Click-Engineered Magnetic Fusogenic Cell-Derived Nanocarriers for Enhanced Magnetic-Field-Assisted Drug Delivery

Click-Engineered Magnetic Fusogenic Cell-Derived Nanocarriers for Enhanced Magnetic-Field-Assisted Drug Delivery | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Click-Engineered Magnetic Fusogenic Cell-Derived Nanocarriers for Enhanced Magnetic-Field-Assisted Drug Delivery Manuel Rodríguez-Pérez, Sajid Fazal, Martina Migliavacca, Ramón Iglesias-Rey, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8712225/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Cell membrane-derived nanoparticles (NP) have emerged as a transformative platform in nanomedicine, offering unique advantages for therapeutic delivery and immune modulation. By harnessing the native biological properties of source cells such as red blood cells, platelets, and cancer cells, these biomimetic NP exhibit prolonged circulation, enhanced biocompatibility, and specific targeting capabilities. Membranes retain functional proteins and receptors, enabling precise interfacing with biological environments and providing inherent ability to evade immune detection and to interact with target tissues. Hybrid and engineered membrane platforms that integrate components of different origins (e.g. cell-derived and inorganic NP) hold strong potential for next-generation targeted therapies in oncology, as they can synergistically combine the advantages of both biological and inorganic components. Therefore, we proposed a new NP design based on biomimetic fusogenic nanomembranes decorated through click chemistry with iron oxide (IO) NPs for enhanced homotypic targeting and chemotherapeutic drug delivery under magnetic guidance in different in vitro models of non-small cell lung cancer. Results Our findings present an efficient preparation of a hybrid nanoplatform (magFSMs), based on nanomembranes and IO NPs coupled through strain-promoted azide-alkyne click chemistry (SPAAC) reaction. Our system preserved the magnetic properties of IO NPs as well as the homotypic targeting capabilities of the parental cancer cell membranes. This was evaluated both in adherent cell mono and co-cultures as well as in 3D heterotypic spheroids where magFSMs exhibited preferential recognition for tumoral cells. Our nanoplatform also showed versatile drug-loading capacity, as proved by the incorporation of different small anticancer molecules like carboplatin and doxorubicin, leading to enhanced antitumor activity compared with free drugs. Moreover, those observations were also preserved and even increased in lung cancer spheroids under magnetic guidance, underscoring the feasibility of using magFSMs as magnetically guided drug-delivery nanocarriers. Conclusions MagFSMs prepared within this work has been successfully employed as anticancer nanosystem in several in vitro models of lung cancer, representing a promising tool in personalized and targeted nanomedicine. Our results support the potential of magFSMs as a modular, biomimetic, and magnetically responsive drug-delivery platform, encouraging us to further explore their applicability in additional disease models and to expand their evaluation toward more complex in vivo scenarios. biomimetic nanocarriers cell membrane-derived nanoparticles nano-bio interfaces fusogenic nanovesicles magnetic targeting intracellular drug delivery stimuli-responsive nanomedicine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Drug delivery nanoplatforms have received increasing attention in recent years due to the vast range of approaches offered by nanostructured materials in countering roadblocks associated with conventional drug delivery. Back in the 1980s, liposomal formulations constituted a groundbreaking advance in drug delivery, increasing the efficacy of anticancer (Doxorubicin-DOXIL)[ 1 ] or antifungal (Amphotericin B-AMBISOME)[ 2 ] treatments as well as decreasing undesired side effects. Currently, lipid-based systems have evolved exponentially, with outstanding translation to human clinical practice as revealed by the new vaccines developed during the SARS-CoV-2 outbreak in 2019 [ 3 , 4 ]. These systems rely on the combination of lipids ( i.e. , 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP), cholesterol, etc.), which provide stability and biocompatibility to the payload [ 5 ], while enabling its efficient loading and delivery by responding effectively to the external environment [ 6 ]. Moreover, such systems exhibit great loading efficiency and are synthetized following simple and straightforward procedures, leading to the high-yield production of homogeneous NPs [ 7 , 8 ]. Despite the significant advances achieved in preclinical studies and clinical applications, these nanomedicines still need to overcome major biological barriers before in vivo administration including, but not limited to, rapid clearance from the bloodstream by the mononuclear phagocytic system [ 9 ] and unspecific activation of the immune cells [ 10 ] leading to undesired allergic reactions, and non-specific targeting of action sites [ 11 ]. As medicine is evolving towards a new era of precision strategies, one of the main concerns during nanomaterial design is to achieve active (and/or enhance the passive) targeting to the desired disease site [ 12 ]. In this sense, numerous approaches have been explored based on the engineering of NP´s surface for coupling different active molecules such as polyethylene glycol (PEG) [ 13 ], antibodies [ 14 ], glycans [ 15 – 17 ] as well as other ligands [ 18 ]. These new functionalities influence NP´s biodistribution, promoting their accumulation in the desired organ or tissue [ 12 ]. In this sense, the use of fragments of cell membranes to produce biomimetic NP coatings has emerged in the last decade as a revolutionary approach for active targeting [ 19 – 21 ]. Many different cell types have been used as a source to produce biomimetic NPs, including platelets [ 22 ], leukocytes [ 22 ], erythrocytes [ 23 ], macrophages [ 24 ], etc., exploiting the presence of receptors, glycans, and a plethora of ligands in the cell membrane, which play a key role in cell-cell interactions. Moreover, since they are obtained from biological entities, they exhibit low immunogenicity, thus bypassing the surveillance of the immune system. These membranes have also been employed for cloaking NPs, providing a new identity to the initial NPs. This has been done by employing surface coating strategies such as co-extrusion [ 25 ], sonication [ 26 ], electroporation [ 27 ], and in situ loading [ 28 ] to physically force the NPs and cell membrane surface to interact with each other. Since NPs eventually interact with cells, the mechanism by which the NP payload is released into cells has given rise to a vast discussion. In general, once recognized by cells NPs are considered to be internalized following the endocytic pathway, i.e. , NPs interacting with cell membrane are engulfed in vesicles forming endosomes [ 29 ]. These endosomal vesicles evolve to late endosomes and ultimately to lysosomes, where the highly acidic pH degrades the engulfed materials within [ 30 ]. During this process, a significant fraction of the NPs will remain in the lysosomal pathway, suffering from potential degradation and thus minimizing the available dose of the molecules of interest. To enhance NP-mediated drug delivery, researchers have been tailoring NP features to facilitate effective endosomal disruption or evasion, thereby allowing NPs to be released into the cell cytoplasm through endosomal escape mechanisms (similar to the mRNA LNP vaccines) [ 31 , 32 ]. However, novel strategies are still needed to protect NP payloads from degradation, minimizing side effects, and increasing the drug dose delivered to target cells. In this regard, approaches addressing the direct cytosolic delivery of NP payload could contribute to overcoming the main drawbacks in this field [ 33 – 36 ]. In addition, it could constitute a new opportunity for classical chemotherapeutic drugs that usually suffer from undesired side effects. Recently, our group has proposed a new approach using fusogenic biomimetic NPs for the delivery of small molecules, proteins, and synthetic NPs [ 37 , 38 ]. This work employed a combination of cell membranes and a combination of aromatic-labeled and cationic lipids, constituting a proof of concept of the feasibility and the potential of engineering cell-derived NPs for enhanced active drug delivery. Over and above enhanced cargo delivery, clinical practice now emphasizes the demand for new approaches combining therapy and diagnosis [ 39 ]. As part of this evolution, researchers have developed theranostic nanomaterials that are able to be traced using current medical diagnosis techniques (Magnetic Resonance Imaging-MRI, Computed Tomography-CT, Positron Emission Tomography-PET, etc.) and simultaneously provide a therapeutic response [ 40 – 43 ]. To address these unmet limitations, in this work, we aim to design a theranostic hybrid nanocarrier for cytosolic delivery of cargo by combining stimuli-responsive inorganic NP and cell-derived membranes in a single nanoplatform for magnetically controlled drug delivery. Unlike other bio-mimetic inorganic nanoplatforms, the novelty of our system design relies in the anchoring of inorganic NPs onto the biomimetic cell-membrane derived vesicles covalently using a click-chemistry approach which is a simple, fast and efficient loading strategy relative to common energy-intensive processes. In this work, we focus on the rational design and in vitro validation of a multifunctional nano-bio interface rather than on preclinical in vivo translation. METHODS Preparation of nanomembrane vesicles (Cellsomes -CSMs and Fusosomes -FSMs) To prepare CSMs from cell-membranes of A549 cells (ATCC, CCL-185), an already established protocol was utilized [ 44 ]. This method included cell harvesting, hypo-osmotic treatment, freeze-thaw cycles, bath sonication and centrifugation steps to isolate the cellular membranes followed by physical extrusion through a 0.8 µm pore-size polycarbonate membrane using a mini-hand extruder (Avanti Polar Lipids) [ 44 ]. The prepared CSMs were stored in HEPES buffer (25 mM, 7.4 pH) at 4°C until further use. To incorporate an azide functional group (N 3 ) to the CSMs, A549 cells were initially glycometabolically labeled using an azido-modified mannose (N-azidoacetylmannosamine tetraacylated, ManNAz; Thermo Scientific 88904). For that, ManNAz was added to its cell culture growth medium at 50 µM 72 hours pre-cell-membrane isolation and extrusion [ 45 ]. The presence of ManNAz on A549 cell membranes were analyzed through fluorescent imaging of these cells post incubation with Dibenzocyclooctyne-PEG4-Fluor545 (5mM, 1 hour) (see supporting information for more details, Figure S1 ). Additionally, to provide fusogenic properties to CSMs (fusosomes, FSMs), a neutral lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, Avanti Research, 850725), and a cationic lipid DOTAP (1,2-dioleoyl-3-trimethylammonium-propane, Avanti Research, 890890) were used. Additionally, fluorescently labeled DOPE-Atto647 (Sigma, 42247) was also used to fluorescently track the FSMs. In all, a ratio of 90/20/100 µg DOPE/DOPE-Atto647/DOTAP was used for one batch of CSMs obtained from 10 7 A549 cells. After sonication for 10 minutes, a final step of physical extrusion through a 0.8 µm pore-size polycarbonate membrane was employed to promote the insertion of the lipid mixture into the phospholipid bilayer of FSMs [ 37 ]. Preparation of magFSMs and drug loaded magFSMs Dibenzocyclooctyne/dodecylamine-grafted poly(isobutylene-alt-maleic anhydride) polymer (DPMA) was employed as the amphiphilic coating material to transfer hydrophobic IO NPs to aqueous media [ 46 ] while simultaneously providing a DBCO functional group for SPAAC click conjugation. This polymer, hereafter referred to as DPMA, contains both hydrophobic alkyl chains for anchoring onto the nanoparticle surface and DBCO groups enabling covalent coupling with azide-functionalized fusogenic membranes. To prepare the click-chemistry enabled IO@DPMA NP (see supplementary information and Figure S2 ) loaded FSMs (magFSMs), the azide-labeled FSMs and IO@DPMA NPs were mixed together at different (IO@DPMA NP: FSM) ratios, i.e. , 10:1; 50:1 and 100:, in HEPES buffer followed by sonication and incubation at 25°C for 3 hours. The prepared magFSMs were separated from free FSMs and free IO@DPMA NPs by centrifugation at 10000 rcf for 10 minutes. For evaluating and quantifying IO@DPMA NPs loaded onto FSMs, ICP and NTA analysis was carried out. To prepare drug loaded magFSMs, either doxorubicin (DOX) or carboplatin (CbPt) was initially loaded into the azide-labeled FSMs which were then used for click-conjugation with IO@DPMA NPs following the above-mentioned method. For DOX loaded FSMs, 50 µL of 10 mg/mL doxorubicin hydrochloride (Sigma, D1515) prepared in PBS (containing 0.5 mg of drug) was mixed with one batch of FSMs (final volume of 1 mL) and then subsequently subjected to sonication and physical extrusion. Eventually the mixture was subjected to a 30000 RCF centrifugation for 60 minutes at RT to separate out magFSM@DOX. To evaluate drug encapsulation and concentration, absorbance at 480 nm was measured (see supplementary information, Figure S3 ). In case of CbPt-loaded magFSMs, a 50 mM solution of CbPt (216100-M Merck) was initially mixed with one batch of cell derived membranes (final volume of 1 mL) and subsequentially extruded as previously described. After purification through centrifugation, ICP-OES analysis was undertaken to determine the Pt content in the obtained magFSM@CbPt (see supplementary information, Table S1 ). Eventually, dose-response studies were performed which included a concentration range of 0–10 µM and 0-200 µM for DOX and CbPt respectively. Nanoparticle characterization The synthesized FSMs, IO@DPMA NPs and magFSMs were characterized for their physicochemical properties using various techniques. For electron microscopy , 2 µL of the 2% formaldehyde fixed sample was dropped on an amorphous carbon supported 400 mesh copper grids. After leaving the sample to dry for 20 minutes, they were washed with PBS and negatively stained with 2% uranyl acetate. The samples were washed again and left to dry overnight before imaging using a scanning electron microscope ZEISS FESEM ULTRA Plus. To determine Fe concentration, inductively coupled plasma-optical emission spectroscopy (ICP-OES) measurements were carried out. For this, 20 µL of the sample were first dried overnight in a glass container, to which 120 µL HNO 3 and 360 µL HCl along with 10 µL Mn 2+ and Se 2+ (500 ppm) as internal standards were added. After 4 hours of treatment, 500 µL of the digested sample were added to 4.5 mL 2% HCl and analyzed in ICP-OES, wherein atomic emission lines at 239.563 nm and 261.187 nm for Fe were detected to quantify its concentration in the sample. The hydrodynamic diameter and surface charge of the samples were evaluated using a Dynamic Light Scattering (DLS) Malvern Zetasizer Nano ZSP (Malvern Instrument ltd.). Particle size and concentrations were also evaluated using Nanoparticle Tracking Analysis (NTA, NanoSight NS300, Malvern Instrument ltd.) equipped with a 405 nm laser, wherein the samples were diluted (1:200 in PBS) before measurement. To study the magnetic properties of the synthesized sample, COMBI AC HYSTER™ (nanotech solutions, Spain) equipment was utilized. In general, 40 µL of the sample in a glass tube was placed inside a copper coil that produced an alternating magnetic field at 100 kHz and 24 kA m − 1 . Magnetic hysteresis loops were then measured in triplicates to determine the magnetic features including the average specific absorption rate. Additionally, the ability of the NPs as contrast agents in magnetic resonance imaging (MRI) was evaluated by utilizing an in-house designed agarose mold to hold the samples. To prepare the mold, a 2% agarose solution of 100 mL was heated to form a homogenous mix, which was then transferred to a mold containing several holes to host the NP samples. After cooling and solidification, the mold was disassembled, and the holes were filled with a warm mixture of 1:1 liquid agarose and NP suspension. After solidification, the mold was carefully covered with a few more milliliters of the agarose solution and left to solidify. This mold was then scanned with a volume antenna in a Bruker Biospec 9.4 T small animal MR (Bruker, Biospin) equipped with actively shielded gradients (400 mT/m). A quadrature radiofrequency transmit-receive resonator was used for data acquisition. T2-weighted images were acquired using a multi-slice multi-echo (MSME) sequence of 11.32 ms echo time, 3 s repetition time, 16 echoes, 14 slices of 1mm thickness, 1 average, field of view (FOV) of 7.5 cm x 7.5 cm (with saturation bands to suppress the signal outside the FOV) and matrix size of 256 x 256 pixels. After acquiring, images were processed with ImageJ (Rasband WS, National Institutes of Health, Bethesda, MD, USA, https://imagej.net/ij/ ), generating a transverse relaxation time (T 2 ) map archive where the T2 values were measured. Eventually, sample relaxivity (r 2 ) was calculated as the slope of the function relating the inverse of the transverse relaxation times (1/T 2 ) and the Fe concentration in the samples. For their biochemical characterization, flow cytometry analysis was carried out (Millipore Guava EasyCyte HT cytometer). For this FSMs, were labeled with AlexaFluor-488 anti-cadherin antibody (30 minutes incubation; 1 µg Ab/10 7 FSMs) and were compared with Liposomes (see Figure S6 ) as a negative control. All prepared samples were centrifuged and resuspended in PBS before flow cytometry analysis. Cell culture and maintenance A549 human lung epithelial cancer cells and MRC5 human lung fibroblast cells (ATCC CCL-171 ) were cultured and maintained throughout the study in high glucose DMEM media supplemented with 10% FBS, 100 I.U. ml − 1 penicillin and 100 µg mL − 1 streptomycin in a humidified biological incubator maintained at 37 ºC, 5% of CO 2 and 95% relative humidity. The cells were sub-cultured when reaching 80% confluency and were maintained up to passage number 40, after which they were discarded, and new cells were revived. For the development of 3D tumor spheroids, monolayer cell cultures were trypsinized and seeded in a 96 well plate previously coated with 60 µL of 1% agarose. Typically, for homotypic spheroids, 5000 A549 cells in 150 µL cell culture medium were initially seeded. 100 µL of the media was carefully removed every 2 days and replaced with fresh media for maintaining spheroid viability. Experiments on spheroids were undertaken for 5 to 7 days post initial seeding. For developing heterotypic spheroids, a co-culture of A549-GFP and MRC-5 cells at 1200:4000 ratio in 150 µL of medium was seeded initially. Cytotoxicity assay To evaluate potential cytotoxicity arising from sample exposure, cell-viability analysis was carried out using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, M2003) assay. A549 cells were seeded at 7500 cells/100 µL in a 96 well plate overnight and then incubated with the synthesized nanoparticles at different concentrations in cell culture medium for different time periods. Post incubation, the cells were washed and rinsed with PBS and an equal volume of 0.5 mg/mL MTT solution (in cell culture medium) was added to the cells for formazan crystal formation. After 4 hours, the crystals were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, 472301) and absorbance measurements were performed using an Infinite® 200 PRO (TECAN) microplate reader at 540 nm. To evaluate the toxicity effects of drug loaded magFSMs in 3D tumor spheroids, flow cytometry analysis was undertaken. After desired incubation with drug loaded magFSMs, the tumor spheroids were first washed with PBS, disaggregated with trypsin for 10 minutes, and then incubated with 0.1 µM calcein AM for 20 minutes. The cell suspension was then analyzed in a Millipore Guava EasyCyte HT cytometer (using 488 laser line for excitation and the 512/18 nm filter for fluorescence acquisition). In vitro NP uptake studies Different types of 2D and 3D mono and co-culture systems were developed for detailed analysis of samples uptake. For 2D monoculture experiments , A549 lung cancer or MRC5 lung fibroblast cells at 10,000 cells/100 µL in cell culture medium were seeded into an 18 well µ-Slide (Ibidi) overnight. On the other hand, for 2D co-culture experiments , A549-GFP and MRC5 cells were seeded (10000:3000) in an 18 well µ-Slide (Ibidi) overnight. For evaluating homotypic targeting of magFSM@DOX in 2D cocultures of A549 and/or MRC5, the cells were incubated with either magFSM@DOX or equivalent free drug (1 µM) for 24 hours. The cells were then fixed with 4% paraformaldehyde in PBS prior to imaging with a fluorescence microscope, wherein 5 different regions of interest (ROIs) per group were imaged with a minimum of 10 cells per ROI. As mentioned above, for the synthesis of homotypic and heterotypic spheroids either 5000 A549 cells or A549-GFP and MRC-5 cells (4000:1200) were seeded in a 1% agarose gel-coated 96 well plate. The desired concentration of the sample suspension was then added to allow their cellular uptake. After the required incubation period, the cells were washed with PBS and incubated further in fresh culture medium containing 5 µg/mL Hoechst for 20 minutes (Thermo Fischer #62249) for nuclear staining prior to fluorescence microscopy imaging. Fluorescence imaging was undertaken using a Thunder Leica® Microscope type DMI8 equipped with a Leica® DFC9000 camera. All images were further processed using ImageJ software. For flow cytometry analysis , cells were trypsinized to obtain a single cell suspension which was analyzed using a Millipore Guava EasyCyte HT cytometer with the following laser and filter configuration: 488 nm laser with 575/25 filter for DOX and Cy3 and 642 nm laser with 695/50 nm filter for Atto647. Flow cytometry data was further analyzed using Flowjo software. In vitro uptake under magnetic gradient exposure To evaluate particle uptake in heterotypic spheroids of A549 and MRC5 cells under the influence of an external stimuli, we designed a simple experiment trying to simulate the effect of a magnetic field. For this, a neodymium (40 x 40 x 20 mm) magnet (1.29–1.32 T) was placed below the microplate containing cells exposed to free DOX (10 µM) and magFSMs (equivalent to free DOX concentration) for 24 hours. Cells treated with NPs and not exposed to an external magnetic field were used as controls. Fluorescence microscopy was then employed to map the fluorescence signal along the cross-section of the spheroids. RESULTS Synthesis and characterization of magFSMs Nanomaterial design and preparation procedures are depicted in Scheme 1 . First, highly monodisperse IO NPs were synthetized in an organic solvent following a protocol of thermal decomposition [ 47 , 48 ] and subsequently transferred to an aqueous buffer by means of a polymeric coating with DPMA[ 49 ]. TEM imaging (Fig. 1 A) revealed spherical IO NPs with a diameter of 18 nm. For the synthesis of FSMs, the A549 lung cancer cell line was chosen, as this cancer-derived FSMs possess homotypic targeting abilities. For this purpose, we supplemented A549 cell cultures with an azido-modified mannose (ManNAz) sugar to display N 3 groups on the cellular membranes [ 45 ]. Cells effectively incorporated ManNAz to their glycoproteins anabolic pathways through a process known as glycometabolic labeling, eventually translocating N 3 groups in the external layer of the cytoplasmatic membrane as shown in Scheme 1 . The incorporation of the N 3 groups in A549 cells was assessed by the addition of a DBCO-fluorophore (DBCO-fluor 545). It was observed that untreated cells with ManNAz were not reacting to the fluorophore while the azide-expressing A549 could selectively bind this fluorophore ( Figure S1 ). Then azide-labeled CSMs were prepared using the membrane of the azide-expressing A549 cells following an extrusion protocol using polycarbonate membranes of 0.8 µm pore size. During this process, DOPE (neutral), DOPE-Atto647, and DOTAP (cationic) lipids were added and intercalated in toure CSMs to provide them with fusogenic properties to generate FSMs. As revealed by TEM in Fig. 1 B-C, this process yields in the production of spherical NPs (FSMs) with a diameter of ~ 150 nm. Once both components were obtained, FSMs and IO@DPMA NPs were coupled through a SPAAC click reaction leading to the generation of magFSMs. To generate magFSM, three different ratios were explored: 100:1, 50:1 and 10:1 of IO@DPMA NP:FSMs. (Fig. 1 D-G). These magFSMs displayed their surface decorated with a different density of IO@DPMA NPs. The number of IO@DPMA NPs per magFSM was directly related to the ratio used. On analyzing the hydrodynamic diameter of the FSMs and magFSMs through nanoparticle tracking analysis (NTA) (Fig. 1 H and S4 A and B ), no significant change in the hydrodynamic diameter was observed (148.1 ± 2 nm). On the other hand, zeta potential measurements (Fig. 1 I and Figure S4 C and D ) showed an increase in the negative surface charge of magFSMs correlated with a higher incorporation of IO@DPMA NPs (-19.7 ± 0.35 mV). Furthermore, in-depth analysis through ICP-OES showed that for magFSMs, the IO@DPMA NPs loaded per FSMs positively correlated with a higher IO@DPMA NP/FSM ratio ( Figure S4 G ). These results showcase the feasibility of the SPAAC click-reaction to covalently link inorganic NPs to FSMs, providing a straightforward methodology to tailor the FSMs´ surface to modulate their composition with specificity and precision. Then the abilities as MRI contrast agents of the magFSM were tested, since magnetic NPs can act as T2-contrast agents by decreasing the transverse relaxation time (T2) of the nearby water protons providing a hypointense signal in T2-weighted MRI. Therefore, the presence of IO@DPMA NPs in magFSM is expected to provide the ability to respond to stimuli from magnetic nature, constituting a new MRI negative contrast agent. After analysis in magnetic resonance phantoms, magFSMs (10:1 IONPs) showed a significantly higher darker contrast as compared to bare IO@DPMA NPs at the same Fe concentration as revealed in the T2- maps showing hypointense signals (Fig. 2 A-B). Relaxivity measurements from these maps showed that magFSMs exhibited a relaxivity of 164.7 ± 31.4 mM − 1 s − 1 which was significantly higher than that of IO@DPMA NPs (61.2 ± 11.6 mM − 1 s − 1 ). A similar trend in increased relaxivity was also observed for IONPs that were coated with hepatocellular cancer cell membranes as compared to free iron-oxide NPs [ 50 ]. These findings suggest that the IO@DPMA NP arrangement on the surface of magFSMs leads to an increase of the effective magnetic moment thus highlighting the effect of NP clustering on their magnetic features [ 51 ]. Additionally, the arrangement of IO@DPMA NPs on the magFSMs surface may alter the diffusion dynamics of water protons around the NPs compared to bare single IO@DPMA NP suspensions, a factor that has been documented to affect its relaxivity [ 52 ]. Then, dynamic magnetometry measurements were performed to evaluate the magnetic behavior of both systems, IO@DPMA NPs and magFSMs, in solution. These measurements provide information about the magnetic response of materials in more realistic settings ( i.e. , temperature, solvent, viscosity, etc.) compared with other techniques such as superconducting quantum interference devices (SQUID). Dynamic magnetometry measurements revealed that magFSMs exhibited a hysteresis loop similar to that of free IO@DPMA NPs across the same iron concentration range (Fig. 2 C). This indicates that the ability of the IO@DPMA NPs to absorb electromagnetic energy, as quantified by the Specific Absorption Rate (SAR), was not compromised by their attachment to the surface of the FSMs, with SAR of 93.7 ± 7.5 W/g Fe and 84.5 ± 9.7 W/g Fe for magFSMs and IO@DPMA NPs, respectively (Fig. 2 C-D). The SAR values in this case were predominantly governed by Néel relaxation rather than Brownian relaxation. This was further supported by SAR measurements of magFSMs in varying concentrations of glycerol[ 53 ] ( Figure S5 ), in particular three percentages were tested, 5, 10 and 25% v/v of glycerol in water. Increasing the viscosity of the surrounding medium by using higher glycerol concentrations did not result in significant changes in the SAR values of magFSMs. Additionally, no significant differences in magnetization values were observed between magFSMs and free IO@DPMA NPs (Fig. 2 E) in 25 mM HEPES, confirming that the magnetic properties of the synthesized system remained unchanged following the click-coupling process. In-vitro uptake and homotypic targeting of magFSMs in 2D cell cultures It has been widely demonstrated that CSMs produced from cancer cells exhibit a preferential recognition to the same cells from which they originated [ 54 , 55 ]. This ability is known as homotypic targeting. This effect relies on the presence of a plethora of proteins, glycoproteins and ligands involved in cell-cell recognition process involved in cancer cell proliferation, invasion, and metastasis [ 56 ], such as Thomsen–Friedenreich (TF) antigen [ 57 ] and E-cadherin which are retained during the CSM preparation process [ 58 ]. To confirm that our FSMs are presenting these recognition molecules, flow cytometry assays were performed using an anti-cadherin antibody. Figure S6 shows the presence of Cadherin on FSMs as compared to liposome control samples (see supporting information), thus highlighting the presence of cell-membrane specific proteins on the CSMs surface, suggesting its feasibility for homologous binding recognition. To further evaluate the ability of magFSMs to interact with cells and get internalized, fluorescence microscopy and flow cytometry assays were performed. To do so, we first evaluated the cytotoxicity of magFSMs and its individual components. FSMs and IO@DMPA NPs through MTT cytotoxicity assay. From Figure S7 it was observed that FSMs were found to be biocompatible below a concentration of 10 10 NPs mL − 1 and IO@DMPA NPs showed no adverse toxic effects up to a concentration of 25 µg mL − 1 Fe. On the other hand, magFSMs (at 10:1 IO NP:FSM ratio) showed good biocompatibility up to 5 µg mL − 1 of Fe (corresponding to ~ 10 10 FSMs mL − 1 ). Thus, for all our in vitro experiments involving magFSMs, a concentration of less than 5 µg mL − 1 Fe or 10 10 FSMs mL − 1 was set. Using fluorescence microscopy, co-localized fluorescence signals were observed for both the FSM (labeled with DOPE-Atto647) and the cargo (IO@DPMA NPs, labeled with Cy3) in cells. This colocalization corroborates the hybrid nature of magFSMs and confirmed its stability in cell culture conditions, meaning they are stable at biologically relevant salt concentrations and to the presence of several enzymes with protease activity that could impair the linkages formed through electrostatic or other non-covalent mechanisms, at least for the observed timepoint (3h). Furthermore, the fusogenic behavior was studied for the two samples prepared, magFSMs and magCSMs with the same ratio of IO@DMPA NPs (1:10). The fusogenic abilities of magFSMs were confirmed by the localization of the fluorescence signal at the cell-membrane, together with a diffuse intracellular fluorescence throughout the cytoplasm (Fig. 3 A and Figure S8) . This contrasts sharply with the behavior of magCSMs, which are not expected to display fusogenic properties ( Figure S8 A-B) . Instead, magCSMs required longer incubation times to be internalized and exhibited a characteristic punctate distribution, consistent with their uptake through endocytic pathways. These two different behaviors are in agreement with previous results of our group [ 37 ] and confirm that neither the incorporation of azide groups via metabolic engineering nor the decoration with IO@DPMA NPs compromise the fusogenic properties of FSMs, at least at the ratios tested. Considering that the presence of IO@DPMA NPs on FSMs could potentially hinder their interaction with target cells by masking membrane-associated receptors, different IO@DPMA NPs:FSM ratios were tested to determine optimum conditions for minimal interference on cellular uptake as compared to the inherent features of FSMs. Flow cytometry analysis revealed that, when using equal carrier concentrations, magFSMs synthesized at a 10:1 IO NP:FSM ratio exhibited an uptake comparable to that of bare FSMs, in contrast to the higher ratios tested (Fig. 3 B ) . Based on these results, magFSMs prepared at the 10:1 ratio demonstrated the most favorable balance of magnetic performance, cytocompatibility, and uptake efficiency, and were therefore selected for all subsequent experiments. Following this initial comparative assessment on in vitro uptake, the homotypic targeting of magFSMs was further compared in A549 and MRC5 (lung fibroblast) mono- and co-cultures in an attempt for a more accurate resemblance of tumor microenvironment [ 59 – 62 ], as a result of the combination of healthy and cancerous lung cells. To facilitate the identification of the two different cell lines, GFP-expressing A549 cells were selected to distinguish them from MRC5. Remarkably, fluorescence microscopy of co-cultures revealed that magFSMs not only clearly fused with cell membranes of A549-GFP cells to a greater extent as compared with MRC5 cells, but the fusion was similar to what was observed in the case of FSM with no IONP decoration (Fig. 3 C). This was further corroborated through flow cytometry analysis, which allowed the precise quantification of the homotypic recognition of cancer cells in mono and co-cultures (Fig. 3 D). From Fig. 3 E and F , it is evident that magFSMs showed a significantly higher uptake in A549 cells as compared to MRC5 cells, when quantified through fluorescence signals from both Cy3 (IO NP) and Atto647 (FSM), which was similar to FSMs alone. This trend was also maintained in co-cultures of A549-GFP and MRC5 cells wherein both FSM and magFSMs were more efficiently internalized (~ 2-fold) compared with MRC5 fibroblasts (Fig. 3 E and F ). Specifically, MFI values (Fig. 3 E) at the Cy3 channel, confirmed that magFSMs are specific vehicles for the delivery of IO@DMPA NPs as cargo. Overall, these findings were consistent with those observed in monocultures of A549 or MRC5. Taken together, these results corroborate the feasibility of click-chemistry conjugation of IO@DPMA NPs to FSMs as a strategy to engineer hybrid carriers without compromising fusogenicity or homotypic specificity [ 63 ]. This provides solid evidence of the preferential recognition of cancer cells by magFSMs even in a multicellular tumor-like scenario. Homotypic targeting of magFSMs in 3D cell cultures. After assessing its targeting capability in 2D cell cultures, we evaluated the magFSMs´ capabilities to target cancer cells in 3D models. Since the tumor microenvironment (TME) comprises vasculature cells, cancer stem cells, immune cells, and tumor-associated fibroblasts (that have been linked with cancer progression) that add to its complexity, it is essential for a drug delivery vehicle to navigate through this environment to effectively target cancer cells [ 64 , 65 ]. Within the TME, the stromal component, usually known as cancer associated fibroblasts (CAFs) are one of the most vastly studied components of the TME due to their implication in the promotion of the growth and invasion of cancer cells by various mechanisms [ 66 – 68 ]. CAFs are able to produce fibroblast secreted protein-1 (FSP1) [ 59 ] and Insulin-like growth factor 1 (IGF-1) [ 69 ] which are related to cell growth and migration. Moreover, stromal cell-derived factor 1 (SDF-1) derived from CAFs was found to promote angiogenesis through the recruitment of endothelial cell precursors (EPCs) for the formation of new blood vessels [ 60 ]. Having this in mind, our aim was to model this complex environment by using heterotypic spheroids where fibroblast constitutes an additional cell barrier to overcome for common nanomedicines. In this study, heterotypic spheroids composed of A549-GFP and MRC5 ( Figure S9 ), were produced to assess the homotypic specificity of magFSMs in a multi-cellular lung cancer model. Different ratios of A459-GFP and MRC5 cells were tested in order to optimize the reproducibility of the spheroid formation. Three initial ratios of 1:1, 1.5:5 and 1:5 A549-GFP:MCR5 were tested. Ratio 1:5 showed a very poor reproducibility while ratios 1:1 and 1.5:5 showed a similar size of approximately 400 µm ( Figure S9 A ). Based on these results, the ratio 1.5:5 was selected for further analysis. Fluorescence imaging of heterotypic spheroids 24 hours after treatment with 10 9 FSM ml − 1 or magFSMs, revealed that the magFSMs distribution within spheroids was similar to that found in FSMs (Fig. 4 A). Importantly, the incorporation of the IO@DPMA NPs within the magFSMs did not significantly interfere with their targeting capability in this system. It was clearly observed that the fluorescence signal from Atto647 (FSM) was comparable in both groups, while the Cy3 fluorescence of the IO@DPMA NPs was only present in the magFSMs (Fig. 4 A), and it appears spread within the whole spheroid. When treated spheroids were observed at higher magnification, we could distinguish areas with a clear colocalization of A549-GFP cells (green) and the FSMs and magFSMs (Fig. 4 B), confirming the preferential internalization by cancer cells. These results depicting effective homotypic recognition at 3D cultures were later confirmed by means of flow cytometry (Fig. 4 C) where MFI at 24 hours showed an uptake of magFSMs at least 4-fold in A549 cells compared with MCR5. In vitro drug delivery and therapy in 2D and 3D cell cultures. After demonstrating the homotypic targeting properties of our systems, we then evaluated their ability to deliver anti-cancer drugs. For this, magFSMs were loaded with two different classical anticancer drugs: doxorubicin (DOX) or carboplatin (CbPt) (Figure S4 E and F, Table S2). These two molecules are both capable of interacting with DNA to induce cell apoptosis. At the molecular level, both are small molecules (MW below 550 g/mol). Meanwhile, DOX is a planar aromatic anthraquinone system with a sugar moiety attached, CbPt is a coordination organometallic platinum complex. As shown in Fig. 5 A-B, flow cytometry assays in 2D monocultures of A549 cells, even for short incubation periods of 10 minutes, the loading of DOX in FSMs or magFSMs (FSM@DOX and magFSM@DOX, respectively) leads to an increased uptake of DOX as compared to the same dose (1 µM) of unencapsulated drug. This enhancement was found to be consistent for higher incubation times. Both FSM@DOX and magFSM@DOX showed a significant 2.6-fold enhancement of doxorubicin delivery in A549 cells after 24 hours. Furthermore, to take advantage of the homotypic targeting abilities of the FSMs, DOX uptake was studied in 2D co-cultures of A549-GFP and MRC5 cells (Fig. 5 C-D). Since DOX is a DNA intercalator [ 70 ], its uptake was evaluated and quantified through fluorescence measurements from cell nuclei fluorescence microscopy. From the fluorescent images obtained after incubation with 1µM of DOX equivalents for 24 hours, it can be clearly seen that both FSM@DOX and magFSM@DOX display targeted binding to A549 lung cancer cells (Atto647 fluorescence), and their cell fusion led to the delivery of DOX intracellularly into the nucleus (Fig. 5 C). This is in stark opposition to the non-specific accumulation of free drug in both A549 and MRC5. By quantifying relative fluorescence intensity of DOX in cell nuclei (Fig. 5 D), it was observed that magFSM@DOX showed a specific drug delivery effect similar to FSM@DOX. For both FSMs, the DOX accumulation was 2-fold for the A549 cells compared with the accumulation in MRC5. This confirms that the incorporation of IO@DPMA NPs did not negatively affect the targeting dynamics of the drug-loaded FSMs. This specific targeting of cancer cells should be translated to increased cytotoxicity of these nanoformulations. Results revealed an enhanced cytotoxic effect for both FSMs at short (10 minutes) and long (24 hours) incubation periods. Specifically, both FSM@DOX and magFSM@DOX exhibited an increased delivery of DOX inside cancer cells (Fig. 6 A) compared with free drug, which was found notably lower over a 24 hours incubation period. Those observations were confirmed in dose-response experiments at both 10 minutes and 24 hours of exposure time (Fig. 6 B) where FSM@DOX and magFSM@DOX significantly outperformed free DOX in exerting cytotoxic effects on cancer cells. Similar results were observed when magFSMs were loaded with another chemotherapeutic drug, CbPt, where FSM@CbPt and magFSM@CbPt showed an increased cytotoxic activity, as compared to free CbPt, both for 10 minutes and 24 hours of drug exposure (Fig. 6 C and Figure S10 ). To further test the robustness of drug-loaded magFSMs as cytotoxic agents, their effects in a model of A549 spheroid were evaluated. For that, we take into consideration that the dynamics of its uptake were different as compared to 2D cell cultures, as previously discussed (Fig. 4 ). We are modeling this by selecting the DOX-loaded magFSM as a therapeutic tool. After 24 hours of A549 3D spheroids exposure to magFSM@DOX, we found a significantly higher percentage of dead cells (51.4%) as compared to free drug (10 µM) alone (29.5%), proving the feasibility of magFSMs to efficiently deliver DOX inside a 3D structure where the drug cargo was able to exert their function (Fig. 6 D). Once the feasibility of magFSM@DOX as drug delivery vehicle was assessed, we aimed to combine the features displayed in 2D to a 3D model, by taking into account not only its biological attributes, but also its physico-chemical properties. As explained earlier, 3D heterotypic spheroids used within this work were composed of lung fibroblasts (MRC5 cells) and lung cancer cells (A549-GFP), and these were further used to explore the use of magFSMs as a magnetic-field guided delivery vehicle. Here, the IO@DPMA NPs decorated on the FSM surface provide our vehicle with magnetic properties as shown in Fig. 2 . Therefore, we explore the magnetically-driven accumulation of magFSMs within heterotypic spheroids. Figure 7 and Figure S11 highlight the impact of the use of a magnetic gradient to promote the accumulation of the magFSMs versus the free DOX within a spheroid as observed by fluorescence microscopy studies. There is a noticeable greater penetration for the magFSMs (Atto647) and cargo (DOX) when the magnetic field is used (Fig. 7 A -II ). This demonstrates that the presence of a magnetic field drove the in-depth penetration of magFSM over and above the inherent ability of FSM to penetrate into the spheroids ( Figure S11 ). Even though overcoming physical barriers for tumor penetration is advantageous, drug accumulation must continue to occur preferentially in tumoral cells within the tumor microenvironment. Thus, it is essential that the enhanced accumulation synergistically reinforces homotypic targeting rather than causing nonspecific or uncontrolled NP uptake. To evaluate this, we undertook flow cytometry analysis on heterotypic spheroids to study the DOX accumulation in the different cell types used at different times ( i.e. , 1 hour and 24 hours) (Fig. 7 B). Looking at the DOX accumulation profile (Fig. 7 B I-II ), for the free DOX we did not observe any increment by the presence of the magnetic gradient in any condition, as expected. This was consistent for both incubation times, 1 hour (Fig. 7 B I ) and 24 hours (Fig. 7 B II ). Interestingly, no preferential drug accumulation was observed in cancer cells in any condition, with A549 and MRC5 cells internalizing DOX at a comparable rate. Focusing on magFSMs, we observed that 1 hour after treatment, both in the presence and absence of a magnetic gradient, there was preferential drug accumulation in A549 lung cancer cells compared with MRC5 fibroblasts, consistent with their inherent homotypic targeting abilities. This accumulation was approximately twice as high in A549 cells as in MRC5 cells. Interestingly, this preferential uptake was maintained upon application of the magnetic gradient, which produced an additional ~ 2-fold increase in the accumulation of magFSM@DOX (Fig. 7 B). Moreover, comparing the signal of the internalized DOX and that of the internalized FSMs after 1 hour, a clear correlation was observed, confirming that magFSMs effectively delivered the drug. Over a period of 24 hours, the combined effect of homotypic targeting and magnetic field guidance resulted in a clearly enhanced accumulation of magFSMs within cancer cells in a complex 3D tumor spheroid. This was evidenced by the distribution patterns of the nanocarriers, which closely resembled those of the drugs under magnetic field exposure (Fig. 7 B). Notably, fluorescence imaging and spatial mapping across the spheroid width revealed that both drug and vehicle penetration were substantially higher when magFSMs were subjected to an external magnetic field (Figure S11). This synergy between the physical and biological properties of magFSMs, enabling simultaneous stimuli responsiveness, imaging, and active targetability within a single platform, highlights their strong potential for future theranostic applications. DISCUSSION Tailoring a personalized therapeutic regimen for cancer therapy is one of the most important and challenging goals for cancer nanomedicine. To achieve this, the materials chosen for nanomedicine design should exhibit biocompatibility as well as targetability to specific cancer sites when administered inside the body. Here, endogenous biomaterials provide a unique opportunity since they inherently possess biocompatibility and bio-recognition capability. When assembled to form nanostructures they form biomimetic cargo delivery vehicles that possess unique advantages as compared to other conventional nanoparticle systems. One of the most widely used biomaterials for the synthesis of biomimetic drug delivery vehicles are phospholipid cell membrane components. We have previously synthesized biomimetic fusogenic nanovesicles using cancer cell membranes in our group [ 37 , 44 , 71 ], which unlike other reported biomimetic nanostructures demonstrates direct cytoplasmic delivery of cargo inside cells [ 37 ]. The ability to directly fuse with cell membranes to deliver cargo is highly advantageous as it circumvents the possibility of lysosomal degradation of cargo components. In this report, we add an additional approach to this repertoire by demonstrating the conjugation of stimuli-responsive inorganic IO NPs to the surface of cell membrane nanovesicles using a robust click-chemistry approach. Unlike other widely reported methods of cargo loading using energy intensive techniques (such as extrusion, sonication, electroporation, etc .), this method of functionalizing biomimetic vesicles demonstrated here is simple and straightforward that could easily be scaled up and could theoretically be applied to a wide variety of cell membrane sources and NPs/cargo. This combined approach, integrating cancer cell membrane-derived recognition molecules, fusogenic membrane components, and magnetic IO NPs within a single platform, renders the resulting nanostructure (magFSMs) unique, as it enables active targeting further enhanced by magnetic field-assisted delivery, while promoting direct cytosolic delivery of the cargo. With this development, we demonstrate the synthesis of magFSMs, their magnetic properties, and homotypic recognition capabilities, as well as stimuli-responsive active targeting under an external magnetic field. The robustness of this system was extensively validated across multiple in vitro 2D and 3D homo- and heterotypic tumor models. The simple copper-free click-conjugation employed here was facilitated by utilizing a DBCO functional group on the IO NP and an azide-labeled sugar incorporated nanovesicle leading to the generation of magFSMs of ~ 200 nm diameter. One of the main advantages of functionalizing a biomimetic nanovesicle on its surface is that it makes possible the loading of drugs/cargo separately into its empty lumen, thus maximizing the available volume per NP for drug delivery applications. Importantly, the labeling of FSMs with IO NPs was not found to significantly change the physicochemical properties of the developed biomimetic nanosystem but rather led to the combination of their individual properties. When imaged in MRI, magFSMs depicted good T2-contrast enhancement effects and a correspondingly high relaxivity of 164.8 mM −1 s − 1 , implying that when used in vivo , their accumulation at a diseased site should be easily detectable. This is especially important when the homotypic targeting capability of magFSMs is needed, since they would actively recognize and bind to cancer cells, thus opening up the possibility of imaging tumor location using MRI. Additionally, this particular configuration of decorating IO NPs on cell-derived NPs was found to be advantageous as no significant changes in the SAR values of magFSMs were observed as compared to free IO NPs. In theory, this would imply that the magFSMs could also be used for magnetic hyperthermia, and the exposure of such a system to an alternating magnetic field would lead to an on-demand drug release. In the future, the use of this system for magnetic hyperthermia induced cancer therapy is envisioned. Moreover, the robustness of this system also implies that other small sized stimuli-responsive NPs (such as gold nanorods, quantum dots etc.) could be covalently conjugated on a biomimetic NPs without significantly altering its biological properties. Of particular importance in this study is the ability of magFSMs to bind specifically to A549 cancer cells, the parent cells from which the cell membrane phospholipids were isolated for preparing FSMs. Since the coupling of IO NPs to FSM surface could spatially hinder the functioning of homotypic recognition receptors on the nanocarrier, an optimum ratio of IO NP:FSM was determined (10:1) that would provide adequate vehicle as well as cargo uptake in A549 cancer cells. Thus, while utilizing a conjugating technique to load cargo on FSM surface is a simple process, the amount of cargo that can be loaded is overall limited through this route, as higher surface-loaded cargo can limit the normal biological function of the FSMs. When tested in vitro in 2D heterotypic co-cultures of A549 and MRC5 cells, magFSMs were found to preferentially bind to A549 cells as compared to MRC5 fibroblast cells. This ability to recognize and preferentially bind to the parent cell from which the nanocarriers were derived has been widely reported in literature [ 72 , 73 ]. However, within this work, we demonstrated that FSMs homotypic recognition capability could be exploited in conjunction with additional non-native properties by optimizing the surface conjugation of IONPs onto FSMs. Moreover, this beneficial property of magFSM continued to be retained when tested in a more complex 3D environment of tumor spheroids comprising of A549 and MRC5 cells thus showing its robustness and capability to penetrate deep into a tumor microenvironment. When utilized for the delivery of DOX which are loaded as an additional cargo into its lumen (unlike IO NPs conjugated on FSM surface), magFSM@DOX showed preferential binding and drug accumulation in A549 cancer cells. Importantly, it was observed that in a 2D culture, magFSMs were able to deliver DOX to A549 cancer cells much more rapidly within a shorter incubation time of 10 minutes, that was significantly faster than the use of free drugs. This shows that homotypic targeting using biomimetic FSMs actively drives the targeting of drugs into cancer cells, unlike in the case of passive uptake. This is especially useful when used in vivo , where a constantly changing dynamic environment is encountered, within which biochemically driven active targeting processes would show more efficacy for drug delivery over passive targeting strategies. Furthermore, over a period of 24 hours, magFSMs also enabled a > 2-fold drug accumulation in A549 cells. This is especially relevant as indiscriminate cytotoxicity, once administered in clinics, is a major challenge for many of the drugs currently used for anti-cancer therapy. The feasibility of this system to be adaptable to different drugs was also demonstrated with the loading of CbPt. Upon evaluation in 3D tumor spheroids, even though a higher concentration of doxorubicin was consequently needed as compared to 2D culture, magFSM@DOX treatment led to a significantly greater loss in tumor cell viability as compared to the use of free drugs, thus highlighting its potential as a robust drug delivery system even in a complex tumor environment. Even though magFSMs demonstrated excellent homotypic targeting ability, good biological stability, improved drug-delivery capability, and robust activity in a complex tumor microenvironment, the most significant property of magFSMs is their ability to respond to an external magnetic field stimulus. This was envisaged due to the excellent magnetic properties demonstrated by magFSMs owing to the covalent binding of IO NPs on their surface. As a proof of concept, the stimuli-responsive capability of magFSMs for targeted drug delivery under an external magnetic field was evaluated in heterotypic spheroids, wherein its capability to specifically deliver drugs actively to cancer cells was found to increase many-fold further as compared to conditions where no external magnetic field was applied. Within a clinical setting, the use of a non-invasive, highly penetrating magnetic field to direct drug-loaded nanovehicles to the tumor site could be highly advantageous. Thus, in this report, we show for the first time the use of a simple click-chemistry approach for surface loading stimuli-responsive IO NPs on the surface of fusogenic biomimetic nanocarriers, that can be used for combined homotypic and magnetic targeting for enhanced and specific drug delivery. For future studies, we envisage the use of such systems for magnetic hyperthermia or photothermal drug delivery. While the present study demonstrates the robust performance of magFSMs across advanced in vitro mono- and heterotypic 3D tumor models, further investigations will be required to evaluate their in vivo biodistribution, pharmacokinetics, and long-term safety. Additionally, the scalability and standardization of cell membrane-derived nanocarriers remain crucial aspects for future translational development. Nevertheless, the modularity of the click-chemistry approach described here provides a flexible framework to adapt this platform to different biological membranes, inorganic nanocomponents, and therapeutic cargos. Conclusions In this study, we developed a new hybrid bio-mimetic drug-delivery system, termed magFSMs, consisting of fusogenic cell-membrane derived nanovesicles covalently coupled to magnetic-field responsive IO NPs. Unlike other hybrid biomimetic systems this structure was unique because the IO NPs were covalently conjugated on the surface of the biomimetic nanovesicle through click-chemistry. The magFSMs successfully integrate the advantageous properties of both components combined in a single platform by exhibiting (a) preferential homotypic targeting, enabling selective recognition of cancer cells, and (b) stimuli responsiveness to an external magnetic field, enabling remote manipulation capability. Apart from exhibiting good biocompatibility, magFSMs not only showed enhanced uptake and good targeting capability in 2D co-culture systems, but also in a complex 3D heterotypic tumor spheroid environment, wherein it preferentially associated to cancer cells. When loaded with an anti-cancer therapeutic payload, specific drug accumulation was observed in cancer cells in both 2D and 3D cell culture environments, and this effect was further amplified by the use of an external magnetic field owing to its stimuli-responsive behavior. This study demonstrates an efficient and simple strategy for generating theranostic organic-inorganic hybrid NPs, and opens the possibility for the rational development of customizable biomimetic targeted drug delivery systems. Declarations Competing interests The authors declare that they have no competing interests Funding The authors thank the financial support of the European Research Council (starting grant #950421), the European Commission for the HORIZON-MSCA-2021-PF (#101063372), the MICIU/AEI/ 10.13039/501100011033 (PID2023-152844NB-I00; PID2022-142338OB-100, PID2020-119206RB-I00, PID2023-151448NB-I00, CNS2023-144318), and the Xunta de Galicia (#ED431C 2022/18, #ED431B2023/19, #2021-CP090 and Centro de Investigación do Sistema Universitario de Galicia accreditation 2023–2027 #ED431G 2023/03). R. Iglesias-Rey (CP22/00061) from the Miguel Servet Program of ISCIII and Co-financed by the EU. Author Contribution M.R-P., S.F. and M.M. conducted the experiments. R. I-R conducted the MRI experiments. E.P, P.d.P and B.P designed the experiments and secured the funding necessary for this work. 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Lehto V-P: Cell membrane coating integrity affects the internalization mechanism of biomimetic nanoparticles. Nat Commun. 2021;12:5726. Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files RodrguezPrezSuplementary.docx floatimage1.png Scheme 1. Schematic illustration depicting the synthetic steps to produce magFSMs from nanovesicles obtained from glycometabolically modified A549 cells with functional azide-groups and their subsequent labeling through click-chemistry with IO@DPMA NPs. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8712225","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":584686255,"identity":"95140093-0af4-42ad-b30c-85e77c3c0998","order_by":0,"name":"Manuel Rodríguez-Pérez","email":"","orcid":"","institution":"University of Santiago de Compostela","correspondingAuthor":false,"prefix":"","firstName":"Manuel","middleName":"","lastName":"Rodríguez-Pérez","suffix":""},{"id":584686257,"identity":"879bf7ca-b7cd-4c6b-b2cd-0503f5be58d1","order_by":1,"name":"Sajid Fazal","email":"","orcid":"","institution":"University of Santiago de Compostela","correspondingAuthor":false,"prefix":"","firstName":"Sajid","middleName":"","lastName":"Fazal","suffix":""},{"id":584686259,"identity":"35ce3f64-f49b-44c3-9585-02c5479cd696","order_by":2,"name":"Martina Migliavacca","email":"","orcid":"","institution":"University of Santiago de Compostela","correspondingAuthor":false,"prefix":"","firstName":"Martina","middleName":"","lastName":"Migliavacca","suffix":""},{"id":584686260,"identity":"8920ea42-b69d-4270-a981-583863b647bd","order_by":3,"name":"Ramón Iglesias-Rey","email":"","orcid":"","institution":"Instituto de Investigación Sanitaria de Santiago","correspondingAuthor":false,"prefix":"","firstName":"Ramón","middleName":"","lastName":"Iglesias-Rey","suffix":""},{"id":584686261,"identity":"d9c2558d-80ed-4595-a133-81152718abf1","order_by":4,"name":"Ester Polo","email":"","orcid":"","institution":"University of Santiago de Compostela","correspondingAuthor":false,"prefix":"","firstName":"Ester","middleName":"","lastName":"Polo","suffix":""},{"id":584686264,"identity":"09e7e4c3-1a84-4aa2-8296-ae50c2544747","order_by":5,"name":"Pablo del Pino","email":"","orcid":"","institution":"University of Santiago de Compostela","correspondingAuthor":false,"prefix":"","firstName":"Pablo","middleName":"del","lastName":"Pino","suffix":""},{"id":584686265,"identity":"f3bc4cc8-afa9-4cde-8749-1d2655bf57d4","order_by":6,"name":"Beatriz Pelaz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYBACAwTJ3MDAUEGcFsYGiBYgzXCGaC0MUC2MbURoMWdvfv64oqAOyGhs/Phz3mF7/gbmwx/wabHsOWbYeMaADcg42Cwhue1w4owDbGkSeB12I8GwscGAB8hIbJAw3HY4wYCBxwy/X+4//wjUIgFkPGz+kTjnsL0BA/9nvA4zuMEDssUAyGBskzjYcJhxAwMPA16HWfbkFM5sMEjgsexJbLNsOJaeOOMwmxleLebsxzd8bPhTJ2fOfvjwzR811vb87c2P8ToMBngM4ExmYtSDgAFhJaNgFIyCUTBSAQAePklEgZYWrQAAAABJRU5ErkJggg==","orcid":"","institution":"University of Santiago de Compostela","correspondingAuthor":true,"prefix":"","firstName":"Beatriz","middleName":"","lastName":"Pelaz","suffix":""}],"badges":[],"createdAt":"2026-01-27 15:25:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8712225/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8712225/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101900632,"identity":"04aa5523-9cea-4f79-adac-3592643418c4","added_by":"auto","created_at":"2026-02-04 19:04:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":805490,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of nanomaterials. \u003cstrong\u003eA\u003c/strong\u003e) Transmission electron micrographs of IONPs as synthesized. \u003cstrong\u003eB-C\u003c/strong\u003e) FSMs as synthetized after extrusion. \u003cstrong\u003eD\u003c/strong\u003e) magFSMs after “click” coupling of FSMs and IO@DPMA NPs. Different ratios of magFSMs: \u003cstrong\u003eE\u003c/strong\u003e) 100:1, \u003cstrong\u003eF)\u003c/strong\u003e 50:1, and \u003cstrong\u003eG\u003c/strong\u003e) 10:1. \u003cstrong\u003eH)\u003c/strong\u003e Hydrodynamic diameter of FSMs and magFSMs measured through NTA. \u003cstrong\u003eI\u003c/strong\u003e) z-potential values of the different nanomaterials. \u0026nbsp;Scale bars correspond to \u003cstrong\u003eA-B\u003c/strong\u003e: 50 nm; \u003cstrong\u003eC\u003c/strong\u003e: 50 nm; \u003cstrong\u003eD-G\u003c/strong\u003e: 200 nm.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8712225/v1/ea06b47e1f2e6061e325dab6.png"},{"id":101900638,"identity":"3db864e9-a2af-4b78-b631-b7340908d0df","added_by":"auto","created_at":"2026-02-04 19:04:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":294539,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMagnetic properties of magFSMs (10:1 IO NP:FSM)\u003c/strong\u003e. \u003cstrong\u003eA)\u003c/strong\u003e Representative T2- maps of IO@DPMA NPs and magFSMs at different concentrations in agar phantoms. \u003cstrong\u003eB)\u003c/strong\u003e Corresponding relaxivity values.\u003cstrong\u003e C)\u003c/strong\u003e Magnetic hysteresis loop of magFSMs and IO@DPMA NPs under a magnetic field of 24 kA/m and a frequency of 100 KHz. \u003cstrong\u003eD\u003c/strong\u003e) Specific absorption rate values and \u003cstrong\u003eE\u003c/strong\u003e) Magnetization values at 24 kA/m.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8712225/v1/543c1c47525d44a909d5d15d.png"},{"id":101900634,"identity":"65e80fc5-22a9-4071-891e-6f692c2fa84c","added_by":"auto","created_at":"2026-02-04 19:04:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1134659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHomotypic targeting in 2D\u003c/strong\u003e. \u003cstrong\u003eA\u003c/strong\u003e) Fluorescence microscopy images of A549 cells exposed for 1h to magFSMs at 10\u003csup\u003e9\u003c/sup\u003e NP mL\u003csup\u003e-1\u003c/sup\u003e. IO@D\u003cu\u003ePMA NPs\u003c/u\u003e (Cy3, yellow), FSMs (Atto647, magenta) and GFP (green) are observed. Scale bars correspond to 50 µm. \u003cstrong\u003eB\u003c/strong\u003e). Flow cytometry plots showing cellular uptake of magFSMs with different ratios of IO@DMPA NPs (\u003cem\u003ei.e.\u003c/em\u003e, 10, 50, 100, and untreated cells) after 1 h of incubation.\u003cstrong\u003e C\u003c/strong\u003e). Fluorescence microscopy imaging of co-cultures of A549-GFP and MRC5 cells exposed to 10\u003csup\u003e9\u003c/sup\u003e FSM ml\u003csup\u003e-1\u003c/sup\u003e (above) and magFSMs (below) for 1h. Scale bars: 20 µm. \u003cstrong\u003eD\u003c/strong\u003e) Flow cytometry charts depicting Atto647 fluorescence found in single A549 or MRC5 cultures (top) as well as in cocultures of both cell types (bottom). \u003cstrong\u003eE\u003c/strong\u003e) Normalized mean fluorescence intensity from IONPs labeled (Cy3) of different cell cultures exposed to FSM and magFSMs for 1 hour \u003cstrong\u003eF\u003c/strong\u003e) Normalized mean fluorescence intensity from FSMs label (Atto647). Error bars indicate SEM. *P ≤ 0.05, **P ≤ 0.005, and ***P ≤ 0.001. n.s., no significant difference.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8712225/v1/515b917e8dfb3fa53a34755b.png"},{"id":101944175,"identity":"8214dbd9-fb72-41f0-bfa9-e791e032624b","added_by":"auto","created_at":"2026-02-05 09:49:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1232068,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHomotypic targeting 3D\u003c/strong\u003e. \u003cstrong\u003eA\u003c/strong\u003e) Maximum projections of fluorescence microscopy imaging of A549-MRC5 heterotypic spheroids exposed to FSM and magFSMs for 24 hours including fluorescence channels for GFP (A549 cells), Cy3 (IONPs) and Atto647 (FSMs). \u003cstrong\u003eB\u003c/strong\u003e) High magnification image of NP uptake (as revealed by Atto647 and Cy3 signal) by A549 cells after exposure to FSMs and magFSMs. \u003cstrong\u003eC\u003c/strong\u003e) MFI of Cy3 and Atto647 found in both cell populations of heterotypic spheroids after 24 hours of NP exposure as measured by flow cytometry. Scale bars: 200 µm in A; 50 µm in B. Error bars indicate SEM. *P ≤ 0.05, **P ≤ 0.005, and ***P ≤ 0.001. n.s., no significant difference.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8712225/v1/2c5aa03edbc67ed21b5d2599.png"},{"id":101900639,"identity":"184167eb-95fc-41c1-80a6-cb30ac699e53","added_by":"auto","created_at":"2026-02-04 19:04:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":980980,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHomotypic targeting 3D\u003c/strong\u003e. \u003cstrong\u003eA\u003c/strong\u003e) Maximum projections of fluorescence microscopy imaging of A549-MRC5 heterotypic spheroids exposed to FSM and magFSMs for 24 hours including fluorescence channels for GFP (A549 cells), Cy3 (IONPs) and Atto647 (FSMs). \u003cstrong\u003eB\u003c/strong\u003e) High magnification image of NP uptake (as revealed by Atto647 and Cy3 signal) by A549 cells after exposure to FSMs and magFSMs. \u003cstrong\u003eC\u003c/strong\u003e) MFI of Cy3 and Atto647 found in both cell populations of heterotypic spheroids after 24 hours of NP exposure as measured by flow cytometry. Scale bars: 200 µm in A; 50 µm in B. Error bars indicate SEM. *P ≤ 0.05, **P ≤ 0.005, and ***P ≤ 0.001. n.s., no significant difference.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8712225/v1/82beda85d658ddf04aad8769.png"},{"id":101900636,"identity":"afcb7bef-17d8-46fd-8513-60764c0392b2","added_by":"auto","created_at":"2026-02-04 19:04:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":847052,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTherapy 2D and 3D. A\u003c/strong\u003e) Therapeutic effect of DOX (left), DOX-loaded FSMs (center) and DOXO-loaded magFSMs (right). Fluorescence microscopy imaging of A549 cells exposed to 1 µM of Dox for 10 minutes. Channels represent DOX cargo (red), Atto647 vehicle (magenta) and cell nuclei (cyan). Scale bars: 50 µm. \u003cstrong\u003eB\u003c/strong\u003e) Dose-response curves of A549 cells after 10 minutes (I) and 24 hours (II) of NP exposure. \u003cstrong\u003eC\u003c/strong\u003e) Dose-response curves of A549 cells after 10 minutes (I) and 24 hours (II) of A549 cells exposure to CbPt, CbPt-loaded FSMs and CbPt-loaded magFSMs.\u003cstrong\u003e D\u003c/strong\u003e) Flow cytometry pooled plots of damaged cells found in A549 spheroids after 24 hours of treatment exposure (I: control cells, II: DOX (10 µM), III: magFSM@DOX, IV: quantification). Calcein AM cell viability assay quantifies live cells through intracellular esterase activity and membrane integrity. N= 3 independent spheroids per group. Error bars indicate SEM. *P ≤ 0.05, **P ≤ 0.005, and ***P ≤ 0.001. n.s., no significant difference\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8712225/v1/c696381ca0c8d2cdf40b39bd.png"},{"id":101900635,"identity":"165e8345-6274-4555-99a8-6220972a3c0d","added_by":"auto","created_at":"2026-02-04 19:04:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1112266,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDOX delivery in heterotypic 3D cultures. A\u003c/strong\u003e) Fluorescence microscopy imaging of DOX delivery in heterotypic spheroids in the absence (I) and presence (II) of a magnetic field after exposure to 10 µM of DOX equivalents for 24 hours. Fluorescence channels include: Atto647-vehicle (magenta), DOX-cargo (red), A549 cells (green) and nuclei (blue). Scale bar: 100 µm. \u003cstrong\u003eB\u003c/strong\u003e) Flow cytometry plots of DOX signal found in spheroids after exposure to DOX and magFSM@DOX for 1 hour (I) and 24 hours (II); and Atto647 signal in magFSM@DOX after an exposure period of 1 hour (III) and 24 hours (IV) in the absence and presence of magnetic field. Values indicates relative MFI of vehicle fluorescence (Atto647), and cargo (DOX) fluorescence as found in the cell components (MRC5 or A549) of heterotypic spheroids. Error bars indicate SEM. *P ≤ 0.05, **P ≤ 0.005, and ***P ≤ 0.001. n.s., no significant difference\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8712225/v1/e337b25a1de52874e16ed965.png"},{"id":102749361,"identity":"ff708e30-2d43-42b2-a949-30e58dceedd0","added_by":"auto","created_at":"2026-02-16 09:12:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7556142,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8712225/v1/7b4aa9a8-1811-46e3-9222-eea3e0d649d0.pdf"},{"id":102294930,"identity":"c57869b5-9286-4267-876d-0b8b46d0ce4e","added_by":"auto","created_at":"2026-02-10 10:04:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5751894,"visible":true,"origin":"","legend":"","description":"","filename":"RodrguezPrezSuplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-8712225/v1/0686f588b106da48fae61797.docx"},{"id":101943338,"identity":"f71029d1-398c-46c2-b528-4a8d32ed2742","added_by":"auto","created_at":"2026-02-05 09:41:40","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":291794,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e. Schematic illustration depicting the synthetic steps to produce magFSMs from nanovesicles obtained from glycometabolically modified A549 cells with functional azide-groups and their subsequent labeling through click-chemistry with IO@DPMA NPs.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8712225/v1/8815e0de66fa289dd6e2c895.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Click-Engineered Magnetic Fusogenic Cell-Derived Nanocarriers for Enhanced Magnetic-Field-Assisted Drug Delivery","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDrug delivery nanoplatforms have received increasing attention in recent years due to the vast range of approaches offered by nanostructured materials in countering roadblocks associated with conventional drug delivery. Back in the 1980s, liposomal formulations constituted a groundbreaking advance in drug delivery, increasing the efficacy of anticancer (Doxorubicin-DOXIL)[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] or antifungal (Amphotericin B-AMBISOME)[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] treatments as well as decreasing undesired side effects. Currently, lipid-based systems have evolved exponentially, with outstanding translation to human clinical practice as revealed by the new vaccines developed during the SARS-CoV-2 outbreak in 2019 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These systems rely on the combination of lipids (\u003cem\u003ei.e.\u003c/em\u003e, 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP), cholesterol, etc.), which provide stability and biocompatibility to the payload [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], while enabling its efficient loading and delivery by responding effectively to the external environment [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Moreover, such systems exhibit great loading efficiency and are synthetized following simple and straightforward procedures, leading to the high-yield production of homogeneous NPs [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Despite the significant advances achieved in preclinical studies and clinical applications, these nanomedicines still need to overcome major biological barriers before \u003cem\u003ein vivo\u003c/em\u003e administration including, but not limited to, rapid clearance from the bloodstream by the mononuclear phagocytic system [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and unspecific activation of the immune cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] leading to undesired allergic reactions, and non-specific targeting of action sites [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs medicine is evolving towards a new era of precision strategies, one of the main concerns during nanomaterial design is to achieve active (and/or enhance the passive) targeting to the desired disease site [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In this sense, numerous approaches have been explored based on the engineering of NP\u0026acute;s surface for coupling different active molecules such as polyethylene glycol (PEG) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], antibodies [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], glycans [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] as well as other ligands [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These new functionalities influence NP\u0026acute;s biodistribution, promoting their accumulation in the desired organ or tissue [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In this sense, the use of fragments of cell membranes to produce biomimetic NP coatings has emerged in the last decade as a revolutionary approach for active targeting [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Many different cell types have been used as a source to produce biomimetic NPs, including platelets [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], leukocytes [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], erythrocytes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], macrophages [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], etc., exploiting the presence of receptors, glycans, and a plethora of ligands in the cell membrane, which play a key role in cell-cell interactions. Moreover, since they are obtained from biological entities, they exhibit low immunogenicity, thus bypassing the surveillance of the immune system. These membranes have also been employed for cloaking NPs, providing a new identity to the initial NPs. This has been done by employing surface coating strategies such as co-extrusion [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], sonication [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], electroporation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and \u003cem\u003ein situ\u003c/em\u003e loading [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] to physically force the NPs and cell membrane surface to interact with each other.\u003c/p\u003e \u003cp\u003eSince NPs eventually interact with cells, the mechanism by which the NP payload is released into cells has given rise to a vast discussion. In general, once recognized by cells NPs are considered to be internalized following the endocytic pathway, \u003cem\u003ei.e.\u003c/em\u003e, NPs interacting with cell membrane are engulfed in vesicles forming endosomes [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. These endosomal vesicles evolve to late endosomes and ultimately to lysosomes, where the highly acidic pH degrades the engulfed materials within [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. During this process, a significant fraction of the NPs will remain in the lysosomal pathway, suffering from potential degradation and thus minimizing the available dose of the molecules of interest. To enhance NP-mediated drug delivery, researchers have been tailoring NP features to facilitate effective endosomal disruption or evasion, thereby allowing NPs to be released into the cell cytoplasm through endosomal escape mechanisms (similar to the mRNA LNP vaccines) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, novel strategies are still needed to protect NP payloads from degradation, minimizing side effects, and increasing the drug dose delivered to target cells. In this regard, approaches addressing the direct cytosolic delivery of NP payload could contribute to overcoming the main drawbacks in this field [\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In addition, it could constitute a new opportunity for classical chemotherapeutic drugs that usually suffer from undesired side effects. Recently, our group has proposed a new approach using fusogenic biomimetic NPs for the delivery of small molecules, proteins, and synthetic NPs [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This work employed a combination of cell membranes and a combination of aromatic-labeled and cationic lipids, constituting a proof of concept of the feasibility and the potential of engineering cell-derived NPs for enhanced active drug delivery.\u003c/p\u003e \u003cp\u003eOver and above enhanced cargo delivery, clinical practice now emphasizes the demand for new approaches combining therapy and diagnosis [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. As part of this evolution, researchers have developed theranostic nanomaterials that are able to be traced using current medical diagnosis techniques (Magnetic Resonance Imaging-MRI, Computed Tomography-CT, Positron Emission Tomography-PET, etc.) and simultaneously provide a therapeutic response [\u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. To address these unmet limitations, in this work, we aim to design a theranostic hybrid nanocarrier for cytosolic delivery of cargo by combining stimuli-responsive inorganic NP and cell-derived membranes in a single nanoplatform for magnetically controlled drug delivery. Unlike other bio-mimetic inorganic nanoplatforms, the novelty of our system design relies in the anchoring of inorganic NPs onto the biomimetic cell-membrane derived vesicles covalently using a click-chemistry approach which is a simple, fast and efficient loading strategy relative to common energy-intensive processes. In this work, we focus on the rational design and \u003cem\u003ein vitro\u003c/em\u003e validation of a multifunctional nano-bio interface rather than on preclinical \u003cem\u003ein vivo\u003c/em\u003e translation.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of nanomembrane vesicles (Cellsomes -CSMs and Fusosomes -FSMs)\u003c/h2\u003e \u003cp\u003eTo prepare CSMs from cell-membranes of A549 cells (ATCC, CCL-185), an already established protocol was utilized [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. This method included cell harvesting, hypo-osmotic treatment, freeze-thaw cycles, bath sonication and centrifugation steps to isolate the cellular membranes followed by physical extrusion through a 0.8 \u0026micro;m pore-size polycarbonate membrane using a mini-hand extruder (Avanti Polar Lipids) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The prepared CSMs were stored in HEPES buffer (25 mM, 7.4 pH) at 4\u0026deg;C until further use. To incorporate an azide functional group (N\u003csub\u003e3\u003c/sub\u003e) to the CSMs, A549 cells were initially glycometabolically labeled using an azido-modified mannose (N-azidoacetylmannosamine tetraacylated, ManNAz; Thermo Scientific 88904). For that, ManNAz was added to its cell culture growth medium at 50 \u0026micro;M 72 hours pre-cell-membrane isolation and extrusion [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The presence of ManNAz on A549 cell membranes were analyzed through fluorescent imaging of these cells post incubation with Dibenzocyclooctyne-PEG4-Fluor545 (5mM, 1 hour) (see supporting information for more details, \u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Additionally, to provide fusogenic properties to CSMs (fusosomes, FSMs), a neutral lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, Avanti Research, 850725), and a cationic lipid DOTAP (1,2-dioleoyl-3-trimethylammonium-propane, Avanti Research, 890890) were used. Additionally, fluorescently labeled DOPE-Atto647 (Sigma, 42247) was also used to fluorescently track the FSMs. In all, a ratio of 90/20/100 \u0026micro;g DOPE/DOPE-Atto647/DOTAP was used for one batch of CSMs obtained from 10\u003csup\u003e7\u003c/sup\u003e A549 cells. After sonication for 10 minutes, a final step of physical extrusion through a 0.8 \u0026micro;m pore-size polycarbonate membrane was employed to promote the insertion of the lipid mixture into the phospholipid bilayer of FSMs [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of magFSMs and drug loaded magFSMs\u003c/h3\u003e\n\u003cp\u003eDibenzocyclooctyne/dodecylamine-grafted poly(isobutylene-alt-maleic anhydride) polymer (DPMA) was employed as the amphiphilic coating material to transfer hydrophobic IO NPs to aqueous media [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] while simultaneously providing a DBCO functional group for SPAAC click conjugation. This polymer, hereafter referred to as DPMA, contains both hydrophobic alkyl chains for anchoring onto the nanoparticle surface and DBCO groups enabling covalent coupling with azide-functionalized fusogenic membranes.\u003c/p\u003e \u003cp\u003eTo prepare the click-chemistry enabled IO@DPMA NP (see supplementary information and \u003cb\u003eFigure S2\u003c/b\u003e) loaded FSMs (magFSMs), the azide-labeled FSMs and IO@DPMA NPs were mixed together at different (IO@DPMA NP: FSM) ratios, \u003cem\u003ei.e.\u003c/em\u003e, 10:1; 50:1 and 100:, in HEPES buffer followed by sonication and incubation at 25\u0026deg;C for 3 hours. The prepared magFSMs were separated from free FSMs and free IO@DPMA NPs by centrifugation at 10000 rcf for 10 minutes. For evaluating and quantifying IO@DPMA NPs loaded onto FSMs, ICP and NTA analysis was carried out. To prepare drug loaded magFSMs, either doxorubicin (DOX) or carboplatin (CbPt) was initially loaded into the azide-labeled FSMs which were then used for click-conjugation with IO@DPMA NPs following the above-mentioned method. For DOX loaded FSMs, 50 \u0026micro;L of 10 mg/mL doxorubicin hydrochloride (Sigma, D1515) prepared in PBS (containing 0.5 mg of drug) was mixed with one batch of FSMs (final volume of 1 mL) and then subsequently subjected to sonication and physical extrusion. Eventually the mixture was subjected to a 30000 RCF centrifugation for 60 minutes at RT to separate out magFSM@DOX. To evaluate drug encapsulation and concentration, absorbance at 480 nm was measured (see supplementary information, \u003cb\u003eFigure S3\u003c/b\u003e). In case of CbPt-loaded magFSMs, a 50 mM solution of CbPt (216100-M Merck) was initially mixed with one batch of cell derived membranes (final volume of 1 mL) and subsequentially extruded as previously described. After purification through centrifugation, ICP-OES analysis was undertaken to determine the Pt content in the obtained magFSM@CbPt (see supplementary information, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Eventually, dose-response studies were performed which included a concentration range of 0\u0026ndash;10 \u0026micro;M and 0-200 \u0026micro;M for DOX and CbPt respectively.\u003c/p\u003e\n\u003ch3\u003eNanoparticle characterization\u003c/h3\u003e\n\u003cp\u003eThe synthesized FSMs, IO@DPMA NPs and magFSMs were characterized for their physicochemical properties using various techniques.\u003c/p\u003e \u003cp\u003eFor \u003cb\u003eelectron microscopy\u003c/b\u003e, 2 \u0026micro;L of the 2% formaldehyde fixed sample was dropped on an amorphous carbon supported 400 mesh copper grids. After leaving the sample to dry for 20 minutes, they were washed with PBS and negatively stained with 2% uranyl acetate. The samples were washed again and left to dry overnight before imaging using a scanning electron microscope ZEISS FESEM ULTRA Plus.\u003c/p\u003e \u003cp\u003eTo determine Fe concentration, \u003cb\u003einductively coupled plasma-optical emission spectroscopy (ICP-OES)\u003c/b\u003e measurements were carried out. For this, 20 \u0026micro;L of the sample were first dried overnight in a glass container, to which 120 \u0026micro;L HNO\u003csub\u003e3\u003c/sub\u003e and 360 \u0026micro;L HCl along with 10 \u0026micro;L Mn\u003csup\u003e2+\u003c/sup\u003e and Se\u003csup\u003e2+\u003c/sup\u003e (500 ppm) as internal standards were added. After 4 hours of treatment, 500 \u0026micro;L of the digested sample were added to 4.5 mL 2% HCl and analyzed in ICP-OES, wherein atomic emission lines at 239.563 nm and 261.187 nm for Fe were detected to quantify its concentration in the sample. The \u003cb\u003ehydrodynamic diameter and surface charge\u003c/b\u003e of the samples were evaluated using a Dynamic Light Scattering (DLS) Malvern Zetasizer Nano ZSP (Malvern Instrument ltd.). Particle size and concentrations were also evaluated using Nanoparticle Tracking Analysis (NTA, NanoSight NS300, Malvern Instrument ltd.) equipped with a 405 nm laser, wherein the samples were diluted (1:200 in PBS) before measurement.\u003c/p\u003e \u003cp\u003eTo study the \u003cb\u003emagnetic properties\u003c/b\u003e of the synthesized sample, COMBI AC HYSTER\u0026trade; (nanotech solutions, Spain) equipment was utilized. In general, 40 \u0026micro;L of the sample in a glass tube was placed inside a copper coil that produced an alternating magnetic field at 100 kHz and 24 kA m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Magnetic hysteresis loops were then measured in triplicates to determine the magnetic features including the average specific absorption rate.\u003c/p\u003e \u003cp\u003eAdditionally, the ability of the NPs as contrast agents in \u003cb\u003emagnetic resonance imaging (MRI)\u003c/b\u003e was evaluated by utilizing an in-house designed agarose mold to hold the samples. To prepare the mold, a 2% agarose solution of 100 mL was heated to form a homogenous mix, which was then transferred to a mold containing several holes to host the NP samples. After cooling and solidification, the mold was disassembled, and the holes were filled with a warm mixture of 1:1 liquid agarose and NP suspension. After solidification, the mold was carefully covered with a few more milliliters of the agarose solution and left to solidify. This mold was then scanned with a volume antenna in a Bruker Biospec 9.4 T small animal MR (Bruker, Biospin) equipped with actively shielded gradients (400 mT/m). A quadrature radiofrequency transmit-receive resonator was used for data acquisition. T2-weighted images were acquired using a multi-slice multi-echo (MSME) sequence of 11.32 ms echo time, 3 s repetition time, 16 echoes, 14 slices of 1mm thickness, 1 average, field of view (FOV) of 7.5 cm x 7.5 cm (with saturation bands to suppress the signal outside the FOV) and matrix size of 256 x 256 pixels. After acquiring, images were processed with ImageJ (Rasband WS, National Institutes of Health, Bethesda, MD, USA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.net/ij/\u003c/span\u003e\u003cspan address=\"https://imagej.net/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), generating a transverse relaxation time (T\u003csub\u003e2\u003c/sub\u003e) map archive where the T2 values were measured. Eventually, sample relaxivity (r\u003csub\u003e2\u003c/sub\u003e) was calculated as the slope of the function relating the inverse of the transverse relaxation times (1/T\u003csub\u003e2\u003c/sub\u003e) and the Fe concentration in the samples.\u003c/p\u003e \u003cp\u003eFor their biochemical characterization, flow cytometry analysis was carried out (Millipore Guava EasyCyte HT cytometer). For this FSMs, were labeled with AlexaFluor-488 anti-cadherin antibody (30 minutes incubation; 1 \u0026micro;g Ab/10\u003csup\u003e7\u003c/sup\u003eFSMs) and were compared with Liposomes (see \u003cb\u003eFigure S6\u003c/b\u003e) as a negative control. All prepared samples were centrifuged and resuspended in PBS before flow cytometry analysis.\u003c/p\u003e\n\u003ch3\u003eCell culture and maintenance\u003c/h3\u003e\n\u003cp\u003eA549 human lung epithelial cancer cells and MRC5 human lung fibroblast cells (ATCC CCL-171\u003cb\u003e)\u003c/b\u003e were cultured and maintained throughout the study in high glucose DMEM media supplemented with 10% FBS, 100 I.U. ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e penicillin and 100 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e streptomycin in a humidified biological incubator maintained at 37 \u0026ordm;C, 5% of CO\u003csub\u003e2\u003c/sub\u003e and 95% relative humidity. The cells were sub-cultured when reaching 80% confluency and were maintained up to passage number 40, after which they were discarded, and new cells were revived.\u003c/p\u003e \u003cp\u003eFor the development of 3D tumor spheroids, monolayer cell cultures were trypsinized and seeded in a 96 well plate previously coated with 60 \u0026micro;L of 1% agarose. Typically, for homotypic spheroids, 5000 A549 cells in 150 \u0026micro;L cell culture medium were initially seeded. 100 \u0026micro;L of the media was carefully removed every 2 days and replaced with fresh media for maintaining spheroid viability. Experiments on spheroids were undertaken for 5 to 7 days post initial seeding. For developing heterotypic spheroids, a co-culture of A549-GFP and MRC-5 cells at 1200:4000 ratio in 150 \u0026micro;L of medium was seeded initially.\u003c/p\u003e\n\u003ch3\u003eCytotoxicity assay\u003c/h3\u003e\n\u003cp\u003eTo evaluate potential cytotoxicity arising from sample exposure, cell-viability analysis was carried out using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, M2003) assay. A549 cells were seeded at 7500 cells/100 \u0026micro;L in a 96 well plate overnight and then incubated with the synthesized nanoparticles at different concentrations in cell culture medium for different time periods. Post incubation, the cells were washed and rinsed with PBS and an equal volume of 0.5 mg/mL MTT solution (in cell culture medium) was added to the cells for formazan crystal formation. After 4 hours, the crystals were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, 472301) and absorbance measurements were performed using an Infinite\u0026reg; 200 PRO (TECAN) microplate reader at 540 nm.\u003c/p\u003e \u003cp\u003eTo evaluate the toxicity effects of drug loaded magFSMs in 3D tumor spheroids, flow cytometry analysis was undertaken. After desired incubation with drug loaded magFSMs, the tumor spheroids were first washed with PBS, disaggregated with trypsin for 10 minutes, and then incubated with 0.1 \u0026micro;M calcein AM for 20 minutes. The cell suspension was then analyzed in a Millipore Guava EasyCyte HT cytometer (using 488 laser line for excitation and the 512/18 nm filter for fluorescence acquisition).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eNP uptake studies\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDifferent types of 2D and 3D mono and co-culture systems were developed for detailed analysis of samples uptake.\u003c/p\u003e \u003cp\u003eFor \u003cb\u003e2D monoculture experiments\u003c/b\u003e, A549 lung cancer or MRC5 lung fibroblast cells at 10,000 cells/100 \u0026micro;L in cell culture medium were seeded into an 18 well \u0026micro;-Slide (Ibidi) overnight. On the other hand, for \u003cb\u003e2D co-culture experiments\u003c/b\u003e, A549-GFP and MRC5 cells were seeded (10000:3000) in an 18 well \u0026micro;-Slide (Ibidi) overnight. For evaluating homotypic targeting of magFSM@DOX in 2D cocultures of A549 and/or MRC5, the cells were incubated with either magFSM@DOX or equivalent free drug (1 \u0026micro;M) for 24 hours. The cells were then fixed with 4% paraformaldehyde in PBS prior to imaging with a fluorescence microscope, wherein 5 different regions of interest (ROIs) per group were imaged with a minimum of 10 cells per ROI.\u003c/p\u003e \u003cp\u003eAs mentioned above, for the synthesis of \u003cb\u003ehomotypic and heterotypic spheroids\u003c/b\u003e either 5000 A549 cells or A549-GFP and MRC-5 cells (4000:1200) were seeded in a 1% agarose gel-coated 96 well plate. The desired concentration of the sample suspension was then added to allow their cellular uptake. After the required incubation period, the cells were washed with PBS and incubated further in fresh culture medium containing 5 \u0026micro;g/mL Hoechst for 20 minutes (Thermo Fischer #62249) for nuclear staining prior to fluorescence microscopy imaging.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFluorescence imaging\u003c/b\u003e was undertaken using a Thunder Leica\u0026reg; Microscope type DMI8 equipped with a Leica\u0026reg; DFC9000 camera. All images were further processed using ImageJ software.\u003c/p\u003e \u003cp\u003eFor \u003cb\u003eflow cytometry analysis\u003c/b\u003e, cells were trypsinized to obtain a single cell suspension which was analyzed using a Millipore Guava EasyCyte HT cytometer with the following laser and filter configuration: 488 nm laser with 575/25 filter for DOX and Cy3 and 642 nm laser with 695/50 nm filter for Atto647. Flow cytometry data was further analyzed using Flowjo software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003euptake under magnetic gradient exposure\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo evaluate particle uptake in heterotypic spheroids of A549 and MRC5 cells under the influence of an external stimuli, we designed a simple experiment trying to simulate the effect of a magnetic field. For this, a neodymium (40 x 40 x 20 mm) magnet (1.29\u0026ndash;1.32 T) was placed below the microplate containing cells exposed to free DOX (10 \u0026micro;M) and magFSMs (equivalent to free DOX concentration) for 24 hours. Cells treated with NPs and not exposed to an external magnetic field were used as controls. Fluorescence microscopy was then employed to map the fluorescence signal along the cross-section of the spheroids.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis and characterization of magFSMs\u003c/h2\u003e \u003cp\u003eNanomaterial design and preparation procedures are depicted in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. First, highly monodisperse IO NPs were synthetized in an organic solvent following a protocol of thermal decomposition [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] and subsequently transferred to an aqueous buffer by means of a polymeric coating with DPMA[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTEM imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) revealed spherical IO NPs with a diameter of 18 nm. For the synthesis of FSMs, the A549 lung cancer cell line was chosen, as this cancer-derived FSMs possess homotypic targeting abilities. For this purpose, we supplemented A549 cell cultures with an azido-modified mannose (ManNAz) sugar to display N\u003csub\u003e3\u003c/sub\u003e groups on the cellular membranes [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Cells effectively incorporated ManNAz to their glycoproteins anabolic pathways through a process known as glycometabolic labeling, eventually translocating N\u003csub\u003e3\u003c/sub\u003e groups in the external layer of the cytoplasmatic membrane as shown in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The incorporation of the N\u003csub\u003e3\u003c/sub\u003e groups in A549 cells was assessed by the addition of a DBCO-fluorophore (DBCO-fluor 545). It was observed that untreated cells with ManNAz were not reacting to the fluorophore while the azide-expressing A549 could selectively bind this fluorophore (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Then azide-labeled CSMs were prepared using the membrane of the azide-expressing A549 cells following an extrusion protocol using polycarbonate membranes of 0.8 \u0026micro;m pore size. During this process, DOPE (neutral), DOPE-Atto647, and DOTAP (cationic) lipids were added and intercalated in toure CSMs to provide them with fusogenic properties to generate FSMs. As revealed by TEM in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C, this process yields in the production of spherical NPs (FSMs) with a diameter of ~\u0026thinsp;150 nm. Once both components were obtained, FSMs and IO@DPMA NPs were coupled through a SPAAC click reaction leading to the generation of magFSMs. To generate magFSM, three different ratios were explored: 100:1, 50:1 and 10:1 of IO@DPMA NP:FSMs. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-G). These magFSMs displayed their surface decorated with a different density of IO@DPMA NPs. The number of IO@DPMA NPs per magFSM was directly related to the ratio used. On analyzing the hydrodynamic diameter of the FSMs and magFSMs through nanoparticle tracking analysis (NTA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH and S4 A \u003cb\u003eand B\u003c/b\u003e), no significant change in the hydrodynamic diameter was observed (148.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2 nm). On the other hand, zeta potential measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI \u003cb\u003eand Figure S4 C and D\u003c/b\u003e) showed an increase in the negative surface charge of magFSMs correlated with a higher incorporation of IO@DPMA NPs (-19.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35 mV). Furthermore, in-depth analysis through ICP-OES showed that for magFSMs, the IO@DPMA NPs loaded per FSMs positively correlated with a higher IO@DPMA NP/FSM ratio (\u003cb\u003eFigure S4 G\u003c/b\u003e). These results showcase the feasibility of the SPAAC click-reaction to covalently link inorganic NPs to FSMs, providing a straightforward methodology to tailor the FSMs\u0026acute; surface to modulate their composition with specificity and precision.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThen the abilities as MRI contrast agents of the magFSM were tested, since magnetic NPs can act as T2-contrast agents by decreasing the transverse relaxation time (T2) of the nearby water protons providing a hypointense signal in T2-weighted MRI. Therefore, the presence of IO@DPMA NPs in magFSM is expected to provide the ability to respond to stimuli from magnetic nature, constituting a new MRI negative contrast agent. After analysis in magnetic resonance phantoms, magFSMs (10:1 IONPs) showed a significantly higher darker contrast as compared to bare IO@DPMA NPs at the same Fe concentration as revealed in the T2- maps showing hypointense signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). Relaxivity measurements from these maps showed that magFSMs exhibited a relaxivity of 164.7\u0026thinsp;\u0026plusmn;\u0026thinsp;31.4 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e which was significantly higher than that of IO@DPMA NPs (61.2\u0026thinsp;\u0026plusmn;\u0026thinsp;11.6 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). A similar trend in increased relaxivity was also observed for IONPs that were coated with hepatocellular cancer cell membranes as compared to free iron-oxide NPs [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. These findings suggest that the IO@DPMA NP arrangement on the surface of magFSMs leads to an increase of the effective magnetic moment thus highlighting the effect of NP clustering on their magnetic features [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Additionally, the arrangement of IO@DPMA NPs on the magFSMs surface may alter the diffusion dynamics of water protons around the NPs compared to bare single IO@DPMA NP suspensions, a factor that has been documented to affect its relaxivity [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThen, dynamic magnetometry measurements were performed to evaluate the magnetic behavior of both systems, IO@DPMA NPs and magFSMs, in solution. These measurements provide information about the magnetic response of materials in more realistic settings (\u003cem\u003ei.e.\u003c/em\u003e, temperature, solvent, viscosity, etc.) compared with other techniques such as superconducting quantum interference devices (SQUID). Dynamic magnetometry measurements revealed that magFSMs exhibited a hysteresis loop similar to that of free IO@DPMA NPs across the same iron concentration range (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This indicates that the ability of the IO@DPMA NPs to absorb electromagnetic energy, as quantified by the Specific Absorption Rate (SAR), was not compromised by their attachment to the surface of the FSMs, with SAR of 93.7\u0026thinsp;\u0026plusmn;\u0026thinsp;7.5 W/g\u003csub\u003eFe\u003c/sub\u003e and 84.5\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7 W/g\u003csub\u003eFe\u003c/sub\u003e for magFSMs and IO@DPMA NPs, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D). The SAR values in this case were predominantly governed by N\u0026eacute;el relaxation rather than Brownian relaxation. This was further supported by SAR measurements of magFSMs in varying concentrations of glycerol[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] (\u003cb\u003eFigure S5\u003c/b\u003e), in particular three percentages were tested, 5, 10 and 25% v/v of glycerol in water. Increasing the viscosity of the surrounding medium by using higher glycerol concentrations did not result in significant changes in the SAR values of magFSMs. Additionally, no significant differences in magnetization values were observed between magFSMs and free IO@DPMA NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) in 25 mM HEPES, confirming that the magnetic properties of the synthesized system remained unchanged following the click-coupling process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn-vitro\u003c/b\u003e \u003cb\u003euptake and homotypic targeting of magFSMs in 2D cell cultures\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIt has been widely demonstrated that CSMs produced from cancer cells exhibit a preferential recognition to the same cells from which they originated [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. This ability is known as homotypic targeting. This effect relies on the presence of a plethora of proteins, glycoproteins and ligands involved in cell-cell recognition process involved in cancer cell proliferation, invasion, and metastasis [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], such as Thomsen\u0026ndash;Friedenreich (TF) antigen [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] and E-cadherin which are retained during the CSM preparation process [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. To confirm that our FSMs are presenting these recognition molecules, flow cytometry assays were performed using an anti-cadherin antibody. \u003cb\u003eFigure S6\u003c/b\u003e shows the presence of Cadherin on FSMs as compared to liposome control samples (see supporting information), thus highlighting the presence of cell-membrane specific proteins on the CSMs surface, suggesting its feasibility for homologous binding recognition.\u003c/p\u003e \u003cp\u003eTo further evaluate the ability of magFSMs to interact with cells and get internalized, fluorescence microscopy and flow cytometry assays were performed. To do so, we first evaluated the cytotoxicity of magFSMs and its individual components. FSMs and IO@DMPA NPs through MTT cytotoxicity assay. From \u003cb\u003eFigure S7\u003c/b\u003e it was observed that FSMs were found to be biocompatible below a concentration of 10\u003csup\u003e10\u003c/sup\u003e NPs mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and IO@DMPA NPs showed no adverse toxic effects up to a concentration of 25 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Fe. On the other hand, magFSMs (at 10:1 IO NP:FSM ratio) showed good biocompatibility up to 5 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of Fe (corresponding to ~\u0026thinsp;10\u003csup\u003e10\u003c/sup\u003e FSMs mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Thus, for all our \u003cem\u003ein vitro\u003c/em\u003e experiments involving magFSMs, a concentration of less than 5 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Fe or 10\u003csup\u003e10\u003c/sup\u003e FSMs mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was set. Using fluorescence microscopy, co-localized fluorescence signals were observed for both the FSM (labeled with DOPE-Atto647) and the cargo (IO@DPMA NPs, labeled with Cy3) in cells. This colocalization corroborates the hybrid nature of magFSMs and confirmed its stability in cell culture conditions, meaning they are stable at biologically relevant salt concentrations and to the presence of several enzymes with protease activity that could impair the linkages formed through electrostatic or other non-covalent mechanisms, at least for the observed timepoint (3h). Furthermore, the fusogenic behavior was studied for the two samples prepared, magFSMs and magCSMs with the same ratio of IO@DMPA NPs (1:10). The fusogenic abilities of magFSMs were confirmed by the localization of the fluorescence signal at the cell-membrane, together with a diffuse intracellular fluorescence throughout the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cb\u003eFigure S8)\u003c/b\u003e. This contrasts sharply with the behavior of magCSMs, which are not expected to display fusogenic properties (\u003cb\u003eFigure S8 A-B)\u003c/b\u003e. Instead, magCSMs required longer incubation times to be internalized and exhibited a characteristic punctate distribution, consistent with their uptake through endocytic pathways. These two different behaviors are in agreement with previous results of our group [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and confirm that neither the incorporation of azide groups via metabolic engineering nor the decoration with IO@DPMA NPs compromise the fusogenic properties of FSMs, at least at the ratios tested.\u003c/p\u003e \u003cp\u003eConsidering that the presence of IO@DPMA NPs on FSMs could potentially hinder their interaction with target cells by masking membrane-associated receptors, different IO@DPMA NPs:FSM ratios were tested to determine optimum conditions for minimal interference on cellular uptake as compared to the inherent features of FSMs. Flow cytometry analysis revealed that, when using equal carrier concentrations, magFSMs synthesized at a 10:1 IO NP:FSM ratio exhibited an uptake comparable to that of bare FSMs, in contrast to the higher ratios tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Based on these results, magFSMs prepared at the 10:1 ratio demonstrated the most favorable balance of magnetic performance, cytocompatibility, and uptake efficiency, and were therefore selected for all subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFollowing this initial comparative assessment on \u003cem\u003ein vitro\u003c/em\u003e uptake, the homotypic targeting of magFSMs was further compared in A549 and MRC5 (lung fibroblast) mono- and co-cultures in an attempt for a more accurate resemblance of tumor microenvironment [\u003cspan additionalcitationids=\"CR60 CR61\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], as a result of the combination of healthy and cancerous lung cells. To facilitate the identification of the two different cell lines, GFP-expressing A549 cells were selected to distinguish them from MRC5. Remarkably, fluorescence microscopy of co-cultures revealed that magFSMs not only clearly fused with cell membranes of A549-GFP cells to a greater extent as compared with MRC5 cells, but the fusion was similar to what was observed in the case of FSM with no IONP decoration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). This was further corroborated through flow cytometry analysis, which allowed the precise quantification of the homotypic recognition of cancer cells in mono and co-cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). From Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cb\u003eF\u003c/b\u003e, it is evident that magFSMs showed a significantly higher uptake in A549 cells as compared to MRC5 cells, when quantified through fluorescence signals from both Cy3 (IO NP) and Atto647 (FSM), which was similar to FSMs alone. This trend was also maintained in co-cultures of A549-GFP and MRC5 cells wherein both FSM and magFSMs were more efficiently internalized (~\u0026thinsp;2-fold) compared with MRC5 fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE \u003cb\u003eand F\u003c/b\u003e). Specifically, MFI values (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) at the Cy3 channel, confirmed that magFSMs are specific vehicles for the delivery of IO@DMPA NPs as cargo. Overall, these findings were consistent with those observed in monocultures of A549 or MRC5. Taken together, these results corroborate the feasibility of click-chemistry conjugation of IO@DPMA NPs to FSMs as a strategy to engineer hybrid carriers without compromising fusogenicity or homotypic specificity [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. This provides solid evidence of the preferential recognition of cancer cells by magFSMs even in a multicellular tumor-like scenario.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHomotypic targeting of magFSMs in 3D cell cultures.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter assessing its targeting capability in 2D cell cultures, we evaluated the magFSMs\u0026acute; capabilities to target cancer cells in 3D models. Since the tumor microenvironment (TME) comprises vasculature cells, cancer stem cells, immune cells, and tumor-associated fibroblasts (that have been linked with cancer progression) that add to its complexity, it is essential for a drug delivery vehicle to navigate through this environment to effectively target cancer cells [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Within the TME, the stromal component, usually known as cancer associated fibroblasts (CAFs) are one of the most vastly studied components of the TME due to their implication in the promotion of the growth and invasion of cancer cells by various mechanisms [\u003cspan additionalcitationids=\"CR67\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. CAFs are able to produce fibroblast secreted protein-1 (FSP1) [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] and Insulin-like growth factor 1 (IGF-1) [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e] which are related to cell growth and migration. Moreover, stromal cell-derived factor 1 (SDF-1) derived from CAFs was found to promote angiogenesis through the recruitment of endothelial cell precursors (EPCs) for the formation of new blood vessels [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Having this in mind, our aim was to model this complex environment by using heterotypic spheroids where fibroblast constitutes an additional cell barrier to overcome for common nanomedicines. In this study, heterotypic spheroids composed of A549-GFP and MRC5 (\u003cb\u003eFigure S9\u003c/b\u003e), were produced to assess the homotypic specificity of magFSMs in a multi-cellular lung cancer model. Different ratios of A459-GFP and MRC5 cells were tested in order to optimize the reproducibility of the spheroid formation. Three initial ratios of 1:1, 1.5:5 and 1:5 A549-GFP:MCR5 were tested. Ratio 1:5 showed a very poor reproducibility while ratios 1:1 and 1.5:5 showed a similar size of approximately 400 \u0026micro;m (\u003cb\u003eFigure S9 A\u003c/b\u003e). Based on these results, the ratio 1.5:5 was selected for further analysis.\u003c/p\u003e \u003cp\u003eFluorescence imaging of heterotypic spheroids 24 hours after treatment with 10\u003csup\u003e9\u003c/sup\u003e FSM ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e or magFSMs, revealed that the magFSMs distribution within spheroids was similar to that found in FSMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Importantly, the incorporation of the IO@DPMA NPs within the magFSMs did not significantly interfere with their targeting capability in this system. It was clearly observed that the fluorescence signal from Atto647 (FSM) was comparable in both groups, while the Cy3 fluorescence of the IO@DPMA NPs was only present in the magFSMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), and it appears spread within the whole spheroid. When treated spheroids were observed at higher magnification, we could distinguish areas with a clear colocalization of A549-GFP cells (green) and the FSMs and magFSMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), confirming the preferential internalization by cancer cells. These results depicting effective homotypic recognition at 3D cultures were later confirmed by means of flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) where MFI at 24 hours showed an uptake of magFSMs at least 4-fold in A549 cells compared with MCR5.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003edrug delivery and therapy in 2D and 3D cell cultures.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAfter demonstrating the homotypic targeting properties of our systems, we then evaluated their ability to deliver anti-cancer drugs. For this, magFSMs were loaded with two different classical anticancer drugs: doxorubicin (DOX) or carboplatin (CbPt) (Figure S4 E and F, Table S2). These two molecules are both capable of interacting with DNA to induce cell apoptosis. At the molecular level, both are small molecules (MW below 550 g/mol). Meanwhile, DOX is a planar aromatic anthraquinone system with a sugar moiety attached, CbPt is a coordination organometallic platinum complex. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B, flow cytometry assays in 2D monocultures of A549 cells, even for short incubation periods of 10 minutes, the loading of DOX in FSMs or magFSMs (FSM@DOX and magFSM@DOX, respectively) leads to an increased uptake of DOX as compared to the same dose (1 \u0026micro;M) of unencapsulated drug. This enhancement was found to be consistent for higher incubation times. Both FSM@DOX and magFSM@DOX showed a significant 2.6-fold enhancement of doxorubicin delivery in A549 cells after 24 hours.\u003c/p\u003e \u003cp\u003eFurthermore, to take advantage of the homotypic targeting abilities of the FSMs, DOX uptake was studied in 2D co-cultures of A549-GFP and MRC5 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D). Since DOX is a DNA intercalator [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e], its uptake was evaluated and quantified through fluorescence measurements from cell nuclei fluorescence microscopy. From the fluorescent images obtained after incubation with 1\u0026micro;M of DOX equivalents for 24 hours, it can be clearly seen that both FSM@DOX and magFSM@DOX display targeted binding to A549 lung cancer cells (Atto647 fluorescence), and their cell fusion led to the delivery of DOX intracellularly into the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). This is in stark opposition to the non-specific accumulation of free drug in both A549 and MRC5. By quantifying relative fluorescence intensity of DOX in cell nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), it was observed that magFSM@DOX showed a specific drug delivery effect similar to FSM@DOX. For both FSMs, the DOX accumulation was 2-fold for the A549 cells compared with the accumulation in MRC5. This confirms that the incorporation of IO@DPMA NPs did not negatively affect the targeting dynamics of the drug-loaded FSMs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis specific targeting of cancer cells should be translated to increased cytotoxicity of these nanoformulations. Results revealed an enhanced cytotoxic effect for both FSMs at short (10 minutes) and long (24 hours) incubation periods. Specifically, both FSM@DOX and magFSM@DOX exhibited an increased delivery of DOX inside cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) compared with free drug, which was found notably lower over a 24 hours incubation period. Those observations were confirmed in dose-response experiments at both 10 minutes and 24 hours of exposure time (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) where FSM@DOX and magFSM@DOX significantly outperformed free DOX in exerting cytotoxic effects on cancer cells. Similar results were observed when magFSMs were loaded with another chemotherapeutic drug, CbPt, where FSM@CbPt and magFSM@CbPt showed an increased cytotoxic activity, as compared to free CbPt, both for 10 minutes and 24 hours of drug exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and \u003cb\u003eFigure S10\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further test the robustness of drug-loaded magFSMs as cytotoxic agents, their effects in a model of A549 spheroid were evaluated. For that, we take into consideration that the dynamics of its uptake were different as compared to 2D cell cultures, as previously discussed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We are modeling this by selecting the DOX-loaded magFSM as a therapeutic tool. After 24 hours of A549 3D spheroids exposure to magFSM@DOX, we found a significantly higher percentage of dead cells (51.4%) as compared to free drug (10 \u0026micro;M) alone (29.5%), proving the feasibility of magFSMs to efficiently deliver DOX inside a 3D structure where the drug cargo was able to exert their function (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eOnce the feasibility of magFSM@DOX as drug delivery vehicle was assessed, we aimed to combine the features displayed in 2D to a 3D model, by taking into account not only its biological attributes, but also its physico-chemical properties. As explained earlier, 3D heterotypic spheroids used within this work were composed of lung fibroblasts (MRC5 cells) and lung cancer cells (A549-GFP), and these were further used to explore the use of magFSMs as a magnetic-field guided delivery vehicle. Here, the IO@DPMA NPs decorated on the FSM surface provide our vehicle with magnetic properties as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Therefore, we explore the magnetically-driven accumulation of magFSMs within heterotypic spheroids.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and \u003cb\u003eFigure S11\u003c/b\u003e highlight the impact of the use of a magnetic gradient to promote the accumulation of the magFSMs versus the free DOX within a spheroid as observed by fluorescence microscopy studies. There is a noticeable greater penetration for the magFSMs (Atto647) and cargo (DOX) when the magnetic field is used (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u003cb\u003e-II\u003c/b\u003e). This demonstrates that the presence of a magnetic field drove the in-depth penetration of magFSM over and above the inherent ability of FSM to penetrate into the spheroids (\u003cb\u003eFigure S11\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eEven though overcoming physical barriers for tumor penetration is advantageous, drug accumulation must continue to occur preferentially in tumoral cells within the tumor microenvironment. Thus, it is essential that the enhanced accumulation synergistically reinforces homotypic targeting rather than causing nonspecific or uncontrolled NP uptake. To evaluate this, we undertook flow cytometry analysis on heterotypic spheroids to study the DOX accumulation in the different cell types used at different times (\u003cem\u003ei.e.\u003c/em\u003e, 1 hour and 24 hours) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Looking at the DOX accumulation profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB \u003cb\u003eI-II\u003c/b\u003e), for the free DOX we did not observe any increment by the presence of the magnetic gradient in any condition, as expected. This was consistent for both incubation times, 1 hour (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB \u003cb\u003eI\u003c/b\u003e) and 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB \u003cb\u003eII\u003c/b\u003e). Interestingly, no preferential drug accumulation was observed in cancer cells in any condition, with A549 and MRC5 cells internalizing DOX at a comparable rate.\u003c/p\u003e \u003cp\u003eFocusing on magFSMs, we observed that 1 hour after treatment, both in the presence and absence of a magnetic gradient, there was preferential drug accumulation in A549 lung cancer cells compared with MRC5 fibroblasts, consistent with their inherent homotypic targeting abilities. This accumulation was approximately twice as high in A549 cells as in MRC5 cells. Interestingly, this preferential uptake was maintained upon application of the magnetic gradient, which produced an additional\u0026thinsp;~\u0026thinsp;2-fold increase in the accumulation of magFSM@DOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Moreover, comparing the signal of the internalized DOX and that of the internalized FSMs after 1 hour, a clear correlation was observed, confirming that magFSMs effectively delivered the drug.\u003c/p\u003e \u003cp\u003eOver a period of 24 hours, the combined effect of homotypic targeting and magnetic field guidance resulted in a clearly enhanced accumulation of magFSMs within cancer cells in a complex 3D tumor spheroid. This was evidenced by the distribution patterns of the nanocarriers, which closely resembled those of the drugs under magnetic field exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Notably, fluorescence imaging and spatial mapping across the spheroid width revealed that both drug and vehicle penetration were substantially higher when magFSMs were subjected to an external magnetic field (Figure S11). This synergy between the physical and biological properties of magFSMs, enabling simultaneous stimuli responsiveness, imaging, and active targetability within a single platform, highlights their strong potential for future theranostic applications.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eTailoring a personalized therapeutic regimen for cancer therapy is one of the most important and challenging goals for cancer nanomedicine. To achieve this, the materials chosen for nanomedicine design should exhibit biocompatibility as well as targetability to specific cancer sites when administered inside the body. Here, endogenous biomaterials provide a unique opportunity since they inherently possess biocompatibility and bio-recognition capability. When assembled to form nanostructures they form biomimetic cargo delivery vehicles that possess unique advantages as compared to other conventional nanoparticle systems. One of the most widely used biomaterials for the synthesis of biomimetic drug delivery vehicles are phospholipid cell membrane components. We have previously synthesized biomimetic fusogenic nanovesicles using cancer cell membranes in our group [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e], which unlike other reported biomimetic nanostructures demonstrates direct cytoplasmic delivery of cargo inside cells [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The ability to directly fuse with cell membranes to deliver cargo is highly advantageous as it circumvents the possibility of lysosomal degradation of cargo components. In this report, we add an additional approach to this repertoire by demonstrating the conjugation of stimuli-responsive inorganic IO NPs to the surface of cell membrane nanovesicles using a robust click-chemistry approach. Unlike other widely reported methods of cargo loading using energy intensive techniques (such as extrusion, sonication, electroporation, \u003cem\u003eetc\u003c/em\u003e.), this method of functionalizing biomimetic vesicles demonstrated here is simple and straightforward that could easily be scaled up and could theoretically be applied to a wide variety of cell membrane sources and NPs/cargo. This combined approach, integrating cancer cell membrane-derived recognition molecules, fusogenic membrane components, and magnetic IO NPs within a single platform, renders the resulting nanostructure (magFSMs) unique, as it enables active targeting further enhanced by magnetic field-assisted delivery, while promoting direct cytosolic delivery of the cargo. With this development, we demonstrate the synthesis of magFSMs, their magnetic properties, and homotypic recognition capabilities, as well as stimuli-responsive active targeting under an external magnetic field. The robustness of this system was extensively validated across multiple in vitro 2D and 3D homo- and heterotypic tumor models.\u003c/p\u003e \u003cp\u003eThe simple copper-free click-conjugation employed here was facilitated by utilizing a DBCO functional group on the IO NP and an azide-labeled sugar incorporated nanovesicle leading to the generation of magFSMs of ~\u0026thinsp;200 nm diameter. One of the main advantages of functionalizing a biomimetic nanovesicle on its surface is that it makes possible the loading of drugs/cargo separately into its empty lumen, thus maximizing the available volume per NP for drug delivery applications. Importantly, the labeling of FSMs with IO NPs was not found to significantly change the physicochemical properties of the developed biomimetic nanosystem but rather led to the combination of their individual properties. When imaged in MRI, magFSMs depicted good T2-contrast enhancement effects and a correspondingly high relaxivity of 164.8 mM\u003csup\u003e\u0026minus;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, implying that when used \u003cem\u003ein vivo\u003c/em\u003e, their accumulation at a diseased site should be easily detectable. This is especially important when the homotypic targeting capability of magFSMs is needed, since they would actively recognize and bind to cancer cells, thus opening up the possibility of imaging tumor location using MRI. Additionally, this particular configuration of decorating IO NPs on cell-derived NPs was found to be advantageous as no significant changes in the SAR values of magFSMs were observed as compared to free IO NPs. In theory, this would imply that the magFSMs could also be used for magnetic hyperthermia, and the exposure of such a system to an alternating magnetic field would lead to an on-demand drug release. In the future, the use of this system for magnetic hyperthermia induced cancer therapy is envisioned. Moreover, the robustness of this system also implies that other small sized stimuli-responsive NPs (such as gold nanorods, quantum dots etc.) could be covalently conjugated on a biomimetic NPs without significantly altering its biological properties.\u003c/p\u003e \u003cp\u003eOf particular importance in this study is the ability of magFSMs to bind specifically to A549 cancer cells, the parent cells from which the cell membrane phospholipids were isolated for preparing FSMs. Since the coupling of IO NPs to FSM surface could spatially hinder the functioning of homotypic recognition receptors on the nanocarrier, an optimum ratio of IO NP:FSM was determined (10:1) that would provide adequate vehicle as well as cargo uptake in A549 cancer cells. Thus, while utilizing a conjugating technique to load cargo on FSM surface is a simple process, the amount of cargo that can be loaded is overall limited through this route, as higher surface-loaded cargo can limit the normal biological function of the FSMs.\u003c/p\u003e \u003cp\u003eWhen tested \u003cem\u003ein vitro\u003c/em\u003e in 2D heterotypic co-cultures of A549 and MRC5 cells, magFSMs were found to preferentially bind to A549 cells as compared to MRC5 fibroblast cells. This ability to recognize and preferentially bind to the parent cell from which the nanocarriers were derived has been widely reported in literature [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. However, within this work, we demonstrated that FSMs homotypic recognition capability could be exploited in conjunction with additional non-native properties by optimizing the surface conjugation of IONPs onto FSMs. Moreover, this beneficial property of magFSM continued to be retained when tested in a more complex 3D environment of tumor spheroids comprising of A549 and MRC5 cells thus showing its robustness and capability to penetrate deep into a tumor microenvironment.\u003c/p\u003e \u003cp\u003eWhen utilized for the delivery of DOX which are loaded as an additional cargo into its lumen (unlike IO NPs conjugated on FSM surface), magFSM@DOX showed preferential binding and drug accumulation in A549 cancer cells. Importantly, it was observed that in a 2D culture, magFSMs were able to deliver DOX to A549 cancer cells much more rapidly within a shorter incubation time of 10 minutes, that was significantly faster than the use of free drugs. This shows that homotypic targeting using biomimetic FSMs actively drives the targeting of drugs into cancer cells, unlike in the case of passive uptake. This is especially useful when used \u003cem\u003ein vivo\u003c/em\u003e, where a constantly changing dynamic environment is encountered, within which biochemically driven active targeting processes would show more efficacy for drug delivery over passive targeting strategies. Furthermore, over a period of 24 hours, magFSMs also enabled a\u0026thinsp;\u0026gt;\u0026thinsp;2-fold drug accumulation in A549 cells. This is especially relevant as indiscriminate cytotoxicity, once administered in clinics, is a major challenge for many of the drugs currently used for anti-cancer therapy. The feasibility of this system to be adaptable to different drugs was also demonstrated with the loading of CbPt. Upon evaluation in 3D tumor spheroids, even though a higher concentration of doxorubicin was consequently needed as compared to 2D culture, magFSM@DOX treatment led to a significantly greater loss in tumor cell viability as compared to the use of free drugs, thus highlighting its potential as a robust drug delivery system even in a complex tumor environment.\u003c/p\u003e \u003cp\u003eEven though magFSMs demonstrated excellent homotypic targeting ability, good biological stability, improved drug-delivery capability, and robust activity in a complex tumor microenvironment, the most significant property of magFSMs is their ability to respond to an external magnetic field stimulus. This was envisaged due to the excellent magnetic properties demonstrated by magFSMs owing to the covalent binding of IO NPs on their surface. As a proof of concept, the stimuli-responsive capability of magFSMs for targeted drug delivery under an external magnetic field was evaluated in heterotypic spheroids, wherein its capability to specifically deliver drugs actively to cancer cells was found to increase many-fold further as compared to conditions where no external magnetic field was applied. Within a clinical setting, the use of a non-invasive, highly penetrating magnetic field to direct drug-loaded nanovehicles to the tumor site could be highly advantageous.\u003c/p\u003e \u003cp\u003eThus, in this report, we show for the first time the use of a simple click-chemistry approach for surface loading stimuli-responsive IO NPs on the surface of fusogenic biomimetic nanocarriers, that can be used for combined homotypic and magnetic targeting for enhanced and specific drug delivery. For future studies, we envisage the use of such systems for magnetic hyperthermia or photothermal drug delivery.\u003c/p\u003e \u003cp\u003eWhile the present study demonstrates the robust performance of magFSMs across advanced \u003cem\u003ein vitro\u003c/em\u003e mono- and heterotypic 3D tumor models, further investigations will be required to evaluate their \u003cem\u003ein vivo\u003c/em\u003e biodistribution, pharmacokinetics, and long-term safety. Additionally, the scalability and standardization of cell membrane-derived nanocarriers remain crucial aspects for future translational development. Nevertheless, the modularity of the click-chemistry approach described here provides a flexible framework to adapt this platform to different biological membranes, inorganic nanocomponents, and therapeutic cargos.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we developed a new hybrid bio-mimetic drug-delivery system, termed magFSMs, consisting of fusogenic cell-membrane derived nanovesicles covalently coupled to magnetic-field responsive IO NPs. Unlike other hybrid biomimetic systems this structure was unique because the IO NPs were covalently conjugated on the surface of the biomimetic nanovesicle through click-chemistry. The magFSMs successfully integrate the advantageous properties of both components combined in a single platform by exhibiting (a) preferential homotypic targeting, enabling selective recognition of cancer cells, and (b) stimuli responsiveness to an external magnetic field, enabling remote manipulation capability. Apart from exhibiting good biocompatibility, magFSMs not only showed enhanced uptake and good targeting capability in 2D co-culture systems, but also in a complex 3D heterotypic tumor spheroid environment, wherein it preferentially associated to cancer cells. When loaded with an anti-cancer therapeutic payload, specific drug accumulation was observed in cancer cells in both 2D and 3D cell culture environments, and this effect was further amplified by the use of an external magnetic field owing to its stimuli-responsive behavior. This study demonstrates an efficient and simple strategy for generating theranostic organic-inorganic hybrid NPs, and opens the possibility for the rational development of customizable biomimetic targeted drug delivery systems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors thank the financial support of the European Research Council (starting grant #950421), the European Commission for the HORIZON-MSCA-2021-PF (#101063372), the MICIU/AEI/\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.13039/501100011033\u003c/span\u003e\u003cspan address=\"10.13039/501100011033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (PID2023-152844NB-I00; PID2022-142338OB-100, PID2020-119206RB-I00, PID2023-151448NB-I00, CNS2023-144318), and the Xunta de Galicia (#ED431C 2022/18, #ED431B2023/19, #2021-CP090 and Centro de Investigaci\u0026oacute;n do Sistema Universitario de Galicia accreditation 2023\u0026ndash;2027 #ED431G 2023/03). R. Iglesias-Rey (CP22/00061) from the Miguel Servet Program of ISCIII and Co-financed by the EU.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.R-P., S.F. and M.M. conducted the experiments. R. I-R conducted the MRI experiments. E.P, P.d.P and B.P designed the experiments and secured the funding necessary for this work.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBarenholz Y. Doxil(R)--the first FDA-approved nano-drug: lessons learned. J Control Rel. 2012;160:117\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStone NR, Bicanic T, Salim R, Hope W, Liposomal Amphotericin B. (AmBisome((R))): A Review of the Pharmacokinetics, Pharmacodynamics, Clinical Experience and Future Directions. 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Nat Commun. 2021;12:5726.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme ","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"biomimetic nanocarriers, cell membrane-derived nanoparticles, nano-bio interfaces, fusogenic nanovesicles, magnetic targeting, intracellular drug delivery, stimuli-responsive nanomedicine","lastPublishedDoi":"10.21203/rs.3.rs-8712225/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8712225/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCell membrane-derived nanoparticles (NP) have emerged as a transformative platform in nanomedicine, offering unique advantages for therapeutic delivery and immune modulation. By harnessing the native biological properties of source cells such as red blood cells, platelets, and cancer cells, these biomimetic NP exhibit prolonged circulation, enhanced biocompatibility, and specific targeting capabilities. Membranes retain functional proteins and receptors, enabling precise interfacing with biological environments and providing inherent ability to evade immune detection and to interact with target tissues. Hybrid and engineered membrane platforms that integrate components of different origins (e.g. cell-derived and inorganic NP) hold strong potential for next-generation targeted therapies in oncology, as they can synergistically combine the advantages of both biological and inorganic components. Therefore, we proposed a new NP design based on biomimetic fusogenic nanomembranes decorated through click chemistry with iron oxide (IO) NPs for enhanced homotypic targeting and chemotherapeutic drug delivery under magnetic guidance in different \u003cem\u003ein vitro\u003c/em\u003e models of non-small cell lung cancer.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur findings present an efficient preparation of a hybrid nanoplatform (magFSMs), based on nanomembranes and IO NPs coupled through strain-promoted azide-alkyne click chemistry (SPAAC) reaction. Our system preserved the magnetic properties of IO NPs as well as the homotypic targeting capabilities of the parental cancer cell membranes. This was evaluated both in adherent cell mono and co-cultures as well as in 3D heterotypic spheroids where magFSMs exhibited preferential recognition for tumoral cells. Our nanoplatform also showed versatile drug-loading capacity, as proved by the incorporation of different small anticancer molecules like carboplatin and doxorubicin, leading to enhanced antitumor activity compared with free drugs. Moreover, those observations were also preserved and even increased in lung cancer spheroids under magnetic guidance, underscoring the feasibility of using magFSMs as magnetically guided drug-delivery nanocarriers.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eMagFSMs prepared within this work has been successfully employed as anticancer nanosystem in several \u003cem\u003ein vitro\u003c/em\u003e models of lung cancer, representing a promising tool in personalized and targeted nanomedicine. Our results support the potential of magFSMs as a modular, biomimetic, and magnetically responsive drug-delivery platform, encouraging us to further explore their applicability in additional disease models and to expand their evaluation toward more complex \u003cem\u003ein vivo\u003c/em\u003e scenarios.\u003c/p\u003e","manuscriptTitle":"Click-Engineered Magnetic Fusogenic Cell-Derived Nanocarriers for Enhanced Magnetic-Field-Assisted Drug Delivery","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-04 19:04:39","doi":"10.21203/rs.3.rs-8712225/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d92900b3-90fb-4f34-af41-5e91c9c586e4","owner":[],"postedDate":"February 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T07:26:11+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-04 19:04:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8712225","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8712225","identity":"rs-8712225","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}