Recent developments in synthesis, physicochemical characterization and molecular recognition of MNPs for molecular imaging and therapeutic applications of the MNPs make them as sound theranostics for targeted delivery of drugs and genes, in large part due to their toxicological and metabolic reactions are predictable.2,67-70 MNPs with distinctive features (e.g., high surface, quantum specificities, and small size) are significantly useful for optical and magnetic imaging. In addition, these NSs prevent from accumulation even in intense magnetic platform and have favorable capability to use as magnetic fluid preparation.71 MNPs, especially as small or ultra-small superparamagnetic iron oxide NPs (SPIONs and USPIONs, respectively) offer unique external tuning that is very important for biomedical applications and improvement of thermal therapy with low toxicity.72,73 Effective surface conjugation of biocompatible MNPs with Abs, Aps, proteins and enzymes offers the possibility of using these NPs in specific targeting of diseased tissues such as tumors.74,75 It should be pointed out that the SPIONs with hydrophobic surface can be accumulated within the healthy liver tissue as a consequence of their rapid absorption by macrophages and Kuppfer cells of reticuloendothelial system (RES). Minimized RES assimilation is achieved by conjugating of SPIONs with different biomaterials and hydrophilic polymers (e.g., PEG) that improves their blood half-life and enhanced permeability and retention (EPR) effect and consequently particular accumulation in tumor cell may occur.57,76
PET or SPECT imaging with high quantification is achieved by radio labeling SPIONs with nuclear medicine isotopes as imaging agent.77,78 Further, high relaxivity in MRI imaging with higher safety than gadolinium based MRI allows SPIONs to be applied as contrast enhancement agents in MRI.79
MNPs for PET imaging
PET as a nuclear imaging method provides the opportunity for monitoring different biological and/or physiological phenomena in living systems through administration of radiolabeled probes.80 To date, various types of magnetic NPs have been functionalized with suitable radionuclides for PET imaging.81 A cyclotron like18F, 11C, 64Cu, 124I, 86Y, 15O and 13N, or a generator like 68Ga as positron-emitting radioisotopes have been used in PET imaging technique to monitor emitted γ rays.
Peptide receptors have potential to overexpress in human cancer cells and could be used as molecular targets for radiolabeled peptides for early detection of tumor angiogenesis. Atrial natriuretic peptide with anticancer feature interacts with cell surface natriuretic peptide receptor A and natriuretic peptide clearance receptor, which expressed in tumor angiogenesis and consequently could be used in therapy and PET imaging. PET imaging of C-type atrial natriuretic factor (CANF) peptide conjugated with polymeric NPs as amphiphilic comb-like NPs (64Cu-CANF-Comb) has the potential to apply as a prognostic marker to target the prostate cancer cells.82 Given that the tissue macrophages are very important for standard physiological and disease circumstances because of their wide distribution, a number of investigations have been conducted to analyze the macrophages functions in cancer.83-85 In anticipation that coordinated NPs are able to prolong the circulation times to assimilate into macrophages, NP-based PET/CT and PET/MRI have been developed.85-87 In a study, Zirconium-89 (89Zr) with long half-life has been opted as PET isotopes for imaging. Quantitation of tumor-associated macrophages have been accomplished by injection of radiolabeled dextran-coated MNPs that have significant macrophage avidity.88 Fig. 3 illustrates the in vivo micro-images of PET and CT and multimodal NPs used for PET imaging.
Fig. 3. The in vivo micro-images of PET and CT and multimodal nanoparticles used for PET imaging. Panels A and B represent PET and CT images, respectively (our unpublished data). Panels C and D show multifunctional magnetic and gold nanosystem, respectively. Positron emission tomography. CT, computed tomography; MNP, magnetic nanoparticle; GNP, gold nanoparticle; Ab, antibody; Ap, aptamer.
Given that the tumor-associated macrophages (TAMs) are extremely important in solid tumors with great diagnostic and prognostic as well as targeting values,89 in a study, 89Zr-labeled high-density lipoprotein (HDL) NPs-facilitated PET imaging has been performed for detection of TAMs in orthotopic mouse model of breast cancer. HDL NPs were formulated using phospholipids and apolipoprotein A-I and conjugated with 89Zr complexed with deferoxamine. Intravenous administration of these NSs resulted in profound tumor radioactivity 24 h after injection, which was revalidated with histologic analysis showing high colocalization in the TAM-rich tumor sections. Based on such findings, the researchers recommended these NSs for quantitative detection of TAMs as noninvasive monitoring strategy in solid tumors.85
MNPs as dual modality for imaging and targeted therapy
In addition to imaging techniques (e.g., MRI, PET and CT), combination of molecular imaging modalities have been developed and shown to provide synergistic advantages in early-stage diagnosis of malignancies with more detailed information to clinicians in comparison with any single imaging modalities. Overall, MRI is considered as a noninvasive imaging strategy with wide applications in clinic, in large part because of plausibility to modify SPIONs with different biomolecules and decorate them with specific agents (Fig. 3). In addition to being suitable tissue contrast agent, they can be used for monitoring of tumor progression, while providing possibility for accumulation of NPs in tumor side and on-demand liberation of anticancer drug molecules using an external stimuli. All these feature are due to large surface of SIONPs, and possibility for surface functionalization make them unique NSs.91 It should be stated that the dual- modality imaging NPs can be employed through either passive or active targeting mechanism. The passive targeting is the accumulation of drug at a tissue with physicochemical or pharmacological aspects through the EPR effect, while the uptake of NPs by the kupffer cells of the liver or RES can affect the end point aim of the treatment. In the active targeting mechanism, NPs are armed with a homing agent to specifically target the designated diseased cells. In fact, the delivery of NPs to the metastatic solid tumors demands specific and active targeting approach using Abs/Aps to target the cancer antigens.92 In the following context, we will discuss some of these approaches.
In vivo integrity of stabilized SPIONs in the systemic circulation directed researchers to use them in nuclear medical imaging for dual modality imaging purposes. Radiolabeled MNPs like MRI contrast agents have longer blood shelf life, superior sensitivity, fewer adverse effect, and larger numbers of radionuclides in comparison with gadolinium-based pharmaceuticals. Designing MNPs with various physicochemical properties make development of multimeric NSs possible, which can be further modified with different moieties. MNPs, as robust core, offer significant plausibility for surface modification while showing excellent optical properties, which make them one of the best candidates for dual molecular imaging and targeted therapy.93 Development of Cu(II)-labeled MNPs covered by porous silica shell (SPION@SiO2) have been considered as PET/MRI contrast agent, which has been tested as stem cells labeling agent. This NS displayed great biocompatibility, relaxivity, low toxicity in long-term in vivo uses. These SIPON-based NS was successfully used for the detection and tracking of stem cells, which was also reported as robust technique for the diagnosis of anomalous cells.94 In a study, MNPs were modified to display greater degrees of hydrophilicity through deposition of Al(OH)3 layer, which resulted in production of a bimodal contrast agents to be used in PET/MRI imaging after radiolabeling with copper-64 (64Cu). High affinity of aluminum hydroxide with fluoride anions and bisphosphonate groups (64Cu-bisphosphonate) offers a simple method of radiolabeling and functionalization with high biocompatibility. Small size, controllable surface potential, superior colloidal stability and fine transverse relaxivity were the properties of this NS as a theranostic probe.95
Recently, MNPs were modified and with PEG decorated with 125I radionuclide and 3H11 Ab, which were examined for detection of xenografted tumors. As a dual-modality molecular probe, this MNP-based NS was successfully used for the MRI/SPECT anatomical and functional images.96 In another study, PEGylated MNPs were stabilized with oleic acid and phospholipids to improve its aqueous dispersibility. To engineer MRI/SPECT/PET three-modality imaging probe, they were radiolabeled with Indium-111 (111In), 59Fe (to label the iron oxide core) and 14C to label the oleic acid used in shell. The bio-distribution studies showed that despite detection of 111In in reticuloendothelial organs, 59Fe analysis showed a greater level than111In in liver and spleen, while analysis of 14C demonstrated lower levels.97
As another case, PEGylated SPION probe was developed for dual modality PET/MRI imaging by modification of MNPs with phospholipids and 64Cu radioisotope. The 64Cu-SPION probes appeared to offer great imaging possibility in detection of atherosclerosis and cancers, showing desirable stability in mouse serum.98 A multifunctional and water-soluble PEGylated SIPON was recently developed and used for PET/MRI dual modality imaging and specific delivery of doxorubicin (DOX) through targeting tumor-associated marker, integrin avb3. The NS offered great PET imaging potential studied through biodistribution and higher accumulation in tumor sites, and also resulted in an enhanced sensitivity in terms of MRI detection while specifically delivering the DOX molecules to the anomalous tumor cells.99
PET/MRI dual-modality molecular probe by specific chelator-free radiolabeling approach and PEGylated SPION with 69Ge (69Ge-SPION@PEG) was fabricated by Chakravarty et al. PEG modification enhanced the in vivo and in vitro stability of the NS in the serum, while notable uptake of NPs in the liver and spleen was observed after intravenous injection in normal BALB/c mice analyzed by PET imaging technique. Additionally, the liver uptake of 69Ge-SPION@PEG was verified by in vivo MRI. Noninvasive PET/MRI dual-modality sentinel lymph nodes mapping was also investigated by PET and MRI scans separately that showed accumulation of 69Ge-SPION@PEG in the popliteal lymph node. Therefore, this chelator-free method was proposed as proper strategy for production of multifunctional theranostics.100
Thorek et al. established a multimodal NS based on 89Zr-ferumoxytol MNPs, and examined its potential in noninvasive mapping of lymph nodes by PET/MRI dual-imaging technique. The desferrioxamine chelate (DFO) was linked to ferumoxytol with a valuable toxicity outline to produce the 89Zr-DFO-labelled ferumoxytol (89Zr-ferumoxytol). The engineered NS was tested through PET/CT, MRI and PET/CT/MRI imaging modalities, which demonstrated high sensitivity and resolution delineation of the nodes following an axillary drainage in naive mice, healthy and prostates tumor suffering mice. Upon such findings, it seems that the engineered NS can be translated to clinical applications towards lymph nodes imaging in deep tissues.101
A MNP-based multimeric NS has recently been developed by Tsiapa et al. In this study, MNPs were modified with aminosilane and conjugated with cyclic Arg-Gly-Asp-D-Phe-Lys (cRGDfK) and an ornithine-modified peptide (cRGDfK-Orn3-CGG), which was labeled with 99mTc. Given that cRGDfK is RGD derivate, 99mTc-cRGDfK-Orn3-CGG nanosystem was examined as a targeted tumor molecular imaging and thermal therapy agent in both normal and alphanubeta3-positive tumor (U87MG glioblastoma) bearing mice. It was found the NS was able to showed high affinity to the integrin αvβ3 receptor with absorbing specificity in several tumors cells. A hyperthermia session was applied in vivo in a U87MG glioblastoma tumor bearing animal model treated with the NS, showing their accumulation and marked impacts in tumor side. This NS with high specific targeting potential was proposed as a suitable agent for dual modality SPECT/MRI imaging through targeting integrin αvβ3.102
For targeted imaging of cancer, Zolata et al. developed a NS based on minosilane-PEG coated SPIONs armed with trastuzumab (an Ab specific to HER2) and loaded with DOX, which was radiolabeled with 111In radionuclide. SPECT/MRI analyses showed that the NS was able to accumulate within the TME through passive targeting mechanism (EPR effect) and target the HER2-possitive cancer cells though active targeting mechanism. SPECT/MRI imaging and therapeutic evaluation in HER2-possitive BALB/c mice bearing breast tumor indicated that the functionalized NS maintained its magnetic properties and ability to target HER2 over expressing in tumors, as a result they could be applied as dual-modality imaging agent. The suitable coating and active targeting by trastuzumab together with 111In-based PET imaging and SPION-based MRI imaging made the NS as a stable and long-circulating robust agent, while the accumulation of NPs in the TME and controlled release of DOX molecules in the tumor site make it a vigorous anticancer agent.90
Bisphosphonates (BPs) are well-known drugs in the osteoporosis and oncology because they could bind avidly to the surface of metabolically active bone. Conjugation of bisphosphonates to the surface of SPIONs and decoration with radionuclides make them suitable for SPECT/PET-MRI imaging. In a study, this NS systems was conjugated with 99mTc-dipicolylamine-alendronate and used as dual-modal imaging agent in comparison with other agents such as Endorem®/Feridex® used as MRI contrast agent. Co-localization of 99mTc and Endorem® in the RES system confirmed by in vivo MRI and SPECT-CT imaging demonstrated that the engineered bimodal imaging nanoprobe displayed high stability in the blood with substantial sensitivity.103 Radiolabeled bisphosphonate-USPIONs armed with 99mTc-DTPA have also been used for in vivo tracking of the NPs by SPECT/PET/MRI imaging. The engineered NS demonstrated a great sensitivity and quantification properties in mice treated with the NS, showing strong T1-effect which is related with longer blood circulation and enhanced the signal.104
Further, the vulnerable atherosclerosis plaque rupture-induced acute obstructive vascular diseases are attributed to relatively high morbidity and mortality worldwide, hence accurate diagnosis and detection of the atherosclerosis onset and/or progression seems to be essential. To tackle this, in a study, USPIONs were decorated with diethylenetriaminepentaacetate acid (DTPA) and PEG and labeled with 99mTc to produce a multimodal NS (99mTc−DTPA−USPION−Annexin V) that is tested by some in vitro examinations and SPECT/MRI imaging. Given that Annexin V is able to target the apoptotic macrophages abundant in vulnerable plaques, the accumulation of the NSs in the plaques was confirmed by both in vitro and in vivo experiments.105
Fluorescence quenching ability of optical dyes and QDs has also been exploited for development of a new class of MNPs. An enzyme responsive SPION-based NSs grafted with optical dyes have also good potential as imaging agents that could be activated during imaging process. Such system as a MRI and NIRF optical imaging probe has been engineered and used for cancer diagnosing based on fluorescence quenching ability of iron oxide.106 Similarly, a hybrid multimeric NS based on MNP has been engineered (64Cu-NOTA-Au-MNP-affibody nanoprobe) by Yang et al. It was used for targeted cancer imaging in the human EGFR expressing cells and tumors, and resulted in high quality micoimages obtained by PET/Optical/ MRI imaging techniques. In vivo and in vitro studies demonstrated that the affibody-targeted Au-MNPs system is a robust nanoprobe that can be further modified with targeting molecules (Abs/Aps specific to cancer antigens such as EGFR) and PET imaging reporters (e.g., 64Cu) and used for target-guided diagnosis.16
To establish a nanoprobe for PET and MRI imaging, Pombo-Garcia et al. reported on development of a bimodal USPIONs probe coated with octylamine-modified polyacrylic acid (OPA) as an amphiphilic polymer with the subsequent conjugation with a new amino pendant-bearing derivative of the 64Cu (II) chelator, N-(4-aminophenyl)-2-[4, 7-bis(2-pyridylmethyl)-1,4,7-triazacyclononan-1-yl]-acetamide (amino-dmptacn). It should be noted that the OPA make MNPs to be water-dispersible with high colloidal stability, which also provides a good potential for further decoration of NPs with different entities. The biocompatibility and cellular uptake of the functionalized USPIONs were evaluated in normal and tumor cell lines. Results showed a cell type and time-dependent internalization of the OPA-USPIONs, and substantial stability in the serum, which indicated that the NS may be considered as a bimodal PET-MRI tumor imaging agent.107
Another radionuclide-labeled NS was formulated based on 64Cu (II) silica-coated MNPs and tested for its potential to serve as an imaging nanoprobe for bimodal recognition of tumors by MRI and PET. Following the covalently binding of MNPs to the synthesized siloxane derivatives of 1,4-bis(2-pyridylmethyl)-1,4,7-triazacyclononane (dmptacn), 1,4,8,11-tetraazacyclotetradecane (cyclam) or 1,4,7,10-tetraazacyclododecane (cyclen), they were factionalized with 64Cu2+. The macrocycle-containing NPs with mean hydrodynamic diameter, aggregated in buffered aqueous solution with the radioactivity persistence in the rat plasma. Nanoparticle tracking analysis of cyclam-functionalized NPs confirmed the radio copper complexes high stability, where the dmptacn-functionalized NPs performed the highest resistance to metal ion leakage that provides a robust nanoprobe for multimodal cancer dual (MRI/PET) imaging and hyperthermia treatment of cancer.108