Ferritin nanocages : A biological platform for drug delivery, imaging and theranostics in cancer

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

Co-administration of H-ferritin-doxorubicin and Trastuzumab in neoadjuvant setting improves efficacy and prevents cardiotoxicity in HER2 + murine breast cancer model

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

Directed self-assembly of a xenogeneic vascularized endocrine pancreas for type 1 diabetes.

Intrahepatic islet transplantation is the standard cell therapy for β cell replacement. However, the shortage of organ donors and an unsatisfactory engraftment limit its application to a selected patients with type 1 diabetes. There is an urgent need to identify alternative strategies based on an unlimited source of insulin producing cells and innovative scaffolds to foster cell interaction and integration to orchestrate physiological endocrine function. We previously proposed the use of decellularized lung as a scaffold for β cell replacement with the final goal of engineering a vascularized endocrine organ. Here, we prototyped this technology with the integration of neonatal porcine islet and healthy subject-derived blood outgrowth endothelial cells to engineer a xenogeneic vascularized endocrine pancreas. We validated ex vivo cell integration and function, its engraftment and performance in a preclinical model of diabetes. Results showed that this technology not only is able to foster neonatal pig islet maturation in vitro, but also to perform in vivo immediately upon transplantation and for over 18 weeks, compared to normal performance within 8 weeks in various state of the art preclinical models. Given the recent progress in donor pig genetic engineering, this technology may enable the assembly of immune-protected functional endocrine organs

Nano-targeting of mucosal addressin cell adhesion molecule-1 identifies bowel inflammation foci in murine model

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

Multivalent exposure of trastuzumab on iron oxide nanoparticles enhances antitumor activity and weakens drug resistance in HER2+ breast cancer cells

Background: The identification of new strategies aimed to optimize the treatment of breast cancer and its metastases represents a great technical and medical challenge. Target\uadspecific therapies, such as Trastuzumab (TZ), have revolutionized the clinical scenario in certain subsets of cancer. However, the huge variability in response to therapy and the frequent onset of drug resistance in patients still hamper the therapeutic success. Antibody\uadconjugated nanoparticles may combine specific recognition of tumor cells with the capability to act as innovative reservoir of active drugs. Here, multivalent TZ\uadconjugated colloidal nanoparticles were developed as target\uadspecific and biologically active nanosystem to enhance the therapeutic potential toward HER2+ breast cancer. Methods: Iron oxide nanoparticles conjugated with multiple half chains of TZ have been developed and tested in different HER2+ breast cancer cell lines, in comparison to free TZ or untargeted nanoparticles. Active targeting and specificity toward HER2 receptor was assessed by flow cytometry and confocal microscopy. Cellular uptake of nanoparticles and HER2 endocytosis were followed by electron or confocal microscopy. Direct anticancer efficacy was assessed by incubation of free or nanoformulated TZ on sensitive breast cancer cells, and analysis of cell viability, cell cycle, and expression of p27kip1. Finally, nanoparticles were tested on TZ\uad-resistant breast cancer cell lines for capability of re\uadsensitization. Results: TZ\uad-conjugated nanoparticles showed specific targeting of HER2, with induction of site\uad-specific phosphorylation in the catalytic domain of the receptor and cellular uptake by endocytosis. Treatment with TZ\uad conjugated nanoparticles dramatically decreased cancer cell viability, by significantly improving the antitumor activity of TZ. This effect was independent from the ADCC mechanism, and associated with marked induction of p27kip1 expression and cell cycle arrest in G1 phase in TZ\uad-sensitive SKBR\uad3 cells. TZ\uad-conjugated nanoparticles also affected viability of breast cancer cells insensitive to TZ, further confirming enhanced potential of the nanoformulation and suggesting interference with some mechanisms of resistance. Conclusions: Our results provide evidence that multivalent exposure of TZ half chain on iron oxide nanoparticles affords enhanced antitumor potential and target-\uadspecific activity in HER2+ breast cancer cells. Powerful inhibition of HER2 signaling by TZ\uad-conjugated nanoparticles could favor responsiveness of drug resistant cells, thus suggesting novel therapeutic strategies to overcome resistance

Targeting fibroblast activation protein in Crohn’s disease intestinal strictures restores balanced deposition of extracellular matrix and reduces fibrosis

Background Bowel fibrosis is a chronic and progressive process characterised by excessive deposition of extracellular matrix (ECM) and imbalance between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). This phenomenon represents a severe end-stage of some chronic inflammatory disorders such as Crohn\u2019s disease (CD), for which effective medical therapy is currently unavailable. Key players in CD fibrosis are intestinal myofibroblasts, whose activation and function are characterised by a number of elements, including the overexpression of fibroblast activation protein (FAP). We investigated the effects of FAP-targeted therapy on ECM remodelling in CD strictures ex vivo. Methods Stenotic and non-stenotic bowel specimens were collected from ileal resections from 30 patients with fibrostenotic CD. Mucosal myofibroblasts were isolated and analysed for FAP expression by immunoblotting and flow cytometry. Bowel tissues were treated with anti-FAP antibody in culture medium; total soluble collagen, type I and III collagen, TIMP-1 and MMPs were measured in the supernatants, while inflammatory and fibrogenic pathways were investigated by immunoblotting on tissue protein extracts. Effects of anti-FAP treatment on the migratory potential and survival of intestinal myofibroblasts were evaluated by wound healing assay and Annexin V staining, respectively. Results Stenotic CD myofibroblasts showed upregulation of FAP protein. Treatment with anti-FAP antibody induced a significant dose-dependent decrease in collagen production by stenotic bowel tissues, while it did not affect non-stenotic bowel areas. Treatment on CD strictures particularly reduced type I collagen and TIMP-1 production, and did not change MMP-3 and MMP-12 secretion. Moreover, anti-FAP treatment on myofibroblasts from stenotic CD mucosa inhibited TIMP-1 expression and enhanced myofibroblast migration, without affecting their survival. Conclusions Our results demonstrated a role for FAP targeting in reducing type I collagen and TIMP-1 production by CD strictures ex vivo, thus suggesting a valuable reconstitution of ECM homeostasis in fibrostenotic CD

Inhibition of Fibroblast Activation Protein Restores a Balanced Extracellular Matrix and Reduces Fibrosis in Crohn's Disease Strictures Ex Vivo

Background: Crohn\u2019s disease (CD) is a chronic bowel inflammation that ultimately leads to fibrosis, for which medical therapy is currently unavailable. Fibrotic strictures in CD are characterized by excessive extracellular matrix (ECM) deposition, altered balance between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), and overexpression of fibroblast activation protein (FAP), a marker of active fibroblasts. Here we investigated the role of FAP-targeted therapy in ECM remodeling in CD strictures ex vivo. Methods: Bowel specimens were obtained from stenotic and nonstenotic ileal segments from 30 patients with fibrostenotic CD undergoing surgery. FAP expression was evaluated in isolated mucosal myofibroblasts by immunoblotting and flow cytometry. Bowel tissue cultures were treated with anti-FAP antibody, and soluble collagen, TIMP-1, and MMPs were measured in tissue culture supernatants by immunoblotting. Anti-FAP-treated myofibroblasts were analyzed for TIMP-1 expression by immunoblotting, for migratory potential by wound healing assay, and for apoptosis by Annexin V staining. Results: Myofibroblasts from stenotic CD mucosa showed upregulation of FAP expression when compared with nonstenotic mucosa. Treatment of stenotic tissues with anti-FAP antibody induced a dose-dependent decrease in collagen production, particularly affecting type I collagen. The treatment also reduced TIMP-1 production in CD strictures, without altering MMP-3 and MMP-12 secretion. Accordingly, anti-FAP treatment inhibited TIMP-1 expression in stenotic CD myofibroblasts and enhanced myofibroblast migration without affecting survival. Conclusions: FAP inhibition reduced type I collagen and TIMP-1 production by CD strictures ex vivo without compromising uninvolved bowel areas. These results suggest that targeting FAP could reconstitute ECM homeostasis in fibrostenotic CD

Half-Chain Cetuximab Nanoconjugates Allow Multitarget Therapy of Triple Negative Breast Cancer

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 reduced minimal effective dosages in both sensitive and resistant tumors

Nanoparticle‐Mediated Suicide Gene Therapy for Triple Negative Breast Cancer Treatment

Systemic chemotherapy has not significantly reduced clinical demand for triple negative breast cancer (TNBC) treatments. To address the need for more effective therapy, the use of nonviral nanoparticles is explored to deliver suicide gene therapy as valuable alternative to protect nucleic acids in the bloodstream and improve their tumor uptake. Biocompatible cationic lipid nanoparticles are developed as a novel delivery system of a suicide plasmid gene encoding saporin. Active targeting is accomplished by taking advantage of nanoparticle functionalization with U11 peptide, designed to be directed toward urokinase plasminogen activator receptor, limiting off\u2010target toxicity. The antitumor effect of U11\u2010lipid\u2010protamine\u2010DNA (U11\u2010LPD) nanoparticles are tested in TBNC cells, showing a strong prevalence of targeted versus nontargeted nanoparticles in terms of uptake kinetics and proliferation inhibition. Transfection of green fluorescent protein (GFP) plasmid in MDA\u2010MB\u2010231 cells is demonstrated. U11\u2010LPD nanoparticles administered by retro bulbar injection exhibit excellent tumor tropism in TNBC orthotopic xenograft mice and effectively transfect TNBC cells with saporin plasmid resulting in tumor mass reduction. No systemic toxicity or organ damage is discovered after repeated treatments with nanoparticles. The findings suggest that systemic administration of targeted LPD nanoparticles to deliver saporin safely allows for active inhibition of cancer progression even in the absence of specific promoter gene sequences

Anti-MAdCAM-1-Conjugated Nanocarriers Delivering Quantum Dots Enable Specific Imaging of Inflammatory Bowel Disease

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