Dual-Energy CT Imaging of Tumor Liposome Delivery After Gold Nanoparticle-Augmented Radiation Therapy

Abstract

Gold nanoparticles (AuNPs) are emerging as promising agents for both cancer therapy and computed tomography (CT) imaging. AuNPs absorb x-rays and subsequently release low-energy, short-range photoelectrons during external beam radiation therapy (RT), increasing the local radiation dose. When AuNPs are near tumor vasculature, the additional radiation dose can lead to increased vascular permeability. This work focuses on understanding how tumor vascular permeability is influenced by AuNP-augmented RT, and how this effect can be used to improve the delivery of nanoparticle chemotherapeutics. Methods: Dual-energy CT was used to quantify the accumulation of both liposomal iodine and AuNPs in tumors following AuNP-augmented RT in a mouse model of primary soft tissue sarcoma. Mice were injected with non-targeted AuNPs, RGD-functionalized AuNPs (vascular targeting), or no AuNPs, after which they were treated with varying doses of RT. The mice were injected with either liposomal iodine (for the imaging study) or liposomal doxorubicin (for the treatment study) 24 hours after RT. Increased tumor liposome accumulation was assessed by dual-energy CT (iodine) or by tracking tumor treatment response (doxorubicin). Results: A significant increase in vascular permeability was observed for all groups after 20 Gy RT, for the targeted and non-targeted AuNP groups after 10 Gy RT, and for the vascular-targeted AuNP group after 5 Gy RT. Combining targeted AuNPs with 5 Gy RT and liposomal doxorubicin led to a significant tumor growth delay (tumor doubling time ~ 8 days) compared to AuNP-augmented RT or chemotherapy alone (tumor doubling time ~3-4 days). Conclusions: The addition of vascular-targeted AuNPs significantly improved the treatment effect of liposomal doxorubicin after RT, consistent with the increased liposome accumulation observed in tumors in the imaging study. Using this approach with a liposomal drug delivery system can increase specific tumor delivery of chemotherapeutics, which has the potential to significantly improve tumor response and reduce the side effects of both RT and chemotherapy.

Keywords: dual-energy CT; nanoparticles; radiation therapy; small animal imaging; tumor drug delivery; vascular imaging.

Figure 1

Imaging protocol timeline. RT occurred 24 hours after gold nanoparticle injection, which was followed 24 hours later by injection of liposomal iodine. Dual-energy CT imaging was performed 3 days after iodine injection to allow sufficient time for iodine accumulation in the tumors.

Figure 2

Characterization of gold nanoparticles and liposomes. (A) TEM of bare gold nanoparticles, (B) TEM of liposomal iodine, (C) DLS average diameter and zeta potential measurements for each nanoparticle type with the standard error, (D) DLS size distribution of gold nanoparticles, and (E) DLS size distribution of liposomes.

Figure 3

Darkfield microscopy of endothelial cell targeting by gold nanoparticles. RGD-AuNPs bind to the endothelial cells to a very high degree at both 1 mg Au/mL and 6 mg Au/mL. PEG-AuNPs show no cell binding at 1 mg Au/mL and only minimal binding at 6 mg Au/mL. The scale bar represents 50 µm and is the same in all panels.

Figure 4

In vivo gold distribution after IV injection in healthy mice. (A) Measured concentration for PEG-AuNPs and RGD-AuNPs in the blood over time, along with an exponential fit curve for each nanoparticle type. (B) Organ gold accumulation at 48 hours for each nanoparticle type. The RGD-AuNPs showed more rapid clearance from the blood than PEG-AuNPs as well as increased accumulation in the liver and spleen at 48 hours.

Figure 5

Maximum intensity projections of the dual-energy CT iodine map for hindlimb sarcomas in each RT treatment group. Dual-energy CT was performed 3 days after liposomal iodine injection (5 days after gold nanoparticle injection). The iodine map is overlaid on the grayscale 80 kVp dataset. Iodine (red) is present in all the tumors, but is visibly lowest in the no AuNP (0 Gy, 5 Gy, and 10 Gy) groups as well as the PEG-AuNP 5 Gy group. All the other tumors appear to have increased tumor liposomal iodine accumulation as a result of RT. The iodine map is windowed from 2-35 mg iodine/mL. The grayscale images (80 kVp dataset) are windowed from -100-3500 HU.

Figure 6

Tumor liposomal iodine accumulation after gold nanoparticle-augmented radiation therapy. Concentrations are expressed in both mg iodine/mL (primary axis) and %ID/g (secondary axis). The addition of non-targeted PEG-AuNPs increased the iodine accumulation in the 10 Gy group, but made no change at the 5 Gy or 20 Gy doses. The addition of vascular-targeted RGD-AuNPs led to a large increase in accumulation at 5 Gy (compared to both control and PEG-AuNP groups). At 10 Gy, the addition of RGD-AuNPs did not statistically increase the iodine accumulation compared to PEG-AuNPs, but RGD-AuNPs did increase iodine accumulation compared to the control group with no AuNPs. * represents a statistically significant difference between groups (p<0.05).

Figure 7

Maximum intensity projections of the dual-energy CT gold map for hindlimb tumors in each AuNP-RT treatment group. Dual-energy CT was performed 3 days after liposomal iodine injection (5 days after gold injection). Gold nanoparticle accumulation (shown in green) appears to be the highest in the PEG-AuNP groups that also had higher iodine accumulation (10 Gy and 20 Gy). Gold accumulation in the PEG-AuNP 5 Gy group is visibly lower than the other PEG-AuNP groups. Both RGD-AuNP groups show very low gold accumulation in the tumors, despite the increased vascular permeability seen in these groups in the iodine imaging. The gold map (green) is windowed between 2.5 mg Au/mL and 40 mg Au/mL. The grayscale images (80 kVp dataset) are windowed from -100 HU to 3500 HU.

Figure 8

Tumor gold accumulation after gold nanoparticle-augmented radiation therapy. Concentrations are expressed in both mg Au/mL (primary axis) and %ID/g (secondary axis). Gold accumulation was quantified by dual-energy CT 3 days after liposomal iodine injection (5 days after gold injection). The highest gold accumulation was seen in the PEG-AuNP 10 Gy and 20 Gy groups, which also demonstrated high vascular permeability to liposomal iodine. The lowest gold accumulation was seen in the RGD-AuNP groups, despite the increased vascular permeability to liposomal iodine seen in these groups. Accumulation in the 20 Gy group was significantly higher than that of all the other groups except 10 Gy PEG-AuNP. The accumulation in the 10 Gy PEG-AuNP group was significantly higher than that of the 10 Gy RGD-AuNP group. * represents a statistically significant difference between groups (p<0.05).

Figure 9

Darkfield and immunofluorescence microscopy of tumor sections. Endothelial cells are shown in green (CD31), cell nuclei are shown in blue (DAPI) and gold nanoparticles are shown in orange. (A) Tumor section from a mouse injected with PEG-AuNPs. (B) Tumor section from a mouse injected with RGD-AuNPs. Nanoparticles are readily visualized in both groups, but the overall distribution differs. The PEG-AuNPs are spread throughout the tissue relatively uniformly, while the RGD-AuNPs are grouped together in smaller regions. In some cases, the nanoparticles appear to be located intracellularly (white arrows). The scale bar represents 50 µm and is the same in both panels.

Figure 10

Tumor volume doubling time for mice treated with RT and liposomal doxorubicin. The combined therapy with vascular-targeted RGD-AuNPs, RT, and liposomal doxorubicin showed a significant growth delay (higher doubling time) compared to all other treatment groups. The other treatment groups showed a small increase in tumor doubling time compared to the non-treated control mice, but these differences were not statistically significant.