1.4.2. Silica-Based Nanoparticles

Silica-based NPs are widely applied in drug delivery, bio-imaging, and cell targeting as they are considered an ideal biocompatible matrix to integrate imaging probes. There are two major classes of silica-based NPs: solid (SiNPs) and mesoporous (MSNs). SiNPs are extensively used as optical imaging agents, while MSNs are often used in CT, MRI, PET, molecular, and multimodal imaging. MSNs are synthesized by a surfactant templated sol–gel method [98] and have attractive properties such as an extremely large surface area, a tunable structure in terms of size, morphology, and porosity, and ease of functionalization through synthetic approaches [99]. In bimodal PET/MRI imaging, MSNs are used as a coating material for metallic NPs or as carrier for MRI CAs and PET radioisotopes. CAs can be encapsulated in channels and protected from the environment, together with drugs or genes for theranostic purpose. A porous silica shell improves MRI contrast enhancement since the pores allow intimate contact between water molecules and the iron-oxide NPs [100]. We report two examples of MSNs in PET/MRI for cancer imaging. Burke and colleagues [101] described silica-coated

iron-oxide-based nanorods radiolabeled with 68Ga. In detail, nanorods were coated with various ratios of a siloxane-terminated tetraazamacrocycle (siloxane-DO3A) and a siloxane PEG derivative. Nanorods offer some advantages over nanospheres such as improved T2 MRI contrast and direct uptake in the liver via phagocytosis. Moreover, thanks to the silica coating, a macrocyclic chelator for highly stable radiolabeled nanoconstructs was not required. Huang and coworkers [102] reported a mesoporous silica-based triple modal imaging nanoprobe to map and track tumor metastatic sentinel lymph nodes (T-SLNs). In this system, three imaging probes including near-infrared (NIR) dye ZW800, T1 CA Gd-DTTA, and the positron-emitting radionuclide 64Cu were integrated into MSNs via different conjugation strategies. PET and MRI imaging probes were located on the surface and in the mesoporous channel of NPs. A faster uptake rate and higher uptake of the multifunctional MSN probes were observed in T-SLNs compared with normal SLNs, confirming the feasibility of these MSN probes as CAs to map SLNs and identify tumor metastasis. Images revealed that NP accumulation in T-SLNs was much higher than in normal controlateral SLNs (N-SLNs), where almost no signal was observed.

#### 1.4.3. Organic Nanoparticles

Over the last decade, a number of organic NPs, such as dendrimers, polymeric micelles, liposomes, and proteins were used in various applications for cancer diagnosis. These organic NPs carry imaging moieties such as radionuclides and show potential for tumor diagnosis [78]. Liposomes are spherical phospholipid bilayers similar to a cell membrane. Phospholipids are amphiphilic molecules as they have a hydrophilic head group and two hydrophobic tails; thus, they present an inner aqueous compartment that can encapsulate hydrophilic molecules, while hydrophobic agents can be inserted in the lipid shell. Liposomes can be classified by size or by the number of bilayers. In fact, they can also present more than one bilayer; these multilamellar constructs are characterized by an onion structure where each bilayer of phospholipids is separated from the adjacent by a water layer [82]. Among the various protocols for preparing liposomes with different size and number of layers, the most established are based upon sonication and extrusion [82]. Functional moieties can be attached on the bilayer membrane surface. They are biocompatible, non-toxic, and biodegradable, and they are extensively used for drug delivery. After PEGylation, the blood circulation time of liposomes can be prolonged for sustained release or targeted delivery of imaging and therapeutic agents [44]. Liposomes can exploit the EPR effect or active targeting with antibodies, peptides, and vitamins to reach cancer cells [103]. To date, several liposomal formulations were approved for cancer therapy, mainly loaded with doxorubicin, and treatment of infections such as fungal infections; a few anticancer-loaded liposomes are currently undergoing clinical trials [104].

In MRI imaging, liposomes can be used as a coating material to prevent iron-oxide NPs from aggregating and to target tumor cells. They are an excellent platform for multimodal imaging and theranostic application. As an example, Malinge et al. [85] realized magnetic liposomes by incorporation of iron-oxide NPs in the liposomal aqueous core. Liposomes were radiolabeled through a 68Ga-based radiotracer allowing a dual-modality tracking of particle in vivo distribution through MRI and PET imaging; in addition, glucose was grafted onto the NP surface. On U87MG-bearing mice, the magnetic characteristic of the liposomes and the superficial presence of the glucose enabled a dual tumor-targeting mechanism. Through an external magnet, particles were driven in the tumoral region, and the Warburg mechanism allowed their preferential interaction with tumoral cells. In addition, this study confirmed the role of the lipid bilayer in regulating the exchange of water molecules from the external environment to the aqueous core and the consequent increase of the iron-oxide NPs relaxivity r2, improving MRI performance. In another study, Li and colleagues [105] constructed a multifunctional theranostic liposomal drug delivery system; liposomes encapsulated doxorubicin and were conjugated with Gd-DOTA for MRI and IRDye for near-infrared fluorescence. Liposomes were also radiolabeled with 99mTc and 64Cu for SPECT and PET imaging. After intratumoral injection, MR images displayed, with high resolution, the micro-intratumoral distribution of the liposomes in squamous cell carcinoma of head and neck tumor xenografts in nude rats. NIR fluorescent, SPECT, and PET images confirmed MRI findings. In addition, these multifunctional liposomes have the potential for the accurate monitoring and in vivo delivery of liposomal chemotherapeutic drugs or therapeutic radionuclides such as 186Re/ 188Re. Mitchell et al. [106] prepared liposomal formulations with short *n*-ethylene glycol spacers of varying length; multifunctional imaging was gained through a chelator (DOTA) in the head group of lipids, thereby chelating Gd3<sup>+</sup> for MRI, 111In for SPECT, and 64Cu for PET. Compared to conventional PEG shielded liposomes (DSPE-PEG2000), this system showed good cellular internalization in tumor cells and similar distribution and blood half-lives. Abou and colleagues [107] radiolabeled preformed paramagnetic (Gd) liposomes with 89Zr (positron emitter). The authors used a chelator-free strategy thanks to the radiometal affinity for the lipid phosphate head groups; this dual mode CA was conjugated with octreotide to selectively target neuroendocrine tumors via human somatostatin receptor subtype 2 (SSTr2). MR and PET images revealed significantly greater accumulation of octreotide liposomes to SSTr2-expressing cells compared to control liposomes.

Like liposomes, micelles are also characterized by a core/shell structure but, unlike liposomes, the core can also be hydrophobic while the shell is hydrophilic. Micelles can be made of nonionic surfactants (surfactant micelles) or of amphiphilic block copolymers (polymeric micelles). In polymeric micelles, the length of the hydrophilic block exceeds that of the hydrophobic one, thus resulting in spherical shapes [88]. Polymeric micelles were greatly investigated for delivering hydrophobic drugs; moreover, they have a smaller size compared to liposomes, and the hydrophilic shell reduces interactions with macrophages [108]. Because of the hydrophilic nature of CAs, they can be bound to the hydrophilic blocks or covalently conjugated to the hydrophobic lipid chain in order to be incorporated into micelles [109]. A special group of polymeric micelles can be synthesized by the conjugation of water-soluble copolymers with lipids constituting the hydrophobic blocks (such as polyethylene glycol–phosphatidyl ethanolamine, PEG–PE). The main feature that makes PEG–lipid micelles attractive for diagnostic imaging applications is their size [88]; in fact, due to the lipid bilayer curvature limitation, it is not possible to prepare liposomes that are smaller than a certain minimal diameter (usually, 70–100 nm) [110]. Such a vector was realized by Trubetskoy and colleagues [111]; Gd-DTPA-PE and 111In-DTPA-SA were incorporated into 20-nm PEG–PE micelles to visualize lymph nodes during percutaneous lymphography using gamma scintigraphy and MRI imaging in rabbits. A recent study by Starmans et al. [112] provided a PET/MRI dual imaging polymeric micellar system consisting of self-assembling amphiphilic diblock copolymers functionalized with 89Zr deferoxamine and Fe3<sup>+</sup> deferoxamine. In vivo PET and MRI images clarified tumor visualization thanks to the EPR effect. However, both liposomes and micelles are unstable, especially in the presence of serum, and, for this reason, many authors crosslink them to achieve better stability [94,95].

Dendrimers are a group of highly branched spherical polymers with a tree-like internal structure. They are characterized by an inner core surrounded by a number of branches called *generations*. Depending on the number of generations, they vary in size and molecular weight. CAs or drugs can be encapsulated in the inner spaces or anchored on the external terminations [113]. To date, dendrimers as dual modal agents are used for MRI and fluorescence [114,115], optical imaging and nuclear medicine [116], CT, and MRI [117].

However, few studies on positron-emitting radionuclide-labeled dendrimers were reported [118,119], and when such dendrimer platforms are used to develop PET/MRI or SPECT/MRI agents, it is challenging to achieve precise control of radioisotope loading into specific chelating moieties [120]. Indeed, to our knowledge, studies about dendritic formulations for combined PET/MRI remain to be published.

In recent years, the biomimetic approach gained increasing interest in the scientific community, and many scientists are trying to mimic what naturally occurs in the body in order to obtain more biocompatible and biodegradable materials for medical applications. The crucial idea behind the biomimetic approach is that a biopolymer naturally occurring in living organs can be modified for diagnostic and therapeutic purposes, improving probe efficiency and reducing immunogenicity and inflammatory potential. Biological polymers such as alginate, hyaluronic acid, and chitosan, as well

as proteins, antibodies, enzymes, lipoproteins, and viral capsids (protein cages), are becoming very attractive for diagnostic and therapeutic applications [121].

Maham et al. synthesized engineered platforms for drug delivery systems of different types and shapes (NPs, microspheres, films, minirods, hydrogels) using gelatin, albumin, collagen, elastin, ferritin/apoferritin, gliadin, casein, zein (corn protein), whey protein, and soy protein [103] highlighting promises and challenges.

Vecchione et al. [122] proposed a fully biocompatible platform for dual MRI/PET imaging with improved relaxometric properties. The core–shell nanocarriers made of chitosan and hyaluronic acid entrapped Gd-DTPA, boosting its relaxometric properties up to five times, and carried the adsorbed 18F-FDG without any modification of both FDA-approved CAs.

Fan and coworkers [123] produced a water-soluble melanin NP formulation; this system, after PEGylation, naturally bound 64Cu and Fe3<sup>+</sup> for PET and MRI imaging, and its surface was functionalized with RGD. Shukla et al. [124] proposed a virus-based synthesis, where bacteriophages and plant viruses were used as a scaffold to carry 18F and iron oxide or Gd3<sup>+</sup>. These virus-based NPs resulted homogenous and monodisperse, representing a promising delivery system for CAs. However, there are still several open concerns related to their immunogenicity and loading efficiency. In Table 4, a comprehensive overview of nanoparticulate constructs used in PET/MRI imaging and related properties is provided.


**Table 4.** Overview of multimodal PET/MRI nanoparticles.

\* HSA = human serum albumin; RGD = Arg–Gly–Asp; PEG = polyethylene glycol; PLGA = polylactic-*co*-glycolic acid; nEG = *n*-ethylene glycol spacers; MnMEIO = Mn-doped magnetism engineered iron oxide; PASP = polyaspartic acid; DTCBP = dithiocarbamatebisphosphonate; EGFR = epidermal growth factor receptor; NOTA = 1,4,7-triazacyclonane-1,4,7-triacetic acid; DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; DOTAGA = 1,4,7,10-tetraazacyclododecane-1-glutaric anhydride-4,7,10-triacetic acid; NODAGA = 2,2- -(7-(1-carboxy-4-((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl) diacetic acid; DO3A = 1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetylamide. FDG = fluorodeoxyglucose.
