Evaluating the immunogencinity of YFe2O3@Ag nanocomposite as a platform for the discovery and screening of vaccine adjuvants

Project leaders

Stefano Lupi, Asmaa Mohamed Salman

Agreement

EGITTO - NRC - National Research Centre of Egypt

Call

CNR/NRC biennio 2018-2019 2018-2019

Department

Physical sciences and technologies of matter

Thematic area

Physical sciences and technologies of matter

Status of the project

New

Research proposal

Background: The unique physicochemical properties of Nanoparticles (NPs) enable their potential applications in several biomedical fields. In particular, super paramagnetic NPs have been applied in drug discovery, gene delivery, imaging, photodynamic therapy and tissue engineering. Standard toxicity studies of NPs in biological systems are essential component of NPs safety profiling. To this end, the Egyptian group synthesized super paramagnetic YFe2O3@Ag NPs and assessed their toxicity both in vitro and preclinically in vivo, using traditional toxicological assessment and biochemical parameters [the study was funded by Science and Technology Development Fund (STDF), project title: Optimization and characterization of the biosynthesis of metal nanoparticles for medical application, ID number 12649]. The results showed that 100 mg/kg is a safe NP dose as assessed by pathological and biochemical parameters. However, preliminary studies with Fourier transform infrared imaging of tissue sections of mice treated with the aforementioned NPs provided evidence for detectable changes in the biochemical profile of the tissues (including alterations of protein folding, nucleic acids and carbohydrates content) mainly in organs of the immune system (namely spleen, bone marrow and blood) at sub-cytotoxic concentrations. This work has been presented as a poster at the 9th International Conference on Advanced Vibrational Spectroscopy; ICAVS 2017, in Victoria (Canada) by Dr. Eid. Thus, the in vivo and in vitro biochemical and pathological studies together with ex-vivo FTIR Imaging suggest that YFe2O3@Ag nanocomposite could possibly behave as a safe immunomodulator. Relying on purely biochemical signatures rather than on labeling with exogenous dyes and stains, infrared chemical imaging has the potential to greatly enhance the reliability of micro-morphological and/or biochemical assays.This demand is particularly severe for NPs, since their nano-size induces unpredictable interaction with the most common dyes, leading to contradictory and often unreliable results. Conversely, label-free FTIR imaging provides in a single spectrum and comprehensive overview of the biochemical sample profile, giving information on concentration and structure of biological relevant macromolecules and also on their spatial distribution at the micrometer scale.The use of dimensional Focal Plane Array detectors allow for the fast imaging of large samples, such as tissues, while Synchrotron Radiation (SR) FTIR point mapping enhance the spectral quality for minute and low-absorbing samples, such as individual cells.
The present project aims to gain insight into the effect of YFe2O3@Ag NPs at sub-cytotoxic concentrations on the biochemical profile of immune organs (inguinal, axillary, brachial and cervical lymph nodes, and thymus), combining ex-vivo FTIR Imaging with the investigation of cytokine secretions and standard in vivo and in vitro assays. In addition, we aim to validate the hypothesis that YFe2O3@Ag NPs can be used as a carrier for in vitro screening of vaccine adjuvants, exploiting single cell Synchrotron Radiation FTIR microscopy, and correlating information from conventional cell-based assays on lymphocytes and peritoneal macrophages.
The project goal will be achieved through a collaborative research that involves complementary scientific team from Egyptian and Italian CNR Institutions. The pharmacology and physics team from National Research Centre, Egypt will be responsible for preparation of the NPs and their biological evaluation in vitro and in vivo. The staff of the infrared beamline at Elettra Sincrotrone Trieste, from both CNR and Elettra branches, will contribute to this project providing instrumentation for performing both tissue ex vivo and cellular in vitro vibrational analyses. Expertise in bio-spectroscopy and data analysis will be shared with the Egyptian team, aiming to create a synergy that will last beyond the project limits.
Methodology: Specifically the objectives of the present proposal will be addressed in three steps. Step 1: In vivo and ex vivo testing of the effect of administering 100 mg/kg YFe2O3@Ag NPs on the immune system of mice: i. Animal treatment and isolation of immune organs. All animal procedure of the proposed work is enclosed under ethical approval sought for ongoing project (Identifying novel natural immunomodulatory therapeutics using in vitro and in vivo approaches) funded by National Research Centre (NRC) (project number: 11010310, ethical permit number: 16090); ii.Evaluation of inflammatory markers and immunoglobulins. Step2: Evaluation of spleen derived lymphocytes and peritoneal macrophage functions in response to YFe2O3@Ag NPs treatment in vitro based on viability, reactive oxygen species (ROS) production and cytokine levels. Step 3: Explore the use of YFe2O3@Ag NPs as a carrier for vaccine adjuvants (bacterial lipopolysaccharide and Polyinosinic-polycytidylic acid) by in vitro screening of NP effects on peritoneal macrophages using SR-FTIR microspectroscopy. i.Loading of lipopolysaccharide (LPS) and Polyinosinic-polycytidylic acid (Poly I:C) onto YFe2O3@Ag; ii. Validating the use of YFe2O3@Ag NPs as a carrier of vaccine adjuvants (LPS and Poly I:C) by SR-FTIR microspectroscopy.
Expected outcomes: 1. Demonstration of the immunological response induced by YFe2O3@Ag NPs in in vivo and in vitro models. 2. Assessment of the potentials of YFe2O3@Ag NPs as a carrier of vaccine adjuvants. 3. Strengthening the international collaboration between Egypt and Italy in the framework of NRC-CNR agreement that goes beyond the specific projects.

Research goals

The project goal will be achieved by the fulfillment of the following objectives:
1. Assessment of the impact on mice immune organs - including inguinal, axillary, brachial and cervical lymph nodes, thymus and spleen - of administering 100 mg/kg ³Fe2O3@Ag NPs by complementing in vivo and ex vivo studies, using biochemical markers and FTIR imaging.
2. Evaluation of the immune responses to ³Fe2O3@Ag NPs treatment in spleen derived lymphocytes and peritoneal macrophage functions in vitro
3. Investigation of the potential use of ³Fe2O3@Ag NPs as a carrier of vaccine adjuvants (namely, bacterial lipopolysaccharide and Polyinosinic-polycytidylic acid) and their potential of immunogenicity, through in vitro screening of the effects of adjuvant-NP conjugates on peritoneal macrophages using SR-FTIR microscopy complemented by cytokine signature analysis.

Last update: 13/05/2024