Despite the efforts and advances done in the last few decades, cancer still remains one of the main leading causes of death worldwide. Nanomedicine and in particular extracellular vesicles are one of the most potent tools to improve the effectiveness of anticancer therapies. In these attempts, the aim of this work is to realize a hybrid nanosystem through the fusion between the M1 macrophages-derived extracellular vesicles (EVs-M1) and thermoresponsive liposomes, in order to obtain a drug delivery system able to exploit the intrinsic tumor targeting capability of immune cells reflected on EVs and thermoresponsiveness of synthetic nanovesicles. The obtained nanocarrier has been physicochemically characterized, and the hybridization process has been validated by cytofluorimetric analysis, while the thermoresponsiveness was in vitro confirmed through the use of a fluorescent probe. Tumor targeting features of hybrid nanovesicles were in vivo investigated on melanoma-induced mice model monitoring the accumulation in tumor site through live imaging and confirmed by cytofluorimetric analysis, showing higher targeting properties of hybrid nanosystem compared to both liposomes and native EVs. These promising results confirmed the ability of this nanosystem to combine the advantages of both nanotechnologies, also highlighting their potential use as effective and safe personalized anticancer nanomedicine. Graphical Abstract

Triple-negative breast cancer (TNBC) is an aggressive malignancy characterized by the lack of expression of estrogen and progesterone receptors and amplification of human epidermal growth factor receptor 2 (HER2). Being the Epidermal Growth Factor Receptor (EGFR) highly expressed in mesenchymal TNBC and correlated with aggressive growth behavior, it represents an ideal target for anticancer drugs. Here, we have applied the phage display for selecting two highly specific peptide ligands for targeting the EGFR overexpressed in MDA-MB-231 cells, a human TNBC cell line. Molecular docking predicted the peptide-binding affinities and sites in the extracellular domain of EGFR. The binding of the FITC-conjugated peptides to human and murine TNBC cells was validated by flow cytometry. Confocal microscopy confirmed the peptide binding specificity to EGFR-positive MDA-MB-231 tumor xenograft tissues and their co-localization with the membrane EGFR. Further, the peptide stimulation did not affect the cell cycle of TNBC cells, which is of interest for their utility for tumor targeting. Our data indicate that these novel peptides are highly specific ligands for the EGFR overexpressed in TNBC cells, and thus they could be used in conjugation with nanoparticles for tumor-targeted delivery of anticancer drugs.

Clinical differentiation between Parkinson’s disease (PD) and essential tremor (ET) may be challenging, highlighting the need for easily assessable diagnostic biomarkers. Neuronally derived extracellular vesicles (NDEVs) have been proposed as a peripheral matrix that can well recapitulate the cellular composition of neurons. We investigated the clinical usefulness of NDEV oligomeric and p129-α-synuclein levels in discriminating between patients with PD and those with ET. NDEV oligomeric and p129-α-synuclein species were assessed using an ELISA in 43 patients with PD, 21 patients with ET, and 45 healthy controls (HCs). NDEV oligomeric α-synuclein levels were significantly higher in PD in comparison with ET and HCs, while p129-α-synuclein values were significantly lower in HCs compared to other groups. By using a receiver operator characteristic (ROC) analysis, oligomeric-α-synuclein achieved an excellent classification performance in distinguishing PD from both ET and HCs (AUC: 0.976 and 0.997, respectively), while lower performance was obtained in differentiating ET from HCs (AUC: 0.85). On the other hand, p129-α-synuclein accurately discriminated both PD and ET from HCs (AUC: 0.997 and 0.952, respectively) but had very low performance in differentiating PD from ET (AUC: 0.47). Our study suggests that NDEV oligomeric α-synuclein is an accurate blood-derived biomarker to differentiate PD from ET, while p129-α-synuclein may be useful in distinguishing ET from HCs.

Parkinson’s disease (PD) and essential tremor with resting tremor (rET) show a remarkable symptom overlap that complicates the clinical differentiation and highlights the need for easily assessable diagnostic biomarkers. Neuronally derived extracellular vesicles (NDEVs) have been suggested as a peripheral substrate providing insights on the central nervous system pathology. Here, we assessed the accuracy of NDEV neurofilament light chain (Nf-L) levels in discriminating between PD and rET patients. Thirty-three PD and 13 rET patients and 30 healthy controls (HC) were consecutively enrolled in the study. NDEVs were obtained from serum through size exclusion chromatography followed by immunocapture based on L1CAM expression. NDEV Nf-L levels were quantified by ELISA on Ella Platform. Receiver operator characteristic (ROC) analysis was performed to investigate the NDEV Nf-L classification performance. Associations between NDEV Nf-L and demographical or clinical variables were investigated with Spearman’s method. NDEV Nf-L levels were significantly higher in PD compared to rET patients and HC, and achieved an excellent classification performance in distinguishing PD from both rET and HC (AUC: 0.902 and 0.987, respectively). No differences were observed between rET and HC. A significant negative association between NDEV Nf-L and sex was detected in rET patients. Our pilot study suggests that NDEV Nf-L might be a useful blood-derived biomarker helping the differential diagnosis between PD and rET patients. Future studies are needed to validate these findings in larger independent cohorts.

Chronic Lymphocytic Leukemia (CLL) is a heterogeneous disease characterized by variable clinical courses among different patients. This notion was supported by the possible coexistence of two or more independent CLL clones within the same patients, identified by the characterization of the B cell receptor immunoglobulin (BcR IG) idiotypic sequence. By using the antigen-binding site of the BcR IG as bait, the identification and isolation of aggressive and drug-resistance leukemic B-cell clones could allow a deeper biological and molecular investigation. Indeed, by the screening of phage display libraries, we previously selected a peptide binder of the idiotypic region of CLL BCR IGs expressing the unmutated rearrangement IGHV1-69 and used it as a probe to perform a peptide-based cell sorting by flow cytometry in peripheral blood samples from patients with CLL. Since the IGHV1-69 clones persisted during the follow-up time in both patients, we explored the possibility of these clones having acquired an evolutive advantage compared to the other coexisting clones in terms of a higher expression of genes involved in the survival and apoptosis escape processes. To this end, we studied the expression patterns of a panel of genes involved in apoptosis regulation and in NF-kB-dependent pro-survival signals by comparative qRT-PCR assays. According to the results, IGHV1-69 clones showed a higher expression of pro-survival and anti-apoptotic genes as compared to the other CLL clones with different immunogenetic characteristics. Moreover, these IGHV1-69 clones did not carry any characteristic genetic lesions, indicating the relevance of our approach in performing a comprehensive molecular characterization of single tumor clones, as well as for designing new personalized therapeutic approaches for the most aggressive and persistent tumor clones.