Chemical conjugation of aptamer-sphingomyelin nanosystems and their potential as inhibitors of tumour cell proliferation in breast cancer cells.

Breast cancer is a complex and heterogeneous disease with a high mortality rate due to non-specific cytotoxicity, low intratumoral accumulation and drug resistance associated with the ineffectiveness of chemotherapy. In recent years, all efforts have been focused on finding new markers and therapeutic targets, protein kinase MNK1b being a promising candidate. Recently, an aptamer known as apMNK2F showed a highly specific interaction with this protein kinase, leading to a significant reduction in tumour cell proliferation, migration and colony formation. However, as aptamers are unable to penetrate the cell membrane and reach the target, these small biomolecules need to be conjugated to suitable vectors that can transport and protect them inside the cells. In this work, covalent conjugation between biocompatible and non-harmful nanoemulsions of vitamin E and sphingomyelin and the aptamer was performed to facilitate intracellular delivery of the therapeutic aptamer apMNK2F. All strategies employed were based on 2-step bioconjugation and optimized to get the simplest and most reproducible vehicle with the highest association efficiency (about 70% in all cases). The ability of the nanosystems to successfully deliver the conjugated therapeutic aptamer was demonstrated and compared to other commercial transfection agents such as Lipofectamine 2000, leading to an effective decrease of breast cancer cell proliferation in the MDA-MB-231 cell line. The proliferation inhibition of the aptamer nanoconjugates compared to the non-conjugated aptamer provides evidence that the antitumoral capacity derived from kinase interaction is improved in a dose-dependent manner. Furthermore, various experiments including cell migration and colony formation assays, along with apoptosis induction experiments, emphasize the significant antitumoral potential. Overall, the obtained results indicate that the developed formulation could be a promising therapy for the treatment of breast cancer.

Peptide-Functionalized Nanoemulsions as a Promising Tool for Isolation and Ex Vivo Culture of Circulating Tumor Cells.

Circulating Tumor Cells (CTCs) are shed from primary tumors and travel through the blood, generating metastases. CTCs represents a useful tool to understand the biology of metastasis in cancer disease. However, there is a lack of standardized protocols to isolate and culture them. In our previous work, we presented oil-in-water nanoemulsions (NEs) composed of lipids and fatty acids, which showed a benefit in supporting CTC cultures from metastatic breast cancer patients. Here, we present Peptide-Functionalized Nanoemulsions (Pept-NEs), with the aim of using them as a tool for CTC isolation and culture in situ. Therefore, NEs from our previous work were surface-decorated with the peptides Pep10 and GE11, which act as ligands towards the specific cell membrane proteins EpCAM and EGFR, respectively. We selected the best surface to deposit a layer of these Pept-NEs through a Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) method. Next, we validated the specific recognition of Pept-NEs for their protein targets EpCAM and EGFR by QCM-D and fluorescence microscopy. Finally, a layer of Pept-NEs was deposited in a culture well-plate, and cells were cultured on for 9 days in order to confirm the feasibility of the Pept-NEs as a cell growth support. This work presents peptide-functionalized nanoemulsions as a basis for the development of devices for the isolation and culture of CTCs in situ due to their ability to specifically interact with membrane proteins expressed in CTCs, and because cells are capable of growing on top of them.

A multifunctional drug nanocarrier for efficient anticancer therapy.

So far, the success of anticancer nanomedicines has been moderate due to their lack of adequate targeting properties and/or to their difficulties for penetrating tumors. Here we report a multifunctional drug nanocarrier consisting of hyaluronic acid nanocapsules conjugated with the tumor homing peptide tLyp1, which exhibits both, dual targeting properties (to the tumor and to the lymphatics), and enhanced tumor penetration. Data from a 3D co-culture in vitro model showed the capacity of these nanocapsules to interact with the NRP1 receptors over-expressed in cancer cells. The targeting capacity of the nanocapsules was evidenced in orthotopic lung cancer-bearing mice, using docetaxel as a standard drug. The results showed a dramatic accumulation of docetaxel in the tumor (37-fold the one achieved with Taxotere®). This biodistribution profile correlated with the high efficacy shown in terms of tumor growth regression and drastic reduction of metastasis in the lymphatics. When efficacy was validated in a pancreatic patient-derived tumor, the nanocapsule's activity was comparable to that of a dose ten times higher of Abraxane®. Multi-functionality was found to be the key to the success of this new therapy.