Biofabrication PAPER • OPEN ACCESS You may also like - Experience in radiological risk assessment All-in-one biofabrication and loading of of a surface waste disposal facility in Chiinu, Moldova recombinant vaults in human cells Shulan Xu, Ryk Kos, Björn Dverstorp et al. - Effect of materials and manufacturing on the bending stiffness of vaulting poles To cite this article: Fernando Martín et al 2022 Biofabrication 14 025018 C L Davis and S N Kukureka - Recombinant protein scaffolds for tissue engineering Jerome A Werkmeister and John A M Ramshaw View the article online for updates and enhancements. This content was downloaded from IP address 84.78.139.232 on 18/07/2022 at 17:55 Biofabrication 14 (2022) 025018 https://doi.org/10.1088/1758-5090/ac584d Biofabrication PAPER All-in-one biofabrication and loading of recombinant vaults in OPEN ACCESS human cells RECEIVED 9 February 2021 Fernando Martín1,7, Aida Carreño1,8, Rosa Mendoza1,2, Pablo Caruana3, Francisco Rodriguez3, REVISED Marlon Bravo4,5, Antoni Benito4,5, Neus Ferrer-Miralles1,2,6, M Virtudes Céspedes3,∗ 15 February 2022 and José Luis Corchero1,2,6,∗ ACCEPTED FOR PUBLICATION 1 24 February 2022 Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain 2 PUBLISHED CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, 08193 Barcelona, Spain 3 9 March 2022 Grup d´Oncologia Ginecològica i Peritoneal. Institut d’Investigacions Biomédiques Sant Pau, Hospital de Santa Creu i Sant Pau, 08041 Barcelona, Spain 4 Laboratori d’Enginyeria de Proteïnes, Departament de Biologia, Universitat de Girona, 17003 Girona, Spain Original content from 5 this work may be used Institut d’Investigació Biomèdica de Girona Josep Trueta, (IdIBGi), 17190 Salt, Spain 6 under the terms of the Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain Creative Commons 7 Present address: Nanobioengineering group, Institute for Bioengineering of Catalonia (IBEC), Helix building, C/ Baldiri Reixac 15-21, Attribution 4.0 licence. Barcelona 08028, Spain. Any further distribution 8 Present address: Institut de Ciència de Materials de Barcelona, ICMAB-CSIC, Campus UAB, 08193 Bellaterra, Spain. of this work must ∗ Authors to whom any correspondence should be addressed. maintain attribution to the author(s) and the title E-mail: MCespedes@santpau.cat and jlcorchero@ciber-bbn.es of the work, journal citation and DOI. Keywords: vault particles, protein nanocages, drug delivery systems, self-assembling protein nanoparticles, eukaryotic cell factories Abstract One of the most promising approaches in the drug delivery field is the use of naturally occurring self-assembling protein nanoparticles, such as virus-like particles, bacterial microcompartments or vault ribonucleoprotein particles as drug delivery systems (DDSs). Among them, eukaryotic vaults show a promising future due to their structural features, in vitro stability and non-immunogenicity. Recombinant vaults are routinely produced in insect cells and purified through several ultracentrifugations, both tedious and time-consuming processes. As an alternative, this work proposes a new approach and protocols for the production of recombinant vaults in human cells by transient gene expression of a His-tagged version of the major vault protein (MVP-H6), the development of new affinity-based purification processes for such recombinant vaults, and the all-in-one biofabrication and encapsulation of a cargo recombinant protein within such vaults by their co-expression in human cells. Protocols proposed here allow the easy and straightforward biofabrication and purification of engineered vaults loaded with virtually any INT-tagged cargo protein, in very short times, paving the way to faster and easier engineering and production of better and more efficient DDS. 1. Introduction delivery to the desired biological site. In this line, a perfect nanocarrier should be able to deliver its cargo Current therapies based on the administration of at the right moment, place and concentration. Thus, recombinant proteins in their native form usually the use of smart drug delivery systems (DDSs), act- face several drawbacks, such as short in vivo half-life, ing as a depot for high therapeutic concentrations and low membrane permeability, rapid elimination from providing a solubilizing and protective environment blood stream, and undesired side effects caused by is a promising strategy [1–3]. Nanoparticles (NPs) the multiple or high doses administered in order to have been deeply explored as DDS, either for local reach the desirable concentration in the target cell or release, where they are retained at the site of deliv- organ. As a result, these proteins need to be protec- ery, or for systemic delivery, increasing the circulation ted, typically by using nanocarriers that, at the same lifetime. NPs with self-assembly properties, good sta- time, can be engineered to improve their targeted bility and specificity have already been used clinically © 2022 The Author(s). Published by IOP Publishing Ltd Biofabrication 14 (2022) 025018 F Martín et al to provide targeted cellular/tissue delivery of chemo- confer vaults specific targeting by adding a tag at the therapeutics, to improve drug bioavailability, to sus- C-terminus of the MVP monomer. Such recombinant tain drug effect in target tissue and for diagnostic vaults expose these peptides on their exterior surface. purposes [4, 5]. This targeting strategy has been successfully applied Protein nano-DDSs are protein structures formed to target cancerous cells [14]. Finally, vaults can be by the assembly of multiple copies of one or several internalized in vitro via macropinocytosis or phago- different proteins. Nature already offers such func- cytosis [15]. Taking into account all these properties, tional macromolecular structures that can be eas- vaults have become one of the most promising nan- ily manipulated for nanobiotechnology applications. ovehicles as controlled DDS due to all their advant- Such naturally occurring structures, as virus capsids, ages (particle size, lumen large enough to encapsu- bacterial microcompartments or ferritin cages, serve late hundreds of protein molecules, protection of the as excellent templates for functional biomaterials with cargo molecules from external proteases, biodegrad- precise architectures, unattainable by synthetic pro- ability, biocompatibility, and non-immunogenicity). cesses [3, 6, 7]. Advantages of protein NPs for drug In this context, recombinant vaults have already been delivery applications include their abundance in nat- successfully explored for vaccination [16–18], pro- ural sources, biocompatibility, biodegradability, easy teins delivery [13, 19], targeting of cell surface recept- synthesis process, and cost-effectiveness. In contrast, ors [14] and delivery of therapeutics for lung cancer other particulate systems like metallic NPs show sev- [16] or glioblastoma [20]. eral drawbacks like potential toxicity, large size, accu- Current production of recombinant vault NPs mulation, or rapid clearance from the body. In addi- is mainly performed in Spodoptera frugiperda (Sf 9) tion, protein-based NPs offer the opportunity for insect cells [12], where expression of only MVP pro- surface modification by standard genetic engineer- tein is capable of directing the assembly of vault- ing techniques, or by conjugation of other protein/s like particles on polyribosomes [21]. However, this and carbohydrate ligands. The regular arrangement approach is complex and costly for industrial scale of protein subunits within protein cage structures applications. allows for engineering of specific regions and surfaces On the other hand, in order to develop an eco- (inner and/or outer) of the cage. Protein engineering nomically competitive platform based on recom- has been extensively used to redesign structure and binant vaults, efficient down-stream processes need function, yielding particles with very narrow size dis- also to be set-up and optimized, since vault size tributions and multiple functionalities [8, 9]. In this complicates its purification. Current vault purific- context, eukaryotic vaults have emerged as an attract- ation protocol (based in several ultracentrifuga- ive nanocarrier for diverse types of biomolecules. tion steps and additional gradient centrifugations) Vaults are ribonucleoprotein NPs large enough is complex, tedious and labor intensive [12]. Thus, to encapsulate therapeutic molecules, and constitute much effort is being devoted to the development a new and attractive approach for DDS as their use of faster and easier down-stream procedures for could reduce systemic side effects and immunogeni- recombinant vaults, mainly focused in the substi- city. This feature, together with its homogeneity and tution of centrifugation by special chromatographic versatility to be modified and specifically delivered columns. to target cells, make vaults powerful nano-DDSs. In order to offer more efficient and fast up-stream These nanocapsules are the largest known ribonuc- and down-stream strategies for vaults production and leoprotein particles (∼40 × 70 nm, 13 MDa) found purification, we describe in this work for the first time in eukaryotic cells. Vaults show a hollow, barrel- the production of recombinant vaults in human cells shaped structure with two protruding caps and an by means of transient expression of a His-tagged MVP invaginated waist, based on a hierarchical protein protein (MVP-H6). This strategy also allows, by co- self-assembly [10, 11]. Analysis of eukaryotic vaults expressing MVP-H6 with INT-tagged cargo proteins, revealed that each particle is composed by multiple the spontaneous NPs biofabrication and their loading copies of three proteins. Among them, the ‘major within the producing cell factory. Combined with the vault protein’ (MVP, 96 kDa) makes up 75% of the development of immobilized metal affinity chroma- vault mass. Studies of recombinant vaults produced tography (IMAC)-based purification methods, our by MVP expression in insect cells confirmed that all approach renders a straightforward, ‘all-in-one’ pro- the information needed to form the vault is encoded cedure for easy and fast biofabrication of ‘ready-to- by this protein [12]. use’ loaded DDS. One interesting feature of vaults is their ability to encapsulate virtually any cargo protein, by two 2. Material and methods independent strategies: (a) fusing the cargo protein to the N-terminus of MVP protein or (b) fusing the 2.1. Design, synthesis and cloning of recombinant cargo protein to the INT peptide (a vault targeting genes peptide from the C-terminus of the vault interact- The sequence of the human major vault protein ing protein VPARP) [13]. Moreover, it is possible to (MVP, UniProt Q14764, MVP_HUMAN) was used 2 Biofabrication 14 (2022) 025018 F Martín et al to design the recombinant MVP-H6 gene. At the 2.3. Cell fractionation 3′ end of its native sequence, extra triplets encod- Cells were harvested, five days post-transfection ing a six-histidine tag (6xHis) were added for fur- (unless otherwise stated), by centrifugation ther purification purposes. This sequence was cloned (15 000 rpm, 10 min). Supernatants were discarded, into the pTriEx1.1-Hygro vector (Novagen, cat. n◦ and cell pellets were resuspended with phosphate 70928-3, 6951 bp), between NcoI and EcoRI restric- buffered saline (PBS) supplemented with a protease tion sites. This expression vector enables optimal pro- inhibitor cocktail (cOmplete, EDTA-free, Roche Life tein expression in bacterial, insect and mammalian Sciences, ref. 05056489001) and kept at −20 ◦ C until cells from a single plasmid. The resulting plasmid cell lysis. To obtain soluble and insoluble fractions, pTriEx1.1-MVP-H6 encodes the protein MVP-H6, samples were lysed by gentle sonication. Lysates were with 899 aminoacids in length and a molecular weight centrifuged (15 000 rpm, 15 min) and supernatants of 100.1 kDa. containing soluble proteins were separated from pel- With the aim of testing the putative encap- lets (insoluble fraction), which were further resus- sulation of a model protein within vault nano- pended in PBS. capsules, an ‘enhanced green fluorescence protein’ (eGFP) was used. For that, the INT domain sequence 2.4. SDS-PAGE and western blot analyses was fused at the 3′ end of the eGFP gene. The To detect and quantify the recombinant proteins, INT domain is defined as the 163-aa region of SDS-PAGE was performed by using TGX Stain- the C-terminus (amino acids 1562-1724) of poly- Free™ FastCast™ acrylamide 12% (Bio-Rad, ref. 161- (ADP-ribose) polymerase 4 (PARP4 protein, UniProt 0185), and further visualization of proteins with a Q9UKK3, PARP4_HUMAN), which is the smallest ChemiDoc™ Touch Imaging System (Bio-Rad). In region identified for PARP4 interaction with MVP some cases, further western-blot analyses were per- protein in natural vaults. This GFP-INT sequence formed. To detect MVP-H6 protein, a rabbit poly- was also cloned into pTriEx1.1-Hygro vector between clonal anti-MVP (Abcam, ref. Ab90009), or anti-His NcoI and EcoRI restriction sites. The resulting plas- mouse monoclonal antibodies (from GenScript, ref. mid pTriEx1.1-GFP-INT encodes the protein GFP- A00186-100, or from Clontech, ref. 631212), were INT, with 401 aminoacids in length and a molecular used as primary antibodies. GFP-INT fusion pro- weight of 45.2 kDa. tein was detected with an anti-GFP rabbit polyclonal All these genes were synthesized and cloned by (Santa Cruz Biotechnology, ref. sc-8334). GeneArt® (Life Technologies), and Escherichia coli MVP-H6 protein amounts were densitometric- DH5α strain was used for the maintenance and amp- ally estimated by comparison, after SDS-PAGE and lification of the expression plasmids. western-blot analyses, with known amounts of a control His-tagged protein produced and purified 2.2. Recombinant protein expression in-house, and quantified by the bicinchoninic acid Recombinant MVP-H6 and GFP-INT proteins were (BCA) method. Samples and standards to be quant- produced in a human cell line by transient gene itatively compared were run in the same gel and pro- expression (TGE). Briefly, the suspension-adapted cessed as a set. Densitometric analyses of the immun- human embryonic kidney (HEK) cell line FreeStyle oreactive bands were performed with the Image Lab™ 293F (Invitrogen, Life Technologies, ref R790-07) was software (version 5.2.1., Bio-Rad). used to produce the proteins by polyethylenimine (PEI)-mediated transfection and further TGE. Trans- 2.5. Protein purification fection conditions for this cell line had been previ- Purification of MVP-H6 protein was carried out from ously set and optimized [22] at 1 µg DNA ml−1 of soluble fractions of transfected HEK 293F cells by culture and a ratio DNA:PEI of 1:3 (w/w). Valproic commercial magnetic particles (MPs) (‘Dynabeads acid (VPA) was added to cells (at 4 mM final con- His-tag isolation and Pull-down’, Novex, Life Tech- centration) 4 h post-transfection in order to improve nologies, ref. 10103D), a platform based on IMAC recombinant protein expression. In co-expression chemistry. These MPs are superparamagnetic beads experiments, both plasmids pTriEx1.1-MVP-H6 and that use a cobalt-based IMAC chemistry to selectively pTriEx1.1-GFP-INT were mixed at different ratios capture His-tagged proteins. MPs were separated with (before their mixing with PEI), always maintaining a magnetic separator rack (New England Biolabs, the final amount of 1 µg plasmid DNA ml−1 and a ref. S1506S). Prior to purification, MPs were washed ratio DNA:PEI of 1:3 (w/w). twice with PBS. Then, MPs were mixed with soluble To check protein expression, 1 ml samples from fractions containing MVP-H6 and/or GFP-INT pro- cultures were taken at different time-points after teins, and incubated on a roller for 15 min at room transfection, centrifuged (13 400 rpm, 10 min) and temperature. After protein capture, MPs were mag- after that, supernatants were discarded. Cell pellets netically immobilized and supernatant was collec- were resuspended in Laemmli buffer in order to ana- ted (flow-through fraction, FT). Subsequently, MPs lyze them by sodium dodecyl sulfate polyacrylamide were washed with PBS, placed on the magnet and the gel electrophoresis (SDS-PAGE). supernatant collected as wash (W) fraction. Finally, 3 Biofabrication 14 (2022) 025018 F Martín et al MVP-H6 protein was eluted from MPs with imidazole vaults containing the same concentration of GFP- 500 mM in PBS and collected as eluted (EL) frac- INT and incubated for 2 or 4 h at 37 ◦ C in complete tion. Eluted samples were dialyzed against PBS, used medium without phenol red. After incubation, cells as storage buffer. Purified vaults were stored in PBS at were washed with cold PBS, trypsinized and centri- 4 ◦ C until use. fuged. Cells were resuspended in 0.5 ml of cold PBS and analyzed by flow cytometry. Ten thousand events 2.6. Physico-chemical characterization of were acquired in a Novocyte flow cytometer (Acea recombinant vaults Biosciences, Inc.). Samples were analyzed using the Before transmission electronic microscopy (TEM), NovoExpress software (Acea Biosciences, Inc.). Res- samples containing purified MVP-H6 were dialyzed ults are shown as mean and standard error of the in dialysis cassettes (Slide-A-Lyzer 3.5 K MWCO mean (SEM) of two independent samples. Dyalisis Cassettes, Thermo Scientific, ref. 66330) against buffer A (Tris-HCl 50 mM pH 8; NaCl 75 mM; 2.9. Cytotoxicity of recombinant vaults MgCl2 0.75 mM). To visualize recombinant vaults Using the ovarian cancer model OVCAR-8 cell line, through TEM, samples (5 µl) were prepared by cytotoxicity of recombinant vaults was analyzed as absorption onto Cu-C grids for 2 min at room tem- follows. OVCAR8 cells were exposed for 48 h to differ- perature. Following sample adsorption, grids were ent concentrations (up to 0.8 µM) of purified MVP- dried on filter paper, covered for 2 min with 5 µl of 2% H6 vault-like structures. After that, cell viability was uranyl acetate at room temperature and dried prior to determined using the Cell Proliferation Kit II (XTT) viewing in a JEOL JEM 1400 microscope. (Roche, ref. 11465015001), following the manufac- Size distribution of vault-based NPs was meas- turer’s specifications. Absorbance was measured at ured by dynamic light scattering (DLS) at 633 nm 450 nm on a spectrophotometer TECAM 2000. Data wavelength, combined with non-invasive backscatter are expressed as cell viability relative (in percentages) technology in a DLS analyzer (Zetasizer Nano ZS, to control cells (cells not exposed to recombinant pro- Malvern Instruments Ltd, Malvern, U.K.). Disper- tein). Results are shown as mean and SEM of two sions of purified NPs were filtered before DLS analysis independent samples. to avoid putative interferences by remaining traces of MPs used for purification. Mean value of three 2.10. MVP-H6 protein stability in human serum measurements was taken as the hydrodynamic NP Stability of MVP-H6 NPs in human serum was also diameter. explored. For that, purified vaults were diluted in commercial human serum (Sigma, ref. S2257-5ML) and immediately incubated at 37 ◦ C in a DLS ana- 2.7. GFP-INT protein expression and spontaneous lyzer. At different times (0, 5, 10, 15, 30 and 45 min) encapsulation DLS measurements (by triplicate) were taken to fol- Expression and spontaneous encapsulation of GFP- low NPs size. As controls, same experiment was per- INT protein inside MVP-H6 based vaults was formed but using phosphate buffer as vaults diluent checked by co-transfecting HEK 293F cells with both or using vaults diluted in serum but kept at room pTriEx1.1-MVPH6 and pTriEx1.1-GFPINT expres- temperature. sion vectors as described below. As controls, transfec- tions with only one of the plasmids were performed. 2.11. Statistical analysis Five days post-transfection, recombinant proteins The normal distribution of the data was tested using were purified following the magnetic procedure pre- the Kolmogorov-Smirnov test. Multiple comparis- viously described. Finally, samples obtained during ons with Kruskal–Wallis (SPSS software, version 19.0, the process were submitted to SDS-PAGE and west- IBM, New York, NY, USA) were used to compare ern blot analyses, developed with monoclonal Abs cytotoxicity, and the pair-wise Student’s t-test (Excel anti-His or a polyclonal Ab anti-GFP. software, Microsoft Corporation), was used to com- pare NP internalization. The quantitative data of the 2.8. GFP-INT cell internalization mediated by experiments is reported as mean ± SEM. Differences recombinant vaults among groups were considered significant at p < 0.05, Internalization of vaults loaded with GFP-INT in and relevant divergences were labeled as ∗ p < 0.05, ∗∗ OVCAR-8 ovarian cancer cells was explored, by p < 0.01. reproducing experiments described by Galbiati et al [23]. This cell line is representative of high- 3. Results grade serous ovarian cancer, the most common and deadly form of ovarian cancer. OVCAR-8 cells 3.1. MVP-H6 protein expression and solubility (7.5 × 104 ) were seeded in a 24-well plate and incub- Recombinant MVP-H6 protein was produced in HEK ated overnight with 0.5 ml Roswell Park Memorial 293F cells by means of PEI-mediated transfection Institute (RPMI) complete media. Then, they were of plasmid pTriEx1.1-MVP-H6, and further TGE. treated with 12 nM GFPH6 or with recombinant Expression and accumulation of MVP-H6 into cells 4 Biofabrication 14 (2022) 025018 F Martín et al Figure 1. Expression levels and solubility of MVP-H6 protein expressed in HEK 293F cells. Expression of MVP-H6 protein in transfected HEK 293F cells (panels (a) and (b)) was monitored at days 0 (before transfection, D0) and two, four and five days post-transfection (D2, D4 and D5 lanes). Cells were analyzed by SDS-PAGE and further western blot detection with either a polyclonal antibody against MVP protein (panel (a)) or a monoclonal antibody anti-His tag (panel (b)). Effect of VPA on MVP-H6 expression was also checked (panel (c)). For that, duplicates of untreated (C1, C2) and VPA-treated (V1, V2) cultures taken two and five days post-transfection were analyzed by western blot developed with a monoclonal anti-His tag. Finally, effect of VPA on MVP-H6 solubility was explored (panel (d)) by comparing soluble (S) and insoluble (I) fractions of untreated (C1 and C2) of VPA-treated (V1 and V2) cultures taken 5 d post-transfection and analyzed by western blot developed with a monoclonal anti-His tag antibody. were checked by SDS-PAGE and western-blot of of transfected cells taken at two and five days total cell pellets taken at two, four and five days post-transfection, results showed that cultures treated post-transfection, showing a progressive accumula- with VPA produced 5.4- and 3.9-fold more pro- tion of the recombinant MVP-H6 protein within the tein than untreated cells, at two and five days post- cells (figures 1(a) and (b)). This protein, as expected, transfection respectively (figure 1(c)). Solubility of is recognized by antibodies directed to MVP protein any recombinant protein is of paramount importance (figure 1(a)) or to the His-tag (figure 1(b)), in a band regarding its putative application. For this reason, corresponding to the expected MVP-H6 monomer of solubility of recombinant MVP-H6 protein was ana- 100 kDa. In sample of day 0 (cells before transfec- lyzed. HEK 293F cells were transfected and treated tion) no endogenous MVP protein is detected. This with VPA, or kept untreated as a control. Five days is indicative that, eventhough human cells contain post-transfection, cell pellets were lysed and fractions endogenous MVP protein, they would only repres- analyzed by SDS-PAGE and further western blot. Res- ent a small percentage (not detected by the anti-MVP ults (figure 1(d)) showed that MVP-H6 protein is pro- protein antibody) compared to the amount of recom- duced mainly in its soluble form. In untreated, con- binant MVP-H6 protein. trol cells, 90.4 ± 0.9% of the protein remained within In an attempt to improve protein yield, effect of the soluble fraction, while in VPA-treated cells, the VPA was checked. After analyzing total cell pellets percentage of soluble MVP-H6 was of 86.2 ± 0.9%, 5 Biofabrication 14 (2022) 025018 F Martín et al Figure 2. Magnetic purification of MVP-H6 protein. Initially, 1.2 mg of MPs and 40 µg of MVP-H6 (2 ml of initial sample, (I) were used. Flowthrough (F) and wash (W) fractions were also of 2 ml. Captured MVP-H6 was eluted (EL) in a final volume of 0.5 ml. Samples were analyzed by SDS-PAGE and TGX visualization (panel (a)) and western blot developed with a monoclonal antibody against His-tag (panel (b)). To optimize the efficiency of the process, a second experiment (panel (c)) was performed in which 30 µg of MVP-H6 protein were used as initial sample and mixed with 1.2, 2.4 or 4.8 mg of MPs. Samples were analyzed by western blot developed with a monoclonal antibody against His-tag. Numbers indicate the percentage of protein found in each sample according to densitometric analysis of the membranes. indicating that increase in production due to VPA to a significant degree of purity. This indicates that addition does not have a significant effect on protein His-tag is correctly expressed and exposed, and able solubility. to interact with metal ions on the MPs surface. In Finally, amounts of soluble MVP-H6 protein were addition, no relevant proteins were detected in the estimated by western-blot. These amounts were of ‘wash’ fraction, indicating that capture through the 20 ± 0.3 µg ml−1 or 86 ± 5 µg ml−1 in untreated His-tag is highly specific. However, when analyzing or VPA-treated cells, respectively. VPA addition rep- the corresponding western blot (figure 2(b)), it was resents a 4.3-fold increase, in agreement with the clear that a significant percentage of the initial MVP- previously detected increase. Taking all these res- H6 protein still remained in the ‘flow-through’ frac- ults together, addition of VPA 4 h post-transfection tion, which indicates that the amount of MPs used was included in the production protocol for further was not enough to capture all the initial His-tagged experiments. protein. To address this issue and further improve the process efficiency, the experiment was repeated, 3.2. MVP-H6 protein purification mixing a constant initial amount of 30 µg of MVP- MVP-H6 protein was purified by means of MPs, using H6 protein with increasing amounts (1.2, 2.4 and as starting material soluble fractions of transfected 4.8 mg) of MPs. Under these conditions, 1.2 mg of HEK 293F cells. In a preliminary experiment, sol- MPs was again unable to capture the total amount uble fraction containing 40 µg of MVP-H6 was mixed of initial MVP-H6, rendering in the ‘eluted’ fraction with 1.2 mg of MPs. Samples (initial, flow-through, only a 43% of the initial protein. However, when wash and eluted) obtained during the process were increasing the amount of MPs to 2.4 and 4.8 mg, analyzed by SDS-PAGE and western-blot (figure 2). percentages of MVP-H6 recovered increased to 56% TGX-based visualization (figure 2(a)) showed that and 96%, respectively (figure 2(c)). Taking all these MVP-H6 can be captured by MPs, and further eluted results together, we could estimate that 5 mg of 6 Biofabrication 14 (2022) 025018 F Martín et al Figure 3. Nanoarchitecture of MVP-H6 based vaults. Transmission electron microscopy (TEM) micrographs (panel (a)) and DLS (panel (b)) of magnetically purified MVP-H6 protein. Both techniques show that MVP-H6 monomers self-assemble rendering nanocages with size (between 70 and 90 nm according to microscopy images) and shape (barrel) indistinguishable from that of native vaults or recombinant vaults expressed in other cell factories. MPs should be able and enough to capture 30 µg low polydispersity indexes obtained (0.28 ± 0.007), of MVP-H6 protein, with a high degree (>85%) of also indicative of their suitability for DLS ana- purity. lysis. The mean hydrodynamic diameters of our vaults, expressed as zeta-average mean size (as the 3.3. Physico-chemical characterization of primary parameter obtained by the technique) were recombinant vaults of 92 ± 0.7 nm. Purified MVP-H6 protein was visualized by TEM to check if this protein displays self-assembly properties 3.4. GFP-INT protein expression and form vaults (or ‘vault-like’ structures). Results To determine if these NPs were able to encapsu- (figure 3(a)) confirmed the presence of NPs, almost late a cargo protein, the fusion protein GFP-INT identical in shape (a barrel-like nanocage formed by was used as a model. For that, co-transfections of two symmetrical halves) and size (∼70 × 40 nm) HEK 293F cells with plasmids pTriEx1.1-MVP-H6 to the native or to recombinant vaults produced and/or pTriEx1.1-GFP-INT were performed. In a first either in insect cells [12] or in Pichia cells [24]. This experiment, both plasmids were mixed at a ratio result confirms that MVP-H6 monomer produced 1:1 (0.5 µg of each plasmid ml−1 , keeping the final in mammalian cells is correctly folded and main- amount of 1 µg DNA ml−1 ). With this strategy, we tains its self-assembling properties to form vault NPs. wanted to check (a) the possibility to express both The size of these nanocages was estimated by DLS proteins simultaneously, and (b) the effect of the (figure 3(b)). When checking the size distribution amount of each plasmid in the protein yield obtained. of samples, results showed the presence of one main Cell pellets were collected five days post-transfection peak (corresponding to more than 95% of the sample and analyzed by western-blot either with antibodies intensity). Moreover, data showed a unique, single against His-tag or GFP proteins. Results (figures 4(a) exponential decay in the auto-correlation functions. and (b)) showed that both proteins are simultan- Taken together, these results are indicative of the eously produced when co-transfecting cells with both presence of a single, main population. The samples plasmids. Decreasing in a 50% the amount of each were highly monodisperse as determined by the plasmid does not result in a directly proportional 7 Biofabrication 14 (2022) 025018 F Martín et al Figure 4. Co-expression MVP-H6 and GFP-INT proteins in HEK 293F cells. Co-expression of MVP-H6 and GFP-INT genes was driven by transfection of HEK 293F with the indicated concentrations of their respective plasmids (from 1 to 0.05 µg ml−1 ). Five days post-transfection, cells were analyzed by SDS-PAGE and further western blot developed with either monoclonal antibodies anti-His tag (panels (a) and (c)) to detect MVP-H6 protein, or with a polyclonal antibody against GFP (panels (b) and (d)) to detect GFP-INT. decrease in the yield of each protein: productions developed with a monoclonal anti-His (figure 5(a)) when co-expressing both proteins yielded >70% of showed that, as checked before, MPs used were each protein compared to controls (yields of pro- enough to capture all the MVP-H6 protein present in tein expressed individually in single-plasmid trans- the samples, that is further eluted by imidazol addi- fections). tion. Most important, the presence of GFP-INT did In a second experiment, we checked the pos- not interfere in the interaction of vaults with MPs, sibility to further modulate the expression of each since vaults were correctly purified when GFP-INT protein by varying plasmids ratio. For that, we was present in the initial sample. increased amount of pTriEx1.1-MVP-H6 (from 0.5 to On the other hand, results from membranes 0.95 µg ml−1 ) and decreased amount of pTriEx1.1- developed with a monoclonal anti-GFP (figure 5(b)) GFP-INT (from 0.5 to 0.05 µg ml−1 ). Results showed, showed that when GFP-INT was expressed alone, it again, that both proteins were correctly expressed did not interact with MPs and was mainly found in simultaneously (figures 4(c) and (d)). Transfecting the FT fraction. However, when both MVP-H6 and cells with only 0.05 µg ml−1 of plasmid pTriEx1.1- GFP-INT proteins were co-expressed and present in GFP-INT was enough to direct expression of GFP- the initial sample, a significant percentage (54%) of INT protein at significant levels (31.6% compared to GFP-INT was found in the eluted fraction. Since GFP- those obtained by transfecting cells with 0.5 µg ml−1 ). INT has no His-tag and therefore it cannot directly Results of MVP-H6 protein (figure 4(c)) confirmed interact with MPs (as seen before), results indicate those of previous experiment: transfecting with only that GFP-INT protein was co-purified through its 0.5 µg of pTriEx1.1-MVP-H6 plasmid ml−1 still interaction with MVP-H6 protein. rendered >70% of protein compared to transfection However, almost 45% of GFP-INT protein still using 0.95 µg of pTriEx1.1-MVP-H6 ml−1 . remained in the FT fraction, indicating that vaults present in the sample were not enough to encapsu- 3.5. Encapsulation and co-purification of GFP-INT late all the cargo protein. With the aim to improve protein the efficiency of the encapsulation process, purifica- Soluble fractions from co-transfection experiments tion protocol was repeated using a sample from a co- and controls (see above) were used to check the transfection in which plasmids pTriEx1.1-GFP-INT putative encapsulation of GFP-INT into vaults. For and pTriEx1.1-MVP-H6 had been used at 0.05 and that, MVP-H6 protein was purified by using MPs, to 0.95 µg ml−1 , respectively. With this strategy, we check if GFP-INT protein was co-purified through expected to increase the ratio vault versus cargo pro- its interaction (mediated by the INT peptide) with tein, thus favoring the complete encapsulation of all MVP-H6 protein. Samples of each fraction harves- the synthesized GFP-INT protein. Under these new ted during purification were analyzed by SDS-PAGE conditions, cargo protein found in the FT fraction and western-blot (figure 5). Results from membranes decreased to 14% (data not shown), corroborating 8 Biofabrication 14 (2022) 025018 F Martín et al Figure 5. Co-purification of MVP-H6 and GFP-INT proteins in HEK 293F cells. Magnetic co-purification of MVP-H6 and GFP-INT proteins was performed as described in section 2. For each purification, initial (I), flowthrough (F), wash (W) and eluted (E) fractions were obtained and analyzed by western blot with a monoclonal antibody anti-His tag (panel (a)) or a polyclonal antibody against GFP (panel (b)). Symbols (+) and (−) indicate presence or absence of each protein in each sample. Figure 6. Cell internalization of GFP-INT mediated by vaults. The ability to internalize in OVCAR-8 cancer cells for a control protein GFPH6 (light grey bars) and for GFP-INT encapsulated within MVP-H6 based vaults (dark grey bars) was checked. Results showed here correspond to mean and SEM of two independent samples. that after a careful adjustment and optimization of 3.6. GFP-INT cell internalization mediated by ratios vault/cargo protein, it should be possible to recombinant vaults encapsulate within vaults all the cargo protein sim- The ability to internalize in cancer cells of a control ultaneously produced within the cell factory. protein GFPH6 and of GFP-INT encapsulated within Finally, we explored the amount of GFP-INT MVP-H6 based vaults was checked by means of flow cargo protein entrapped by purified vaults. By cytometry technique. Results (figure 6) showed that means of SDS-PAGE and further western-blots the control GFPH6 protein is hardly internalized into (using as standards a commercial MVP protein and OVCAR-8 cells during the time of the assay. On the a recombinant GFP), we estimated that 1 mg of other hand, when the counterpart GFP-INT protein MVP-H6 protein self-assembled as vault could encap- is encapsulated within vaults, it is able to internal- sulate approx. 15 µg of GFP-INT. ize in a time-dependent fashion. According to this, 9 Biofabrication 14 (2022) 025018 F Martín et al Figure 7. Vault cytotoxicity. Vaults cytotoxicity in OVCAR8 cancer cells was checked after 48 h of incubation with different MVP-H6 protein concentrations. Data are expressed as cell viability relative (in percentages) to control cells (cells not exposed to recombinant protein), and correspond to the mean and SEM of two independent samples. the percentage of OVCAR-8 cells that become fluor- 750 nm and 100 nm for peaks ‘one’ and ‘two’, respect- escent due to GFP-INT internalization increases to ively. Such sizes seem to stabilize and remain con- approximately 12% and 24% after 2 and 4 h of incub- stant after 30–45 min of incubation at 37 ◦ C. Inter- ation, respectively. This confirms the ability of vaults estingly, vaults diluted in human serum, but kept at to enter cells (even without any specific cell targeting, room temperature, maintain their hydrodynamic dia- as in our case) and thus, the well described functional- meter even after 45 min of incubation. ity [16–20] of these nanocarriers as an efficient DDS. 4. Discussion 3.7. Cytotoxicity of recombinant vaults Cytotoxicity of MVP-H6 based vaults was checked Currently, many efforts are devoted to the develop- by determining cell viability of OVCAR8 cells after ment of new drug delivery strategies to overcome the their exposure for 48 h to different concentrations shortcomings of ongoing drug-based therapies such of recombinant MVP-H6 protein. Results (figure 7) as poor biocompatibility, lack of targeting specificity, showed no cytotoxic effect after 48 h of incubation, inability to control release and low efficiency. In this indicating that self-assembling process does not con- context, eukaryotic vaults are a very promising DDS fer any toxicity to proteins that, like MVP-H6, are as they could overcome most, if not all, of these draw- endogenous to human cells and, therefore, intrinsic- backs. ally innocuous. Recombinant vaults are produced as empty pro- tein nanocapsules and have been extensively explored 3.8. Stability of recombinant vaults in human in the last years as efficient DDS due to their biocom- serum patibility, large inner lumen, simple composition Stability of MVP-H6 based vaults when incubated (multiple copies of only one self-assembling pro- in human serum under physiological conditions was tein), and the possibility (by means of easy, stand- checked by following NPs size by DLS technique. Res- ard genetic engineering procedures) to tailor their ults (figure 8) showed that, when diluted in phosphate sequence/structure. Recombinant vaults are currently buffer, NPs basically remain unchanged and main- produced in Sf 9 insect cells [12], one of the few tain their original hydrodynamic diameter. On the eukaryotic cells lacking endogenous vaults. This contrary, when incubated in human serum, two sim- baculovirus-insect cell system is rather complex and ultaneous phenomena seem to occur. In one hand, costly for industrial scale applications. Obtention original structure (of about 195 nm, ‘peak one’) rap- of a recombinant baculovirus requires tedious and idly starts to dissociate, rendering a smaller particle time-consuming (3–6 months) processes, together of originally 42 nm of diameter (‘peak 2’). On the with further growing, titration and maintenance of other hand, and following incubation, both struc- the baculovirus stocks. Moreover, continuous pro- tures increase their hydrodynamic diameter up to tein production is hampered by insect cell lysis during 10 Biofabrication 14 (2022) 025018 F Martín et al Figure 8. Stability of vault-based nanoparticles in human serum. Architectonic stability of vault-based nanoparticles in human serum at 37 ◦ C was explored by DLS analysis. After dilution in human serum, DLS measurements were taken at different times (panel (A)). As controls, samples diluted in serum but kept at room temperature (panel (B)) and diluted in phosphate buffer at 37 ◦ C (panel (C)) were also analyzed. infection. Release of intracellular proteins from lysed without further genetic selection of the encoding cells, or removal or inactivation of progeny baculovir- DNA [32]. Key TGE features are its simplicity and uses released by budding off from infected cells, may short time (normally, days) needed for the expres- result in protein degradation by proteases and com- sion of the product, allowing the simultaneous study plicate the downstream process [25–27]. of many genes or their variants. This eukaryotic One significant change in the vault production expression system provides fully homologous post- process was the proposal of the replacement of the translational modifications to address biosafety con- Sf 9 insect cells for insect larvae, which allows higher cerns [33, 34]. productions [28], or the production of recombinant In the present work, we have successfully pro- vaults in the yeast Pichia pastoris [24]. In these two duced a His-tagged MVP protein in HEK 293F cells alternative cell factories, expression of MVP alone also by PEI-mediated transfection and further TGE. The led to the formation of intact vaults, morphologically protein was correctly produced and identified by similar to endogenous vaults isolated from other euk- antibodies against both MVP and His-tag domains. aryotes. Nevertheless, due to a general unfamiliarity In our system, and after optimization of the pro- or restricted access to insect larvae culture, or putative tocol by VPA addition to transfected cells, MVP- immune response due to dissimilar post-translational H6 production reached (according to our prelimin- modifications [29], these systems have not reached ary studies) values of approx. 80 µg ml−1 , higher widespread popularity. than those reported for P. pastoris (between 7 and As a promising alternative, we propose the pro- 11 mg l−1 ). No endogenous MVP protein was detec- duction of recombinant vaults in a human cell line ted in non-transfected cells. This suggests that, even (HEK 293F) by means of PEI-mediated transfection though human cells are known to contain endo- and further TGE. TGE is a well-suited strategy to fill genous MVP protein, this represents only a small this purpose, because of its ability to produce signific- percentage (not detected by the anti-MVP protein ant amounts of proteins within a short period of time antibody) compared to the amount of recombin- [30, 31]. TGE involves short-term protein produc- ant MVP-H6 protein. In this line, a model has been tion (typically for up to ten days post-transfection) described [21] for MVP synthesis and self-assembly 11 Biofabrication 14 (2022) 025018 F Martín et al into vaults whereby the polyribosome acts like a 3D very similar in architecture (if not exactly the same) ‘nanoprinter’ to direct the ordered translation and and size than natural human vaults. assembly of the multi-subunit vault homopolymer, In the past, INT-tagged proteins have been suc- a process referred to as ‘polyribosome templating’. cessfully encapsulated within recombinant vaults by According to this model, polyribosome would allow co-infection of Sf 9 cells with recombinant bacu- MVP monomers to self-assemble and form the vault lovirus encoding MVP and the INT-tagged cargo structure within the crowded cytoplasm environment protein [13]. Again, this approach needs the pre- despite an extremely low overall monomer concen- vious construction of the respective recombinant tration. Taking all these results together, it is reas- baculovirus. To overcome this drawback, we have onable to predict that a single vault NP would be co-expressed MVP-H6 protein with an INT-tagged mainly composed by either endogenous MVP or by GFP by co-transfection of HEK 293F cells with their recombinant MVP-H6 protein, depending on the respective encoding plasmids. As described previ- mRNA used as template in the polyribosomes. In this ously [44], in TGE procedures a percentage of the scenario, only vaults containing His-tagged MVP-H6 encoding plasmid can be replaced with ‘filler DNA’ would be purified, since our down-stream process without a proportional reduction in the volumetric relies on platforms designed to capture His-tagged yield of the recombinant protein. In this line, our res- polypeptides, being endogenous, untagged vaults dis- ults indicate that the strategy presented here allows regarded during this downstream process. to co-express both MVP-H6 and INT-cargo proteins, As described recently using the yeast P. pastoris and to modulate their yields by adjusting ratio of both as cell factory [24], expression of MVP-H6 in human encoding plasmids. Maintaining DNA total amount cells also led to the formation of vault particles, at 1 µg ml−1 during co-transfection, 0.05 µg ml−1 of with size (between 70 and 90 nm) and shape (bar- GFP-INT encoding plasmid was still enough to pro- rel) indistinguishable from that of native vaults or duce significant amounts of this protein. By this ‘all- recombinant vaults expressed in other cell factor- in-one’ approach, substitution of a small percentage ies. It is well known that NP size is a key parameter of MVP-coding DNA by a plasmid encoding for INT- affecting applicability as DDS. Even within the nano- tagged cargo proteins renders an easy, fast and effi- scale range, particle size strongly affects bioavailabil- cient way to encapsulate proteins with putative thera- ity and blood circulation time [35, 36]. Following sys- peutic or biotechnological applications. temic administration, particles with diameters below Despite the increasing interest raised by vaults 10 nm are rapidly eliminated through extravasation in the era of nanotechnology, current purification and renal clearance [37], while particles with dia- protocol is still rather complex and labor intensive meters greater than 200 nm are usually sequestered [12], requiring three ultracentrifugation steps and by the spleen and eventually removed by phago- two additional gradient centrifugations. Sucrose or cytes [38]. Particles with diameters ranging from 10 cesium chloride gradient ultracentrifugation is gen- to 70 nm can penetrate even very small capillaries erally considered appropriate for purification of dif- [39], and particles with diameters ranging from 70 ferent protein-based NPs, like virus-like particles to 200 nm demonstrate the most prolonged circula- (VLPs) [45, 46], but this approach is labor-intensive, tion times [35]. Therefore, vaults produced in mam- time-consuming and scale-restricted [47], and can be malian cells (as those expressed in other cell factor- associated with unexpected batch-to-batch variation. ies) would represent an excellent DDS candidate in Although several reports have shown that gradient terms of size parameter, since such NP dimensions ultracentrifugation can be used for VLPs purification, (between 70 and 90 nm) fall within the optimal range it provided low yields and failed to remove impurities described to interact with and internalize within tar- (including recombinant baculoviruses) from the final get cells [40, 41]. Moreover, our TEM images showed products [48]. Taking all these facts together, the need the typical ‘barrel’ structure previously described for for the development of faster and easier down-stream both natural and recombinant vaults. In this line, procedures for recombinant vaults is clear, and much when cryoelectron microscopy and single-particle effort is being devoted to such purpose. A simpler image reconstruction methods were used to determ- procedure has been reported, which however requires ine the structure of nine recombinant vaults of vari- baculovirus-infected insect larvae as starting mater- ous composition, all of them resulted in the typical ial, not easily accessible to many research laborator- vault ‘barrel’ structure, with a 48-fold rotational sym- ies [28]. More recently, vault purification was pro- metry [42]. Also, cryo-tomography images of nat- posed by means of a dialysis step and a subsequent size ural vaults in murine, simian and human cells were exclusion chromatography [23]. However, and des- morphologically very similar, suggesting that vault pite all these efforts and advances, vault purification structure is highly conserved among mammals [43]. is still performed by the original protocol based on Altogether, with such previous literature, our TEM several ultracentrifugations. and DLS results suggest that MVP-H6 proteins self- In this line, another significant result of our study assemble spontaneously to form a recombinant vault is the possibility to purify recombinant vaults by 12 Biofabrication 14 (2022) 025018 F Martín et al means of magnetic procedures, in an attempt to sub- deliver the cargo protein to cells, even in the absence stitute currently used vaults down-stream proced- of specific targeting peptides. These results confirm ures. The application of magnetic separation tech- previous ones showing that non functionalized vaults niques has received considerable attention in recent are taken up unspecifically by HeLa [13, 14] and glio- years. Magnetic separation is fast, gentle, scalable, eas- blastoma cell lines [23]. In the latter case, the pro- ily automated, can achieve separations that would cess was demonstrated to be mediated by clathrin- be impossible or impractical to achieve by other mediated endocytosis. To improve cell internalization techniques, and have demonstrated credibility in a of recombinant vaults, their engineering by adding wide range of fields, including wastewater treatment, specific targeting peptides is one possibility that can molecular biology, cell sorting or clinical diagnostics be easily explored by the methods proposed here. [49, 50]. The properties of MPs allow their select- Transfection of human cells and transient expression ive manipulation and separation in the presence of of engineered MVP proteins as proposed in this work other suspended solids, making possible to magnet- is an easy and fast strategy to obtain recombinant ically separate target proteins directly out from crude vaults to be explored as new, improved DDS. biological samples (e.g. fermentation broths, cell lys- Finally, stability of vault-based NPs in human ates, plasma, milk, whey or plant extracts). By this, the serum was addressed. Results show that vaults incub- several stages of sample pretreatment (especially cent- ated at 37 ◦ C in serum evolve from a monodisperse rifugation, filtration and membrane separation) that solution with one single NP population to the rapid are normally required to prepare samples before its appearance of some higher order structures, that sta- loading onto packed bed chromatography columns, bilize after 30 min of incubation. Such higher ordered may be eliminated. It is also possible to integrate the structures have already been observed and described disintegration and separation steps and thus shorten in vaults resulting from the overexpression of MVP the total separation time [51]. Moreover, magnetic protein in insect cells [12]. Those ordered structures separation is usually very gentle to the target proteins were referred to as ‘vaultimers’ and appeared to con- or peptides. Even large protein complexes that tend to tain three to more than a dozen half-vaults. Moreover, be broken up by traditional column chromatography these larger structures appeared to be enriched at techniques may remain intact when using more gentle higher sucrose concentrations in the gradient used for magnetic separation procedures [52]. Finally, MPs purification. In our case, when incubating purified can be used for protein concentration instead of ultra- vaults in a phosphate buffer also at 37 ◦ C, formation filtration or precipitation techniques [53] in the cases of vaultimers was not detected. All these results could where standard chromatography techniques render be indicative that complex matrixes or diluents might high volumes of diluted protein. promote or facilitate vaultimers formation. However, Following this rationale, we used commercial diluent seems not to be the only factor affecting MPs to purify recombinant vaults from lysates of this phenomenon, since incubation in human serum mammalian cells. This approach allowed us to obtain but at room temperature does not result in vault- vaults with a high degree of purity (>85%) in a imers formation. The tendency of half-vaults to self- very short time by a fast, easy protocol. However, assemble into higher order structures is supported by our results indicate that 5 mg of MPs would capture the fact that the smaller structures (around 40 nm) only 30 µg of MVP-H6 protein, a rather low cap- initially appearing rapidly grow into bigger structures ture efficiency compared to other purification sys- of up to 100 nm. Therefore, a combination of several tems like affinity chromatography. This issue need factors (that should be further explored in detail in further study and optimization in the future if such the future) seems to determine the final architecture magnetic strategy wants to substitute current down- of vault-based NPs. stream procedures for vault purification. Alternat- ively, innovative approaches based on affinity-based 5. Conclusions chromatography columns able to handle particles within the nanometric range could be explored in the In this work, we have produced for the first time in future in order to substitute current vaults purific- a human cell line recombinant NPs resulting from ation protocols. In this line, ion exchange columns the self-assembly of a His-tagged MVP protein (main Fractogel® EMD SO3 − [54] or Fractogel® EMD component of eukaryotic vaults), and a model pro- TMAE followed by a polishing step by a gel filtra- tein (GFP-INT) could be encapsulated within such tion chromatography column (Superdex 200) [28] protein-based NPs. By using an approach combin- rendered final overall purities higher than 99%. ing MPs and western blot, we proved interaction of Moreover, a two-steps protocol for vaults purification the INT-tagged GFP with vaults according to their was recently described based on a dialysis step and co-purification profile. In addition, we have proved further size exclusion chromatography [23]. that GFP-INT cell internalization is clearly promoted By using magnetically purified vaults loaded with when such protein is encapsulated within MVP-H6 the cargo protein GFP-INT, we have demonstrated based vaults, with no cytotoxicity detected for the that recombinant vaults produced in human cells can nanovehicle. Altogether, these results prompt us to 13 Biofabrication 14 (2022) 025018 F Martín et al propose human cells as cell factory, in combination Conflict of interest with well-known TGE procedure, as a promising way to produce (in a very short period of time) signific- The authors declare no commercial or financial con- ant amounts of recombinant vaults and variants of flict of interest. it. This all-in-one process in which nanovehicle and its therapeutic cargo are simultaneously produced ORCID iDs provide a new promising approach to improve pro- duction processes. Such material can be an excellent Fernando Martín  https://orcid.org/0000-0002- tool for structural studies, or to develop improved 3496-9093 DDS by addition of targeting peptides to the vault. Aida Carreño  https://orcid.org/0000-0001-7725- Further work is still needed to optimize purification 7515 protocols of such new vehicles before they finally can Rosa Mendoza  https://orcid.org/0000-0002-0488- be used as therapeutic DDS. In this context, large scale 4814 and reproducible particle purification methodologies Pablo Caruana  https://orcid.org/0000-0002-0170- need to be developed, together with the production 4355 of vaults under GMP, and the extensive toxicology, Francisco Rodriguez  https://orcid.org/0000-0001- absorption, biodistribution, pharmacokinetics, and 7909-932X excretion studies required for all new drugs. Marlon Bravo  https://orcid.org/0000-0002-0065- 9812 Data availability statement Antoni Benito  https://orcid.org/0000-0002-9776- 1003 All data that support the findings of this study are Neus Ferrer-Miralles  https://orcid.org/0000-0003- included within the article (and any supplementary 2981-3913 files). M Virtudes Céspedes  https://orcid.org/0000- 0003-2956-5833 José Luis Corchero  https://orcid.org/0000-0002- Acknowledgments 6109-144X We acknowledge the financial support to our research References in the field of vault nanoparticles as DDS received from Instituto de Salud Carlos III, through ‘Acciones [1] Qi B, Wang C, Ding J and Tao W 2019 Editorial: CIBER’. The Networking Research Center on Bioen- Applications of nanobiotechnology in pharmacology Front. 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