Method for the uptake of magnetic nanoparticles (MNPS) by immune cells

Abstract

Method for the uptake / internalization of magnetic nanoparticles (MNPs) by immune cells, comprising the steps of preparing a mixture of a given amount of immune cells with a given amount of MNPs, and then exposing that mixture to an alternating magnetic field, at a given frequency and a given magnetic field strength, in order to heat that mixture to a temperature between 38° and 43° C, for a given time.

Description

[0001] Method for the uptake of magnetic nanoparticles (MNPs) by immune cells

[0002] The present invention relates to the therapeutic use of the magnetic nanoparticles (MNPs), and particularly it relates to the use of MNPs in immunotherapy for treatment of cancer; more specifically, the invention relates to a method for the uptake of MNPs by immune cells.

[0003] Magnetic nanoparticles (MNPs) play a crucial role in the field of Nanomedicine thanks to their magnetic properties. These properties make them valuable for purposes such as serving as carriers for drug delivery, tracking agents capable of functioning in diverse magnetic fields, and as heat sources when subjected to alternating magnetic fields (AMF). Magnetic Hyperthermia Treatment (MHT) harnesses-controlled temperature elevation to achieve therapeutic values, such as 45 °C, as a precise method for applying heat-based therapy to combat cancer. As is already known, temperature elevation, originating from either endogenous or exogenous sources, triggers the activation of the intratumoral immune system. The inflammation stage at the tumor results in an increased recruitment of immune cells to the tumor site, thanks to the expansion of blood vessels surrounding the tumor mass. Furthermore, the temperature elevation induced by heat encourages the expression of damage-associated molecular pattern antigens on the surface of cancer cells. This, in turn, facilitates their recognition by tumor-associated dendritic cells, macrophages, as well as other immune cells, including natural killer (NK) cells and T-lymphocytes.

[0004] The potential advantages of combining immunotherapy with hyperthermia are further supported by ongoing clinical trials. The benefits offered by MNPs stem from their ability to selectively increase the temperature exclusively at the tumor site, through remote activation using an AMF. This leads to a more efficient heat release and localized recruitment of immune cells. Specifically, the research group of the present applicant has demonstrated that mild MHT at approximately 43°C has the ability to modulate the immunological profile of glioblastoma cancer cells (U87). This modulation is achieved through the upregulation of stress-associated ligands, resulting in an enhanced phagocytosis and killing of cancer cells by macrophages (Persano et al, Pharmaceutics 2021 , 13 (10), 1668).

[0005] Furthermore, mild MHT has been observed to have a modulatory effect on the expression of inhibitory and activating ligands on NK cells, leading to an increased susceptibility of U87 cells to NK cell-mediated killing. Our study, along with other relevant research in the literature, highlights the immune system's activation through heat generated by MNPs when they are administered to cancer cells in in vitro studies, [(Persano, Stefano, et al. Pharmaceutics 13.10 (2021 ): 1668)]. However, the dose of nanoparticles (NPs) to accumulate to the tumor remains a critical issue in nanomedicine. However, none of these studies use MNPs as heat mediators to promote internalization / uptake of the nanoparticles themselves into cells of the immune system.

[0006] Among the various solutions proposed in literature, a subset of studies is focusing on harnessing immune cells as physiological carriers for nanoparticle delivery to the tumor, eliminating the need for additional targeting moieties. To accomplish this objective, current approaches rely on various methods to regulate the interaction between cells and MNPs. Most of those methods exploit either the simple incubation of cells with NPs, or the chemical interactions (nonspecific or covalent chemical bond- mediated adsorption) between the NPs and the cells, or the application of a static magnetic field used during the cell culturing to push the MNPs on the cells and enhance magnetic uptake [(Sanz-Ortega, Laura, et al. Frontiers in Immunology 10 (2019): 2073), Li, Kangan, et al. Nanomedicine 10.11 (2015): 1761-1774), Sim, Taehoon, et al. ACS nano 15.8 (2021 ): 12780-12793)]. For instance, Sanz-Ortega et al. demonstrated the labeling of NK cells by incubating NK-92MI cells or murine primary NK cells with iron oxide NPs coated with (3-aminopropyl) triethoxysilane (APS) polymer for a prolong time lapse (generally, at least 2h). The demonstrated nonspecific association between NK cells and MNPs (14 pg Fe / cell in the case of NK92) did not affect cell viability, allowing the cells to maintain their primary effector functions, such as degranulation, IFN-y production, and migration in response to chemotactic signals. The same group showed the versatility of their protocol by successfully labeling T cells with the same material (Sanz- Ortega, Laura, et al. Frontiers in Immunology 10 (2019): 2073).

[0007] In a separate investigation, Kangan Li and colleagues employed ultrasmall superparamagnetic iron oxide nanoparticles (USPIO) measuring 17-31 nm in size to construct a nanocomplex comprising heparin-protamine-ferumoxytol (HPF), a iron oxide NP based drug used in anemia treatment in patient as labelling agent for natural killer cells (NK-92MI). The association of ultra small iron oxide USPIO NPs with NK cells was achieved by exposing the cells to varying concentrations of nanocomplexes during a 4-hour incubation period (25, 50, or 100 pg / ml ferumoxytol based NPs). Following this protocol, the researchers attained an iron content per cell of 0 pg / ml HPF = 0.03±0.01 pg; 25 pg / ml HPF = 1 .72 ± 0.32 pg; 50 pg / ml HPF = 2.46 ± 0.39 pg; and 100 pg / ml HPF = 3.47 ± 0.45 pg, respectively. Although these conditions maintained satisfactory viability and cytolysis activity, the iron content per cell remained relatively low, and at the highest concentration, some toxicity was observed (Li, Kangan, et al. Nanomedicine 10.11 (2015): 1761 -1774). Following the footsteps of these authors, Taehoom Sim and colleagues developed magnetic complexes made from hyaluronic acid, protamine, and ferumoxytol, denoted as HAPF, for labeling NK-92 cells. To optimize the protocol, they incubated NK cells with the nanocomplexes for 1 , 2, or 4 h time exposure at different concentrations (12.5, 25, 50 and 100 pg / mL). They showed that both the exposure duration and the initial seeding concentration of HAPF are significant for the optimization of internalization on NK cells. Notably, an increase in incubation time corresponded to a higher percentage of labeled cells.

[0008] Similarly, elevated HAPF seeding concentrations correlated with increased labeling of NK cells. However, an initial feeding concentration of 100 pg / mL adversely affected cell viability. Consequently, the conditions of 50 pg / mL initial feeding of HAPF and a 4-hour co-incubation period were selected. This achieved an iron content per cell equivalent to 9.8 ± 0.8 pg without impairing cells viability and functionality (Sim, Taehoon, et al. ACS nano 15.8 (2021 ): 12780-12793). These ferumoxytol based product although has been used as MRI contrast agents having an iron oxide core below 8 nm, cannot be applied as magnetic hyperthermia heating agents, because at this size iron oxide nanoparticles do not produce any heating under the AMF conditions of clinical use and therefore are not usable in MHT.

[0009] In another study by Wu L. et al., the authors detailed the loading of polydopamine-coated iron oxide NPs onto NK cells derived from peripheral blood. However, their approach involves co-incubating the NPs with NK cells for a duration of at least 12 hours to achieve intracellular NP uptake of unknown amount since it is not reported (Wu, Liya, et al. Biomaterials science 6.10 (2018): 2714-2725). The non-specific nature of this co-incubation-based method has prompted the exploration of alternative innovative solutions.

[0010] To address this point, Burga R.A. and colleagues developed a "biohybrid" method in which iron oxide NPs were attached to the cell membrane of cord blood-derived NK cells by streptavidin-biotin high association constant binding. The authors showed that the highest IONP binding occurred when using 50 picograms of streptavidin-coated lONPs per NK cell. However, under these incubation conditions, they achieved slightly more than 2% IONP loading per NK cell on a gram-to-gram basis, while higher concentrations (100 pg / cell) resulted in observable aggregations. Functionalization with streptavidin allowed stable anchoring of NPs to the cell surface in less than 1 hour, successfully preserving the internal structure of the cells. Indeed, the labeled NK cells remained viable, retaining both their phenotype as well as their ability to degranulate in the presence of tumor cells. However, in this approach, the NK cells must be pretreated in orderto anchor the biotin, which then binds to the streptavidin present on the MNPs (Burga, Rachel A., et al. Bioconjugate chemistry 30.3 (2019): 552-560). Jang E.S. et al. proposed a distinct strategy for labeling cells exploiting a static magnetic field. They demonstrated that Cy5.5 conjugated FesC / SiC^ core / shell NPs (20 pg / mL) could be loaded onto NK-92MI cells, when exposed to a static magnet with a field gradient of 159 gauss / mm placed beneath the cell dish in the incubator for 30 min. In this case, the magnetic attraction of magnetic NPs to a magnet has been exploited as the accumulating principle rather than the MHT under AMF. However, as a protocol based on cells in suspension (and not monolayer), the accumulation of MNPs toward the bottom of the well is not optimal to promote maximum interaction between MNPs and cells. In addition, the article lacks any reference to the quantification of MNPs associated with NK cells and also to structural images such as, for example, images taken by transmission electron microscope (TEM) or scanning electron microscope (SEM) that can illustrate the exact location of NPs within cells (Jang, Eue-Soon, et al. Biomaterials 33.22 (2012): 5584-5592).

[0011] The current body of literature primarily focuses on MNPs labeling protocols that involve the simple co-incubation of NPs with cells for a specified incubation period (typically up to 12 hours) to facilitate MNP attachment to the cell membrane. Furthermore, most of these studies have employed cell lines such as NK-92MI, which although in some clinical trials are derived from tumor human patients and, due to their immortalized nature, possess somatic characteristics which are significantly different in comparison to primary NK cells from healthy donors (Williams, Brent A., et al. Oncotarget 8.51 (2017): 89256).

[0012] Only a limited number of studies discuss intracellular uptake methods on NK cells (Jang, Eue-Soon, et al. Biomaterials 33.22 (2012): 5584-5592), and even fewer investigations are centered on the use of primary immune cells obtained from Human healthy donors. NK cells from human donors are smaller in size than NK-cells derived from immortalized cell lines and do not have the tendency to uptake the NPs. Both parameters make the development of protocols more difficult for MNPs association to the same cells.

[0013] The research leading to the present invention thus proposes a novel approach to promote immune cells / MNPs interaction. Therefore, an aim of the present invention is to create an immune-delivery system loaded at the maximum dose of magnetic materials and at the lowest cytotoxicity for the immune cells for the delivery and accumulation of both MNPs and NK cells at the target tumor site to combine MHT with immunotherapy, or to track NK cells once injected systemically.

[0014] It is an object of the present invention to provide a method for the uptake / internalization of magnetic nanoparticles by immune cells, comprising the steps of preparing a mixture of a given amount of immune cells with a given amount of MNPs, and subjecting that mixture to an alternating magnetic field, having a given frequency and a given magnetic field strength, in order to heat that mixture to a temperature in the range of 38° to 43° C, for a given time interval of a few minutes to a few hours. The mixture comprises a few million immune cells (preferably 1.2 million) and MNPs so as to achieve a final concentration in the range of 1 to 4 gFe / L and preferably 3 gFe / L.

[0015] In a preferred executive form, immune cells are NK cells, and specifically among this type of cells are chosen primary NK cells, i.e. , NK cells derived from a healthy donor, and NK92-MI cells, i.e., an immortalized strain of immune cells. However, the method developed here is adaptable to other cells of the immune system as, for example demonstrated, with T-cells or as other cells such as monocytes.

[0016] The method is applicable to any magnetic nanoparticles that show the ability to heat under a clinically safe kHz-based radiofrequency exposure and with NPs that can be stably dispersed in physiological medium suitable for immune cells.

[0017] Specifically, Magnetic nanoparticles preferably exhibit superparamagnetic properties so that they are non-interacting with each other and thus stable in the cellular medium in which they are added to and mixed with cells.

[0018] In addition, their formulation must be free of toxic effects to cells so that they are biocompatible in the physiological environment in which they will be used. Preferably, MNPs are nanoparticles of iron oxide (FesC ).

[0019] It is emphasized that iron oxide-based magnetic nanoparticles are FDA (Food and Drug Administration) approved and can be used in clinical trials of magnetic hyperthermia therapy.

[0020] As will be described later, alternatively, other mixed iron ferrites, such as Zinc-Ferrite of composition ZnxFes-xC , can be used, which have not been found to be toxic to cells.

[0021] According to one possible embodiment, MNPs are iron oxide (FesO4) nanoparticles with dimensions between 14-25 nm and cube shaped.

[0022] Alternatively, MNPs are zinc-doped iron oxide (ZnFe2O4) nanocubes with the edge of the cube in the range of 14-25 nm.

[0023] To ensure their stability and compatibility in biological substrates, MNPs are coated with amphiphilic polymers of various kinds. In this method, two polymers were taken as examples; polymer 1 or Poly (maleic anhydride-alt-1 -octadecene) and functionalized with diaminopolyethylene glycol; or polymer 2, a polymer derivative poly(maleic anhydride-iso-butylene) equipped with dopamine molecules for multianchoring to the surface of MNPs and some amino-polyethyleneglycol derivative molecules (amino-PEG derivatives) for stabilization and further anchoring of other molecules.

[0024] The alternating magnetic field is applied to the mixture at a frequency in the range of 50 kHz to 1 MHz with a preference for the range of 175-185 kHz and with a field strength ranging from 5 to 47 kA / m with a preference for the range of 15-25 kA / m; the duration of the mixture treatment is in the range of a few to 120 minutes with a preference for the range of 30-80 minutes and preferably for experiments set at 60 minutes.

[0025] The NK cells have been considered as preferred immune cells due to their crucial role in immune defense against transformed cells, particularly against tumor cells. Unlike other types of immune system cells, they are allogeneic in that cells from a healthy donor can be used and not necessarily those obtained from the patient himself. Moreover, the proliferation rate of NK cells is much faster than other immune cell types, such as T cells (days for NK cells versus weeks for T cells) making the expansion much quicker at a production cost much lower.

[0026] Moreover, NK cells possess the unique ability to selectively eliminate cancer cells without prior tumor antigen exposure, but instead relying on a variety of activating and inhibitory receptors to govern tumor recognition and cytotoxic functions. All these characteristics have made them a valuable component in adoptive immunotherapy, where cells obtained from peripheral blood are expanded, activated, and reintroduced into the patient. As here shown, the use of mild-MHT at approximately 41 °C, while speeding the loading process to only 1 hour, does not compromise the physiology of the immune-response of NK cells ensuring their killing action against tumor cells. However, to demonstrate the implementation of the method proposed here of uptake / internalization of MNPs mediated by magnetic hyperthermia, the experiment was extended to T cells of the immune system.

[0027] The present invention relates to a rapid and effective approach to facilitate the internalization and membrane association of magnetic NPs in NK-cells, as well as primary NK cells and NK-92MI cells, T cells, and monocytes without compromising their viability and functionality. This method relies on the enhanced diffusion of nanoparticles and enhanced permeability of immune cell membrane when exposed to mild temperature induced byAMF in mild-MHT treatment. To demonstrate this concept, the present inventors have opted for iron oxide nanoparticles (lONCs) and other mixed ferrites such as zinc ferrite (Zn-Fe) in the form of nanocubes coated with different types of polymers for their transfer and stabilization in physiological media but also nanoparticles with other shapes, such as iron oxide clusters in the form of multi-domains or hexagonal or spherical nanoparticles, as heat mediators, to demonstrate, through the following examples, the use of this method with any kind of MNPs that have the right physical parameters for heating in MHT These MNPs, together with the application of AMFs under conditions that are biologically safe for immune cells, thus causing no adverse effects on NK cells or other cells of the immune system, and the maintenance of a maximum temperature of about 41 -43°C fora maximum duration of about 60-120 minutes, have been exploited to enhance the internalization / absorption of NPs in a time interval of just 1 hour or so.

[0028] Notably, for both primary NK cells and NK-92MI cells, from a quantitative point of view, the iron uptake and, in turn, the NPs uptake induced by mild-MHT was always significantly higher for NK cells incubated with the same NPs dose and exposed to mild-MHT than for the same cells incubated with NPs but not exposed to MHT. Proof of NPs internalization with localization within intracellular vesicles and on the cell membrane was provided by Transmission electron microscopy (TEM) analysis suggesting the effective action of heat on permeabilization of the cell membrane and promotion of endocytosis under heating as well as cellular surface adhesion. Compared to other existing methods in literature, the method according to the present invention induces enhanced MNPs uptake not only in NK-92MI cells, more commonly used in most of the works, but also on the most difficult primary NK cells from healthy donor in a much shorter time compared to the other methods reported.

[0029] Additionally, as demonstrated by the viability results and phenotype analysis, the presence of MNPs within the immune cells had minimal impact on viability and functionality. Specifically, the receptors expression levels that regulate the migratory properties of NK cells remained similar between (untreated) control cells and NK cells loaded with lONCs with MHT.

[0030] This method enables the rapid internalization and association of iron oxide nanoparticles into NK92 and primary NK cells by mild-MHT in the temperature range of 38°C to 43°C and under magnetic field and frequency conditions in the range of 80 kHz to 1 Mhz. that do not affect the viability and function of NK cells. In the following examples the method of the invention is carried out with cubic-shape iron oxide NPs, prepared by a synthesis method according to the patent IT 102019000006469. However, as here shown with the case of zinc-iron oxide NPs, the method is applicable to any magnetic nanoparticle that exhibits the ability to heat up upon exposure to AMF and with MNPs that can be stably dispersed in the physiological substrate of NKs, regardless also of their size, shape, and type of coating used to stabilize them in the physiological medium.

[0031] Specifically, the method according to the present invention is able to induce both internalization and adhesion to the cell surface of nanoparticles in a short MHT time, thereby drastically reducing the time required for uptake due to the cell membrane permeabilization achieved during mild-MHT and the rapid diffusion of NPs at a temperature of about 38-43°C with preference for the temperature of 40-41 °C.

[0032] In the initial phase of the study addressed to the present invention, the resistance of NK cells, to different temperatures for different time duration was established, by placing the vial in which NK-92MI cells were contained in a thermomixer at fixed temperature and recording the viability of NK-92MI cells. The results have suggested that a duration of 1 hour at 41 °C was the optimal thermal treatment which did not show any sign of sufferance on NK cells.

[0033] Next, these optimal selected thermal conditions of temperature and time duration were implemented for the mild-MHT on both NK-92MI and NK cells.

[0034] To do so, for the mild-MHT, the working concentration of nanocubes was set at 3gFe / L added (MNPs concentration range between 1 and 4 gFe / L is reasonable) to 1 .2 million of NK cells and, by fixing the working frequency preferably at 182 kHz, we could vary the field amplitude up to 17-20 kA / m values to reach the final temperature of 40- 41 °C which was maintained for 1 hour. Under these field amplitude and frequency conditions, eddy currents are not significant and therefore no heat or local field damage are recorded on cells treated with MHT and in absence of AMF.

[0035] In the experiments carried out during the development of the present invention, lONCs (16 nm) coated with Poly (maleic anhydride-alt- 1 -octadecane) polymer and functionalized with diamine-polyethylene glycol (NH2-PEG-NH2, MW 2000) were prepared (Figure 3), and they were showing a surface charge of -28±7 mV in cell culture media. These NPs rapidly reached the set temperature of 40-41 °C in mild MHT under chosen radiofrequency conditions and they were well-dispersed and stable in the NK cell medium. Through elemental analysis, the inventors compared the iron uptake (thus the NP uptake) in NK-92MI cells in presence or absence of the AMF. In the presence of AMF, a clearly elevated iron dose (and then of MNPs) was measured on cells which was equivalent to 14 pgFe / cell. This uptake was clearly higher than the iron dose of 8 pgFe / cell measured on NK cells exposed to MNPs for the same incubation period but in absence of AMF.

[0036] Furthermore, the preservation of the structural integrity of the cells was maintained under mild-MHT as observed by TEM analysis and the presence of NPs with the cells was observed by identifying the presence of cubic shaped NPs on the cell membrane and also inside endosomal cellular bodies.

[0037] Mild-MHT did not even adversely affect both cell viability (Figure 5) and degranulation-mediated cytotoxic activity of NK-92MI cells against glioblastoma cancer cells (U87).

[0038] Using the same experimental setup, it has been examined the intracellular uptake provided by the zinc-iron oxide NPs instead of iron oxide coated with the same polymer as the aforementioned NPs. The sole distinction lies in their composition, as these nanoparticles are doped with Zinc. Similarly, also in this case we observed a variation in the iron uptake (and then also that of Zinc) between the sample exposed to nanoparticles and then to MHT and the one incubated with nanoparticles at 37°C without being exposed to AMF.

[0039] With the successful development of this method to NK-92MI cell line, it has been implemented its application to primary NK cells obtained from a healthy human donor.

[0040] As expected, considering the smaller volumetric size of primary NK cells compared with NK-92MI cells, iron quantification analysis, using the same experimental setup applied to NK-92, revealed that primary NK cells internalized less iron and, consequently, fewer NPs than NK-92MI cells. Nevertheless, a significant difference in iron uptake on primary NK with or without the exposure to the mild-MHT was still measured for both NC- PEG and ZN-Fe NPs (Figure 9-10). In fact, the iron values (measured by quantitative optical emission spectrometry technique also called inductively coupled plasma-optical emission spectrometry) per NK cell are 6.5 pgFe / cell in the presence of AMF versus 3 pgFe / cell in the absence of AMF indicate the crucial role of mild MHT in doubling the amount of NPs internalization in primary NK cells.

[0041] Also, for primary NK cells, the AMF and internalization and / or association of NPs, did not impact the structural integrity of the cells and no cell toxic effects were recorded by viability assay. Moreover, even the physiological functionality of primary NK cells was preserved as measured by the degranulation activity measured using primary NK cells (after exposure to NPs and MHT) on two distinct tumor cell lines: the glioblastoma cancer cells (U87) and the neuroblastoma cancer cells (SHSY5Y). Finally, on primary NK cells, it is established that the phenotypic profile of NK cells, particularly the ligands featuring their migratory abilities, which are crucial for circulation and targeting functions, remained largely unaffected after exposure to the NPs and mild-MHT. Lastly, some of the above-mentioned MNPs were used in experiments with or without the application of AMF in the presence of different cells of the immune system (T cells), as an example of protocol extension beyond NK cells to other immune cells.

[0042] Further advantages and features of the method according to the present invention will become apparent from the following description of some embodiments of the same, with reference to the following examples, and to the figures of the appended tables of illustrations, in which:

[0043] Figure 1 shows the viability of NK-92MI cells under different combination of temperature (T: 37°C, 41°C or43°C) and time duration (60 min or 90 min) when using a thermomixer as a heating source;

[0044] Figure 2 shows the optical images of NK-92MI taken at 24h after the exposure to different thermal treatment conditions when using the thermomixer as heating source; Figure 3 illustrates the characterization of lONCs used for the mild-

[0045] MHT;

[0046] Figure 4 illustrates the NPs Cellular uptake evaluation of NC-PEG NPs after mild-MHT through ICP and TEM imaging;

[0047] Figure 5 illustrates the evaluation of NK-92MI cells viability with or without NPs and with or without the subsequent application of mild-MHT;

[0048] Figure 6 illustrates the FACS (Fluorescence-activated cell sorting) evaluation of the degranulation activity of NK-92MI cells against LI87 cancer cells;

[0049] Figure 7 shows the characterization of Zn-Fe NPs used as alternative magnetic NPs to iron oxide NPs;

[0050] Figure 8 shows the demonstration of the generalization of the use of the mild-MHT protocol on NK cells when using Zn-Fe NPs as another type of magnetic NPs;

[0051] Figure 9 shows the characterization of MNPs both cubes and clusters in the shape of Rubik's cubes and both based on iron oxide coated by another polymer, polymer 2 as an example of nanoparticles stabilized by any other amphiphilic coating for transfer and stabilization in water and internalized by NK-92 cells with or without exposure to mild- MHT.

[0052] Figure 10 shows the Mild-MHT experiment carried out on primary NK cells derived from healthy donor exploiting Zn / Fe NPs;

[0053] Figure 11 illustrates the Mild-MHT experiment on primary NK with or without MHT and NC-PEG NPs;

[0054] Figure 12 illustrates the degranulation activity measured by percentage of CD107a positive on primary NK cells with NC-PEG NPs with and without MHT application;

[0055] Figure 13 illustrates the functional and Phenotypic profiles of primary NK cells through the analysis of activating, inhibitory and chemokines receptors expression when using NC-PEG NPs; Figure 14 illustrates the Mild-MHT experiment conducted on T cells and monocytes with MNPs of IONC with or without the application of MHT as an example of protocol extension to any other type of immune system cells.

[0056] Setting of the conditions of MHT using NK-92MI cells

[0057] To set up the mild magnetic hyperthermia treatment (MHT) protocol as a way to promote quick intracellular uptake of nanoparticles (NPs) by enhancing the permeabilization of the cell membrane while maintaining cell viability, several preliminary analysis were conducted to optimize the thermal treatment (thus MHT) conditions in terms of temperature, duration, and NPs concentration.

[0058] The initial step involved determining the hyperthermia temperatures that NK cells could tolerate without compromising their viability or functionality. To facilitate comparison with previously published works and for ease of manipulation, the NK-92MI cell line was utilized for these preliminary experiments. Approximately, one million NK-92MI cells were counted and subjected to two different temperatures (41 °C or 43°C) for two duration periods, 60 minutes or 90 minutes using a thermomixer as a heat source. Following the incubation, the cells were collected, seeded in 96-well plates, and analysed for viability after 24 hours. Cell viability was assessed using a dead staining dye (FVS540) and flow cytometry analysis, (Figure 1). The viability results were then compared to control NK-92MI cells maintained at 37°C for the same duration time of the thermal treatment.

[0059] The analysis revealed that the optimal combination of temperature and heating duration, which had no adverse effect on cell viability with less than 90 percent viability, was that set at 41 °C for 60 minutes. Under these culture conditions, the highest percentage of cell survival was observed, with 94% of the NK-92MI cells remaining alive (Figure 1 A). This viability percentage was comparable to that of the control NK-92MI cells maintained at 37°C (93% viable cells) and to the NK-92MI sample incubated at 43°C (92% viable cells) for the same duration of exposure. In Figure 1 are summarized the data relating to the viability of NK- 92MI cells under different combination of temperature (T: 37°C, 41 °C or 43°C) and time duration (60 min or 90 min) when using a thermomixer as a heating source. Cell viability was assessed using a dead staining marker (FVS450). The portion (A) of the Figure 1 refers to viability collected for NK-92MI cells incubated at different temperatures for a time exposure equal to 60 min at 37°C (left upper plot), 41 °C (middle upper plot) and 43°C (right upper plot). The portion (B) refers to the viability data collected on NK-92MI cells incubated for an exposure of 90 min using a thermomixer at 37°C (left lower plot), 41 °C (middle lower plot) and 43°C (right lower plot), respectively. In all plots, a square box highlights the negative cells, namely the alive cells. The Side Scatter Area (SSC-A) and log-fluorescence intensity (FVS450) are reported in each dot plot. The (C) portion of the image, on the other hand, represents cell proliferation measured 72h after the application of mild MHT treatment.

[0060] This observation indicates that a 60-m inute exposure time at the chosen temperatures of 41 °C did not significantly impact cell viability. However, when the incubation was prolonged to 90 minutes, viability decreased with increasing also exposure time (Figure 1 ). Specifically, at the highest temperature of 43°C after 90 minutes exposure, only 41 % of the cells remained viable.

[0061] These findings were further supported by optical images (Figure 2). In culture, a high density of NK-92MI cells promotes their proliferation, resulting in the formation of cell clusters that are easily visible under an optical microscope. After 24 hours from the thermal experiment, the cells appeared healthy and alive, exhibiting a macroscopic morphology similar to that of the control samples kept at 37°C for 60 or 90 minutes in most of the tested conditions. Conversely, the incubation of cells at 43°C for 90 minutes resulted in a high mortality rate. This is reflected in the optical images, which show a distinct distribution of cells compared to the controls and other treatment samples. Based on the results reported in Figure 1 and 2, we selected 41 °C for 60 minutes as the optimal conditions for hyperthermia application, ensuring minimal impact on cell viability. In Figure 2 are shown the Optical images of NK-92MI taken at 24h after the exposure to different thermal treatment conditions when using the thermomixer as heating source. In the left side of the panels are shown NK-92MI cells exposed at 37°C for 60 or 90 min; in the middle part those at 41 °C for 60 or 90 min; and in the right side those at 43°C for 60 or 90 min. The morphology of the cells at 41 °C looks very similar to that at 37°C while it start to progressively change at 43°C and with increase of the thermal treatment from 60 to 90 minutes. Magnification used 10X.

[0062] Impact of surface coatings on cellular uptake mechanisms

[0063] After determining the optimal hyperthermia conditions, the next step involved repeating the experiment using MNPs as the heat mediators. In this case, we selected lONCs nanocubes having 16 nm edge. To ensure their stability and compatibility in biological media, the prepared nanocubes were coated with an amphiphilic Poly (maleic anhydridealt-1 -octadecane) polymer using a well-established coating procedure (R Di Corato et al., Journal of Materials Chemistry, 2008 18 (17), 1991-1996). Subsequently, the polymer-coated nanoparticles were further functionalized with diamine-polyethylene glycol (NH2-PEG-NH2, MW 2000) using standard EDC chemistry (RA Sperling et al., Advanced Functional Materials, 2006 16 (7), 943-948). TEM imaging (Figure 3A) confirmed the successful coating of the NPs by the thin polymer layer visible on some nanocubes.

[0064] The dynamic light scattering (DLS) measurements (Figure 3C) revealed that the hydrodynamic size (Hd) of the lONCs coated with polymaleic polymer and PEG (NC-PEG) was determined to be 130 ± 24 nm by intensity weight. The surface charge, express in zeta potential ( ) indicated that NC-PEG possessed a charge of -28 ± 7 mV. This data was also supported by the electrophoresis run, where the migration distance of the PEG-functionalized sample was lower compared to the polymer- coated (PC) sample indicating a decrease in charge compared with polymer-only coated particles not functionalized with amino-PEG (Figure 3B). Furthermore, the stability of the NC-PEG sample in cell culture medium, measured in terms of Hd stability overtime, was unaffected up to 24 hours (Figure 3D). The lONCs showed magnetic heating performance express in terms of specific adsorption rate (SAR) values when exposed to AMF of clinical conditions (at a fixed field of 24 kA / m at a frequency of 70 or 170 or 250 kHz) that are among the highest for iron oxide NPs of these size(Figure 3E). Indeed, a lONCs solution of only 3mgFe / mL can reach and maintain a temperature of 41 °C at 170 kHz with a varying field intensities of 17-24 kA / m, field conditions that are biological safe (Figure 3F).

[0065] The Figure 3 shows the data relating to the characterization of iron oxide nanocubes NC-PEG NPs used for the mild-MHT. The portion (A) of the Figure comprises TEM images of iron oxide nanocubes (NC-PEG) NPs at different magnifications. NPs appeared well dispersed on the grid and coated by a thin and visible polymer layer. The portion (B) show the electrophoretic run of NC-PEG NPs at two distinct steps of their functionalization, after the polymer coating (PC) as first inner coating and the further PEG functionalization (PC PEG) after covalent attachment of PEG to the PC. The portion (C) illustrates the DLS profile weighted by intensity of the sample of NC-PEG in water. The portion (D) shows NC- PEG stability analysis of hydrodynamic size weighted by intensity in NK cells culture medium. The portion (E) illustrates SAR values measured on NC-PEG NPs at a fixed field of 24 kA / m and frequencies of 70, 170 and 250 kHz, respectively. The portion (F) shows the heating profiles under the exposure of an AMF (at fixed frequency of 182 kHz and constant temperature of 41 °C obtained under an oscillating field amplitude between 17-20 kA / m).

[0066] Consequently, the mild / MHT experiment has been carried out, this time using lONCs (15 nm), and replacing the thermomixer heater with the addition of lONCs and the application of MHT heating. In brief, to 1.2 million NK-92MI cells UV-sterilized NC-PEG were added in a volume of 150 pl media to obtain a final nanoparticle concentration of 3gFe / L, and then subjected to mild-MHT to reach 41 °C as the one set above (Figure 3F) were applied with the only difference that the MHT was applied in two cycles of 30 minutes each, at a fixed temperature of 41 °C and a fixed frequency of 182 KHz. The magnetic field strength (H) during the experiment ranged from 15 to 17 kA / m, and was automatically adjusted by the AMF applicator to maintain the desired temperature (Figure 3F).

[0067] The Figure 4 illustrates the cellular uptake evaluation of NC-PEG NPs after mild-MHT through ICP and TEM imaging. The portion (A) of the Figure is a scheme of the MHT experiment; 1 .2 million of NK-92MI cells were mixed with NC-PEG (3 gFe / L) in 150 pl of cell culture medium. The sample was exposed to 2 cycles of mild-MHT (30 min each, at fixed T of 41 °C and f of 182 kHz while field amplitude varied between 15 to 17 kA / m (adjusted automatically by the AMF applicator to maintain the selected temperature of 41 °C). The portion (B) relates to the elemental analysis of Iron evaluated as the pg of Fe per cell for NK-92MI cells incubated with NC-PEG NPs at 37°C for 60 min with no AMF exposure (internalization of ~8 pg Fe / cell) and for NK-92MI incubated with NC-PEG NPs and exposed to 2 cycles of MHT at 41 °C (internalization of ~13 pg Fe / cell). The upper portion (C) shows representative TEM images of NK-92MI cells after NPs and mild-MHT (NK-92MI cells upper panels) with highlighted with arrows the cellular invaginations with the nanocubes with a darker contrast. The lower portion (C) shows TEM images of the NK-92MI cells after NPs incubation at 37°C with no AMF application. The image captures nanocubes mostly on the cell membrane.

[0068] After the MHT treatment, cells were washed to remove any excess of NPs and then replated with fresh medium. After 24 hours, cell viability was assessed using the FVS450 dead staining dye on different cells samples: untreated cells which did not receive NPs nor MHT (Ctrl); cells in which NPs were added but no MHT was applied (NC-PEG); cells that were exposed to MHT but which did not receive any NPs (AMF) and cells exposed to both NPs and MHT (NC-PEG + MHT). The MHT-treated sample exhibited a 12.87% cell death fraction, (Figure 5A). However, this percentage decreased to 0% after 72 hours post-treatment, indicating that the cells were able to recover from the acute and mild MHT stress without any signs of toxicity. The control samples, including cells maintained at 37°C, cells exposed to AMF for 60 minutes (at a fixed frequency of 182 KHz and field strength of 17 kA / m), and cells incubated with NC-PEG at 37°C, displayed mortality rates of 7.1 %, 8.4%, and 6.1 %, respectively (Figure 5B-C-D). Furthermore, to demonstrate the nontoxicity of the alternating magnetic field in the absence of MNPs, we conducted an experiment in which both NK92-MI cells and T cells were exposed to the maximum magnetic field (40 kA / m) and frequency (182 kHz) conditions, (see Figure 4F-G). As reported, the viability values between the control, i.e., cells maintained at 37°C for 1 hour, and the sample exposed to AMF for the same exposure, showed comparable values of cell viability with no significant differences.

[0069] To quantify and assess the internalization of NC-PEG within NK- 92MI cells following MHT, elemental analysis (ICP-OES) and TEM images were conducted. The cells were exposed to AMF (f=182 kHz, H=17 kA / m) for 60 minutes, and immediately after the treatment, excess NPs were removed through a washing step allowing the evaluation of only the NPs associated with the cells. Subsequently, the cells were pelleted and processed differently depending on the analysis to be performed.

[0070] From elemental analysis, consistent with the hypothesis of the present inventors, MHT induced a higher internalization of NPs within the cells compared to standard incubation at 37°C. The iron uptake in the sample exposed to NC-PEG and MHT was 1.5 times higher (~13 pgFe / cell) than that in the sample of NK-92MI cells exposed to NC-PEG at 37°C without application of MHT, which exhibited an iron amount of 8 pgFe / cell (Figure 4B). These ICP results were further supported by TEM images, revealing NPs with their distinct cubic shape both on the NK-92MI cell membrane and inside intracellular vesicles (Figure 4C). Moreover, by visual inspection of the cell pellet, a brownish color appeared in both cases due to the NPs uptake but the one exposed to MHT appears slightly darker than that not exposed to MHT but exposed to MNPs (inset Figure 4B).

[0071] The Figure 5 illustrates the evaluation of NK-92MI cells viability with or without NPs and with or without the application of mild-MHT. Viability results by flow cytometry analysis of the dead staining marker FVS540 of A) NK-92MI cells exposed to NPs and MHT (NC-PEG + MHT), B) NK-92MI cells incubated with NPs and kept at 37°C (NC-PEG), C) NK- 92MI cells not treated with NPs and exposed to an AMF (fixed f : 182 kHz and H:17 kA / m)(AMF), and D) NK-92MI cells alone incubated at 37°C with no exposure to NPs and MHT (Ctrl). The Side Scatter Area (SSC-A) and log-fluorescence intensity (FVS450) are reported in each dot plot. On the right part of the figure is shown the histohgram relative to three independent experiments. Portions F and G of Figure show the viability results obtained on both NK92-MI cells and T cells when exposed to AMF under the maximum conditions (182 kHz, 40 kA / m) in the absence of nanoparticles.

[0072] Next aim was to assess the impact of this internalization on the physiologycal activity of NK-92MI cells against the U87 glioblastoma cell line. When NK cells are exposed to cancer cells, they are typically activated, leading to cell degranulation. This degranulation involves the fusion of cytotoxic vesicles containing granzymes and perforin with the plasma membrane. As a result, lysosome-associated membrane protein- 1 (such as CD107a), which characterizes the membrane of cytotoxic vesicles, is transferred to the cell surface, making it accessible for antibody binding.

[0073] To gain insight into the cytotoxic activity of NK cells with or without exposure to MHT, we measured the membrane expression of CD107a, which serves as a marker for NK cell degranulation.

[0074] Briefly, after washing the cells exposed to MHT and their corresponding controls, as described above, they were plated back for 24 hours. Subsequently, NK-92MI cells were co-cultured with U87 cells in a 1 :1 ratio, and the levels of CD107a on the NK cell surface were analyzed using flow cytometry (Figure 6). Consistently with the viability data collected 24 hours post-MHT, the sample exposed to NC-PEG and MHT exhibited a slight decrease in degranulation activity (22.52%) compared to the untreated cells incubated with the target (Ctrl), which showed a positive expression of CD107a at 26.39%. The simple incubation of cells with NPs (NC-PEG) and exposure to an AMF in the absence of NPs (AMF) did not affect the functionality of the cells, with a percentage of CD107a+ / CD56+ NK cells at 27.11 % and 27.80%, respectively. A sample of NK-92MI cells alone without cancer cells was used as a blank to observe the basal level of CD107a expression.

[0075] Figure 6 illustrates the degranulation activity of NK-92MI cells against U87 cancer cells measured through FACS analysis. NK-92MI cells were co-cultured with U87 cells (at a ratio 1 :1 ) in the presence of anti-CD107a antibody to detect its presence on the cell membrane as a marker of degranulation. The percentage of degranulating CD107a+NK cells was evaluated through flow cytometry and was represented as individual plot (left panel) and as histograms of the percentage of CD107a positive cells for: untreated NK cells incubated with the target U87 cancer cells (Ctrl); NK cells alone exposed to an AMF (fixed f: 182 kHz and H: 17-20 kA / m) in the absence of NPs and incubated with the U87 target (NK-92 + AMF); a sample of NK cells incubated with NC-PEG NPs (but no exposure to MHT) and U87 cells (NK-92 + NC-PEG); the treatment group of NK cells with NC-PEG NPs and MHT exposure and then incubated with U87 cells (NK-92 + NC-PEG + MHT). A sample composed of only NK-92MI cells was used as a blank. Standard deviations on the histograms are derived by three independent experiments.

[0076] Impact of MNPs composition on cellular uptake mechanisms

[0077] To generalize the mild-MHT method here proposed to any magnetic NPs suitable for MHT, it has been decided to test nanocubes composed of ferrite nanoparticles doped with Zinc, the so-called Zinc ferrite (Zn-Fe) NPs. The choice was focused on Zn-Fe NPs sharing the same uniform cubic shape to that of lONCs, tested above and almost a comparable size (18 nm). The Zn-Fe NPs have been coated with the same amphiphilic polymer, Poly (maleic anhydride-alt-1 -octadecane), and then further functionalized with diamine-polyethylene glycol (NH2-PEG- NH2, MW 2000) using standard EDC chemistry employed for iron oxide nanocubes. TEM imaging confirmed the successful nanoparticle coating, while dynamic light scattering measurements indicated a hydrodynamic size of 18 nm for the Zn-Fe NPs based on intensity weight. The potential analysis revealed a charge of -39 mV for the Zn-Fe NPs. Further support for this data was found in the electrophoresis run, where Zn-Fe NPs migrate towards the positive pole and the migration distance of the PEG- functionalized sample was observed to be less than that of the polymer- coated sample, (Figure 7).

[0078] The Figure 7 illustrates the characterization of Zn-Fe NPs used as alternative magnetic NPs to iron oxide NPs. The portion (A) of the figure shows TEM images of Zn-Fe nanocubes at different magnifications. NPs appeared well dispersed on the grid and coated by a thin and visible polymer layer. The portion (B) shows the electrophoretic run of Zn-Fe NPs at two distinct steps of their functionalization, after the polymer coating (PC) as first inner coating and the further PEG functionalization (PC PEG) after covalent attachment of PEG to the PC. The portion (C) shows the DLS profile weighted by intensity of the sample of NC-PEG in water. The portion (D) shows the SAR values measured on Zn-Fe NPs at a fixed frequency 170 kHz and a field amplitude of 12, 16, and 24 kA / m, respectively.

[0079] For the experiment, a very similar protocol to that of lONCs was used, a mixture of 1 .2 million NK-92MI cells and UV-sterilized Zn-Fe NPs, totaling 150 pL, achieving a final NPs concentration of 3gFe / L, followed by exposure to mild MHT. The MHT treatment encompassed two 30- m inute cycles, maintaining a constant temperature of 41°C and a frequency of 182 KHz. The magnetic field intensity, ranging from 16 to 18 kA / m, was automatically adjusted by the AMF applicator to sustain the specified temperature. After the treatment, the cells underwent a thorough washing process to eliminate any excess of Zn-Fe NPs before replating NK-92MI cells in fresh medium. After a 24-hour incubation at 37°C, cell viability was evaluated using the dead cells staining dye FVS540. As shown in Figure 8A, after the exposure to MHT NK-92MI cells maintain a good viability, comparable to untreated cells incubated at 37°C for the same time exposure.

[0080] The Figure 8 illustrates the demonstration of the generalization of the mild-MHT protocol on NK-92MI cells when using Zn-Fe NPs as another type of MNPs. The portion (A) shows the viability of NK-92MI when using Zn-Fe NPs in presence or absence of after mild-MHT. The portion (B) shows the cellular uptake by iron elemental analysis by ICP expressed in picogram of Fe uptake per cell (pg / cell). For the NK-92MI cells incubated with Zn-Fe NPs at 37°C for 60 min an internalization of ~5 pg Fe / cell was measured, while an internalization of ~9 pg Fe / cell was measured for NK-92MI incubated with Zn-Fe NPs and exposed to AMF for 2 cycles of MHT at 41 °C. The portion (C) shows the evaluation of Zn- Fe NPs internalization at different magnifications, upon mild-MHT protocol by TEM analysis.

[0081] To evaluate the internalization of Zn-Fe NPs within NK-92MI cells post-MHT, both ICP-OES and TEM analyses have been utilized. The cells underwent exposure to an alternating magnetic field (frequency = 182 kHz, field strength = 16-18 kA / m) for 60 minutes in two cycles of 30 minutes each. After this treatment, excess NPS were eliminated through washing, facilitating the quantification and visualization of only the percentage associated with the cells. Following the washing step, the cell pellet exhibited a brownish tint in both cases due to nanoparticle uptake, with the MHT-exposed pellet appearing slightly darker than the non-MHT- exposed pellet but exposed to the nanoparticles (inset Figure 8B). This observation was also confirmed by the ICP analysis.

[0082] As expected, MHT resulted in an enhanced internalization of NPs within the cells compared to standard incubation at 37°C. The iron uptake in the Zn-Fe NPs and MHT-exposed sample was greater (approximately 9 pgFe / cell) than that of the sample of NK-92MI cells exposed to Zn-Fe NPs at 37°C without MHT, which exhibited an iron amount of 5 pgFe+Zn / cell (see Figure 8B). These ICP results found support in TEM images, showing NPs with their characteristic cubic shape both on the cell membrane and within intracellular vesicles (see Figure 8C).

[0083] Impact of MNPs coating on cellular uptake mechanisms

[0084] To generalize the mild-MHT method proposed here to any MNPs suitable for MHT, it was decided to test iron oxide nanoparticles that share the same composition and cubic shape as the NPs previously tested but were coated with a different polymer. In this particular case, it was decided to coat the lONCs instead of polymer 1 , with polymer 2 (see Figure 9A- B). TEM images confirmed the successful coating of the nanoparticles, while dynamic light scattering measurements indicated a hydrodynamic size, dH, of 16 nm for the NPs based on intensity weight (Figure 9).

[0085] In addition, the same procedure used previously was also adopted here. Briefly, 1 .2 million cells were exposed to NPs at a concentration of 3g / L in a volume of 150 pL and subjected to two cycles of mild MHT, for 60 minutes at a frequency of 182 kHz while reaching a temperature of 38°C as indicated in the temperature profile (see Figure 9C). After mild- MHT treatment, the cells were subjected to a washing step to remove excess nanoparticles that did not interact with the cells. Of the cells used, 200,000 cells were replicated in a 96-well plate to assess viability and proliferation after 24 hours, while the remaining portion of cells were processed to assess and quantify the presence of iron within the cells.

[0086] Viability assessments conducted after 24h revealed that cell samples exposed to NPs in the absence and presence of AMF behaved similarly to untreated cells, i.e. , those incubated in the same volume of cell medium only and maintained at 37°C for the same duration (see Figure 9D). To determine the presence of NPs in the cell pellet, the cells were treated for ICP-OES after the washing step. Analysis showed that exposure to mild MHT treatment induced greater internalization of NPs in the NK-92MI cells compared with the sample maintained at 37°C, showing an iron value of 8 pgFe / cell compared with 4 pgFe / cell (see Figure 9E).

[0087] Figure 9 shows the demonstration of the generalization of the mild- MHT protocol on NK-92MI cells when using lONCs coated with a polymer 2 as another type of coating. Portions (A) and (B) of the Figure show the characterization of the new sample of NPs used. Specifically, portion (A) shows the TEM images of the NPs while portion (B) shows the hydrodynamic radius size of the NPs after transfer to water with polymer 2 using DLS. Portion (C) of the image shows the temperature profile obtained with the NPs coated with polymer 2. Portion (D) shows the viability of NK-92MI when using NPs in the presence or absence of mild- MHT. Portion (E) shows cellular uptake by elemental iron analysis with ICP expressed in picograms of Fe uptake per cell (pg / cell). An internalization of ~4 pg Fe / cell was measured for NK-92MI cells incubated with NPs at 37°C for 60 min, while an internalization of ~8 pg Fe / cell was measured for NK-92MI cells incubated with NPs and exposed to 2 cycles of MHT for a total of 60 min of AMF exposure at 38°C. The use of this polymer for the transfer of iron oxide clusters in the shape of Rubik's cubes (Figure 9F) was also tested under similar conditions, and a difference in iron internalization was observed on the same cells with and without the application of mild-MHT in a range of iron values comparable to that of iron oxide nanocubes.

[0088] Implementation of the MHT protocol for IQNCs internalization using NK primary cells

[0089] Having set the mild-MHT treatment at a temperature of 41°C for 60 minutes as a successful method to speed the internalization of NC- PEG / Zn-Fe NPs without significantly impacting the viability or functionality of NK-92MI cells, the next step focused on validating the developed protocol using primary NK cells derived from healthy donors. The primary NK cells were isolated from peripheral blood and cultured in the presence of interleukin ( IL)-2 as a first step.

[0090] Impact of the composition on cellular uptake mechanism

[0091] As for NK-92MI, the same mild-MHT procedure were also adopted for primary NK cells. Similarly, 1.2 million cells were exposed to Zn-Fe NPs at a concentration of 3g / L in a volume of 150 pL and subjected to two cycles of mild MHT, for 60 minutes at a fixed frequency and temperature (182 kHz and 41 °C). Following treatment, cells underwent a washing step to eliminate excess nanoparticles that did not interact with the cells. Subsequently, 200,000 cells were replated into a 96-well plate to assess viability and proliferation after 24 hours, while the remaining cell portion was divided and treated in different ways to assess and quantify the presence of iron and zinc within the cells. Viability assessments revealed that the cells behaved similarly to untreated cells, comparable to cells incubated in the same volume of medium only and kept at 37°C (see Figure 10A). To determine the presence of NPs in the cell pellet, cells were processed for ICP-OES after the washing step. As also observed for NK-92MI cells, MHT induced an enhanced NPs uptake in primary cells (expressed in pgFe / cell), being twofold higher than the sample of cells incubated with nanoparticles and kept at 37°C for the same exposure time (60 minutes) (see Figure 10B). In addition, TEM analysis to check the cell structure immediately after MHT confirmed the presence of NPs within and on the surface of the cells (see Figure 10C).

[0092] The Figure 10 summarizes the Mild-MHT experiment carried out on primary NK cells derived from healthy donor with Zn-Fe NPs. The portion (A) shows the viability results of primary NK cells treated with Zn- Fe NPs with or without the exposure to the AMF for 1 hour and compared to control cells (CTRL) kept for 1 h at room temperature with no exposure to NPs and to MHT field. The portion (B) shows the uptake of Zn-Fe NPs by iron elemental analysis per cell (pg / cell): The samples represented are: NK cells incubated with only Zn-Fe NPs (no application of MHT) at 37°C for 60 min with an internalization of ~3 pg Fe / cell;NK cells incubated with Zn-Fe NPs and exposed to 2 cycles of 30 minutes of MHT at 41 °C with an internalization of ~7 pg Fe / cells. Portion (C) shows TEM images of the sample of NK cells exposed to Zn-Fe NPs and exposed to 2 cycles of MHT at 41 °C.

[0093] On the same primary NK cells, NC-PEG NPs were also tested. In brief, 1.2 million cells were mixed with NC-PEG (3gFe / L) and exposed to two cycles of MHT (30 minutes each), reaching a mild temperature of 41 °C using a clinical-use frequency (f=182 kHz) and a field amplitude of H=17-20 kA / m. After the MHT application, NK cells were washed from the NPs in excess and then plated back into a 96-well plate, and their viability was assessed after 24 hours. To investigate whether the NPs induced any toxicity, cell viability was analyzed using the FVS450 dead cells staining dye assay and flow cytometry (Figure 11 A). Overall, NC-PEG treatment did not significantly affect NK cell viability at 24 hours post-MHT, as evidenced by the comparable percentages of viable cells (-90%) to the controls used. This suggests that the incubation of primary NK cells with MNPs at the concentration employed (3gFe / L) and mild-MHT application do not have a significant impact on the cells.

[0094] Furthermore, to evaluate the internalization of lONCs by NK cells, elemental analysis for iron quantification and TEM analysis for cellular structure inspections were conducted immediately after MHT (Figure 11 B- C). The results from the elemental analysis revealed that the primary NK cells with NC-PEG sample exposed to MHT (Figure 11 B) exhibited an iron uptake corresponding to 6.5 pgFe / cell, while the sample incubated at 37°C had an iron content of only 2 pgFe / cell, nearly three times lower than that observed for the primary NK cells exposed to MHT. The ICP-OES results for NC-PEG NPs were confirmed by TEM images, which allowed for the localization of the NPs inside the cells, appearing in cellular invaginations in the cytoplasm resembling endocytic vesicles, as well as along the cellular membrane (indicated by black arrows in Figure 11 C for the NC-PEG sample).

[0095] Additionally, ICP-OES analysis was performed 24 hours after mild- MHT to examine whether the NPs were retained or released by the cells over time. The iron amount per cells measured just after MHT was applied was found of 6 pgFe / cell and it did not change on a sample of NK cells exposed to the same treatment and kept for additional 24 hours in culture before repeating the iron measurement. These data suggest the complete preservation of lONCs by NK cells 24 hours post MHT treatment.

[0096] In Figure 11 are illustrated the results of the Mild-MHT experiment on primary NK with or without MHT and NC-PEG NPs. The portion (A) of the figure shows the viability of primary NK cells after treatment with NC- PEG using FVS staining. The portion (B) shows the quantification by ICP analysis of the elemental iron associated with primary NK cells with or without MHT exposure. The portion (C) shows representative images obtained by TEM at different magnification. In the images are shown depicting primary NK cells after NC-PEG NPs treatment in the case of cells exposed to MHT incubated with NC-PEG. Further, the functionality of the primary NK cells with or without MHT application has been analyzed. Like NK-92MI cells, we examined the degranulation activity of primary NK cells (CD107a+ NK cells) in this case in presence of two cancer cell lines: human neuroblastoma (SHSY5Y) and human glioblastoma (U87). Controls consisted of primary NK cells without the anti-CD107a antibody and primary NK cells with anti- CD107a antibody (to assess the basal expression of CD107a) in the absence of a target. For the full analysis NK cells samples were analysed and compared under various culture conditions: cells alone incubated at 37°C as a control (Ctrl); NK cells exposed to an AMF without NC-PEG NPs (NK+AMF); NK cells incubated with NC-PEG NPs at 37°C and no exposure to MHT (NK+NC-PEG); and cells incubated with NC-PEG NPs and exposed to MHT (NK+NC-PEG+MHT).

[0097] After MHT or incubation at 37°C, cells were washed to remove the excess of nanoparticles and then suspended in medium without IL-2 on ice. This step aimed to freeze the cell status immediately after MHT, which represents the most aggressive time point during the whole process. In culture, cells tend to recover from the stress induced by the treatment, so it has been chosen to analyze cell functionality immediately after the treatment, corresponding to the highest stress point for the NK cells if any dysfunctional effect may be induced.

[0098] Primary NK cells exposed to NC-PEG and to two cycles of 30 minutes each of MHT (light grey bar) exhibited significant degranulation against both U87 and SHSY5Y cell lines, with degranulation percentages of 41 % and 31 %, respectively, (Figure 12). These results were comparable to the degranulation observed in the Ctrl, NK+AMF, and NK+NC-PEG samples against both SHSY5Y and U87 cells, with degranulation values ranging from 35% to 40% of CD107a-positive cells. The graph highlights that the uptake of NPs alone induces a weak degranulation, as evident from the higher basal expression of CD107a on the surface of NK cells in the NK+NC-PEG and NK+NC-PEG+MHT samples compared to Ctrl and NK+AMF samples (refer to Figure 12B for the NK+CD107a condition). The Figure 12 illustrates the degranulation activity measured by percentage of CD107a positive on primary NK cells with NC-PEG NPs with and without MHT application. A representative schematic of the cytotoxic degranulation activity carried out by NK cells in the presence of tumor cells is shown in portion (A). In portion (B) the quantification of degranulation for NK cells associated with NC-PEG NPs against the neuroblastoma target SHSY5Y or glioblastoma (U87) is shown. Four different conditions were analyzed for all these samples: cells only (first bar), cells exposed to AMF in the absence of NPs (second bar); cells incubated with NC-PEG alone (third bar); cells incubated with NC-PEG NPs and exposed to 2 cycles of MHT (fourth bar). Data (mean ± SEM) were obtained from three independent experiments, each performed in triplicate. Portion (C) of the image shows reference histograms obtained from the degranulation analysis performed at FACS.

[0099] Furthermore, the phenotypic profile of NK cells has been examined by evaluating the expression of their primary activating, inhibitory, and chemokines receptors, (Figure 13A). Unlike T cells, NK cell activity is not triggered by MHC-restricted antigen recognition but rather by the integration of signals mediated by activating and inhibitory receptors upon binding to specific ligands on target cells (Figure 13A). Inhibitory receptors play a crucial role in regulating NK cell activity against normal cells by recognizing Human Leukocyte Antigen-class I (HLA-I), which is expressed by nearly all nucleated cells. This interaction dampens NK cell activation to prevent cytotoxic reactions against self-cells. Inhibitory receptors include killer immunoglobulin-like receptors (KIRs) specific to classical HLA-I alleles (HLA-A, -B, and -C), as well as C-type lectin receptors (e.g. NKG2A / B) specific to non-classical HLA-E.

[0100] On the other hand, activating receptors identify pathological cells and initiate a response against them. These receptors recognize ligands induced or upregulated on pathological cells, such as tumour cells, thereby tilting the balance between positive and negative signals towards NK cell activation. The activating receptors involved in killing tumour cells include Natural Cytotoxicity Receptors (NCR) NKp30, NKp46, and NKp44, NKG2D, D NAM Accessory Molecule-1 (DNAM-1 ), and NKp80. Upon activation, NK cells can express CD69, which is an activating receptor and a typical marker of NK cell activation. The exposure of NK cells to pathological cells occurs because NK cells are capable of migrating and circulating in the blood. This process relies on the expression of chemokines receptors, such as CXCR3 and CXCR4, which guide NK cells towards peripheral tissues in response to chemokines gradients. Therefore, if NK cells are used as physiological carriers of NPs to the tumour site, it is essential for these NK cells to maintain their functionality, especially their ability to migrate within the circulation and reach the tumour. To assess this, following MHT or incubation at 37°C, cells were washed and incubated with different antibodies to evaluate the expression of activating, inhibitory, and chemokines receptors (Figure 13A).

[0101] To assess the impact of NPs and MHT on NK cells, an analysis was carried out of activating, inhibitory, and chemokines receptors by washing the primary NK cells and incubating them with specific antibodies. In the case of the NC-PEG NPs (refer to Figure 13 C-D-E), the bar charts clearly indicate that the presence of NPs, whether through simple co-incubation or co-incubation with NC-PEG NPs and application of MHT, led to a slight decrease in the expression of certain activating and inhibitory receptors in NK cells. This effect was more pronounced for DNAM-1 and CD69 and for CD16 (Figure 13C), while for other markers such as NKG2D, NKp80, NKp30 (activating receptors), and KIRs and NKG2A (inhibitory receptors), the impact was less pronounced and comparable to the expression levels observed in the control samples. However, the expression of chemokine receptors, CXCR3 and CXCR4, remained unaffected by the presence of NPs, showing similar fluorescence intensity across all conditions (Figure 13E).

[0102] Overall, the presence of NPs inside the cells had only a marginal effect on their viability and functionality. After MHT, NK cells were still capable of degranulation similar to the control samples. It is worth considering that the activity of NK cells relies on the cooperation of multiple activating and inhibitory receptors, which may diminish the modulatory effects induced by MHT. Importantly, the data collected in this study demonstrate that our NPs and MHT treatments do not impede NK cell "patrolling" in the body, as no apparent downregulation of chemokine receptor expression was observed, in the specific case of the receptor CXCR3.

[0103] The Figure 13 illustrates the functional and Phenotypic profiles of primary NK cells through the analysis of activating, inhibitory and chemokine receptors expression when using NC-PEG NPs. The portion (A) of the figure shows a scheme of NK cells activation through activators, inhibitors and chemokines signaling when in presence of cancer cells. Portion (B) shows representative histograms for each molecule analyzed during FACS analysis. The portions (C)-(E) show the quantification of relevant surface markers expression, including activating receptors (CD16, NKp30, NKp46, NKG2D, DNAM-1 , NKp80, CD69), inhibitory receptors (KIRs and NKG2A) and chemokine receptors (CXCR3 and CXCR4). These analysis were performed for all the conditions tested (bar at different colors), such as control cells, primary NK cells in presence of MHT field (NK+AMF), primary NK cells in presence of NPs only with no exposure to MHT (NK+NC-PEG) primary NK cells in presence of NPs and exposed to mild-MHT (NK+NC-PEG+MHT).

[0104] Implementation of the MHT protocol for internalization of IQNCs using other cells of the immune system (e.g., T cells)

[0105] Having established that mild-MHT treatment at a temperature of 41 °C for 60 minutes is an effective method to accelerate internalization in NK cells (NK-92MI and donor NK) of different types of NPs, which differentiate in composition, coating and size, without significantly impacting cell viability or functionality, it was decided to take a step further by going to validate the protocol used on other cells of the immune system, such as T cells.

[0106] As an initial test to validate the uptake / internalization protocol for MNPs in T cells, it was decided to test cubic-shaped iron oxide nanoparticles with a size of 16 nm and coated with polymer 2, previously tested with NK-92MI. As before, the protocol adopted involved 1 .2 million cells that were exposed to NPs at a concentration of 3g / L in a volume of 150 pL and subjected to two cycles of mild MHT, for 60 minutes at a frequency of 182 kHz while reaching a temperature of 38°C. After mild-MHT treatment, the cells were subjected to a washing step to remove excess nanoparticles that did not interact with the cells. Subsequently, 200,000 cells were replicated in a 96-well plate to assess viability and proliferation after 24 hours, and the remaining portion of cells were processed to assess and quantify the presence of iron within the cells.

[0107] Viability assessments conducted 24h after application of the mild- MHT protocol showed that cell samples exposed to MNPs in the absence and presence of AMF behaved similarly to untreated cells, (see Figure 14). In addition, to determine the presence of NPs in the cell pellet, cells were treated for ICP-OES after the washing step (Figure 14). As observed for both NK-92MI and donor NK cells, mild MHT induced a higher uptake of NPs expressed in (pgFe / cell) in T cells with approximately two-fold higher values (~6 pgFe / cell) than in T cells maintained at 37°C for the same exposure time (60 min) in the absence of AMF(~3 pgFe / cell), (see Figure 14B).

[0108] Figure 14 illustrates the extension of the mild MHT protocol to another type of immune system cells, specifically T cells. Portion (A) of the image shows the viability of T cells after being treated with NPs coated with polymer 2 with or without exposure to AMF for 1 hour. Portion (B) of the image shows NPs uptake expressed as (pgFe / cell) in T cells downstream of mild MHT treatment after a washout. The samples represented are: T cells incubated with MNPs only (without application of MHT) at 37°C for 60 minutes with an internalization of ~3 pgFe / cell; T cells incubated with MNPs and exposed to 2 cycles of 30 minutes of MHT at 41 °C with an internalization of ~6 pg Fe / cell. In addition, by visual inspection of the cell pellet, it appeared brownish in both cases due to MNPs uptake, but the one exposed to MHT appeared slightly darker than the one not exposed to MHT but exposed to MNPs (inset of Figure 14B).

Claims

CLAIMS1. Method for the uptake of magnetic nanoparticles (MNPs) by immune cells, comprising the steps of preparing a mixture of a given quantity of immune cells with a given quantity of MNPs, characterized in that it is provided a step of subjecting the said mixture to an alternating magnetic field, having a given frequency and a given intensity, in order to heat the said mixture at a temperature comprised between 38° and 43° C, for a given time.

2. Method according to claim 1 , in which the mixture comprises between 1 and 2 millions of immune cells and MNPs so as to reach final concentration of about 0, 5-4,0 gFe / L.

3. Method according to claim 1 or 2, in which the immune cells are NK cells.

4. Method according to claim 1 or 2, in which the immune cells are T cells.

5. Method according to claim 1 or 2, in which the immune cells are monocytes.

6. Method according to claim 3, in which the NK cells are primary NK cells, that is to say NK cells derived from a healthy donor.

7. Method according to claim 3, in which the NK cells are NK92-MI cells, that is to say an immortalized strain of immune cells.

8. Method according to anyone of the preceding claims 1 to 7, in which the magnetic nanoparticles (MNPs) show the ability to heat under a clinically safe kHz-based radiofrequency exposure and can be stably dispersed in physiological medium suitable for immune cells.

9. Method according to claim 8, in which the MNPs are iron oxide based.

10. Method according to claim 9, in which MNPs are nanocubes of iron oxide (FesC ) having the cube edge in the range of 14-15 nm.

11. Method according to claim 8, in which the MNPs are ferrite nanoparticles containing other metals.

12. Method according to claim 11 , in which MNPs are nanocubes of iron oxide doped with Zinc (ZnFe2O4) having the cube edge in the range of 14-25 nm.

13. Method according to anyone of the preceding claims 8 to 12, in which the MNPs are coated with an amphiphilic polymer.1 . Method according to claim 13, in which the amphiphilic polymer is Poly (maleic anhydride-alt-1 -octadecane) polymer and functionalized with diamine-polyethylene glycol (NH2-PEG-NH2).

15. Method according to claim 13, in which the amphiphilic polymer is Poly (maleic anhydride iso-butylene) equipped with dopamine molecules and amino-polyethylene glycol derivative molecules (amino-PEG derivatives).

16. Method according to anyone of the preceding claim 1 to 15, in which the alternating magnetic field is applied to the mixture at frequency in the range of 80 kHz-1 MHz, and preferably of 175- 185 kHz, and with a field intensity of 5-45 kA / m.

17. Method according to anyone of the preceding claims 1 to 16, in which the duration of the treatment of the mixture is in the range of 30-120 minutes, and preferably is 60-80 minutes or less.

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