www.acsami.org Research Article Probing the Biosafety of Implantable Artificial Secretory Granules for the Sustained Release of Bioactive Proteins Patricia Á lamo,◆ Eloi Parladé,◆ Marianna T. P. Favaro, Alberto Gallardo, Rosa Mendoza, Luís C. S. Ferreira, Nerea Roher, Ramón Mangues,* Antonio Villaverde,* and Esther Vázquez Cite This: ACS Appl. Mater. Interfaces 2023, 15, 39167−39175 Read Online ACCESS See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. Metrics & More Article Recommendations Downloaded via 79.117.19.253 on October 11, 2023 at 07:44:39 (UTC). ABSTRACT: Among bio-inspired protein materials, secretory protein microparticles are of clinical interest as self-contained, slow protein delivery platforms that mimic secretory granules of the human endocrine system, in which the protein is both the drug and the scaffold. Upon subcutaneous injection, their progressive disintegration results in the sustained release of the building block polypeptides, which reach the bloodstream for systemic distribution and subsequent biological effects. Such entities are easily fabricated in vitro by Zn-assisted cross-molecular coordination of histidine residues. Using cationic Zn for the assembly of selected pure protein species and in the absence of any heterologous holding material, these granules are expected to be nontoxic and therefore adequate for different clinical uses. However, such presumed biosafety has not been so far confirmed and the potential protein dosage threshold not probed yet. By selecting the receptor binding domain (RBD) from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein as a model protein and using a mouse lab model, we have explored the toxicity of RBD-made secretory granules at increasing doses up to ∼100 mg/kg of animal weight. By monitoring body weight and biochemical blood markers and through the histological scrutiny of main tissues and organs, we have not observed systemic toxicity. Otherwise, the bioavailability of the material was demonstrated by the induction of specific antibody responses. The presented data confirm the intrinsic biosafety of artificial secretory granules made by recombinant proteins and prompt their further clinical development as self-contained and dynamic protein reservoirs. KEYWORDS: recombinant protein, protein engineering, self-assembling, protein materials, drug delivery ■ INTRODUCTION The potential toxicity of materials to be in contact with fabrication of supramolecular entities with increasing levels of complexity from nano to microscales. The coordination of biological interfaces is a great matter of concern in diverse cationic Zn with histidine (his)-rich peptides displayed on clinical fields.1−8 In this context, novel biodegradable and selected proteins allows their controlled cross-molecular biomimetic materials are under continuous exploration, interactions,18−20 resulting in nanoparticles or microparticles tailoring, and development to avoid undesired side damage in depending on the metal/his ratio used for coordination.21 While the patient.9−13 Aiming at the delivery of bioactive proteins, including drugs and antigens, protein-based constructs benefit Received: June 15, 2023 from the intrinsic lack of toxicity of polypeptides as universal Accepted: July 27, 2023 biomacromolecules. This is in addition to their functional and Published: August 10, 2023 structural versatility that can be adjusted by rather simple genetic engineering.14−17 Among emerging protein materials, those based on self-assembling architectonic principles allow the © 2023 The Authors. Published by American Chemical Society https://doi.org/10.1021/acsami.3c08643 39167 ACS Appl. Mater. Interfaces 2023, 15, 39167−39175 ACS Applied Materials & Interfaces www.acsami.org Research Article nanoparticles are useful as nanostructured drugs or drug from inclusion bodies by a previously described purification protocol.44 vehicles,22,23 microparticles show an intriguing amyloidal In summary, cell pellets were sonicated (8 min at 40% amplitude, 1 s architecture and slow protein-leaking properties.24−26 This ON, 4 s OFF, Branson digital sonifier, MO) and centrifuged (8228g, 30 type of protein depot resembles, in both structure and function, min). The sediment, including inclusion bodies, was washed and resuspended in a solubilization buffer (1 mM EDTA, 15 mM DTT, 6 M secretory granules of peptide hormones occurring in the guanidine hydrochloride) for protein refolding (0.18 mM EDTA, 0.5 M mammalian endocrine system27−30 in which the proteins are L-arginine, 1.9 mM reduced glutathione, 0.9 mM oxidized glutathione, 2 self-contained as mechanically stable, amyloid-like protein-only M urea in 20 mM phosphate buffer, pH 8.0). Finally, the protein was structures.28,31 Regarding both their rough and irregular surfaces dialyzed against a battery of intermediate buffers for a soft transition to and their intrinsic molecular organization, they also resemble the storage buffer (75 mM L-arginine, 233 mM sucrose, 20 mM bacterial inclusion bodies32 that also show intriguing protein- phosphate buffer, pH 8.0). The integrity of the refolded protein was releasing properties.33−35 Secretory granules and bacterial assessed via SDS-PAGE and matrix-assisted laser desorption/ inclusion bodies are considered examples of functional amyloids ionization−time of flight (MALDI-TOF). in nature.27,30,36−39 The in vitro fabrication of microparticles out of refolded protein was based on a modified version of a previously described method.22 Briefly, Such synthetic microscale secretory granules have been the microscale granules were formed by adding a molar excess of ionic observed as long-term dynamic reservoirs of functional Zn to protein samples, relative to the number of histidine residues in the proteins24,40 that are made available to the bloodstream during H6 tag. Such a tag, placed at the carboxy terminus, was then used for a the progressive disintegration of the granules after subcutaneous cross-molecular Zn-mediated anchorage between homologous poly- administration.26 The disintegration process is progressive, and peptides.18 Specifically, 30, 100, 300, or 1000 μg of soluble protein was it results in the sustained leakage of the forming protein.26 This mixed with ZnCl2 in a molar excess ratio of 1:300 (protein/cation) in event is probably mediated by a physiological dilution of Zn potassium−sodium phosphate-buffered saline with a pH value of 7.4, cations from the complexes that result in the separation of within a physiological range and optimal for in vivo studies, with a final monomeric or oligomeric polypeptides from the bulk material. protein concentration of 1 mg/mL in a final volume of 100 μL. After Such artificial granules, fabricated in the laboratory by exploiting mixing the components and allowing them to react for 10 min, the samples were centrifuged at 15,000g for 15 min to separate the the interactivity between cationic metals and histidine residues, microgranules, which remain insoluble and tend to sediment from the differ from other slow-release platforms in that the protein is self- unreacted soluble protein in the supernatant. High-resolution electron contained in the particle, in the absence of any scaffolding, microscopy images of the microparticles were obtained using field porous, or matrix-like agents to hold it.41 Using several in vitro emission scanning electron microscopy (FESEM Zeiss Merlin, and in vivo models, these emerging materials have been proved Oberkochen, Germany) operating at 1 kV and equipped with a high- useful and promising in the sustained delivery of bioactive resolution secondary electron detector. proteins in different fields, including regenerative medicine,42 In Vivo Experiments. Experiments involving animals were cancer treatment,24 or as drugs for neurodegenerative conducted through procedures according to the European Council disorders.43 Despite their potential applicability as a generic directives and approved by the Animal Ethics Committee at the Hospital de la Santa Creu i Sant Pau (procedure 9721). Male and platform with a wide spectrum of applications in clinics, the female BALB/c mice, aged 6−8 weeks and weighing between 16−27 g, potential toxicity of the system, when using elevated protein were obtained from Charles River (L’Arbresle, France) and housed in amounts, remains unexplored, and the upper threshold dosage pathogen-free conditions. The administration regimen included 1 or 2 has not been established yet. The usual working doses of such repeated doses of different amounts of microgranules (1000, 300, 100, protein-releasing materials, proven useful for many applications, or 30 μg) or phosphate-buffered saline (PBS) in the control groups. are expected to contain Zn amounts below the daily The mice were randomly allocated in each group, with doses recommended intake,21 but specific conditions and applications administered on days 0 and 21. The administration was done involving elevated local amounts of a given polypeptide drug subcutaneously in the lumbar region of the mice using a pellet of might require high dosage. In the present study, we have protein antigen granules suspended in 150 μL of PBS buffer. Mice were weighed 3 times a week to monitor for any weight loss. subcutaneously administered increasing amounts of microscale Humoral Response and Histology. On day 35 after the initial secretory granules in a mouse model to challenge the biological injection, blood samples were collected from the mice to measure the limits of the system and to explore potential adverse effects. The humoral response to the protein antigen. The collected sera were obtained results reveal the absence of systemic toxicity when up analyzed for antibody levels using the Recombivirus Mouse Anti SARS- to ∼100 mg/kg of animal weight has been administered, while CoV-2 (COVID) Spike 1 RBD IgG ELISA Kit (RV-405420 α the results confirm the bioavailability of the protein after Diagnostics, San Antonio, Texas) following the manufacturer′s administration. The whole set of data endorses artificial instructions. Mice were euthanized after day 35, and their liver, kidney, secretory granules as promising biosafe drug delivery systems and spleen were resected. The tissues were fixed in 4% paraformalde- that in their development toward clinics should not pose hyde, embedded in paraffin, sectioned, and stained with hematoxylin regulatory issues linked to toxicity. and eosin (H&E) for histological analysis, which was performed by two independent observers using an Olympus BX53 microscope. ■ MATERIALS AND METHODS Protein Synthesis and Preparation of Granules. The gene Biochemistry. The liver function and renal function were evaluated by measuring the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CRE), and uric acid (UA) encoding eRBD-H6, an extended version of the receptor binding using commercial kits (20764949322, 20764957322, 03183807190, domain (RBD) from the severe acute respiratory syndrome coronavirus and 04810716190, respectively, Roche, Basel, Switzerland) adapted for 2 (SARS-CoV-2) spike protein,44 was inserted into pET22b and a COBAS 6000 autoanalyzer (Roche, Basel, Switzerland). expressed in Escherichia coli BL21 DE3 (Novagen-Merck, Darmstadt, Statistical Analysis. Data are presented as mean ± standard Germany). Culture samples were routinely analyzed using sodium deviation. Statistical analyses were performed using GraphPad Prism dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE) and version 8.0.2 (GraphPad Software, San Diego, California). Differences Western blot with anti-His mouse monoclonal antibodies to confirm between groups were analyzed using a one-way ANOVA or Kruskal− protein production. Cell pellets were harvested and resuspended in 50 Wallis test, followed by Dunnett’s or Dunn’s multiple comparisons test, mM Tris, pH 8.0, in the presence of protease inhibitors (cOmplete respectively. Statistical significance levels were set at p < 0.05 (*) and p EDTA-Free, Roche, Basel, Switzerland). eRBD-H6 was solubilized < 0.001(***). 39168 https://doi.org/10.1021/acsami.3c08643 ACS Appl. Mater. Interfaces 2023, 15, 39167−39175 ACS Applied Materials & Interfaces www.acsami.org Research Article Figure 1. Composition of protein secretory granules. (A) eRBD-H6 amino acid sequence including the H6 tag used for both purification and metal- based clustering. At the bottom, the molecular model of the trimeric spike protein (PDB: 6VXX) is shown. The inset shows the tridimensional structure of the eRBD-H6 construct as predicted by AlphaFold2 with the H6 tag solvent-exposed region highlighted in yellow. Additional structural details can be found elsewhere.44 (B) SDS-PAGE showing eRBD-H6 resulting from IB protein refolding and from the concentrate version ready for clustering. The apparent mobility was consistent with the expected molecular mass of the protein, also confirmed by mass spectrometry (28.6 kDa, not shown). (C) A FESEM micrograph shows the detail of two microgranules randomly picked among those occurring in the main size range (around 10 μm, see panel D). (D) Size distribution of the material population upon clustering determined by FESEM image analysis. (E) General functioning of the self-contained, self-disintegrating protein depots. Polypeptides (purple) are clustered together by the coordination of divalent Zn cations (red balls) with histidine-rich end terminal tags into mechanically stable microparticles (left). Upon in vivo administration, these materials slowly disintegrate along time (dashed blue line), releasing monomeric or oligomeric forms of the protein (right, linked to the loss of ionic Zn from the material), which are then available for desired functionalities. Figure 2. Body weight evolution of experimental animal groups administered with secretory granules. Body weight tracking of male (A) and female (B) mice groups upon administration of different amounts of protein microgranules in either a single or a double (×2) dose. All weight measurements were individually normalized by the initial weight on day 0, with values representing the increase in body weight along time. Days 0 and 21 are the administration times. Differences between group pairs were seen as not significant (ns) in any case. ■ RESULTS Protein microgranules made of the RBD domain from the size of 20.3 ± 17.0 μm (n = 18, Figure 1D) and a median of 12.4 μm. As determined in several model proteins of different functional and structural complexities, including SARS-CoV-2 spike protein (extended version, eRBD, Figure eRBD,24,26,40,42−45 such microparticles are self-disintegrating 1A44) were fabricated with pure recombinant proteins produced under physiological conditions, probably by a progressive loss of in E. coli (Figure 1B). The formation of protein clusters was Zn under dilution. This fact results in a consequent time- triggered by the addition of ZnCl2 to protein solutions that prolonged leakage of monomeric or oligomeric forms of the resulted in discrete microparticles (Figure 1C) with an average protein (Figure 1E) that are properly folded and functional.24 39169 https://doi.org/10.1021/acsami.3c08643 ACS Appl. Mater. Interfaces 2023, 15, 39167−39175 ACS Applied Materials & Interfaces www.acsami.org Research Article Figure 3. Analysis of blood biochemical markers. Determination of AST, ALT, CRE, and UA in animals administered with microscale secretory granules. Blood samples were collected at day 35. Differences between equivalent groups (treated and nontreated) were seen as not significant (ns) in all cases. Figure 4. Histology of main organs in treated animals. Representative hematoxylin and eosin stain of the liver, kidney, and spleen, collected 35 days after the administration of different doses of secreting granules. Bar: 50 μm. Then, the material, once administered in vivo, acts as an Aiming to assess the impact of secretory granules on liver and intriguing protein delivery platform in which the building block kidney function, we measured biochemical markers in blood polypeptides are self-contained and finally self-delivered from (AST, ALT, CRE, and UA) at the end of the experimental time those dynamic, protein-only depots. in those mice groups administered with the highest protein Different amounts of the granules were subcutaneously amounts (i.e., one or two doses of 1000 μg) to evaluate damage administered in a mouse model, in both female and male in main organs. Data presented in Figure 3 showed no groups. In one set of groups, one single protein bolus was statistically significant alterations in those markers compared administered at day 0, and in another, a second bolus was to the saline-administered control. In any case, a second dose administered three weeks later. In that case, not only were the enhanced the level of those markers that in general were slightly doses doubled but we also reproduced the 1−3 weeks of lower (although at a nonsignificant level) upon such second administration regimes for enzyme replacement therapies46,47 administration. While a significant increase in these markers (as a model of conditions treatable by protein administration) or would be associated with hepatic or renal damage, the slight conventional vaccination to amplify any potential inflammatory tendency toward a reduction of AST and ALT in treated animals cannot be linked to any condition, as low levels of these markers or immunotoxic response. Any external sign of inflammation or are in general considered protective.48 In any case, all of the irritation at the administration site was observed upon injection. obtained values are within those considered normal for healthy Also, the whole-body weight of the animals was monitored for BALB/c mice, namely, between 55−352 U/L for AST and 35 days. As observed, the administration of protein did not between 41−131 U/L for ALT (www.criver.com; retrieved on significantly affect the gain of body weight that was progressive July 10, 2023). Furthermore, the absence of organ toxicity was in all of the groups and during the whole experimental time finally confirmed through the histopathological analysis of the (Figure 2). The weight gain of the groups receiving two doses spleen, kidney, and liver (Figure 4). was comparable with that shown by saline-injected animal In previous studies, the protein leakage from artificial groups. This first set of data suggested the absence of toxicity secretory granules obtained by Zn-his coordination24,26 and in linked to the administered protein as formulated in the form of particular from eRDB-H6 microgranules44 had been robustly microgranules even at high doses (2000 μg). For 20 g-weight described. To fully confirm that the absence of toxicity described animals, such an upper dose represents approximately 100 mg of above occurred in a truly secretory system, the bioavailability of protein/kg of body weight, far over any envisaged clinical use in the protein was assessed through the triggered immune humans. response. Indeed, anti-protein antibodies were detected in 39170 https://doi.org/10.1021/acsami.3c08643 ACS Appl. Mater. Interfaces 2023, 15, 39167−39175 ACS Applied Materials & Interfaces www.acsami.org Research Article administered animals, with a response generically tending to be In contrast to such holder−drug dual systems, functional higher at higher doses (Figure 5). It must be noted that the amyloids in nature are efficient protein storage systems in which the functional polypeptides are self-clustered together as microgranules with the assistance of cationic metals.27−29,36,37,63 The disassembling of these materials is progressive, and it involves the leakage of individual polypeptides by metal chelation or dilution (Figure 1E). Therefore, the protein is the functional agent and also the building block of a mechanically stable structure that hosts itself. This mechanism is the basis of the human endocrine system that stores and releases a diversity of peptide hormones.28−30 Many secretory hormones from the mammalian endocrine system form functional amyloids in secretory granules.27−31 Importantly, irrespective of their sequence and structure, they aggregate as amyloid fibrils.28,30 Thus, protein aggregation and amyloid formation follows a polymerization mechanism where protein or peptide species slowly associate to form aggregation competent nuclei. These cores further recruit the monomeric counterpart to grow into mature amyloid fibrils, eventually reaching a steady-state Figure 5. Humoral immune response triggered against subcutaneously equilibrium between the fibrils and monomeric proteins.64 administered protein granules. Blood samples were collected 35 days Based on this equilibrium, the protein storage as an amyloid after administration and tested in an anti SARS-CoV-2 ELISA IgG kit. depot is also able to release the functional monomeric proteins upon dilution.64 Mimetic versions of these natural systems have been recently developed, which has resulted in promising delivery systems, second administration of the protein material only enhanced the fully functional and operational in different experimental antibody titer moderately. Altogether, these data confirmed the settings in vitro and in vivo.24,26,42,65 The protein is then self- effective protein release from granules in all tested doses while contained in vitro when exposed to cationic Zn and self-delivered also suggesting that immunotoxicities or exacerbated immune in vivo, upon administration, when the metal is diluted in the responses did not occur. physiological medium. Consequently, these granules follow a ■ DISCUSSION In conventional nanoparticle-based drug delivery, the need for a similar mechanism as described above for the secretory hormones; thus, sustained release of the building block bioactive proteins may happen because of the progressive disintegration of drug vehicle is a matter of concern as it contributes to the system the amyloids to reach a systemic distribution and achieve the with a bulk of nontherapeutic material that usually represents subsequent biological effects. The aggregating polypeptides are the majority fraction of the whole drug complex mass.49 Such a then, at the same time, the bioactive agents and the mechanically carrier system might display individual and environmental stable scaffolds that act as a dynamic protein depot. Although the toxicities. It represents additional production costs, and it also platform itself is progressively understood, becoming suited to adds important complexity to the formulation process and specific tailoring, toxicity remains an unexplored issue. Even if regulatory challenges to the approval for clinical uses.49 In this unexpected, the possibility of undesired side effects linked to regard, the concept of self-assembling, self-targeted drugs high local concentrations of foreign protein needs to be lacking heterologous vehicles has gained interest in nano- evaluated before further development. In this regard, the biotechnology.50 Using bioactive proteins organized as tumor- evolution of the whole-body weight (Figure 2) and the targeted nanoparticles, this vehicle-free principle has been fully histological analysis of main organs (Figure 4) revealed the demonstrated in several models of human cancer,51−53 and such absence of toxicity, even when the dose of the administered a concept can be extended to any bioactive protein usable in protein (in a granular format) is up to ∼2 mg. This amount, for a clinics, irrespective of the function to be performed in the body. reference 20 g animal, represents 100 mg/kg of body weight, This includes antimicrobial peptides, cytotoxic proteins, which is much beyond the employed doses of therapeutic targeting agents, imaging agents, immunomodulators, antigens, proteins. The dosage of protein drugs is usually akin to body enzymes, hormones, and growth factors, among others. weight.66,67 As a paradigmatic example, in enzyme replacement On the other hand, the development of slow-release systems, therapies, protein doses usually range between 0.2 and 20 mg/ for both chemicals and proteins, faces a similar bottleneck. kg, given systemically every 1−2 weeks.47 Novel delivery systems based on the sustained release of the The moderate antibody response triggered against the payload agent are highly desired to reduce its level oscillations granulated model protein (Figure 5) indicates bioavailability and to keep constant concentrations at the site of action.54,55 of the building block, but it also suggests the absence of a potent Porous and hollowed materials, hydrogels, and different immune reaction that could derive into immunopathogenic polymeric matrices are among the most explored holding damage. Importantly, the potential immunotoxicity of ther- systems for time-prolonged release.56−61 In them, the active apeutic proteins, especially in aggregated forms, is a generic molecule is stored for further release under physiological matter of worry that relies on mechanisms not completely conditions. Upon administration, such materials are expected understood.68,69 Such adverse events are mostly expected during to be mechanically and chemically stable and biologically systemic administration in blood, 70,71 a fact that can functional in contact with organic tissues during prolonged dramatically reduce the therapeutic or in general the biological periods. Toxicity, then, is again a matter of concern.62 impact of the protein by triggering protein-binding antibodies.72 39171 https://doi.org/10.1021/acsami.3c08643 ACS Appl. Mater. Interfaces 2023, 15, 39167−39175 ACS Applied Materials & Interfaces www.acsami.org Research Article Although the T-cell response has not been particularly analyzed Barcelona, 08193 Bellaterra, Spain; orcid.org/0000-0001- here, the local, subcutaneous administration approached in the 5750-550X present study has not represented any acute impact on the Marianna T. P. Favaro − Institut de Biotecnologia i de animals. In this regard, the histopathological observation of the Biomedicina, Universitat Autònoma de Barcelona, 08193 spleen is fully normal (Figure 4) and the modest immune Bellaterra, Spain; Instituto de Ciências Biomédicas, response tends to be dose-dependent, although without a robust Universidade de São Paulo, São Paulo 05508-000, Brazil statistical confirmation (Figure 5). On the other hand, no Alberto Gallardo − Institut d’Investigació Biomèdica Sant Pau alterations in the blood levels of several organ-damage markers (IIB SANT PAU), 08041 Barcelona, Spain; Department of have been observed (Figure 3), in agreement with the Pathology, Hospital de la Santa Creu i Sant Pau, 08025 histological analyses of the spleen, kidney and liver (Figure 4). Barcelona, Spain Although deeper toxicological studies should be done during the Rosa Mendoza − CIBER de Bioingeniería, Biomateriales y clinically oriented adaptation of protein-only secretory granules Nanomedicina (CIBER-BBN, ISCIII), Universitat Autònoma as slow delivery systems, the study presented here allows de Barcelona, 08193 Bellaterra, Spain; Institut de discarding any acute organic and tissue damage or immunotox- Biotecnologia i de Biomedicina, Universitat Autònoma de icities associated with the aggregated status of the high-dose Barcelona, 08193 Bellaterra, Spain protein depots administered through the subcutaneous route. Luís C. S. Ferreira − Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo 05508-000, Brazil ■ CONCLUSIONS Self-contained, self-disintegrating protein microparticles are Nerea Roher − CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN, ISCIII), Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; Institut de artificial protein depots that act as convenient platforms for the Biotecnologia i de Biomedicina, Universitat Autònoma de slow delivery of the building block protein in a bioactive form. Barcelona, 08193 Bellaterra, Spain; Department of Cell Despite their interest in several clinical contexts, their potential Biology, Animal Physiology and Immunology, Universitat toxicity has not been so far explored. Here, we have Autònoma de Barcelona, 08193 Bellaterra, Spain demonstrated that these materials, upon subcutaneous admin- Esther Vázquez − CIBER de Bioingeniería, Biomateriales y istration in mice, do not show any sign of toxicity measured Nanomedicina (CIBER-BBN, ISCIII), Universitat Autònoma through body weight evolution, histological analyses of main de Barcelona, 08193 Bellaterra, Spain; Institut de organs, and levels of biochemical markers of hepatic and renal Biotecnologia i de Biomedicina and Departament de Genètica i damage. Since the present analysis has been extended up to de Microbiologia, Universitat Autònoma de Barcelona, 08193 ∼100 mg of protein administered/kg of body weight, much over Bellaterra, Spain any potential clinical use in humans, we conclude that the proposed material is intrinsically biosafe and suited for use in Complete contact information is available at: biological interfaces. This information definitely supports its https://pubs.acs.org/10.1021/acsami.3c08643 adaptation to specific applications, and it will for sure find its regulatory route to clinics. Author Contributions ◆ P.A. and E.P. contributed equally to this work. The manuscript ■ AUTHOR INFORMATION Corresponding Authors was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Ramón Mangues − Institut d’Investigació Biomèdica Sant Pau Funding (IIB SANT PAU), 08041 Barcelona, Spain; Josep Carreras The authors are indebted to AGAUR for the project Leukaemia Research Institute (IJC), 08916 Badalona, Spain; 2020PANDE00003 granted to A.V., to the Agencia Española CIBER de Bioingeniería, Biomateriales y Nanomedicina de Investigación (AEI) for granting projects on the construction (CIBER-BBN, ISCIII), Universitat Autònoma de Barcelona, of protein materials of clinical interest (PID2019-105416RB- 08193 Bellaterra, Spain; Email: RMangues@santpau.cat Antonio Villaverde − CIBER de Bioingeniería, Biomateriales y I00/AEI/10.13039/501100011033 and PDC2022-133858-I00 Nanomedicina (CIBER-BBN, ISCIII), Universitat Autònoma to E.V. and PID2020-116174RB-I00 to A.V.), to AGAUR for de Barcelona, 08193 Bellaterra, Spain; Institut de projects SGR 2021 00092 to A.V. and SGR 2021 01140 to R.M., Biotecnologia i de Biomedicina and Departament de Genètica i and to ISCIII for the project P21/00150 to R.M. This work was de Microbiologia, Universitat Autònoma de Barcelona, 08193 also supported by the Fundação de Amparo à Pesquisa do Bellaterra, Spain; orcid.org/0000-0002-2615-4521; Estado de São Paulo (FAPESP�grants 2020/05204-7 and Email: Antoni.villaverde@uab.cat 2020/10700-3). We also appreciate the support from the Authors CERCA Programme, Generalitat de Catalunya and from the Patricia Á lamo − Institut d’Investigació Biomèdica Sant Pau CIBER-Consorcio Centro de Investigación Biomédica en Red- (IIB SANT PAU), 08041 Barcelona, Spain; Josep Carreras (projects NANOSARS and NANOREMOTE, CB06/01/0014 Leukaemia Research Institute (IJC), 08916 Badalona, Spain; and CB06/01/1031), Instituto de Salud Carlos III, Ministerio CIBER de Bioingeniería, Biomateriales y Nanomedicina de Ciencia e Innovación. A.V. received an ICREA ACADEMIA (CIBER-BBN, ISCIII), Universitat Autònoma de Barcelona, award. 08193 Bellaterra, Spain Notes Eloi Parladé − CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN, ISCIII), Universitat Autònoma The authors declare the following competing financial de Barcelona, 08193 Bellaterra, Spain; Institut de interest(s): PA, RM, AV and EV appear as inventors in a patent Biotecnologia i de Biomedicina, Universitat Autònoma de covering the biomedical uses of artificial secretory granules. 39172 https://doi.org/10.1021/acsami.3c08643 ACS Appl. Mater. Interfaces 2023, 15, 39167−39175 ACS Applied Materials & Interfaces www.acsami.org Research Article ■ ACKNOWLEDGMENTS The in vivo studies were done at the Nanotoxicology Unit of the (18) López-Laguna, H.; Voltà-Durán, E.; Parladé, E.; Villaverde, A.; Vázquez, E.; Unzueta, U. Insights on the Emerging Biotechnology of Histidine-Rich Peptides. Biotechnol. Adv. 2022, 54, No. 107817. ICTS-141007 Nanbiosis Platform (http://www.nanbiosis.es/ (19) Khare, E.; Holten-Andersen, N.; Buehler, M. J. Transition-Metal portfolio/u18-nanotoxicology-unit/). The production of the Coordinate Bonds for Bioinspired Macromolecules with Tunable therapeutic proteins was assisted, in part, by the Protein Mechanical Properties. Nat. Rev. Mater. 2021, 6, 421−436. Production (PPP) Unit of the ICTS Nanbiosis Platform of the (20) Kluska, K.; Adamczyk, J.; Krężel, A. Metal Binding Properties, CIBER-BBN/IBB (http://www.nanbiosis.es/unit/u1-protein- Stability and Reactivity of Zinc Fingers. Coord. Chem. Rev. 2018, 367, production-platform-ppp/). 18−64. ■ ABBREVIATIONS AST, aspartate aminotransferase (21) López-Laguna, H.; Sánchez, J.; Unzueta, U.; Mangues, R.; Vázquez, E.; Villaverde, A. Divalent Cations: A Molecular Glue for Protein Materials Trends in Biochemical Sciences An Official Publication of the International Union of Biochemistry and Molecular ALT, alanine aminotransferase Biology. Trends Biochem. Sci. 2020, 45, 992−1003. CRE, creatinine (22) López-Laguna, H.; Sánchez, J. M.; Carratalá, J. V.; Rojas-Peña, DTT, dithiothreitol EDTA ethylenediaminetetraacetic acid M.; Sánchez-García, L.; Parladé, E.; Sánchez-Chardi, A.; Voltà-Durán, UA, uric acid E.; Serna, N.; Cano-Garrido, O.; Flores, S.; Ferrer-Miralles, N.; Nolan, ■ REFERENCES (1) Gokul, G. L.; Lakshmi, T.; Don, K. R. Biocompatibility of Dental V.; de Marco, A.; Roher, N.; Unzueta, U.; Vazquez, E.; Villaverde, A. Biofabrication of Functional Protein Nanoparticles through Simple His-Tag Engineering. ACS Sustainable Chem. Eng. 2021, 9, 12341− Materials-a Review. Int. J. Res. Pharm. Sci. 2020, 11, 1634−1637. 12354. (2) Yang, Y.; Qin, Z.; Zeng, W.; Yang, T.; Cao, Y.; Mei, C.; Kuang, Y. (23) López-Laguna, H.; Unzueta, U.; Conchillo-Solé, O.; Sánchez- Toxicity Assessment of Nanoparticles in Various Systems and Organs. Chardi, A.; Pesarrodona, M.; Cano-Garrido, O.; Voltà, E.; Sánchez- Nanotechnol. Rev. 2017, 6, 279−289. García, L.; Serna, N.; Saccardo, P.; Mangues, R.; Villaverde, A.; (3) Zheng, J.; Li, J.; Zhang, L.; Chen, X.; Yu, Y.; Huang, H. Post- Vázquez, E. Assembly of Histidine-Rich Protein Materials Controlled Graphene 2D Materials-Based Antimicrobial Agents: Focus on through Divalent Cations. Acta Biomater. 2019, 83, 257−264. Fabrication Strategies and Biosafety Assessments. J. Mater. Sci. 2020, (24) Sánchez, J. M.; López-Laguna, H.; Á lamo, P.; Serna, N.; Sánchez- 55, 7226−7246. Chardi, A.; Nolan, V.; Cano-Garrido, O.; Casanova, I.; Unzueta, U.; (4) Yu, Y.; Bu, F.; Zhou, H.; Wang, Y.; Cui, J.; Wang, X.; Nie, G.; Xiao, Vazquez, E.; Mangues, R.; Villaverde, A. Artificial Inclusion Bodies for H. Biosafety Materials: An Emerging New Research Direction of Clinical Development. Adv. Sci. 2020, 7, No. 1902420. Materials Science from the COVID-19 Outbreak. Mater. Chem. Front. (25) López-Laguna, H.; Parladé, E.; Á lamo, P.; Sánchez, J. M.; Voltà- 2020, 4, 1930−1953. Durán, E.; Serna, N.; Sánchez-García, L.; Cano-Garrido, O.; Sánchez- (5) Chen, Y.; Chen, H.; Shi, J. In Vivo Bio-Safety Evaluations and Chardi, A.; Villaverde, A.; Mangues, R.; Unzueta, U.; Vázquez, E.; Diagnostic/Therapeutic Applications of Chemically Designed Meso- López-Laguna, H.; Parladé, E.; Sánchez, J. M.; Voltà-Durán, E.; Serna, porous Silica Nanoparticles. Adv. Mater. 2013, 25, 3144−3176. N.; Sánchez-García, L.; Villaverde, A.; Vázquez, E.; Á lamo, P.; Unzueta, (6) Li, Z.; Zhang, L.; Jiang, K.; Zhang, Y.; Liu, Y.; Hu, G.; Song, J. U.; Mangues, R. In Vitro Fabrication of Microscale Secretory Granules. Biosafety Assessment of Delivery Systems for Clinical Nucleic Acid Adv. Funct. Mater. 2021, 31, No. 2100914. Therapeutics. Biosaf. Health 2022, 4, 105−117. (26) Á lamo, P.; Parladé, E.; López-Laguna, H.; Voltà-Durán, E.; (7) Wan, M.; Li, T.; Chen, H.; Mao, C.; Shen, J. Biosafety, Unzueta, U.; Vazquez, E.; Mangues, R.; Villaverde, A. Ion-dependent Functionalities, and Applications of Biomedical Micro/Nanomotors. slow protein release from in vivo disintegrating micro-granules. Drug Angew. Chem., Int. Ed. 2021, 60, 13158−13176. Delivery 2021, 28, 2383−2391. (8) Parkinson, A. An Overview of Current Cytochrome P450 (27) Soragni, A.; Maji, S. K.; Riek, R. Toward a Comprehension of Technology for Assessing the Safety and Efficacy of New Materials. Functional Aggregation into Amyloids in Pituitary Secretory Granules. Toxicol. Pathol. 1996, 24, 45−57. Amyloid 2010, 17, No. 41. (9) Amukarimi, S.; Mozafari, M. Biodegradable Magnesium (28) Jacob, R. S.; Anoop, A.; Maji, S. K. Protein Nanofibrils as Storage Biomaterials�Road to the Clinic. Bioengineering 2022, 9, No. 107. Forms of Peptide Drugs and Hormones. Adv. Exp. Med. Biol. 2019, (10) Martina, M.; Hutmacher, D. W. Biodegradable Polymers Applied 1174, 265−290. in Tissue Engineering Research: A Review. Polym. Int. 2007, 56, 145− (29) Jacob, R. S.; Das, S.; Ghosh, S.; Anoop, A.; Jha, N. N.; Khan, T.; 157. Singru, P.; Kumar, A.; Maji, S. K. Amyloid Formation of Growth (11) Kumar, N. S.; Suvarna, R. P.; Naidu, K. C. B.; Banerjee, P.; Ratnamala, A.; Manjunatha, H. A Review on Biological and Biomimetic Hormone in Presence of Zinc: Relevance to Its Storage in Secretory Materials and Their Applications. Appl. Phys. A 2020, 126, No. 445. Granules. Sci. Rep. 2016, 6, No. 23370. (12) Ma, P. X. Biomimetic Materials for Tissue Engineering. Adv. Drug (30) Maji, S. K.; Perrin, M. H.; Sawaya, M. R.; Jessberger, S.; Delivery Rev. 2008, 60, 184−198. Vadodaria, K.; Rissman, R. A.; Singru, P. S.; Nilsson, K. P. R.; Simon, R.; (13) Cao, D.; Chen, L.; Zhang, Z.; Luo, Y.; Zhao, L.; Yuan, C.; Lu, J.; Schubert, D.; Eisenberg, D.; Rivier, J.; Sawchenko, P.; Vale, W.; Riek, R. Liu, X.; Li, J. Biodegradable Nanomaterials for Diagnosis and Therapy Functional Amyloids as Natural Storage of Peptide Hormones in of Tumors. J. Mater. Chem. B 2023, 11, 1829−1848. Pituitary Secretory Granules. Science 2009, 325, 328−332. (14) Shen, Y.; Levin, A.; Kamada, A.; Toprakcioglu, Z.; Rodriguez- (31) Seuring, C.; Verasdonck, J.; Gath, J.; Ghosh, D.; Nespovitaya, N.; Garcia, M.; Xu, Y.; Knowles, T. P. J. From Protein Building Blocks to Wälti, M. A.; Maji, S. K.; Cadalbert, R.; Güntert, P.; Meier, B. H.; Riek, Functional Materials. ACS Nano 2021, 15, 5819−5837. R. The Three-Dimensional Structure of Human β-Endorphin Amyloid (15) Miserez, A.; Yu, J.; Mohammadi, P. Protein-Based Biological Fibrils. Nat. Struct. Mol. Biol. 2020, 27, 1178−1184. Materials: Molecular Design and Artificial Production. Chem. Rev. (32) García-Fruitós, E.; Rodríguez-Carmona, E.; Díez-Gil, C.; Ferraz, 2023, 123, 2049−2111. R. M.; Vázquez, E.; Corchero, J. L.; Cano-Sarabia, M.; Ratera, I.; (16) Corchero, J. L.; Vázquez, E.; García-Fruitós, E.; Ferrer-Miralles, Ventosa, N.; Veciana, J.; Villaverde, A. Surface Cell Growth N.; Villaverde, A. Recombinant Protein Materials for Bioengineering Engineering Assisted by a Novel Bacterial Nanomaterial. Adv. Mater. and Nanomedicine. Nanomedicine 2014, 9, 2817−2828. 2009, 21, 4249−4253. (17) Jang, Y.; Champion, J. A. Self-Assembled Materials Made from (33) Unzueta, U.; Cespedes, M. V.; Sala, R.; Alamo, P.; Sánchez- Functional Recombinant Proteins. Acc. Chem. Res. 2016, 49, 2188− Chardi, A.; Pesarrodona, M.; Sánchez-García, L.; Cano-Garrido, O.; 2198. Villaverde, A.; Vázquez, E.; Mangues, R.; Seras-Franzoso, J. Release of 39173 https://doi.org/10.1021/acsami.3c08643 ACS Appl. Mater. Interfaces 2023, 15, 39167−39175 ACS Applied Materials & Interfaces www.acsami.org Research Article Targeted Protein Nanoparticles from Functional Bacterial Amyloids: A (51) Zhao, Z.; Huang, Y.; Wang, J.; Lin, H.; Cao, F.; Li, S.; Li, Y.; Li, Death Star-like Approach. J. Controlled Release 2018, 279, 29−39. Z.; Liu, X. A Self-Assembling CXCR4-Targeted Pyroptosis Nanotoxin (34) Céspedes, M. V.; Fernández, Y.; Unzueta, U.; Mendoza, R.; for Melanoma Therapy. Biomater. Sci. 2023, 11, 2200−2210. Seras-Franzoso, J.; Sánchez-Chardi, A.; Á lamo, P.; Toledo-Rubio, V.; (52) Rioja-Blanco, E.; Arroyo-Solera, I.; Á lamo, P.; Casanova, I.; Ferrer-Miralles, N.; Vázquez, E.; Schwartz, S.; Abasolo, I.; Corchero, J. Gallardo, A.; Unzueta, U.; Serna, N.; Sánchez-García, L.; Quer, M.; L.; Mangues, R.; Villaverde, A. Bacterial Mimetics of Endocrine Villaverde, A.; Vázquez, E.; León, X.; Alba-Castellón, L.; Mangues, R. Secretory Granules as Immobilized in Vivo Depots for Functional CXCR4-Targeted Nanotoxins Induce GSDME-Dependent Pyroptosis Protein Drugs. Sci. Rep. 2016, 6, No. 35765. in Head and Neck Squamous Cell Carcinoma. J. Exp. Clin. Cancer Res. (35) Céspedes, M. V.; Cano-Garrido, O.; Á lamo, P.; Sala, R.; Gallardo, 2022, 41, No. 49. A.; Serna, N.; Falgàs, A.; Voltà-Durán, E.; Casanova, I.; Sánchez-Chardi, (53) Núñez, Y.; Garcia-León, A.; Falgàs, A.; Serna, N.; Sánchez- A.; López-Laguna, H.; Sánchez-García, L.; Sánchez, J. M.; Unzueta, U.; García, L.; Garrido, A.; Sierra, J.; Gallardo, A.; Unzueta, U.; Vázquez, E.; Vázquez, E.; Mangues, R.; Villaverde, A. Engineering Secretory Villaverde, A.; Mangues, R.; Casanova, I. T22-PE24-H6 Nanotoxin Amyloids for Remote and Highly Selective Destruction of Metastatic Selectively Kills CXCR4-High Expressing AML Patient Cells In Vitro Foci. Adv. Mater. 2020, 32, No. 1907348. and Potently Blocks Dissemination In Vivo. Pharmaceutics 2023, 15, (36) Balistreri, A.; Goetzler, E.; Chapman, M. Functional Amyloids No. 727. Are the Rule Rather than the Exception in Cellular Biology. (54) Bhowmik, D.; Gopinath, H.; Pragati Kumar, B.; Duraivel, S.; Microorganisms 2020, 8, No. 1951. Sampath Kumar, K. P. THE PHARMA INNOVATION Controlled (37) Sergeeva, A. V.; Galkin, A. P. Functional Amyloids of Eukaryotes: Release Drug Delivery Systems. Pharma Innovation 2012, 1 (10), 24− Criteria, Classification, and Biological Significance. Curr. Genet. 2020, 32. 66, 849−866. (55) Huang, X.; Brazel, C. S. On the Importance and Mechanisms of (38) Rubel, M. S.; Fedotov, S. A.; Grizel, A. V.; Sopova, J. V.; Burst Release in Matrix-Controlled Drug Delivery Systems. J. Controlled Malikova, O. A.; Chernoff, Y. O.; Rubel, A. A. Functional Mammalian Release 2001, 73, 121−136. Amyloids and Amyloid-like Proteins. Life 2020, 10, No. 156. (56) Han, L.; Peng, K.; Qiu, L. Y.; Li, M.; Ruan, J. H.; He, L. L.; Yuan, (39) de Marco, A.; Ferrer-Miralles, N.; Garcia-Fruitós, E.; Mitraki, A.; Z. X. Hitchhiking on Controlled-Release Drug Delivery Systems: Peternel, S.; Rinas, U.; Trujillo-Roldán, M. A.; Valdez-Cruz, N. A.; Opportunities and Challenges for Cancer Vaccines. Front. Pharmacol. Vázquez, E.; Villaverde, A. Bacterial Inclusion Bodies Are Industrially 2021, 12, No. 679602. Exploitable Amyloids. FEMS Microbiol. Rev. 2019, 43, 53−72. (57) Kass, L. E.; Nguyen, J. Nanocarrier-Hydrogel Composite (40) Chen, T. Y.; Cheng, W. J.; Horng, J. C.; Hsu, H. Y. Artificial Delivery Systems for Precision Drug Release. WIREs Nanomed. Peptide-Controlled Protein Release of Zn2+-Triggered, Self-As- Nanobiotechnol. 2022, 14, No. e1756. sembled Histidine-Tagged Protein Microparticle. Colloids Surf., B (58) Vigata, M.; Meinert, C.; Hutmacher, D. W.; Bock, N. Hydrogels 2020, 187, No. 108032. as Drug Delivery Systems: A Review of Current Characterization and (41) Cano-Garrido, O.; Serna, N.; Unzueta, U.; Parladé, E.; Mangues, Evaluation Techniques. Pharmaceutics 2020, 12, No. 1188. R.; Villaverde, A.; Vázquez, E. Protein Scaffolds in Human Clinics. (59) Diksha, S.; Dhruv, D.; Prasad, D. N.; Mansi, H. Sustained Release Biotechnol. Adv. 2022, 61, No. 108032. Drug Delivery System with the Role of Natural Polymers: A Review. J. (42) Serna, N.; Cano-Garrido, O.; Sánchez, J. M.; Sánchez-Chardi, A.; Drug Delivery Ther. 2019, 9, 913−923. Sánchez-García, L.; López-Laguna, H.; Fernández, E.; Vázquez, E.; (60) Ciolacu, D. E.; Nicu, R.; Ciolacu, F. Cellulose-Based Hydrogels Villaverde, A. Release of Functional Fibroblast Growth Factor-2 from as Sustained Drug-Delivery Systems. Materials 2020, 13, No. 5270. Artificial Inclusion Bodies. J. Controlled Release 2020, 327, 61−69. (61) Idrees, H.; Zaidi, S. Z. J.; Sabir, A.; Khan, R. U.; Zhang, X.; (43) Zhao, L.; Luo, L.; Meng, F. Metal-Mediated Nanobody Hassan, S. U. A Review of Biodegradable Natural Polymer-Based Assemblies as Potent Alleviators of Human Islet Amyloid Polypeptide Nanoparticles for Drug Delivery Applications. Nanomaterials 2020, 10, Aggregation. Mater. Chem. Front. 2023, 7, 2068−2077. No. 1970. (44) Corchero, J. L.; Favaro, M. T. P.; Márquez-Martínez, M.; Lascorz, (62) Ghasemiyeh, P.; Mohammadi-Samani, S. Hydrogels as Drug J.; Martínez-Torró, C.; Sánchez, J. M.; López-Laguna, H.; de Souza Delivery Systems; Pros and Cons. Trends Pharm. Sci. 2019, 5, 7−24. Ferreira, L. C.; Vázquez, E.; Ferrer-Miralles, N.; Villaverde, A.; Parladé, (63) Levkovich, S. A.; Gazit, E.; Bar-Yosef, D. L. Two Decades of E. Recombinant Proteins for Assembling as Nano- and Micro-Scale Studying Functional Amyloids in Microorganisms. Trends Microbiol. Materials for Drug Delivery: A Host Comparative Overview. 2021, 29, 251−265. Pharmaceutics 2023, 15, No. 1197. (64) Chatterjee, D.; Jacob, R. S.; Ray, S.; Navalkar, A.; Singh, N.; (45) Serna, N.; Falgàs, A.; García-León, A.; Unzueta, U.; Núñez, Y.; Sengupta, S.; Gadhe, L.; Kadu, P.; Datta, D.; Paul, A.; Sakunthala, A.; Sánchez-Chardi, A.; Martínez-Torró, C.; Mangues, R.; Vazquez, E.; Mehra, S.; Pindi, C.; Kumar, S.; Singru, P. S.; Senapati, S.; Maji, S. K. Casanova, I.; Villaverde, A. Time-Prolonged Release of Tumor- Co-Aggregation and Secondary Nucleation in the Life Cycle of Human Targeted Protein−MMAE Nanoconjugates from Implantable Hybrid Prolactin/Galanin Functional Amyloids. eLife 2022, 11, No. e73835. Materials. Pharmaceutics 2022, 14, No. 192. (65) Sanchez, J. M.; López-Laguna, H.; Serna, N.; Unzueta, U.; Clop, (46) Abasolo, I.; Seras-Franzoso, J.; Moltó-Abad, M.; Díaz-Riascos, P. D.; Villaverde, A.; Vazquez, E. Engineering the Performance of V.; Corchero, J. L.; Pintos-Morell, G.; Schwartz, S. Nanotechnology- Artificial Inclusion Bodies Built of Catalytic β-Galactosidase. ACS Based Approaches for Treating Lysosomal Storage Disorders, a Focus Sustainable Chem. Eng. 2021, 9, 2552−2558. on Fabry Disease. WIRES Nanomed. Nanobiotechnol. 2021, 13, (66) Zhang, S.; Shi, R.; Li, C.; Parivar, K.; Wang, D. D. Fixed Dosing No. e1684. versus Body Size-Based Dosing of Therapeutic Peptides and Proteins in (47) Grabowski, G. A. Treatment Perspectives for the Lysosomal Adults. J. Clin. Pharmacol. 2012, 52, 18−28. Storage Diseases. Expert Opin. Emerging Drugs 2008, 13, 197−121. (67) Wang, D. D.; Zhang, S.; Zhao, H.; Men, A. Y.; Parivar, K. Fixed (48) Hu, C.; Zhao, L.; Zhang, F.; Li, L. Regulation of Autophagy Dosing versus Body Size-Based Dosing of Monoclonal Antibodies in Protects against Liver Injury in Liver Surgery-Induced Ischaemia/ Adult Clinical Trials. J. Clin. Pharmacol. 2009, 49, 1012−1024. Reperfusion. J. Cell. Mol. Med. 2021, 25, 9905−9917. (68) Lundahl, M. L. E.; Fogli, S.; Colavita, P. E.; Scanlan, E. M. (49) Shen, J.; Wolfram, J.; Ferrari, M.; Shen, H. Taking the Vehicle out Aggregation of Protein Therapeutics Enhances Their Immunogenicity: of Drug Delivery. Mater. Today 2017, 20, 95−97. Causes and Mitigation Strategies. RSC Chem. Biol. 2021, 2, 1004−1020. (50) Sánchez-García, L.; Serna, N.; Á lamo, P.; Sala, R.; Céspedes, M. (69) Moussa, E. M.; Panchal, J. P.; Moorthy, B. S.; Blum, J. S.; Joubert, V.; Roldan, M.; Sánchez-Chardi, A.; Unzueta, U.; Casanova, I.; M. K.; Narhi, L. O.; Topp, E. M. Immunogenicity of Therapeutic Mangues, R.; Vázquez, E.; Villaverde, A. Self-Assembling Toxin-Based Protein Aggregates. J. Pharm. Sci. 2016, 105, 417−430. Nanoparticles as Self-Delivered Antitumoral Drugs. J. Controlled Release (70) Rosenberg, A. S. Effects of Protein Aggregates: An Immunologic 2018, 274, 81−92. Perspective. AAPS J. 2006, 8, E501−E507. 39174 https://doi.org/10.1021/acsami.3c08643 ACS Appl. Mater. Interfaces 2023, 15, 39167−39175 ACS Applied Materials & Interfaces www.acsami.org Research Article (71) Jawa, V.; Cousens, L. P.; Awwad, M.; Wakshull, E.; Kropshofer, H.; De Groot, A. S. T-Cell Dependent Immunogenicity of Protein Therapeutics: Preclinical Assessment and Mitigation. Clin. Immunol. 2013, 149, 534−555. (72) Sauerborn, M.; Brinks, V.; Jiskoot, W.; Schellekens, H. Immunological Mechanism Underlying the Immune Response to Recombinant Human Protein Therapeutics. Trends Pharmacol. Sci. 2010, 31, 53−59. Recommended by ACS Investigating BioCaRGOS, a Sol–Gel Matrix for the Stability of Heme Proteins under Enzymatic Degradation and Low pH Chinmay S. Potnis, Gautam Gupta, et al. AUGUST 21, 2023 ACS OMEGA READ Exploring the Extreme Acid Tolerance of a Dynamic Protein Nanocage Jesse A. Jones, Tobias W. Giessen, et al. FEBRUARY 16, 2023 BIOMACROMOLECULES READ Formation of Protein Nanoparticles in Microdroplet Flow Reactors Qi Zhang, Tuomas P. J. Knowles, et al. JUNE 12, 2023 ACS NANO READ Rational Design of Virus-like Particles for Nanomedicine Wenjun Shan, Lei Ren, et al. AUGUST 25, 2023 ACCOUNTS OF MATERIALS RESEARCH READ Get More Suggestions > 39175 https://doi.org/10.1021/acsami.3c08643 ACS Appl. Mater. Interfaces 2023, 15, 39167−39175