Hybrid Self-Assembling Nanoparticles Encapsulating Zoledronic Acid: A Strategy for Fostering Their Clinical Use.

Self-assembling nanoparticles (SANPs) promise an effective delivery of bisphosphonates or microRNAs in the treatment of glioblastoma (GBM) and are obtained through the sequential mixing of four components immediately before use. The self-assembling approach facilitates technology transfer, but the complexity of the SANP preparation protocol raises significant concerns in the clinical setting due to the high risk of human errors during the procedure. In this work, it was hypothesized that the SANP preparation protocol could be simplified by using freeze-dried formulations. An in-depth thermodynamic study was conducted on solutions of different cryoprotectants, namely sucrose, mannitol and trehalose, to test their ability to stabilize the produced SANPs. In addition, the ability of SANPs to deliver drugs after lyophilization was assessed on selected formulations encapsulating zoledronic acid in vitro in the T98G GBM cell line and in vivo in an orthotopic mouse model. Results showed that, after lyophilization optimization, freeze-dried SANPs encapsulating zoledronic acid could retain their delivery ability, showing a significant inhibition of T98G cell growth both in vitro and in vivo. Overall, these results suggest that freeze-drying may help boost the industrial development of SANPs for the delivery of drugs to the brain.

Hybrid lipid self-assembling nanoparticles for brain delivery of microRNA.

Hybrid self-assembling nanoparticles (SANPs) have been previously designed as novel drug delivery system that overcomes stability issues following long-term storage and with an easy scale-up. This system has been successfully used to deliver anionic-charged agents, e.g. bisphosphonates, in different types of tumors, such glioblastoma (GBM). Here, SANPs were tested and optimized for the delivery of nucleic acids, in particular of a specific microRNA, e.g. miR603, used for its potential role in controlling the chemoresistance in different forms of cancer, e.g. (GBM). To this aim, SANPs with different lipids were prepared and characterized, in terms of size, polydispersity index, zeta potential, miRNA encapsulation, stability in BSA, serum and hemolytic activity. Then, SANPs were tested in vitro on two different cell lines of GBM. Finally, miRNA biodistribution was tested in vivo in an orthotopic model of GBM. The majority of the formulations showed good technological characteristics and were stable in BSA and serum with a low hemolytic activity. The intracellular uptake studies on GBM cell lines showed that SANPs allow to achieve a higher miRNA delivery compared to others transfection agents, e.g. lipofectamine. Finally, in vivo biodistribution studies in an orthotopic of GBM demonstrated that the optimized SANP formulations, were able to deliver miRNA in different organs, e.g. the brain.

ABCA1/ABCB1 Ratio Determines Chemo- and Immune-Sensitivity in Human Osteosarcoma.

The ATP Binding Cassette transporter B1 (ABCB1) induces chemoresistance in osteosarcoma, because it effluxes doxorubicin, reducing the intracellular accumulation, toxicity, and immunogenic cell death induced by the drug. The ATP Binding Cassette transporter A1 (ABCA1) effluxes isopentenyl pyrophosphate (IPP), a strong activator of anti-tumor Vγ9Vδ2 T-cells. Recruiting this population may represent an alternative strategy to rescue doxorubicin efficacy in ABCB1-expressing osteosarcoma. In this work, we analyzed how ABCA1 and ABCB1 are regulated in osteosarcoma, and if increasing the ABCA1-dependent activation of Vγ9Vδ2 T-cells could be an effective strategy against ABCB1-expressing osteosarcoma. We used 2D-cultured doxorubicin-sensitive human U-2OS and Saos-2 cells, their doxorubicin-resistant sublines (U-2OS/DX580 and Saos-2/DX580), and 3D cultures of U-2OS and Saos-2 cells. DX580-sublines and 3D cultures had higher levels of ABCB1 and higher resistance to doxorubicin than parental cells. Surprisingly, they had reduced ABCA1 levels, IPP efflux, and Vγ9Vδ2 T-cell-induced killing. In these chemo-immune-resistant cells, the Ras/Akt/mTOR axis inhibits the ABCA1-transcription induced by Liver X Receptor α (LXRα); Ras/ERK1/2/HIF-1α axis up-regulates ABCB1. Targeting the farnesylation of Ras with self-assembling nanoparticles encapsulating zoledronic acid (NZ) simultaneously inhibited both axes. In humanized mice, NZ reduced the growth of chemo-immune-resistant osteosarcomas, increased intratumor necro-apoptosis, and ABCA1/ABCB1 ratio and Vγ9Vδ2 T-cell infiltration. We suggest that the ABCB1highABCA1low phenotype is indicative of chemo-immune-resistance. We propose aminobisphosphonates as new chemo-immune-sensitizing tools against drug-resistant osteosarcomas.

In Vitro Biophysical and Biological Characterization of Lipid Nanoparticles Co-Encapsulating Oncosuppressors miR-199b-5p and miR-204-5p as Potentiators of Target Therapy in Metastatic Melanoma.

Uncontrolled MAPK signaling is the main oncogenic driver in metastatic melanomas bearing mutations in BRAF kinase. These tumors are currently treated with the combination of BRAF/MEK inhibitors (MAPKi), but this therapy is plagued by drug resistance. In this context we recently discovered that several microRNAs are involved in the development of drug resistance. In particular miR-204-5p and miR-199b-5p were found to function as antagonists of resistance because their enforced overexpression is able to inhibit melanoma cell growth in vitro either alone or in combination with MAPKi. However, the use of miRNAs in therapy is hampered by their rapid degradation in serum and biological fluids, as well as by the poor intracellular uptake. Here, we developed lipid nanoparticles (LNPs) encapsulating miR-204-5p, miR-199b-5p individually or in combination. We obtained LNPs with mean diameters < 200 nm and high miRNA encapsulation efficiency. These formulations were tested in vitro on several melanoma cell lines sensitive to MAPKi or rendered drug resistant. Our results show that LNPs encapsulating combinations of the two oncosuppressor miRNAs are highly efficient in impairing melanoma cell proliferation and viability, affect key signaling pathways involved in melanoma cell survival, and potentiate the efficacy of drugs inhibiting BRAF and MEK. These results warrant further assessment of the anti-tumor efficacy of oncosuppressor miRNAs encapsulating LNPs in in vivo tumor models.

Urotensin-II-Targeted Liposomes as a New Drug Delivery System towards Prostate and Colon Cancer Cells.

Urotensin-II (UT-II) and its receptor (UTR) are involved in the occurrence of different epithelial cancers. In particular, UTR was found overexpressed on colon, bladder, and prostate cancer cells. The conjugation of ligands, able to specifically bind receptors that are overexpressed on cancer cells, to liposome surface represents an efficient active targeting strategy to enhance selectivity and efficiency of drug delivery systems. The aim of this study was to develop liposomes conjugated with UT-II (LipoUT) for efficient targeting of cancer cells that overexpress UTR. The liposomes had a mean diameter between 150 nm and 160 nm with a narrow size distribution (PI ≤ 0.1) and a doxo encapsulation efficiency of 96%. Moreover, the conjugation of UT-II to liposomes weakly reduced the zeta potential. We evaluated UTR expression on prostate (DU145, PC3, and LNCaP) and colon (WIDR and LoVo) cancer cells by FACS and western blotting analysis. UTR protein was expressed in all the tested cell lines; the level of expression was higher in WIDR, PC3, and LNCaP cells compared with LoVo and DU145. MTT cell viability assay showed that LipoUT-doxo was more active than Lipo-doxo on the growth inhibition of cells that overexpressed UTR (PC3, LNCaP, and WIDR) while in LoVo and DU145 cell lines, the activity was similar to or lower than that one of Lipo-doxo, respectively. Moreover, we found that cell uptake of Bodipy-labeled liposomes in PC3 and DU145 was higher for LipoUT than the not-armed counterparts but at higher extent in UTR overexpressing PC3 cells (about 2-fold higher), as evaluated by both confocal and FACS. In conclusion, the encapsulation of doxo in UT-II-targeted liposomes potentiated its delivery in UTR-overexpressing cells and could represent a new tool for the targeting of prostate and colon cancer.