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Simple Summary Aimed at minimizing side toxicities cancer therapies require appropriate functional vehicles at the nanoscale, for receptor-mediated tumor-targeted drug delivery. The aim of the present study was to explore the human peptide EPI-X4 as a CXCR4-targeting agent in self-assembled, protein-only nanoparticles. While the systemic tumor biodistribution of EPI-X4-based materials is modest, this peptide shows potent proapoptotic effects on CXCR4+ cancer cells. Interestingly, the in vivo selectivity of EPI-X4 was dramatically improved, once combined into biparatopic nanoparticles, with a second CXCR4 ligand, the peptide T22. Biparatopic nanoparticles promote a highly selective tumor destruction in a mouse model of human colorectal cancer, probably associated to the CXCR4 antagonist role of EPI-X4. This study not only validates a new human ligand of the tumoral marker CXCR4, but it also offers a novel strategy for the combination, in protein nanoparticles, of dual acting ligands of tumoral markers for highly selective drug delivery. Abstract The accumulated molecular knowledge about human cancer enables the identification of multiple cell surface markers as highly specific therapeutic targets. A proper tumor targeting could significantly avoid drug exposure of healthy cells, minimizing side effects, but it is also expected to increase the therapeutic index. Specifically, colorectal cancer has a particularly poor prognosis in late stages, being drug targeting an appropriate strategy to substantially improve the therapeutic efficacy. In this study, we have explored the potential of the human albumin-derived peptide, EPI-X4, as a suitable ligand to target colorectal cancer via the cell surface protein CXCR4, a chemokine receptor overexpressed in cancer stem cells. To explore the potential use of this ligand, self-assembling protein nanoparticles have been generated displaying an engineered EPI-X4 version, which conferred a modest CXCR4 targeting and fast and high level of cell apoptosis in tumor CXCR4+ cells, in vitro and in vivo. In addition, when EPI-X4-based building blocks are combined with biologically inert polypeptides containing the CXCR4 ligand T22, the resulting biparatopic nanoparticles show a dramatically improved biodistribution in mouse models of CXCR4+ human cancer, faster cell internalization and enhanced target cell death when compared to the version based on a single ligand. The generation of biparatopic materials opens exciting possibilities in oncotherapies based on high precision drug delivery based on the receptor CXCR4.

Green fluorescent protein (GFP) is a widely used scaffold for protein-based targeted nanomedicines because of its high biocompatibility, biological neutrality and outstanding structural stability. However, being immunogenicity a major concern in the development of drug carriers, the use of exogenous proteins such as GFP in clinics might be inadequate. Here we report a human nidogen-derived protein (HSNBT), rationally designed to mimic the structural and functional properties of GFP as a scaffold for nanomedicine. For that, a GFP-like β-barrel, containing the G2 domain of the human nidogen, has been rationally engineered to obtain a biologically neutral protein that self-assembles as 10nm-nanoparticles. This scaffold is the basis of a humanized nanoconjugate, where GFP, from the well-characterized protein T22-GFP-H6, has been substituted by the nidogen-derived GFP-like HSNBT protein. The resulting construct T22-HSNBT-H6, is a humanized CXCR4-targeted nanoparticle that selectively delivers conjugated genotoxic Floxuridine into cancer CXCR4+ cells. Indeed, the administration of T22-HSNBT-H6-FdU in a CXCR4-overexpressing colorectal cancer mouse model results in an even more efficient selective antitumoral effect than that shown by its GFP-counterpart, in absence of systemic toxicity. Therefore, the newly developed GFP-like protein scaffold appears as an ideal candidate for the development of humanized protein nanomaterials and successfully supports the tumor-targeted nanoscale drug T22-HSNBT-H6-FdU., Patricia Álamo and Juan Cedano contributed equally to this work. The authors are indebted to Agencia Estatal de Investigación (AEI) and to Fondo Europeo de Desarrollo Regional (FEDER) (grant BIO2016-76063-R, AEI/FEDER, UE), to AGAUR (2017SGR-229) and CIBER-BBN (project NANOPROTHER), granted to AV, to CIBER-BBN (project NANOSCAPE and NANOLINK) and ISCIII (PI20/00400 co-funding FEDER) granted to UU, to ISCIII (PI15/00272 co-founding FEDER) granted to EV and to ISCIII (PIE15/00028 and PI18/00650, co-funding FEDER) and AGAUR (2017 SGR 865 GRC) granted to RM. We are also indebted to CERCA programme (Generalitat de Catalunya) and to the Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) that is an initiative funded by the VI National R&D&I Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III, with assistance from the European Regional Development Fund. We also appreciate the support from the COST-Action Nano2Clinics. Protein production has been partially performed by the ICTS “NANBIOSIS”, more specifically by the Protein Production Platform of CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN)/ IBB, at the UAB sePBioEs scientific-technical service (http://www.nanbiosis.es/portfolio/u1-protein-production-platform-ppp/), and the nanoparticle size analysis by the Biomaterial Processing and Nanostructuring Unit. Synthesis of thiolated oligo-FdU was performed by the ICTS NANBIOSIS Oligonucleotide Synthesis Platform (CIBER-BBN). The in vivo work was performed by the ICTS NANBIOSIS of the CIBER-BBN Nanotoxicology Unit (http://www.nanbiosis.es/portfolio/u18-nanotoxicology-unit/). We are indebted to Servei de Microscopia from UAB for their excellent confocal and electronic microscopy services. We are also indebted to Servei de Cultius Celulars i Anticossos (SCAC) form UAB for their excellent cell culture and flow cytometry facilities and especially to Fran Cortes for his excellent technical support. We are thankful to Dra. Marta Taulés from CCiT-UB for her help in SPR experiments and analysis. We are also thankful to Luis Carlos Navas from Institut d'Investigacions biomèdiques Sant Pau (IIB Sant Pau) for his technical support in immunohistochemistry experiments. UU and LMCD were supported by Miguel Servet (CP19/00028) and PFIS (FI19/00148) contracts respectively from ISCIII co-funded by European Social Fund (ESF investing in your future). NS was supported by a pre-doctoral fellowship from the Government of Navarra and LAC was supported by AECC Scientific Foundation grant postdoctoral fellow. AV received an ICREA ACADEMIA award.

Aïda Falgàs,1– 3 Victor Pallarès,1– 3 Ugutz Unzueta,1– 4 Yáiza Núñez,1,2 Jorge Sierra,2,5 Alberto Gallardo,1 Lorena Alba-Castellón,1,2 Maria Antonia Mangues,3,6 Patricia Álamo,1– 3 Antonio Villaverde,3,4,7 Esther Vázquez,3,4,7 Ramon Mangues,1– 3 Isolda Casanova1– 3 1Biomedical Research Institute Sant Pau (IIB-Sant Pau), Hospital de la Santa Creu i Sant Pau, Barcelona, 08025, Spain; 2Josep Carreras Leukaemia Research Institute (IJC), Barcelona, 08916, Spain; 3CIBER de Bioingeniería Biomateriales y Nanomedicina (CIBER-BBN), Madrid, 28029, Spain; 4Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Barcelona, 08193, Spain; 5Department of Hematology, Hospital de la Santa Creu i Sant Pau, Barcelona, 08025, Spain; 6Department of Pharmacy, Hospital de la Santa Creu i Sant Pau, Barcelona, 08025, Spain; 7Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, Barcelona, 08193, SpainCorrespondence: Esther VázquezInstitute of Biotechnology and Biomedicine (IBB) and Department of Genetics and Microbiology, Universitat Autònoma de Barcelona and CIBER-BBN, Barcelona, SpainTel +34 935812148Email Esther.Vazquez@uab.esRamon ManguesBiomedical Research Institute Sant Pau (IIB-Sant Pau), Josep Carreras Leukaemia Research Institute (IJC) and CIBER-BBN, Barcelona, SpainTel +34 935537918Email RMangues@santpau.catBackground and Purpose: Around 40– 50% of diffuse large-B cell lymphoma (DLBCL) patients suffer from refractory disease or relapse after R-CHOP first-line treatment. Many ongoing clinical trials for DLBCL patients involve microtubule targeting agents (MTAs), however, their anticancer activity is limited by severe side effects. Therefore, we chose to improve the therapeutic window of the MTA monomethyl auristatin E developing a nanoconjugate, T22-AUR, that selectively targets the CXCR4 receptor, which is overexpressed in many DLBCL cells (CXCR4+) and associated with poor prognosis.Methods: The T22-AUR specificity towards CXCR4 receptor was performed by flow cytometry in different DLBCL cell lines and running biodistribution assays in a subcutaneous mouse model bearing CXCR4+ DLBCL cells. Moreover, we determined T22-AUR cytotoxicity using cell viability assays, cell cycle analysis, DAPI staining and immunohistochemistry. Finally, the T22-AUR antineoplastic effect was evaluated in vivo in an extranodal CXCR4+ DLBCL mouse model whereas the toxicity analysis was assessed by histopathology in non-infiltrated mouse organs and by in vitro cytotoxic assays in human PBMCs.Results: We demonstrate that the T22-AUR nanoconjugate displays CXCR4-dependent targeting and internalization in CXCR4+ DLBCL cells in vitro as well as in a subcutaneous DLBCL mouse model. Moreover, it shows high cytotoxic effect in CXCR4+ DLBCL cells, including induction of G2/M mitotic arrest, DNA damage, mitotic catastrophe and apoptosis. Furthermore, the nanoconjugate shows a potent reduction in lymphoma mouse dissemination without histopathological alterations in non-DLBCL infiltrated organs. Importantly, T22-AUR also exhibits lack of toxicity in human PBMCs.Conclusion: T22-AUR exerts in vitro and in vivo anticancer effect on CXCR4+ DLBCL cells without off-target toxicity. Thus, T22-AUR promises to become an effective therapy for CXCR4+ DLBCL patients.Keywords: nanomedicine, targeted drug delivery, MMAE, DLBCL

Nanomedicine has opened an opportunity to improve current clinical practice by enhancing the selectivity in the delivery of antitumor drugs to specific cancer cells. These new strategies are able to bypass toxicity on normal cells increasing the effectiveness of current anticancer treatments. In acute myeloid leukemia (AML) current chemotherapy treatments generate a relevant toxic impact in normal cells and severe side effects or even patient death. In this study, we have designed a self-assembling protein nanoparticle, T22-DITOX-H6, which incorporates a ligand (T22) targeting CXCR4-overexpressing (CXCR4+) cells, and a potent cytotoxic diphtheria toxin domain. CXCR4 is overexpressed in AML leukemic cells and associates with poor prognosis, being, therefore, a relevant clinical target. We demonstrate here that T22-DITOX-H6 induces apoptosis in CXCR4+ leukemic cells through CXCR4-dependent internalization. In addition, repeated T22-DITOX-H6 treatment (10 μg/dose per 10 doses, intravenously injected) in a disseminated AML mouse model (NSG mice intravenously injected with THP-1-Luci cells, n = 10 per group) potently blocks the dissemination of AML cells in bone marrow, spleen and liver of treated mice, without inducing toxicity in healthy tissues. In conclusion, our strategy of selectively ablating CXCR4 positive leukemic cells by administering the T22-DITOX-H6 nanoparticle could be a promising treatment, especially in patients undergoing AML relapse after chemotherapy, in which leukemic cells overexpress CXCR4.

Fluorescent dye labeling is a common strategy to analyze the fate of administered nanoparticles in living organisms. However, to which extent the labeling processes can alter the original nanoparticle biodistribution has been so far neglected. In this work, two widely used fluorescent dye molecules, namely, ATTO488 (ATTO) and Sulfo‐Cy5 (S‐Cy5), have been covalently attached to a well‐characterized CXCR4‐targeted self‐assembling protein nanoparticle (known as T22‐GFP‐H6). The biodistribution of labeled T22‐GFP‐H6‐ATTO and T22‐GFP‐H6‐S‐Cy5 nanoparticles has been then compared to that of the non‐labeled nanoparticle in different CXCR4+ tumor mouse models. We observed that while parental T22‐GFP‐H6 nanoparticles accumulated mostly and specifically in CXCR4+ tumor cells, labeled T22‐GFP‐H6‐ATTO and T22‐GFP‐H6‐S‐Cy5 nanoparticles showed a dramatic change in the biodistribution pattern, accumulating in non‐target organs such as liver or kidney while reducing tumor targeting capacity. Therefore, the use of such labeling molecules should be avoided in target and non‐target tissue uptake studies during the design and development of targeted nanoscale drug delivery systems, since their effect over the fate of the nanomaterial can lead to considerable miss‐interpretations of the actual nanoparticle biodistribution. Fil: Álamo, Patricia. Centro de Investigacion Biomedica En Red.; España. Biomedical Research Institute Sant Pau; España Fil: Pallarès, Victor. Centro de Investigacion Biomedica En Red.; España. Biomedical Research Institute Sant Pau; España Fil: Céspedes, María Virtudes. Centro de Investigacion Biomedica En Red.; España. Biomedical Research Institute Sant Pau; España Fil: Falgàs, Aïda. Centro de Investigacion Biomedica En Red.; España. Biomedical Research Institute Sant Pau; España Fil: Sanchez, Julieta Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Universitat Autònoma de Barcelona; España. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Ciencias y Tecnología de los Alimentos; Argentina Fil: Serna, Naroa. Universitat Autònoma de Barcelona; España. Centro de Investigacion Biomedica En Red.; España Fil: Sánchez-García, Laura. Universitat Autònoma de Barcelona; España. Centro de Investigacion Biomedica En Red.; España Fil: Voltà-Duràn, Eric. Universitat Autònoma de Barcelona; España. Centro de Investigacion Biomedica En Red.; España Fil: Morris, Gordon A.. University of Huddersfiel; Reino Unido Fil: Sánchez-Chardi, Alejandro. Universitat Autònoma de Barcelona; España Fil: Casanova, Isolda. Biomedical Research Institute Sant Pau; España. Centro de Investigacion Biomedica En Red.; España Fil: Mangues, Ramón. Biomedical Research Institute Sant Pau; España. Centro de Investigacion Biomedica En Red.; España Fil: Vazquez, Esther. Universitat Autònoma de Barcelona; España. Centro de Investigacion Biomedica En Red.; España Fil: Villaverde Corrales, Antonio. Universitat Autònoma de Barcelona; España. Centro de Investigacion Biomedica En Red.; España Fil: Unzueta, Ugutz. Universitat Autònoma de Barcelona; España. Centro de Investigacion Biomedica En Red.; España

Current therapies fail to eradicate colorectal Cancer Stem Cells (CSCs). One of the proposed reasons for this failure is the selection, by chemotherapy exposure, of resistant cells responsible for tumor recurrence. In this regard, CXCR4 overexpression in tumor associates with resistance and poor prognosis in colorectal cancer (CRC) patients. In this study, the effectiveness of engineered CXCR4-targeted self-assembling toxin nanoparticles has been explored in the selective killing of CXCR4(+) human colon-CSCs compared to 5-Fluorouracil and Oxaliplatin, both classical CRC chemotherapeutic agents. To assess this, 3D spheroid colon-CSCs cultures directly derived from CRC patients and CRC-CSC spheroid-derived tumor mouse models were developed. In these animal models, nanostructured toxins show highly selective induction of pyroptosis in the absence of apoptosis, thus having a great potential to overcome tumor resistance, since the same tumor models show resistance to chemotherapeutics. Results set the basis for further development of more efficient therapies focused on selective CXCR4+ CSCs elimination activating non-apoptotic mechanisms and represent a pre-clinical proof of concept for the use of CSCs-targeted nanostructured toxins as protein drugs for CRC therapy.

Altres ajuts: Fondo Europeo de Desarrollo Regional (FEDER) (grant PID2019-105416RB-I00 to EV. U COST Action CA 17140. AV received an ICREA ACADEMIA award. Miguel Servet (CP19/00028) A functional 29 amino acid-segment of the helix α5 from the human BAX protein has been engineered for production in recombinant bacteria as self-assembling, GFP-containing fluorescent nanoparticles, which are targeted to the tumoral marker CXCR4. These nanoparticles, of around 34 nm in diameter, show a moderate tumor biodistribution and limited antitumoral effect when systemically administered to mouse models of human CXCR4 colorectal cancer (at 300 μg dose). However, if such BAX nanoparticles are co-administered in cocktail with equivalent nanoparticulate versions of BAK and PUMA proteins at the same total protein dose (300 μg), protein biodistribution and stability in tumor is largely improved, as determined by fluorescence profiles. This fact leads to a potent and faster destruction of tumor tissues when compared to individual pro-apoptotic factors. The analysis and interpretation of the boosted effect, from both the structural and functional sides, offers clues for the design of more efficient nanomedicines and theragnostic agents in oncology based on precise cocktails of human proteins. Statement of significance: Several human pro-apoptotic peptides (namely BAK, BAX and PUMA) have been engineered as self-assembling protein nanoparticles targeted to the tumoral marker CXCR4. The systemic administration of the same final amounts of those materials as single drugs, or as combinations of two or three of them, shows disparate intensities of antitumoral effects in a mouse model of human colorectal cancer, which are boosted in the triple combination on a non-additive basis. The superiority of the combined administration of pro-apoptotic agents, acting at different levels of the apoptotic cascade, opens a plethora of possibilities for the development of effective and selective cancer therapies based on the precise cocktailing of pro-apoptotic nanoparticulate agents. ing Research Center on Bioengineering, Biomaterials and Nanomedicine

Bacterial inclusion bodies (IBs) are mechanically stable protein particles in the microscale, which behave as robust, slow‐protein‐releasing amyloids. Upon exposure to cultured cells or upon subcutaneous or intratumor injection, these protein materials secrete functional IB polypeptides, functionally mimicking the endocrine release of peptide hormones from secretory amyloid granules. Being appealing as delivery systems for prolonged protein drug release, the development of IBs toward clinical applications is, however, severely constrained by their bacterial origin and by the undefined and protein‐to‐protein, batch‐to‐batch variable composition. In this context, the de novo fabrication of artificial IBs (ArtIBs) by simple, cell‐free physicochemical methods, using pure components at defined amounts is proposed here. By this, the resulting functional protein microparticles are intriguing, chemically defined biomimetic materials that replicate relevant functionalities of natural IBs, including mammalian cell penetration and local or remote release of functional ArtIB‐forming protein. In default of severe regulatory issues, the concept of ArtIBs is proposed as a novel exploitable category of biomaterials for biotechnological and biomedical applications, resulting from simple fabrication and envisaging soft developmental routes to clinics., Functional microscale protein granules can be obtained by simple physicochemical methods. These materials successfully mimic appealing properties of bacterial inclusion bodies, including amyloidal architecture, biological activity, and the release functional protein in vitro and in vivo. The applicability of these novel materials in biotechnology and in biomedicine is fully supported by their synthetic nature and defined chemical composition.

Altres ajuts: we are indebted to CIBER de Bioingeniería, Biomateriales y Nanomedicina (projects NANOREMOTE and VENOM4CANCER) to EV and AV respectively, Marató de TV3 foundation (TV32013-132031) and CIBER (NanoMets3) to RM. Protein production has been partially performed by the ICTS "NANBIOSIS", more specifically by the Protein Production Platform of CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN)/IBB, at the UAB SepBioES scientific-technical service (http://www.nanbiosis.es/unit/u1-protein-production-platform-ppp/), whereas the in vivo biodistribution assays were performed in the NANBIOSIS Nanotoxicology platform (http://www.nanbiosis.es/unit/u18-nanotoxicology-unit/). We are also indebted to Fran Cortes from the Cell Culture and Cytometry Units of the Servei de Cultius Cel·lulars, Producció d'Anticossos i Citometria (SCAC), and to the Servei de Microscòpia at the UAB. AV received an ICREA ACADEMIA award. U.U received a Sara Borrell postdoctoral fellowship from ISCIII, MVC was supported by Miguel Servet contract from ISCIII, and JSF received and AECC postdoctoral fellowship. Sustained release of drug delivery systems (DDS) has the capacity to increase cancer treatment efficiency in terms of drug dosage reduction and subsequent decrease of deleterious side effects. In this regard, many biomaterials are being investigated but none offers morphometric and functional plasticity and versatility comparable to protein-based nanoparticles (pNPs). Here we describe a new DDS by which pNPs are fabricated as bacterial inclusion bodies (IB), that can be easily isolated, subcutaneously injected and used as reservoirs for the sustained release of targeted pNPs. Our approach combines the high performance of pNP, regarding specific cell targeting and biodistribution with the IB supramolecular organization, stability and cost effectiveness. This renders a platform able to provide a sustained source of CXCR4-targeted pNPs that selectively accumulate in tumor cells in a CXCR4 colorectal cancer xenograft model. In addition, the proposed system could be potentially adapted to any other protein construct offering a plethora of possible new therapeutic applications in nanomedicine.

Loading capacity and drug leakage from vehicles during circulation in blood is a major concern when developing nanoparticle-based cell-targeted cytotoxics. To circumvent this potential issue it would be convenient the engineering of drugs as self-delivered nanoscale entities, devoid of any heterologous carriers. In this context, we have here engineered potent protein toxins, namely segments of the diphtheria toxin and the Pseudomonas aeruginosa exotoxin as self-assembling, self-delivered therapeutic materials targeted to CXCR4(+) cancer stem cells. The systemic administration of both nanostructured drugs in a colorectal cancer xenograft mouse model promotes efficient and specific local destruction of target tumor tissues and a significant reduction of the tumor volume. This observation strongly supports the concept of intrinsically functional protein nanoparticles, which having a dual role as drug and carrier, are designed to be administered without the assistance of heterologous vehicles.

A single chain polypeptide containing the low density lipoprotein receptor (LDLR) ligand Seq-1 with blood-brain barrier (BBB) crossing activity has been successfully modified by conventional genetic engineering to self-assemble into stable protein-only nanoparticles of 30 nm. The nanoparticulate presentation dramatically enhances in vitro, LDLR-dependent cell penetrability compared to the parental monomeric version, but the assembled protein does not show any enhanced brain targeting upon systemic administration. While the presentation of protein drugs in form of nanoparticles is in general advantageous regarding correct biodistribution, this principle might not apply to brain targeting that is hampered by particular bio-physical barriers. Irrespective of this fact, which is highly relevant to the nanomedicine of central nervous system, engineering the cationic character of defined protein stretches is revealed here as a promising and generic approach to promote the controlled oligomerization of biologically active protein species as still functional, regular nanoparticles. (C) 2016 Elsevier Inc. All rights reserved.