Protease-Mediated T1 Contrast Enhancement of Multilayered Magneto-Gadolinium Nanostructures for Imaging and Magnetic Hyperthermia.

In this work, we constructed a multifunctional composite nanostructure for combined magnetic hyperthermia therapy and magnetic resonance imaging based on T1 and T2 signals. First, iron oxide nanocubes with a benchmark heating efficiency for magnetic hyperthermia were assembled within an amphiphilic polymer to form magnetic nanobeads. Next, poly(acrylic acid)-coated inorganic sodium gadolinium fluoride nanoparticles were electrostatically loaded onto the magnetic nanobead surface via a layer-by-layer approach by employing a positively charged enzymatic-cleavable biopolymer. The positive-negative multilayering process was validated through the changes occurring in surface ζ-potential values and structural characterization by transmission electron microscopy (TEM) imaging. These nanostructures exhibit an efficient heating profile, in terms of the specific absorption rates under clinically accepted magnetic field conditions. The addition of protease enzyme mediates the degradation of the surface layers of the nanostructures with the detachment of gadolinium nanoparticles from the magnetic beads and exposure to the aqueous environment. Such a process is associated with changes in the T1 relaxation time and contrast and a parallel decrease in the T2 signal. These structures are also nontoxic when tested on glioblastoma tumor cells up to a maximum gadolinium dose of 125 µg mL-1, which also corresponds to a iron dose of 52 µg mL-1. Nontoxic nanostructures with such enzyme-triggered release mechanisms and T1 signal enhancement are desirable for tracking tumor microenvironment release with remote T1-guidance and magnetic hyperthermia therapy actuation to be done at the diseased site upon verification of magnetic resonance imaging (MRI)-guided release.

CuFeS2 Nanoparticles Functionalized with a Thermoresponsive Polymer for Photothermia and Externally Controlled Drug Delivery.

CuFeS2 chalcopyrite nanoparticles (NPs) can generate heat under exposure to near-infrared laser irradiation. Here, we develop a protocol to decorate the surface of CuFeS2 NPs (13 nm) with a thermoresponsive (TR) polymer based on poly(ethylene glycol methacrylate) to combine heat-mediated drug delivery and photothermal heat damage. The resulting TR-CuFeS2 NPs feature a small hydrodynamic size (∼75 nm), along with high colloidal stability and a TR transition temperature of 41 °C in physiological conditions. Remarkably, TR-CuFeS2 NPs, when exposed to a laser beam (in the range of 0.5 and 1.5 W/cm2) at NP concentrations as low as 40-50 µg Cu/mL, exhibit a high heating performance with a rise in the solution temperature to hyperthermia therapeutic values (42-45 °C). Furthermore, TR-CuFeS2 NPs worked as nanocarriers, being able to load an appreciable amount of doxorubicin (90 µg DOXO/mg Cu), a chemotherapeutic agent whose release could then be triggered by exposing the NPs to a laser beam (through which a hyperthermia temperature above 42 °C could be reached). In an in vitro study performed on U87 human glioblastoma cells, bare TR-CuFeS2 NPs were proven to be nontoxic at a Cu concentration up to 40 µg/mL, while at the same low dose, the drug-loaded TR-CuFeS2-DOXO NPs displayed synergistic cytotoxic effects due to the combination of direct heat damage and DOXO chemotherapy, under photo-irradiation by a 808 nm laser (1.2 W/cm2). Finally, under a 808 nm laser, the TR-CuFeS2 NPs generated a tunable amount of reactive oxygen species depending on the applied power density and NP concentration.

Fe3 O4 @Au@Cu2-x S Heterostructures Designed for Tri-Modal Therapy: Photo- Magnetic Hyperthermia and 64 Cu Radio-Insertion.

Here, the synthesis and proof of exploitation of three-material inorganic heterostructures made of iron oxide-gold-copper sulfide (Fe3 O4 @Au@Cu2-x S) are reported. Starting with Fe3 O4 -Au dumbbell heterostructure as seeds, a third Cu2-x S domain is selectively grown on the Au domain. The as-synthesized trimers are transferred to water by a two-step ligand exchange procedure exploiting thiol-polyethylene glycol to coordinate Au and Cu2-x S surfaces and polycatechol-polyethylene glycol to bind the Fe3 O4 surface. The saline stable trimers possess multi-functional properties: the Fe3 O4 domain, of appropriate size and crystallinity, guarantees optimal heating losses in magnetic hyperthermia (MHT) under magnetic field conditions of clinical use. These trimers have indeed record values of specific adsorption rate among the inorganic-heterostructures so far reported. The presence of Au and Cu2-x S domains ensures a large adsorption which falls in the first near-infrared (NIR) biological window and is here exploited, under laser excitation at 808 nm, to produce photo-thermal heat alone or in combination with MHT obtained from the Fe3 O4 domain. Finally, an intercalation protocol with radioactive 64 Cu ions is developed on the Cu2-x S domain, reaching high radiochemical yield and specific activity making the Fe3 O4 @Au@Cu2-x S trimers suitable as carriers for 64 Cu in internal radiotherapy (iRT) and traceable by positron emission tomography (PET).

Magnetic nanoparticles and clusters for magnetic hyperthermia: optimizing their heat performance and developing combinatorial therapies to tackle cancer.

Magnetic hyperthermia (MHT) is a therapeutic modality for the treatment of solid tumors that has now accumulated more than 30 years of experience. In the ongoing MHT clinical trials for the treatment of brain and prostate tumors, iron oxide nanoparticles are employed as intra-tumoral MHT agents under a patient-safe 100 kHz alternating magnetic field (AMF) applicator. Although iron oxide nanoparticles are currently approved by FDA for imaging purposes and for the treatment of anemia, magnetic nanoparticles (MNPs) designed for the efficient treatment of MHT must respond to specific physical-chemical properties in terms of magneto-energy conversion, heat dose production, surface chemistry and aggregation state. Accordingly, in the past few decades, these requirements have boosted the development of a new generation of MNPs specifically aimed for MHT. In this review, we present an overview on MNPs and their assemblies produced via different synthetic routes, focusing on which MNP features have allowed unprecedented heating efficiency levels to be achieved in MHT and highlighting nanoplatforms that prevent magnetic heat loss in the intracellular environment. Moreover, we review the advances on MNP-based nanoplatforms that embrace the concept of multimodal therapy, which aims to combine MHT with chemotherapy, radiotherapy, immunotherapy, photodynamic or phototherapy. Next, for a better control of the therapeutic temperature at the tumor, we focus on the studies that have optimized MNPs to maintain gold-standard MHT performance and are also tackling MNP imaging with the aim to quantitatively assess the amount of nanoparticles accumulated at the tumor site and regulate the MHT field conditions. To conclude, future perspectives with guidance on how to advance MHT therapy will be provided.

Successful Application of CuAAC Click Reaction in Constructing 64Cu-Labeled Antibody Conjugates for Immuno-PET Imaging.

Immuno-positron emission tomography (immuno-PET) is a rapidly growing imaging technique in which antibodies are radiolabeled to monitor their in vivo behavior in real time. However, effecting the controlled conjugation of a chelate-bearing radioactive atom to a bulky antibody without affecting its immunoreactivity at a specific site is always challenging. The in vivo stability of the radiolabeled chelate is also a key issue for successful tumor imaging. To address these points, a facile ultra-stable radiolabeling platform is developed by using the propylene cross-bridged chelator (PCB-TE2A-alkyne), which can be instantly functionalized with various groups via the click reaction, thus enabling specific conjugation with antibodies as per choice. The PCB-TE2A-tetrazine derivative is selected to demonstrate the proposed strategy. The antibody trastuzumab is functionalized with the trans-cyclooctene (TCO) moiety in the presence or absence of the PEG linker. The complementary 64Cu-PCB-TE2A-tetrazine is synthesized via the click reaction and radiolabeled with 64Cu ions, which then reacts with the aforementioned TCO-modified antibody via a rapid biorthogonal ligation. The 64Cu-PCB-TE2A-trastuzumab conjugate is shown to exhibit excellent in vivo stability and to maintain a higher binding affinity toward HER2-positive cells. The tumor targeting feasibility of the radiolabeled antibody is evaluated in tumor models. Both 64Cu-PCB-TE2A-trastuzumab conjugates show high tumor uptakes in biodistribution studies and enable unambiguous tumor visualization with minimum background noise in PET imaging. Interestingly, the 64Cu-PCB-TE2A-PEG4-trastuzumab containing an additional PEG linker displays a much faster body clearance compared to its counterpart with less PEG linker, thus affording vivid tumor imaging with an unprecedentedly high tumor-to-background ratio.

Cation Exchange Protocols to Radiolabel Aqueous Stabilized ZnS, ZnSe, and CuFeS2 Nanocrystals with 64Cu for Dual Radio- and Photo-Thermal Therapy.

Here, cation exchange (CE) reactions are exploited to radiolabel ZnSe, ZnS, and CuFeS2 metal chalcogenide nanocrystals (NCs) with 64Cu. The CE protocol requires one simple step, to mix the water-soluble NCs with a 64Cu solution, in the presence of vitamin C used to reduce Cu(II) to Cu(I). Given the quantitative cation replacement on the NCs, a high radiochemical yield, up to 99%, is reached. Also, provided that there is no free 64Cu, no purification step is needed, making the protocol easily translatable to the clinic. A unique aspect of the approach is the achievement of an unprecedentedly high specific activity: by exploiting a volumetric CE, the strategy enables to concentrate a large dose of 64Cu (18.5 MBq) in a small NC dose (0.18 µg), reaching a specific activity of 103 TBq g-1. Finally, the characteristic dielectric resonance peak, still present for the radiolabeled 64Cu:CuFeS2 NCs after the partial-CE reaction, enables the generation of heat under clinical laser exposure (1 W cm-2). The synergic toxicity of photo-ablation and 64Cu ionization is here proven on glioblastoma and epidermoid carcinoma tumor cells, while no intrinsic cytotoxicity is seen from the NC dose employed for these dual experiments.