Structural Stabilization of Clinically Oriented Oligomeric Proteins During their Transit through Synthetic Secretory Amyloids.

Developing time-sustained drug delivery systems is a main goal in innovative medicines. Inspired by the architecture of secretory granules from the mammalian endocrine system it has generated non-toxic microscale amyloid materials through the coordination between divalent metals and poly-histidine stretches. Like their natural counterparts that keep the functionalities of the assembled protein, those synthetic structures release biologically active proteins during a slow self-disintegration process occurring in vitro and upon in vivo administration. Being these granules formed by a single pure protein species and therefore, chemically homogenous, they act as highly promising time-sustained drug delivery systems. Despite their enormous clinical potential, the nature of the clustering process and the quality of the released protein have been so far neglected issues. By using diverse polypeptide species and their protein-only oligomeric nanoscale versions as convenient models, a conformational rearrangement and a stabilization of the building blocks during their transit through the secretory granules, being the released material structurally distinguishable from the original source is proved here. This fact indicates a dynamic nature of secretory amyloids that act as conformational arrangers rather than as plain, inert protein-recruiting/protein-releasing granular depots.

Nanoparticle-Based Secretory Granules Induce a Specific and Long-Lasting Immune Response through Prolonged Antigen Release.

Developing prolonged antigen delivery systems that mimic long-term exposure to pathogens appears as a promising but still poorly explored approach to reach durable immunities. In this study, we have used a simple technology by which His-tagged proteins can be assembled, assisted by divalent cations, as supramolecular complexes with progressive complexity, namely protein-only nanoparticles and microparticles. Microparticles produced out of nanoparticles are biomimetics of secretory granules from the mammalian hormonal system. Upon subcutaneous administration, they slowly disintegrate, acting as an endocrine-like secretory system and rendering the building block nanoparticles progressively bioavailable. The performance of such materials, previously validated for drug delivery in oncology, has been tested here regarding the potential for time-prolonged antigen release. This has been completed by taking, as a building block, a nanostructured version of p30, a main structural immunogen from the African swine fever virus (ASFV). By challenging the system in both mice and pigs, we have observed unusually potent pro-inflammatory activity in porcine macrophages, and long-lasting humoral and cellular responses in vivo, which might overcome the need for an adjuvant. The robustness of both innate and adaptive responses tag, for the first time, these dynamic depot materials as a novel and valuable instrument with transversal applicability in immune stimulation and vaccinology.

Analysis of cardiovascular risk factors for the viability of cornea donors: a case-control study.

OBJECTIVE: The main objective of the study was to evaluate the influence of arterial hypertension, diabetes, dyslipidaemia, smoking, alcoholism and COPD (chronic obstructive pulmonary disease) on the viability of the extracted tissue as well as the donor. DESIGN: Observational case-control study. SETTING: Regional hospital in Northern Spain. PARTICIPANTS: 1517 corneas were registered. INTERVENTIONS: Patients' medical history was reviewed after corneal donation and evaluation. Previous medical information (age, sex and cardiovascular risk factors (CVRFs)) and data related to the donor (type of donor), the corneal tissue and its evaluation, and the viability of the implant were collected. RESULTS: A total of 1517 corneas were registered and 81.5% of the donors presented at least one CVRF. In relation to the viability of the donor, it was observed that having suffered from COPD reduced the viability of the donor (no COPD: 93.8% vs COPD: 88%; OR=0.49; 95% CI: 0.28 to 0.84) while alcohol consumption increased it (drinker or ex-drinker: 95.8% vs non-drinker: 92.5%; OR=1.84; 95% CI: 1.01 to 3.33). Regarding tissue viability, decreased viability was observed in the presence of COPD (no COPD: 72.5% vs COPD: 64; OR=0.67; 95% CI: 0.47 to 0.96) and diabetes mellitus (no diabetes: 72.9% vs diabetes: 67.2%; OR=0.76; 95% CI: 0.58 to 0.99). As regards the viability of the implant, a total of 1039 corneas (68.9%) were suitable, observing decreased viability when suffering from COPD (no COPD: 69.8% vs COPD: 60.7%; OR=0.67; 95% CI: 0.47 to 0.94) and increased when having an active smoking habit (no habit: 65.3% vs habit: 74.1%; OR=1.52; 95% CI: 1.21 to 1.91). CONCLUSIONS: Through this study, it can be concluded that in the absence of absolute exclusion criteria for donors, the assessment of how CVRF, alcoholism and COPD may affect the donor provides details about the quality of the tissue to be obtained.

Efficient Delivery of Antimicrobial Peptides in an Innovative, Slow-Release Pharmacological Formulation.

Both nanostructure and multivalency enhance the biological activities of antimicrobial peptides (AMPs), whose mechanism of action is cooperative. In addition, the efficacy of a particular AMP should benefit from a steady concentration at the local place of action and, therefore, from a slow release after a dynamic repository. In the context of emerging multi-resistant bacterial infections and the urgent need for novel and effective antimicrobial drugs, we tested these concepts through the engineering of four AMPs into supramolecular complexes as pharmacological entities. For that purpose, GWH1, T22, Pt5, and PaD, produced as GFP or human nidogen-based His-tagged fusion proteins, were engineered as self-assembling oligomeric nanoparticles ranging from 10 to 70 nm and further packaged into nanoparticle-leaking submicron granules. Since these materials slowly release functional nanoparticles during their time-sustained unpacking, they are suitable for use as drug depots in vivo. In this context, a particular AMP version (GWH1-NIDO-H6) was selected for in vivo validation in a zebrafish model of a complex bacterial infection. The GWH1-NIDO-H6-secreting protein granules are protective in zebrafish against infection by the multi-resistant bacterium Stenotrophomonas maltophilia, proving the potential of innovative formulations based on nanostructured and slowly released recombinant AMPs in the fight against bacterial infections.

High-precision targeting and destruction of cancer-associated PDGFR-ß+ stromal fibroblasts through self-assembling, protein-only nanoparticles.

The need for more effective and precision medicines for cancer has pushed the exploration of new materials appropriate for drug delivery and imaging, and alternative receptors for targeting. Among the most promising strategies, finding suitable cell surface receptors and targeting agents for cancer-associated platelet derived growth factor receptor ß (PDGFR-ß)+ stromal fibroblasts is highly appealing. As a neglected target, this cell type mechanically and biologically supports the growth, progression, and infiltration of solid tumors in non-small cell lung, breast, pancreatic, and colorectal cancers. We have developed a family of PDGFR-ß-targeted nanoparticles based on biofabricated, self-assembling proteins, upon hierarchical and iterative selective processes starting from four initial candidates. The modular protein PDGFD-GFP-H6 is well produced in recombinant bacteria, resulting in structurally robust oligomeric particles that selectively penetrates into PDGFR-ß+ stromal fibroblasts in a dose-dependent manner, by means of the PDGFR-ß ligand PDGFD. Upon in vivo administration, these GFP-carrying protein nanoparticles precisely accumulate in tumor tissues and enlighten them for IVIS observation. When GFP is replaced by a microbial toxin, selective tumor tissue destruction is observed associated with a significant reduction in tumor volume growth. The presented data validate the PDGFR-ß/PDGFD pair as a promising toolbox for targeted drug delivery in the tumor microenvironment and oligomeric protein nanoparticles as a powerful instrument to mediate highly selective biosafe targeting in cancer through non-cancer cells. STATEMENT OF SIGNIFICANCE: We have developed a transversal platform for nanoparticle-based drug delivery into cancer-associated fibroblasts. This is based on the engineered modular protein PDGFD-GFP-H6 that spontaneously self-assemble and selectively penetrates into PDGFR-ß+ stromal fibroblasts in a dose-dependent manner, by means of the PDGFR-ß ligand PDGFD. In vivo, these protein nanoparticles accumulate in tumor and when incorporating a microbial toxin, they destroy tumor tissues with a significant reduction in tumor volume, in absence of side toxicities. The data presented here validate the PDGFR-ß/PDGFD pair as a fully versatile toolbox for targeted drug delivery in the tumor microenvironment intended as a synergistic treatment.

Probing the Biosafety of Implantable Artificial Secretory Granules for the Sustained Release of Bioactive Proteins.

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.

Protein features instruct the secretion dynamics from metal-supported synthetic amyloids.

Hexahistidine-tagged proteins can be clustered by divalent cations into self-containing, dynamic protein depots at the microscale, which under physiological conditions leak functional protein. While such protein granules show promise in clinics as time-sustained drug delivery systems, little is known about how the nature of their components, that is, the protein and the particular cation used as cross-linker, impact on the disintegration of the material and on its secretory performance. By using four model proteins and four different cation formulations to control aggregation, we have here determined a moderate influence of the used cation and a potent impact of some protein properties on the release kinetics and on the final fraction of releasable protein. In particular, the electrostatic charge at the amino terminus and the instability and hydropathicity indexes determine the disintegration profile of the depot. These data offer clues for the fabrication of efficient and fully exploitable secretory granules that being biocompatible and chemically homogenous allow their tailored use as drug delivery platforms in biological systems.

Lymphocyte infiltration and antitumoral effect promoted by cytotoxic inflammatory proteins formulated as self-assembling, protein-only nanoparticles.

Two human proteins involved in the inflammatory cell death, namely Gasdermin D (GSDMD) and the Mixed Lineage Kinase Domain-Like (MLKL) protein have been engineered to accommodate an efficient ligand of the tumoral cell marker CXCR4, and a set of additional peptide agents that allow their spontaneous self-assembling. Upon production in bacterial cells and further purification, both proteins organized as stable nanoparticles of 46 and 54 nm respectively, that show, in this form, a moderate but dose-dependent cytotoxicity in cell culture. In vivo, and when administered in mouse models of colorectal cancer through repeated doses, the nanoscale forms of tumor-targeted GSDMD and, at a lesser extent, of MLKL promoted CD8+ and CD20+ lymphocyte infiltration in the tumor and an important reduction of tumor size, in absence of systemic toxicity. The potential of these novel pharmacological agents as anticancer drugs is discussed in the context of synergistic approaches to more effective cancer treatments.

Recombinant Proteins for Assembling as Nano- and Micro-Scale Materials for Drug Delivery: A Host Comparative Overview.

By following simple protein engineering steps, recombinant proteins with promising applications in the field of drug delivery can be assembled in the form of functional materials of increasing complexity, either as nanoparticles or nanoparticle-leaking secretory microparticles. Among the suitable strategies for protein assembly, the use of histidine-rich tags in combination with coordinating divalent cations allows the construction of both categories of material out of pure polypeptide samples. Such molecular crosslinking results in chemically homogeneous protein particles with a defined composition, a fact that offers soft regulatory routes towards clinical applications for nanostructured protein-only drugs or for protein-based drug vehicles. Successes in the fabrication and final performance of these materials are expected, irrespective of the protein source. However, this fact has not yet been fully explored and confirmed. By taking the antigenic RBD domain of the SARS-CoV-2 spike glycoprotein as a model building block, we investigated the production of nanoparticles and secretory microparticles out of the versions of recombinant RBD produced by bacteria (Escherichia coli), insect cells (Sf9), and two different mammalian cell lines (namely HEK 293F and Expi293F). Although both functional nanoparticles and secretory microparticles were effectively generated in all cases, the technological and biological idiosyncrasy of each type of cell factory impacted the biophysical properties of the products. Therefore, the selection of a protein biofabrication platform is not irrelevant but instead is a significant factor in the upstream pipeline of protein assembly into supramolecular, complex, and functional materials.

Enhanced recombinant protein capture, purity and yield from crude bacterial cell extracts by N-Lauroylsarcosine-assisted affinity chromatography.

BACKGROUND: Recombinant proteins cover a wide range of biomedical, biotechnological, and industrial needs. Although there are diverse available protocols for their purification from cell extracts or from culture media, many proteins of interest such as those containing cationic domains are difficult to purify, a fact that results in low yields of the final functional product. Unfortunately, this issue prevents the further development and industrial or clinical application of these otherwise interesting products. RESULTS: Aiming at improving the purification of such difficult proteins, a novel procedure has been developed based on supplementing crude cell extracts with non-denaturing concentrations of the anionic detergent N-Lauroylsarcosine. The incorporation of this simple step in the downstream pipeline results in a substantial improvement of the protein capture by affinity chromatography, an increase of protein purity and an enhancement of the overall process yield, being the detergent not detectable in the final product. CONCLUSION: By taking this approach, which represents a smart repurposing of N-Lauroylsarcosine applied to protein downstream, the biological activity of the protein is not affected. Being technologically simple, the N-Lauroylsarcosine-assisted protein purification might represent a critical improvement in recombinant protein production with wide applicability, thus smothering the incorporation of promising proteins into the protein market.

T22-PE24-H6 Nanotoxin Selectively Kills CXCR4-High Expressing AML Patient Cells In Vitro and Potently Blocks Dissemination In Vivo.

Despite advances in the development of targeted therapies for acute myeloid leukemia (AML), most patients relapse. For that reason, it is still necessary to develop novel therapies that improve treatment effectiveness and overcome drug resistance. We developed T22-PE24-H6, a protein nanoparticle that contains the exotoxin A from the bacterium Pseudomonas aeruginosa and is able to specifically deliver this cytotoxic domain to CXCR4+ leukemic cells. Next, we evaluated the selective delivery and antitumor activity of T22-PE24-H6 in CXCR4+ AML cell lines and BM samples from AML patients. Moreover, we assessed the in vivo antitumor effect of this nanotoxin in a disseminated mouse model generated from CXCR4+ AML cells. T22-PE24-H6 showed a potent, CXCR4-dependent antineoplastic effect in vitro in the MONO-MAC-6 AML cell line. In addition, mice treated with nanotoxins in daily doses reduced the dissemination of CXCR4+ AML cells compared to buffer-treated mice, as shown by the significant decrease in BLI signaling. Furthermore, we did not observe any sign of toxicity or changes in mouse body weight, biochemical parameters, or histopathology in normal tissues. Finally, T22-PE24-H6 exhibited a significant inhibition of cell viability in CXCR4high AML patient samples but showed no activity in CXCR4low samples. These data strongly support the use of T22-PE24-H6 therapy to benefit high-CXCR4-expressing AML patients.

Endosomal escape for cell-targeted proteins. Going out after going in.

Protein-based nanocarriers are versatile and biocompatible drug delivery systems. They are of particular interest in nanomedicine as they can recruit multiple functions in a single modular polypeptide. Many cell-targeting peptides or protein domains can promote cell uptake when included in these nanoparticles through receptor-mediated endocytosis. In that way, targeting drugs to specific cell receptors allows a selective intracellular delivery process, avoiding potential side effects of the payload. However, once internalized, the endo-lysosomal route taken by the engulfed material usually results in full degradation, preventing their adequate subcellular localization, bioavailability and subsequent therapeutic effect. Thus, entrapment into endo-lysosomes is a main bottleneck in the efficacy of protein-drug nanomedicines. Promoting endosomal escape and preventing lysosomal degradation would make this therapeutic approach clinically plausible. In this review, we discuss the mechanisms intended to evade lysosomal degradation of proteins, with the most relevant examples and associated strategies, and the methods available to measure that effect. In addition, based on the increasing catalogue of peptide domains tailored to face this challenge as components of protein nanocarriers, we emphasize how their particular mechanisms of action can potentially alter the functionality of accompanying protein materials, especially in terms of targeting and specificity in the delivery process.

The Diphtheria Toxin Translocation Domain Impairs Receptor Selectivity in Cancer Cell-Targeted Protein Nanoparticles.

Protein-based materials intended as nanostructured drugs or drug carriers are progressively gaining interest in nanomedicine, since their structure, assembly and cellular interactivity can be tailored by recruiting functional domains. The main bottleneck in the development of deliverable protein materials is the lysosomal degradation that follows endosome maturation. This is especially disappointing in the case of receptor-targeted protein constructs, which, while being highly promising and in demand in precision medicines, enter cells via endosomal/lysosomal routes. In the search for suitable protein agents that might promote endosome escape, we have explored the translocation domain (TD) of the diphtheria toxin as a functional domain in CXCR4-targeted oligomeric nanoparticles designed for cancer therapies. The pharmacological interest of such protein materials could be largely enhanced by improving their proteolytic stability. The incorporation of TD into the building blocks enhances the amount of the material detected inside of exposed CXCR4+ cells up to around 25-fold, in absence of cytotoxicity. This rise cannot be accounted for by endosomal escape, since the lysosomal degradation of the new construct decreases only moderately. On the other hand, a significant loss in the specificity of the CXCR4-dependent cellular penetration indicates the unexpected role of the toxin segment as a cell-penetrating peptide in a dose-dependent and receptor-independent fashion. These data reveal that the diphtheria toxin TD displayed on receptor-targeted oligomeric nanoparticles partially abolishes the exquisite receptor specificity of the parental material and it induces nonspecific internalization in mammalian cells.

Selectively Targeting Breast Cancer Stem Cells by 8-Quinolinol and Niclosamide.

Cancer maintenance, metastatic dissemination and drug resistance are sustained by cancer stem cells (CSCs). Triple negative breast cancer (TNBC) is the breast cancer subtype with the highest number of CSCs and the poorest prognosis. Here, we aimed to identify potential drugs targeting CSCs to be further employed in combination with standard chemotherapy in TNBC treatment. The anti-CSC efficacy of up to 17 small drugs was tested in TNBC cell lines using cell viability assays on differentiated cancer cells and CSCs. Then, the effect of 2 selected drugs (8-quinolinol -8Q- and niclosamide -NCS-) in the cancer stemness features were evaluated using mammosphere growth, cell invasion, migration and anchorage-independent growth assays. Changes in the expression of stemness genes after 8Q or NCS treatment were also evaluated. Moreover, the potential synergism of 8Q and NCS with PTX on CSC proliferation and stemness-related signaling pathways was evaluated using TNBC cell lines, CSC-reporter sublines, and CSC-enriched mammospheres. Finally, the efficacy of NCS in combination with PTX was analyzed in vivo using an orthotopic mouse model of MDA-MB-231 cells. Among all tested drug candidates, 8Q and NCS showed remarkable specific anti-CSC activity in terms of CSC viability, migration, invasion and anchorage independent growth reduction in vitro. Moreover, specific 8Q/PTX and NCS/PTX ratios at which both drugs displayed a synergistic effect in different TNBC cell lines were identified. The sole use of PTX increased the relative presence of CSCs in TNBC cells, whereas the combination of 8Q and NCS counteracted this pro-CSC activity of PTX while significantly reducing cell viability. In vivo, the combination of NCS with PTX reduced tumor growth and limited the dissemination of the disease by reducing circulating tumor cells and the incidence of lung metastasis. The combination of 8Q and NCS with PTX at established ratios inhibits both the proliferation of differentiated cancer cells and the viability of CSCs, paving the way for more efficacious TNBC treatments.

Recombinant vaccines in 2022: a perspective from the cell factory.

The last big outbreaks of Ebola fever in Africa, the thousands of avian influenza outbreaks across Europe, Asia, North America and Africa, the emergence of monkeypox virus in Europe and specially the COVID-19 pandemics have globally stressed the need for efficient, cost-effective vaccines against infectious diseases. Ideally, they should be based on transversal technologies of wide applicability. In this context, and pushed by the above-mentioned epidemiological needs, new and highly sophisticated DNA-or RNA-based vaccination strategies have been recently developed and applied at large-scale. Being very promising and effective, they still need to be assessed regarding the level of conferred long-term protection. Despite these fast-developing approaches, subunit vaccines, based on recombinant proteins obtained by conventional genetic engineering, still show a wide spectrum of interesting potentialities and an important margin for further development. In the 80's, the first vaccination attempts with recombinant vaccines consisted in single structural proteins from viral pathogens, administered as soluble plain versions. In contrast, more complex formulations of recombinant antigens with particular geometries are progressively generated and explored in an attempt to mimic the multifaceted set of stimuli offered to the immune system by replicating pathogens. The diversity of recombinant antimicrobial vaccines and vaccine prototypes is revised here considering the cell factory types, through relevant examples of prototypes under development as well as already approved products.

Protein scaffolds in human clinics.

Fundamental clinical areas such as drug delivery and regenerative medicine require biocompatible materials as mechanically stable scaffolds or as nanoscale drug carriers. Among the wide set of emerging biomaterials, polypeptides offer enticing properties over alternative polymers, including full biocompatibility, biodegradability, precise interactivity, structural stability and conformational and functional versatility, all of them tunable by conventional protein engineering. However, proteins from non-human sources elicit immunotoxicities that might bottleneck further development and narrow their clinical applicability. In this context, selecting human proteins or developing humanized protein versions as building blocks is a strict demand to design non-immunogenic protein materials. We review here the expanding catalogue of human or humanized proteins tailored to execute different levels of scaffolding functions and how they can be engineered as self-assembling materials in form of oligomers, polymers or complex networks. In particular, we emphasize those that are under clinical development, revising their fields of applicability and how they have been adapted to offer, apart from mere mechanical support, highly refined functions and precise molecular interactions.

Novel Endometrial Cancer Models Using Sensitive Metastasis Tracing for CXCR4-Targeted Therapy in Advanced Disease.

Advanced endometrial cancer (EC) lacks therapy, thus, there is a need for novel treatment targets. CXCR4 overexpression is associated with a poor prognosis in several cancers, whereas its inhibition prevents metastases. We assessed CXCR4 expression in EC in women by using IHC. Orthotopic models were generated with transendometrial implantation of CXCR4-transduced EC cells. After in vitro evaluation of the CXCR4-targeted T22-GFP-H6 nanocarrier, subcutaneous EC models were used to study its uptake in tumor and normal organs. Of the women, 91% overexpressed CXCR4, making them candidates for CXCR4-targeted therapies. Thus, we developed CXCR4+ EC mouse models to improve metastagenesis compared to current models and to use them to develop novel CXCR4-targeted therapies for unresponsive EC. It showed enhanced dissemination, especially in the lungs and liver, and displayed 100% metastasis penetrance at all clinically relevant sites with anti-hVimentin IHC, improving detection sensitivity. Regarding the CXCR4-targeted nanocarrier, 60% accumulated in the SC tumor; therefore, selectively targeting CXCR4+ cancer cells, without toxicity in non-tumor organs. Our CXCR4+ EC models will allow testing of novel CXCR4-targeted drugs and development of nanomedicines derived from T22-GFP-H6 to deliver drugs to CXCR4+ cells in advanced EC. This novel approach provides a therapeutic option for women with metastatic, high risk or recurrent EC that have a dismal prognosis and lack effective therapies.

Self-assembling protein nanocarrier for selective delivery of cytotoxic polypeptides to CXCR4+ head and neck squamous cell carcinoma tumors.

Loco-regional recurrences and distant metastases represent the main cause of head and neck squamous cell carcinoma (HNSCC) mortality. The overexpression of chemokine receptor 4 (CXCR4) in HNSCC primary tumors associates with higher risk of developing loco-regional recurrences and distant metastases, thus making CXCR4 an ideal entry pathway for targeted drug delivery. In this context, our group has generated the self-assembling protein nanocarrier T22-GFP-H6, displaying multiple T22 peptidic ligands that specifically target CXCR4. This study aimed to validate T22-GFP-H6 as a suitable nanocarrier to selectively deliver cytotoxic agents to CXCR4+ tumors in a HNSCC model. Here we demonstrate that T22-GFP-H6 selectively internalizes in CXCR4+ HNSCC cells, achieving a high accumulation in CXCR4+ tumors in vivo, while showing negligible nanocarrier distribution in non-tumor bearing organs. Moreover, this T22-empowered nanocarrier can incorporate bacterial toxin domains to generate therapeutic nanotoxins that induce cell death in CXCR4-overexpressing tumors in the absence of histological alterations in normal organs. Altogether, these results show the potential use of this T22-empowered nanocarrier platform to incorporate polypeptidic domains of choice to selectively eliminate CXCR4+ cells in HNSCC. Remarkably, to our knowledge, this is the first study testing targeted protein-only nanoparticles in this cancer type, which may represent a novel treatment approach for HNSCC patients.

GSDMD-dependent pyroptotic induction by a multivalent CXCR4-targeted nanotoxin blocks colorectal cancer metastases.

Colorectal cancer (CRC) remains the third cause of cancer-related mortality in Western countries, metastases are the main cause of death. CRC treatment remains limited by systemic toxicity and chemotherapy resistance. Therefore, nanoparticle-mediated delivery of cytotoxic agents selectively to cancer cells represents an efficient strategy to increase the therapeutic index and overcome drug resistance. We have developed the T22-PE24-H6 therapeutic protein-only nanoparticle that incorporates the exotoxin A from Pseudomonas aeruginosa to selectively target CRC cells because of its multivalent ligand display that triggers a high selectivity interaction with the CXCR4 receptor overexpressed on the surface of CRC stem cells. We here observed a CXCR4-dependent cytotoxic effect for T22-PE24-H6, which was not mediated by apoptosis, but instead capable of inducing a time-dependent and sequential activation of pyroptotic markers in CRC cells in vitro. Next, we demonstrated that repeated doses of T22-PE24-H6 inhibit tumor growth in a subcutaneous CXCR4+ CRC model, also through pyroptotic activation. Most importantly, this nanoparticle also blocked the development of lymphatic and hematogenous metastases, in a highly aggressive CXCR4+ SW1417 orthotopic CRC model, in the absence of systemic toxicity. This targeted drug delivery approach supports for the first time the clinical relevance of inducing GSDMD-dependent pyroptosis, a cell death mechanism alternative to apoptosis, in CRC models, leading to the selective elimination of CXCR4+ cancer stem cells, which are associated with resistance, metastases and anti-apoptotic upregulation.

An In Silico Methodology That Facilitates Decision Making in the Engineering of Nanoscale Protein Materials.

Under the need for new functional and biocompatible materials for biomedical applications, protein engineering allows the design of assemblable polypeptides, which, as convenient building blocks of supramolecular complexes, can be produced in recombinant cells by simple and scalable methodologies. However, the stability of such materials is often overlooked or disregarded, becoming a potential bottleneck in the development and viability of novel products. In this context, we propose a design strategy based on in silico tools to detect instability areas in protein materials and to facilitate the decision making in the rational mutagenesis aimed to increase their stability and solubility. As a case study, we demonstrate the potential of this methodology to improve the stability of a humanized scaffold protein (a domain of the human nidogen), with the ability to oligomerize into regular nanoparticles usable to deliver payload drugs to tumor cells. Several nidogen mutants suggested by the method showed important and measurable improvements in their structural stability while retaining the functionalities and production yields of the original protein. Then, we propose the procedure developed here as a cost-effective routine tool in the design and optimization of multimeric protein materials prior to any experimental testing.