Scientists have developed magnetoferritin nanoparticles (M-HFn) that they claim can be used to target and visualize a range of tumor types without the need for targeting ligands or contrast agents. The diagnostic imaging technology, devised by a multidisciplinary research team at the Chinese Academy of Sciences, effectively involves encapsulating iron oxide nanoparticles in a recombinant human heavy-chain ferritin (HFn) protein shell, which binds to tumors that overexpress the transferrin receptor 1 (TfR1). Once bound, the iron oxide catalyzes the oxidation of an added peroxidase substrates in the presence of hydrogen peroxide (H2O2), resulting in a color-forming reaction that can be visualized.
Reporting their technique in Nature Nanotechnology, Xiyun Yan, Ph.D., and colleagues say tests on 474 clinical specimens from patients with nine different types of tumors showed that the nanoparticles could distinguish cancerous cells from normal cells with a sensitivity of 98% and specificity of 95%. Their paper is titled “Magnetoferritin nanoparticles for targeting and visualizing tumor tissues.”
Ferritin is composed of multiple subunits of HFn and light-chain ferritin and has a spherical structure with an inner cavity. Recent studies have shown that HFn binds to human cells via TfR1, which is overexpressed in tumor cells and thus represents a targeting marker for tumor diagnosis and therapy, the authors explain. Current HFn-based tumor detection methods involve functionalizing the particles with recognition ligands and signal molecules.
In contrast, the Chinese Academy scientists devised an approach that would negate the need for ligands and signaling molecules, by combining the tumor-targeting feature of HFn with their own previous work that had demonstrated how iron oxide nanoparticles can catalyze the oxidation of peroxidase substrates in the presence of hydrogen peroxide to generate colour reactions. The overall concept was that magnetoferritin nanoparticles produced by encapsulating iron oxide nanoparticles inside an HFn shell should be able to target tumor-expressed TfR1 without needing to add tumor targeting ligands and highlight the tumor tissues through the peroxidase activity of the iron oxide core.
The team first generated recombinant human HFn in E. coli. and loaded the protein shells with iron, which they then oxidized using H2O2. Analyses indicated the resulting M-HFn particles comprised an intact protein shell of about 12–16 nm diameter, with an inner iron oxide core of about 4.7 nm. They then demonstrated that the M-HFn nanoparticles catalyzed the oxidation of the peroxidase substrate di-azo-aminobenzene (DAB) in the presence of H2O2 to give a brown color.
The team’s initial tests on a wide range of different cancer cell lines and xenograft tumors showed that HFn itself binds specifically to tumors that express TfR1 but not to those that don’t express the transferrin receptor. In support of the specificity of binding the researchers confirmed that HFn bound to TfR1 immunoprecipitated from cancer cell lysates and that the binding of HFn to TfR1 on cancer cells could be blocked completely when an anti-TfR1 antibody was added.
To demonstrate the potential utility of M-HFn nanoparticles for clinical cancer diagnostic applications, the team first carried out fluorescence staining of xenograft tumors with FITC-conjugated HFn. They then loaded the HFn particles with iron and added the DAB substrate and H2O2 to oxidize the iron. As hoped, the M-HFn staining co-localized with the fluorescence staining. This indicated that neither the iron loading nor fluorescence labeling affected the tumor-binding activity of the HFn protein and added further support to the feasibility of using M-HFn staining as an assay for tumor detection.
Notably, when the investigators compared the M-HFn nanoparticle method with traditional immunohistochemical staining using anti-TfR1 antibodies on xenograft tumor tissues, they found that both methods generated an almost identical intensity and pattern of staining “demonstrating the accuracy of tumor detection by the M-HFn nanoparticles.”
The team then used the M-HFn nanoparticles to screen 247 clinical tumor tissue samples and 227 normal tissue samples. They found that while M-HFn either didn’t stain or only very slightly stained normal or lesion tissues, the magnetoferritin nanoparticles strongly stained tumor cells, and there was a clear distinction between cancerous cells and adjacent normal cells. Again, the staining patterns were consistent with those obtained when the tissue specimens were further stained with FITC-conjugated HFn protein shells.
Importantly, M-HFn showed distinct staining reactions in different grades and growth patterns of a number of tumor types including hepatocellular carcinoma, lung squamous cell carcinoma, cervical squamous cell carcinoma, prostate adenocarcinoma, ovarian serous papillary carcinoma, and colonic adenocarcinoma.
“This study suggests that the easily synthesized M-HFn nanoparticles have the potential to become a diagnostic tool for rapid, low-cost, and universal assessment of cell cancerization,” the authors conclude. They claim that the approach has a number of advantages when compared with conventional antibody-based histological methods for cancer detection. These include much high sensitivity and specificity, improved ability to distinguish tumor tissue from normal tissues, and the requirement for far fewer manipulation steps, as the M-HFn nanoparticle-based method requires just a one-step incubation of one reagent.
Cost is another major advantage, given that HFn can be produced in E. coli at high yield, and the M-HFn nanoparticles can be mass-produced by simply oxidizing the encapsulated iron using H2O2. “Our method also has the advantage of a rapid examination time, taking one hour, rather than the four hours required for immunohistochemistry, which generally involves multistep incubation of primary antibody, secondary antibody, or enzyme-labeled third antibody,” the researchers add. “Our studies show that one-step tumor targeting and visualization with low-cost and mass-produced M-HFn nanoparticles is feasible for convenient and sensitive monitoring and analysis of tumor cells in tissue specimens."