Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Summary

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

Abstract

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.

Introduction

Over the past decade, various plasmonic particles such as gold nanorods, nanoshells and nanocages, have received considerable attention for applications in biomedical optics^1,2,3,4. 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 ....

Protocol

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 µM 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 biocompatib....

Results

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 .......

Discussion

The notion to target tumor-associated macrophages is emerging as a powerful concept to combat cancer^34,35,36. 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.......

Materials

List of materials used in this article

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