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 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.
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 200̇,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.
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 biocompatibility and affinity for plasmatic membranes, by the combination of polyethylene glycol strands10,11,27,28 and quaternary ammonium compounds22.
2. Loading of Murine Macrophages with Gold Nanorods
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.
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.
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 massive accumulation in macrophages, which maintain their normal morphology. Particles are found to be confined within tight endocytic vesicles.
Figure 2a displays an optical transmission image of macrophages containing cationic gold nanorods and dispersed in a chitosan phantom after step 3. As proven by this micrograph, the inclusion in the chitosan hydrogel does not affect the cellular morphology. Cells are well dispersed throughout the sample. Controls of chitosan films containing gold nanorods without cells are homogeneous. Figure 2b shows that the typical plasmonic band of gold nanorods is retained when the particles are taken up by the macrophages, consistent with our previous work14,32. Therefore, effects such as plasmonic coupling on segregation in endocytic vesicles and a differential uptake of particles with different size and shape that coexist in a polydisperse colloid28,33 do not play a substantial role in these protocols. Figure 2b also proves the feasibility of chitosan as an optical phantom.
Figure 3 shows the trend of R as a function of Fexc measured according to step 4 and gives an idea of the data and the analysis that are required for the determination of Fth as per step 4.5.5. Fth was found to be (11 ± 1) mJ/cm2 in this example. PA measurements on this sample gave signals with signal to noise ratio (SNR) greater than 20 when averaged over 500 pulses at few mJ/cm2, which provides high accuracy for the investigation of the stability of PA conversion from the cellular vehicles.
Figure 1. Cationic gold nanorods and macrophages characterization. a: (650 × 500) nm2 TEM image of as-synthesized gold nanorods; b, c and d: respectively (13 × 8.6) µm2, (2.3 × 1.7) µm2 and (870 × 650) nm2 TEM images of macrophages treated with cationic gold nanorods. The appearance of the particles in panel d is affected by their inclination in the cell. Please click here to view a larger version of this figure.
Figure 2. Chitosan film characterization. a: Optical transmission image of macrophages containing cationic gold nanorods and dispersed in a chitosan phantom. b: Optical extinction spectra of chitosan phantoms containing gold nanorods without cells (solid black line, control sample) and macrophages containing gold nanorods (broken red line). Please click here to view a larger version of this figure.
Figure 3. Cellular vehicles photostability. Ratio R of the intensities of ILO taken after and before irradiation at each Fexc versus Fexc. The error bars originate from the signal fluctuations at FLO. The reshaping threshold is extracted from these data as R falls below unity. The red solid line serves as a guide to the eye. Please click here to view a larger version of this figure.
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 cationic gold nanorods does not suffer from the confinement of particles within endocytic vesicles, which implies that their plasmonic coupling and cross-overheating are not critical. We hypothesize that the PEG strands and the incidence of shapes that are off-resonance, such as gold nanospheres, prevent the particles to get into too tight contact, which is about their diameters17 (around 10 nm) and one thermal diffusion length (around 30 nm in 5 nsec) for plasmonic coupling and cross-overheating, respectively.
The protocol is innovative with respect to existing methods in the design of the particles and of the investigation of their photostability. The design of gold nanorods according to step 1 combines the observation by Vigderman et al.22 that quaternary ammonium compounds are capable to drive a massive uptake of gold nanorods into endocytic vesicles and the notion that cell penetrating agents with a cationic profile24,37 may be embedded within a PEG shell38 and remain functional, while gaining colloidal stability and biocompatibility28. Indeed step 1 resembles the method by Yuan et al.38, with the replacement of cell penetrating peptides with smaller and cheaper quaternary ammonium compounds. With these modifications, cationic gold nanorods are multifunctional and sustainable. Since these particles are intended as contrast agents for PA imaging, a PA probe is ideal to test their functional features. The measurement of the stability of PA conversion by the definition of a reshaping threshold in step 4 is quantitative and reproducible, which are unique features in the frame of the scientific literature. Besides, we note that this method does not require a calibration of the PA equipment.
Critical steps within the protocol include the fabrication of the chitosan films to inspect the cellular vehicles in terms of their efficiency as contrast agents for PA imaging. Chitosan is a linear chain biopolymer comprising glucosamine and N-acetyl glucosamine residues joined together by 1,4-glycosidic bonds. Some physiochemical features of chitosan, such as its pore size, porosity and mechanical properties, as well as its versatility and handiness, make it an ideal option for the fabrication of hydrogels in the form of thin films or porous membranes29,39,40. Moreover, the polysaccharide backbone of chitosan is structurally similar to glycosaminoglycans, the major component of the extra-cellular matrix of connective tissues, which has promoted its use for engineering biomimetic and cell-supporting scaffolds41. Overall, chitosan hydrogels exhibit thermal and elastic moduli that are representative of connective tissue29,41,42, which is ideal for PA tests. Care should be taken to achieve films with the proper thickness (50 µm), low optical turbidity and good homogeneity. Note that the doses given in step 3 have been optimized. The viscous suspension of murine macrophages in chitosan should be mixed with diligence according to step 3.2. With these instructions, these films have already been used to compare the stability of PA conversion of gold nanorods of different size6.
Possible modifications of the protocol include the preparation of the cationic gold nanorods and cellular vehicles in steps 1 and 2. The method in step 1 may be subject to incremental improvements, e.g., by the substitution of the cell penetrating agent or the length of the PEG strands etc., in order to minimize any interference with the physiology of the tumor-associated macrophages. The use of ligands that are specific for macrophages may be an option43. Other parameters that affect the uptake of the particles include their size and shape33 and their inorganic coating. For instance, a shell of silica may give a combination of high internalization44, optical stability against aggregation17 and PA stability7, at the expense of more sophistication and more foreign material. We conjecture that the endosomal pathway of internalization may be a common effect33,43,44,45. A critical examination of the cells from step 2 is underway and the best arrangement of optical contrast, viability and chemotactic activity in vitro and in vivo may still require to adjust the dosage of the plasmonic particles in terms of concentration and duration of the incubation. Although the morphology of the cells and the preliminary evidence in our hands suggest a low cytotoxicity, the investigation of these parameters is beyond the scope of this work. Another perspective would be to reproduce step 2 with other cells of the immune system46 and primary cells, which may be chosen on a case-by-case basis. Indeed, we speculate that the notion to modulate the uptake of the particles with their electrokinetic potential is most versatile.
Limitations of the protocol include the need to use fixed cells rather than live cells, because the prescriptions in step 3 are incompatible with the preservation of cellular viability. Other restrictions relate to the need of a sufficient SNR in the determination of a probe fluence in step 4.4, which translates into a sufficient combination of optical absorbance and photostability of the film.
In conclusion, we have described an innovative protocol to prepare and to perform a functional characterization of cellular vehicles that are feasible as contrast agents for photoacoustic imaging. We have found that the introduction of gold nanorods into murine macrophages does not negatively affect their photostability. The focus of our current work is on the physiology of these cells, with a special attention for their viability and chemotactic activity. In the future, this method shall be tested to investigate the preparation of different cellular vehicles containing different solutions of optical contrast agents. The PA probe may also serve to test the targetability of optical contrast agents to malignant cells in vitro, without the use of cellular vehicles.
The authors have nothing to disclose.
This work was partially supported by Regione Toscana and European Community within the frame of the ERANET+ Projects LUS BUBBLE and BI-TRE.
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 |