Periodic Reporting for period 1 - MAGNAMED (Novel magnetic nanostructures for medical applications)
Période du rapport: 2017-04-01 au 2019-03-31
Cancer is one of the main causes of death in Europe. An early stage detection and an effective treatment are keystones to reduce cancer mortality. Current clinical procedures fail to detect small amounts of tumoral biomarkers (molecules that appear in higher concentrations in patients with disease). For some kinds of cancer, tumors are detected at advanced stages when aggressive and conventional treatments are not effective. Moreover, existing cancer treatments like chemotherapy are not selective and destroy both healthy and unhealthy cells. Thus, the development of high-sensitive instruments able to detect cancer at a very initial stage and localized treatments that act on tumoral cells only would have an important impact to cure and reduce the effects of this disease in the society. MAGNAMED proposes a novel approach to improve the detection limits of cancer biomarkers and a remote actuation to selectively annihilate cancer cells in-vitro experiments. Both strategies are based on magnetic nanostructures, elements that look like magnetic coins smaller than one micron. On the one hand, these nanocoins, functionalized with the tumoral biomarker to be detected, can provide a high signal in magnetic-based sensors, which can detect small amounts of analyte. On the other hand, the nanocoins oscillate with a magnetic field. The oscillation of the nanostructures attached to the membrane of a cancer cell will produce its death. This remote action only affects cells loaded with nanostructures and keep safe the rest of the tissue.
The main objective of the project is to fabricate different types of magnetic nanostructures (MNS) with novel spin configurations and demonstrate their potential in medical diagnostics and cancer therapy. Thus, the progress of the project requires several steps, some of them can be done in parallel but others require the completion of previous steps. The project is organized into four scientific work packages (WP). The scientific progress of each WP is described below.
WP1
This WP is responsible for the fabrication of MNS that will be used in the biomedical applications. It is the basis of the project as must provide the bricks to build up the rest of the building. It is important to attain significant progress in this part to guarantee on-time execution of the subsequent scientific tasks. In this context, the progress during the first periodic report has been very positive.
Several lithography techniques were proposed to achieve different sizes of MNS. Starting with bigger sizes, a photolithography mask with disk diameters of 2 and 4 microns was designed by the partner CNEA.
The lithography technique proposed to yield MNS with feature sizes smaller than one micron is the laser interference lithography (IL). Using this technique, UPV has produced MNS in a vortex state with diameters between 500 and 700 nm.
Smaller feature sizes can be obtained by anodic aluminum oxide (AAO) membranes. Two methods were originally planned to achieve MNS with sub-100 nm in diameter. i) The group at UTSA has transferred AAO membranes onto silicon substrates. This procedure allows a further deposit of the magnetic materials by thermal evaporation. ii) The second method to fabricate MNS consists of the electrodeposition of the magnetic material inside the pores of the AAO membrane. The group at UP has fabricated AAO membranes with pore diameter between 40 and 70 nm.
An important part of this WP is the deposit of magnetic materials to achieve a specific spin configuration suitable for bio-applications: vortex, synthetic antiferromagnets (SAF) with in-plane (IP) and out-of-plane (OoP) anisotropy. SAF with IP magnetization were deposited at USM, but nanodisks show a ridge on the border due to the angular deposit of the material. In the case of SAF with OoP anisotropy, UCM has fabricated and optimized multilayers of Co/Pd with strong out-of-plane anisotropy to fabricate SAF structures.
WP2
In order to selectively detect cancer biomarkers and improve the efficiency of the magneto-mechanical actuation of MNS in tumoral cell cultures, a functionalization process is required. The aim of WP2 is to develop protocols for the functionalization of i) Giant magnetoresistance (GMR) sensors used for cancer diagnostics, and ii) MSN employed in-vitro assays for cell annihilation using alternating magnetic fields.
i) Functionalization of GMR sensors.
The functionalization of these new types of sensor substrates was optimized at IMG by using specific protocols for the activation of the nanostructured supports with streptavidin detectors. Immobilization protocol efficiencies have been characterized, but more geometries and sizes should be explored.
ii) Functionalization of MNS.
In order to functionalize MNS and improve their uptake by cancer cells, DSPE-PEG2000-FA conjugate synthesis has been optimized by ICETA and Nanovex. Another route to improve the biocompatibility and enhance cellular uptake by target cells is the lipid coating of MNS, which is been developed by UP and ICETA.
WP3
WP3 is devoted to the diagnostic part of the project using MNS and giant magnetoresistance (GMR) sensors. The first advance in this line was carried out during a secondment of an INESC-MN staff member to IMG Pharma. The aim was to use this technology on the surface of GMR sensors fabricated by INESC, and test the magneto-electrical signal of the sensor with magnetic nanoparticles. A screening of a panel of commercial antibodies against the CEA was carried out in order to select the pair of antibodies for the sandwich type immunodetection. The printing condition were set using the microarray technology.
WP4
WP4 comprises the therapeutic approach of MNS in-vitro assays and the toxicity analysis of the metallic materials used in the MNS. Few experiments were accomplished to assess the cytotoxicity of Permalloy and iron disks protected by a thin titanium layer. No toxicity effects were observed.
The interaction between cells and MNS was also investigated in leukemia and breast cancer cell lines. Protocols for the assays with MNS and human leukemia monocyte THP1 cells were established. This cell line has further been used to perform interaction studies with MNS using electron microscopy techniques. Images show that cell morphology seems not to be affected by MNS uptake.
WP1
This WP is responsible for the fabrication of MNS that will be used in the biomedical applications. It is the basis of the project as must provide the bricks to build up the rest of the building. It is important to attain significant progress in this part to guarantee on-time execution of the subsequent scientific tasks. In this context, the progress during the first periodic report has been very positive.
Several lithography techniques were proposed to achieve different sizes of MNS. Starting with bigger sizes, a photolithography mask with disk diameters of 2 and 4 microns was designed by the partner CNEA.
The lithography technique proposed to yield MNS with feature sizes smaller than one micron is the laser interference lithography (IL). Using this technique, UPV has produced MNS in a vortex state with diameters between 500 and 700 nm.
Smaller feature sizes can be obtained by anodic aluminum oxide (AAO) membranes. Two methods were originally planned to achieve MNS with sub-100 nm in diameter. i) The group at UTSA has transferred AAO membranes onto silicon substrates. This procedure allows a further deposit of the magnetic materials by thermal evaporation. ii) The second method to fabricate MNS consists of the electrodeposition of the magnetic material inside the pores of the AAO membrane. The group at UP has fabricated AAO membranes with pore diameter between 40 and 70 nm.
An important part of this WP is the deposit of magnetic materials to achieve a specific spin configuration suitable for bio-applications: vortex, synthetic antiferromagnets (SAF) with in-plane (IP) and out-of-plane (OoP) anisotropy. SAF with IP magnetization were deposited at USM, but nanodisks show a ridge on the border due to the angular deposit of the material. In the case of SAF with OoP anisotropy, UCM has fabricated and optimized multilayers of Co/Pd with strong out-of-plane anisotropy to fabricate SAF structures.
WP2
In order to selectively detect cancer biomarkers and improve the efficiency of the magneto-mechanical actuation of MNS in tumoral cell cultures, a functionalization process is required. The aim of WP2 is to develop protocols for the functionalization of i) Giant magnetoresistance (GMR) sensors used for cancer diagnostics, and ii) MSN employed in-vitro assays for cell annihilation using alternating magnetic fields.
i) Functionalization of GMR sensors.
The functionalization of these new types of sensor substrates was optimized at IMG by using specific protocols for the activation of the nanostructured supports with streptavidin detectors. Immobilization protocol efficiencies have been characterized, but more geometries and sizes should be explored.
ii) Functionalization of MNS.
In order to functionalize MNS and improve their uptake by cancer cells, DSPE-PEG2000-FA conjugate synthesis has been optimized by ICETA and Nanovex. Another route to improve the biocompatibility and enhance cellular uptake by target cells is the lipid coating of MNS, which is been developed by UP and ICETA.
WP3
WP3 is devoted to the diagnostic part of the project using MNS and giant magnetoresistance (GMR) sensors. The first advance in this line was carried out during a secondment of an INESC-MN staff member to IMG Pharma. The aim was to use this technology on the surface of GMR sensors fabricated by INESC, and test the magneto-electrical signal of the sensor with magnetic nanoparticles. A screening of a panel of commercial antibodies against the CEA was carried out in order to select the pair of antibodies for the sandwich type immunodetection. The printing condition were set using the microarray technology.
WP4
WP4 comprises the therapeutic approach of MNS in-vitro assays and the toxicity analysis of the metallic materials used in the MNS. Few experiments were accomplished to assess the cytotoxicity of Permalloy and iron disks protected by a thin titanium layer. No toxicity effects were observed.
The interaction between cells and MNS was also investigated in leukemia and breast cancer cell lines. Protocols for the assays with MNS and human leukemia monocyte THP1 cells were established. This cell line has further been used to perform interaction studies with MNS using electron microscopy techniques. Images show that cell morphology seems not to be affected by MNS uptake.
Some of the most relevant findings have already been published in high impact journals. There are other results pointing out to novel approaches beyond the state of the art, although they still require deeper studies. For example, the fabrication of sub-100 nm MNS by transferring alumina oxide membranes or the use of the patented technology of IMG Pharma to imprint microarrays of specific antigens on a GMR sensor, which might yield a multiplexing detection in a small lab-on-a-chip device.
We expect to fulfill the original plan in the second half of the project; consequently, the expected results are those of the proposal. Namely, the detection of cancer biomarkers using MNS on functionalized GMR sensors and the annihilation of cancer cells in-vitro assays by magneto-mechanical actuation of MNS.
A proof of the potential impact of MAGNAMED might be the two innovations identified by the European Commission's Innovation Radar during the Mid-term meeting.
We expect to fulfill the original plan in the second half of the project; consequently, the expected results are those of the proposal. Namely, the detection of cancer biomarkers using MNS on functionalized GMR sensors and the annihilation of cancer cells in-vitro assays by magneto-mechanical actuation of MNS.
A proof of the potential impact of MAGNAMED might be the two innovations identified by the European Commission's Innovation Radar during the Mid-term meeting.