Final Report Summary - PSY-NANO-SI (NANOSILICON-BASED PHOTOSYNTHESIS FOR CHEMICAL AND BIOMEDICAL APPLICATIONS)
The PSY-NANO-SI project explored the possible applications of porous silicon particles containing luminescent Si nanocrystals (nano-Si particles, or nano-Si) as efficient generators of singlet molecular oxygen (1O2). The project aimed to develop novel biologically tolerable and environmentally friendly materials and prototype systems, able to compete with existing photo-sensitisers in photochemical, biological and medical applications. The work packages (WP) of the project are as follows:
WP1. Synthesis of nano-Si with size and properties adequate for sensitising applications.
WP2. Modification of the composition and properties of nano-Si.
WP3. Kinetics and physical mechanisms of photo-stimulated generation of singlet oxygen by nano-Si.
WP4. Exploration of the possibilities to use nano-Si photo-sensitisers in photodynamic therapy of cancer.
WP5. Standardisation, management and control.
Some of the main overviews and conclusions lined to the most important work packages can be found below.
Conclusions for synthesis of nano-Si with size and properties adequate for sensitising applications.
Nano-Si particles, suitable for photo-sensitising applications, can be produced in large quantities by direct stain-etching of crystalline Si powders. The size of the photoactive particles produced in that way can be varied in the micrometre to nanometre range and depends mainly on the grain size of the raw Si material. The stain-etching process and the equipment, developed in the course of this project, can be easily rescaled to industrial production.
Immediately after preparation by stain-etching the nano-Si particles contain a significant amount of toxic impurities HF and H2SiF6. Thus, they are not appropriate for direct bio-medical applications. On the other hand, these impurities stabilise the photo-activity of the stain-etched material in air ambient for a prolonged time (up to years), which is an advantage for non-biological applications.
An efficient cleaning procedure for removal of the toxic substances from nano-Si particles was developed. The procedure results in a reduction of the concentration of HF and H2SiF6 residua in stain-etched particles by more than three orders of magnitude. Thus, the purified particles, initially produced by stain-etching, become acceptable for bio-medical applications too.
Ways to increase the productivity of electrochemical production of nano-Si via non-steady-state processes were demonstrated in the course of the project. A large flexibility in respect to the structure and properties of the obtained nano-porous material has been achieved by applying pulsed current regimes. A significant added advantage of these regimes is that particles with electrochemically predetermined sizes can be produced by segregation of the so prepared nano-porous layer.
The nano-Si particles, prepared by electrochemical etching show much lower, but still measurable, concentrations HF and H2SiF6 residua. A simplified purification procedure outgasing in vacuum at 150-180 degrees Celsius - can be recommended for successful bio-medical applications of electrochemically prepared photo-sensitisers.
Conclusions for modification of the composition and properties of nano-Si
Modification of nano-Si surface through physical adsorption of non-ionic surfactants renders the surface hydrophilic. In de-ionised water the surface remains stable against erosion during many days if the modification is realised with linear unsaturated hydrocarbons such as undecylenic acid, oleic alcohol. In physiological solutions the nano-Si surface modified by physical adsorption of non-ionic surfactants is not protected against fast erosion and dissolution.
Chemical (thermal) modification of nano-Si by same unsaturated hydrocarbons via Si-C bond formation results in hydrophylicity of the surface and protects it against fast dissolution. Partial oxidation of the modified nano-Si surface during modification can result in formation of such a specific protective layer which allows effective interaction between excited Si nanocrystals and molecular oxygen (that is a basis for photosensitising activity) without visible corrosion/dissolution of nano-Si during many days. On the other hand, chemical modification of nano-Si surface in oxygen- and water-free conditions results in full protection of the excited Si nanocrystals against interaction with O2 that well stabilises nano-Si luminescence but excludes a use of such material as a photosensitiser.
It is found that in case of both physical and chemical modification of nano-Si surface by hydrocarbons with polar end groups specific luminescent surface states are formed on Si nanocrystal surface as a result of its contact with water. These surface states but not confined excitons are then effectively quenched by molecular oxygen.
Classical molecular photosensitisers used for singlet oxygen production, such as porphyrin-type molecules, adsorbed on oxidised nano-Si matrix can effectively generate 1O2 but, at the same time, the generated 1O2 is effectively quenched by the same surface.
Combination of immobilised porphyrin and luminescent (non-oxidised) nano-Si chemically modified by unsaturated hydrocarbons and simultaneously oxidised by water can be effectively used for controllable release of the photosensitiser into physiological solution because the rate of the nano-Si dissolution (and therefore of the photosensitiser release) can be easy governed by conditions of nano-Si chemical modification.
Exploration of the possibilities to use nano-Si photosensitizers in photodynamic therapy (PDT) of cancer
Photodynamic therapy (PDT) of tumours involves the systemic or topical administration of a photosensitiser (PS) to patients followed by irradiation of the tumour mass with light of an appropriate wavelength. In the presence of oxygen, photoactivated sensitisers generate highly reactive oxygen species, especially singlet oxygen (1O2). The oxidative damage to various cellular organelles and functions induced by singlet oxygen leads to direct cytotoxicity on tumour cells. Indirect effects, however, have also been shown to contribute to tumour destruction. These effects originate from the PDT-induced severe vascular damage and result in the collapse of the entire microcirculation system. Consequently, persistent tumour ischemia contributes to the overall tumour ablation. Moreover, an indirect antitumoral-specific immune reaction may also be induced by PDT as a result of the activation of inflammatory cells. It is likely that this phenomenon plays a role in long-term tumour control.
A number of PS are already in clinical practice. The first PS approved was Photofrin, a porfimer sodium, followed by Visudyne (benzoporphyrin derivative), Levulan (5-aminolevulinic acid), and most recently Foscan (m-tetra hydroxyphenyl chlorin) and Hexvix (hexyl-5-aminolevulenic acid). 5-Aminolevulinic acid (5-ALA) and its hexyl-ester are precursors of protoporphyrin IX (PpIX), a PS which is formed especially in targeted malignant tissue. All these compounds have a long triplet state lifetime which is considered to be a requirement for efficient photosensitisation.
In the framework of the present project the first solid-state PDT photosensitiser is developed, which is a powder of micro- and submicrometer-size particles of the luminescent porous silicon (hereafter nanosilicon, or nano-Si) that can produce singlet oxygen when illuminated in O2 ambience. Compared with the above-mentioned molecular photosensitisers, nano-Si can prove to be a more efficient source of 1O2 as the life times of photo-created excitons in it are longer. It also has an extremely large inner surface, where exciton-to-O2 energy transfer can occur. And finally, nano-Si is biocompatible and can be easy and controllably decomposed in physiological solutions down to simple innocent compounds of silicic acid.
Main results for exploration of the possibilities to use nano-Si photosensitisers in photodynamic therapy (PDT) of cancer
To increase the chance of success the consortium partners were solicited to investigate time and money in finding different nano-Si preparations, and over the entire project period, a total of 64 samples were received and investigated into detail on their (photo)cytotoxic characteristics. As PDT efficacy might depend on certain conditions used, specific attention was paid to the following parameters:
a) cell culture related issues (extracellular concentration, dark cytotoxicity, intracellular accumulation, cell type, incubation time/conditions, presence/absence of serum constituents)
b) nano-Si particle related issues (preparation method, size, coating or not, chemical stability), and
c) light related issues (fluence rate, fluence, activation spectrum).
Standardisation, management and control and design of central server for the project control
Structural design, development of software, connection protocols via internet, anti-theft measures, testing of the central server and its launching in the internet have been fulfilled in time and, during all the time of the PSY-NANO-Si project implementation, all materials of the project were completely available at the WEB site: http://www.mtm.upv.es/psy-nano-si(s’ouvre dans une nouvelle fenêtre)
Summary
On the basis of comprehensive physico-chemical model investigations, photocytotoxic and PDT activities of the nano-Si materials prepared and functionalised by the different methods have been studied. The conditions are found at which nano-Si exhibits in vitro photocytotoxic and in vivo PDT activities, but at the same time no any toxicity is observed without special illumination. Those conditions are, however, yet far from the demands placed on approved PDT drugs, first of all from the point of view of maximum admissible drug concentration and intensity of illumination that makes one to conclude that the present generation of nano-Si particles is not yet ready to be tested preclinically in in vivo conditions and that much more in vitro work has to be performed including development of new (and not existing at the moment) methods of (semi-)industrial down-up synthesis of a few-nanometer size Si nanocrystals as a new nano-Si material.
Different methods of porous silicon particles (nano-Si material) fabrication, such as stain etching of initial metallurgical-grade Si powder and electrochemical anodisation of polycrystalline Si wafers, as well as corresponding equipment have been developed to produce from tens milligrams to hundreds grams of different porous nano-Si material. Various methods of nano-Si surface modification and functionalisation have been also applied to make the surface hydrophilic and stable in de-ionised water as well as in physiological solutions (electrolytes). An ability and efficiency of nano-Si materials to produce singlet molecular oxygen (1O2) in different environments, from gas phase to organic liquids and then aqueous suspensions, have been studied by direct optical detection of 1O2 as well as by use of 1O2 chemical traps.
The work performed enabled us to develop the methods of scalable production of the luminescent porous-Si based nano-Si materials as a new type of solid-state photosensitisers as well as a new carrier for drug delivery, to investigate transformation of nano-Si surface and its photosensitising ability as a result of interaction with water, to develop new methods of full and partial protection of nano-Si surface against corrosion in physiological solutions. These new possibilities are developed now in the consortium laboratories and will be used for practical applications.
WP1. Synthesis of nano-Si with size and properties adequate for sensitising applications.
WP2. Modification of the composition and properties of nano-Si.
WP3. Kinetics and physical mechanisms of photo-stimulated generation of singlet oxygen by nano-Si.
WP4. Exploration of the possibilities to use nano-Si photo-sensitisers in photodynamic therapy of cancer.
WP5. Standardisation, management and control.
Some of the main overviews and conclusions lined to the most important work packages can be found below.
Conclusions for synthesis of nano-Si with size and properties adequate for sensitising applications.
Nano-Si particles, suitable for photo-sensitising applications, can be produced in large quantities by direct stain-etching of crystalline Si powders. The size of the photoactive particles produced in that way can be varied in the micrometre to nanometre range and depends mainly on the grain size of the raw Si material. The stain-etching process and the equipment, developed in the course of this project, can be easily rescaled to industrial production.
Immediately after preparation by stain-etching the nano-Si particles contain a significant amount of toxic impurities HF and H2SiF6. Thus, they are not appropriate for direct bio-medical applications. On the other hand, these impurities stabilise the photo-activity of the stain-etched material in air ambient for a prolonged time (up to years), which is an advantage for non-biological applications.
An efficient cleaning procedure for removal of the toxic substances from nano-Si particles was developed. The procedure results in a reduction of the concentration of HF and H2SiF6 residua in stain-etched particles by more than three orders of magnitude. Thus, the purified particles, initially produced by stain-etching, become acceptable for bio-medical applications too.
Ways to increase the productivity of electrochemical production of nano-Si via non-steady-state processes were demonstrated in the course of the project. A large flexibility in respect to the structure and properties of the obtained nano-porous material has been achieved by applying pulsed current regimes. A significant added advantage of these regimes is that particles with electrochemically predetermined sizes can be produced by segregation of the so prepared nano-porous layer.
The nano-Si particles, prepared by electrochemical etching show much lower, but still measurable, concentrations HF and H2SiF6 residua. A simplified purification procedure outgasing in vacuum at 150-180 degrees Celsius - can be recommended for successful bio-medical applications of electrochemically prepared photo-sensitisers.
Conclusions for modification of the composition and properties of nano-Si
Modification of nano-Si surface through physical adsorption of non-ionic surfactants renders the surface hydrophilic. In de-ionised water the surface remains stable against erosion during many days if the modification is realised with linear unsaturated hydrocarbons such as undecylenic acid, oleic alcohol. In physiological solutions the nano-Si surface modified by physical adsorption of non-ionic surfactants is not protected against fast erosion and dissolution.
Chemical (thermal) modification of nano-Si by same unsaturated hydrocarbons via Si-C bond formation results in hydrophylicity of the surface and protects it against fast dissolution. Partial oxidation of the modified nano-Si surface during modification can result in formation of such a specific protective layer which allows effective interaction between excited Si nanocrystals and molecular oxygen (that is a basis for photosensitising activity) without visible corrosion/dissolution of nano-Si during many days. On the other hand, chemical modification of nano-Si surface in oxygen- and water-free conditions results in full protection of the excited Si nanocrystals against interaction with O2 that well stabilises nano-Si luminescence but excludes a use of such material as a photosensitiser.
It is found that in case of both physical and chemical modification of nano-Si surface by hydrocarbons with polar end groups specific luminescent surface states are formed on Si nanocrystal surface as a result of its contact with water. These surface states but not confined excitons are then effectively quenched by molecular oxygen.
Classical molecular photosensitisers used for singlet oxygen production, such as porphyrin-type molecules, adsorbed on oxidised nano-Si matrix can effectively generate 1O2 but, at the same time, the generated 1O2 is effectively quenched by the same surface.
Combination of immobilised porphyrin and luminescent (non-oxidised) nano-Si chemically modified by unsaturated hydrocarbons and simultaneously oxidised by water can be effectively used for controllable release of the photosensitiser into physiological solution because the rate of the nano-Si dissolution (and therefore of the photosensitiser release) can be easy governed by conditions of nano-Si chemical modification.
Exploration of the possibilities to use nano-Si photosensitizers in photodynamic therapy (PDT) of cancer
Photodynamic therapy (PDT) of tumours involves the systemic or topical administration of a photosensitiser (PS) to patients followed by irradiation of the tumour mass with light of an appropriate wavelength. In the presence of oxygen, photoactivated sensitisers generate highly reactive oxygen species, especially singlet oxygen (1O2). The oxidative damage to various cellular organelles and functions induced by singlet oxygen leads to direct cytotoxicity on tumour cells. Indirect effects, however, have also been shown to contribute to tumour destruction. These effects originate from the PDT-induced severe vascular damage and result in the collapse of the entire microcirculation system. Consequently, persistent tumour ischemia contributes to the overall tumour ablation. Moreover, an indirect antitumoral-specific immune reaction may also be induced by PDT as a result of the activation of inflammatory cells. It is likely that this phenomenon plays a role in long-term tumour control.
A number of PS are already in clinical practice. The first PS approved was Photofrin, a porfimer sodium, followed by Visudyne (benzoporphyrin derivative), Levulan (5-aminolevulinic acid), and most recently Foscan (m-tetra hydroxyphenyl chlorin) and Hexvix (hexyl-5-aminolevulenic acid). 5-Aminolevulinic acid (5-ALA) and its hexyl-ester are precursors of protoporphyrin IX (PpIX), a PS which is formed especially in targeted malignant tissue. All these compounds have a long triplet state lifetime which is considered to be a requirement for efficient photosensitisation.
In the framework of the present project the first solid-state PDT photosensitiser is developed, which is a powder of micro- and submicrometer-size particles of the luminescent porous silicon (hereafter nanosilicon, or nano-Si) that can produce singlet oxygen when illuminated in O2 ambience. Compared with the above-mentioned molecular photosensitisers, nano-Si can prove to be a more efficient source of 1O2 as the life times of photo-created excitons in it are longer. It also has an extremely large inner surface, where exciton-to-O2 energy transfer can occur. And finally, nano-Si is biocompatible and can be easy and controllably decomposed in physiological solutions down to simple innocent compounds of silicic acid.
Main results for exploration of the possibilities to use nano-Si photosensitisers in photodynamic therapy (PDT) of cancer
To increase the chance of success the consortium partners were solicited to investigate time and money in finding different nano-Si preparations, and over the entire project period, a total of 64 samples were received and investigated into detail on their (photo)cytotoxic characteristics. As PDT efficacy might depend on certain conditions used, specific attention was paid to the following parameters:
a) cell culture related issues (extracellular concentration, dark cytotoxicity, intracellular accumulation, cell type, incubation time/conditions, presence/absence of serum constituents)
b) nano-Si particle related issues (preparation method, size, coating or not, chemical stability), and
c) light related issues (fluence rate, fluence, activation spectrum).
Standardisation, management and control and design of central server for the project control
Structural design, development of software, connection protocols via internet, anti-theft measures, testing of the central server and its launching in the internet have been fulfilled in time and, during all the time of the PSY-NANO-Si project implementation, all materials of the project were completely available at the WEB site: http://www.mtm.upv.es/psy-nano-si(s’ouvre dans une nouvelle fenêtre)
Summary
On the basis of comprehensive physico-chemical model investigations, photocytotoxic and PDT activities of the nano-Si materials prepared and functionalised by the different methods have been studied. The conditions are found at which nano-Si exhibits in vitro photocytotoxic and in vivo PDT activities, but at the same time no any toxicity is observed without special illumination. Those conditions are, however, yet far from the demands placed on approved PDT drugs, first of all from the point of view of maximum admissible drug concentration and intensity of illumination that makes one to conclude that the present generation of nano-Si particles is not yet ready to be tested preclinically in in vivo conditions and that much more in vitro work has to be performed including development of new (and not existing at the moment) methods of (semi-)industrial down-up synthesis of a few-nanometer size Si nanocrystals as a new nano-Si material.
Different methods of porous silicon particles (nano-Si material) fabrication, such as stain etching of initial metallurgical-grade Si powder and electrochemical anodisation of polycrystalline Si wafers, as well as corresponding equipment have been developed to produce from tens milligrams to hundreds grams of different porous nano-Si material. Various methods of nano-Si surface modification and functionalisation have been also applied to make the surface hydrophilic and stable in de-ionised water as well as in physiological solutions (electrolytes). An ability and efficiency of nano-Si materials to produce singlet molecular oxygen (1O2) in different environments, from gas phase to organic liquids and then aqueous suspensions, have been studied by direct optical detection of 1O2 as well as by use of 1O2 chemical traps.
The work performed enabled us to develop the methods of scalable production of the luminescent porous-Si based nano-Si materials as a new type of solid-state photosensitisers as well as a new carrier for drug delivery, to investigate transformation of nano-Si surface and its photosensitising ability as a result of interaction with water, to develop new methods of full and partial protection of nano-Si surface against corrosion in physiological solutions. These new possibilities are developed now in the consortium laboratories and will be used for practical applications.
