Transferring biomarker into molecular probe: melanin nanoparticle as a naturally active platform for multimodality imaging

Abstract

Developing multifunctional and easily prepared nanoplatforms with integrated different modalities is highly challenging for molecular imaging. Here, we report the successful transfer of an important molecular target, melanin, into a novel multimodality imaging nanoplatform. Melanin is abundantly expressed in melanotic melanomas and thus has been actively studied as a target for melanoma imaging. In our work, the multifunctional biopolymer nanoplatform based on ultrasmall (<10 nm) water-soluble melanin nanoparticle (MNP) was developed and showed unique photoacoustic property and natural binding ability with metal ions (for example, (64)Cu(2+), Fe(3+)). Therefore, MNP can serve not only as a photoacoustic contrast agent, but also as a nanoplatform for positron emission tomography (PET) and magnetic resonance imaging (MRI). Traditional passive nanoplatforms require complicated and time-consuming processes for prebuilding reporting moieties or chemical modifications using active groups to integrate different contrast properties into one entity. In comparison, utilizing functional biomarker melanin can greatly simplify the building process. We further conjugated αvβ3 integrins, cyclic c(RGDfC) peptide, to MNPs to allow for U87MG tumor accumulation due to its targeting property combined with the enhanced permeability and retention (EPR) effect. The multimodal properties of MNPs demonstrate the high potential of endogenous materials with multifunctions as nanoplatforms for molecular theranostics and clinical translation.

Figure 1

Multimodality molecular imaging of MNPs. The melanin granules were first dissolved in 0.1 N NaOH aqueous solution, and then neutralized under sonication to obtain melanin nanoparticles in high water monodispersity and homogeneity. After PEG surface-modification, RGD was further attached to the MNP for tumor targeting. Then Fe³⁺ and/or ⁶⁴Cu²⁺ were chelated to the obtained MNPs for PAI/MRI/PET multimodal imaging.

Figure 2

Characterization of physical properties of MNPs. (A) From left to right: pictures of (1) pristine melanin granule in H₂O, (2) melanin neutralized without sonication in H₂O, (3) freeze-dried PWS-MNP, (4) freeze-dried PWS-MNP redissolved in PBS (pH = 7.4), (5) freeze-dried PEG-MNP, (6) freeze-dried PEG-MNP redissolved in PBS (pH = 7.4). (B) TEM of PWS-MNP (left) and PEG-MNP (right), scale bar = 20 nm. (C) The plot of the relationship between the number of metal ions attached on one MNP with feed ratio (W_ions: W_MNP). (D) Stability study of metal ion-chelated MNPs in PBS (pH = 7.4).

Figure 3

In vitro and in vivo study of PAI of MNPs. (A) The photoacoustic signal produced by PEG-MNPs at concentrations of 0.625, 1.25, 2.5, 5.0, 10, and 20 μM, and it was observed to be linearly dependent on its concentration (R² = 0.995). (B) Photoacoustic detection of PEG-MNP in living mice. Mice were injected subcutaneously (region enveloped by blue dotted line) with PEG-MNP at concentrations of 0, 5, 10 (from left to right in top row), and 20, 40, 80 (from left to right in bottom row) μM. One vertical slice in the photoacoustic image (red) was overlaid on the corresponding slice in the ultrasound image (gray). (C) The photoacoustic signal from each inclusion was calculated. The background level represents the endogenous signal measured from tissues. The linear regression is calculated on the five most concentrated inclusions (R² = 0.998). (D) The overlaying of ultrasonic (gray) and photoacoustic (red) imagings of U87MG tumor (region enveloped by yellow dotted line) before and after tail-vein injection of 250 μL of 200 μM RGD-PEG-MNP in living mice (n = 3) and their subtraction imagings. (E) Quantitative analysis of enhanced PA signal of U87MG tumor after tail-vein injection with RGD-PEG-MNP at 4 h, compared with at 0 h.

Figure 4

In vitro and in vivo study of MRI of Fe³⁺-chelated MNPs. (A) T₁ relaxation rates (1/T₁, s^–1) as a function of Fe-RGD-PEG-MNP (mM) in agarose gel (1.0 T, 25 °C). (B) MRI detection of Fe-RGD-PEG-MNPs in living mice. Mice were injected subcutaneously (region enveloped by red dotted line) with Fe-RGD-PEG-MNPs at concentrations of 0, 1.25, 2.5 (from left to right in upper layer), and 5, 10, 20 (from left to right in bottom layer) μM. (C) Quantitative analysis of enhanced MR signal of U87MG tumor after tail-vein injection with RGD-PEG-MNP at 4 h, compared with at 0 h. (D) MRI images of U87MG tumors (region enveloped by yellow dotted line) before and after tail-vein injection of 250 μL of 200 μM RGD-PEG-MNP in living mice (n = 3) (TR: 700 ms, TE: 5.2 ms). Top row shows black and white images, and bottom row shows the pseudocolored images.

Figure 5

In vitro and in vivo study of PET of ⁶⁴Cu-labeled MNPs. (A) Uptake of ⁶⁴Cu-RGD-PEG-MNP with and without blocking in U87MG cells at 37 °C for 1, 2 and 4 h incubation. All results, expressed as percentage of cellular uptake, are mean of triplicate measurements ± SD. (B) Representative decay-corrected coronal (top) and transaxial (bottom) small animal PET images (left three images) and the overlaying of CT (gray) and PET (color) images (right three images) of U87MG tumors (region enveloped by yellow dotted line) acquired at 2, 4, and 24 h after tail vein injection of ⁶⁴Cu-RGD-PEG-MNP. (C) Biodistribution of ⁶⁴Cu-RGD-PEG-MNP in mice (n = 3) at 2, 4, and 24 h after injection. The radioactive signal from each organ was calculated using a region of interest drawn over the whole organ region.

Figure 6

In vivo multimodality imaging of tumor (region enveloped by yellow dotted line) bearing mice with PAI and MRI/PET respectively. (A) Photographic images of U87MG tumor bearing mice. (B) The overlaying of ultrasonic (gray) and photoacoustic (red) imaging of U87MG tumor before and after tail-vein injection of ⁶⁴Cu-Fe-RGD-PEG-MNP (200 μL of 10 μM) in living mice and their subtraction imaging. (C) The overlaying of representative decay-corrected coronal (top) and transaxial (bottom) small animal CT (gray) and PET (color) images of U87MG tumors acquired at 2, 4, and 24 h after tail vein injection of ⁶⁴Cu-Fe-RGD-PEG-MNP and Fe-RGD-PEG-MNP (250 μL of 200 μM). (D) MRI images of U87MG tumor before and after tail-vein injection of ⁶⁴Cu-Fe-RGD-PEG-MNP and Fe-RGD-PEG-MNP (250 μL of 200 μM) in living mouse. Top row shows black and white images, and bottom row shows the pseudocolored images. White arrow refers to the tumor position.