Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

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.