Final Report Summary - DINAMIT (Dual-Imaging Nano/Micro-sized Theranostics (against cancer))
Dual-Imaging Nano/Micro-sized Theranostics (against cancer) DINaMIT
Magnetic and metallic nanoparticles represent fundamentals of modern nanotechnology area. They open opportunities for controlling physico-chemical properties at the nanoscale. Current exchange program was set to explore potential of such nanotechnology building blocks for designing state-of-the art drug delivery system capable of imaging, remote activation and release. Functionalization of delivery carriers by nanoparticles was an important prerequisite of successfully achieving the goals of the project. Optical imaging is the technique of choice for working with cell cultures and for routine fluorescent sample characterization. Commercial microscopes are readily available for coupling of additional laser sources. That allows for remote activation and release from carriers functionalized with metal nanoparticles. Simplicity and flexibilities are the biggest advantage of the optical imaging / remote activation system. A significant advancement of the scientific research program is addition of magnetic response capabilities for such carriers, which results into dual optical / magnetically responsive system.
Work performed since the beginning of the project
Novel chitosan-based nanofibrous composite materials containing different amount of the photosensitizer Photosens (PS) were obtained by electrospinning process and characterized by scanning electron (SEM) and confocal laser scanning microscopy (CLSM). The release of PS from the material was investigated in water and phosphate buffered saline (PBS). Non-cancerous (murine osteoblasts MC3T3-E1) and cancerous (breast; mammary gland T-47D) cell lines were cultivated on PS-containing scaffolds.
Two kinds of bovine serum albumin-g-porphyrin submicron particles (BSA-g-Por/ BSA-g-PorCu SMPs) were prepared by a Schiff base reaction between 2-formyl-5,10,15,20-tetraphenylporphyrin /2-formyl-5,10,15,20-tetraphenylporphyrin Copper(Ⅱ) (Por/ PorCu) and BSA doped 800 nm MnCO3 particles, followed by crosslinking with glutaraldehyde (GA), quenching with glycine and template removal with ethylenediamine tetraacetic acid (EDTA). The immunogenicity of the particles was tested with phagotest and phagoburst assays.
BSA submicron particles were obtained by using 4-Bromomethyl-3-nitrobenzoic acid (BNBA) to cross-link BSA doped in MnCO3 particles followed removal of MnCO3. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the structure of the particles.
Red blood cells of different species have been investigated in solutions of different osmotic pressure before and after surface treatment with neuraminidase and trypsin in respect of their surface functionalization. Additionally, the RBC have been loaded with superparamagnetic nanoparticles (SPIONs).
Remote release of encapsulated material within living cells was first shown by a cw laser, where polymeric capsules with a model-system molecule, AF-488 (Alexa Fluorophore) labeled dextran, was released at a desired point in time and at a desired location. The walls of the capsules contained gold nanoparticles. Subsequently, the same principles were applied to release small peptides inside living cells.
Functionalization of theranostic NanoParticles (NPs) with specific ligands for targeted delivery of imaging (diagnostic) probe and curative drug requires the presence on the external surface of the NP of reactive groups (usually amino and/or carboxylic group). The functionalization with the ligand can result in the generation of free charged group on the NPs.
Uptake and biocompatibility of NPs are greatly influenced by the presence of charged reactive groups on their surface.
In the last period, we have extensively analyzed the cellular stress response to NPs bearing either free amino or carboxylic groups. Cells with different genetic background showing different efficiency in the endocytic machinery have been tested for their ability to endocytose and retain fluorescent Polystyrene NPs of different size (30 and 50 nm) bearing either NH2 or COOH free groups on the surface.
Main results achieved so far:
Electrospinning can be effectively used for the fabrication of chitosan-based nanofibrous materials with different amount of PS. In aqueous environment PS is released with a two-stage profile corresponding to a burst release of physically adsorbed PS during swelling of the fibers and a slow sustained release due to dissociation of chitosan/PS complexes. The scaffolds, independently of their PS content, are suitable as a support for cultivation of cells.
The BSA-g-Por/ BSA-g-PorCu SMPs with relatively smooth surface were stable and well dispersed in water, emitting red fluorescence. The two kinds of porphyrin particles can be used for imaging as well as photodynamic therapy.
The dissociation behavior of BSA submicron particles was initially observed by microscopy in situ. For better observation, the particles were labeled by RBITC. Under UV irradiation, since the cross-linking points formed by benzyl bromide and amino groups can be cleaved, the particles began to swell slightly and decomposed completely after 60 min.
Functionalized RBC have been loaded with SPIONS. The success can be seen by forming a bridge of RBC between two magnets. Using a centrifugation technique it is possible to determine the force, which is necessary to break the brifge. This provides an information about the dipolmoment.
NPs were fully characterized for their physical-chemical characteristics (including solubility and stability in different physiological media; SEM and HRTEM for structure/dimension; etc). The uptake, intracellular retention and cellular toxicity of these NPs in different cell types have been quantified. Additionally, the proper conditions were found to solubilize and maintain soluble the NPs and well dispersed in biological fluid.
The cell toxicity and cellular uptake of PLGA/NR@PEI-DTPA-Gd NPs by SKOV3 cells were measured by cell tracker methods and found the conditions that the NPs entered the cells. After exposure to these NPs for 24 h the cells manifested signs of toxicity.
The imaging set-up for both Optical-fluorescence and MRI has been proven suitable to perform the ‘in vitro’ pre-test of dual-probe nanotheranostic in order to monitor the uptake and intracellular fate of the NPs. We show that NPs can induce autophagy as a stress response which can lead to degradation of NPs or to cell death (necrosis) depending on the surface charge of Ps-NPs.
Magnetic and metallic nanoparticles represent fundamentals of modern nanotechnology area. They open opportunities for controlling physico-chemical properties at the nanoscale. Current exchange program was set to explore potential of such nanotechnology building blocks for designing state-of-the art drug delivery system capable of imaging, remote activation and release. Functionalization of delivery carriers by nanoparticles was an important prerequisite of successfully achieving the goals of the project. Optical imaging is the technique of choice for working with cell cultures and for routine fluorescent sample characterization. Commercial microscopes are readily available for coupling of additional laser sources. That allows for remote activation and release from carriers functionalized with metal nanoparticles. Simplicity and flexibilities are the biggest advantage of the optical imaging / remote activation system. A significant advancement of the scientific research program is addition of magnetic response capabilities for such carriers, which results into dual optical / magnetically responsive system.
Work performed since the beginning of the project
Novel chitosan-based nanofibrous composite materials containing different amount of the photosensitizer Photosens (PS) were obtained by electrospinning process and characterized by scanning electron (SEM) and confocal laser scanning microscopy (CLSM). The release of PS from the material was investigated in water and phosphate buffered saline (PBS). Non-cancerous (murine osteoblasts MC3T3-E1) and cancerous (breast; mammary gland T-47D) cell lines were cultivated on PS-containing scaffolds.
Two kinds of bovine serum albumin-g-porphyrin submicron particles (BSA-g-Por/ BSA-g-PorCu SMPs) were prepared by a Schiff base reaction between 2-formyl-5,10,15,20-tetraphenylporphyrin /2-formyl-5,10,15,20-tetraphenylporphyrin Copper(Ⅱ) (Por/ PorCu) and BSA doped 800 nm MnCO3 particles, followed by crosslinking with glutaraldehyde (GA), quenching with glycine and template removal with ethylenediamine tetraacetic acid (EDTA). The immunogenicity of the particles was tested with phagotest and phagoburst assays.
BSA submicron particles were obtained by using 4-Bromomethyl-3-nitrobenzoic acid (BNBA) to cross-link BSA doped in MnCO3 particles followed removal of MnCO3. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the structure of the particles.
Red blood cells of different species have been investigated in solutions of different osmotic pressure before and after surface treatment with neuraminidase and trypsin in respect of their surface functionalization. Additionally, the RBC have been loaded with superparamagnetic nanoparticles (SPIONs).
Remote release of encapsulated material within living cells was first shown by a cw laser, where polymeric capsules with a model-system molecule, AF-488 (Alexa Fluorophore) labeled dextran, was released at a desired point in time and at a desired location. The walls of the capsules contained gold nanoparticles. Subsequently, the same principles were applied to release small peptides inside living cells.
Functionalization of theranostic NanoParticles (NPs) with specific ligands for targeted delivery of imaging (diagnostic) probe and curative drug requires the presence on the external surface of the NP of reactive groups (usually amino and/or carboxylic group). The functionalization with the ligand can result in the generation of free charged group on the NPs.
Uptake and biocompatibility of NPs are greatly influenced by the presence of charged reactive groups on their surface.
In the last period, we have extensively analyzed the cellular stress response to NPs bearing either free amino or carboxylic groups. Cells with different genetic background showing different efficiency in the endocytic machinery have been tested for their ability to endocytose and retain fluorescent Polystyrene NPs of different size (30 and 50 nm) bearing either NH2 or COOH free groups on the surface.
Main results achieved so far:
Electrospinning can be effectively used for the fabrication of chitosan-based nanofibrous materials with different amount of PS. In aqueous environment PS is released with a two-stage profile corresponding to a burst release of physically adsorbed PS during swelling of the fibers and a slow sustained release due to dissociation of chitosan/PS complexes. The scaffolds, independently of their PS content, are suitable as a support for cultivation of cells.
The BSA-g-Por/ BSA-g-PorCu SMPs with relatively smooth surface were stable and well dispersed in water, emitting red fluorescence. The two kinds of porphyrin particles can be used for imaging as well as photodynamic therapy.
The dissociation behavior of BSA submicron particles was initially observed by microscopy in situ. For better observation, the particles were labeled by RBITC. Under UV irradiation, since the cross-linking points formed by benzyl bromide and amino groups can be cleaved, the particles began to swell slightly and decomposed completely after 60 min.
Functionalized RBC have been loaded with SPIONS. The success can be seen by forming a bridge of RBC between two magnets. Using a centrifugation technique it is possible to determine the force, which is necessary to break the brifge. This provides an information about the dipolmoment.
NPs were fully characterized for their physical-chemical characteristics (including solubility and stability in different physiological media; SEM and HRTEM for structure/dimension; etc). The uptake, intracellular retention and cellular toxicity of these NPs in different cell types have been quantified. Additionally, the proper conditions were found to solubilize and maintain soluble the NPs and well dispersed in biological fluid.
The cell toxicity and cellular uptake of PLGA/NR@PEI-DTPA-Gd NPs by SKOV3 cells were measured by cell tracker methods and found the conditions that the NPs entered the cells. After exposure to these NPs for 24 h the cells manifested signs of toxicity.
The imaging set-up for both Optical-fluorescence and MRI has been proven suitable to perform the ‘in vitro’ pre-test of dual-probe nanotheranostic in order to monitor the uptake and intracellular fate of the NPs. We show that NPs can induce autophagy as a stress response which can lead to degradation of NPs or to cell death (necrosis) depending on the surface charge of Ps-NPs.