Use of new nanogold materials as delivery systems for modulation of MCL-1/p53 siRNA in ALL

Pagina web NanoLAL

Project title: Use of new nanogold materials as delivery systems for modulation of MCL-1/p53 siRNA in ALL
Acronim: NanoLAL
Bilateral Cooperation Romania –South Africa
Executive Agency for Higher Education, Research, Development and Innovation (UEFISCDI)
Project Code 7BM/2016
Period: 2016-december 2017
Budget: 10.000 euro – 45.000 lei
Coordinator: The Oncology Institute Prof.Dr. I.Chiricuta Cluj Napoca
Partner: “”Iuliu Hatieganu” University of Medicine and Pharmacy


Nanotechnology and its potential for clinical translation in order efficiently and safely and efficacy represent an interesting scientific field that request multidisciplinary approaches. To sustain a rapid progress in the case of the nanoparticles formulation the selected molecules and to implement into the clinic, basic and mechanistic data must first be assessed in preclinical studies, integrating an understanding of the complex action, in selected ALL pathology.Gold nanoparticles (GNPs) have reached considerable attention as targeted delivery systems, due to their beneficial surface characteristics that permit a simple functionalization with a wide range of biological molecules in parallel with a reduced yield of cytotoxicity. The project will be focused on the developing of novel functionalized agents with rituximab along with siRNA for MCL-1/p53 and miRNA modulators in order to achieve a high bioavailability and internalization rate. At this point molecular investigations, combined with genomic and proteomic profiling of cells will contribute to understand interactions and molecular mechanisms that occur in treated ALL cells as result to exposure to multifunctionalized nanoparticles drugs.In parallel will be performed profiling studies in order to emphasis the differences and common signature between the two different regions.The project will also focus on developing of trainings in order to give the opportunity to young investigators to fulfill their research approaches.

Objectives and hypothesis
Our goal is to use the combination between teaching and experimental approaches to boost the interest and students understanding for the new concept novel nanodrugs (nanostructure functionalized with rituximab + siRNA for MCL-1/p53 and/or miRNA mimics/mimetics for selected genes) for ALL. The scientific hypothesis to be tested is that the combination between classical chemoterapeutics and siRNA inhibition will affect by different pathways the cell survival and therefore will induce more profound antitumor effects.
Preliminary results supporting the new concept

Phase 1 – september 2016 – December 5, 2016
Acute lymphoblastic leukemia (ALL) despite an ≈ 85 % cure rate, remains the second most common cause of cancer- related mortality in children young adults. Despite tremendous progress in the past few years, its prognosis is poor, in part due to diagnosis at late disease stages. A better understanding of the molecular mechanisms underlying ALL genesis, leading to the development of better early diagnostic tools, are thus imperative. Nanotechnology is emerging as a new and highly promising solution for the management of hematological malignancies, including ALL is a suitable candidate for novel nanotechnology-based therapeutics.
One of the biggest obstacles interfacing in the case of hematological diseases is improving that are efficient and have therapeutic selectivity. The implementation of gold nanoparticles (GNPs) into medical research as drug-delivery systems has quickly spreading out. GNPs can functionalized with a wide range of biocompatible polymers, like polyethylene glycol (PEG) and can be linked on the surface can multiple natural or synthetic chemotherapeutics agents, including siRNA.

Both siRNAs and miRNAs are single-stranded noncoding RNAs of 21–23 nucleotides that negatively regulate gene expression at the post-transcriptional level. . miRNAs/siRNA can regulate the expression of genes involved in development, growth, proliferation and apoptosis. siRNA targeted therapies have improved cancer drugs development over the past few years in terms of efficiency and safety. Researchers identified some small molecules that interact with miRNA pathways, from which, for the present study, we selected to test different scenarios of functionalized nanostructure (lipolex/liposemes versus gold nanoparticles) along with rituximab and siRNA for MCL-1/p53.

Myeloid cell leukaemia-2 (MCL-1) is a highly regulated member of the anti-apoptotic B-cell lymphoma-2 (Bcl-2) family of proteins. This protein was initially observed in the case of ML-1 human myeloid leukemia cells for the period of differentiation process, and then was demonstrated to have a vital representation in the cell cycle regulation and apoptosis. Some investigations have displayed that MCL-1 is overexpressed in varied hematological malignancies, and the inhibition of MCL-1 using specific RNAi or using miRNA mimics/mimetics leads to the modulation and activation of apoptosis,in parallel with a reduced yield of tumour cell proliferation (Karami H, Baradaran B, Esfahani A, Sakhinia M, Sakhinia E. Therapeutic Effects of Myeloid Cell Leukemia-1 siRNA on Human Acute Myeloid Leukemia Cells. Adv Pharm Bull. 2014;4(3):243-8. doi: 10.5681/apb.2014.035.).

Another significant therapeutic target represents p53. According to a common view, p53 should sensitize tumor cells to therapy, as p53 is expected to trigger apoptotic events. p53 could favor the recovery of cells damaged by therapy, thus acting as a survival factor preventing mitotic catastrophe, and so p53 inhibitory therapies could be ( Braicu C, Pileczki V, Irimie A, Berindan-Neagoe I. p53siRNA therapy reduces cell proliferation, migration and induces apoptosis in triple negative breast cancer cells. Mol Cell Biochem. 2013 Sep;381(1-2):61-8. doi: 10.1007/s11010-013-1688-5).

MCL-1/p53 siRNA theraphy could promote the convergence of the extrinsic and intrinsic pathways in a synergic manner with classical chemotherapeutics agents, leading to an increase response to therapy.

In parallel will be identified small molecules to target specific miRNA that modulate MCL-1/p53 regulation, then functionalized with nanostructured and testest in vitro.

Consequently, the principal goal of this project is the development of functionalized nanoparticles with an improved chemotherapeutic effect and targeted delivery, demonstrated in preclinical ALL models. Our project will be focused on the developing a novel class of nanotherapeutic agents based on siRNA, which synergistically incorporate multiple functionalities targeting altered multiple mechanisms altered in ALL.

At this stage of the project it was performed the synthesis and characterization of gold nanoparticles and was tested their internalization and bioavailability capacity in vitro on cell cultures.

Phase 2 – January 2017 - December 2017

Remember: The principal goal of this project was the development of a novel class of nanotherapeutic agents based on siRNA, which synergistically incorporate multiple functionalities targeting altered multiple mechanisms altered in ALL.

The next studies were conducted in vitro too, this time using two B-cell cell lines, an ALL cell line and a CLL cell line to make a comparative study. At this phase of the project, regarding the studies on rituximab conjugated nanogold were performed in vitro experiments to monitor the MCL-1 and p53 genes relative expression during treatment. These experiments involved a combination between rituximab nanogold and siRNA transfection procedures. Then the effects were analyzed using molecular biology techniques like internalization assays, apoptosis assay, fluorescence microscopy, flow cytometry, cell viability assay and qRT-PCR technique. The internalization of the GNP-rituximab complex was studied using darkfield microscopy. As gold nanoparticles scatter light strongly at the wavelength of their plasmonic resonance they can be visualized in dark field mode as shiny dots of a specific color. Apoptosis assay was performed using Annexin V-FITC and PI staining under fluorescence microscopy. Flow cytometry assay was used for apoptosis, necrosis and cell cycle analysis. Cell viability assays were performed using MTS a suitable test for cells that grow in suspension like leukemia cells. In fact, all these studies led to establishing the effective dose of transfection reagent and rituximab nanoconstructs. Rituximab conjugated gold nanoparticles have proved to be a cell viability reducing system which stimulates apoptosis.

As part of the exchanges between partners, professor Bert Klumperman from Stellenbosch University and Cristina Ștefan, from South Africa came to Cluj-Napoca. During his visits, professor Bert Klumpermann presented at the Iuliu Hatieganu University of Medicine and Pharmacy a workshop titled ,,End functional polymers for bioconjugation’’ in 2016 and another one titled „Nanotechnology in the service of personalized medicine” in 2017. Also, from Romania, the visit of Dr. Mihnea Zdrenghea and Dr. Ciprian Tomuleasa was carried out in 2016. The project results were included in a poster titled: „Rituximab functionalized gold nanoparticles as molecular therapy in b cell acute lymphoblastic leukemia”, which was presented at the Macromolecules & Materials Workshop & Conference at Stellenbosch University, South Africa (11-13 April 2017), at the poster presentation section during Anca Onaciu and professor Rareș Buigă visits in South Africa.

The experiments carried out in this project, as well as the training of the Romanian physicians and scientists in South Africa has led to the submission of three papers, two of them already published (as E-pub ahead of print, online) and another accepted for publication.

The papers are:
1. THE NEW ERA OF NANOTECHNOLOGY, AN ALTERNATIVE TO CHANGE CANCER TREATMENT. Drug Design, Development and Therapy, in press. impact factor: 2.881.
3. THE ROLE OF PATHOLOGY DEPARTMENT IN THE PRE-ANALYTICAL PHASE OF MOLECULAR ANALYSES. Cancer Management and Research, in press, impact factor: 3.851

Final conclusions
This project represents a basic step in B-cell ALL therapy by offering a new perspective of treatment. Nanogold functionalized with rituximab seems to be an efficient molecular therapy for B-cell ALL that express the CD20 receptor. Moreover, combining with siRNA molecules for MCL-1 and siRNA molecules for p53 can improve and enhance the chemotherapeutic effects of these nanosystems.

This grant brought together young researchers, students, Ph.D. students, different partners and Postdoctoral fellows. They had the chance to develop or to enhance their skills in in vitro and in vivo studies, and also to collaborate and to perform in nanotechnology and cell cultures field. Further research projects will focus on ALL animal models, immunophenotyping techniques, nanoconstructs synthesis, thus improving the laboratory management.