Name: Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods (Video) | JoVE Journal
Uploaded: 2016-05-02T13:00:00.000+00:00
Duration: 10 min 46 s
Description: Learn to prepare cellular vehicles loaded with gold nanorods for targeted photoacoustic imaging of tumors using tumor-associated macrophages. This method enhances delivery efficiency and improves optical stability for cancer diagnostics.

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5 minutes

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.25 and adapted according to Ratto et al.26. Then these gold nanorods are modified in order to gain more biocompatibility and affinity for plasmatic membranes, by the combination of polyethylene glycol strands10,11,27,28 and quaternary ammonium compounds22.
<ol>
	<li>Purify 24 ml CTAB-capped gold nanorods at a concentration of 450 &#181;M Au by two cycles of centrifugation (12,000 x g, 30 min) and decantation. Make sure that the ratio of dead volume to initial volume is around 1/200 or lower for all centrifugation steps in this protocol. Use 500 &#181;M aqueous CTAB as a washing solution and finally transfer the particles into 6 ml 100 mM acetate buffer at pH 5 containing 500 &#181;M CTAB and 0.005% (v/v) polysorbate 20.</li>
	<li>Add 30 &#181;l 10 mM aqueous alpha-methoxy-omega-mercapto-poly(ethylene glycol) (MW ~ 5,000) and leave to react for 30 min at 37 &#176;C.</li>
	<li>Add 30 &#181;l 100 mM (11-Mercaptoundecyl)-N,N,N-trimethylammonium bromide in dimethyl sulfoxide and leave at rest for 24 hr at 37 &#176;C.</li>
	<li>Next, add 18 ml 0.005% (v/v) polysorbate 20 in water and purify these particles by four cycles of centrifugation (12,000 x g, 30 min) and decantation. Use 0.005% (v/v) polysorbate 20 in water as washing solution and finally transfer the particles into 2.4 ml sterile PBS at pH 7.4. The final nominal concentration of gold is 4.5 mM.</li>
</ol>
2. Loading of Murine Macrophages with Gold Nanorods
<ol>
	<li>Use the monocyte/macrophagic cell line J774a.1 and DMEM supplemented with 10% fetal bovine serum, 1 mM glutamine, 100 units/ml penicillin and 100 &#181;g/ml streptomycin as culture medium. Plate 5 x 105 cells in four Petri dishes of 60 mm diameter and allow them to grow for 24 hr, so as to be subconfluent at the time of detachment (see step 2.2).
	<ol>
		<li>Throughout the protocol, maintain the cells under standard culture conditions (37 &#176;C, 5% CO2, 95% air and 100% relative humidity). Use a laminar flow cabinet and appropriate personal protective equipment to manipulate the cells.</li>
	</ol>
	</li>
	<li>After 24 hr, load the cells with cationic gold nanorods and prepare them to be embedded into chitosan films:
	<ol>
		<li>In order to allow the particles to be taken up by the cells, add an aliquot of 4.5 mM Au cationic gold nanorods in PBS into each Petri dish, so as to achieve a final concentration of 100 &#181;M Au. Leave the Petri dishes in incubation for 24 hr.</li>
		<li>Next, observe the cells under an optical microscope to confirm their good conditions and the upload. Cells should exhibit their normal morphology and a number of dark intracellular vesicles. Detach the cells by a scraper, merge those from two Petri dishes, in order to achieve a suitable amount of cells for the following steps (at least 2 x 106 cells), and centrifuge them (120 x g, 6 min) to eliminate any excess of cationic gold nanorods. The cellular pellet should look almost black.</li>
		<li>Suspend the pellet in 2 ml PBS and count the cells by the use of a B&#252;rker chamber. Centrifuge a suspension containing 2 x 106 cells (120 x g, 6 min) and fix their pellet in 2 ml 3.6% (w/v) formaldehyde in PBS for ten minutes at room temperature. Finally, wash this pellet three times by centrifugation (120 x g, 6 min) in order to remove the fixative. Use PBS as washing solution.</li>
	</ol>
	</li>
</ol>
3. Embedment of Macrophages into Chitosan Films
Note: The peculiar properties of chitosan26,27,28,29 are exploited to produce biomimetic phantoms containing macrophages stained with cationic gold nanorods. With respect to other hydrogels such as agarose, chitosan enables films that are much stronger and thinner, which is critical for PA microscopy6. The fabrication of these phantoms is carried out according to previous protocols29,30,31 with some modifications as prescribed in the followings30.
<ol>
	<li>Prepare an acidified (pH 4.5, obtained by the addition of acetic acid) and viscous 3% (w/v) low molecular weight chitosan (average MW 120 kDa) solution, thoroughly mix it and let it homogenize for 24 hr at 40 &#176;C.</li>
	<li>Next, mix murine macrophages containing cationic gold nanorods (2 x 106 cells) with 500 &#181;l of chitosan solution.</li>
	<li>In order to obtain ~50 &#181;m thick phantoms, pour 250 mg of the mixture into 1.91 cm2 polystyrene molds and leave them under a nitrogen stream for 24 hr. Thereafter, treat these samples with 500 &#181;l 1 M NaOH, in order to induce cross-linking, and rinse them with 10 ml of ultrapure water.</li>
</ol>
4. Test of the Stability of Photoacoustic Conversion
Note: The stability of PA conversion is investigated by means of PA experiments with the home-made setup that is described in ref 6.
<ol>
	<li>Suspend a chitosan film containing the macrophages in DI water, for instance by the use of a plastic holder immersed in a Petri dish, so as to keep a distance of ~5 mm from the bottom of the plate. Put this plate onto a micrometric XY stage in order to control the sample position.</li>
	<li>Focus a laser beam with ~5 nsec pulse duration in resonance with the longitudinal plasmonic band of the gold nanorods (e.g., from an optical parametric oscillator pumped by the third harmonic of a Q-switched Nd:YAG laser with wavelength range of 400 - 2,500 nm and pulse duration of 5 nsec) perpendicular to the film surface with a ~300 &#181;m spot diameter.
	<ol>
		<li>Place an attenuator in front of the laser exit to tune the laser fluence and use a beam splitter to focus part of the laser beam to an energy meter (e.g., a pyroelectric detector) and monitor fluence fluctuations. Maintain the optical fluence below ~1 mJ/cm2 per pulse during the alignment. Use appropriate laser safety eyewear whenever the laser is on.</li>
	</ol>
	</li>
	<li>Dip an ultrasound transducer (frequency range of 1 - 20 MHz) into the Petri dish ~2 mm off of the film surface and adjust its position by using micrometric translations and rotational stages to maximize the PA signal emitted from the film.</li>
	<li>Determine a probe fluence FLO that does not damage the sample6:
	<ol>
		<li>Irradiate a random point of the sample at a fluence around 1 mJ/cm2 per pulse for at least 500 pulses. For each pulse, acquire the corresponding PA signal from the ultrasound transducer and laser fluence from the energy meter with an oscilloscope. Name the average fluence as FLOtrial.</li>
		<li>Calculate the intensity of the PA signal as its peak-to-peak amplitude as a function of pulse number. In order to counterweight the laser intensity fluctuations, normalize the amplitude of each PA signal to the ratio of its own fluence to FLOtrial. Analyze the trend of the normalized PA intensity as a function of pulse number and verify its stability over time.</li>
		<li>In the case of instability, repeat steps 4.4.1 to 4.4.3 with a lower value of FLOtrial. In the case of stability, repeat them with a value of FLOtrial higher by ~10%, until a loss of stability arises. Set the probe fluence FLO as the second highest value of FLOtrial that ensures stability.</li>
	</ol>
	</li>
	<li>Measure a reshaping threshold fluence:
	<ol>
		<li>Choose another random point of the sample and probe an average PA intensity (ILOa) over 500 pulses at FLO.</li>
		<li>Set a nominal fluence greater than FLO and deliver 50 pulses. Name their average fluence as Fexc.</li>
		<li>Probe an average PA intensity (ILOb) over 500 pulses at FLO once again. Calculate the ratio R = ILOb / ILOa. A value of R below unity gives evidence of an irreversible change of the optical properties of the sample.</li>
		<li>Use the micrometric XY stage to move the film and change the point of the sample at random. Repeat steps 4.5.1 to 4.5.4 with different values of Fexc, so as to take a few values of R below, around and above unity. 15 points are reasonable.</li>
		<li>Plot R as a function of Fexc and identify the reshaping threshold fluence Fth as that value when R begins to differ from one beyond statistical uncertainty.6</li>
	</ol>
	</li>
</ol>

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<li>Oh, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+uptake+and+fate+of+PEGylated+gold+nanoparticles+is+dependent+on+both+cell-penetration+peptides+and+particle+size%5BTitle%5D)+AND+%22ACS+Nano%22%5BJournal%5D)">Cellular uptake and fate of PEGylated gold nanoparticles is dependent on both cell-penetration peptides and particle size.</a> ACS Nano. 5 (8), 6434-6448 (2011).</li>
<li>Yuan, H., Fales, A. M., Vo-Dinh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TAT+peptide-functionalized+gold+nanostars%3A+Enhanced+intracellular+delivery+and+efficient+NIR+photothermal+therapy+using+ultralow+irradiance%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">TAT peptide-functionalized gold nanostars: Enhanced intracellular delivery and efficient NIR photothermal therapy using ultralow irradiance.</a> J. Am. Chem. Soc. 134 (28), 11358-11361 (2012).</li>
<li>Ladest, S., Fales, A. M., Domard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-membrane+hydrogels%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Multi-membrane hydrogels.</a> Nature. 452, 76-79 (2008).</li>
<li>Matteini, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photothermally+activated+hybrid+films+for+quantitative+confined+release+of+chemical+species%5BTitle%5D)+AND+%22Angew.+Chem.+Int.+Ed.+%28English%29%22%5BJournal%5D)">Photothermally activated hybrid films for quantitative confined release of chemical species.</a> Angew. Chem. Int. Ed. (English). 52, 5956-5960 (2013).</li>
<li>Khor, E., Lim, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Implantable+applications+of+chitin+and+chitosan%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Implantable applications of chitin and chitosan.</a> Biomaterials. 24 (13), 2339-2349 (2003).</li>
<li>Kennedy, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((T+cells+enhance+gold+nanoparticle+delivery+to+tumors+in+vivo%5BTitle%5D)+AND+%22Nanoscale+Res.+Lett%22%5BJournal%5D)">T cells enhance gold nanoparticle delivery to tumors in vivo.</a> Nanoscale Res. Lett. 6 (1), 283(2011).</li>
<li>Pissuwan, D., Valenzuela, S. M., Killingsworth, M. C., Xu, X., Cortie, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+destruction+of+murine+macrophage+cells+with+bioconjugated+gold+nanorods%5BTitle%5D)+AND+%22J.+Nanopart.+Res%22%5BJournal%5D)">Targeted destruction of murine macrophage cells with bioconjugated gold nanorods.</a> J. Nanopart. Res. 9 (6), 1109-1124 (2007).</li>
<li>Jokerst, J. V., Thangaraj, M., Kempen, P. J., Sinclair, R., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photoacoustic+imaging+of+mesenchymal+stem+cells+in+living+mice+via+silica-coated+gold+nanorods%5BTitle%5D)+AND+%22ACS+Nano%22%5BJournal%5D)">Photoacoustic imaging of mesenchymal stem cells in living mice via silica-coated gold nanorods.</a> ACS Nano. 6 (7), 5920-5930 (2012).</li>
<li>Ding, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gold+nanorods+coated+with+multilayer+polyelectrolyte+as+contrast+agents+for+multimodal+imaging%5BTitle%5D)+AND+%22J.+Phys.+Chem.+C%22%5BJournal%5D)">Gold nanorods coated with multilayer polyelectrolyte as contrast agents for multimodal imaging.</a> J. Phys. Chem. C. 111 (34), 12552-12557 (2007).</li>
<li>Esposito, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((et+al.+In+vivo+laser+assisted+microvascular+repair+and+end-to-end+anastomosis+by+means+of+indocyanine+green-infused+chitosan+patches%3A+A+pilot+study%5BTitle%5D)+AND+%22Lasers+Surg.+Med%22%5BJournal%5D)">et al. In vivo laser assisted microvascular repair and end-to-end anastomosis by means of indocyanine green-infused chitosan patches: A pilot study.</a> Lasers Surg. Med. 45 (5), 318-325 (2013).</li>
</ol>

The authors declare that they have no competing financial interests.

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

preparation and photoacoustic analysis of cellular vehicles containing gold nanorods

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 ...

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 ...

The overall goal of this protocol is to use cellular vehicles containing gold nanorods as an innovative strategy to deliver plasmonic particles into tumors. Our protocol can help answer key questions on the development of photoacoustic and photothermal approaches to image and treat cancer by the use of exogenous contrast agents. This method rests on the tropism of tumor-associated microphages to deliver plasmonic particles to tumors.The idea is to isolate these cells from a patient, load them with plasmonic particles in vitro, and then inject them back into their host with the intent to use them as estrogen hosts. One of the advantages of this approach is the possibility of gaining more control over the optical and biochemical stability of the particles, because their biological interface is fully constructed in vitro. To begin, purify six milliliters of cetrimonium-capped gold nanorods at a gold concentration of 450 micromolar, using two centrifugations, followed by decanting and washing in 500 micromolar aqueous cetrimonium bromide.Then, add 1.5 milliliters of 100 millimolar acetate buffer with cetrimonium bromide and polysorbate 20 into the vessel containing the gold particle pellet. Then, add 7.5 microliters of 10 millimolar alpha-Methoxy-omega-mercapto polyethylene glycol in water and leave it to react for 30 minutes at 37 degrees Celsius. Next, add 7.5 microliters of 100 millimolar mercaptoundecyl trimethyl ammonium bromide in dimethyl sulfoxide.Then, let the reaction rest for 24 hours at 37 degrees Celsius. After 24 hours, add 4.5 milliliters of 0.005%polysorbate 20 in water. Now, using this mild polysorbate 20 solution, purify the particles using four cycles of centrifugation and decantation.Finally, transfer the particles into 600 microliters of sterile phosphate-buffered saline. The nominal concentration of gold should now be 4.5 millimolar. To prepare the cells, plate two 60 millimeter dishes with half a million monocyte macrophagic cells in supplemented Dulbecco's Modified Eagle Medium and grow them for 24 hours.After 24 hours, replace the cell medium with fresh medium, supplemented with the prepared cationic gold nanorods for a final concentration of 100 micromolar gold and solution in each Petri dish. Then continue the incubation for another 24 hours. The next day, check the cells under a microscope.They should have normal morphology and many dark intracellular vesicles. If the cells look good, wash them with fresh medium, detach them using a scraper, and merge the cells from two dishes to have at least two million cells in one collection. Now, centrifuge the collections at 120 Gs for 6 minutes to remove any excess cationic gold nanorods.Discard the supernates. The cell pellets should look almost black. Suspend the pellets in two milliliters of phosphate-buffered saline, and count the cells by the use of a Neubauer chamber.Then, mix suspensions containing two million cells and spin them down at 120 Gs for six minutes. Bring the pellets up in two milliliters of 3.6%formaldehyde and phosphate-buffered saline, and let the cells fix for 10 minutes at room temperature. Finally, remove the fixative using three phosphate-buffered saline washes.One day in advance of starting this procedure, prepare 500 microliters of an acidified 3%weight-by-volume, low-molecular-weight chitosan solution. Achieve a pH of 4.5 by the addition of acetic acid. To thoroughly mix the solution, let it homogenize for 24 hours at 40 degrees Celsius.The next day, mix the murine macrophages prepared with gold nanorods with all 500 microliters of the chitosan solution. In order to get 50 micron thick phantoms, load 250 milligrams of the mixture into 1.9 square centimeter polystyrene molds. Let the samples rest under a hood for 24 hours.The next day, treat the samples with 500 microliters of 1 molar aqueous sodium hydroxide, in order to induce cross-linking and rinse them with 10 milliliters of ultra-pure water. Analyze the stability of the photoacoustic conversion using a customized photoacoustic microscope, which features an optical parametric oscillator, an attenuator to tune the optical fluence, and an ultrasound transducer. Begin with suspending the chitosan film containing the macrophages in deionized water using a plastic holder immersed in a water tank.Determine a probe fluence that does not damage the sample and that conveys a photoacoustic emission with sufficient signal-to-noise ratio. Set a trial fluence and acquisition parameters. For each pulse from the laser, acquire the corresponding photoacoustic signal from the ultrasound transducer.Calculate the ratio of photoacoustic intensity to laser fluence for each pulse. Analyze its trend as a function of pulse number and verify its stability over time. In the case of instability, repeat this measurement with a lower trial fluence.In the case of stability, the trial fluence may be used as a probe fluence, provided that its photoacoustic emission exhibits a suitable signal-to-noise ratio. Next, measure a reshaping threshold fluence. Choose a random point of the sample, set the laser at the probe fluence, and measure an average photoacoustic intensity, A, over 500 pulses.Then, increase the nominal fluence above the probe fluence and deliver 50 pulses. Name their average fluence as F excitation. Reset the laser at the probe fluence and repeat the measurement, taking the average photoacoustic intensity, B, over 500 pulses.Now, calculate the ratio R of B to A.An R value below unity indicates an irreversible change has occurred in the optical properties of the sample. Next, use the micrometric stage to move the film to change the measurement point of the sample at random. Then, repeat the measurement of R for different values of F excitation, so as to take a few values of R around and below unity.About 15 R points is a reasonable collection of data. Then, plot R as a function of F excitation and identify the reshaping threshold as that fluence when R departs from unity without uncertainty. Typically, cationic gold nanorods undergo a massive accumulation in macrophages, which maintain their normal morphology.The particles are found to be confined within tight endocytic vesicles. Later in the process, the inclusion in the chitosan hydrogel does not appear to affect cellular morphology. Cells are well dispersed throughout the sample, as viewed with a micrograph.Controls of chitosan films containing gold nanorods without cells are homogeneous. Plasmonic bands of gold nanorods are preserved in their cellular vehicles. Effects such as plasmonic coupling or a different uptake of particles with different size and shape did not play a substantial role in these protocols.After collecting data, the trend of R as a function of F excitation gives an idea of the data and the analysis required to determine the F threshold. Here, the F threshold was found to be about 11 millijoules per square centimeter. Photoacoustic measurements were highly accurate with a signal-to-noise ratio greater than 20 over 500 pulses.Our protocol is innovative with respect to existing methods in the design of the particles and the investigation of their photostability. The modification of pegylated gold nanorods with quaternary ammonium cations provides for high efficiency of cellular uptake while keeping colloidal stability. The measurement of the stability of photoacoustic conversion by the definition of the reshaping threshold, is quantitative and reproducible.Since these particles are intended as contrast agents for photoacoustic imaging, a photoacoustic probe is ideal to test their functional features. Cellular vehicles of plasmonic particles are visible as contrast agents for photoacoustic imaging. Their photostability is the same as that of free plasmonic particles.The focus of our current work is on the physiology of these cells with special attention for their viablility and chemotactic activity.

preparation-photoacoustic-analysis-cellular-vehicles-containing-gold

Research

JoVE Journal

Bioengineering

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

In this video, we describe the darkfield microscopy (DFM) technique combined with single-particle tracking (SPT) analysis to monitor the interaction of plasmonic gold nanorods with the cell membrane of a specific cell line of interest. Combined DFM and SPT analysis allows the visualization of the nanorod movement through the cell membrane with high spatiotemporal resolution.

Darkfield Microscopy to Visualize Diffusional Dynamics of Nanorods on Cell Membrane

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

We describe a simple method for producing highly stable oligomeric clusters of gold nanoparticles via the reduction of chloroauric acid (HAuCl4) with sodium thiocyanate (NaSCN). The oligoclusters have a narrow size distribution and can be produced with a wide range of sizes and surface coats.

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

This paper describes a protocol for the synthesis of gold nanorods, based on the use of hydroquinone as reducing agent, plus the different mechanisms for controlling their size and aspect ratio.

Hydroquinone Based Synthesis of Gold Nanorods

We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes.

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Plasmonic gold nanorods can be trapped in liquids and rotated at kHz frequencies using circularly-polarized optical tweezers. Introducing tools for Brownian dynamics analysis and light scatteringspectroscopy leads to a powerful system for research and application in numerous fields of science.

Construction and Operation of a Light-driven Gold Nanorod Rotary Motor System

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Learn to prepare cellular vehicles loaded with gold nanorods for targeted photoacoustic imaging of tumors using tumor-associated macrophages. This method enhances delivery efficiency and improves optical stability for cancer diagnostics.

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

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