Colloids and Surfaces B: Biointerfaces 208 (2021) 112123 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb Tolerability to non-endosomal, micron-scale cell penetration probed with magnetic particles Eugènia Ruiz-Cánovas a, 1, Rosa Mendoza b, a, Antonio Villaverde a, c, b, José L. Corchero b, a, c, * a Institute for Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain b CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, 08193 Barcelona, Spain c Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain A R T I C L E I N F O A B S T R A C T Keywords: The capability of HeLa cells to internalize large spherical microparticles has been evaluated by using inorganic, Magnetic particles magnetic microparticles of 1 and 2.8 µm of diameter. In both absence but especially under the action of a magnet, Cell penetration both types of particles were uptaken, in absence of cytotoxicity, by a significant percentage of cells, in a non- Cellular therapy endosomal process clearly favored by the magnetic field. The engulfed particles efficiently drive inside the Recombinant protein Human alpha-galactosidase A cells chemically associated proteins such as GFP and human alpha-galactosidase A, without any apparent loss of protein functionalities. While 1 µm particles are completely engulfed, at least a fraction of 2.8 µm particles remain embedded into the cell membrane, with only a fraction of their surface in cytoplasmic contact. The detected tolerance to endosomal-independent cell penetration of microscale objects is not then restricted to organic, soft materials (such as bacterial inclusion bodies) as previously described, but it is a more general phenomenon also applicable to inorganic materials. In this scenario, the use of magnetic particles in combination with external magnetic fields can represent a significant improvement in the internalization efficiency of such agents optimized as drug carriers. This fact offers a wide potential in the design and engineering of novel par­ ticulate vehicles for therapeutic, diagnostic and theragnostic applications. 1. Introduction using magnetic fields. Moreover, after reaching their targets, MPs exposure to a high frequency alternating magnetic field (AMF) results in Intracellular drug delivery is on the basis of precision medicines. a localized temperature increase in target cells (hyperthermia) by MPs Capability to drive the controlled internalization in target cells of ma­ activation, inducing localized cell death. Such properties have shown a terials with therapeutic potential has been explored by a wide spectrum significant therapeutic value in, for instance, cancer therapy. Other of nanomedical approaches [1]. The use of cell-penetrating peptides for promising biomedical applications for MPs include magnetically a fast and efficient internalization or of ligands of cell surface molecules enhanced transfection (magnetofection), biomolecules purification, acting as selective receptors [2] is accompanied by the development of gene therapy, or tissue engineering, among others [6,7]. appropriate nanocarriers for payload drugs and/or imaging agents [3]. Once at the target location, DDS face cellular uptake, an essential In a prototypic nanoconjugate, a nanoscale vehicle is functionalized event required to deliver their therapeutic cargo. Apart from other with such peptides and chemically coupled or loaded with the cargo [4, relevant physicochemical parameters, there is a consensus in the fact 5]. that size determines both nanovehicle fate and its cell uptake. Even Multiple materials and vehicle types are being explored as drug de­ within the nano-scale range, particle size strongly affects bioavailability livery systems (DDS), as well as different sizes, geometries and surface and blood circulation time [8,9]. Following systemic administration, charges of the final conjugates. Among them, nano- and micro- particles nanoparticles with diameters below the renal cut-off (approx. 7–8 nm) with magnetic properties (MPs) are increasingly being used in a wide are rapidly eliminated through extravasation and renal clearance [10]. spectrum of biomedical applications. After functionalization to enable A nanoscale size over 10 nm prevents renal clearance and also benefits specificity, MPs can be conveniently positioned both in vitro and in vivo from the enhanced permeability and retention (EPR) effect, if the drug is * Corresponding author at: Institute for Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain. E-mail address: jlcorchero@ciber-bbn.es (J.L. Corchero). 1 Present address: GAT Therapeutics SL, Parc Científic de Barcelona, C/ Baldiri Reixac 4–8, 08028 Barcelona, Spain. https://doi.org/10.1016/j.colsurfb.2021.112123 Received 5 February 2021; Received in revised form 11 September 2021; Accepted 16 September 2021 Available online 20 September 2021 0927-7765/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). E. Ruiz-Cánovas et al. Colloids and Surfaces B: Biointerfaces 208 (2021) 112123 intended for oncology. Usually, optimal sizes for cell penetration have four of cobalt’s six coordination sites are occupied. The imidazole rings been determined to be between 20 and 80 nm, with some variability of histidine residues present in a 6xHis tag are able to occupy the two depending on the experimental setting up. Particles with such size remaining coordination sites, resulting in protein binding. ranges can penetrate even very small capillaries [11] and ensure smooth Magnetic particles were magnetically captured either with a mag­ endosomal-mediated cell penetration as well as the maintenance of cell netic rack (New England Biolabs, rack S1506S) or with a precision integrity and viability during the process. Particles with diameters magnetophoresis system from Sepmag Systems (STEPMAG 125). ranging from 70 to 200 nm demonstrate the most prolonged circulation times [8], while particles with diameters greater than 200 nm are usu­ 2.2. Recombinant proteins used as “cargo” attached to magnetic particles ally sequestered by the spleen and eventually removed by phagocytes [12]. Two recombinant proteins (with either fluorescent or enzymatic Interaction of nanoparticles with the cell membrane plays a key role properties) were coated onto the different magnetic particles and used as in the internalization mechanics [13–16], which consequently impacts a cargo to check the ability of these magnetic vehicles to deliver a in the efficiency of the process or application, such as biomedical im­ protein to a target cell. A 6xHistidine-tagged recombinant green fluo­ aging and drug delivery. Two important steps of these processes are the rescence protein (GFP-H6) was produced in Rosetta BL21(DE3) Escher­ adhesion of the particles to the membrane and their subsequent ichia coli as previously described [34]. As a second model protein, a engulfment. Recently, we have demonstrated that a special type of 6xHistidine-tagged human alpha-galactosidase A (GLA) was chosen. proteinaceous material, namely bacterial inclusion bodies (IBs), can The enzymatic activity of GLA enzyme can be easily determined by in penetrate mammalian cells in absence of toxicity [17–24]. Being a soft vitro assays and, in addition, it has therapeutic value, functioning here matter, IBs are mechanically stable protein clusters that, in form of as a model of a protein drug. Its deficiency causes Fabry disease, a rare functional non-toxic amyloids, show a size range between 0.5 and 1 µm. pathology currently treated by periodic administration of the recombi­ The penetration of bacterial IBs into mammalian cells seems to be nant enzyme. Recombinant GLA used here was produced by favored by a strong membrane affinity shown by such protein materials, PEI-mediated transient gene expression in mammalian HEK 293F cells but it can be additionally targeted to specific cell receptors by incor­ as previously described [35]. Both GFP and GLA proteins were purified porating appropriate ligands of cell surface receptors [23,25–27]. The by affinity chromatography using HiTrap Chelating HP columns (GE same penetrability in absence of toxicity has been very recently Healthcare) following the manufacturer instructions. In some experi­ demonstrated for artificial IBs, IB mimetics generated in vitro but ments, a commercial bovine serum albumin (BSA, bovine serum albu­ showing a diameter ranging between 2 and 3 µm [28–30]. When either min fraction V, Roche, ref. 001,10,078,735) was used as a negative IBs or artificial IBs contain a protein with therapeutic value, target cells control (protein without fluorescence or enzymatic activity). benefit from such biological activity, demonstrating, in a functional way, the efficient internalization of the material [31–33]. 2.3. Conjugation of proteins to magnetic particles Irrespective of the mechanisms that allow cell penetration of micron- scale materials, still not completely solved, this possibility is promising, Tosylactivated (M-280 and MyOne) particles were coated as previ­ as it would largely increase the drug delivery potential of particulate ously described [36]. Briefly, particles to be GLA-coated (10 mg unless systems. However, it is not clear if the cell penetrability described for IBs otherwise stated) were washed twice with coating/storage buffer (0.01 is linked to their soft matter nature, or to other particular properties such M acetic acid, pH 4.5) and resuspended in the same buffer with 200 as membrane avidity. Therefore, a better understanding of the mecha­ µg/ml GLA (20 µg GLA/mg particles). After conjugation (overnight, in nisms involved in the cell uptake processes could help in the future to agitation at 25 rpm, 37ºC), particles were magnetically captured, and increase the delivery efficiency of therapeutic compounds by micron the supernatant removed. Coated particles were then washed for 15 min particles. In this context, we have tested here the tolerance of cultured with phosphate buffered saline (PBS) plus Tween-20 0.05% (in order to cells to the penetration of large microparticles, by using a hard, inor­ remove only-adsorbed, non-covalently attached protein), for 15 min ganic material system based on magnetic particles. The obtained data with 0.2 M Tris-HCl pH = 8 (to block the remaining free tosyl groups), reveal a cell tolerability higher than expected regarding the size of and twice with PBS. Finally, they were diluted in coating/storage buffer particulate materials than cells can uptake, in absence of any detectable to 1.5 mg MPs/ml and kept at 2–8 ºC. For GFP-His and BSA coating, cytotoxicity. The results are discussed under the light of potential novel protocol was essentially the same, but using PBS instead of acetic acid as applications of these new concepts in drug delivery and imaging. coating/storage buffer. On the other hand, TALON MPs were coated with GLA enzyme as follows. After washing with PBS, particles were 2. Material and methods diluted in PBS with 200 µg/ml of protein (20 µg protein/mg particles). After protein capture (in agitation at room temperature for 30 min, as 2.1. Magnetic particles (MPs) recommended by the supplier), supernatant was removed. MPs were washed 4 times with PBS, diluted in PBS to 1.5 mg MPs/ml and stored at Commercial Dynabeads® MyOne™ Tosylactivated (ref. 655.01, 1 2–8 ºC. µm diameter), M-280 Tosylactivated (ref. 301.01, 2.8 µm diameter) and Dynabeads® TALON™ (ref. 101.01, 1 µm diameter) were obtained from 2.4. Cell culture, magnetic-driven internalization studies and particles Dynal® (Invitrogen). Tosylactivated particles are polystyrene beads cytotoxicity coated with a polyurethane layer. The hydroxy groups are activated by reaction with p-toluensulphonyl chloride. The resulting sulphonyl ester HeLa cells (human cervical carcinoma, from the American Type can react covalently with the amino groups present in the cargo protein. Culture Collection, reference CCL-2) were grown as monolayers in Thus, any ligand (e.g. antibody, protein, peptide or glycoprotein) con­ Dulbecco’s modified Eagles’s medium (DMEM) with 50 units/ml peni­ taining amino groups can be covalently coupled to the beads surface. cillin, 50 μg/ml streptomycin, and supplemented with 10% fetal bovine Tosylactivated MyOne and M-280 magnetic particles do not need serum (FBS). Cell cultures were performed in a 5% CO2 humidified at­ additional activation prior to protein immobilization procedures, and mosphere at 37ºC. their handling and protein coupling was done according to the manu­ For internalization experiments, cells were seeded into two 24-well facturer instructions. Dynabeads® TALON™ MPs use cobalt-based plates at an initial density of 50,000 cells/well. Cells exposure to mag­ Immobilized Metal Affinity Chromatography (IMAC) using magnetic netic particles was performed 24 h after plating, at approximately beads on which the BD TALON™ chemistry has been immobilized. The 60–70% cell confluence. Before exposure to magnetic particles, cell TALON technology is based on a tetradentate metal chelator in which culture supernatants were removed and replaced by 500 µL of fresh 2 E. Ruiz-Cánovas et al. Colloids and Surfaces B: Biointerfaces 208 (2021) 112123 medium. One plate was kept as a “Control” and the other (“Magnet the final trypsinization step. Then, and before confocal observation, cell plate”) was placed over a BioMag® Flask Separator (Bangs Laboratories, nuclei were labeled with 5 μg/ml Hoechst 33342 (Life Technologies) Inc., ref. MS004). The BioMag® Flask Separator is a 12.5 cm × 6 cm and plasma membranes with 2.5 μg/ml CellMask™ Deep Red (Life rectangular magnetic separation unit designed for use with tissue cul­ Technologies) for 10 min in the dark. Micrographs were then taken by ture flasks or plates. The unit consists of 24 permanent magnets encased TCS-SP5 confocal laser scanning microscopy (Leica Microsystems) using in a plastic frame. The magnetic strength for each disc magnet is 27 a Plan Apo 63 × /1.4 (oil HC × PL APO lambda blue) objective. Z-se­ megagauss Oersteds. Then, cells and magnetic particles were incubated ries were collected at 0.5 mm intervals, and images processed using together (at room temperature) in both “Control” and “Magnet” plates, Imaris version 6.1.0 software (Bitplane, Zürich, Switzerland). Z-stack by adding at different time-points 25 µL of protein-coated MPs to the function was applied. Multiple images were taken along the z-direction corresponding wells. After incubation, magnet was removed, and su­ to study the cells in depth. The in-depth imaging allowed the cells and pernatants from both plates were gently aspirated. Cells were washed MPs interactions to be visualized in 3-dimensions. This was used to twice with 500 µL of cold PBS, and trypsinized with 500 µL of trypsin figure out whether the MPs were completely internalized, just next to 0.05%, during 10 min at 37ºC. Then, cells were transferred to micro­ the cell or membrane embedded. centrifuge tubes, centrifuged at 1500 rpm for 10 min, supernatant was discarded, and cells were resuspended in 1 ml of PBS. After that, per­ 2.6. Enzymatic assay of GLA activity centage of fluorescent cells was determined by flow cytometry (FACS­ Calibur, BD Biosciences, San José, CA). This protocol is schematically Enzymatic GLA activity was assayed fluorometrically as described by outlined in Fig. 1. In the case of internalization of GLA-coated particles, Desnick et al. [37] with the modifications of Mayes et al. [38] Basically, protocol was essentially the same, but with the following modifications: enzymatic GLA activity was assayed with 4-methylumbelliferyl after internalization and washing, cells were resuspended in 500 µL of α-D-galactoside (4-MUG, ref. M7633, Sigma Chemical) as substrate, at distilled H2O, gently sonicated, and lysates were directly submitted to a concentration of 2.46 mM in 0,01 M acetic buffer (pH 4.5). A typical the GLA enzymatic assay. assay was performed with a reaction mixture containing 100 µL of For cytotoxicity studies, protein-coated MPs were added to cells as substrate and 25 µL of cells lysates. Enzymatic reactions took place in previously described. Internalization of MPs was magnetically driven by agitation, at 37ºC for 1 h, followed by their termination by addition of 10 min incubation with the magnet. Then, cells were washed, and fresh 1.25 ml of 0.2 M glycine-NaOH buffer (pH 10.4). The released 4-meth­ medium was added. After 24 h of incubation, cell viability was esti­ ylumbelliferone (4-MU) was determined by fluorescence measurement mated with the EZ4U Cell Proliferation and Cytotoxicity Assay (Bio­ at 365 and 450 nm as excitation and emission wavelengths, respectively. medica, ref. BI-5000), a cell proliferation and cytotoxicity assay based Samples ranging from 5 to 500 ng/ml of 4-MU (ref. M1381, Sigma, St. on the reduction of tetrazolium salt to colored formazan. Louis, MO) in 0.2 M glycine-NaOH buffer (pH 10.4), were used to cali­ brate the readings. Results are expressed as ng 4-MU/ml. 2.5. Confocal microscopy analysis 2.7. Statistical analysis For confocal analysis, cells were grown to 100,000 cells/ml on MatTek culture dishes (MatTek Corporation) for 24 h at 37 ◦ C at 5% The quantitative data of the experiments is reported as mean­ CO2. Incubation with MPs was done as previously described, but without ± standard error of the mean (SEM). Pairwise comparisons between Fig. 1. General strategy to deliver MP-coated proteins into cells. A: MPs are coated with a protein (e.g. GFP) as described in Materials and Methods. B: Coated MPs are added to the wells and forced to enter into HeLa cells by exposure (during different times) to magnetic force, by contact with magnets placed under the cell culture plate. C: After magnetic exposure, cells are washed, trypsinized and recovered. By flow cytometry, the percentage of fluorescent cells (due to previous uptake of fluorescent MPs) is determined. 3 E. Ruiz-Cánovas et al. Colloids and Surfaces B: Biointerfaces 208 (2021) 112123 groups were determined by paired t-test with Excel software (Microsoft of the materials in presence of a constant unidirectional magnetic field, Corporation). Probability values (p) < 0.05 were regarded as statisti­ which might also remove the particles from cells. The fact that the cally significant, and relevant divergences were labeled as *p < 0.05, fluorescent fading is softer in absence of the magnet fully supports this **p < 0.01 and ***p < 0.001. hypothesis, that we would favor over an intracellular degradation of the cargo protein once inside the cells, that is presumed to require longer 3. Results and discussion times. To assess if the fluorescence emitted by cells is due to a real inter­ Initially, internalization of GFP-coated MPs of two different di­ nalization of MPs instead of a mere cell surface anchorage, confocal ameters (1 and 2.8 µm) in cultured HeLa cells was checked, using a microscopy images of cells exposed to both types of MPs for 10 min were magnet to force MPs to contact and penetrate into the cells. Initially, obtained (Fig. 3). GFP was covalently attached to MPs by the tosyl groups present in MPs Most of the smaller MyOne-GFP particles were detected inside the surface and was used as a convenient reporter of internalization but also cells, clearly separated from cell membrane (left panels). The uptake was as a model of a potentially attached drug, imaging or any other func­ confirmed by 3D reconstructions (left, upper panel) and the lateral tional agent, to evaluate its stability during the process. projections (left, lower panel), that rendered images compatible with the In absence of a magnetic force, 1 µm nanoparticles are able to full uptake of the material. On the other hand, M280-GFP particles (right spontaneously penetrate within a significant fraction of cultured cells, panels) were mainly found on the cell surface, embedded in the cell resulting in ~10% of fluorescent cells after 30 min of exposure (Fig. 2, membrane but not being fully engulfed. Lateral projections revealed a left panel). A smaller, but still detectable fraction of cells becomes fraction of the particles remaining exposed to the media and other areas fluorescent when exposed to 2.8 µm particles (Fig. 2, right panel). In this partially internalized and facing the cell cytoplasm. In this respect, it has case, the bigger particle size clearly hampers their spontaneous inter­ been reported that a greater cellular association of paclitaxel into 4T1, nalization without the external aid of the magnetic force. Interestingly, Caco-2, and Cor-L23/R cells was achieved when delivered in micro­ cell exposure to 1-micron particles in absence of magnetic force for more particles compared to nanoparticles [40]. There, authors hypothesized than 30 min does not result in better percentage of internalization, but it that larger microparticles adhere to mucus on the cell surface, enhancing results in a slight decrease in the final percentage of fluorescent cells. the cellular association of paclitaxel. In a more recent study, further This effect is not evident for 2.8 µm MPs, most probably due to the very experiments supported the hypothesis that microparticles adhere to, and low levels of internalization observed under these experimental are retained in the mucus on the cell surface, and are not internalized by conditions. clathrin-mediated endocytosis or macropinocytosis [41]. This fact un­ On the other hand, when a magnetic field is applied, MPs are forced expectedly indicates that the cell penetration previously described for to enter cells, reaching a peak of fluorescent cell population after 5 or soft protein materials, as bacterial IBs or their biomimetic particles, is 10 min of exposure to 1 µm and 2.8 µm MPs, respectively. In this line, not restricted to soft materials, but it seems to be a general event. and for both MPs types, percentages of fluorescent cells with or without Interestingly, these visualizations are compatible with the observation the magnetic field are statistically different, corroborating the positive of HeLa cells exposed to GFP IBs that were also, in some cases, partially effect of the magnetic field in MPs internalization. Moreover, the internalized and tightly attached to the cell membrane [22]. The process magnetically mediated penetration of 1 µm nanoparticles is faster and by which bacterial IBs interact with mammalian cells has been recently slightly more efficient than those of larger ones, as well as their apparent explored. Such process starts with an early and tight cell membrane stability or permanence inside cells. In a previous study, barium sul­ anchorage of IBs, followed by cellular uptake of single or grouped IBs of phate nanoparticles with three different sizes (40 nm, 420 nm, and variable sizes by micropinocytosis. Despite the high affinity of IBs for 1 mm) were all efficiently taken up by bone marrow-derived phagocy­ cell membranes, the first completely internalized particles were not tosing cells (bmPCs) within a few hours. The amount of uptaken nano­ observed by TEM until 3 h after their addition to cell cultures. At longer particles increased with increasing particle concentration in the medium incubation times, a significant number of IBs could be detected and with increasing incubation time [39]. In contrast, using magnetic completely inside the cells in periods of time significantly shorter than materials as DDS, in combination with an appropriate magnetic force, those described in previous studies [19,21,22,32]. could greatly decrease the time needed to interact and enter target cells. In the context of such deep interaction but partial internalization This shows a clear advantage of magnetic vehicles over other types of observed for large particles, we decided to evaluate the availability to DDS. The decreasing fraction of cells with intracellular material, exposed cells of a functional protein (other than GFP) linked to the observed when increasing the magnetic exposure longer than magnetic, inorganic materials. For that, a recombinant human alpha- 10–15 min, is probably indicative of a transient intracellular lastingness galactosidase A (GLA, a lysosomal enzyme with therapeutic uses in Fig. 2. Effect of MPs diameter in their magnetically mediated entrance within HeLa cells. Left: MyOne particles (1 µm diameter). Right: M280 particles (2.8 µm diameter). Percentages of fluorescent cells after different incubation times in the absence (blue bars) or presence (green bars) of magnet are shown. Values are given as mean and SEM of duplicates for each condition and time tested, performed in 3 independent experiments. 4 E. Ruiz-Cánovas et al. Colloids and Surfaces B: Biointerfaces 208 (2021) 112123 Fig. 3. Internalization of GFP-coated MPs in HeLa cells, by confocal microscopy. Left panels: MyOne-GFP particles. Right panels: M280-GFP particles. Bar: 5 µm. enzyme replacement therapy) [42], was selected as payload. Fabry chemistries to coat GLA enzyme onto different TALON and tosyl-based disease is an X-linked disease characterized by the accumulation of MPs, and the effects of such immobilization process in both structural globotriaosylceramide (Gb3) and other glycosphingolipids in cellular and enzymatic GLA parameters [36]. GLA enzyme could be successfully lysosomes, caused by a deficient activity of the GLA enzyme. Enzyme immobilized by means of alternative coupling chemistries (covalent and replacement therapy (ERT) was the first disease-specific treatment for metal affinity adsorption), and results showed that such immobilized Fabry patients, providing a clear therapeutic benefit [43]. Nowadays, GLA had higher specific activity and enhanced stability than its soluble recombinant GLA-based ERT remains the main treatment for most Fabry counterpart, as well as the possibility of reusability due to the immo­ patients. Moreover, GLA enzyme might have interesting applications in bilization process. TALON chemistry seemed to provide better results in other fields, like in the food industry. There are several reports on the terms of better GLA specific enzymatic activity, suggesting a clear in­ use of GLA from plants [44], bacteria [45], yeast [46] and fungal sources fluence of the chemistry used for immobilization on the final structure [47] for the removal of raffinose family sugars (major factors respon­ and performance of the enzyme. GLA enzyme is mainly attached to the sible for flatulence due to soybean and legumes ingestion). GLA is also TALON MPs surface by its C-terminal hexahistidine tag, rendering a known to remove galactose moieties from guar (Cyamopsis tetragono­ more homogeneous, well-ordered disposition of the protein onto the loba) gum, improving its gelling properties, and GLA-modified gal­ surface. On the contrary, for tosyl-activated MPs (MyOne and M-280), actomannan has been used to improve the gelling properties of some any amino group (either at the N-terminus or in the lateral chains of any polysaccharides [48]. In a more biomedical field, GLA has shown a very solvent-exposed amino acid residue) exposed at the protein surface can appealing application in blood transfusion by removing the immuno­ be theoretically covalently coupled to the tosyl groups present in MPs. dominant α-1,3-linked galactose residues from group B red blood cells This means that GLA can be covalently attached to the surface by several [49–51]. In this context, enzymatic conversion of group B red blood cells different amino groups present at its surface, therefore acquiring using purified or recombinant coffee bean GLA has already been ach­ different conformations (some of them being more favorable than others ieved [52,53], eventhough the high amounts of enzyme needed consti­ to the enzymatic reaction). These two strategies of GLA coupling are tuted an economic obstacle for routine use in blood transfusions. outlined in Fig. 4. Moreover, in a previous work we explored different procedures and Improvement of key characteristics like specific activity and/or 5 E. Ruiz-Cánovas et al. Colloids and Surfaces B: Biointerfaces 208 (2021) 112123 Fig. 4. Influence of coupling strategies in the final disposition of GLA molecules onto MPs surface. The TALON technology (left side) is based on a tetradentate metal chelator in which four of cobalt’s six coordination sites are occupied. The imidazole rings of histidine residues present in the His-tag occupy the two remaining coordination sites, resulting in protein binding. Thus, GLA enzyme is mainly coupled to TALON MPs surface by its C-terminal His-tag, rendering a more homoge­ neous, well-ordered disposition of the protein onto the surface. On the other hand, tosyl groups (right side) can react covalently with any amino group present in the cargo protein. This means that GLA can be covalently attached to the surface by several different amino groups present at its surface, therefore acquiring different conformations. thermal stability after enzyme immobilization is not exclusive of GLA. As shown (Fig. 5), cells treated with GLA-coated MPs showed a sig­ On the contrary, protein immobilization seems to result, as a general nificant intracellular GLA enzymatic activity, as trypsinization is ex­ trend, in a stabilization of the protein structure and therefore a better pected to remove externally attached enzyme. As presumed, only biological performance. This has been already described for different background GLA activity was detected in HeLa cells treated with M280- proteins, like glycosyltransferases [54], antibodies [55], alkaline phos­ GLA MPs but not submitted to magnetic force. Such residual GLA ac­ phatase [56], amyloglucosidase [57], or α-chymotrypsin [58]. All these tivity cannot be explained by the action of endogenous GLA, since cells results together make magnetic bioconjugates excellent platforms to not treated or treated with control MPs did not show any background display highly active versions of human GLA for both in vitro catalysis value. In this line, such GLA activity is statistically higher than that and therapeutic applications. found in cells not confronted to MPs. On the other hand, intracellular In this context, we first tested the ability of M-280 MPs to deliver levels of GLA activity were clearly enhanced when the magnetic force enzymatically active GLA into HeLa cells. As a control, no MPs and M- was applied. This result was indicative of the bioavailability of the 280 MPs coated with irrelevant proteins (GFP and BSA) were included. enzyme even under the partial penetration of such larger particles suggested by confocal 3D reconstructions (Fig. 3). In a further experiment (Fig. 6), the kinetics of intracellular GLA Fig. 5. Delivery of GLA enzyme by means of M-280 MPs. M-280 particles coated with different proteins (GFP, GLA or BSA) were incubated with HeLa Fig. 6. Delivery of GLA enzyme by means of TALON particles coated with GLA. cells for 5 min in the absence (blue bars) or presence (green bars) of magnet. TALON-GLA MPs were incubated with HeLa cells for different times in the Then, cells were recovered by trypsinization, lysed and submitted to the GLA absence (blue bars) or presence (green bars) of magnet. Then, cells were enzymatic assay. Values are given as mean and SEM of at least 4 indepen­ recovered, lysated and submitted to the GLA enzymatic assay. Values are given dent samples. as mean and SEM of two independent samples. 6 E. Ruiz-Cánovas et al. Colloids and Surfaces B: Biointerfaces 208 (2021) 112123 activity was monitored in GLA-loaded TALON microparticles (1-micron diameter). These MPs allowed spontaneous, time-dependent entrance into cells in the absence of a magnet at levels comparable to those of larger particles. On the other hand, the use of a magnet significantly accelerated MPs entrance as well as the transient nature of the intra­ cellular permanence, as indicated by data shown in Fig. 2. Again, the levels of internalized GLA were similar to those reached by larger MPs in presence of the magnet. To fully confirm such membrane flexibility, we performed an anal­ ysis of cytotoxicity potentially caused by all types of used MPs. As observed (Fig. 7), none of the used MPs caused a significant decrease in cell viability when compared to untreated cells, or to cells exposed to MPs storage buffers (PBS pH 7.4, or acetic acid pH 4.5). 4. Conclusion Fig. 7. Cytotoxicity of MPs. M-280 and MyOne particles coated with different proteins (GFP or GLA) were incubated with HeLa cells for 10 min in the pres­ Microparticles, and specially nanoparticles, have been extensively ence of magnet. Then, cells were washed and incubated 24 h in fresh medium. explored as drug delivery systems. However, success of such vehicles Finally, cells were submitted to the MTT metabolic assay. Values are given as relies on the ability to overcome two main key bottlenecks in the mean and SEM of at least 3 independent samples. administration pathway, namely a poor biodistribution through an appropriate site-specific cell targeting, and the external localization of Research Center on Bioengineering, Biomaterials, and Nanomedicine the drug, through an efficient cellular uptake of the drug-material con­ (CIBER-BBN) is an initiative funded by the VI National R&D&I Plan jugates. In this sense, magnetic vehicles are very attractive for the de­ 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Ac­ livery of therapeutic agents as they can be targeted to specific locations tions and financed by the Instituto de Salud Carlos III with assistance in the body through the application of a magnetic field gradient. Such from the European Regional Development Fund. We are indebted to ability has been explored in processes like magnetofection or hyper­ AGAUR (2017SGR-229, to AV). AV received an ICREA ACADEMIA thermia therapy. However, once the target cell has been reached, suc­ award. cessful applicability of nano- and microparticles as DDS depends, among other factors, on their ability to bind to and cross cell membranes. Proteins are generally unable to cross the membrane barrier of CRediT authorship contribution statement eukaryotic cells. Thus, interaction of the drug carrier (nano- and mi­ croparticles) with cell membranes is of paramount importance. Two Eugènia Ruiz-Canovas: Investigation, Data curation. Rosa Men­ main steps of this interaction between vehicle and membrane are the doza: Investigation. Antonio Villaverde: Conceptualization, Supervi­ adhesion of the particles to the membranes and their subsequent sion, Writing – review & editing. José L. Corchero: Conceptualization, engulfment by these membranes. Investigation, Supervision, Data curation, Writing – original draft, The capability of large magnetic particles to intracellularly deliver Writing – review & editing. cargo proteins in a functional way, demonstrated in this study, opens a plethora of possibilities regarding the use of hard materials as vehicles for cell internalization. The control of the particle size in the range Declaration of Competing Interest 1–2.8 µm allows to determine the localization of the material upon the application of magnetic fields. While smaller particles penetrate with The authors declare that they have no known competing financial high efficacy, most of the larger particles remain associated to the outer interests or personal relationships that could have appeared to influence side of the cell membrane, or deeply embedded in the lipidic bilayer. the work reported in this paper. Such a discrimination cannot be achieved by conventional homing materials such as nanoparticles functionalized with peptidic ligands of Acknowledgments cell surface molecules, that inexorably determine the endosomal route of cell penetration. Interestingly, the engulfment of MPs with or without We thank Dr. Lluís M. Martínez and Sepmag Systems for their tech­ payload, as driven with a magnet, does not result in any loss of cell nical help and kindly providing the Stepmag 125 magnetic separation viability or in morphological aberrations of the target cells. We have system, and to Dr. Escarlata Rodríguez-Carmona for their support and proved that large MPs used as internalizing drug carriers are fully technical help regarding protocols for magnetic particles coating. Cell biocompatible, as determined by a cell viability assay, and under the culture experiments and confocal microscopy analysis were performed conditions tested in this study, some of them quite harsh and intrinsi­ at the Cell Culture Service (SCAC) and at the Servei de Microscòpia (both cally challenging cell viability. at the UAB), respectively. We also acknowledge the ICTS “NANBIOSIS”, The membrane-linked localization of larger materials would be more specifically the support from the Protein Production Platform of especially appealing to deliver hormones, growth factors, antagonists CIBER-BBN/IBB, at the UAB SepBioES scientific-technical service (http and other cell signaling effectors with complicated delivery processes. 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