Name: preparation and photoacoustic analysis of cellular vehicles containing gold nanorods
Uploaded: 2016-05-02T13:00:00
Description: Watch this Scientific Journal Video about Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods at JoVE.com

This work was partially supported by Regione Toscana and European Community within the frame of the ERANET+ Projects LUS BUBBLE and BI-TRE.

Note: All concentrations of gold nanorods are expressed in terms of nominal Au molarities. For comparison with other works, note that 1 M Au roughly corresponds to 20 &#181;M gold nanorods, in our case.
1. Preparation of Cationic Gold Nanorods
Note: The method begins with the synthesis of cetrimonium bromide (CTAB)-capped gold nanorods by the autocatalytic reduction of HAuCl4 with ascorbic acid, according to the protocol introduced by Nikoobakht et al.<...

The notion to target tumor-associated macrophages is emerging as a powerful concept to combat cancer34,35,36. Here, instead of their destruction, these cells are recruited as cellular vehicles to bring gold nanorods into a tumor, by the exploitation of their tropism. This perspective requires a thoughtful design of the particles, their integration into the cells and their characterization. We have found that the photostability of murine macrophages loaded with cation...

Over the past decade, various plasmonic particles such as gold nanorods, nanoshells and nanocages, have received considerable attention for applications in biomedical optics1,2,3,4. At variance with standard gold nanospheres, these newer particles resonate in the near infrared (NIR) window that provides for deepest optical penetration through the body and highest optical contrast over endogenous components1. This feature has aroused interest for innovative applications, such as the photoacoustic (PA) imaging and the photothermal ablation of cancer. However, several issues restrain the clinical penetration of these particles. For instance, their optical activation tends to induce their overheating and to modify their functional shapes towards more spherical profiles, which drives a photoinstability5,6,7,8,9. Another issue that dominates the scientific debate is their systemic delivery into tumors. In particular, gold nanorods combine sizes that are ideal to pervade tumors that display enhanced permeability and retention and ease of conjugation with specific probes of malignant markers. Therefore, their preparation for a direct injection into the bloodstream is perceived as a feasible scheme10,11,12,13. However, this route remains problematic, with most of the particles becoming captured by the mononuclear phagocyte system10,11,12. In addition, another concern is the optical and biochemical stability of the particles after circulation through the body14. When particles lose their colloidal stability and aggregate, their plasmonic features and heat transfer dynamics may suffer from plasmonic coupling15,16,17 and cross-overheating18.
More recently, the notion to exploit the tropism of tumor-associated macrophages has emerged as a smart alternative19,20,21. These cells hold an innate ability to detect and pervade tumors with high specificity. Therefore, one perspective may be to isolate these cells from a patient, load them with gold nanorods in vitro and then inject them back into the patient, with the intent to use them as cellular vehicles in charge of the delivery. Another advantage would be to gain more control over the optical and biochemical stability of the particles, because their biological interface would be constructed in vitro. Still, the performances of these cellular vehicles as optical contrast agents need a critical analysis.
In this work, we describe the preparation and critical issues of cellular vehicles containing gold nanorods for the PA imaging of cancer. PEGylated gold nanorods are modified with quaternary ammonium compounds22, in order to achieve a cationic profile that is expected to promote their interactions with plasmatic membranes23,24. These particles undergo efficient and unspecific uptake from most cellular kinds, hopefully without interfering much with their biological functions. Murine macrophages are loaded with up to as many as 200&#775;,000 cationic gold nanorods per cell, which become confined within tight endocytic vesicles. This configuration should arise concern, because of the threat of plasmonic coupling and cross-overheating inside these vesicles. Therefore, the macrophages are embedded in biopolymeric hydrogels that mimic biological tissues, in order to verify that most of the stability of PA conversion of the particles is retained in the transfer from the growth medium to the endocytic vesicles. Effective measurement criteria are worked out in order to measure the stability of PA conversion under conditions of immediate interest for PA imaging. A reshaping threshold is set at the very onset of optical instability after a train of 50 laser pulses with the typical repetition rate of 10 Hz.
We are confident that these results may provide momentum for the development of novel strategies to deliver plasmonic particles to tumors.

<table border="0" cellpadding="0" cellspacing="0" width="760">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hexadecyltrimethylammonium bromide</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">H6269</td>
			<td style="width:321px;">To synthesize gold nanorods</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gold(III) chloride trihydrate</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">520918</td>
			<td style="width:321px;">To synthesize gold nanorods</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Silver nitrate</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">S6506</td>
			<td style="width:321px;">To synthesize gold nanorods</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-ascorbic acid</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">A5960</td>
			<td style="width:321px;">To synthesize gold nanorods</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium borohydride</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:321px;">To synthesize gold nanoseeds</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MeO-PEG-SH</td>
			<td style="width:104px;">Iris Biotech</td>
			<td style="width:119px;">PEG1171</td>
			<td style="width:321px;">To PEGylate gold nanorods. Molecular weight about 5,000 Da</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Acetic acid</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">320099</td>
			<td style="width:321px;">To PEGylate gold nanorods and solubilize chitosan</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium acetate</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">S8750</td>
			<td style="width:321px;">To PEGylate gold nanorods</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">(11-Mercaptoundecyl)-N,N,N-trimethylammonium bromide</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">733305</td>
			<td style="width:321px;">To modify gold nanorods with quaternary ammonium compounds</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dimethyl sulfoxide</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">276855</td>
			<td style="width:321px;">To solubilize (11-mercaptoundecyl)-N,N,N-trimethylammonium bromide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;&#160;Polysorbate 20</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2287</td>
			<td style="width:321px;">To centrifuge PEGylated gold nanorods</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PBS</td>
			<td style="width:104px;">Lonza</td>
			<td style="width:119px;">BE17-516F</td>
			<td style="width:321px;">To suspend gold nanorods before incubation with cells and to treat pellets of cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">J774a.1</td>
			<td style="width:104px;">ATCC</td>
			<td style="width:119px;">TIB-67</td>
			<td style="width:321px;">Monocyte/macrophage murine cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:104px;">Lonza</td>
			<td style="width:119px;">BE12-707F</td>
			<td style="width:321px;">Cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:104px;">Lonza</td>
			<td style="width:119px;">DE14-801F</td>
			<td style="width:321px;">To be added to cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-glutamine</td>
			<td style="width:104px;">Lonza</td>
			<td style="width:119px;">BE17-605E</td>
			<td style="width:321px;">To be added to cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin/streptomycin</td>
			<td style="width:104px;">Lonza</td>
			<td style="width:119px;">DE17-602E</td>
			<td style="width:321px;">To be added to cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dish</td>
			<td style="width:104px;">NEST</td>
			<td style="width:119px;">705001</td>
			<td style="width:321px;">Cell culture dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell scraper</td>
			<td style="width:104px;">EuroClone</td>
			<td style="width:119px;">ES7018</td>
			<td style="width:321px;">To detach cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Formaldehyde</td>
			<td style="width:104px;">Fluka</td>
			<td style="width:119px;">47630</td>
			<td style="width:321px;">To fix cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Chitosan, low molecular weight&#160;</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">448869</td>
			<td style="width:321px;">75-85% deacetylated. Molecular weight about 120,000 Da</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium hydroxyde</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">306576</td>
			<td style="width:321px;">To insolubilize chitosan and generate the hydrogel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polystyrene cell culture plates</td>
			<td style="width:104px;">NEST</td>
			<td style="width:119px;">702011</td>
			<td style="width:321px;">Used as molds to fabricate chitosan hydrogels</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Optical parametric oscillator pumped by the third harmonic of a Q-switched Nd:YAG laser</td>
			<td style="width:104px;">Continuum, Santa Clara, USA</td>
			<td style="width:119px;">&#160;Surelite OPO plus</td>
			<td style="width:321px;">Source of optical excitation for photoacoustic tests</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pyroelectric detector&#160;</td>
			<td style="width:104px;">Gentec, Quebec, Canada</td>
			<td style="width:119px;">QE8SP</td>
			<td style="width:321px;">To monitor optical fluence for photoacoustic tests</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Pre amplified needle hydrophone</td>
			<td style="width:104px;">Precision Acoustic, Dorset, UK</td>
			<td style="width:119px;">Model with 1 mm sensor diameter and 1-20 MHz frequency range</td>
			<td style="width:321px;">To measure photoacoustic signals</td>
		</tr>
	</tbody>
</table>

preparation and photoacoustic analysis of cellular vehicles containing gold nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

Here, the feasibility of cellular vehicles containing gold nanorods for the PA imaging of cancer is shown together with typical outcomes of the protocol.
The TEM images in Figure 1 show the usual appearance of the particles after step 1 and their cellular vehicles after step 2. The preparation of the particles and of the cells for TEM imaging is described elsewhere17. Cationic gold nanorods undergo a ...

Watch this Scientific Journal Video about Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods at JoVE.com

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

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