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Despite the advent of numerous targeted therapies in clinical practice, anthracyclines, including doxorubicin (DOX), continue to play a pivotal role in breast cancer (BC) treatment. DOX directly disrupts DNA replication, demonstrating remarkable efficacy against BC cells. However, its non-specificity toward cancer cells leads to significant side effects, limiting its clinical utility. Interestingly, DOX can also enhance the antitumor immune response by promoting immunogenic cell death in BC cells, thereby facilitating the presentation of tumor antigens to the adaptive immune system. However, the generation of an adaptive immune response involves highly proliferative processes, which may be adversely affected by DOX-induced cytotoxicity. Therefore, understanding the impact of DOX on dividing T cells becomes crucial, to deepen our understanding and potentially devise strategies to shield anti-tumor immunity from DOX-induced toxicity. Our investigation focused on studying DOX uptake and its effects on human lymphocytes. We collected lymphocytes from healthy donors and BC patients undergoing neoadjuvant chemotherapy (NAC). Notably, patient-derived peripheral blood mononuclear cells (PBMC) promptly internalized DOX when incubated in vitro or isolated immediately after NAC. These DOX-treated PBMCs exhibited significant proliferative impairment compared to untreated cells or those isolated before treatment initiation. Intriguingly, among diverse lymphocyte sub-populations, CD8 + T cells exhibited the highest uptake of DOX. To address this concern, we explored a novel DOX formulation encapsulated in ferritin nanocages (FerOX). FerOX specifically targets tumors and effectively eradicates BC both in vitro and in vivo. Remarkably, only T cells treated with FerOX exhibited reduced DOX internalization, potentially minimizing cytotoxic effects on adaptive immunity. Our findings underscore the importance of optimizing DOX delivery to enhance its antitumor efficacy while minimizing

Brain metastasis (BM) represents a clinical challenge for patients with advanced HER2 + breast cancer (BC). The monoclonal anti-HER2 antibody trastuzumab (TZ) improves survival of BC patients, but it has low central nervous system penetrance, being ineffective in treating BM. Previous studies showed that ferritin nanoparticles (HFn) may cross the blood brain barrier (BBB) through binding to the transferrin receptor 1 (TfR1). However, whether this has efficacy in promoting the trans-BBB delivery of TZ and combating BC BM was not studied yet. Here, we investigated the potential of HFn to drive TZ brain delivery and promote a targeted antitumor response in a murine model of BC BM established by stereotaxic injection of engineered BC cells overexpressing human HER2. HFn were covalently conjugated with TZ to obtain a nanoconjugate endowed with HER2 and TfR1 targeting specificity (H-TZ). H-TZ efficiently achieved TZ brain delivery upon intraperitoneal injection and triggered stable targeting of cancer cells. Treatment with H-TZ plus docetaxel significantly reduced tumor growth and shaped a protective brain microenvironment by engaging macrophage activation toward cancer cells. H-TZ-based treatment also avoided TZ-associated cardiotoxicity by preventing drug accumulation in the heart and did not induce any other major side effects when combined with docetaxel. These results provided in vivo demonstration of the pharmacological potential of H-TZ, able to tackle BC BM in combination with docetaxel. Indeed, upon systemic administration, the nanoconjugate guides TZ brain accumulation, reduces BM growth and limits side effects in off-target organs, thus showing promise for the management of HER2 + BC metastatic to the brain.

Background: Bisdemethoxycurcumin (BDC) might be an inflammation inhibitor in Alzheimer's Disease (AD). However, BDC is almost insoluble in water, poorly absorbed by the organism, and degrades rapidly. We thus developed a new nanoformulation of BDC based on H-Ferritin nanocages (BDC-HFn). Methods: We tested the BDC-HFn solubility, stability, and ability to cross a blood-brain barrier (BBB) model. We tested the effect of BDC-HFn on AD and control (CTR) PBMCs to evaluate the transcriptomic profile by RNA-seq. Results: We developed a nanoformulation with a diameter of 12 nm to improve the solubility and stability. The comparison of the transcriptomics analyses between AD patients before and after BDC-HFn treatment showed a major number of DEG (2517). The pathway analysis showed that chemokines and macrophages activation differed between AD patients and controls after BDC-HFn treatment. BDC-HFn binds endothelial cells from the cerebral cortex and crosses through a BBB in vitro model. Conclusions: Our data showed how BDC-Hfn could improve the stability of BDC. Significant differences in genes associated with inflammation between the same patients before and after BDC-Hfn treatment have been found. Inflammatory genes that are upregulated between AD and CTR after BDC-HFn treatment are converted and downregulated, suggesting a possible therapeutic approach.

The design and development of contrast agents (CAs) for magnetic resonance imaging (MRI) in clinical analysis is expected to improve the image spatial resolution and to increase the detection sensitivity, especially regarding neurological disorders and cancer disease. In particular, advanced CAs for T1-weighted images are investigated to achieve the sensitive detection of early-stage primary tumors or brain metastases. In this study, we present a strategy toward diagnostic T1 CAs for MRI, based on polymer-modified MnO nanoparticles (NPs). Two different nanosystems were synthesized, consisting of (1) colloidal MnO nanocrystals wrapped by a multidentate amphiphilic polymer and (2) MnO nanocrystals embedded within poly(lactic-co-glycolic acid) (PLGA) NPs. These nanosystems were compared in terms of their MRI contrast power and biological safety. The latter system combines the excellent biocompatibility of PLGA with the unique magnetic properties of MnO NPs and allows sustained contrast enhancement over time. Longitudinal relaxivities of both MnO composite nanomaterials proved to be higher than those of commercial Gd-based CAs and Teslascan, both in phosphate-buffered saline and in plasma, also exhibiting low cytotoxicity. The high relaxation rates achieved with these contrast enhancers are promising toward future application in in vivo imaging.

Purpose: Assessment of inflammatory bowel disease (IBD) currently relies on aspecific clinical signs of bowel inflammation. Specific imaging of the diseased bowel regions is still lacking. Here, we investigate mucosal addressin cell adhesion molecule 1 (MAdCAM-1) as a reliable and specific endothelial target for engineered nanoparticles delivering imaging agents to obtain an exact mapping of diseased bowel foci. Materials and Methods: We generated a nanodevice composed of PLGA-PEG coupled with anti-MAdCAM-1 antibody half-chains and loaded with quantum dots (P@QD-MdC NPs). Bowel localization and systemic biodistribution of the nanoconjugate were analyzed upon injection in a murine model of chronic IBD obtained through repeated administration of dextran sulfate sodium salt. Specificity for diseased bowel regions was also assessed ex vivo in human specimens from patients with IBD. Potential for development as contrast agent in magnetic resonance imaging was assessed by preliminary study on animal model. Results: Synthesized nanoparticles revealed good stability and monodispersity. Molecular targeting properties were analyzed in vitro in a cell culture model. Upon intravenous injection, P@QD-MdC NPs were localized in the bowel of colitic mice, with enhanced accumulation at 24 h post-injection compared to untargeted nanoparticles (p<0.05). Nanoparticles injection did not induce histologic lesions in non-target organs. Ex vivo exposure of human bowel specimens to P@QD-MdC NPs revealed specific recognition of the diseased regions vs uninvolved tracts (p<0.0001). After loading with appropriate contrast agent, the nanoparticles enabled localized contrast enhancement of bowel mucosa in the rectum of treated mice. Conclusion: P@QD-MdC NPs efficiently detected bowel inflammation foci, accurately following the expression pattern of MAdCAM-1. Fine-tuning of this nanoconjugate with appropriate imaging agents offers a promising non-invasive tool for specific IBD diagnosis.

Neoadjuvant chemotherapy has been established as the standard of care for HER2-positive breast cancer since it allows cancer down-staging, up to pathological complete response. The standard of care in the neoadjuvant setting for HER2-positive breast cancer is a combination of highly cytotoxic drugs such as anthracyclines and the anti-HER2 monoclonal antibody. Despite this cocktail allows a pathological complete response in up to 50%, their co-administration is strongly limited by intrinsic cardiotoxicity. Therefore, only a sequential administration of anthracyclines and the anti-HER2 treatment is allowed. Here, we propose the anthracycline formulation in H-Ferritin nanocages as promising candidate to solve this unmet clinical need, thanks to its capability to increase anthracyclines efficacy while reducing their cardiotoxicity. Treating a murine model of HER2-positive breast cancer with co-administration of Trastuzumab and H-Ferritin anthracycline nanoformulation, we demonstrate an improved tumor penetration of drugs, leading to increased anticancer efficacy and reduced of cardiotoxicity.

Monoclonal antibodies (mAbs) have been developed to improve the treatment of cancer patients. They are able to target certain cancer antigens and possess multiple relevant mechanisms of action (i) modifying the host response to the malignant cells, (ii) delivering cytotoxic moieties, (iii) redirecting T cells towards the cancer to reduce cancer progression inducing an anti-tumour immune response [1, 2]. The research of different formats of antibody fragments, such as single chain variable fragment (scFv), Fab, nanobody, bispecific antibody, bifunctional antibody and half-chain antibodies (HC-mAbs) has been focused on the discovery of valuable alternatives to the nanoconjugation of entire mAbs, which could be able to maintain their functional potential [3]. Many monoclonal antibodies approved for therapeutic purposes or are under clinical investigation. Among them, Cetuximab (CTX): a recombinant human/mouse chimeric monoclonal antibody which binds to the tyrosine-kinase Epidermal Growth Factor Receptor (EGFR). It acts by blocking the dimerization of EGFR thus inhibiting downstream signalling pathways. CTX-based treatments suffer of several limitations: inadequate pharmacokinetics, short circulation half-life, low tumour penetration and activation of the autoimmune response [4]. In this context, the rise of nanotechnology has offered new opportunities of treatment with mAbs, exploiting new strategies of conjugation to nanoparticles (NPs) or of incorporation within nanocarriers in order to overcome the major limitations of the mAbs-based conventional therapy [5]. Here, we investigated the effect of a CTX-nanoconjugate on triple negative breast cancer (TNBC). TNBC is a particularly aggressive subtype of breast cancer characterized by high heterogeneity and different molecular expression profiles, which make even more difficult the establishment of targeted therapy to control tumour growth [6]. The majority of TNBCs cell lines are characterized by the overexpression of E

Brain cancers are a group of neoplasms that can be either primary, such as glioblastoma multiforme (GBM), or metastatic, such as the HER2+ breast cancer brain metastasis. The brain represents a sanctuary for cancer cells thanks to the presence of the blood brain barrier (BBB) that controls trafficking of molecules, protecting the brain from toxic substances including drugs. Considering that GBM and HER2+ breast cancer brain metastases are characterized by EGFR and HER2 over-expression respectively, CTX- and TZ-based treatment could be effective. Several studies show that these monoclonal antibodies (mAbs) exert both a cytostatic activity interfering with the transduction pathways of EGFR family and a cytotoxic activity mainly through the immune system activation via the antibody dependent cell-mediated cytotoxicity (ADCC). Since the major limitation to therapeutic mAbs application is the presence of the BBB, here we use a recombinant form of human apoferritin (HFn) as a nanovector to promote the delivery of mAbs to the brain for the activation of the ADCC response. Using a transwell model of the BBB we proved the crossing ability of HFn-mAb. Cellular uptake of HFn-mAb by human cerebral microvascular endothelial cells (hCMEC/D3) was demonstrated by confocal microscopy. Moreover, after crossing the endothelial monolayer, HFn-conjugated mAbs retain their biological activity against targets, as assessed by MTS and ADCC assays. Our data support the use of HFn as efficient carrier to enhance the BBB crossing of mAbs, without affecting their antitumoral activity.

Cancer-associated fibroblasts (CAFs) are key actors in regulating cancer progression. They promote tumor growth, metastasis formation, and induce drug resistance. For these reasons, they are emerging as potential therapeutic targets. Here, with the aim of developing CAF-targeted drug delivery agents, we functionalized H-ferritin (HFn) nanocages with fibroblast activation protein (FAP) antibody fragments. Functionalized nanocages (HFn-FAP) have significantly higher binding with FAP+ CAFs than with FAP cancer cells. We loaded HFn-FAP with navitoclax (Nav), an experimental Bcl-2 inhibitor pro-apoptotic drug, whose clinical development is limited by its strong hydrophobicity and toxicity. We showed that Nav is efficiently loaded into HFn (HNav), maintaining its mechanism of action. Incubating Nav-loaded functionalized nanocages (HNav-FAP) with FAP+ cells, we found significantly higher cytotoxicity as compared to non-functionalized HNav. This was correlated with a significantly higher drug release only in FAP+ cells, confirming the specific targeting ability of functionalized HFn. Finally, we showed that HFn-FAP is able to reach the tumor and to target CAFs in a mouse syngeneic model of triple negative breast cancer after intravenous administration. Our data show that HNav-FAP could be a promising tool to enhance specific drug delivery into CAFs, thus opening new therapeutic possibilities focused on tumor microenvironment.

Poly(ADP-ribose) polymerase (PARP) inhibitors are a novel promising strategy toward triple-negative breast cancer (TNBC). TNBC is a breast cancer subtype that represents one of the main clinical challenges in cancer treatment due to its heterogeneity, biological behavior, invasiveness and poor prognosis. Moreover, it often shows genomic instability and/or BRCA mutations. However, clinical results obtained with PARP inhibitors are controversial, and no benefits were demonstrated in case of wild type BRCA, possibly due to poor bioavailability and inadequate nuclear delivery [1-4]. Nanotechnology could overcome these major limitations with the development of nanocaged-drugs. Among these, the most promising is represented by H-ferritin (HFn): natural protein-based nanoparticles with native capability of targeting tumor cells thanks to its specific interaction with the TfR1, which is overexpressed in tumor cells. HFn are constituted of self-assembling human H-ferritin monomers arranged in a hollow structure. HFn also possess some other unique features very appealing from the clinical point of view: i) uniform cage architecture, which allows the precise control of the amount of encapsulated drugs; ii) negligible toxicity and immunogenicity; iii) stability in physiological environment as a result of their protein nature, which increases circulation time, protects the cargo molecule from degradation and improves bioavailability[5-7]. The aim of this study was to assess the anticancer efficacy of H-Ferritin nanoformulated Olaparib (HOla) vs. free Olaparib (Ola) on BRCA-mutated and non mutated TNBC cells exploiting both the natural targeting of H-ferritin toward the transferrin receptor-1 and also its physiological tropism toward the nuclear compartment. BRCA-mutated HCC1937 cells and BRCA-wild type MDA MB-231 and MDA MB-468 cells were treated with HOla or Ola in vitro. All TNBC cell lines over-expressed TfR-1 and were succesfully recognized by H-Ferritin, displaying a fast i

Evaluation of acute colitis relies on invasive endoscopic examination to assess the presence and the severity of the disease, and on aspecific radiological signs of bowel inflammation, such as contrast enhancement of bowel wall thickening. Consequently, the current best clinical management only offers an approximation of the real clinical scenario, and has a negative impact on the quality of life of patients. An accurate stadiation of inflammatory bowel diseases for assessment of response to therapies or indication for surgery remains challenging. Here, mucosal addressin cell-adhesion molecule-1 (MAdCAM-1) was investigated as a reliable and specific target of active bowel inflammation, to be exploited for site-specific nanotheranostics in a preclinical murine model of colitis. MAdCAM-1 is a cell adhesion molecule, which is upregulated on gut endothelium in case of inflammation at very early stages of the disease, and is responsible for T cells recruitment in involved bowel tracts. Moreover, it is finely related to activity of inflammatory bowel diseases and correlates with response to therapy. Anti-MAdCAM-1 antibodies were conjugated to the surface of smart and safe delivery nanoparticles, consisting of a manganese oxide core endowed with paramagnetic properties. The aim of the study was to assess the in vivo targeting efficacy and the accuracy of these innovative low-toxic MAdCAM-1-targeted nanoparticles, specifically designed to detect active bowel inflammation foci at early stages of the disease. Manganese oxide nanoparticles revealed good stability and polydispersity. Their negligible citotoxicity was demonstrated on endothelial cell cultures by analysis of viability and apoptosis, and on blood samples by hemolysis assay. Colitis was induced in mice by oral treatment with dextran sulfate sodium, and MAdCAM-1 overexpression was assessed on the intestinal vascular endothelium by immunohistochemistry and western blotting. Fluorescent anti-MAdCAM-1 nanoparticles wer

Poly(ADP-ribose) polymerase (PARP) inhibitors represent a promising strategy toward the treatment of triple-negative breast cancer (TNBC), which is often associated to genomic instability and/or BRCA mutations. However, clinical outcome is controversial and no benefits have been demonstrated in wild type BRCA cancers, possibly due to poor drug bioavailability and low nuclear delivery. In the attempt to overcome these limitations, we have developed H-Ferritin nanoformulated olaparib (HOla) and assessed its anticancer efficacy on both BRCA-mutated and non-mutated TNBC cells. We exploited the natural tumor targeting of H-Ferritin, which is mediated by the transferrin receptor-1 (TfR1), and its physiological tropism toward cell nucleus. TNBC cell lines over-expressing TfR-1 were successfully recognized by H-Ferritin, displaying a fast internalization into the cells. HOla induced remarkable cytotoxic effect in cancer cells, exhibiting 1000-fold higher anticancer activity compared to free olaparib (Ola). Accordingly, HOla treatment enhanced PARP-1 cleavage, DNA double strand breaks and Ola delivery into the nuclear compartment. Our findings suggest that H-Ferritin nanoformulation strongly enhances cytotoxic efficacy of Ola as a stand-alone therapy in both BRCA-mutated and wild type TNBC cells, by promoting targeted nuclear delivery.

Nowadays cancer represents a prominent challenge in clinics. Main achievements in cancer management would be the development of highly accurate and specific diagnostic tools for early detection of cancer onset, and the generation of smart drug delivery systems for targeted chemotherapy release in cancer cells. In this context, protein-based nanocages hold a tremendous potential as devices for theranostics purposes. In particular, ferritin has emerged as an excellent and promising protein-based nanocage thanks to its unique architecture, surface properties and high biocompatibility. By exploiting natural recognition of the Transferrin Receptor 1, which is overexpressed on tumor cells, ferritin nanocages may ensure a proper drug delivery and release. Moreover, researchers have applied surface functionalities on ferritin cages for further providing active tumor targeting. Encapsulation strategies of non metal-containing drugs within ferritin cages have been explored and successfully performed with encouraging results. Various preclinical studies have demonstrated that nanoformulation within ferritin nanocages significantly improved targeted therapy and accurate imaging of cancer cells. Aims of this review are to describe structure and functions of ferritin nanocages, and to provide an overview about the nanotechnological approaches implemented for applying them to cancer diagnosis and treatment.

Targeted therapies have profoundly changed the clinical prospect in human epidermal growth factor receptor 2 (HER2)-positive breast cancer. In particular, the anti-HER2 monoclonal antibody trastuzumab represents the gold standard for the treatment of HER2+ breast cancer patients. Its contribution in dampening cancer progression is mainly attributed to the antibody-dependent cell-mediated cytotoxicity (ADCC) rather than HER2 blockade. Here, multiple half chains of trastuzumab were conjugated onto magnetic iron oxide nanoparticles (MNP-HC) to develop target-specific and biologically active nanosystems to enhance anti-HER2 therapeutic potential. HER2 targeting was assessed in different human breast cancer cell lines, where nanoparticles triggered site-specific phosphorylation in the catalytic domain of the receptor and cellular uptake by endocytosis. MNP-HC induced remarkable antiproliferative effect in HER2+ breast cancer cells, exhibiting enhanced activity compared to free drug. Accordingly, nanoparticles induced p27kip1 expression and cell cycle arrest in G1 phase, without loosing capability to prime ADCC. Finally, MNP-HC affected viability of trastuzumab-resistant cells, suggesting interference with the resistance machinery. Our findings indicate that multiple arrangement of trastuzumab half chain on the nanoparticle surface enhances anticancer efficacy in HER2+ breast cancer cells. Powerful inhibition of HER2 signaling could promote responsiveness of resistant cells, thus suggesting ways for drug sensitization.

The use of therapeutic monoclonal antibodies (mAbs) has revolutionized cancer treatment. The conjugation of mAbs to nanoparticles has been broadly exploited to improve the targeting efficiency of drug nanocarriers taking advantage of high binding efficacy and target selectivity of antibodies for specific cell receptors. However, the therapeutic implications of nanoconjugation have been poorly considered. In this study, half-chain fragments of the anti-EGFR mAb cetuximab were conjugated to colloidal nanoparticles originating stable nanoconjugates that were investigated as surrogates of therapeutic mAbs in triple negative breast cancer (TNBC). Three TNBC cell lines were selected according to EGFR expression, which regulates activation of MAPK/ERK and PI3K/Akt pathways, and to distinctive molecular profiling including KRAS, PTEN, and BRCA1 mutations normally associated with diverse sensitivity to treatment with cetuximab. The molecular mechanisms of action of nanoconjugated half-chain mAb, including cell targeting, interference with downstream signaling pathways, proliferation, cell cycle, and apoptosis, along with triggering of ADCC response, were investigated in detail in sensitive and resistant TNBC cells. We found that half-chain mAb nanoconjugation was able to enhance the therapeutic efficacy and improve the target selectivity against sensitive, but unexpectedly also resistant, TNBC cells. Viability assays and signaling transduction modulation suggested a role of BRCA1 mutation in TNBC resistance to cetuximab alone, whereas its effect could be circumvented using half-chain cetuximab nanoconjugates, suggesting that nanoconjugation not only improved the antibody activity but also exerted different mechanisms of action. Our results provide robust evidence of the potential of half-chain antibody nanoconjugates in the treatment of TNBC, which could offer a new paradigm for therapeutic antibody administration, potentially allowing improved curative efficiency and reduce

Aim: We investigate MAdCAM-1 as a reliable target to detect active bowel inflammation for selective noninvasive nanodiagnostics. Materials & methods: We coupled anti-MAdCAM-1 antibodies to manganese oxide nanoparticles, and analyzed nanoconjugate biodistribution and safety in murine model of inflammatory bowel disease by imaging and histology. Results: Nanoparticles were stable and nontoxic. Upon administration in colitic mice, anti-MAdCAM-1 functionalized nanoparticles preferentially localized in the inflamed bowel, whereas untargeted nanoparticles were more rapidly washed out. Nanoparticles did not induce lesions in nontarget organs. Conclusion: Anti-MAdCAM-1 functionalized nanoparticles detected active bowel inflammation foci, accurately following MAdCAM-1 expression pattern. These nanoconjugates could be a promising noninvasive imaging system for an early and accurate follow-up in patients affected by acute colitis.

Chemotherapeutic treatment of breast cancer is based on maximum tolerated dose (MTD) approach. However, advanced stage tumors are not effectively eradicated by MTD owing to suboptimal drug targeting, onset of therapeutic resistance and neoangiogenesis. In contrast, "metronomic" chemotherapy is based on frequent drug administrations at lower doses, resulting in neovascularization inhibition and induction of tumor dormancy. Here we show the potential of H-ferritin (HFn)-mediated targeted nanodelivery of metronomic doxorubicin (DOX) in the setting of a highly aggressive and metastatic 4T1 breast cancer mouse model with DOX-inducible expression of chemoresistance. We find that HFn-DOX administered at repeated doses of 1.24 mg kg-1 strongly improves the antitumor potential of DOX chemotherapy arresting the tumor progression. We find that such a potent antitumor effect is attributable to multiple nanodrug actions beyond cell killing, including inhibition of tumor angiogenesis and avoidance of chemoresistance. Multiparametric assessment of heart tissues, including histology, ultrastructural analysis of tissue morphology, and measurement of markers of reactive oxygen species and hepatic/renal conditions, provided evidence that metronomic HFn-DOX allowed us to overcome cardiotoxicity. Our results suggest that HFn-DOX has tremendous potential for the development of "nanometronomic" chemotherapy toward safe and tailored oncological treatments.

In this study, insulin-containing nanoparticles were loaded into pellet cores and orally administered to diabetic rats. Polyethylene imine-based nanoparticles, either placebo or loaded with insulin, were incorporated by extrusion and spheronization technology into cores that were subsequently coated with three overlapping layers and a gastroresistant film. The starting and coated systems were evaluated in vitro for their physico-technololgical characteristics, as well as disintegration and release performance. Nanoparticles-loaded cores showed homogeneous particle size distribution and shape. When a superdisintegrant and a soluble diluent were included in the composition enhanced disintegration and release performance were observed. The selected formulations, coated either with enteric or three-layer films, showed gastroresistant and release delayed behavior in vitro, respectively. The most promising formulations were finally tested for their hypoglycemic effect in diabetic rats. Only the nanoformulations loaded into the three-layer pellets were able to induce a significant hypoglycemic activity in diabetic rats. Our results suggest that this efficient activity could be attributed to a retarded release of insulin into the distal intestine, characterized by relatively low proteolytic activity and optimal absorption.

Metastatic breast cancer represents a diagnostic and therapeutic challenge due to tumor heterogeneity and to various physiological barriers that hinder drug delivery to the metastatic sites. To overcome these limitations, nanoformulated drugs have been developed and tested in preclinical studies, and few of them have been successfully translated into clinical practice. In particular, liposomal anthracyclines and nanoformulated albumin-bound paclitaxel have revealed an improved therapeutic index when compared to conventional chemotherapy, with significant reduction of drugs toxicity. Several strategies for nanoparticles engineering have more recently been explored to increase selectivity for tumor cells and to reach poorly accessible metastatic districts. Targeted nanoparticles, directed toward tumor markers and tissue-specific metastases, may provide effective devices in case of low-vascularized and small-sized metastases, thus paving the way for a real change in the natural history of metastatic disease. A number of targets have been identified and exploited for surface functionalization of different types of nanoparticles, which are currently undergoing preclinical studies. The aim of this review is to provide an overview of current nanotechnology applied to metastatic breast cancer diagnosis and treatment. Promising results encourage an upcoming translation of this research into clinical practice for an effective management of the disease in the near future.

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs through the blood-brain barrier (BBB), causing difficulties for effective pharmacological therapies. In recent years, different strategies for promoting brain targeting have aimed to improve drug delivery and activity at this site, including innovative nanosystems for drug delivery across the BBB. In this context, an in vitro approach based on a simplified cellular model of the BBB provides a useful tool to investigate the effect of nanoformulations on the trans-BBB permeation of molecules. This study describes the development of a double-layer BBB, consisting of co-cultured commercially available primary rat brain microvascular endothelial cells and astrocytes. A multiparametric approach for the validation of the model, based on the measurement of the transendothelial electrical resistance and the apparent permeability of a high molecular weight dextran, is also described. As proof of concept for the employment of this BBB model to study the effect of different nanoformulations on the translocation of fluorescent molecules across the barrier, we describe the use of fluorescein isothiocyanate (FITC), loaded into ferritin nanoparticles. The ability of ferritins to improve the trans-BBB permeation of FITC was demonstrated by flux measurements and confocal microscopy analyses. The results suggest this is a useful system for validating nanosystems for delivery of drugs across the BBB.

Eradication of virus by sanctuary sites is a main goal in HIV management. The central nervous system (CNS) is a classic model of sanctuary where viral replication occurs despite a complete viral suppression in peripheral blood. In recent years, nanotechnologies have provided a great promise in the eradication of HIV from the CNS. We hereby demonstrate for the first time that the structurally complex antiretroviral drug enfuvirtide (Enf), which normally is unable to penetrate the cerebrospinal fluid, is allowed to cross the blood brain barrier (BBB) in mice by conjugation with a nanoconstruct. Iron oxide nanoparticles coated with an amphiphilic polymer increase Enf translocation across the BBB in both in vitro and in vivo models. The mechanism involves the uptake of nanoconjugated-Enf in the endothelial cells, the nanocomplex dissociation and the release of the peptide, which is eventually excreted by the cells in the brain parenchyma. From the Clinical Editor: Despite the success of cocktail therapy of antiretroviral drugs, the complete eradication of HIV remains elusive, due to existence of viral sanctuary sites. The authors showed in this study that an antiretroviral drug complexed with iron oxide nanoparticles and coated with PMA amphiphilic polymer crosses the blood brain barrier. Furthermore, there was significant anti-viral activity. The results would aid further drug designs to eradicate HIV.

A genetically engineered apoferritin variant consisting of 24 heavy-chain subunits (HFn) was produced to achieve a cumulative delivery of an antitumor drug, which exerts its cytotoxic action by targeting the DNA at the nucleus of human cancer cells with subcellular precision. The rationale of our approach is based on exploiting the natural arsenal of defense of cancer cells to stimulate them to recruit large amounts of HFn nanoparticles loaded with doxorubicin inside their nucleus in response to a DNA damage, which leads to a programmed cell death. After demonstrating the selectivity of HFn for representative cancer cells compared to healthy fibroblasts, doxorubicin-loaded HFn was used to treat the cancer cells. The results from confocal microscopy and DNA damage assays proved that loading of doxorubicin in HFn nanoparticles increased the nuclear delivery of the drug, thus enhancing doxorubicin efficacy. Doxorubicin-loaded HFn acts as a "Trojan Horse": HFn was internalized in cancer cells faster and more efficiently compared to free doxorubicin, then promptly translocated into the nucleus following the DNA damage caused by the partial release in the cytoplasm of encapsulated doxorubicin. This self-triggered translocation mechanism allowed the drug to be directly released in the nuclear compartment, where it exerted its toxic action. This approach was reliable and straightforward providing an antiproliferative effect with high reproducibility. The particular self-assembling nature of HFn nanocage makes it a versatile and tunable nanovector for a broad range of molecules suitable both for detection and treatment of cancer cells.