Characterization of a novel MR-detectable nanoantioxidant that mitigates the recall immune response

Abstract

In many human diseases, the presence of inflammation is associated with an increase in the level of reactive oxygen species (ROS). The resulting state of oxidative stress is highly detrimental and can initiate a cascade of events that ultimately lead to cell death. Thus, many therapeutic attempts have been focused on either modulating the immune system to lower inflammation or reducing the damaging caused by ROS. Berlin et al. reported the development of a novel nanoantioxidant known as poly(ethylene glycol)-functionalized-hydrophilic carbon clusters (PEG-HCCs). They showed that PEG-HCCs could be targeted to cancer cells, utilized as a drug delivery vector, and can even be visualized ex vivo. Our work here furthers this work and characterizes Gd-DTPA conjugated PEG-HCCs and explores the potential for in vivo tracking of T cells in live mice. We utilized a mouse model of delayed-type hypersensitivity (DTH) to assess the immunomodulatory effects of PEG-HCCs. The T1 -agent Gd-DTPA was then conjugated to the PEG-HCCs and T1 measurements, and T1 -weighted MRI of the modified PEG-HCCs was done to assess their relaxivity. We then assessed if PEG-HCCs could be visualized both ex vivo and in vivo within the mouse lymph node and spleen. Mice treated with PEG-HCCs showed significant improvements in the DTH assay as compared to the vehicle (saline)-treated control. Flow cytometry demonstrated that splenic T cells are capable of internalizing PEG-HCCs whereas fluorescent immunohistochemistry showed that PEG-HCCs are detectable within the cortex of lymph nodes. Finally, our nanoantioxidants can be visualized in vivo within the lymph nodes and spleen of a mouse after addition of the Gd-DTPA. PEG-HCCs are internalized by T cells in the spleen and can reduce inflammation by suppression of a recall immune response. PEG-HCCs can be modified to allow for both in vitro and in vivo visualization using MRI. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.

Keywords: MRI; PEG-HCCs; T1 agents; antioxidant; cell labeling; inflammation; nanotechnology; oxidative stress.

Figure 1

PEG‐HCCs are immunomodulating. C57Bl/6 J mice were treated chronically for a period of 5 weeks with either PEG‐HCC or vehicle. Ear swelling after OVA challenge shows that chronic treatment with PEG‐HCCs resulted in significantly reduced inflammation compared to those treated with vehicle. *p < 0.05.

Figure 2

[Gd]DTPA‐PEG‐HCCs have increased signal intensity compared to PEG‐HCCs alone. T ₁‐weighted scans and T ₁‐times of [Gd]DTPA‐PEG‐HCCs, PEG‐HCCs and appropriate controls were assessed in 0.2‐mL tubes at room temperature. The signal intensity of phantoms containing [Gd]DTPA‐PEG‐HCC is comparable to that of Magnavist® compared to that of phosphate‐buffered (PBS), water or PEG‐HCC alone. T ₁‐times were also measured, and the T ₁‐times of [Gd]DTPA‐PEG‐HCC is also decreased compared to PEG‐HCC alone. 1‐PEG‐HCC, 2‐[Gd]DTPA‐PEG‐HCC, 3‐Water, 4‐PBS, 5‐Magnevist (5 mM). **p < 0.01, ***p < 0.001.

Figure 3

[Gd]DTPA‐PEG‐HCCs have increased relaxivity compared to PEG‐HCC alone. Relaxivity was measured using two different batches of [Gd]DTPA‐PEG‐HCC. There was little variation to be seen within the two samples, and the relaxivity was measured to be approximately 9.5 mM ⁻¹  s ⁻¹ with a correlation coefficient of 0.9895. PEG‐HCC alone, however, did not show any concentration dependent changes in T ₁‐times.

Figure 4

[Gd]DTPA‐PEG‐HCCs accumulate in draining lymph nodes. Twenty‐four hours post‐injection, [Gd]DTPA‐PEG‐HCCs can be seen visually to accumulate in the draining lymph nodes (axiliary lymph nodes) after subcutaneous injection at the base of the tail in C57Bl/6 J mice.

Figure 5

[Gd]DTPA‐PEG‐HCC‐treated lymph nodes can be visualized ex vivo with MRI. Lymph nodes were embedded in 1% agarose and were imaged using T ₁‐weighted MRI. Shown above are the [Gd]DTPA‐PEG‐HCC treated lymph node (top), vehicle‐treated lymph node (bottom), which were both embedded in 1% agarose, and water in a PCR tube (middle). The first column of images demonstrate that the [Gd]DTPA‐PEG‐HCC treated lymph node shows increased T ₁‐signal intensity along where there is accumulation of [Gd]DTPA‐PEG‐HCC, while this increase in signal is not observed in the vehicle‐treated lymph node. This signal intensity increase is further demonstrated with the application of a color map (second column) has been applied to indicate increased levels of signal intensity. The hotter (red) colors indicate higher T ₁‐signal intensity, wherea colder colors (blue) indicate a lower T ₁‐signal intensity.

Figure 6

[Gd]DTPA‐PEG‐HCC accumulation can be seen in lymph nodes. [Gd]DTPA‐PEG‐HCC‐treated lymph nodes show a bright band along their edge. Twenty‐four hours post‐injection, mice were imaged using T1‐weighed scans through their inguinal lymph nodes. Image (a.) is is the lymph node of a vehicle‐treated mouse, whereas image (b.) is the lymph nodes from a [Gd]DTPA‐PEG‐HCC‐ treated mouse. Increased signal intensity can be visualized along the edge of the [Gd]DTPA‐PEG‐HCC‐ treated lymph node, however, no such signal intensity increase can be discerned in the vehicle‐treated lymph node.

Figure 7

Plot profile through the lymph node shows increased signal intensity (SI) on the edge of the lymph node. An increase in SI can be seen along the edge of the lymph node treated with [Gd]DTPA‐PEG‐HCC on the bottom compared to the vehicle‐treated lymph node shown on the top. Images were initially normalized to water phantom and then a Gaussian filter was applied to increase the contrast of the lymph nodes.

Figure 8

Accumulation of [Gd]DTPA‐PEG‐HCCs is seen in the lymph node cortex around the germination centers. [Gd]DTPA‐PEG‐HCCs (red) accumulate in the cortex, including the area surrounding germination centers within lymph nodes as shown in (a). This area is adjacent to the T‐cell zone as evidenced by positive CD3 staining (green). Positive [Gd]DTPA‐PEG‐HCC staining is absent in images taken from vehicle treated lymph nodes as shown in (b). All images are 20X magnification. A schematic of the lymph node is shown in (c) to indicate relevant regions where positive [Gd]DTPA‐PEG‐HCC staining was observed.

Figure 9

T ₁ maps of the spleen demonstrate lower T ₁ values in animals treated with [Gd]DTPA‐PEG‐HCC compared with saline. T ₁ maps of [Gd]DTPA‐PEG‐HCC‐treated mice show lower values in the spleens compared with those treated with saline. Twenty‐four hours post‐injection, mice were imaged using T ₁‐weighed scans through the spleens. Image (a) two mice treated with saline, (b) two mice treated with [Gd]DTPA‐PEG‐HCC. In column 1, anatomical scans are presented. In column 2, T ₁ maps are presented. The insets are zoomed in views of the spleen for both sets of data. Column 3 is a histogram generated in PV 5.1 within a region of interest in the spleen only. Note that the T ₁ values are shifted lower in the PEG‐HCC‐treated animals. Decreases in the T ₁ can be visualized throughout the [Gd]DTPA‐PEG‐HCC‐treated spleens although it is not uniform. The T ₁ maps do not demonstrate nearly the degree of decreased T ₁ values in the vehicle‐treated spleens as shown in the histograms.

Figure 10

Splenic T cells take up PEG‐HCCs. As shown in (a), the T cell surface marker CD3 was used to isolate a purified population of T cells from vehicle and PEG‐HCC treated animals. These T cells were then assessed for the presence of PEG‐HCCs as shown in (b) Among PEG‐HCC‐treated samples, permeabilized samples had significantly higher PEG positive cells than what was observed in intact samples. n = 3 per group. *p < 0.05.