ITMI20090887A1 - "NEW HYBRID PARTICLES AND THEIR USE IN DIAGNOSIS AND THERAPY" - Google Patents

"NEW HYBRID PARTICLES AND THEIR USE IN DIAGNOSIS AND THERAPY" Download PDF

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ITMI20090887A1
ITMI20090887A1 IT000887A ITMI20090887A ITMI20090887A1 IT MI20090887 A1 ITMI20090887 A1 IT MI20090887A1 IT 000887 A IT000887 A IT 000887A IT MI20090887 A ITMI20090887 A IT MI20090887A IT MI20090887 A1 ITMI20090887 A1 IT MI20090887A1
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particles
particles according
diagnosis
nanoparticles
agents
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IT000887A
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Italian (it)
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Fausto Elisei
Loredana Lattarini
Luigi Tarpani
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Uni Degli Studi Perugia
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Priority to ITMI2009A000887A priority Critical patent/IT1394302B1/en
Priority to PCT/IB2010/001145 priority patent/WO2010133941A2/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

Description

DESCRIZIONE DESCRIPTION

La presente invenzione h per oggetto delle nuove particelle che trovano impiego nella diagnosi e nella terapia di alcune patologie, in particolare di nanoparticelle da utilizzarsi nella diagnosi e nella terapia di tumori. The present invention relates to new particles which are used in the diagnosis and therapy of some pathologies, in particular of nanoparticles to be used in the diagnosis and therapy of tumors.

Sono note da tempo le tecniche di diagnosi basate sulla raccolta di immagini (in inglese “imaging†) che permettono di effettuare esami diagnostici altamente affidabili e non invasivi. A titolo di esempio sì possono citare la risonanza magnetica, la tomografia a emissione di positroni, ia tomografia assiale computerizzata, Pecotomografia, i’angiografia e fa tomografia ottica a coerenza di fase. Diagnosis techniques based on the collection of images (in English â € œimagingâ €) have been known for some time, allowing highly reliable and non-invasive diagnostic tests to be carried out. As an example, we can mention magnetic resonance, positron emission tomography, computed axial tomography, Pecotomography, angiography and phase coherence optical tomography.

Alcune di queste tecniche impiegano agenti marcanti, che permettono la visualizzazione selettiva dei diversi tessuti durante l'elaborazione delle immagini. Detti agenti marcanti sono spesso adsorbiti su nanoparticelle di silice, che sono considerate dei nuclei particolarmente adatti per supportare tali agenti diagnostici. Sono note infatti nanoparticelle con agenti fuoruscenti o degli agenti magnetici, che vengono comunemente impiegate in diagnostica come mezzi dì contrasto per la visualizzazione di tessuti e cellule. Some of these techniques employ marking agents, which allow selective visualization of different tissues during image processing. Said labeling agents are often adsorbed on silica nanoparticles, which are considered nuclei particularly suitable for supporting such diagnostic agents. In fact, nanoparticles with leaking agents or magnetic agents are known, which are commonly used in diagnostics as contrast media for the visualization of tissues and cells.

Sono altresì note delle nanostruttnre che comprendono dei metalli, in particolare dejl’oro, che possono essere utilizzate per applicazioni biomcdiche (non diagnostiche) grazie alia loro bassa tossicità generale e alle loro proprietà ottiche. In particolare, à ̈ noto che delle particelle comprendenti dell'oro, quando adeguatamente eccitate da una fonte luminosa a particolari lunghezze d’onda, possono essere usate per convertire in modo efficace la luce assorbita in calore e possono così aumentare la temperatura deH’ambiente in cui si trovano. Nanostructures are also known which include metals, in particular gold, which can be used for biomedical (non-diagnostic) applications thanks to their low general toxicity and optical properties. In particular, it is known that particles comprising gold, when adequately excited by a light source at particular wavelengths, can be used to effectively convert the absorbed light into heat and can thus increase the temperature of the € ™ environment in which they are found.

Uno scopo della presente invenzione à ̈ di fornire delle particelle di duplice uso, cioà ̈ delie particelle qui definite “ibridi†, che permettano di svolgere delle attività diagnostiche e, quando necessario, anche degli interventi terapeutici, che consentano cioà ̈ di effettuare le due operazioni (diagnostica e terapeutica) con la somministrazione di un solo tipo di particelle. An aim of the present invention is to provide dual-use particles, that is to say the particles here defined as â € œhybridâ €, which allow to carry out diagnostic activities and, when necessary, also therapeutic interventions, which allow to carry out the two operations (diagnostic and therapeutic) with the administration of only one type of particles.

Così, secondo uno dei suoi aspetti, l'invenzione ha per oggetto delle particelle di materiale inerte che comprendono almeno un agente fluorescente e che sono rivestite da uno strato di almeno un metallo che rilasci calore quando eccitato da una fonte di luce ad opportune lunghezze d’onda. Secondo la presente invenzione, per “particelle" si intende designare delie nanopartieellc aventi una dimensione media di 30-70 nm, preferibilmente 40-60 nin, ad esempio circa 50-60 nm, detta dimensione essendo riferita alla particella finale, ivi compreso lo strato metallico. Thus, according to one of its aspects, the invention relates to particles of inert material which include at least one fluorescent agent and which are coated with a layer of at least one metal which releases heat when excited by a light source at suitable lengths wave. According to the present invention, by â € œparticles "is meant to designate nanoparticles having an average size of 30-70 nm, preferably 40-60 nm, for example about 50-60 nm, said size referring to the final particle, including the metal layer.

Secondo la presente invenzione, per “materiale inerte†si intende indicare un materiale che non interviene attivamente nelle operazioni di diagnosi per immagini e di terapia durante l'uso delle dette particelle. Un materiale inerte particolarmente preferito à ̈ la silice. According to the present invention, by â € œinert materialâ € is meant to indicate a material which does not actively intervene in the imaging and therapy operations during the use of said particles. A particularly preferred inert material is silica.

Secondo la presente invenzione, per “agente fluorescente†si intende indicare una o più molecole fluorofore, cioà ̈ molecole in grado di emettere fluorescenza quando eccitate da una fonte di luce ad opportune lunghezze d’onda. A titolo illustrativo, come fonte di luce per fare emettere fluorescenza ai detti agenti fuorescemi à ̈ possibile utilizzare una lampada o laser convenzionale, che emetta luce nel UV-visibile, ad esempio che emetta luce a lunghezze d'onda comprese tra 380 e 500 nm, vantaggiosamente intorno a 400-450 nm, per esempio intomo ai 440 nm, dove lo strato metallico non assorbe o assorbe in maniera trascurabile. Le nanoparti celle dell'invenzione cosi eccitate divengono fluorescenti consentendo la visualizzazione delle cellule tumorali in cui sì sono accumulate. According to the present invention, by â € œfluorescent agentâ € is meant to indicate one or more fluorophoric molecules, ie molecules capable of emitting fluorescence when excited by a light source at suitable wavelengths. By way of illustration, as a light source to make said fluorescent agents emit fluorescence, it is possible to use a conventional lamp or laser, which emits light in the UV-visible, for example that emits light at wavelengths between 380 and 500 nm. , advantageously around 400-450 nm, for example around 440 nm, where the metal layer does not absorb or absorbs in a negligible way. The nanoparticles of the invention thus excited become fluorescent allowing the visualization of the tumor cells in which they are accumulated.

Diversi materiali sono comunemente utilizzati come agenti di contrasto nelle tecniche di diagnosi che si basano sulla diagnostica ottica, ad esempio nella tomografia a emissione di positroni (PET), nella risonanza magnetica, la tomografia a emissione di positroni, la tomografia assiale computerizzata e la tomografìa ottica a coerenza di fase. Degli agenti fuorescenti adatti includono delle molecole organiche, ad esempio ma non solo, con funzioni amminiehe, come Pamminoacridina, l’amminoacridone, ramminoperilene, aminoantracene e loro derivati. Several materials are commonly used as contrast agents in diagnostic techniques based on optical diagnostics, for example in positron emission tomography (PET), magnetic resonance, positron emission tomography, computed axial tomography and tomography. Phase coherence optics. Suitable extinguishing agents include organic molecules, for example but not limited to, with amino functions, such as paminoacridine, aminoacridone, rhamminoperylene, aminoanthracene and their derivatives.

Secondo la presente invenzione, l’almeno un “metallo†utilizzato nelle particelle à ̈ un metallo non tossico, che non si degradi, e che sia in grado di rilasciare calore dopo assorbimento di luce. Possono essere altresì utilizzate delle leghe metalliche. Un metallo preferito à ̈ Poro, che quando eccitato a lunghezze d’onda ne! vicino infrarosso (Near-InfraRed) rilascia calore nell'ambiente circostante. According to the present invention, the at least one â € œmetalâ € used in the particles is a non-toxic metal, which does not degrade, and which is capable of releasing heat after absorption of light. Metal alloys can also be used. A favorite metal is Pore, which when excited at ne wavelengths! near infrared (Near-InfraRed) releases heat into the surrounding environment.

Lo strato di metallo secondo la presente invenzione ha uno spessore di circa 5-15 nm, preferibilmente di circa 7-10 nm. The metal layer according to the present invention has a thickness of about 5-15 nm, preferably about 7-10 nm.

Le particelle dell’invenzione possono comprendere inoltre altri componenti utili, ad esempio possono essere funzionalizzate con molecole che favoriscono l'attacco dello strato metallico al nucleo della particella, o ancora possono comprendere agenti adatti al targeting delia particella verso il tessuto o verso le cellule bersaglio. Tali agenti per il targeting sono noti alla tecnica, in particolare nel campo della diagnosi e terapia del cancro. The particles of the invention can also comprise other useful components, for example they can be functionalized with molecules that favor the attachment of the metal layer to the nucleus of the particle, or they can also comprise agents suitable for targeting the particle towards the tissue or towards the cells. target. Such targeting agents are known in the art, particularly in the field of cancer diagnosis and therapy.

Te particelle dell’invenzione sono destinate all’impiego nella diagnosi e, quando necessario, nella immediata successiva terapia di vari tipi di tumori. The particles of the invention are intended for use in the diagnosis and, when necessary, in the immediate subsequent therapy of various types of tumors.

A titolo illustrativo, come fonte di luce à ̈ possibile utilizzare un laser convenzionale, che emetta luce nel vicino infrarosso (dove il nucleo fluorescente non assorbe), ad esempio che emetta luce a lunghezze d’onda comprese tra 800 e 1200 nm, vantaggiosamente intorno a 800-1000 nm, per esempio intorno ai 970 nm. Le nanoparticelie dell'invenzione così irradiale convertiranno la luce assorbita in calore e provocheranno la morte delle cellule tumorali in cui si sono concentrate. By way of illustration, as a light source it is possible to use 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 800 and 1200 nm, advantageously around 800-1000 nm, for example around 970 nm. The nanoparticles of the invention thus irradiated will convert the absorbed light into heat and cause the death of the tumor cells in which they have concentrated.

Grazie alle loro multiple proprietà, per il fatto cioà ̈ di essere marcate con agenti fluorescenti e di comprendere al contempo un componente (il metallo) in grado dì distruggere le cellule c i tessuti circostanti mediante rilascio di calore, le particelle delTinvenzione rappresentano una nuova e versatile soluzione tecnica nel campo della diagnosi e della terapia di varie forme di cancro e delie altre patologie che richiedono come trattamento la distruzione dei tessuti malati. Thanks to their multiple properties, that is to say that they are marked with fluorescent agents and at the same time include a component (the metal) capable of destroying the cells and surrounding tissues by releasing heat, the particles of the invention represent a new and versatile technical solution in the field of diagnosis and therapy of various forms of cancer and other pathologies that require the destruction of diseased tissues as a treatment.

Così, ad esempio le particelle dell’invenzione che comprendono agenti fluorescenti e uno strato di oro possono essere impiegate come mezzo di contrasto per l’esecuzione di diagnosi mediante tecniche ottiche. In questo caso, grazie aU’eccitazione con luce visibile à ̈ possibile procedere una diagnosi per “imaging†e immediatamente dopo, se necessario, à ̈ possibile attivare la distruzione delle cellule maligne, previamente identificate. Thus, for example, the particles of the invention comprising fluorescent agents and a layer of gold can be used as a contrast medium for diagnosing by optical techniques. In this case, thanks to the excitation with visible light it is possible to carry out a diagnosis by "imaging" and immediately afterwards, if necessary, it is possible to activate the destruction of the malignant cells, previously identified.

Pertanto, con una sola somministrazione delle particelle dell’invenzione à ̈ possibile procedere alla diagnosi e alla terapia, con particolare utilità nelle patologie tumorali, eliminando i tempi di attesa tra la fase di accertamento c la fase di trattamento. Il trattamento à ̈ inoltre reso selettivo, essendo otticamente guidato. Therefore, with a single administration of the particles of the invention it is possible to proceed with the diagnosis and therapy, with particular utility in tumor pathologies, eliminating the waiting times between the assessment phase and the treatment phase. The treatment is also made selective, being optically guided.

Secondo una forma di realizzazione preferita deSÌ’invenzione, le particelle sono delle nanoparticelle di 40-50 mn che comprendono amminoacridma come agente fluorescente e che sono ricoperte da uno strato di oro di circa 7-10 mn. According to a preferred embodiment of the invention, the particles are nanoparticles of 40-50 mn which comprise aminoacryde as a fluorescent agent and which are covered with a layer of gold of about 7-10 mn.

L<'>uso delie particelle dell'invenzione nella diagnosi e/o terapia del cancro costituisce un ulteriore aspetto dell’invenzione. The use of the particles of the invention in the diagnosis and / or therapy of cancer constitutes a further aspect of the invention.

Secondo un altro dei suoi aspetti l’invenzione ha per oggetto una composizione diagnostica e/o farmaceutica che comprende le particelle dell’invenzione, da sole o in combinazione con eccipienti e veicoli farmaceuticamente accettabili. According to another of its aspects, the invention relates to a diagnostic and / or pharmaceutical composition which comprises the particles of the invention, alone or in combination with pharmaceutically acceptable excipients and vehicles.

Delle composizioni adatte, secondo l’invenzione sono ad esempio delle composizioni liquide, preferibilmente a base acquosa, in cui le nanoparticelle sono sospese. Tali composizioni sono vantaggiosamente sterili, in particolare quando se ne prevede la somministrazione per iniezione, infusione o inalazione. Suitable compositions according to the invention are for example liquid compositions, preferably water-based, in which the nanoparticles are suspended. These compositions are advantageously sterile, in particular when their administration by injection, infusion or inhalation is envisaged.

A titolo di esempio, le particelle dell’invenzione possono essere disperse in una soluzione sterile, salina, ad esempio ipotonica e tamponata, di tipo convenzionale. By way of example, the particles of the invention can be dispersed in a sterile, saline solution, for example hypotonic and buffered, of the conventional type.

Le particelle dell’invenzione possono essere preparate miscelando l’agente o gli agenti fluorescenti in una soluzione acquosa e un opportuno derivato silanico come ad esempio trietilsilano, in ambiente alcalino. Dopo agitazione si formano delle particelle di silice marcate con gli agenti fluorescenti. Dette particelle possono essere, se desiderato o necessario, ulteriormente funzionai izzate con gruppi amtninici per facilitare il successivo attacco del metallo. Le particelle inarcate cosi preparate possono essere rivestite di uno strato metallico, ad esempio di oro, per riduzione di sali del detto metallo, ad esempio per riduzione di cloruro di oro idrato usando sodio citrato tribasico, in un solvente opportuno quale l’acqua. The particles of the invention can be prepared by mixing the fluorescent agent or agents in an aqueous solution and a suitable silane derivative such as triethylsilane, in an alkaline medium. After stirring, silica particles marked with the fluorescent agents are formed. Said particles can be, if desired or necessary, further functionalized with aminine groups to facilitate the subsequent etching of the metal. The arched particles thus prepared can be coated with a metallic layer, for example gold, by reduction of salts of said metal, for example by reduction of hydrated gold chloride using tri-sodium citrate, in a suitable solvent such as water.

Dei dettagli di preparazione sono fomiti nella sezione sperimentale che segue e nella figura allegata alla presente descrizione. Preparation details are provided in the experimental section that follows and in the figure attached to the present description.

Ulteriori funzionalizzazioni, ad esempio con agenti adatti per il targeting, ad esempio specìfici anticorpi, verso le differenti cellule e i differenti tessuti da raggiungere, possono essere effettuate secondo i metodi noti alla tecnica. Further functionalizations, for example with agents suitable for targeting, for example specific antibodies, towards the different cells and the different tissues to be reached, can be carried out according to the methods known in the art.

Un metodo diagnostico e/o terapeutico che comprende l’uso delle particelle dell’ invenzione in un soggetto che necessita di un tale trattamento costituisce un ulteriore oggetto della presente invenzione. A diagnostic and / or therapeutic method which includes the use of the particles of the invention in a subject requiring such treatment constitutes a further object of the present invention.

Le nanopartieelle dell'invenzione possono essere marcale con agenti magnetici, invece che con agenti fuorescenti, per essere impiegate nelle tecniche di imaging basate sulla risonanza magnetica nucleare. Alternativamente, le nanopartieelle dell'invenzione possono essere simultaneamente marcate con agenti fluorescenti e con agenti magnetici, in modo da poter essere utilizzate in differenti tecniche di imaging. The nanoparticles of the invention can be marked with magnetic agents, rather than with fuzzing agents, for use in imaging techniques based on nuclear magnetic resonance. Alternatively, the nanoparticles of the invention can be simultaneously labeled with fluorescent and magnetic agents, so that they can be used in different imaging techniques.

Sezione sperimentale Experimental section

Esempio 1 Example 1

Preparazione di nanopartieelle comprendenti un lucro foro e uno stato di oro Preparation of nanoparticles comprising a profit hole and a gold state

Materiali Materials

(3-Aminopropil)trietossisilano (APTES, 98%, Sigma); Cloruro di oro (Ili) idrato (HAuCU, 99,9%, Sigma); Sodio citrato tribasico idrato (NajCit, Fluka); Trietilsilano (UTES, 97%, Aldrieh), Idrossido di ammonio, marcatori tuorescenti come i derivati deH’acridina; Acqua Mil!i-Q nanopurificata (>18.0 ΜΩ) con un sistema gradiente Millipore (3-Aminopropyl) triethoxysilane (APTES, 98%, Sigma); Gold chloride (III) hydrate (HAuCU, 99.9%, Sigma); Tribasic sodium citrate hydrate (NajCit, Fluka); Triethylsilane (UTES, 97%, Aldrieh), Ammonium hydroxide, yolk markers such as the derivatives of Hâ € ™ acridine; Mil! I-Q nanopurified water (> 18.0 ΜΠ©) with a Millipore gradient system

Procedura Procedure

Fase 1 - Preparazione di nanoparticelle marcate Phase 1 - Preparation of labeled nanoparticles

Le nanoparticelle sono state preparate usando il metodo Stober sciogliendo 8.0x10<">M di marcatore e 1.3x10 M di HTES in 50 mL di etanolo. Sono stati poi aggiunti, goccia a goccia e sotto vigorosa agitazione, 3 mL di idrossido di ammonio (32% NED). La soluzione à ̈ stata lasciata in agitazione per una notte, dopodiché à ̈ diventata torbida per la formazione delle particelle di silice. 1 residui non reagiti sono stati rimossi per lavaggio con etanolo. Le particelle sono state ridisperse in 50 mL di etanolo e la loro superficie à ̈ stata am minata per aggiunta di 20 pL di APTES agitando per ulteriori 5 ore. Le particelle amminate sono state centrifugate, lavate con etanolo e poi ridisperse in acqua MilliQ (ca, 50 mi). Si ottiene così una soluzione di particelle comprendenti degli agenti nuore fori, Fase 2 - Preparazione delio strato di metallo The nanoparticles were prepared using the Stober method by dissolving 8.0x10 <"> M of marker and 1.3x10 M of HTES in 50 mL of ethanol. 3 mL of ammonium hydroxide ( 32% NED). The solution was left to stir overnight, after which it became cloudy due to the formation of silica particles. The unreacted residues were removed by washing with ethanol. The particles were redispersed in 50 mL of ethanol and their surface was aminated by adding 20 pL of APTES while stirring for a further 5 hours. The aminated particles were centrifuged, washed with ethanol and then redispersed in MilliQ water (approx. 50 ml). thus a solution of particles comprising of the agents in the holes, Step 2 - Preparation of the metal layer

Dei colloidi di oro sono stati previamente formati per riduzione di cloruro di oro (ili) idrato (6.3X10<0>M) usando sodio citrato tribasico idrato (4.8x1 CT<1>M) in acqua MilliQ a 95-100<?>C. 2 mL della soluzione di particelle sopra preparata sono stati aggiunti a 20 mL della soluzione colloidale di oro sopra descritta sottoagitazione continua per almeno 2 ore . Il colore della soluzione à ̈ virato da un rosso rubino a violaceo. Si aggiunge quindi goccia a goccia una soluzione 5.0χ 10° M di I IauG4 alla miscela e si scalda a 95-100°C. La soluzione vira da un colore violaceo al porpora e inime ad un colore blu scuro. Il procedimento sopra descritto à ̈ schematicamente descritto nella Figura I . Esempio 2 Gold colloids have been previously formed by reduction of gold (ili) chloride hydrate (6.3X10 <0> M) using tri-sodium citrate hydrate (4.8x1 CT <1> M) in MilliQ water at 95-100 <?> C. 2 mL of the solution of particles prepared above was added to 20 mL of the colloidal gold solution described above under continuous stirring for at least 2 hours. The color of the solution has changed from a ruby red to purplish. A 5.0Ï ‡ 10 ° M solution of I IauG4 is then added dropwise to the mixture and heated to 95-100 ° C. The solution changes from a purplish to purple color and finally to a dark blue color. The procedure described above is schematically described in Figure I. Example 2

Caratterizzazione delle nanoparticelle Characterization of nanoparticles

Gli spettri UV-VIS sono stati registrati con uno spettrometro Perkin Elmer Lambda a doppio raggio, il test di fotostabilità à ̈ stato condotto monitorando gli spettri di assorbimento in funzione del tempo di irradiazione (λ^ = 530, 650 and 850 nm per irradiamento continuo e Xcxc= 532 airi per irradiazione pulsata) usando la soluzione di particelle dell’esempio I con 0,4 assorbimenti alla lunghezza d’onda di eccitazione. L<'>irradiamento à ̈ stato effettuato con una lampada Xenon di 450 W o con un laser Nd:YAG (Continuum, Surei ite 11-10, ampiezza impulso ca. 7 ns e energia ≤ 10 mJ impulso<'1>). The UV-VIS spectra were recorded with a double beam Perkin Elmer Lambda spectrometer, the photostability test was conducted by monitoring the absorption spectra as a function of the irradiation time (Î »^ = 530, 650 and 850 nm for irradiation continuous and Xcxc = 532 airi for pulsed irradiation) using the particle solution of Example I with 0.4 absorptions at the excitation wavelength. The irradiation was carried out with a 450 W Xenon lamp or with a Nd: YAG laser (Continuum, Surei ite 11-10, pulse width approx. 7 ns and energy â ‰ ¤ 10 mJ pulse <'1> ).

Si à ̈ usato un microscopio Philips mod. 208 a trasmissione di elettroni (operante a 80 kV di accelerazione del fascio di elettroni) per analizzare la dimensione e la distribuzione dimensionale delle particelle. Le nanoparticelle sciolte in acqua MilliQ sono state depositate su una griglia di rame da 400 mesh, con supporto di forni var e lasciate una notte in essiccatore per evaporare il solvente. A Philips microscope mod. 208 with electron transmission (operating at 80 kV of electron beam acceleration) to analyze the size and dimensional distribution of particles. The nanoparticles dissolved in MilliQ water were deposited on a 400 mesh copper grid, supported by var ovens and left overnight in a desiccator to evaporate the solvent.

Un microscopio a l<'>orza atomica (Solver-Pro P47H, NT-MDT) à ̈ stato usato per registrare la topografia e le immagini delle nanoparticelle. Le misurazioni sono state condotte in condizioni di semi-contatto usando sonde (cantilever) con frequenza di oscillazione di 190-325 kHz. Una goccia del campione sospesa in acqua à ̈ stata posata su mica mediante spìn coating in modo da ottenere una deposizione omogenea e rendere il solvente facilmente evaporabile. An atomic barge microscope (Solver-Pro P47H, NT-MDT) was used to record the topography and images of the nanoparticles. Measurements were conducted under semi-contact conditions using probes (cantilevers) with oscillation frequency of 190-325 kHz. A drop of the sample suspended in water was placed on mica by spìn coating in order to obtain a homogeneous deposition and make the solvent easily evaporable.

Esempio 4 Example 4

Sperimentazione delle particelle delPinvenzione su membrane fosfolipidiche Experimentation of the particles of the invention on phospholipid membranes

L'interazione tra le nanoparticelle e le membrane fosfolipidiche e le cellule sono state testate attraverso imaging ottica registrata con un microscopio confocale a scansione laser (Nikon, PCM20Q0) usando un Ar- laser < λ.Α. = 488 nm) o un He-Ne-laser (λ,_-χε= 543 nm) come fonte di eccitazione. Ue immagini sono state registrate in condizioni di media confocalità e con un obiettivo ad immersione di olio όΠ̧χ, 1 .4 N.A. (512x5 12 pixels). The interaction between the nanoparticles and the phospholipid membranes and the cells was tested by optical imaging recorded with a confocal laser scanning microscope (Nikon, PCM20Q0) using an Ar-laser <Î ».Α. = 488 nm) or a He-Ne-laser (Î », _- Ï ‡ ε = 543 nm) as the excitation source. Ue images were recorded in medium confocal conditions and with an oil immersion objective όΠ̧Ï ‡, 1 .4 N.A. (512x5 12 pixels).

Delle dispersioni di nanoparticelle e membrane fosfolipidiche, entrambe in soluzioni saline ipotoniche tamponate, sono state mescolate e una goccia della miscela stata posata su un vetrino. Le membrane sono state velocemente evidenziate per imaging perché divenivano luminescenti. Dispersions of nanoparticles and phospholipid membranes, both in buffered hypotonic saline solutions, were mixed and a drop of the mixture was placed on a slide. The membranes were quickly highlighted by imaging because they became luminescent.

L’analisi ha mostrato una omogenea distribuzione della parte interna di ogni membrana, dimostrando così una distribuzione uniforme. The analysis showed a homogeneous distribution of the internal part of each membrane, thus demonstrating a uniform distribution.

Esempio 5 Example 5

Sperimentazione deiie particelle dell’invenzione su fìbrobiasti Experimentation of the particles of the invention on fìbrobiasti

Delle cellule di fìbrobiasti sono state incubate una notte con le nanoparticeìle dell’esempio 1 poi fissate su un copri-oggetto e lavate molte volte. 0 confronto con le cellule non trattate indicava che le nanoparticeìle si accumulavano nel nucleo rendendolo molto luminoso. Fìbrobiasti cells were incubated one night with the nanoparticles of example 1 then fixed on a cover and washed many times. Comparison with untreated cells indicated that nanoparticles accumulated in the nucleus making it very bright.

Esempio 6 Example 6

Sperimentazione delle particelle delTinvenzione su cellule tumorali Experimentation of the particles of the invention on tumor cells

Le nanoparticeìle dell’invenzione sono state testate con successo su cellule tumorali secondo la procedura descrìtta in Lapotko, D. O.; Lukianova, E.; Potapnev, M.; Aleinikova, O.; Oraevsky, A. Cancer Leti (2006) 239, 36-45. The nanoparticles of the invention have been successfully tested on tumor cells according to the procedure described in Lapotko, D. O .; Lukianova, E .; Potapnev, M .; Aleinikova, O .; Oraevsky, A. Cancer Leti (2006) 239, 36-45.

Claims (5)

Rivendicazioni 1. Particelle di materiale inerte che comprendono almeno agente fluorescente e che sono rivestite da uno strato di almeno un metallo che rilasci calore quando eccitato da una fonte dì luce ad opportune lunghezze d'onda. Claims 1. Particles of inert material which include at least a fluorescent agent and which are coated with a layer of at least one metal which releases heat when excited by a light source at suitable wavelengths. 2. Particelle secondo la rivendicazione 1, caratterizzate da! fato che la loro dimensione media à ̈ 30-70 nm. 2. Particles according to claim 1, characterized by! given that their average size is 30-70 nm. 3. Particelle secondo le rivendicazioni 1 o 2, caratterizzate dai fatto che detto materiale inerte à ̈ silice. 3. Particles according to claims 1 or 2, characterized in that said inert material is silica. 4. Particelle secondo una qualsiasi delle rivendicazioni da 1 a 3, caraterizzate dal fatto che detti agenti fluorescenti sono scelti tra Pamminoacridina, Pamminoacridone, j'ainminopcriiene, aminoantracene c loro derivati. 4. Particles according to any one of claims 1 to 3, characterized by the fact that said fluorescent agents are selected from among Paminoacridine, Paminoacridone, jainminopriiene, aminoanthracene and their derivatives. 5. Particelle secondo la rivendicazione 4, caratterizzate dal fato che detti agenti fluorescenti sono la ammìnoacridina e i suoi derivati, 6. Particelle secondo una qualsiasi delle rivendicazioni da 1 a 5, caraterizzate da! fatto che detto metallo à ̈ l’oro. 7. Particelle secondo una qualsiasi delle rivendicazioni da 1 a 6, caratterizzate dal fatto che detto strato di deto metallo ha uno spessore di circa 5-15 nm. 8. Particelle secondo una qualsiasi delle rivendicazioni da 1 a 7, caratterizzate da! fiuto che comprendono ammìnoacridina come agente fluorescente e che sono ricoperte da uno strato di oro di circa 7-10 nm. 9. Particelle secondo una qualsiasi delle rivendicazioni da 1 a 8, caratterizzate dal fatto che comprendono molecole adatte per il targeting verso cellule e tessuti specifici. 10. Uso delle particelle secondo una qualsiasi delle rivendicazioni da I a 9, per la preparazione di una composizione diagnostica e terapeutica per la diagnosi e il tratamento del cancro. 1 1. Composizione diagnostica e terapeutica per la diagnosi e il trattamento del cancro che comprende le particelle secondo una qualsiasi delle rivendicazioni da ! a 95. Particles according to claim 4, characterized in that said fluorescent agents are amminoacridine and its derivatives, 6. Particles according to any one of claims 1 to 5, characterized by! the fact that this metal is gold. 7. Particles according to any one of claims 1 to 6, characterized in that said layer of said metal has a thickness of about 5-15 nm. 8. Particles according to any one of claims 1 to 7, characterized by! which include amminoacridine as a fluorescent agent and which are covered with a layer of gold of about 7-10 nm. Particles according to any one of claims 1 to 8, characterized in that they comprise molecules suitable for targeting to specific cells and tissues. Use of the particles according to any one of claims 1 to 9, for the preparation of a diagnostic and therapeutic composition for the diagnosis and treatment of cancer. 1 1. Diagnostic and therapeutic composition for the diagnosis and treatment of cancer that comprises the particles according to any one of claims from! to 9
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