cylindrical shape with PTFE coating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Magnetic stirrer</td>
			<td>IKA</td>
			<td style="width:160px;">RT-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimum Essential Media</td>
			<td>Gibco</td>
			<td style="width:160px;">32561-029</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:255px;">PBS tablets for cell culture</td>
			<td style="width:384px;">VWR Chemicals</td>
			<td>97062-732</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Pen/strep</td>
			<td>VWR Chemicals</td>
			<td>97063-708</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Phosphate Buffer Saline (PBS, pH 7.4)</td>
			<td>Thermo Fisher</td>
			<td>10010031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Rotary Evaporator</td>
			<td>Rotavapor, B&#252;chi Labortechnik AG</td>
			<td style="width:160px;">R-210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Sample concentrator&#160;</td>
			<td>Stuart, Cole-Parmer Instrument Company, LLC</td>
			<td style="width:160px;">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 experim...

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 ...

The fabrication of lipid-coated nanoparticles with the rapid injection method provides a stable drug delivery system for cancer therapy. This technique is a simple, fast, scalable, and reproducible technique for nanoparticle fabrication that encapsulates hydrophobic drugs. This technique is applicable for fabricating diagnostic and therapeutic nanoparticles from hydrphobic materials that can be effectively used in several disease conditions.This fabrication technique, along with DLS, can be used to study the nucleation and growth of hydrophobic materials, such as lipids, proteins, and polymers. Knowing the physical chemical properties of your drug substance is critical before fabricating the nanoparticles. Also, be sure to use the correct concentrations of coating lipids.Visual demonstration of this method is critical to show the simplicity of the fabrication technique and to show the successful delivery of the drug as nanoparticles to the cells. Precaution needs to be taken when working with chloroform and formaldehyde, and working under a fume hood is required. To begin, dispense 2.4 milliliters of 250 micromolar falcarindiol stock solution dissolved in 70%ethanol in a scintillation vial.Using a sample concentrator, evaporate the liquid fraction for approximately four hours to obtain dry falcarindiol. Using the sample concentrator, deliver gas over the sample at room temperature to concentrate the sample. Once dried, add the components shown here to the scintillation vial in a fume hood, making sure to clean the syringe with chloroform after adding each component to avoid cross contamination.Then immediately close the vials containing the lipids so that the solvent does not evaporate and thereby modify the concentration. Wrap the vial with aluminum foil to protect DiI from light and leave the sample overnight in the desiccator to evaporate the chloroform. The next day, dissolve the desiccated sample in absolute ethanol to make a final volume of 1.2 milliliters.This solution represents the organic phase. Take a 12 milliliter glass vial and fill it with nine milliliters of purified water. Then add a magnetic flea into the vial containing nine milliliters of water.Place the vial on a magnetic stirrer and stir at 500 rpm. Next attach a one milliliter glass syringe to the dispensing system and slowly pull chloroform in and out of the glass syringe at least 10 times. Dispense the chloroform into its waste collector each time.Cleaning the syringe with chloroform helps to avoid any contamination. Now prime the syringe with ethanol. Priming replaces the old solvent as well as removes any air bubbles.Using the syringe, aspirate one milliliter of the organic phase, and insert the syringe into the glass vial up to the middle of the nine milliliter water mark. Maintain it steady in the middle of the vial, and inject the solution at 833 microliters per second by pressing the dispense button on the dispensing system. This generates 10 microliters of 50 micromolar lipid coated nanoparticles of falcarindiol in 10%ethanol.This injection speed has been found to achieve finest nanoparticles with narrow particle size distribution. While injecting, it is important to make sure that the needle is inserted in the center, steady and straight. Immediately after the injection, remove the vial from the stirrer and transfer the sample to a 50 milliliter round bottom flask.Attack the flask to the rotary evaporator, and evaporate one milliliter of the organic solvent at room temperature. Be cautious to avoid excess bubble formation. Transfer the nanoparticle suspension from the flask to another 12 milliliter glass vial.Ensure that the volume is nine milliliters, and then split the sample in two 12 milliliter glass vials. Then add 0.5 milliliters of ultra pure water in one of the vials and 0.5 milliliter of 10X phosphate buffered saline in the other vial. Turn on the digital light scattering instrument, and set the desired temperature to 20 degrees Celsius until it stabilizes.Then set the instrument parameters as shown here. Fill the plastic cuvette with one milliliter of nanoparticle suspension, and start the measurement. Report the measured size depending on the solvent that was used.Seed approximately 50, 000 cells on sterile 1.5 cover slips placed in six-well plate to obtain a cell density of approximately 30%Then add culture medium in order to have a final volume of three milliliters in each well. Incubate the cells for 24 hours under standard culture conditions. The next day, add three microliters of the nanoparticle solution for a final falcarindiol concentration of five micromolar.Then return the cells to the incubator for 24 hours. After 24 hours of treatment, wash the cells twice with PBS, and then fix them in 4%formaldehyde for 10 minutes at room temperature. Following fixation, permeabilize the cells with 0.1%Triton X-100 for 30 minutes.Then wash them twice with PBS, and stain them with 250 microliters of 300 nanomolar DAPI for five minutes, protected from light. Place the cover slip onto a slide using a drop of PBS. Turn to a 150x subjective, and transfer the slide to the stage of a wide-field fluorescence microscope equipped with an electron multiplied CCD camera.Image cells using the GFP-LP channel. Additionally, verify the uptake of the nanoparticles into the cells by taking confocal microscopy images using a 63x oil objective with a numerical aperture of 1.4. Image DiI using an argon laser at 514 nanometers and DAPI using a two-photon laser at 780 nanometers.Uncoated nanoparticles of falcarindiol formed in water and measured in PBS showed high polydispersity, with PD index of 0.571. This value indicates broad and nonuniform distribution of particle sizes. In contrast, lipid coated nanoparticles of falcarindiol fabricated in this video were of 74.1 nanometers with a polydispersity index of 0.182, indicated a relatively monodisperse and uniform distribution of particle sizes.As a first observation of the nanoparticles inside the cells, epifluorescence microscopy images were acquired after 24 hours 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. To verify that falcarindiol nanoparticles had entered the cells, confocal microscopy was performed on the cells as well.A large number of nanoparticles are shown here, scattered in the cytoplasm in every cell. These results show that nanoparticles act as a stable drug delivery system for falcarindiol. While attempting this procedure, it's important to inject the solution at the right injection speed, keeping the seeding steady, in the center, to ensure proper mixing.This procedure can be used for any hydrophobic drug to formulate therapeutic nanoparticles or for imaging agents to formulate diagnostic nanoparticles that can be used in various disease conditions, such as cancer. This technique enabled more research to be conducted on the mechanism by which the nanoparticles get taken up by cells, as well as its live cell imaging and on studying the anticancer effect of the drug.

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

Research

JoVE Journal

Bioengineering

8.8K visualizações.  University of Southern Denmark. A fabricação de nanopartículas revestidas de lipídios com o método de injeção rápida fornece um sistema estável de entrega de medicamentos para terapia do câncer. Esta técnica é uma técnica simples, rápida, escalável e reprodutível para fabricação de nanopartículas que encapsula drogas hidrofóbicas. Esta técnica é aplicável para a fabricação de nanopartículas diagnósticas e terapêuticas a partir de materiais hidrofóbicos que podem ser efetivamente utilizados em v?...