WO2010133941A2 - New hybrid particles and their use in diagnostics and therapy - Google Patents

New hybrid particles and their use in diagnostics and therapy Download PDF

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Publication number
WO2010133941A2
WO2010133941A2 PCT/IB2010/001145 IB2010001145W WO2010133941A2 WO 2010133941 A2 WO2010133941 A2 WO 2010133941A2 IB 2010001145 W IB2010001145 W IB 2010001145W WO 2010133941 A2 WO2010133941 A2 WO 2010133941A2
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particles
metal
gold
diagnosis
treatment
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PCT/IB2010/001145
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French (fr)
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WO2010133941A3 (en
Inventor
Loredana Latterini
Fausto Elisei
Luigi Tarpani
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Universita Degli Studi Di Perugia
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Publication of WO2010133941A3 publication Critical patent/WO2010133941A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0026—Acridine dyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0089—Particulate, powder, adsorbate, bead, sphere
    • A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
    • A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention concerns new particles which can be used in the diagnosis and treatment of some pathologies, in particular nanoparticles for use in the diagnosis and treatment of tumours, and their use and the compositions that contain them.
  • Imaging diagnosis techniques which allow highly reliable and non-invasive diagnostic examinations to be performed have been known for some time.
  • MRI magnetic resonance
  • PET scan PET scan
  • CAT scan ectomography
  • angiography angiography
  • OCT optical coherence tomography
  • marking agents which allow selective visualisation of the various tissues during processing of the images.
  • Said marking agents are often adsorbed on silica nanoparticles, which are considered particularly suitable nuclei for supporting said diagnostic agents.
  • Nanoparticles with fluorescent agents or magnetic agents are known, commonly used in diagnosis as contrast media for the visualisation of tissues and cells.
  • Nanostructures which comprise metals, in particular gold, which can be used for biomedical (non-diagnostic) applications due to their low general toxicity and their optical properties are also known.
  • particles comprising gold when appropriately excited by a light source at particular wavelengths, can be used to effectively convert the light absorbed into heat and can thus increase the temperature of the surrounding environment.
  • WO2009/032752 describes particles having a fluorescent silica core and a gold coating.
  • Said coating is not a continuous layer, i.e. it is not complete and uniform (see for example pages 14 and 15).
  • Said incomplete layer entails drawbacks in terms of stability, toxicity and versatility of use, as will be better described below.
  • WO2006/102307 describes particles having an inert core coated with an outer layer of gold to which fluorescent agents are attached. It has been found that also this technical solution, with external fluorescent agent, attached to the metal, involves drawbacks, as will be discussed below.
  • Anal. Chem 81(9):2009 describes particles of silica coated in gold and containing mercury (Hg(O)) for industrial uses. Said particles are suitable for industrial use for analytical purposes but obviously cannot be used for diagnostic and therapeutic purposes, i.e. they cannot be administered to humans or animals, since in order to obtain fluorescence of the mercury, very high levels of energy would have to be provided which would be harmful for the tissues and for the patient.
  • Hg(O) mercury
  • One object of the present invention is to provide particles with a dual use, i.e. particles defined here as "hybrid”, which allow diagnostic activities to be performed and, when necessary, also treatment, i.e. which allow the two operations (diagnosis and treatment) to be carried out with the administration of one single type of particle.
  • a further object of the invention is to provide particles as above, which overcome the disadvantages and drawbacks of the known art.
  • the invention concerns particles which comprise a core of inert material comprising at least one fluorescent agent which absorbs and emits electromagnetic radiation in the region 370 and 500 ran, said core being coated with a continuous layer of at least one metal which releases heat when excited by a light source at appropriate wavelengths.
  • the invention concerns particles which comprise a core of inert material comprising at least one fluorescent agent chosen from aminoacridine, aminoacridone, aminoperylene, aminoanthracene and their derivatives, said core being coated by a continuous layer of at least one metal which releases heat when excited by a light source at appropriate wavelengths.
  • the term “particles” indicates nanoparticles having a mean dimension of 30-70 nm, preferably 40-60 ran, for example approximately 50-60 nm, said dimension referring to the final particle, including the metal layer.
  • the term “inert material” indicates a material which does not actively intervene in the imaging diagnosis operations and treatment during the use of said particles.
  • a particularly preferred inert material is silica.
  • the "fluorescent agent" according to the present invention is able to emit fluorescence when excited by a light source at appropriate wavelengths.
  • Appropriate fluorescent agents are aminoacridine, aminoacridone, aminoperylene and aminoanthracene which absorb and emit electromagnetic radiation in the region 370 and 500 nm. Other fluorescent agents can also be used.
  • derivatives of aminoacridine, of aminoacridone, of aminoperylene and of aminoanthracene indicates derivatives of said compounds which absorb and emit electromagnetic radiation in the region 370 and 500 nm.
  • the fluorescent agent of the invention is physiologically and pharmaceutically acceptable.
  • a light source for emission of fluorescence by said fluorescent agents it is possible to use a conventional lamp or laser, which supplies light in the UV-visible range, for example at wavelengths between 370 and 500 nm, advantageously around 380-450 nm, for example around 400 nm, where the metallic layer does not absorb or absorbs in a negligible manner.
  • the nanoparticles of the invention thus excited become fluorescent and emit in the range between 410 and 500 nm, where the metal layer does not absorb, allowing visualisation of the tumour cells in which they have accumulated, while the metal layer remains inert.
  • the particles of the invention can therefore be well used in the diagnosis techniques based on optical diagnostics, for example in optical tomography.
  • the at least one "metal" used in the particles is a non-toxic metal which does not degrade and which is able to release heat after absorption of light.
  • Metal alloys can also be used.
  • a preferred metal is gold which when excited at wavelengths in the near-infrared releases heat into the surrounding environment.
  • the layer of metal according to the present invention is continuous and advantageously has a thickness of approximately 5-15 run, preferably a thickness of approximately 7-10 nm.
  • continuous layer of metal indicates that the metal entirely covers the inert core comprising the fluorescent agents, thus forming a complete uniform shell.
  • the particles of the invention can furthermore comprise other components useful for diagnosis and/or treatment, for example they can be functionalised with molecules that promote attachment of the metal layer to the core of the particle, or they can comprise agents suitable for targeting the particle towards the tissue or towards the target cells.
  • Said targeting agents are known in the art, in particular in the field of the diagnosis and treatment of cancer.
  • the particles of the invention are intended for use in diagnosis and, when necessary, in the immediate subsequent treatment of various types of tumour.
  • a conventional laser which emits light in the near-infrared (where the fluorescent core does not absorb), for example which emits light at wavelengths between 600 and 1200 nm, advantageously around 800- 1000 nm, for example around 850 nm.
  • the nanoparticles of the invention thus irradiated will convert the light absorbed into heat and will cause the death of the tumour cells in which they are concentrated.
  • the particles of the invention represent a new and versatile technical solution in the field of diagnosis and treatment of various forms of cancer and other pathologies which require, as treatment, destruction of the affected tissues.
  • the particles of the invention which comprise fluorescent agents and a layer of gold can be used as a contrast medium for performing diagnoses by means of optical techniques.
  • the particles of the invention which comprise fluorescent agents and a layer of gold can be used as a contrast medium for performing diagnoses by means of optical techniques.
  • due to excitation with visible light it is possible to perform imaging diagnosis and immediately after, only if necessary, it is possible to activate destruction of the malignant cells previously identified.
  • the particles of the invention overcome the drawbacks of the prior art which feature drawbacks that limit their versatility and therefore their use.
  • the particles described in WO2009/032752 have an incomplete layer of gold.
  • This technical characteristic entails numerous disadvantages, including the possibility of contact between the core comprising the fluorophore agents and the tissues and cells of the patient treated; said compounds are, in fact, more toxic than gold and it is therefore advisable to limit as far as possible their interaction with the tissues and cells.
  • the specific fluorescent agents described therein absorb at wavelengths similar to those of gold, therefore there is no possibility of a clear separation between diagnosis/treatment.
  • a complete layer of metal, especially gold, as in the particles of the invention provides greater particle stability and, even more important, the complete layer moves the absorption band (800-900 run) forward, thus permitting a greater separation between the absorption bands necessary for the fluorescence and for excitation of the gold, therefore a clearer separation between excitation for diagnosis and excitation for treatment.
  • the particles of the invention constitute an important technical improvement.
  • the presence of the fluorescent agent on the outer layer of metal of the particles of WO2006/102307 causes a series of drawbacks.
  • the particles are more toxic, the fluorescence is partially quenched by the proximity to the metal and furthermore, no less important, the fluorescent agent can interfere with further bio-functionalisation of the particle.
  • the particles are nanoparticles of 40-50 run which comprise aminoacridine as fluorescent agent and which are coated in a continuous layer of gold of approximately 7-10 nm.
  • the invention concerns a diagnostic and/or pharmaceutical composition which comprises the particles of the invention, alone or in combination with pharmaceutically acceptable excipients and carriers.
  • compositions are for example liquid compositions, preferably water-based, in which the nanoparticles are suspended.
  • Said compositions are advantageously sterile, in particular when administration is by injection, infusion or inhalation.
  • the particles of the invention can be dispersed in a sterile saline solution, for instance hypotonic and buffered, of conventional type.
  • the particles of the invention can be prepared by mixing the fluorescent agent or agents in an aqueous solution and an appropriate silane derivative such as triethylsilane, in an alkaline environment. After stirring, silica particles form marked with fluorescent agents. Said particles can, if desired or necessary, be further functionalised with amine groups to facilitate subsequent attachment of the metal.
  • the marked particles thus prepared can be coated with a continuous metal layer, for example gold, by reduction of salts of said metal, for example by reduction of gold chloride hydrate using sodium citrate tribasic, in an appropriate solvent such as water.
  • a diagnostic and/or therapeutic method which comprises use of the particles of the invention in a patient requiring said treatment constitutes a further subject-matter of the present invention.
  • the nanoparticles of the invention can be marked with magnetic agents, instead of with fluorescent agents, to be used in the imaging techniques based on NMR.
  • the nanoparticles of the invention can be simultaneously marked with fluorescent agents and with magnetic agents, in order to be used in different imaging techniques.
  • Triethylsilane HTES, 97%, Aldrich
  • ammonium hydroxide fluorescent markers such as the derivatives of acridine
  • Milli-Q water nanopurif ⁇ ed >18.0 M ⁇
  • the nanoparticles were prepared using the St ⁇ ber method, dissolving 8.OxIO "7 M of marker and 1.3x10 3 M of HTES in 50 mL of ethanol. 3 mL of ammonium hydroxide (32% NH 3 ) were then added, dropwise and vigorously stirring. The solution was left under stirring for one night, after which it became turbid due to formation of the particles of silica. The non-reacted residue was removed by washing with ethanol. The particles were re-dispersed in 50 mL of ethanol and their surface was aminated by addition of 20 ⁇ L of APTES, stirring for a further 5 hours.
  • Gold colloids were previously formed by reduction of gold chloride (III) hydrate (6.3x10 "5 M) using sodium citrate tribasic hydrate (4.8XlO "4 M) in MiIIiQ water at 95-100 0 C. 2 mL of the solution of particles prepared as above were added to 20 mL of the gold colloidal solution described above under continuous stirring for at least 2 hours. The colour of the solution turned from a ruby red to violet. A 5.0xl0 "5 M solution of HauC14 is then added dropwise to the mixture and heated to 95-100 0 C. The solution turns from a violet colour to purple and lastly to dark blue.
  • the procedure described above is schematically illustrated in Figure 1.
  • the UV-VIS spectrums were recorded with a dual beam Perkin Elmer Lambda spectrometer.
  • Irradiation was then performed with a 450 W Xenon lamp or with an Nd: YAG laser (Continuum, Surelite 11-10, impulse amplitude approx. 7 ns and energy ⁇ 10 mJ impulse "1 ).
  • a Philips mod. 208 electron transmission microscope was used (operating at 80 kV acceleration of the electron beam) to analyse the dimension and dimensional distribution of the particles.
  • the nanoparticles dissolved in MiIIiQ water were then deposited on a 400 mesh copper grid, with formvar support and left one night in a dryer to evaporate the solvent.
  • Dispersions of nanoparticles and phospholipidic membranes both in buffered hypotonic saline solutions, were mixed and a drop of the mixture was placed on a slide.
  • the membranes were rapidly highlighted by imaging because they became luminescent.
  • the analysis showed an even distribution of the inner part of each membrane, thus demonstrating a uniform distribution.
  • the phospholipidic membranes were irradiated at 750 run where the layer of gold absorbs efficiently and converts the light absorbed into heat.
  • the effect of the irradiation at 750 nm on the phospholipidic structure was monitored by recording images in fluorescence, exciting again at 400 nm. The structure of the membrane was completely destroyed.
  • Fibroblast cells were incubated for one night with the nanoparticles of example 1 , then fixed on a cover slip and washed several times. The comparison with the non- treated cells indicated that the nanoparticles accumulated in the core, making it very luminous.
  • nanoparticles of the invention were successfully tested on tumour cells according to the procedure described in Lapotko, D. O.; Lukianova, E.; Potapnev, M.; Aleinikova, O.; Oraevsky, A. Cancer Lett. (2006) 239, 36-45.

Abstract

The invention concerns new particles which can be used in the diagnosis and treatment of some pathologies, in particular nanoparticles for use in the diagnosis and treatment of tumours, and their use and the compositions that contain them.

Description

"New hybrid particles and their use in diagnostics and therapy"
**************
ABSTRACT
The invention concerns new particles which can be used in the diagnosis and treatment of some pathologies, in particular nanoparticles for use in the diagnosis and treatment of tumours, and their use and the compositions that contain them.
TECHNICAL BACKGROUND
Imaging diagnosis techniques which allow highly reliable and non-invasive diagnostic examinations to be performed have been known for some time. For example MRI, PET scan, CAT scan, ectomography, angiography and optical coherence tomography (OCT) may be cited.
Some of these techniques use marking agents, which allow selective visualisation of the various tissues during processing of the images. Said marking agents are often adsorbed on silica nanoparticles, which are considered particularly suitable nuclei for supporting said diagnostic agents. Nanoparticles with fluorescent agents or magnetic agents are known, commonly used in diagnosis as contrast media for the visualisation of tissues and cells.
Nanostructures which comprise metals, in particular gold, which can be used for biomedical (non-diagnostic) applications due to their low general toxicity and their optical properties are also known. In particular, it is known that particles comprising gold, when appropriately excited by a light source at particular wavelengths, can be used to effectively convert the light absorbed into heat and can thus increase the temperature of the surrounding environment.
WO2009/032752 describes particles having a fluorescent silica core and a gold coating. Said coating is not a continuous layer, i.e. it is not complete and uniform (see for example pages 14 and 15). Said incomplete layer entails drawbacks in terms of stability, toxicity and versatility of use, as will be better described below.
WO2006/102307 describes particles having an inert core coated with an outer layer of gold to which fluorescent agents are attached. It has been found that also this technical solution, with external fluorescent agent, attached to the metal, involves drawbacks, as will be discussed below.
Anal. Chem 81(9):2009 describes particles of silica coated in gold and containing mercury (Hg(O)) for industrial uses. Said particles are suitable for industrial use for analytical purposes but obviously cannot be used for diagnostic and therapeutic purposes, i.e. they cannot be administered to humans or animals, since in order to obtain fluorescence of the mercury, very high levels of energy would have to be provided which would be harmful for the tissues and for the patient.
DESCRIOPTION OF THE INVENTION
One object of the present invention is to provide particles with a dual use, i.e. particles defined here as "hybrid", which allow diagnostic activities to be performed and, when necessary, also treatment, i.e. which allow the two operations (diagnosis and treatment) to be carried out with the administration of one single type of particle.
A further object of the invention is to provide particles as above, which overcome the disadvantages and drawbacks of the known art.
Thus, according to one of its aspects, the invention concerns particles which comprise a core of inert material comprising at least one fluorescent agent which absorbs and emits electromagnetic radiation in the region 370 and 500 ran, said core being coated with a continuous layer of at least one metal which releases heat when excited by a light source at appropriate wavelengths.
According to a preferred embodiment, the invention concerns particles which comprise a core of inert material comprising at least one fluorescent agent chosen from aminoacridine, aminoacridone, aminoperylene, aminoanthracene and their derivatives, said core being coated by a continuous layer of at least one metal which releases heat when excited by a light source at appropriate wavelengths.
According to the present invention, the term "particles" indicates nanoparticles having a mean dimension of 30-70 nm, preferably 40-60 ran, for example approximately 50-60 nm, said dimension referring to the final particle, including the metal layer. According to the present invention, the term "inert material" indicates a material which does not actively intervene in the imaging diagnosis operations and treatment during the use of said particles. A particularly preferred inert material is silica.
The "fluorescent agent" according to the present invention is able to emit fluorescence when excited by a light source at appropriate wavelengths.
Appropriate fluorescent agents are aminoacridine, aminoacridone, aminoperylene and aminoanthracene which absorb and emit electromagnetic radiation in the region 370 and 500 nm. Other fluorescent agents can also be used.
The term "derivatives" of aminoacridine, of aminoacridone, of aminoperylene and of aminoanthracene indicates derivatives of said compounds which absorb and emit electromagnetic radiation in the region 370 and 500 nm.
The fluorescent agent of the invention is physiologically and pharmaceutically acceptable.
By way of example, as a light source for emission of fluorescence by said fluorescent agents, it is possible to use a conventional lamp or laser, which supplies light in the UV-visible range, for example at wavelengths between 370 and 500 nm, advantageously around 380-450 nm, for example around 400 nm, where the metallic layer does not absorb or absorbs in a negligible manner. The nanoparticles of the invention thus excited become fluorescent and emit in the range between 410 and 500 nm, where the metal layer does not absorb, allowing visualisation of the tumour cells in which they have accumulated, while the metal layer remains inert.
The particles of the invention can therefore be well used in the diagnosis techniques based on optical diagnostics, for example in optical tomography.
According to the present invention, the at least one "metal" used in the particles is a non-toxic metal which does not degrade and which is able to release heat after absorption of light. Metal alloys can also be used. A preferred metal is gold which when excited at wavelengths in the near-infrared releases heat into the surrounding environment. The layer of metal according to the present invention is continuous and advantageously has a thickness of approximately 5-15 run, preferably a thickness of approximately 7-10 nm.
The expression "continuous" layer of metal indicates that the metal entirely covers the inert core comprising the fluorescent agents, thus forming a complete uniform shell.
The particles of the invention can furthermore comprise other components useful for diagnosis and/or treatment, for example they can be functionalised with molecules that promote attachment of the metal layer to the core of the particle, or they can comprise agents suitable for targeting the particle towards the tissue or towards the target cells. Said targeting agents are known in the art, in particular in the field of the diagnosis and treatment of cancer.
The particles of the invention are intended for use in diagnosis and, when necessary, in the immediate subsequent treatment of various types of tumour.
By way of example, at the time of the treatment, i.e. to kill the cells containing the particles, it is possible to use as a light source a conventional laser, which emits light in the near-infrared (where the fluorescent core does not absorb), for example which emits light at wavelengths between 600 and 1200 nm, advantageously around 800- 1000 nm, for example around 850 nm. The nanoparticles of the invention thus irradiated will convert the light absorbed into heat and will cause the death of the tumour cells in which they are concentrated.
Thanks to their multiple and selectable properties, i.e. due to the fact that they are marked with fluorescent agents and at the same time comprise a component (the metal) able to destroy the surrounding cells and tissues by release of heat, the particles of the invention represent a new and versatile technical solution in the field of diagnosis and treatment of various forms of cancer and other pathologies which require, as treatment, destruction of the affected tissues.
Thus, for example, the particles of the invention which comprise fluorescent agents and a layer of gold can be used as a contrast medium for performing diagnoses by means of optical techniques. In this case, due to excitation with visible light, it is possible to perform imaging diagnosis and immediately after, only if necessary, it is possible to activate destruction of the malignant cells previously identified.
Therefore, and contrary to what is described in the known art, with one single administration of the particles of the invention it is possible to proceed with diagnosis and treatment, which is particularly useful in the case of tumoural pathologies, eliminating waiting times between the diagnosis and treatment phases. The treatment is furthermore made selective, since it is optically guided.
As said, the particles of the invention overcome the drawbacks of the prior art which feature drawbacks that limit their versatility and therefore their use.
For example the particles described in WO2009/032752 have an incomplete layer of gold. This technical characteristic entails numerous disadvantages, including the possibility of contact between the core comprising the fluorophore agents and the tissues and cells of the patient treated; said compounds are, in fact, more toxic than gold and it is therefore advisable to limit as far as possible their interaction with the tissues and cells. Furthermore, the specific fluorescent agents described therein absorb at wavelengths similar to those of gold, therefore there is no possibility of a clear separation between diagnosis/treatment.
On the contrary, a complete layer of metal, especially gold, as in the particles of the invention, provides greater particle stability and, even more important, the complete layer moves the absorption band (800-900 run) forward, thus permitting a greater separation between the absorption bands necessary for the fluorescence and for excitation of the gold, therefore a clearer separation between excitation for diagnosis and excitation for treatment.
Via the fluorescent agents used in the present invention, it is possible to excite at approximately 400 run and detect the fluorescence for diagnostic purposes; subsequently, only if necessary, excitation can be performed at higher wavelengths, for example 650-800 nm, to destroy any tumour cells present (thanks to the continuous layer of gold) and then, if desired or necessary, again at approximately 400 nm to verify the effect of the treatment. This distinction of operations is not possible with the particles described in WO2009/032752. It is therefore apparent that the particles of the present invention represent considerable technical progress.
Also with respect to the particles described in WO2006/102307 the particles of the invention constitute an important technical improvement. In fact, the presence of the fluorescent agent on the outer layer of metal of the particles of WO2006/102307 causes a series of drawbacks. The particles are more toxic, the fluorescence is partially quenched by the proximity to the metal and furthermore, no less important, the fluorescent agent can interfere with further bio-functionalisation of the particle.
According to a preferred embodiment of the invention, the particles are nanoparticles of 40-50 run which comprise aminoacridine as fluorescent agent and which are coated in a continuous layer of gold of approximately 7-10 nm.
The use of the particles of the invention in diagnosis and/or treatment of cancer constitutes a further embodiment of the invention.
According to another of its aspects, the invention concerns a diagnostic and/or pharmaceutical composition which comprises the particles of the invention, alone or in combination with pharmaceutically acceptable excipients and carriers.
Suitable compositions, according to the invention, are for example liquid compositions, preferably water-based, in which the nanoparticles are suspended. Said compositions are advantageously sterile, in particular when administration is by injection, infusion or inhalation.
By way of example, the particles of the invention can be dispersed in a sterile saline solution, for instance hypotonic and buffered, of conventional type.
The particles of the invention can be prepared by mixing the fluorescent agent or agents in an aqueous solution and an appropriate silane derivative such as triethylsilane, in an alkaline environment. After stirring, silica particles form marked with fluorescent agents. Said particles can, if desired or necessary, be further functionalised with amine groups to facilitate subsequent attachment of the metal. The marked particles thus prepared can be coated with a continuous metal layer, for example gold, by reduction of salts of said metal, for example by reduction of gold chloride hydrate using sodium citrate tribasic, in an appropriate solvent such as water.
Preparation details are provided in the experimental section below and in the figure enclosed with the present description.
Further functionalisations, for example with agents suitable for targeting, for example specific antibodies, towards the various cells and various tissues to be reached, can be performed according to the methods known in the art.
A diagnostic and/or therapeutic method which comprises use of the particles of the invention in a patient requiring said treatment constitutes a further subject-matter of the present invention.
The nanoparticles of the invention can be marked with magnetic agents, instead of with fluorescent agents, to be used in the imaging techniques based on NMR. Alternatively, the nanoparticles of the invention can be simultaneously marked with fluorescent agents and with magnetic agents, in order to be used in different imaging techniques.
EXPERIMENTAL SECTION
Example 1
Preparation of nanoparticles comprising a fluorophore and a layer of gold
Materials
(3-Aminopropyl)triethoxysilane (APTES, 98%, Sigma); gold chloride (III) hydrate
(HAuCl4, 99,9%, Sigma); sodium citrate tribasic hydrate (Na3Qt, Fluka);
Triethylsilane (HTES, 97%, Aldrich), ammonium hydroxide, fluorescent markers such as the derivatives of acridine; Milli-Q water nanopurifϊed (>18.0 MΩ) with a
Millipore gradient system
Procedure
Step 1 - Preparation of marked nanoparticles
The nanoparticles were prepared using the Stδber method, dissolving 8.OxIO"7 M of marker and 1.3x10 3 M of HTES in 50 mL of ethanol. 3 mL of ammonium hydroxide (32% NH3) were then added, dropwise and vigorously stirring. The solution was left under stirring for one night, after which it became turbid due to formation of the particles of silica. The non-reacted residue was removed by washing with ethanol. The particles were re-dispersed in 50 mL of ethanol and their surface was aminated by addition of 20 μL of APTES, stirring for a further 5 hours. The aminated particles were centrifuged, washed with ethanol and then re-dispersed in MiIIiQ water (approx. 50 ml). In this way a solution of particles comprising fluorophore agents is obtained. Step 2 - Preparation of the layer of metal
Gold colloids were previously formed by reduction of gold chloride (III) hydrate (6.3x10"5 M) using sodium citrate tribasic hydrate (4.8XlO"4 M) in MiIIiQ water at 95-1000C. 2 mL of the solution of particles prepared as above were added to 20 mL of the gold colloidal solution described above under continuous stirring for at least 2 hours. The colour of the solution turned from a ruby red to violet. A 5.0xl0"5 M solution of HauC14 is then added dropwise to the mixture and heated to 95-1000C. The solution turns from a violet colour to purple and lastly to dark blue. The procedure described above is schematically illustrated in Figure 1.
Example 2
Characterisation of the nanoparticles
The UV-VIS spectrums were recorded with a dual beam Perkin Elmer Lambda spectrometer. The photostability test was conducted monitoring the absorption spectrums according to the irradiation time (λexc = 530, 650 and 850 nm for continuous irradiation and λexc = 532 nm for pulsed irradiation) using the particle solution of example 1 with 0.4 absorptions at the excitation wavelength. Irradiation was then performed with a 450 W Xenon lamp or with an Nd: YAG laser (Continuum, Surelite 11-10, impulse amplitude approx. 7 ns and energy ≤ 10 mJ impulse"1).
A Philips mod. 208 electron transmission microscope was used (operating at 80 kV acceleration of the electron beam) to analyse the dimension and dimensional distribution of the particles. The nanoparticles dissolved in MiIIiQ water were then deposited on a 400 mesh copper grid, with formvar support and left one night in a dryer to evaporate the solvent.
An atomic-force microscope (Solver-Pro P47H, NT-MDT) was used to record the topography and images of the nanoparticles. The measurements were performed in conditions of semi-contact using probes (cantilever) with oscillation frequency of 190-325 kHz. A drop of the sample suspended in water was placed on mica by means of spin coating in order to obtain a uniform deposition and make the solvent easily evaporable.
Example 3
Testing the particles of the invention on phospholipidic membranes
The interaction between the nanoparticles and the phospholipidic membranes and the cells was tested via optical images recorded with a confocal fluorescence laser scanning microscope (Nikon, PCM2000) using a diode laser (λexc = 400 run). The images were recorded in conditions of medium confocality and with an oil immersion lens 6Ox, 1.4 N. A. (512x512 pixels).
Dispersions of nanoparticles and phospholipidic membranes, both in buffered hypotonic saline solutions, were mixed and a drop of the mixture was placed on a slide. The membranes were rapidly highlighted by imaging because they became luminescent. The analysis showed an even distribution of the inner part of each membrane, thus demonstrating a uniform distribution. Subsequently the phospholipidic membranes were irradiated at 750 run where the layer of gold absorbs efficiently and converts the light absorbed into heat. The effect of the irradiation at 750 nm on the phospholipidic structure was monitored by recording images in fluorescence, exciting again at 400 nm. The structure of the membrane was completely destroyed.
Example 4
Testing the particles of the invention on fibroblasts
Fibroblast cells were incubated for one night with the nanoparticles of example 1 , then fixed on a cover slip and washed several times. The comparison with the non- treated cells indicated that the nanoparticles accumulated in the core, making it very luminous.
Example 5
Testing the particles of the invention on tumour cells
The nanoparticles of the invention were successfully tested on tumour cells according to the procedure described in Lapotko, D. O.; Lukianova, E.; Potapnev, M.; Aleinikova, O.; Oraevsky, A. Cancer Lett. (2006) 239, 36-45.

Claims

1. Particles that comprise a core of inert material comprising at least one fluorescent agent which absorbs and emits electromagnetic radiation in the region 370 and 500 nm, said core being coated by a continuous layer of at least one metal which releases heat when excited by a light source at appropriate wavelengths.
2. Particles as claimed in claim 1, characterised in that said fluorescent agent is chosen from aminoacridine, aminoacridone, aminoperylene, aminoanthracene and their derivatives.
3. Particles as claimed in claim 1 or 2, characterised in that their mean dimension is 30-70 nm.
4. Particles as claimed in the claims from 1 to 3, characterised in that said inert material is silica.
5. Particles as claimed in claim 4, characterised in that said fluorescent agents are aminoacridine and its derivatives.
6. Particles as claimed in any one of the claims from 1 to 5, characterised in that said metal is gold.
7. Particles as claimed in any one of the claims from 1 to 6, characterised in that said layer of said metal has a thickness of approximately 5-15 nm.
8. Particles as claimed in any one of the claims from 1 to 7, characterised in that they comprise aminoacridine as the fluorescent agent and in that they are coated by a continuous layer of gold of approximately 7-10 nm.
9. Particles as claimed in any one of the claims from 1 to 8, characterised in that they also comprise molecules suitable for targeting towards specific cells and tissues.
10. Use of the particles as claimed in any one of the claims from 1 to 9, for the preparation of a diagnostic and therapeutic composition for the diagnosis and treatment of cancer.
1. Diagnostic and therapeutic composition for the diagnosis and treatment of cancer which comprises the particles as claimed in any one of the claims from I to 9.
PCT/IB2010/001145 2009-05-20 2010-05-17 New hybrid particles and their use in diagnostics and therapy WO2010133941A2 (en)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2006102307A2 (en) 2005-03-21 2006-09-28 University Of Louisville Research Foundation, Inc. Target specific nanoparticles for enhancing optical contrast enhancement and inducing target-specific hyperthermia
WO2009032752A2 (en) 2007-08-28 2009-03-12 University Of Florida Research Foundation, Inc. Multimodal nanoparticles for non-invasive bio-imaging

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US8071535B2 (en) * 2003-09-12 2011-12-06 The Regents Of The University Of California Guanidinium derivatives for improved cellular transport
WO2008140624A2 (en) * 2006-12-22 2008-11-20 The Board Of Regents Of The University Of Texas System Methods and compositions related to hybird nanoparticles

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006102307A2 (en) 2005-03-21 2006-09-28 University Of Louisville Research Foundation, Inc. Target specific nanoparticles for enhancing optical contrast enhancement and inducing target-specific hyperthermia
WO2009032752A2 (en) 2007-08-28 2009-03-12 University Of Florida Research Foundation, Inc. Multimodal nanoparticles for non-invasive bio-imaging

Non-Patent Citations (2)

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Title
ANAL. CHEM, vol. 81, no. 9, 2009
LAPOTKO, D. 0.; LUKIANOVA, E.; POTAPNEV, M.; ALEINIKOVA, O.; ORAEVSKY, A., CANCER LETT., vol. 239, 2006, pages 36 - 45

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