nanocubes, namely dimers and trimers, showed a SAR valued almost doubled if compared to individual nanoparticles. When the nanocubes number overcame the threshold of four particles per cluster, an impressive fall of SAR value was observed (Niculaes et al. 2017). Nanocubes assembled with a 2D‐arrangement were also obtained by using an esterase‐sensitive biopolymer as an encapsulating agent. The enzyme activity resulted in the disassembly of the multiparticle cluster and, as confirmation of the above‐described study, in an enhanced SAR performance. In fact, the 2D‐clusters were split into smaller clusters composed of very few particles, in a chain‐like configuration (Avugadda et al. 2019).
Another modified‐amphiphilic polymer, poly(isobutylene‐alt‐maleic anhydride), was used for the fabrication of small nanocluster based on the assembly of hydrophobic nanoparticles. The maleic anhydrides of the polymer backbone acted as an anchor point for the addition of PEG (10%), dopamine (70%) and cystamine, a disulfide linker (20%). The three ligands ensured, respectively, high biocompatibility to the cluster, a high affinity to the nanoparticles surface and an amine for an additional functionalization. Therefore, chlorin e6, a photosensitive drug, was linked to the cystamine for the preparation of this multifunctional polymer. The cluster was prepared by a solvent exchange strategy, mixing the chloroform‐dispersed nanoparticles with a solution of the polymer in DMSO. After ultrasonication and evaporation of the chloroform, the DMSO was changed with water by dialysis. The resulting redox‐responsive nanocluster was suitable for the detection of the high‐reductive intracellular environment (typical of tumors) because of the cleavage of the disulfide bridge and the subsequent release of the drug, for MR imaging and the photodynamic therapy of solid tumors (Yang et al. 2018).
Paquet et al. in 2010 reported the clustering process of hydrophobic nanoparticles in regular assembly assisted by sodium dodecyl sulfate (SDS). This was one of the first paper in which was obtained a fine control over the nanocluster size, ranging from 40 to 200 nm. The method was based on a microemulsion of two components: the fatty acid‐coated nanoparticles dispersed in toluene and an aqueous solution of SDS. By ultrasonication and subsequent ripening at 90 °C, the organic solvent was entirely evaporated, and dense spherical aggregates were obtained. Some key parameters, as surfactant and nanoparticles concentration, the volume ratio of the emulsion, were monitored to control the clustering process and the final diameter of the magnetic nanospheres. As shown in this contribution and also in more recent ones, the SDS clusters need an additional surface coating to stabilize the structure. In this chapter, a 20 nm‐additional shell of polymethacrylate derivates was grafted on the cluster surface (Paquet et al. 2010). Starting from the SDS‐coated cluster, the research group also investigated as the nature and the thickness of the additional polymer shell could vary the relaxivities of the magnetic clusters. By using a precipitation polymerization method, a pH‐sensitive hydrogel coating composed of acrylic acid, N,N'‐methylenebis‐acrylamide and N‐isopropylacrylamide was polymerized onto the clusters. The hydrogel significantly enhances the transverse relaxation rates by lowering the diffusion coefficient of water molecules near the magnetic nanoparticles. By tuning the pH or the initial thickness of the hydrogel, an r2 increase (in comparison to the bare magnetic nanoparticles) was observed from 44% (low pH, the low water content in the thin shell) to 85% (neutral pH, the high water content in the thick shell) (Paquet et al. 2011). Also, Wu et al. in 2015, reported the clustering of hydrophobic magnetic nanoparticles by emulsification method assisted by SDS surfactant. A toluene dispersion of NPs was mixed with a SDS aqueous solution, and the mix was sonicated and kept at 90 °C for two hours. The resulting cluster had a diameter below 200 nm and a fine distribution. To ensure higher nanosystem stability, a shell of polydopamine was polymerized on the cluster surface in alkaline conditions, starting from dopamine monomer. The polydopamine ensured a higher NIR absorption to the cluster, exploitable for photothermal therapy. In a proof of concept magnetophoresis experiment, cancer cells were incubated with polydopamine‐nanocluster with an external magnet and irradiated with a 808 nm laser, achieving a 90% cytotoxicity at the highest concentration (Wu et al. 2015b). Starting from this protocol, Mandriota et al. evaluated many different parameters (choice and concentration of surfactant, size of nanoparticles, choice of organic solvent, oil/water phase ratio, and scalability) to produce clusters with a size around 100 nm. Then, a polydopamine shell was grafted on the cluster (from 4 to 27 nm), and the efficiency of a pH‐sensitive release was assessed, loading a chemotherapy drug as a model, the cisplatin. Below pH 5, an abrupt release of the drug was obtained after 24 hours, with a partial degradation of the nanocluster, and a release of nanoparticles, at pH 3 after 72 hours. In vitro experiments confirmed that nanocluster significantly improved the cellular uptake of the platinum drug, by increasing its cytotoxicity at low dose (Mandriota et al. 2019).
Various encapsulation methods have been employed for the synthesis of iron oxide clusters by using block copolymers, either bihydrophilic or amphiphilic and stabilizing agents. In general, these methods involve the formation of micelles. Ai et al. reported the clustering of monodisperse iron oxide particles inside the hydrophobic core of micelles made of PEG‐modified poly‐caprolactone polymer (Ai et al. 2005). These micelles were obtained by an oil‐in‐water approach performed via sonication; in detail, the nanoparticles and the polymer, dissolved in hexane, were dispersed in an aqueous polymer solution, and the resulting dispersion was sonicated, thus leading to the formation of the micelles, whose mean diameter was of 110 nm. Finally, the organic solvent was removed under reduced pressure. Following a similar approach, also block copolypeptide (Euliss et al. 2003) or copolymers of acrylic acid, styrenesulfonic acid, and vinylsulfonic acid have been studied (Ditsch et al. 2005). More recently, Schmidtke et al. reported a method for clustering colloidal nanoparticles by using a diblock copolymer system. The process was mainly described for magnetic nanoparticles, but a proof of clustering was also provided for semiconductor and plasmonic nanoparticles. First, the native ligand of nanoparticles was exchanged with polymer PI‐DETA; then the coated‐NPs were mixed with PI‐b‐PEG diblock copolymer and transferred in water by different injection approaches (manual, mechanical, and microfluidic). Finally, the polymer was thermally crosslinked by addition of a radical initiator. The resulting nanosphere was ultrastable and with a size ranging from 54 to 750 nm. The size was tuned by modifying the ratio polymer : NP, from 400 : 1 (smaller clusters) to 20 : 1 (larges ones). After magnetic characterizations (relaxometry, magnetization, and magneto‐rheological measurements), the authors observed that the cluster magnetic moment derived by the sum of entrapped NPs moments and that the dipolar interaction between the NPs as the cause of collective effect observer in magnetic clusters (Schmidtke et al. 2014). Another block copolymer, namely poly(aspartic acid)‐b‐poly(ε‐caprolactone), was used for the clustering of 12 nm‐hydrophobic nanoparticles. The clusters showed an average diameter of 125 nm with good dispersion and an excellent r2 relaxivity (335 mM−1 s−1 at 1.5 T). In this study, the nanoclusters were exploited as a contrast agent for the labeling and the in vivo tracking of dendritic cell, achieving fine response for viability, proliferation, and differentiation capacity. The subcutaneous injection of labeled cells in mice footpad allowed to monitor the presence of cells and their migration in lymph nodes up to 72 hours without a significative loss of signal in MRI (Wu et al. 2015a). Recently, Vishwasrao et al. reported an extensive study on the clustering of hydrophobic magnetic nanoparticles by using a modified block copolymer. The clusters were obtained with nonmodified PLE‐b‐PEG block copolymer (via electrostatic binding of carboxylate groups of the PLE blocks and the nanoparticle surface) and with an alendronate sodium trihydrate (ALN)‐modified PLE‐b‐PEG polymer. In the latter, the alendronate acted as an anchor molecule due to the bisphosphonate groups of the molecule. In both cases, the cluster size remained quite small in the range between 40 and 70 nm. Moreover, the cluster was loaded with cisplatin and conjugated with luteinizing hormone‐releasing hormone (LHRH) to target corresponding overexpressed receptors on ovarian cancer cells membranes (Vishwasrao et al. 2016).
Peng et al. described the synthesis of PLGA‐coated nanoclusters for the delivery of siRNA. An aqueous suspension of presynthetized magnetic nanoparticles was mixed with a one‐pot precursor solution, composed of PLGA, siRNA, iron chloride, and citrate acid, and left to react for three hours at 60 °C. Dense clusters from 100 to 300 nm were obtained. The efficacy of these composite to deliver the siRNA was evaluated at 37 °C in a tube, achieving a full