Lenvatinib-Loaded Poly(lactic-co-glycolic acid) Nanoparticles with Epidermal Growth Factor Receptor Antibody Conjugation as a Preclinical Approach to Therapeutically Improve Thyroid Cancer with Aggressive Behavior.

BACKGROUND: Lenvatinib, a tyrosine kinase inhibitor (TKI) approved for the treatment of progressive and radioactive iodine (RAI)-refractory differentiated thyroid cancer (DTC), is associated with significant adverse effects that can be partially mitigated through the development of novel drug formulations. The utilization of nanoparticles presents a viable option, as it allows for targeted drug delivery, reducing certain side effects and enhancing the overall quality of life for patients. This study aimed to produce and assess, both in vitro and in vivo, the cytotoxicity, biodistribution, and therapeutic efficacy of lenvatinib-loaded PLGA nanoparticles (NPs), both with and without decoration using antibody conjugation (cetuximab), as a novel therapeutic approach for managing aggressive thyroid tumors. METHODS: Poly(lactic-co-glycolic acid) nanoparticles (NPs), decorated with or without anti-EGFR, were employed as a lenvatinib delivery system. These NPs were characterized for size distribution, surface morphology, surface charge, and drug encapsulation efficiency. Cytotoxicity was evaluated through MTT assays using two cellular models, one representing normal thyroid cells (Nthy-ori 3-1) and the other representing anaplastic thyroid cells (CAL-62). Additionally, an in vivo xenograft mouse model was established to investigate biodistribution and therapeutic efficacy following intragastric administration. RESULTS: The NPs demonstrated success in terms of particle size, polydispersity index (PDI), zeta potential, morphology, encapsulation efficiency, and cetuximab distribution across the surface. In vitro analysis revealed cytotoxicity in both cellular models with both formulations, but only the decorated NPs achieved an ID50 value in CAL-62 cells. Biodistribution analysis following intragastric administration in xenografted thyroid mice demonstrated good stability in terms of intestinal barrier function and tumor accumulation. Both formulations were generally well tolerated without inducing pathological effects in the examined organs. Importantly, both formulations increased tumor necrosis; however, decorated NPs exhibited enhanced parameters related to apoptotic/karyolytic forms, mitotic index, and vascularization compared with NPs without decoration. CONCLUSIONS: These proof-of-concept findings suggest a promising strategy for administering TKIs in a more targeted and effective manner.

Dopamine receptors D1 and D2 show prognostic significance and potential therapeutic applications for endometrial cancer patients.

OBJECTIVE: Catecholaminergic signaling has been a target for therapy in different type of cancers. In this work, we characterized the ADRß2, DRD1 and DRD2 expression in healthy tissue and endometrial tumors to evaluate their prognostic significance in endometrial cancer (EC), unraveling their possible application as an antitumor therapy. METHODS: 109 EC patients were included. The expression of the ADRß2, DRD1 and DRD2 proteins was evaluated by immunohistochemistry and univariate and multivariate analysis to assess their association with clinic-pathological and outcome variables. Finally, HEC1A and AN3CA EC cell lines were exposed to different concentrations of selective dopaminergic agents alone or in combination to study their effects on cellular viability. RESULTS: ADRß2 protein expression was not associated with clinico-pathological parameters or prognosis. DRD1 protein expression was reduced in tumors samples but showed a significant inverse association with tumor size and stage. DRD2 protein expression was significantly associated with non-endometrioid EC, high grade tumors, tumor size, worse disease-free survival (HR = 3.47 (95%CI:1.35-8.88)) and overall survival (HR = 2.98 (95%CI:1.40-6.34)). The DRD1 agonist fenoldopam showed a reduction of cellular viability in HEC1A and AN3CA cells. The exposure to domperidone, a DRD2 antagonist, significantly reduced cell viability compared to the control. Finally, DRD1 agonism and DRD2 antagonism combination induced a significant reduction in cell viability of the AN3CA cells compared to monotherapy, close to being an additive response than a synergistic effect (CI of 1.1 at 0.5% Fa). CONCLUSION: DRD1 and DRD2 expression levels showed a significant association with clinico-pathological parameters. Both the combined activation of DRD1 and blockage of DRD2 may form an innovative strategy to inhibit tumor growth in EC.

Escherichia coli as a New Platform for the Fast Production of Vault-like Nanoparticles: An Optimized Protocol.

Vaults are protein nanoparticles that are found in almost all eukaryotic cells but are absent in prokaryotic ones. Due to their properties (nanometric size, biodegradability, biocompatibility, and lack of immunogenicity), vaults show enormous potential as a bio-inspired, self-assembled drug-delivery system (DDS). Vault architecture is directed by self-assembly of the "major vault protein" (MVP), the main component of this nanoparticle. Recombinant expression (in different eukaryotic systems) of the MVP resulted in the formation of nanoparticles that were indistinguishable from native vaults. Nowadays, recombinant vaults for different applications are routinely produced in insect cells and purified by successive ultracentrifugations, which are both tedious and time-consuming strategies. To offer cost-efficient and faster protocols for nanoparticle production, we propose the production of vault-like nanoparticles in Escherichia coli cells, which are still one of the most widely used prokaryotic cell factories for recombinant protein production. The strategy proposed allowed for the spontaneous encapsulation of the engineered cargo protein within the self-assembled vault-like nanoparticles by simply mixing the clarified lysates of the producing cells. Combined with well-established affinity chromatography purification methods, our approach contains faster, cost-efficient procedures for biofabrication in a well-known microbial cell factory and the purification of "ready-to-use" loaded protein nanoparticles, thereby opening the way to faster and easier engineering and production of vault-based DDSs.

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.

Nano-Based Approved Pharmaceuticals for Cancer Treatment: Present and Future Challenges.

Cancer is one of the main causes of death worldwide. To date, and despite the advances in conventional treatment options, therapy in cancer is still far from optimal due to the non-specific systemic biodistribution of antitumor agents. The inadequate drug concentrations at the tumor site led to an increased incidence of multiple drug resistance and the appearance of many severe undesirable side effects. Nanotechnology, through the development of nanoscale-based pharmaceuticals, has emerged to provide new and innovative drugs to overcome these limitations. In this review, we provide an overview of the approved nanomedicine for cancer treatment and the rationale behind their designs and applications. We also highlight the new approaches that are currently under investigation and the perspectives and challenges for nanopharmaceuticals, focusing on the tumor microenvironment and tumor disseminate cells as the most attractive and effective strategies for cancer treatments.

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.

Neural plasticity of the uterus: New targets for endometrial cancer?

Endometrial carcinoma is the most common gynecological malignancy in Western countries and is expected to increase in the following years because of the high index of obesity in the population. Recently, neural signaling has been recognized as part of the tumor microenvironment, playing an active role in tumor progression and invasion of different solid tumor types. The uterus stands out for the physiological plasticity of its peripheral nerves due to cyclic remodeling brought on by estrogen and progesterone hormones throughout the reproductive cycle. Therefore, a precise understanding of nerve-cancer crosstalk and the contribution of the organ-intrinsic neuroplasticity, mediated by estrogen and progesterone, of the uterine is urgently needed. The development of new and innovative medicines for patients with endometrial cancer would increase their quality of life and health. This review compiles information on the architecture and function of autonomous uterine neural innervations and the influence of hormone-dependent nerves in normal uterus and tumor progression. It also explores new therapeutic possibilities for endometrial cancer using these endocrine and neural advantages.

Fluorescent Dye Labeling Changes the Biodistribution of Tumor-Targeted Nanoparticles.

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.

Nanostructured toxins for the selective destruction of drug-resistant human CXCR4+ colorectal cancer stem cells.

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.

Engineering Secretory Amyloids for Remote and Highly Selective Destruction of Metastatic Foci.

Functional amyloids produced in bacteria as nanoscale inclusion bodies are intriguing but poorly explored protein materials with wide therapeutic potential. Since they release functional polypeptides under physiological conditions, these materials can be potentially tailored as mimetic of secretory granules for slow systemic delivery of smart protein drugs. To explore this possibility, bacterial inclusion bodies formed by a self-assembled, tumor-targeted Pseudomonas exotoxin (PE24) are administered subcutaneously in mouse models of human metastatic colorectal cancer, for sustained secretion of tumor-targeted therapeutic nanoparticles. These proteins are functionalized with a peptidic ligand of CXCR4, a chemokine receptor overexpressed in metastatic cancer stem cells that confers high selective cytotoxicity in vitro and in vivo. In the mouse models of human colorectal cancer, time-deferred anticancer activity is detected after the subcutaneous deposition of 500 µg of PE24-based amyloids, which promotes a dramatic arrest of tumor growth in the absence of side toxicity. In addition, long-term prevention of lymphatic, hematogenous, and peritoneal metastases is achieved. These results reveal the biomedical potential and versatility of bacterial inclusion bodies as novel tunable secretory materials usable in delivery, and they also instruct how therapeutic proteins, even with high functional and structural complexity, can be packaged in this convenient format.

A CXCR4-targeted nanocarrier achieves highly selective tumor uptake in diffuse large B-cell lymphoma mouse models.

One-third of diffuse large B-cell lymphoma patients are refractory to initial treatment or relapse after rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone chemotherapy. In these patients, CXCR4 overexpression (CXCR4+) associates with lower overall and disease-free survival. Nanomedicine pursues active targeting to selectively deliver antitumor agents to cancer cells; a novel approach that promises to revolutionize therapy by dramatically increasing drug concentration in target tumor cells. In this study, we intravenously administered a liganded protein nanocarrier (T22-GFP-H6) targeting CXCR4+ lymphoma cells in mouse models to assess its selectivity as a nanocarrier by measuring its tissue biodistribution in cancer and normal cells. No previous protein-based nanocarrier has been described as specifically targeting lymphoma cells. T22-GFP-H6 achieved a highly selective tumor uptake in a CXCR4+ lymphoma subcutaneous model, as detected by fluorescent emission. We demonstrated that tumor uptake was CXCR4-dependent because pretreatment with AMD3100, a CXCR4 antagonist, significantly reduced tumor uptake. Moreover, in contrast to CXCR4+ subcutaneous models, CXCR4- tumors did not accumulate the nanocarrier. Most importantly, after intravenous injection in a disseminated model, the nanocarrier accumulated and internalized in all clinically relevant organs affected by lymphoma cells with negligible distribution to unaffected tissues. Finally, we obtained antitumor effect without toxicity in a CXCR4+ lymphoma model by administration of T22-DITOX-H6, a nanoparticle incorporating a toxin with the same structure as the nanocarrier. Hence, the use of the T22-GFP-H6 nanocarrier could be a good strategy to load and deliver drugs or toxins to treat specifically CXCR4-mediated refractory or relapsed diffuse large B-cell lymphoma without systemic toxicity.

Collaborative membrane activity and receptor-dependent tumor cell targeting for precise nanoparticle delivery in CXCR4+ colorectal cancer.

By the appropriate selection of functional peptides and proper accommodation sites, we have generated a set of multifunctional proteins that combine selectivity for CXCR4+ cell binding and relevant endosomal escape capabilities linked to the viral peptide HA2. In particular, the construct T22-GFP-HA2-H6 forms nanoparticles that upon administration in mouse models of human, CXCR4+ colorectal cancer, accumulates in primary tumor at levels significantly higher than the parental T22-GFP-H6 HA2-lacking version. The in vivo application of a CXCR4 antagonist has confirmed the prevalence of the CXCR4+ tumor tissue selectivity over unspecific cell penetration, upon systemic administration of the material. Such specificity is combined with improved endosomal escape, what overall results in a precise and highly efficient tumor biodistribution. These data strongly support the functional recruitment as a convenient approach to generate protein materials for clinical applications. More precisely, they also support the unexpected concept that enhancing the unspecific membrane activity of a protein material does not necessarily compromise, but it can even improve, the selective cell targeting offered by an accompanying functional module. STATEMENT OF SIGNIFICANCE: We have shown here that the combination of cell-penetrating and tumor cell-targeting peptides dramatically enhances precise tumor accumulation of protein-only nanoparticles intended for selective drug delivery, in mouse models of human colorectal cancer. This fact is a step forward for the rational design of multifunctional protein nanomaterials for improved cancer therapies.

Protein-driven nanomedicines in oncotherapy.

Proteins are organic macromolecules essential in life but exploited, mainly in recombinant versions, as drugs or vaccine components, among other uses in industry or biomedicine. In oncology, individual proteins or supramolecular complexes have been tailored as small molecular weight drug carriers for passive or active tumor cell-targeted delivery, through the de novo design of appropriate drug stabilizing vehicles, or by generating constructs with different extents of mimesis of natural cell-targeted entities, such as viruses. In most of these approaches, a convenient nanoscale size is achieved through the oligomeric organization of the protein component in the drug conjugate. Among the different taken strategies, highly cytotoxic proteins such as microbial or plant toxins have been conveniently engineered to self-assemble as self-delivered virus-like, nanometric structures, chemically homogeneous that target metastatic cancer stem cells for the destruction of metastasis in absence of any partner vehicle.

The combined use of EFS, GPX2, and SPRR1A expression could distinguish favorable from poor clinical outcome among epithelial-like head and neck carcinoma subtypes.

BACKGROUND: We aimed at identifying molecular markers predictive of clinical outcome in patients with head and neck cancer based on the expression profile of cells showing epithelial-like (EL) or mesenchymal-like (ML) phenotypes. MATERIALS AND METHODS: We analyzed the association between EL and ML cells and migration, drug resistance, or tumor growth. The differential gene expression profile between cell types was used to build a model to stratify patients according to survival. RESULTS: EL cells were sensitive to cisplatin and cetuximab, showed low migration, and generated squamous differentiated tumors in mouse. A differential 93-gene expression signature between ML and EL cells was used to build a three-gene (EFS, GPX2, and SPRR1A) survival model by analyzing the RNA-seq data of the TCGA-HNSC project. Its prognostic value was confirmed in two independent cohorts. CONCLUSION: EFS, GPX2, and SPRR1A are prognostic markers able to distinguish clinical outcome among subtypes sharing an EL phenotype.

Effect of serpinE1 overexpression on the primary tumor and lymph node, and lung metastases in head and neck squamous cell carcinoma.

BACKGROUND: Serpin Family E Member 1 (SerpinE1) overexpression associates with poor clinical outcome in head and neck squamous cell carcinoma (HNSCC) patients. This study analyzed the role of serpinE1 in HNSCC dissemination. METHODS: We studied the phenotypic characteristics and dissemination of HNSCC cells overexpressing serpinE1 using an orthotopic model and the association between serpinE1 overexpression and clinicopathological variables in patients included in The Cancer Genome Atlas database. RESULTS: SerpinE1 overexpression increased proliferation, tumor budding, and the stromal component, while inhibiting apoptosis in primary tumors. It also enhanced the affectation and metastatic growth in lymph nodes, and the dispersion and growth of metastatic foci in the lung. High serpinE1 expression was associated with larger tumor size, undifferentiated tumors, lymph node metastasis, extracapsular spread, and the presence of perineural and angiolymphatic invasion. CONCLUSION: SerpinE1 overexpression promotes tumor aggressiveness and metastatic dissemination to lymph nodes and lung consistently with its association with poor outcome in HNSCC patients.

Focal Adhesion Genes Refine the Intermediate-Risk Cytogenetic Classification of Acute Myeloid Leukemia.

In recent years, several attempts have been made to identify novel prognostic markers in patients with intermediate-risk acute myeloid leukemia (IR-AML), to implement risk-adapted strategies. The non-receptor tyrosine kinases are proteins involved in regulation of cell growth, adhesion, migration and apoptosis. They associate with metastatic dissemination in solid tumors and poor prognosis. However, their role in haematological malignancies has been scarcely studied. We hypothesized that PTK2/FAK, PTK2B/PYK2, LYN or SRC could be new prognostic markers in IR-AML. We assessed PTK2, PTK2B, LYN and SRC gene expression in a cohort of 324 patients, adults up to the age of 70, classified in the IR-AML cytogenetic group. Univariate and multivariate analyses showed that PTK2B, LYN and PTK2 gene expression are independent prognostic factors in IR-AML patients. PTK2B and LYN identify a patient subgroup with good prognosis within the cohort with non-favorable FLT3/NPM1 combined mutations. In contrast, PTK2 identifies a patient subgroup with poor prognosis within the worst prognosis cohort who display non-favorable FLT3/NPM1 combined mutations and underexpression of PTK2B or LYN. The combined use of these markers can refine the highly heterogeneous intermediate-risk subgroup of AML patients, and allow the development of risk-adapted post-remission chemotherapy protocols to improve their response to treatment.

Selective depletion of metastatic stem cells as therapy for human colorectal cancer.

Selective elimination of metastatic stem cells (MetSCs) promises to block metastatic dissemination. Colorectal cancer (CRC) cells overexpressing CXCR4 display trafficking functions and metastasis-initiating capacity. We assessed the antimetastatic activity of a nanoconjugate (T22-GFP-H6-FdU) that selectively delivers Floxuridine to CXCR4+ cells. In contrast to free oligo-FdU, intravenous T22-GFP-H6-FdU selectively accumulates and internalizes in CXCR4+ cancer cells, triggering DNA damage and apoptosis, which leads to their selective elimination and to reduced tumor re-initiation capacity. Repeated T22-GFP-H6-FdU administration in cell line and patient-derived CRC models blocks intravasation and completely prevents metastases development in 38-83% of mice, while showing CXCR4 expression-dependent and site-dependent reduction in foci number and size in liver, peritoneal, or lung metastases in the rest of mice, compared to free oligo-FdU. T22-GFP-H6-FdU induces also higher regression of established metastases than free oligo-FdU, with negligible distribution or toxicity in normal tissues. This targeted drug delivery approach yields potent antimetastatic effect, through selective depletion of metastatic CXCR4+ cancer cells, and validates metastatic stem cells (MetSCs) as targets for clinical therapy.

CXCR7 expression in diffuse large B-cell lymphoma identifies a subgroup of CXCR4+ patients with good prognosis.

The CXCR4/CXCL12 axis has been extensively associated with different types of cancer correlating with higher aggressiveness and metastasis. In diffuse large B-cell lymphoma (DLBCL), the expression of the chemokine receptor CXCR4 is involved in the dissemination of malignant B cells and is a marker of poor prognosis. CXCR7 is a chemokine receptor that binds to the same ligand as CXCR4 and regulates de CXCR4-CXCL12 axis. These findings together with the report of CXCR7 prognostic value in several tumor types, led us to evaluate the expression of CXCR7 in diffuse large B-cell lymphoma biopsies. Here, we describe that CXCR7 receptor is an independent prognostic factor that associates with good clinical outcome. Moreover, the expression of CXCR7 associates with increased survival in CXCR4+ but not in CXCR4- DLBCL patients. Thus, the combined immunohistochemical evaluation of both CXCR7 and CXCR4 expression in DLBCL biopsies may improve their prognostic value as single markers. Finally, we show that CXCR7 overexpression in vitro is able to diminish DLBCL cell survival and increase their sensitivity to antitumor drugs. Hence, further studies on the CXCR7 receptor may establish its role in DLBCL and the molecular mechanisms that modulate CXCR4 activity.

Release of targeted protein nanoparticles from functional bacterial amyloids: A death star-like approach.

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.

Self-assembling toxin-based nanoparticles as self-delivered antitumoral drugs.

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.