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Assessing the toxic effect in living organisms remains a major issue for the development of safe nanomedicines and exposure of researchers involved in the synthesis, handling and manipulation of nanoparticles. In this study, we demonstrate that Caenorhabditis elegans could represent an in vivo model alternative to superior mammalians for the collection of several physiological functionality parameters associated to both short-term and long-term effects of colloidally stable nanoparticles even in absence of microbial feeding, usually reported to be necessary to ensure appropriate intake. Contextually, we investigated the impact of surface charge on toxicity of superparamagnetic iron oxide coated with a wrapping polymeric envelop that confers them optimal colloidal stability. By finely tuning the functional group composition of this shallow polymer–obtaining totally anionic, partially pegylated, partially anionic and partially cationic, respectively–we showed that the ideal surface charge organization to optimize safety of colloidal nanoparticles is the one containing both cationic and anionic groups. Our results are in accordance with previous evidence that zwitterionic nanoparticles allow long circulation, favorable distribution in the tumor area and optimal tumor penetration and thus support the hypothesis that zwitterionic iron oxide nanoparticles could be an excellent solution for diagnostic imaging and therapeutic applications in nanooncology.

Gold nanoparticles (GNPs) can be exploited for local and targeted treatment for poorly prognosis diseases. Bronchiolitis Obliterans Syndrome (BOS) is the main cause of medium-term failure of lung transplantation and characterized by fibroobliteration of small airways due to uncontrolled proliferation of mesenchymal cells (MCs). Since CD44 receptor is overexpressed by BOS MCs, we developed GNPs decorated with the half chain of moAb against CD44 (GNP-HC). We aimed firslty to assess the in vitro activity of GNP-HC loaded with imatinib (GNP-HCim), a cAbl inhibitor already used in the treatment of chronic pulmonary GVHD. Furthermore we aimed to evaluate efficacy of local delivery of these carrier in heterotopic tracheal transplantation model in mice. For in vitro experiments, we evaluated cytotoxicity of GNP-HC, GNP-HCim and imatinib alone in BOS-derived MCs. Moreover, we performed in vivo experiments in mouse heterotopic trachea transplantation, with continuous intratracheal administration of GNP or vehicle by Alzet Model 2004 miniosmotic pumps, in order to assess their efficacy. GNP-HCim treated MCs showed significantly reduced viability than GNP-HC and imatinib alone, associated to induction of apoptosis (50 ± 0.5 % for GNP-HCim vs. GNP-HC 40 ± 0.35 % and Imatinib alone 30 ± 1.2 %). These results were strengthened by animal model, in which the percentage of tracheal obliterated area was significantly reduced by GNP-HCim (12.99 ± 2.5%) respect to GNP-HC (46.79± 5.7%) and vehicle treated tracheas (30.92 ± 5.8%). GNPs represent a valid vehicle for local and selective drug administration avoiding systemic toxicity.

Interstitial lung involvement in Systemic Sclerosis (SSc-ILD) is a complication with high morbidity and mortality. Specifically, engineered gold nanoparticles (GNPs) are proposed as targeted delivery system increasing efficacy of drugs with antifibrotic effect, such as tyrosine kinases. We aimed to test in vitro and in vivo the activity of targeted Imatinib (Im)-loaded GNP on SSc-ILD patients derived cells and in experimental model of lung fibrosis. GNPs functionalized with anti-CD44 and loaded with Im (GNP-HCIm) were synthesized. Lung fibroblasts (LFs) and alveolar macrophages from bronchoalveolar lavage fluids of SSc-ILD patients were cultured in presence of nanoparticles. GNP-HCIm significantly inhibited proliferation and viability inducing apoptosis of LFs and effectively reduced IL-8 release, viability and M2 polarization in alveolar macrophages. Anti-fibrotic effect of tracheal instilled GNP-HCIm was evaluated on bleomycin lung fibrosis mouse model comparing effect with common route of Im administration. GNP-HCIm were able to reduce significantly lung fibrotic changes and collagen deposition. Finally, electron microscopy revealed the presence of GNPs inside alveolar macrophages. These data support the use of GNPs locally administered in the development of new therapeutic approaches to SSc-ILD.

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 EGFR, which is involved in the control of cell proliferation and survival. EGFR overexpression is associated with poor clinical outcome in patients and has became an emerging therapeutic target for the treatment of TNBC [7]. In the present study, a novel nanosystem consisting of an iron oxide colloidal NPs conjugated with half-chain fragments of CTX (HC-CTX) was developed and tested. The advantage to conjugate HC fragment CTX to colloidal NPs is connected to the presentation of the Fab fragment in the optimal orientation for binding with the receptor in order to improve target recognition. HC-CTX in vitro efficacy was compared to CTX alone on three different TNBC cell lines, selected according to different EGFR expression, distinctive molecular profiling and various CTX sensitivity. HC-CTX-NPs displayed a high binding specificity to EGFR. The antitumor activity was analysed treating all TNBC cell lines in parallel with increasing concentration of CTX or HC-CTX-NPs. In general, the viability assay revealed that HC-CTX-NPs displayed a good capability to inhibit proliferation in TNBC cell lines in comparison with CTX alone, affecting proapoptotic molecular mechanism mediated by CTX. The antitumor efficacy of HC-CTX-NPs observed in CTX-resistant TNBC cell lines could be attributed to an alteration of the EGFR receptor recycling which resulted in decrease of the amount of nuclear EGFR due to its sequestration into endolysosomal pathway, as supported by confocal microscopy and Transmission Electron Microscopy. Besides, we have studied the effect of HC-CTX-NPs on EGFR pathway: CTX-sensitive TNBC cells displayed a high inhibition of EGFR pathway reducing the Y1068 phosphorylation levels, especially at low concentrations. These results reveal that HC-CTX nanoconjugate amplifies the effect of CTX preserving molecular mechanism involved in CTX activity against TNBC cells. In addition we observed the beneficial effects of HC-CTX nanoconjugate in CTX-resistant TNBC cell lines were attributable to the induction of EGFR degradation. This prevents the fast recycling of EGFR, which results in a decrease of nuclear EGFR levels. Moreover, the conjugation of HC-CTX to NPs preserves CTX antibody-dependent cell-mediated cytotoxicity (ADCC) response, resulting in enhanced antitumor activity, attributable to multivalent presentation of HC-CTX that amplify NK activation. In conclusion, this result underlining the potential of this new nanoconjugate in the treatment of TNBC and providing new hope of treatment for this highly aggressive disease.

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.

Purpose The use of gold nanoparticles (GNPs) as local targeted drug delivery system are a promising issue for several orphan diseases. Bronchiolitis obliterans syndrome (BOS) represents about 70% of cases of chronic lung allograft dysfunction limiting long term survival after lung transplant, and occurs also as chronic pulmonary involvement in the context of GVHD. To now treatment strategies in BOS are scarce and poorly effective. In the present work, we investigated the effect of GNP decorated with the half chain of monoclonal antibody against CD44 (GNP-HC), surface receptor overexpressed by primary BOS derived mesenchymal cells (MCs) that cause the partial or total occlusion of alveolar tract. We loaded GNP-HC with imatinib (GNP-HCim), an cAbl inhibitor used in the management of chronic GVHD. Methods In vitro experiments were performed on MCs derived from BOS patients evaluating cell cytotoxicity after incubation with GNP-HC, GNP-HCim and Imatinib alone. We further performed a mouse heterotopic trachea transplantation model modified by the subcutaneous placement of Alzet Model 2004 miniosmotic pumps used to deliver into the trachea different solutions at a constant rate for 28 days. This was performed in order to assess the efficacy of the local intratracheal administration of our nanovectors. Results Thanks to MTT and apoptosis assay we observed that GNP-HCim decrease MCs viability more than GNP-HC and Imatinib alone, due to an induction of apoptosis (50 ± 0.5 % for GNP-HCim in contrast to GNP-HC 40 ± 0.35 % and Imatinib alone 30 ± 1.2 %). Moreover, animal model data confirmed the in vitro results: GNP-HCim significantly reduced the percentage of tracheal obliterative area (12.99 ± 2.5%) respect to GNP-HC (46.79± 5.7%) and vehicle treated mice (30.92 ± 5.8%). Conclusion We conclude that this approach by newly developed nano-based strategies for BOS treatment and especially targeted imatinib gold nanoparticles are promising candidates for further development.