The Au nanocages were prepared via a galvanic replacement reaction between Ag nanocubes and HAuCl4
in an aqueous solution using the procedure that has been optimized in our previous work.[15
] The SPR peak of the Au nanocages was tuned to ~800 nm () to match the central wavelength of the diode laser (λ=808 nm). For the as-synthesized Au nanocages, the surface was covered by poly(vinyl pyrrolidone) (PVP, ~55,000 in molecular weight) and the size was 48±3.5 nm in edge length as measured by TEM ( inset). The hydrodynamic diameter (intensity size, D
) was measured as 99.8 nm with polydispersity index (PDI) of 0.16 by dynamic light scattering. The PVP layer was then replaced by heterofunctional poly(ethylene glycol) with one end terminated in the sulfhydryl group and the other end terminated in the methoxy group (HS-PEG5000
-OMe, ~5,000 in molecular weight). After PEGylation, the surface of each nanocage was covered by HS-PEG5000
-OMe molecules with an average total number on the order of 2×105
polymers per nanocage. This number is in agreement with the value estimated by assuming a footprint of 0.7 nm2
per thiol and an edge length of 48 nm for the nanocage. A previous study has measured the footprint of HS-PEG5000
-OMe on 2.8 nm Au nanoparticle, and a value of 0.35 nm2
per thiol was obtained based on a full monolayer coverage.[19
] The discrepancy can be attributed to two factors: i
) the flat surface of a cubic nanostructure can increase the steric effects between polymer chains as compared to the curved surface of a spherical structure; and ii
) the pores present in the nanocage can reduce the availability of the surface atoms for thiol binding. After PEGylation, the hydrodynamic diameter was reduced to 92.2 nm with PDI of 0.09. This difference in size is due to the conversion of surface coating from PVP to HS-PEG5000
-OMe. Spectroscopic studies showed that the LSPR peak of the PEGylated Au nanocages was red shifted by 5 nm relative to the PVP-coated Au nanocages, while the peak profile remained the same, suggesting that the Au nanocages were well dispersed in phosphate buffered saline (PBS) before and after PEGylation. In addition, the PEGylated Au nanocages were stable in fetal bovine serum (FBS), showing no change to the LSPR peak for months until now (). Using photoacoustic imaging, the absorption cross section of the Au nanocages was measured to be on the order of 6 × 10−15
Figure 1 UV-vis-NIR spectra showing the LSPR peaks of Au nanocages in different media: PVP-coated nanocages in PBS at pH 7.4 (solid line), PEGylated nanocages in PBS at pH 7.4 (dashed line), and PEGylated nanocages in fetal bovine serum (dotted line). The inset (more ...)
To plan for in vivo
photothermal treatment, we measured the temperature increase for a suspension of PEGylated Au nanocages in an aqueous solution under different conditions. For a given nanocage sample, the photothermal effect is determined by the particle concentration, as well as the power density and duration of laser irradiation.[14
] We examined the temperature changes due to 10 min of irradiation by the diode laser at 1 W/cm2
() and 0.5 W/cm2
(), respectively. The temperature profile was recorded by an infrared camera operating at a rate of 10 s per frame. For irradiation at a power density of 1 W/cm2
, the temperature increased rapidly in the first two minutes and gradually reached a plateau after 5 min. When the same series of samples were exposed to the same laser at a power density of 0.5 W/cm2
, the rate of temperature increase became markedly slower. lists the temperature increase (ΔT) for aqueous suspensions of Au nanocages that were exposed to laser irradiation for 10 min. In the absence of nanocages, the temperature only increased by 2–3 °C, indicating that laser irradiation for 10 min at this power density poses minimal risk of adversely affecting cells or tissues. In the presence of 109
nanocage/mL (or ~1 ppm in terms of Au content), the temperature increased dramatically by 5–10 °C. Such a change could increase tissue temperature from 37 °C to >42 °C and cause an irreversible damage to the cells or tissues due to the denaturation of biomolecules.[21
] Unlike the pulsed lasers with immense instantaneous power-per-pulse that can melt the nanocages to nanoparticles,[17
] the continuous-wave (CW) diode laser caused no change to the optical properties of the Au nanocages (), indicating that the nanocages were stable under the irradiation conditions.
Figure 2 Plots of temperature increase for suspensions of Au nanocages at various concentrations as a function of irradiation time using the diode laser at different power densities: A) 1 W/cm2 and B) 0.5 W/cm2. C) UV-vis-NIR spectra of the Au nanocages in an (more ...)
Temperature increase (ΔT) for aqueous suspensions of Au nanocages upon irradiation by the diode laser for 10 min.
We further investigated the photothermal effect of the Au nanocages for selective destruction of the neoplastic tissue using a bilateral tumor model. Athymic mice were subcutaneously injected into the right and left rear flanks with U87wtEGFR cells. After the tumor volume had reached 200–400 mm3, the mice were randomly divided into Group 1 and 2 (n=5 per group). The mice in Group 1 were intravenously administrated with 100 μL of 10 mg/mL (15 nM or 9×1012 particle/mL) PEGylated Au nanocages in PBS. The mice in Group 2 served as control and were injected intravenously with 100 μL of saline. At 72 h post-injection, the tumor on the right rear flank of each mouse was subjected to photothermal treatment by exposure to the diode laser at a power density of 0.7 W/cm2 for 10 min. The spot size of the laser beam was adjusted to cover the entire tumor (). During the laser treatment, full-body thermographic images were captured using an infrared camera, as shown in . The average temperature of the irradiated area was plotted as a function of the irradiation time (). For the nanocage-injected mice, the tumor surface temperature increased rapidly within one minute to reach 50 °C and began to plateau after 2 min at ~54 °C. In the case of saline-injected mouse, the surface temperature remained below 37 °C during the entire treatment.
Figure 3 A) Photograph of a tumor-bearing mouse under the photothermal treatment. 100 μL of PEGylated nanocages at a concentration of 9×1012 particles/mL or saline was administrated intravenously through the tail vein as indicated by an arrow. (more ...)
Changes to tumor metabolism due to photothermal treatment were monitored using18
F-FDG PET. Several human studies have shown that the use of 18
F-FDG as a surrogate marker for tumor metabolism in patients undergoing therapy is superior to the Responsive Evaluation Criteria In Solid Tumors (RECIST), a method that simply evaluates the size of the tumor using an anatomical imaging technique such as computed tomography (CT).[22
] Measurement of tumor metabolism with 18
F-FDG PET/CT imaging was performed before and after laser treatment for mice that had been intravenously injected with either saline or nanocages. Before laser irradiation, the 18
F-FDG PET/CT imaging showed no significant difference between saline-injected mice () and nanocage-injected mice (). At 24 h post-laser treatment, the metabolic activity in tumors of nanocage-injected mice () was significantly reduced as compared to that of saline-injected mice (). We then normalized the PET signal of the laser-treated tumor to that of the untreated tumor to minimize the variation of 18
F-FDG uptake at different time points (). The normalized value is ~0.3 after irradiation for the mice injected with Au nanocages as apposed to ~1 before irradiation, indicating a decrease of metabolic activity by 70%. For the saline-injected mice, the normalized value of 18
F-FDG uptake was close to 1 before and after uptake, suggesting there is no benefit to laser treatment in the absence of Au nanocages.
Figure 4 18F-FDG PET/CT co-registered images of mice intravenously administrated with either saline or Au nanocages, followed by laser treatment: A) a saline-injected mouse prior to laser irradiation; B) a nanocage-injected mouse prior to laser irradiation; C) (more ...)
Photothermal damage to tumor cells in mice injected with Au nanocages was confirmed by histological examination. Marked degenerative changes of coagulative necrosis, including abundant karyorrhectic debris and considerable regions of karyolysis, were found in laser-treated tumor tissue from mice injected with Au nanocages but were absent from tumors not exposed to laser irradiation or from mice injected with saline (). In , a boundary between an area of karyorrhexis and an area of karyolysis is visible as vertical bands. A high magnification view of this boundary reveals extensive pyknosis, karyorrhexis, karyolysis, and interstitial edema ().
Figure 5 Representative histology images of tumor tissues from the two mice intravenously administrated with saline and Au nanocages, respectively, followed by different treatments: A) tumor from saline-injected mouse with no irradiation; B) tumor from saline-injected (more ...)
To quantitatively assess the biodistribution and passive targeting of PEGylated Au nanocages, organs of interest were collected after the photothermal treatment to determine Au content. Inductively coupled plasma mass spectrometry (ICP-MS) was used to analyze the accumulation of exogenously given Au in tissues. plots the distribution of PEGylated-Au nanocages in various organs at 96 h post-injection. The nanocages were completely cleared from the blood (0.04±0.03 %ID/g), and relatively little Au remained in normal tissues (e.g., 0.80±0.12 %ID/g in muscles). Since the size of the nanocage is above the renal filtration limit (<8 nm),[26
] the PEGylated Au nanocages were found to clear via
reticuloendothelial system (RES) uptake with splenic clearance dominating, a pattern similar to that observed previously for PEGylated spherical Au nanoparticles with a diameter of 10 nm.[27
] Most importantly, passive accumulation of the PEGylated Au nanocages within tumors was found to be highly efficient, reaching a particle concentration of 5.1×1010
particles/g (or 5.7% ID/g) at 96 h post-injection. The entire surface of the dissected tumor appeared in dark blue color (inset of ), owing to the presence of Au nanocages. To examine the spatial distribution of the nanocages in a tumor, we cut a rectangular core through each tumor and sectioned them into five pieces. Each small piece was weighed and digested for ICP-MS analysis of Au content. displays the distribution of Au in different portions of the tumor. The edges were found to contain more nanocages than the center portion of the tumor because blood vessels are typically most abundant at the host interface where they may form a prominent, circumferential mantle enveloping tumor and leaving internal portions of tumors less well vascularized.[28
] This result implies that over time the PEGylated Au nanocages penetrated through the leaky blood vessels of the tumor and diffused into the interstitium region of the tumor, allowing for uniform heat generation within the tumor.
Figure 6 A) Tissue distribution of the PEGylated Au nanocages intravenously administrated (100 μL, with a concentration of 9×1012 particles/mL) into tumor-bearing mice. The amount of Au in the tissue sample was analyzed by ICP-MS at 96 h post injection. (more ...)