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Laser Induced Synthesis of Biocompatible Multifunctional Inorganic Nanoparticles: A Novel Route to Produce Multifunctional Contrast Agents for Early Diagnosis of Breast Cancer

Periodic Reporting for period 1 - NIMBLIS (Laser Induced Synthesis of Biocompatible Multifunctional Inorganic Nanoparticles: A Novel Route to Produce Multifunctional Contrast Agents for Early Diagnosis of Breast Cancer)

Reporting period: 2016-02-01 to 2018-01-31

X-ray film mammography (FM) is the most widely used imaging modality for breast cancer screening. However, this technique shows several drawbacks to distinguish malignant tumours from those abnormal masses observed in the mammograms.
To overcome the limitations of FM, this is usually combined with contras enhancement imaging techniques, such as Ultrasonography (US), X-ray computed tomography (CT), digital mammography (DM) and magnetic resonance imaging (MRI) for clarification of mammographically equivocal lesions. However clinical application of this approach is still problematic due the lack of appropriate contrast agents showing capabilities to distinguish between malignant tumours and benign abnormal masses.
This project pursues the fabrication of a new generation of multimodal contract agents (MCAs) for the diagnosis of BC by combining DM, US, MRI and CT techniques. They will consist in colloidal suspensions of nanoparticles (NPs), which will be formed by a magnetic element, to generate contrast in MRI, and radiopaque elements, with different absorption coefficient of X-rays. In doing so, we will develop a new synthesis route consists in the laser ablation of ceramic plates, sowing the same composition of the projected NPs. Once we have synthesized, characterized and stabilized these NPs, we will evaluate their citotoxicity on several cell cultures.
We expect that this project will serve as a springboard for the development of future projects oriented to the functionalization of these NPs with specific biological targets for BC markers, and the in vivo validation and evaluation by the pharmacological industry of the performance of the proposed MCAS in the early diagnosis of BC.
Fabrication of ternary mixed metal oxides and/or binary mixed metal ceramic plates with the same composition of the planned NPs by using a laser melting process.
Nanoparticle synthesis. We have developed a sustainable method to produce multimodal nanoparticles via laser ablation in liquids of the previously hand-made massive targets by using nanosecond or femtosecond laser pulses. We have conducted different tasks to identify and control the factors involved in the generation of these nanoparticles
We have conducted an extensive characterization of the synthesized ceramic plates and nanoparticles by using different analytical techniques, such as X-ray diffractometry, energy dispersive spectroscopy, scanning electron microscopy, transmission electron microscopy, high resolution transmission microscopy, visible and ultraviolet spectroscopy (UV/VIS) and Laser Raman spectroscopy.
In order to produce stable dispersions of these NPs in water or simulated body fluids, we have conducted experiments for enhancing colloidal stability and biocompatibility of the synthesized Nps. In these experiments Nps were coated situ coating with hydrophilic capping molecules, which were dissolved previously in the solvent, where the metallic/ceramic plates were submerged.
We have also determined the ability of the hydrophilic-coated-hybrid-NPs to produce contrast in MRI, US and X-ray imaging as function of their composition and concentration by preparing phantoms of these materials and measuring the contrast enhancement produced in images acquired by using a Preclinical horizontal MRI spectrometer Biospec 3T and Quantum FX CT system.
In addition, we have analysed the in vitro cytotoxicity of the synthesized multimodal NPs by conducting cell viability MTT assays and NPs uptake Prussian blue staining assays in HeLa cells incubated with different concentrations of these hybrid NPs. Moreover, we have also measured the impact of these NPs on the red blood cells by performing haemolysis analysis.
As a result of research conducted in this project, we have synthesized a wide variety of colloidal dispersions of multimodal nanoparticles with different compositions by laser ablation in liquids of hand-made ceramic plates. These nanoparticles were functionalized in situ with citric acid yielded to achieve long-term stable colloidal nanoparticles in water. Moreover, in vitro toxicity and imaging analysis confirmed that these nanoparticles were non-toxic, producing contrast in MRI and CT images at nanoparticles concentrations lower than 2 mg/mL.
Combination of FM with US, DM, MRI and CT medical imaging techniques increases the sensitivity for BC detection of around 30-60% compared to FM. However, the clinical application of this multimodal imaging diagnosis approach is still problematic due to existing commercial contrast agents show low specificity and fails to detect non-vascularised malignant tumours.
Core-shell NPs formed by a magnetic core and shell of a radiopaque element (Au or Bi) are a promising alternative for their use of MCAs in combined MRI-US and X-ray imaging diagnosis, because their high biocompatibility, contrast efficacy and long-term colloidal stability. However, the use of these material is problematic due to: 1) The X-ray attenuation coefficients of Au and Bi limits their use only to high energy CT examinations. 2) Conventional chemical procedures used to synthesize these NPs fail to be transferred from the laboratory to the industry, because they involve numerous parameters difficult to control.
In this state of development of the project, we have achieved: 1) the development and optimization of a laser ablation in liquids process to produce a wide range of multimodal NPs, without the use of high expensive and toxic chemical precursors and surfactants. 2) Synthesis of a new generation of novel multimodal NPs showing contrast in MRI and X-ray imaging. 3) Confirmation that these nanoparticles are not toxic by performing in vitro cell viability assays in HeLa cells.
This proposal pursues the development of new synthesis methods and products for the industry of medical imaging contrast agents. With a global market of $6.2 billion in 2012, it consists mainly in medium size companies, which dedicate limited efforts to product developing due to it takes excessive time and high cost to fabricate new contrast agents (10 years to develop and a cost of $150 million). Thus, in the long term, the successful industrial transfer of the laser driven synthesis process proposed in this project, which allows high production of a broad variety of contrast agents, could position to the European companies, which will opt in the future for this technology, to the head of this industrial area. Moreover, the in vivo validation and clinical application of the multimodal contrast agents proposed in this research will improve breast cancer diagnosis, allowing a selection of patients without the need of a biopsy. In this context, it is important to note that, this research will be continue through a research project of the Spanish Government (MA2015-67354-R), which is focused on the conjugation of these multimodal contrast agents with Trastuzumab (Anti-HER2 antibodies) to obtain anti-HER2-NPS targeted toward breast tumours with HER2 overexpression; the in vivo validation of the biocompatibility and the ability of these anti-HER2-NPs conjugates to generate contrast in US, MRI and X-ray imaging techniques and detect Bc-HER2+ tumours; and 3) The implementation of a proof of concept laser ablation in liquids synthesis reactor to promote their application in production plants of the imaging contrast agents industry.
Image that outlines the process of generation of nanoparticles used in N.I.M.B.L.I.S project