www.acsami.org Research Article Self-Assembled Nanobodies as Selectively Targeted, Nanostructured, and Multivalent Materials Laura Sánchez-García,● Eric Volta-̀ Durán,● Eloi Parladé, Elisa Mazzega, Alejandro Sánchez-Chardi, Naroa Serna, Hec̀ tor López-Laguna, Mara Mitstorfer, Ugutz Unzueta, Esther Vázquez, Antonio Villaverde,* and Ario de Marco* Cite This: ACS Appl. Mater. Interfaces 2021, 13, 29406−29415 Read Online See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. ACCESS Metrics & More Article Recommendations ABSTRACT: Nanobodies represent valuable tools in advanced therapeutic strategies but their small size (∼2.5 × ∼ 4 nm) and Downloaded via 84.78.139.232 on July 18, 2022 at 16:44:04 (UTC). limited valence for interactions might pose restrictions for in vivo applications, especially regarding their modest capacity for multi- valent and cooperative interaction. In this work, modular protein constructs have been designed, in which nanobodies are fused to protein domains to provide further functionalities and to favor oligomerization into stable self-assembled nanoparticles. The nanobody specificity for their targets is maintained in such supramolecular complexes. Also, their diameter around 70 nm and multivalent interactivity should favor binding and penetrability into target cells via solvent-exposed receptor. These concepts have been supported by unrelated nanobodies directed against the ricin toxin (A3C8) and the Her2 receptor (EM1), respectively, that were modified with the addition of a reporter protein and a hexa- histidine tag at the C-terminus that promotes self-assembling. The A3C8-based nanoparticles neutralize the ricin toxin efficiently, whereas the EM1-based nanoparticles enable to selective imaging Her2-positive cells. These findings support the excellent extracellular and intracellular functionality of nanobodies organized in form of oligomeric nanoscale assemblies. KEYWORDS: nanobodies, self-assembling, ricin, nanoparticles, controlled delivery, biomaterials ■ INTRODUCTION The selective delivery of molecules to target cells or organs is and to induce immunogenicity. In addition, their clonality is often unstable, their engineering is complex, their functional- critical to improve the specificity of therapy and image-based ization not reproducible and they are expensive and difficult to diagnosis. Molecular ligands are able to specifically recognize produce due to their highly glycosylated domains.2 The use of and bind cell-surface biomarkers overexpressed in target cells IgG fragments (Fab and scFv formats) can fundamentally solve and they can be then used to confer selectivity to drugs, most of these problems but introduces others, such as a imaging agents, or combined with supramolecular complexes. reduced avidity when compared to multivalent alternatives and Proteins, peptides, antibodies, polysaccharides, aptamers, and a higher propensity to aggregate.3 variety of small molecules have been explored and developed In this scenario, the discovery of camelid heavy-chain as targeting ligands and their comparative potentialities and antibodies (HcAbs) opened interesting possibilities.4 Unlike drawbacks have been deeply described.1 conventional antibodies or IgG fragments, the variable antigen- Antibodies have evolved to recognize their antigens with binding fragment of HcAbs consists of a single structural high affinity and specificity, and therefore, they represent the domain (15 kDa), known as nanobody or VHH (variable most common class of binders used in both research and heavy chain of HcAbs) (Figure 1A). It corresponds to an clinical settings. Specifically, conventional IgG monoclonal antibodies issued from hybridoma cells are currently the most preferred antibody format. However, their large mass (150 Received: May 3, 2021 kDa) and structural complexity make them not suitable for Accepted: June 2, 2021 some applications such as the targeting of solid tumors. These Published: June 15, 2021 properties also hamper the access to dense structures such as tumoral tissues while increasing the probabilities to interact with other molecules in the bloodstream, to accumulate in liver © 2021 American Chemical Society https://doi.org/10.1021/acsami.1c08092 29406 ACS Appl. Mater. Interfaces 2021, 13, 29406−29415 ACS Applied Materials & Interfaces www.acsami.org Research Article Figure 1. Physicochemical properties and structure of nanobodies. A. Representation of the generic nanobody structure. The conserved region (framework) is shown in blue and the variable antigen-binding domain (comprising CDR 1, 2, and 3) in orange. B. Design and formation of VHH self-assembled nanoparticles. C. Properties of selected nanobodies A3C8 and EM1. Abbreviations: BBs: building-blocks, MW: molecular weight, aa: amino acids, R: arginine, K: lysine, and pI: isoelectric point. amino acid sequence that can be easily produced by protein domains, favoring self-assembling, are selected for the recombinant DNA procedures and that preserves the binding construct. This approach would not only contribute to increase selectivity of the whole molecule. The properties of nano- the mass of a VHH but also promote a multivalent bodies, such as small size, good stability and ease of presentation of the ligand. Such a modular design based on engineering and expression made them the preferred domain recruitment might also permit selecting fusion partners components in several biotechnological applications.5−7 They with particular functions that would be combined in single have been successfully produced alone or as fusion proteins in multifunctional particulate entities keeping the precise Gram-negative (Escherichia coli) and Gram-positive bacteria interactivity of the nanobody. (Lactobacillus sp), yeasts, plants, and mammalian cells, according to the required structural and functional features In this context, we have recently generated several categories of the final product.8,9,7 Additionally, VHHs are known to be of protein-only self-assembling nanoparticles following a poorly immunogenic and their immunogenicity can be further semirational protein engineering strategy.28−30 According to reduced by site-directed mutagenesis.5 this approach, the combination of N-terminal domains with In February 2019, the Food and Drug Administration cationic character with C-terminal histidine-rich peptides (such (FDA) approved the use of the first nanobody (Caplacizu- as the hexahistidine H6) favors spontaneous oligomerization mab). Caplacizumab is a recombinant humanized nanobody into regular nanoparticles through a particular distribution of produced in E. coli10 and used as a drug for the treatment of electrostatic charges.29,31 Apart from other protein−protein acquired thrombotic thrombocytopenic purpura (aTTP), a interactions that contribute to the assembly, such as hydrogen rare disease characterized by excessive blood clotting in small bond and van der Waals,28 divalent cations present in the blood vessels.11 Other nanobodies have reached different media stabilize such complexes32 through their coordination phases of clinical trials, such as Ozoralizumab NCT01007175 with overhanging H6 tails.33−35 No external addition is needed and Vobarilizumab NCT02287922 (to treat rheumatoid for such stabilization,36 as Ni2+ traces from the purification arthritis), M1095 NCT02156466 (for psoriasis) and BI 836880 NCT03468426 (for solid tumors), evidencing the columns might be sufficient.33,34 Because of the β barrel potential of nanobodies in different therapeutic areas.12 folding of GFP, GFP-containing constructs are particularly As stated above, the low molecular mass of nanobodies suited for fast and efficient nanoparticle formation following facilitates their tissue penetration.13 However, in contrast, the this principle, as proved by diverse categories of fusion proteins cellular interactivity of VHHs is highly limited by their small of biomedical interest that are based on H6-tagged GFP.37−39 size (around 4 nm),14,15 far from optimal values around 20−80 This engineering approach based on self-oligomerization nm.16,17 In this context, different strategies have been allows enlarging the size of the materials by reaching a developed aiming at enlarging nanobodies by their combina- multimeric organization and it prevents the introduction of tion with other molecules.7,18−23,23 Larger structures (around irrelevant protein domains or other scaffold materials that 24 nm) have been indeed obtained by fusing VHHs to elastin- might represent a load in production and a risk regarding the like peptides and by linking VHHs to the pentameric B-subunit toxicity of the final hybrid material.40 Therefore, we decided to of E. coli verotoxin.24−26 Also, the genetic fusion of the explore the short H6 tag, fused to nanobodies and GFP albumin-binding domain allows a later incorporation of (Figure 1B), as a tool to generate large self-assembled albumin as an external agent for size increase.27 Ideally, size multivalent and multifunctional nanoparticles. For that, two increase should be achieved by oligomerization rather that by the coupling VHHs to any heterologous material. In this way, unrelated nanobodies were selected as models, namely A3C8, the chemical homogeneity of the material would be conserved specific for ricin toxin, and EM1, specific for Her2 (Figure and reproducible, and its functionality would be exploited in 1C),41,42 and engineered to demonstrate the feasibility of the full. Under this concept, the engineering of VHHs by gene proposed approach, that is, controlled oligomerization into fusion to generate modular proteins would confer them the nanoparticles while preserving functionalities of the nano- capability to self-assemble in form of nanoparticles, if proper bodies and of other incorporated protein domains. 29407 https://doi.org/10.1021/acsami.1c08092 ACS Appl. Mater. Interfaces 2021, 13, 29406−29415 ACS Applied Materials & Interfaces www.acsami.org Research Article Figure 2. Physicochemical characterization of VHH-based modular proteins. A. Modular organization of VHHs fused to GFP-H6 and their corresponding amino acid sequence. VHHs A3C8 and EM1 (in dark red and purple, respectively) serve as selective ligands. A short linker (GGSSRSS) in light gray was added between the VHH and GFP for conformational purposes. GFP protein (in green) was incorporated for tracking purposes while H6 (in dark gray) for purification and for assembling purposes. Box sizes are only indicative. B. MALDI-TOF, SDS-PAGE (TGX), and Western Blot (WB) of purified recombinant proteins. Molecular weights are indicated. C. Dynamic light scattering (top) and FESEM images (bottom) of purified assembled materials. Triton was used for disassembling at 0.5%. Bars size: 100 nm. D. Cell viability of HeLa and SK- BR-3 cells after 72 h protein incubation at 1 × 10−7 M. ■ MATERIALS AND METHODS Protein Design, Production, and Purification. The modular 1200 psi (Thermo FA-078A) for optimal lysis. The soluble fraction was collected after centrifugation (45 min, 15 000g at 4 °C) and filtered through a 0.22 μm-diameter filter. Protein purification was constructs A3C8-GFP-H6 and EM1-GFP-H6 (Figure 2A) were performed in an Ä kta Pure FPLC system (GE Healthcare, U.S.) by designed in-house as E. coli codon-optimized genes and synthesized Immobilized Metal Affinity Chromatography (IMAC). After the by GeneArt (ThermoFisher). The nomenclature of fusion proteins selective binding of the protein onto a HisTrap HP column (GE used in this study refers to their modular domain organization. A3C8 Healthcare, U.S.), elution was achieved by applying a linear gradient is a nanobody with high affinity toward the plant toxin ricin, whereas EM1 is specific for Her2 receptor, overexpressed in breast cancer cells. of Elution buffer (20 × 10−3 M Tris-HCl, 500 × 10−3 M NaCl, 500 × Green fluorescent protein (GFP) was incorporated in the constructs 10−3 M imidazole, pH 8.0). The eluted samples were dialyzed against for tracking purposes, and the C-terminal hexa-histidine tag sodium carbonate salt buffer (166 × 10−3 M NaCO3H and 333 × (HHHHHH) was added for cation-coordinated protein oligomeriza- 10−3 M NaCl, pH 8.0) and centrifuged to remove protein aggregates tion and for purification purposes.33 A flexible peptide (GGSSRSS) (15 min, 15 000g at 4 °C). Protein quantification was performed using connecting nanobody and GFP protein was introduced as a spacer to Bradford Assay (BioRad, U.S.). impair undesired structural interactions between the moieties that Physicochemical Characterization. A3C8-GFP-H6 and EM1- could interfere with their functionality. Both fusion sequences A3C8- GFP-H6 proteins were analyzed to assess their degree of purity by GFP-H6 and EM1-GFP-H6 were subcloned in the plasmid pET22b SDS-PAGE using TGX Stain-Free FastCast gels (BioRad, U.S.). and further transformed by heat shock (42 °C for 45 s) in Escherichia Protein integrity was also assessed by Western-blot using an anti-His coli Origami B (BL21, OmpT−, Lon−, TrxB−, Gor−) (Novagen, monoclonal antibody (Santa Cruz Biotechnology, U.S.), and by mass Germany). spectrometry (MALDI-TOF). Additionally, the volume size distribu- Transformed cells (selected through the acquisition of ampicillin tion was determined by dynamic light scattering (DLS) at 633 nm resistance provided by pET22b) were incubated at 37 °C until (Zetasizer Nano ZS, Malvern Instruments Limited, UK), in 50 μL of reaching OD550 = 0.5−0.7. Then, protein production was performed protein storage buffer (in of 166 × 10−3 M NaCO3H and 333 × 10−3 at 20 °C overnight after the addition of IPTG (isopropyl β-D-1- M NaCl). For disassembling, proteins were diluted at 0.5 mg mL−1, thiogalactopyranoside) at a concentration of 0.1 × 10−3 M (A3C8- and Triton X-100 added to 0.5% was used in order to visualize their GFP-H6) and 1 × 10−3 M (EM1-GFP-H6). Finally, cells were respective building blocks by DLS. Size measurements were centrifuged (5000g for 15 min 4 °C) and stored at −80 °C until use. performed in triplicate. Volume data in DLS was representative of Pellets were thawed and resuspended in Wash buffer (20 × 10−3 M the population size distribution while Intensity data provided more Tris-HCl, 500 × 10−3 M NaCl, 10 × 10−3 M imidazole, pH 8.0) accurate hydrodynamic sizes. supplemented with the protease inhibitor cOmplete EDTA-free Ultrastructural Characterization. Ultrastructural morphometry (Roche, U.S.). Bacterial disruption was performed in a French Press at (size and shape) of nanoparticles was visualized at nearly native state 29408 https://doi.org/10.1021/acsami.1c08092 ACS Appl. Mater. Interfaces 2021, 13, 29406−29415 ACS Applied Materials & Interfaces www.acsami.org Research Article with field emission scanning electron microscopy (FESEM) and Surpass Module in Imaris X64 v.7.2.1. software (Bitplane, Switzer- transmission electron microscopy (TEM). Drops of 5 μL of land). nanoparticles resuspended in their buffers were directly deposited Statistical Analysis. The data of the in vitro experiments (cell on silicon wafers (Ted Pella Inc.) for 2 min, excess of liquid blotted, viability and protein internalization) were reported as mean ± SEM air-dried, and immediately observed without coating in a FESEM (Standard Error of the Mean). Results were analyzed using Tukey’s Merlin (Zeiss) operating at 1 kV and equipped with an in-lens pairwise test. Differences between groups were considered significant secondary electron detector. Drops of 5 μL of nanoparticles at p < 0.05. These differences were indicated as * p < 0.05 and ** p < resuspended in their buffers were also deposited on carbon-coated 0.01. Statistical calculations were performed using Past3 software. ■ copper grids (400 mesh, Agar Scientific) for 2 min, excess of liquid blotted, incubated in 2% uranyl acetate (Sigma-Aldrich, Germany) for 5 min, rinsed with deionized water, and air-dried. Samples were RESULTS observed in a TEM JEM-1400 (Jeol Ltd.) operating at 80 kV and Recombinant fusion proteins formed by GFP-H6 and VHHs equipped with an Orius SC 200 CCD camera (Gatan). Representative (A3C8 and EM1) as targeting moieties (Figure 2A) were images of general fields and nanostructure details were captured at successfully produced in E. coli. After purification, protein two high magnifications (2 × 105× and 5× 105× for FESEM, and 1× purity was assessed by SDS-PAGE and protein integrity was 104x and 8× 104× for TEM). Cell Culture and Flow Cytometry. Human cervical adenocarci- confirmed by MALDI-TOF and Western Blot (Figure 2B). noma cells (HeLa cell line, ATCC, CCL-2) were maintained in The molecular weights of A3C8-GFP-H6 and EM1-GFP-H6 MEM-Alpha (Gibco) and incubated at 37 °C and 5% CO2 in a were 42.31 and 41.05 kDa, respectively, in agreement with the humidified atmosphere. Human breast cancer cells (SK-BR-3 cell line, theoretically calculated values. ATCC, HTB-30) were maintained in DMEM (Gibco) at 37 °C and Both VHHs are rich in arginine and lysine residues (over 10% CO2. Both culture media were supplemented with 10% fetal 5%, Figure 1C,A), and we wondered if the positive charge bovine serum (Gibco). HeLa cells (Her2−) and SK-BR-3 cells conferred by these residues would be enough to enable the (Her2+) were scattered in 24-well plates at 6 × 104 and 8 × 104 cells/ protein to self-assemble, as demonstrated for other H6-tagged well, respectively. After 24 h, cells reached 70% confluence and were fusion proteins.29 While a fraction of the protein was purified incubated with 1000 nM of EM1-GFP-H6 for 24 h. Two different trypsinization protocols (harsh and mild) were compared to detach from bacterial cell extracts as monomers (with an hydro- the cells and analyze them through flow cytometry.43 A harsh trypsin dynamic size around 8 nm, not shown), dynamic light (HT) protocol (1 mg mL−1 for 15 min) was aimed to remove cells scattering (DLS) revealed that most of A3C8-GFP-H6 and from the well while stripping the residual protein attached on the cell EM1-GFP-H6 in the eluted protein fraction self-assembled, as membrane surface. Mild trypsin (MT) protocol (0.5 mg mL−1 for 5 expected, into nanoparticles of around 70 nm (Figure 2C). min) was conceived to detach cells from the well without removing Upon detergent-induced nanoparticle disassembly, protein bound protein from the cell surface. Experiments were performed in materials of around 10 nm in diameter were detected (Figure duplicate. 2C), compatible with protein forms that represented the Cell Viability and Neutralization Assay. The CellTiter-Glo building blocks in the protein oligomerization process. Protein Luminescent Cell Viability Assay (Promega) was used to determine the cytotoxicity of the purified recombinant proteins and the nanoparticles showed a regular pseudospherical shape, as neutralizing effect of A3C8-GFP-H6 nanoparticles. HeLa cells observed by FESEM (Figure 2C), and their dimensions, (CXCR4+) and SK-BR-3 cells (Her2+) were scattered at 3500 cells/ determined by FESEM, were in full agreement with DLS well in opaque-walled 96-well plates, until reaching 70% confluence. volume determinations. Then, self-assembled nanobodies were To assess whether A3C8-GFP-H6 and EM1-GFP-H6 nanoparticles studied in vitro for their physiological properties. Cell viability are cytotoxic, HeLa cells and SK-BR-3 cells were treated with a final experiments in HeLa (Her2 −) and SK-BR-3 (Her2+ ) concentration of 1 × 10−7 M for 72 h. In the neutralization assay, ricin mammalian cells showed, as expected, no cytotoxic effect toxin (1 × 10−8 M of the recombinant T22-mRTA-H6) was after 72 h of incubation with any of the nanoparticles (Figure preincubated with increasing amounts of A3C8-GFP-H6 at different ratios (1:1, 1:5, and 1:10) for 72 h. The irrelevant construct EM1- 2D), indicating that the oligomerization event did not confer GFP-H6 (anti-Her2 nanoparticle) was used as a negative control.28 toxicity to proteins that are intrinsically innocuous. After protein incubation, the reagent provided by the manufacturer At this point, we assessed the capacity of nanoparticles to was added to cultured cells and plates were measured in a bind to their target antigens. First, the capacity of A3C8-GFP- conventional luminometer Victor3 (PerkinElmer, U.S.). Experiments H6 to recognize and neutralize ricin was demonstrated. Ricin were performed in triplicate. toxin consists of two chains linked by a disulfide bond. The Confocal Laser Scanning Microscopy. HeLa and SK-BR-3 cells chain A (RTA) corresponds to the catalytic domain with N- were grown on Mat-Tek plates (MatTek Corporation, U.S.) scattering glycosidase enzymatic activity, whereas the chain B (RTB) 1.2 × 105 cells per well. After cellular attachment, protein incubation corresponds to the carbohydrate recognition protein. It has was performed in the presence of EM1-GFP-H6 or GFP-H6 at a final concentration of 1 μM for 1 h. After protein incubation, cell nuclei been described that RTB cell-binding is unspecific and were labeled with 5 μg mL−1 Hoechst 33342 (ThermoFisher, U.S.) presents low affinity, promoting multiple intracellular traffick- and the plasma membrane with 2.5 μg mL−1 CellMask Deep Red ing pathways.44 In order to obtain a finely controlled and (ThermoFisher, U.S.) for 10 min at room temperature. Cells were highly specific version of the toxin, we selected a previously then washed in DPBS buffer (Sigma-Aldrich, Germany). All confocal designed and fully active recombinant chimera (T22-mRTA- images were collected on an inverted TCS SP5 Leica Spectral H6) composed of a mutant A chain (mRTA) fused to the N- confocal microscope (Leica Microsystems, Germany) using 63× (1.4 terminal CXCR4-binding peptide T2245 and to a C-terminal NA) oil immersion objective lenses. Excitation was reached using a histidine-rich tag.46 The CXCR4-binding peptide confers 405 nm blue diode laser (nucleic acids), 488 nm line of an argon ion specificity for such receptor whereas the poly-His, as described laser (nanoparticles) and 633 nm line of a HeNe laser (cell membrane). Optimized emission detection bandwidths were con- above, contributes to the oligomerization of the construct. figured to avoid interchannel crosstalk and multitrack sequential Therefore, the recombinant T22-mRTA-H6, that self- acquisition setting were used. The confocal pinhole was set to 1 Airy assembled as potent cytotoxic nanoparticles of 11 nm, unit and z-stacks acquisition intervals were selected to satisfy Nyquist selectively kills CXCR4+ cells in vitro and in vivo45 and is sampling criteria. Three-dimensional images were processed using the then usable to assess the protective effect of the antiricin 29409 https://doi.org/10.1021/acsami.1c08092 ACS Appl. Mater. Interfaces 2021, 13, 29406−29415 ACS Applied Materials & Interfaces www.acsami.org Research Article Figure 3. Neutralization capacity of antiricin A3C8-GFP-H6 nanoparticles. A. Optical microscopy images of HeLa cells treated with 1 × 10−8 M T22-mRTA-H6 and 1 × 10−7 M A3C8-GFP-H6 separately (top) and in combination at ratio 1:1, 1:5, 1:10 (bottom). Bars size: 100 μm. B. Quantitative data of the neutralization assay performed at 72 h expressed as percentage of cell viability. EM1-GFP-H6 is used as a negative control. Dashed line illustrates cell viability after treatment with 1 × 10−8 M T22-mRTA-H6 under the same conditions. Significant differences between each particular condition and T22-mRTA-H6 alone are indicated as ** p < 0.01. NS: not significant. C. Schematic representation of the neutralization assay and the phenomenon occurring at the different conditions. D. Representative electron microscopy (FESEM and TEM) images of T22-mRTA-H6 toxin treated with A3C8-GFP-H6 showing nanoparticle populations classified by size as free T22-mRTA-H6 (Y), A3C8-GFP- H6 (Z), and complex of the two components (Y+Z). Aggregates compatible with few A3C3-GFP-H6 neutralizing free ricin were shown at right. Bars size: 100 nm. A3C8-GFP-H6 nanoparticles over cultured CXCR4+ HeLa between fluorescence located at the external cell surface or due cells. to nanoparticle internalization. Her2− HeLa cells exposed to The neutralizing activity of A3C8-GFP-H6 was demon- nanoparticles only acquired a low background signal, which strated by measuring the CXCR4+ HeLa cell viability at indicated negligible nanoparticle binding to both cell increasing concentrations of the antidote with reference to subgroups (MT and HT). In contrast, the incubation of those of the toxin (T22-mRTA-H6:A3C8-GFP-H6 molar ratio nanoparticles with Her2+ cells resulted in a significant increase of 1:1, 1:5, 1:10) (Figure 3). Optical microscopy images of the fluorescence linked to these cells. The strong effect of showed that cell confluence was proportional to the increase of neutralizing nanoparticles (Figure 3A). Precisely, cell viability harsh trypsinization (Figure 4B) suggested that EM1-GFP-H6 increased from 35% up to 60% when neutralizing nanoparticles nanoparticles were mostly located at the level of the cellular were used at 1:1 ratio with respect to the toxin alone (T22- membrane. Their precise localization was assessed by confocal mRTA-H6) and reached 100% at a 1:10 ratio (Figure 3B). microscopy (Figure 4C), and the specificity of the cellular Self-assembled control EM1-GFP-H6 nanoparticles were interaction fully confirmed by the absence of green signals in instead unable to neutralize the ricin-induced cytotoxicity at Her2− HeLa cells exposed to EM1-GFP-H6 and in Her2+ cells any concentration (Figure 3B). Moreover, FESEM and TEM exposed to a control GFP-H6 (Figure 4D). The images imaging revealed that the coincubation of nanobody and ricin revealed that important amounts of EM1-GFP-H6 nano- nanoparticles originated a new population of larger nano- particles accumulated in the cell membrane, with preference particles (Figure 3C,D), with a size compatible with for specific foci. However, a moderate green fluorescent signal agglutinated nanobody and ricin constructs. was still observed in the cytoplasm indicative of internalization. The capacity of EM1-GFP-H6 to bind to the Her2 receptor (Figure 4A) was tested by flow-cytometry using Her2− (HeLa) The overall set of such data indicated that EM1-GFP-H6 and Her2+ cells (SK-BR-3) (Figure 4B). Both cell lines were nanoparticles efficiently and specifically interacted with the first incubated with the nanoparticles followed by trypsiniza- Her2 receptor, although its moderate cationic character, tion. Two alternative treatments, referred to as mild trypsin sufficient for assembling, might be an obstacle for efficient (MT) and harsh trypsin (HT), were applied to distinguish internalization.47 29410 https://doi.org/10.1021/acsami.1c08092 ACS Appl. Mater. Interfaces 2021, 13, 29406−29415 ACS Applied Materials & Interfaces www.acsami.org Research Article Figure 4. In vitro assessment of EM1-GFP-H6 binding to Her2 receptor. A. Schematic representation of Her2-targeted nanoparticles (EM1-GFP- H6) binding to Her2+ breast cancer cells (sizes are not representative). B. Flow cytometry of EM1-GFP-H6 nanoparticles in Her2+ (SK-BR-3) and Her2− (HeLa) cells. HT and MT correspond to harsh and mild trypsin protocols, respectively. Significant differences are indicated as ** p < 0.01. C. Confocal images of SK-BR-3 (Her2+) cells after incubation with 1 μM of EM1-GFP-H6 for 1 h. Red signal corresponds to cell membrane, blue to nuclei and green to nanoparticles. At the bottom, orthogonal projections show the localization of EM1-GFP-H6 nanoparticles in yellow, due to the colocalization of red (membrane) and green (nanoparticle) signal. Bars size: 5 μm. D. Wide confocal fields of cultured SK-BR-3 cells and HeLa cells exposed to EM1-GFP-H6 nanoparticles and to a control GFP-H6 protein. Cells are shown without (top) and with (bottom) membrane staining. ■ DISCUSSION Llama-derived nanobodies are small-sized ligands easy to of H6-tagged microbial proteins.36 In the whole category of H6-tagged constructs, divalent cations, even in traces such as produce and engineer. Whereas their reduced dimension is Ni2+ leaking from the purification columns, are proved to very desirable for some applications, for others it would be stabilize the oligomers through cross-interactions between preferable having enlarged versions and desirable, with adjacent solvent-exposed histidine-rich tails.33 Even for self- multivalent interactivity. The results presented in this study assembling constructs such as those based on N-terminal illustrate the development of a new category of nanobody- arginine-rich peptides (R6, R9, etc.),29,48 it has been shown based, self-assembled, multivalent nanoparticles with dimen- that divalent cations have a stabilizing effect.32,33 Further sions larger than the original nanobodies but with preserved details regarding the formation of stable divalent cation- antigen selectivity and newly acquired fluorescence. To mediated materials and the role of histidine-rich peptides in demonstrate this concept, we used model nanobodies with such cross-molecular interactions are available elsewhere.34,35 specificity toward two targets of interest in nanomedicine and Most of the engineered VHHs molecules spontaneously self- designed modular proteins that have been successfully assembled as 70 nm-sized nanoparticles upon purification, well produced, purified and functionally evaluated. The constructs above the renal filtration cutoff (around 8 nm), making them consisted of a nanobody domain fused to GFP and a H6-tag. optimal for potential in vivo applications.49 In this form, the The fluorescent protein simplifies the construct visualization VHHs offer a multivalent presentation of the ligands that whereas the poly-His tag is exploited as a convenient tag for results in highly specific targeting, as demonstrated by Her2+ both protein purification and to assist assembling of the cell labeling by the GFP-containing anti-Her2 nanoparticles. nanobody-based fusion protein. While some H6-tagged The successful characterization of such nanomaterials in terms proteins require the incorporation of additional cationic N- of structural and functional features opens the opportunity to terminal peptides to induce assembling,29,48 the occurrence of test them in applications such as imaging or drug delivery, in a few clustered cationic amino acids in the N-terminal domain which monomeric or multimeric VHHs have already provided of the selected VHHs (Figure 2A) was proved to be sufficient promising results.50−55 While the targeting of EM1-GFP-H6 for oligomerization (Figure 2C). A similar event was materials for the Her2 receptor is efficient and selective (Figure demonstrated elsewhere when producing recombinant versions 4B−D), the internalization of this construct is only moderate 29411 https://doi.org/10.1021/acsami.1c08092 ACS Appl. Mater. Interfaces 2021, 13, 29406−29415 ACS Applied Materials & Interfaces www.acsami.org Research Article (Figure 4C,D). Although specific cell labeling for diagnosis applications but in the self-assembling properties of specifically purposes is a main utility of nanobodies,56 these molecules engineered nanobody versions. have been also described as targeting agents in drug complexes intended for cell-targeted drug delivery.56 However, cell penetrability of nanobodies is a nonconsistent event regarding ■ AUTHOR INFORMATION Corresponding Authors efficacy. It is dramatically enhanced by increasing the cationic Antonio Villaverde − Institut de Biotecnologia i de character of these molecules through selective mutagenesis of Biomedicina, Universitat Autònoma de Barcelona, Barcelona solvent-exposed residues47 or by photochemical induction,57 08193, Spain; Departament de Genètica i de Microbiologia, among other strategies. While the nanobody domain of EM1- Universitat Autònoma de Barcelona, Barcelona 08193, GFP-H6 has a significant positive charge (Figure 1C) sufficient Spain; CIBER de Bioingeniería Biomateriales y for self-assembly, it might be insufficient for efficient Nanomedicina (CIBER-BBN), Barcelona 08193, Spain; internalization. Further protein engineering addressed to orcid.org/0000-0002-2615-4521; introduce additional cationic residues or tails should enhance Email: antoni.villaverde@uab.cat cell penetrability. Ario de Marco − Laboratory for Environmental and Life On the other hand, a specific application for nanobody Sciences, University of Nova Gorica, Nova Gorica 5000, nanoparticles is the exploitation of their agglutinating capacity. Slovenia; Email: ario.demarco@ung.si Toxins are a current threat with a great daily impact, and a lot of effort is being invested toward the detection and treatment Authors of intoxications caused by toxins (botulinum toxin, anthrax Laura Sánchez-García − Institut de Biotecnologia i de toxin, ricin, toxic-shock syndrome toxin-1, Salmonella typhy- Biomedicina, Universitat Autònoma de Barcelona, Barcelona murium toxin) and venoms.21,58−60 Ricin, produced in castor 08193, Spain; Departament de Genètica i de Microbiologia, beans, is one of the most lethal toxins in nature.61 The high Universitat Autònoma de Barcelona, Barcelona 08193, toxicity and the large availability of castor beans has led to a Spain; CIBER de Bioingeniería Biomateriales y significant number of human intoxications. Moreover, no Nanomedicina (CIBER-BBN), Barcelona 08193, Spain postexposure therapeutics are available to reverse the effects of Eric Voltà-Durán − Institut de Biotecnologia i de Biomedicina, intoxication, which can lead to death from poisoning within Universitat Autònoma de Barcelona, Barcelona 08193, 36−72 h of exposure. In this context, we have selected the Spain; Departament de Genètica i de Microbiologia, A3C8 nanobody for the development of neutralizing antiricin Universitat Autònoma de Barcelona, Barcelona 08193, self-assembled nanoparticles, which showed high potency Spain; CIBER de Bioingeniería Biomateriales y against a ricin toxin chimera (T22-mRTA-H6). Even at the Nanomedicina (CIBER-BBN), Barcelona 08193, Spain lowest tested molar ratio (1:1 ratio T22-mRTA-H6:A3C8- Eloi Parladé − Institut de Biotecnologia i de Biomedicina, GFP-H6), a neutralization of 39% was observed, followed by Universitat Autònoma de Barcelona, Barcelona 08193, an 86% at 1:5 ratio and a 99% of neutralization at 1:10 ratio. Spain; Departament de Genètica i de Microbiologia, The use of a functionally unrelated control (EM1-GFP-H6) Universitat Autònoma de Barcelona, Barcelona 08193, demonstrated that the neutralization was not mediated by the Spain; CIBER de Bioingeniería Biomateriales y structure of the nanobody as a nanoparticle, but it is Nanomedicina (CIBER-BBN), Barcelona 08193, Spain specifically due to the selective binding capacity of the A3C8 Elisa Mazzega − Laboratory for Environmental and Life nanobody. The generation of antiricin A3C8-GFP-H6 nano- Sciences, University of Nova Gorica, Nova Gorica 5000, particles opens a wide spectrum of applications in nano- Slovenia medicine, as they may be used as diagnostic agents or be Alejandro Sánchez-Chardi − Servei de Microscòpia, applied prior contact with the toxin to provide passive Universitat Autònoma de Barcelona, Barcelona 08193, immunity. Finally, these materials could be administered as a Spain; Departament de Biologia Evolutiva, Ecologia i neutralizing antidote to intoxicated patients when the toxin is Ciències Ambientals, Facultat de Biologia, Universitat de already circulating in the bloodstream. Similarly, the same Barcelona, 08028 Barcelona, Spain nanoparticle format could be used to agglutinate other targets Naroa Serna − Institut de Biotecnologia i de Biomedicina, by a similar engineering of the corresponding nanobodies. Universitat Autònoma de Barcelona, Barcelona 08193, ■ Spain; Departament de Genètica i de Microbiologia, CONCLUSIONS Universitat Autònoma de Barcelona, Barcelona 08193, Spain; CIBER de Bioingeniería Biomateriales y The rational design of two unrelated nanobodies (A3C8 and Nanomedicina (CIBER-BBN), Barcelona 08193, Spain EM1) fused to a H6-tagged GFP has allowed us to obtain Hèctor López-Laguna − Institut de Biotecnologia i de multivalent protein-only nanoparticles that preserve their Biomedicina, Universitat Autònoma de Barcelona, Barcelona intrinsic binding specificity. A simple recombinant DNA 08193, Spain; Departament de Genètica i de Microbiologia, technology approach is then a suitable tool to obtain highly Universitat Autònoma de Barcelona, Barcelona 08193, specific biocompatible oligomers in a single-step process. In Spain; CIBER de Bioingeniería Biomateriales y this regard, other examples of self-assembling nanobodies have Nanomedicina (CIBER-BBN), Barcelona 08193, Spain been previously developed following different strategies such as Mara Mitstorfer − University of Natural Resources and Life fusion to elastin-like peptides or naturally oligomeric proteins Sciences, Department of Chemistry, Institute of Biochemistry, (B-subunit homopentamer from verotoxin).24−26 Nonetheless, 1190 Vienna, Austria the methodology proposed in the current study does not rely Ugutz Unzueta − Departament de Genètica i de on the presence of any bulky domain that might pose structural Microbiologia, Universitat Autònoma de Barcelona, limitations or safety or immunogenic issues (due to the Barcelona 08193, Spain; CIBER de Bioingeniería presence of nonfunctional material) when moving to in vivo Biomateriales y Nanomedicina (CIBER-BBN), Barcelona 29412 https://doi.org/10.1021/acsami.1c08092 ACS Appl. Mater. Interfaces 2021, 13, 29406−29415 ACS Applied Materials & Interfaces www.acsami.org Research Article 08193, Spain; Biomedical Research Institute Sant Pau (IIB Fab = antigen-binding fragment Sant Pau), 08025 Barcelona, Spain; orcid.org/0000- FDA = food and drug administration 0001-5119-2266 FESEM = field emission scanning electron microscopy Esther Vázquez − Institut de Biotecnologia i de Biomedicina, GFP = green fluorescent protein Universitat Autònoma de Barcelona, Barcelona 08193, H6 = hexahistidine peptide Spain; Departament de Genètica i de Microbiologia, HcAbs = camelid heavy-chain antibodies Universitat Autònoma de Barcelona, Barcelona 08193, IgG = immunoglobulin G Spain; CIBER de Bioingeniería Biomateriales y IPTG = isopropyl β-D-1-thiogalactopyranoside Nanomedicina (CIBER-BBN), Barcelona 08193, Spain SDS = sodium dodecyl sulfate Complete contact information is available at: scFv = single-chain variable fragment https://pubs.acs.org/10.1021/acsami.1c08092 PAGE = polyacrylamide gel electrophoresis VHH = variable heavy chain Author Contributions ● The manuscript was written through contributions of all authors. 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