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WO2022112944A1 - Nanosystème pour le diagnostic et le traitement photothermique de tumeurs - Google Patents

Nanosystème pour le diagnostic et le traitement photothermique de tumeurs Download PDF

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WO2022112944A1
WO2022112944A1 PCT/IB2021/060873 IB2021060873W WO2022112944A1 WO 2022112944 A1 WO2022112944 A1 WO 2022112944A1 IB 2021060873 W IB2021060873 W IB 2021060873W WO 2022112944 A1 WO2022112944 A1 WO 2022112944A1
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nanosystem
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Gaetano Giammona
Nicolò Mauro
Gennara Cavallaro
Fabrizio Messina
Alice SCIORTINO
Gianpiero BUSCARINO
Maurizio MARRALE
Cesare GAGLIARDO
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Universita degli Studi di Palermo
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    • 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
    • A61K47/51—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 non-active ingredient being a modifying agent
    • A61K47/56—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 non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—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 non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
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    • 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
    • A61K47/6927—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 a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • 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
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • A61K49/0065—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle
    • A61K49/0067—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle quantum dots, fluorescent nanocrystals
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1851—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule
    • A61K49/1863—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule the organic macromolecular compound being a polysaccharide or derivative thereof, e.g. chitosan, chitin, cellulose, pectin, starch
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof

Definitions

  • the present invention relates to a nanosystem for the diagnosis, image-guided treatment of tumors and monitoring of the tumor microenvironment.
  • the nanosystem is a contrast medium comprising a polymeric shell based on a hyaluronic acid nanogel and super-paramagnetic nanoparticles based on iron oxide (hereinafter referred to as SPIONs-Superparamagnetic Iron Oxide Nanoparticles) and carbon nanoparticles (hereinafter referred to as such as CDs-Carbon Dots).
  • the invention refers to a nanosystem that fits into two large research areas.
  • the first is the one that describes the use of nanotechnologies for molecular sensing, useful in precision medicine.
  • This research area deals with developing nanotechnologies that respond to very specific endogenous or exogenous stimuli to be able to monitor pathophysiological changes that indicate the course of a disease.
  • the second relates to the development and production of nanodrugs with theranostic (from the combination of therapy and diagnostics) multi-effect (synergistic therapeutic effects) and multimodal (combined diagnosis) action for the diagnosis and monitoring of tumor masses and their simultaneous therapeutic treatment. More particularly, it is a research area aimed at the development of advanced nanotechnologies with multiple therapeutic properties, including the controlled and on-demand release of bioactive molecules and the photothermal treatment induced by infrared lasers, and with contrast multimodal properties (usually magnetic resonance and fluorescence imaging).
  • Nanodrugs Many therapies are currently in use that have already amply demonstrated how the use of nanodrugs can modify the biodistribution profile of anticancer drugs, thus improving their pharmacological effects and decreasing their off-target effects. Concrete examples are the case of Doxil®, Zinostatin®, Myocet® and Nanotherm®. However, to date there are no anticancer nanodrugs in use with multimodal theranostic action, capable of detecting changes in the tumor microenvironment during the course of treatment, and which simultaneously allow targeted photothermal treatment.
  • Some nanodrugs undergoing clinical study are for instance CriPec®, which combines the action of docetaxel with Zirconium-89 as an imaging agent in PET imaging and the NBTXR3® nanosystem, consisting of hafnium nanoparticles as an enhancer of ionizing radiation.
  • MRI magnetic resonance imaging
  • MR imaging Through MR imaging it is possible to acquire high resolution morphological images (spatial and by contrast) as well as information of an ultrastructural, metabolic and functional nature (Chen LQ et al.,
  • paramagnetic contrast agents allows to further improve the performance of diagnostic imaging using MR.
  • the contrast media most commonly used today in this area are gadolinium chelates capable of determining a clear reduction in T1 relaxation times resulting in an increase in the MRI signal in all those tissue areas where gadolinium is deposited (’’Positive" contrast media).
  • These contrast agents are usually administered intravenously to improve the identification of pathological tissues or to detect poorly visible frames using only the MR sequences acquired without contrast medium (for instance meningitis or leptomeningitis).
  • This family of contrast agents is applied by virtue of the ability to determine a decrease in the T2-weighted and T2 * -weighted signal in Spin Echo (SE) and Gradient Echo (GRE) sequences, respectively, with initial evidence in T2-weighted inversion recovery sequences to eliminate the signal from the fluids (Fluid Attenuated Inversion Recovery, FLAIR).
  • SE Spin Echo
  • GRE Gradient Echo
  • Current scientific evidence identifies SPIONs as possible MRI contrast media for diagnostic use to evaluate - among others - inflammatory, oncological, angiogenesis, gene expression, atherosclerosis or ’’stem cell tracking" processes (Zachary, RS et al., Magnetite Nanoparticles for Medical MR Imaging. Mater Today 2011 , 14, 330- 338).
  • SPIONs can also be used in the therapeutic field for ’’targeted drug delivery” if linked to chemotherapeutic, anti inflammatory, anti-infectious, radioactive agents or hyperthermic therapy (Swati Kaushik et al., In Situ Biosynthesized Superparamagnetic Iron Oxide Nanoparticles (SPIONs) Induces Efficient Flyperthermia in Cancer Cells, ACS Appl Biomat 2020, 3, 779-788).
  • SPIONs do not allow to identify changes in the tumor microenvironment in response to therapeutic treatment, since they do not possess pH or temperature sensing properties, all parameters that undergo substantial variations during the healing process. More particularly, SPIONs do not change the contrast properties in MRI based on changes in pH and temperature. It is known that after tumor healing processes, the pH of the tumor microenvironment tends to pass from a weakly acidic condition (5.5 ⁇ pH ⁇ 6.4) to a physiological one (pH 7.4). Therefore, if one had a system capable of noting these differences and capable of responding to changes in pH in this precise range (5.5-7.4) it could be used to monitor the healing process in patients in a non-invasive way and, consequently, it could allow to personalize the treatments for each type of patient.
  • SPIONs cannot be used as local temperature sensors in MRI and therefore in advanced therapeutic applications such as image-guided phototherapy.
  • phototherapy it is essential to know the local temperatures of the tissues to be eradicated by hyperthermia, since tumor cells undergo a selective cell death process at temperatures between 41-43°C, but higher temperatures cause collateral damage to the surrounding normal tissues.
  • the SPIONs are also used to increase the sensitivity to radiotherapy treatments with photon beams. After irradiation, the amount of toxic reactive oxygen species (ROS) in the tumor cells that have incorporated the SPIONs is significantly increased (Klein S, et al. Superparamagnetic iron oxide nanoparticles as novel X-ray enhancer for low-dose radiation therapy. J Phys Chem B. 2014 Jun 12; 118 (23): 6159-66.). Recently, it has also been verified that SPIONs can induce radiosensitization effects on human colon carcinoma cells (HOT 116) irradiated with 150 MeV clinical proton beams. The reduction in cell survival mainly corresponds to the high level of ROS generated by SPIONs which can therefore increase the therapeutic effects of cancer treatment through proton beams (RA Rashid, et al. Radiosensitization effects and ROS generation by high
  • radiotherapy treatments can be made more effective through hyperthermia achieved thanks to the use of SPIONs.
  • poorly perfused tumor nuclei are sensitive to hyperthermia but resistant to ionizing radiation which depends on the formation of toxic oxygen radicals in well perfused areas.
  • cancer cells show radiation resistance but are highly sensitive to heat.
  • the hyperthermia generated by SPIONs can act as a radiosensitizer for radioresistant tumor cells (Chatterjee DK et al., Nanoparticle-mediated hyperthermia in cancer therapy. Ther Deliv. (2011) 2: 1001-14).
  • CDs Carbon Dots
  • They are a family of carbon-based materials that are attracting considerable interest in various fields of application (optoelectronics, imaging, nanomedicine, alternative energy, etc.) and in basic research. Their chemical-physical characteristics and the macroscopic properties that derive from them are strongly influenced by the way in which they are synthesized, but in principle they can be described as nanoparticles with a crystalline or amorphous carbonaceous core, with dimensions ranging from 0.5 to 10 nm, stochastically functionalized on the surface with a variety of polar functional groups (carboxylic, hydroxyl, amide, etc.). Thanks to the high polarity of their surface, CDs are stable in an aqueous environment and are highly biocompatible, as demonstrated by countless in vivo and in vitro studies.
  • CDs in terms of application are ascribable to their extraordinary optical properties, both in terms of fluorescence and induced near infrared (NIR) photothermal properties. In fact, they can emit visible light, and in particular in the biologically transparent area (600-900 nm), with an excellent quantum yield and, at the same time, they can be exploited to produce highly localized after stimulation with infrared light.
  • NIR near infrared
  • CDs have recently been the subject of intense study in the field of nano-medicine for the treatment of solid tumors, with particular reference to applications in theranostics.
  • they have been exploited as contrast agents in fluorescence imaging, combining this property with infrared laser- induced photothermal therapy.
  • it has already been demonstrated that it is possible to produce both red and NIR emitting CDs, in order to obtain high resolution images of tumors in mice xenograft models using fluorescence imaging techniques.
  • CDs have been shown to be potential near infrared conversion tools to produce local heat useful in tumor theranostics.
  • CDs seem very attractive for clinical applications that combine hyperthermia, imaging and drug delivery in a single platform capable of selectively targeting cancer cells.
  • CD-PEG-BTs consist of a passivated carbonaceous core with biotin-terminated PEG2000 chains.
  • irinotecan constitute the active target groups for recognizing cancer cells and are designed to efficiently incorporate a high amount of anticancer drugs such as irinotecan (16-28%) and to act as NIR light-activated nano-heaters capable of trigger local hyperthermia and massive drug release within tumors, thus causing efficient tumor death.
  • anticancer drugs such as irinotecan (16-28%)
  • NIR light-activated nano-heaters capable of trigger local hyperthermia and massive drug release within tumors, thus causing efficient tumor death.
  • the potential of irinotecan-loaded CD-PEG-BTs in fluorescence imaging was investigated on 2D cultures and complex 3D spheroids mimicking tumor architectures in vivo, showing their ability to selectively enter tumor cells via biotin receptors which are overexpressed in the cell membranes of cancer cells.
  • Hyaluronic acid-based nanogels and the related production process are described in patent application WO 2010/061005.
  • the need is also felt to have available a system capable of responding to changes in pH and remarking them in the pH range 5.5-7.4 to be used in the monitoring of healing processes in a non- invasive way, thus allowing to customize treatments for every type of patient.
  • the need is also felt to have particles available that are able to recognize tumor cells and accumulate inside them, allowing them to be monitored through the combination of different imaging techniques such as MRI and FL, so as to obtain information on the tumor microenvironment by means of higher resolution multimodal contrast techniques.
  • the need is also felt to have particles available that can be used simultaneously and in a modulable manner on the basis of clinical needs in the diagnosis and therapy of tumors through a personalized and precision approach, with the possibility of obtaining with a single drug both contrast properties in MRI and FL, the first useful in the case of diagnosis and monitoring of tumors on a millimeter scale and the second in the case of optical monitoring of tumor masses and small metastases even of a few micrometers, and therapeutic properties such as targeted chemotherapy (local drug delivery) and infrared phototherapy.
  • the invention aims to solve the technical problems highlighted by the known art.
  • an aim of the invention is the development of a contrast medium comprising a polymeric shell based on a hyaluronic acid nanogel and super-paramagnetic nanoparticles based on iron oxide (hereinafter referred to as SPIONs-Superparamagnetic Iron Oxide Nanoparticles).
  • the Cn chains are used to hydrophobize hyaluronic acid in such a way as to obtain a random copolymer that gives rise to the formation of thermo-sensitive hydrophobic pockets capable of incorporating hydrophobic drugs. This effect occurs when the alkyl chain is greater than Os.
  • the Cm chains serve as spacer of the alkynic group to subsequently crosslink the copolymer in the presence of the multifunctional CDs and SPIONs nanoparticles. Therefore the length of the chains Cm can be from C3 upwards, with the specifying that it would not in any case be better to exceed C22 because otherwise the material becomes too hydrophobic.
  • the nanosystem is characterized by hydrophobic bonds sensitive to changes in temperature and pH. Therefore, any pH variation that involves protonation/deprotonation of the hyaluronic acid derivative and any temperature variation that involves the formation/breaking of hydrophobic bonds of the hyaluronic acid derivative implies a variation in the swelling degree of the nanosystem in aqueous medium, accompanied by a variation of the average distances between the CDs (fluorescent) and SPIONs (magnetic) components, with consequent variation of the fluorescence and contrast properties in Magnetic Resonance (MRI).
  • CDs fluorescent
  • SPIONs magnetic
  • Another object of the invention is the development of a nanosystem to be used for the diagnosis and treatment of tumors, as well as for monitoring the pH of the tumor microenvironment using magnetic resonance (MRI) and fluorescence imaging (FL). Still another object of the invention is the development of a nanosystem capable of converting infrared light of a frequency between 750 to 900 nm (NIR) into local heat that can be directly used in image-guided photothermal antitumor therapy.
  • NIR infrared light
  • Still further object of the invention is the development of a nanosystem that allows the measurement of the temperature of the microenvironment in which a tumor mass is located (by tumor microenvironment we mean the whole complex of the tumor mass consisting of fibroblasts, macrophages, neutrophils, pericytes, extracellular fluid and extracellular matrix), by fluorescence imaging.
  • a further object of the invention is to obtain a nanosystem capable of monitoring local temperatures through variations in fluorescence intensity, thus allowing highly remotely controllable photoinduced hyperthermic therapies by means of non-invasive measurements with fluorescence imaging techniques.
  • a further object of the invention is the development of a process for the synthesis of the nanosystem defined above.
  • the process includes a mixing step of HA-DA-Cn,Cm, preferably HA-EDA-Cn,Cm, with the SPIONS-N3 and CDS-N3 nanoparticles and the subsequent chemical crosslinking between the polymer chains and the SPIONs- N3 and CDS-N3 nanoparticles via Huisgen azide-alkyne cycloaddition catalyzed with Cu (I) or thermally catalyzed without the use of metal catalysts.
  • intermediate compounds which, through the process of the invention, lead to obtaining the nanosystem of general formula HA-DA-Cn,Cm.
  • the intermediates are, in particular, HA functionalized with alkyne-terminated alkyl chains, the SPIONS-N3 and the CDS-N3.
  • compositions comprising the nanosystem for theranostic uses in the tumor field, both for the treatment of tumors and of relapses and metastases.
  • Figure 1 shows the emission spectrum (dotted line) of a dispersion of CDs (0.1 mg/ml) in water and in the presence of SPIONs (0.1 mg/ml) and the emission spectrum (solid line) of the covalent conjugate CDs- PEG2k-SPIONs. It is noted that after chemical conjugation of the CDs with the SPIONs at a binding distance below 4 nm, the CDs no longer emit light from 600 to 750 nm;
  • Figure 2 shows the IR spectrum of the nanosystem of the invention, crosslinked by azide-alkyne cycloaddition compared with the compound FIA-EDA-Ci8,C5.
  • the disappearance of the triple bonds underlines that the conversion of the Cs alkynic groups into 1 ,2,3, - triazole is complete, underlining that the functionalization is efficient and exhaustive;
  • Figure 3 shows the morphological, structural characterization and diameter distribution of the FIA-Ci8,C5 nanosystem crosslinked with CDS-N3 and SPIONS-N3 obtained by high resolution transmission electron microscopy-FIR TEM (a-a"), atomic force microscopy-AFM
  • Figure 4 shows the variation of the average volume of the nanosystem of the invention as the temperature (a) and the pH of the medium (b) change. As can be clearly seen, the dimensions of the nanosystem increase with increasing temperature and pH;
  • Figure 5 shows the dependence of fluorescence on the pH (a) and temperature (b) of the sample: the insert also shows the quantum yield as the temperature changes;
  • FIG. 6 shows the dependence of Ti (a) and T2 (b) on pH and concentration
  • Figure 7 shows the confocal microscopy measurements of HDFa/MDA-MB-231 organoid treated with the nanosystem of the invention at a concentration of 0.25 mg/ml for 2 and 24 hours.
  • Red fluorescence a-b
  • nuclei with DAPI a'-b'
  • overlap a”- b
  • Figure 8 illustrates the cell vitality of HDFa/MDA-MB-231 organoids treated with the nanosystem of the invention at the concentration of 0.25 mg/ml for 24 hours and followed by photothermal treatment with diode laser at 810 nm for 1 , 2 or 3 consecutive treatment cycles. Temperature of the photothermally treated wells. It is possible to see that vitality decreases as the reached temperature increases as a direct effect of the hypersensitivity of cancer cells to high temperatures. At temperatures over 60 degrees, total photothermal ablation of the organoid is achieved;
  • Figure 9 illustrates Scheme 1. of the synthesis of the amphiphilic derivative of hyaluronic acid HA-EDA-C18-C5 (it is the precursor that is used to make the nanosystem);
  • Figure 10 illustrates Scheme 2. of the synthesis of CDs functionalized with surface azide groups;
  • Figure 11 illustrates Scheme 3. of the synthesis of SPIONs functionalized with surface azide groups
  • Figure 12 illustrates Scheme 4. of the synthesis of the nanosystem obtained through 1 ,3-dipolar azide-alkyne cycloaddition between the HA-EDA-C18-C5, CDS-N3 and SPIONS-N3 derivatives: the reaction takes place between the alkynic and azide groups selectively, quickly and comprehensively.
  • nanosystem refers to the compound of the invention, formed by an amphiphilic derivative of hyaluronic acid HA, crosslinked with superparamagnetic iron oxide nanoparticles (SPIONS-N3) and carbon nanoparticles (CDs- N3), both functionalized on the surface with azide groups;
  • HA means a hyaluronic acid with a weight average molecular weight between 5,000 and 500,000 Da;
  • HA-DA-Cn,Cm identifies a derivative of hyaluronic acid containing side chains deriving from the combination with a diamine (DA) and HA- EDA-Cn,Cm identifies a derivative of hyaluronic acid containing side chains deriving from combination with ethylenediamine (EDA);
  • DA diamine
  • EDA ethylenediamine
  • alkyl indicates, unless otherwise specified, a fully saturated straight, branched or cyclic hydrocarbon radical or a combination of radicals having the designated number of carbon atoms (for instance, Cm indicates an alkyl chain C3-C22 with from 3 to 22 carbon atoms, extremes included).
  • alkyl groups include, without limitation, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)-ethyl, cyclopropyl-methyl and homologs and their isomers, for instance, n-pentyl, n-hexyl, n- heptyl, n-octyl and the like;
  • alkylene indicates a divalent radical derived from an alkyl group, as exemplified by -CH2CH2CH2CH2-;
  • CDs carbon nanodots with a crystalline structure based on carbon in which carbon atoms are occasionally replaced with nitrogen atoms, with emission in the range 500-700 nm and with dimensions between 1 and 8 nm (ACS Appl . Mater. Interfaces 2019, 11 , 22, 19854- 19866; ACS Applied Nano Materials 2020, 3, 7, 6925-6934);
  • SPIONs refers to superparamagnetic nanoparticles of iron oxide with a diameter between 5 and 45 nm and with surface carboxyl groups (Product commercially available at MERCK n. 747335-NACRES NA. 23);
  • CDS-N3 we mean carbon nanodots deriving from the CDs described above and functionalized with surface azide groups as exemplified in scheme 2 of figure 10;
  • SPIONS-N3 we mean superparamagnetic nanoparticles of iron oxide deriving from the SPIONs and functionalized with surface azide groups as exemplified in scheme 3 of figure 11 ;
  • MRI and FL we mean the ability of the nanoparticles of the invention to modify the way in which the aqueous medium in which they are dispersed appears in a medical magnetic resonance image (MRI) and in a fluorescence image (FLI). Particularly, this property allows to change the way in which the region in which the nanoparticles accumulate appears in a medical image, thus allowing the study of changes in an organ, lesion or tissue with respect to what surrounds them;
  • MRI and FL we mean the ability of the nanoparticles of the invention to modify the contrast properties (accentuating or attenuating it) by virtue of their presence in the dispersing medium (for instance the pH);
  • photothermal agent for image-guided phototherapy generally refers to a nanosystem or a molecule capable of simultaneously generating heat when excited with an infrared laser, so as to allow local temperatures above 41 °C to be reached, and to emit light between 500 and 1000 nm when excited with a light source of appropriate wavelength (between 500 and 900 nm).
  • the nanoparticles of the invention have the properties to be defined as photothermal agents for image-guided phototherapy;
  • the term ’’patient means a human or animal subject who obtains an improvement in his conditions when the nanosystem of the invention is administered to him, in particular the human or animal subject is a mammal, more particularly it is a human subject of any age, including the elderly, children and infants.
  • the present invention aims to provide a new method for producing nanosystems based on HA, SPIONs and CDs that can be used directly for the diagnosis and treatment of tumors, as well as for monitoring the pH of the tumor microenvironment by magnetic resonance (MRI) and fluorescence imaging (FL).
  • MRI magnetic resonance
  • FL fluorescence imaging
  • a further object of the present invention is to obtain a nanosystem capable of converting infrared light of frequency between 750 and 900 nm (NIR) into local heat directly usable in image-guided photothermal antitumor therapy.
  • the nanosystem object of the present invention is designed to allow the measurement of the temperature of the tumor microenvironment, by means of fluorescence imaging, by tumor microenvironment meaning the entire complex of the tumor mass which comprises fibroblasts, macrophages, neutrophils, pericytes, extracellular fluid and extracellular matrix.
  • the nanosystem consists of an amphiphilic HA derivative, obtained by covalent crosslinking in a polar solvent, both organic and aqueous, through a functionalization process with multifunctional SPIONs and CDs, as will be explained in detail below.
  • FL fluorescence intensity
  • the nanosystem of the invention is designed to respond to light stimuli (such as for instance diode lasers with a wavelength that can range from 700 to 900 nm or LEDs or other similar light sources) by emitting local heat, thus generating hyperthermia (typically 43-50°C) at the tumor site.
  • light stimuli such as for instance diode lasers with a wavelength that can range from 700 to 900 nm or LEDs or other similar light sources
  • hyperthermia typically 43-50°C
  • This process can be used to eliminate tumor masses usually hypersensitive to localized heat increases (Hyperthermia: Cancer Treatment and Beyond Intech Open, DOI: 10.5772/55795).
  • the present invention also aims to allow the monitoring of local temperatures by means of variations in the fluorescence intensity, thus allowing highly remotely controllable photoinduced hyperthermic therapies by means of non-invasive measurements with fluorescence imaging techniques.
  • the nanosystem contains hydrophobic chains that tend to form thermoreversible Van der Waals bonds between them.
  • the methodology for preparing the nanosystems of the invention is based on a four-step process, wherein:
  • the first step consists in the production of an amphiphilic HA derivative carrying a mixture of Cn and Cm alkyl chains both saturated and with unsaturated end groups of the alkynic type, to give a polymeric intermediate which is able to crosslink by means of 1 ,3-dipolar azide-alkyne cycloaddition,
  • the second step consists in the preparation of SPIONs functionalized on the surface with azide groups, so as to allow the crosslinking of the polymer obtained in the first step by means of
  • the third step consists in the synthesis of surface functionalized CDs with azide groups so as to allow the crosslinking of the polymeric derivative obtained in the first step without however reacting directly with the SPIONs components obtained in the second step,
  • the products obtained in the previous steps are mixed in the presence of Cu(l) (typically 10% w/w) or at temperatures between 60 and 80°C for 2-6 hours, leading to the formation of crosslinked nanosystems wherein the CDs and SPIONs components are indirectly interconnected through the HA derivative prepared in the first step.
  • Cu(l) typically 10% w/w
  • temperatures between 60 and 80°C for 2-6 hours leading to the formation of crosslinked nanosystems wherein the CDs and SPIONs components are indirectly interconnected through the HA derivative prepared in the first step.
  • every pH variation that involves protonation/deprotonation of the HA derivative and every temperature variation that involves the formation/breaking of hydrophobic bonds of the HA derivative implies a variation in the swelling degree of the nanosystem in aqueous medium accompanied by a variation of the average distances between the CDs (fluorescent) and SPIONs (magnetic) components, with consequent variation of the fluorescence and contrast properties in MRI.
  • TAA tetrabutylammonium bromide
  • the activation step can be obtained in solvents such as dimethyl sulfoxide, dimethylformamide, dimethylacetamide and mixtures thereof at temperatures between 10 and 60°C.
  • the reaction can be carried out for a period of between 2 and 6 hours.
  • the functionalization degree whose control is within the reach of the skilled in the art, depends on the reaction time and stoichiometry of the activated HA derivative, the amount of carbonatant used, the reaction time and the temperature.
  • the functionalization degree is between 10 and 95%, much more preferable is the range between 30 and 70%.
  • nitrophenoxycarbonyl leaving group (N02-Ph-0-) is replaced with a mixture of nucleophilic groups, typically bearing NH2 end groups, of general formula CnNH2, and CmNH2 where Cn and Cm have the above mentioned meanings.
  • nucleophilic groups typically bearing NH2 end groups, of general formula CnNH2, and CmNH2 where Cn and Cm have the above mentioned meanings.
  • both monoamines are covalently bonded to the HA by urethane bond and the functionalization degree depends on the amount of amines used, the temperature and the reaction time, conditions that are easily managed by the person skilled in the art.
  • the functionalization degree in CnNH2 is comprised between 30 and 65% mol/mol with respect to the total repetitive units of HA and in CmNH2 it is comprised between 2 and 30% mol/mol.
  • the nucleophilic substitution reaction between the diamine of general structure NH2-RVNH2 and the nitrophenoxycarbonyl groups is carried out at a temperature not exceeding 40°C and using a large excess of diamine to avoid crosslinking of the copolymer following the reaction of both sides of the diamine. In fact, in this reaction the aim is to ensure that one of the two amino groups remains primary and available for further eventual side chain reactions.
  • DA diamine
  • an ion exchange is carried out between the TBA and a suitable cation, such as an alkali or alkaline- earth metal cation, by adding a saturated solution of a salt, such as sodium or potassium chloride, to the copolymer solution and removing the product by precipitation in a suitable mixture of non solvents, typically diethyl-ether/chloroform 1 :1 .
  • a suitable cation such as an alkali or alkaline- earth metal cation
  • the final product is a block copolymer consisting of the following blocks (repeating units): (a) hyaluronic acid unit (D-glucuronic acid subunit and N- acetylglucosamine subunit)
  • the stoichiometric ratio between the repetitive units (a), (b), (c), (d) of HA is equal to:
  • the product obtained in the first step has the general formula HA- DA-Cn,Cm.
  • SPIONs superparamagnetic iron oxide nanoparticles
  • SPIONs superparamagnetic iron oxide nanoparticles
  • MERCK n. 747335- NACRES NA. 23 which are functionalized to contain azide surface groups.
  • SPIONS-N3 superparamagnetic nanoparticles can be obtained by functionalization processes of commercial SPIONs by functionalization of the reactive groups possibly present in commercial SPIONs (including alcohols, carboxylic acids, amines or thiols) and the covalent coupling with heterobifunctional molecules carrying an azide group and a reactive group capable of reacting with the functions present on the selected commercial SPIONs (amine, alkene, acrylamide, thiol, carboxylic acid) by activation with carbodiimides (DCC, EDC, etc.) and N-hydroxy-succinimide (NHS) or similar activators in aqueous solvents (Po-Chiao Lin et al. Surface
  • SPIONs-COOH commercial precursors of SPIONs bearing surface carboxylic groups
  • carbodiimides for instance N,N'-dicyclohexylcarbodiimide- carbodiimide-DCC, 1 -Ethyl-3- (3-dimethylaminopropyl) carbodiimide-EDC, etc.
  • NHS N-hydroxy- succinimide
  • the functionalization degree in azide groups depends on the stoichiometric ratio between the SPIONs and the NH2-R y ”’-N3 derivative used.
  • a mol/mol ratio is used between the amines present in the NH2-R y ”’-N3 derivative and the carboxylic groups contained on the SPIONs and ranging from 0.4 to 2, in order to obtain a degree of surface functionalization ranging from 35 to 100% compared to the carboxy groups originally present on SPIONs.
  • the quantity of surface azide groups determined potentiometrically by titration of the residual carboxy groups following the functionalization processes with the functional group NH2-R y ”’-N3 as described in Example 1 , can be modulated by varying the amount of amine used and the reaction time and can vary from 0.25 to 2.10 meq/mg of product.
  • step 2 is schematically represented in Scheme 2 of Figure 10.
  • the CDs are functionalized with terminal azide groups and in the present invention the CDS-N3 thus obtained are used: - as contrast agents in FL imaging, considering that they emit light in the biologically transparent window (600-1100 nm) and therefore can allow obtaining in vivo images, and
  • hypertemic agents capable of generating local heat once excited, for instance with an 810 nm diode laser with power between 2 and 14 W/cm 2 (Scialabba et al., Highly Homogeneous
  • Biotinylated Carbon Nanodots Red-Emitting Nanoheaters as Theranostic Agents toward Precision Cancer Drug, ACS Applied Materials & Interfaces, 2019 Jun 5; 11 (22): 19854-19866).
  • the third step involves the preparation of already known carbon nanoparticles (Scialabba et al., Highly Homogeneous Biotinylated
  • Carbon Nanodots Red-Emitting Nanoheaters as Theranostic Agents toward Precision Cancer Drug, ACS Applied Materials & Interfaces, 2019 Jun 5; 11 (22) : 19854-19866), called carbon nanodots (CDs), with a diameter that can be from 1.5 to 10 nm and with fluorescence between 500 and 750 nm and ability to convert
  • NIR light 700-900 nm
  • heat photothermal capacity
  • CDs can be prepared by solvothermal reaction by mixing citric acid and urea in polar solvents (preferably dimethylformamide or dimethyl sulfoxide) and using operating pressures between 8 and 100 bar and temperatures between 160 and 300°C (Scialabba et al., Highly Homogeneous Biotinylated Carbon Nanodots: Red-Emitting Nanoheaters as Theranostic Agents toward Precision Cancer Drug, ACS Applied Materials & Interfaces, 2019 Jun 5; 11 (22): 19854-19866).
  • polar solvents preferably dimethylformamide or dimethyl sulfoxide
  • CDs are obtained with a crystalline core of the b- C3N4 type, which can be excited from 400 to 600 nm and which emits red light from 610 to 750 nm and which absorbs light in the near infrared (NIR) transforming it into heat, characterized by the presence of surface carboxy, alcoholic and amino functions, which can be used for further surface functionalization (Scialabba et al., Highly Homogeneous Biotinylated Carbon Nanodots: Red-Emitting Nanoheaters as Theranostic Agents toward Precision Cancer Drug,
  • carbodiimides dicyclohexylurea-DCC or N'- ethylcarbodiimide hydrochloride-EDC, etc.
  • NHS N-hydroxy- succinimide
  • the nucleophilic activation and substitution reaction is performed in situ by dispersing the CDs in aqueous solvents at a concentration between 0.1 and 10 mg/ml and a pH between 5.5 and 7.5 for a reaction time between 4 and 24 hours.
  • the functionalization degree in azide groups depends on the stoichiometric ratio between the NH2-R'-N 3 derivative used and the carboxylic groups present on the CDs, preferably between 3 and 0.5, the pH of the reaction and the reaction time.
  • the derivative with a functionalization degree in azide groups ranging from 70 to 100%, obtained with a stoichiometric ratio between 1 and 2, at a pH preferably between 6.4 and 6.8 and for a reaction time between 6 and 24 hours is preferred.
  • step 3 is schematically represented in Scheme 3 of Figure 11.
  • step (1 ) H-DA-Cn,Cm
  • step (2) SPIONs-N 3
  • step (3) CDs-Na
  • the mixing is generally carried out in a composition by weight ratio which varies respectively from 80 to 95%, from 0.1 to 5% and from 0.1 to 5%
  • a purplish colloidal dispersion is obtained in a mixture of ultrapure water and tetrahydrofuran, preferably 80:20, at a concentration ranging from
  • the dispersion of the three components is then reacted in the presence of Cu(l) (from 1 to 20% w/w), directly added as cuprous salt (for instance cuprous halide, typically CuBr) or generated in situ by adding copper sulphate and a reducing agent, such as ascorbic acid, in excess, for 2-6 hours, in an inert atmosphere by blowing nitrogen or argon or other inert gas, leading to the formation of crosslinked nanosystems in which the components CDS-N3 and SPIONS-N3 are indirectly interconnected through the HA-DA-Cn,Cm derivative by 1-2-3 triazole bond.
  • Cu(l) from 1 to 20% w/w
  • cuprous salt for instance cuprous halide, typically CuBr
  • a reducing agent such as ascorbic acid
  • step 4 the same reaction, although not preferable due to the absence of regioselectivity and the formation of partially degraded nanosystems, can be done in the absence of cuprous catalyst, but thermally catalyzed at temperatures between 60 and 80°C.
  • the CDS-N3 and SPIONS-N3 nanoparticles do not have the possibility of forming reciprocal covalent bonds, since these would turn off the intrinsic fluorescence of the CDs and would not allow for pFI-dependent fluorescence mechanisms.
  • CDS-N3 from 0.1 to 5% w/w with respect to the total of components
  • SPIONS-N3 from 0.1 to
  • a composition ratio expressed as a weight ratio between the CDS-N3 and the SPIONs- N3, which can range from 0.1 to 0.4, as the azide groups of the same nature present on the nanoparticles can react with the alkyne groups present on the hyaluronic acid HA derivative-DA-Cn,Cm (obtained in step 1) only if activated at high temperatures (T> 60°C) or by Cu(l) ions.
  • alkyne groups of HA-DA-Cn,Cm are capable of reacting regioselectively with the surfaces of the nanoparticles dispersed therein only under the conditions indicated above.
  • nanosystems consisting of the amphiphilic HA derivative crosslinked with superparamagnetic nanoparticles of iron oxide (SPIONS-N3) and carbon nanoparticles (CDS-N3) through bonds of the 1 ,3-triazole type obtained by a reaction catalyzed by Cu (I) of the triple bonds -CoC- present on the hyaluronic acid chain with the azide groups of SPIONS-N3 and CDS-N3.
  • This synthetic process is very efficient and allows a fast and thorough conversion of over 99% of the groups involved in the crosslinking ( Figure 2).
  • the final product obtained is a nanogel that can be used for pharmacological uses, as indicated below. More particularly, the process comprises the mixing of HA-DA-Cn,Cm with the SPIONS-N3 and CDS-N3 nanoparticles and the subsequent chemical crosslinking of the polymer chains and nanoparticles by means of Huisgen azide-alkyne cycloaddition catalyzed with Cu(l) (Huisgen, R. (1961 ). ’’Centenary Lecture-1 ,3-Dipolar Cycloadditions". Proceedings of the Chemical Society of London: 357. doi:
  • Nanosystems with an average diameter of about 95 nm are obtained (Figure 3), as can be seen from the AFM (atomic force microscopy) and DLS (dynamic light scattering) measurements.
  • Nanosystems with different average diameters can be obtained by simply increasing or decreasing the concentration of the reaction mixture consisting of HA-DA-Cn,Cm (from 90 to 95% w/w on the total of the components), SPIONS-N3 (from 1 to 5% w/w on the total of the components) and CDS-N3 (from 1 to 5% w/w on the total of the components).
  • the reaction mixture can have a concentration ranging from 0.1 to 10 mg/ml and the average diameter of the nanosystem increases with increasing concentration.
  • the average diameter of the nanosystem can be modulated by subjecting the reaction mixture to sonication cycles after the addition of the catalyst.
  • Nanosystems with an average diameter between 80 and 120 nm can be obtained by modulating the sonication cycles during the reaction from 5 cycles of 5 seconds per minute to 10 cycles of 5 seconds per minute, obtaining smaller nanosystems with increasing cycles, as known to the expert in the field.
  • step 4 is schematically represented in Scheme 4 of Figure 12.
  • the CDs are also used as pH sensors, considering that when the CDs are placed in close contact (distance between 1 and 3 nm) with magnetic nanoparticles such as SPIONs they undergo a significant reduction in fluorescence at 600-750 nm ( Figure 1 ).
  • Figure 1 it is possible to trap the two nanoparticles in a pFI-sensitive polymeric network that changes conformation by virtue of changes in pH in the range 5-7.4 and which therefore determines an approach or separation of the two nanoparticles as a function of pH, with consequent variation of the fluorescence intensity of the signal.
  • amphiphilic HA derivative it is possible to obtain a nanosystem that varies the conformation of the polymeric network depending on the temperatures in the range 37-45°C, with the possibility of collapsing or swelling the nanostructure by varying the temperature, determining a decrease or increase in the fluorescence signal respectively.
  • nanosystems have a variable iron content ranging from 2 to 4% w/w, which can be calculated by means of a colorimetric assay (red-alizarin).
  • red-alizarin a colorimetric assay
  • the nanosystem is made of, it is capable of responding to changes in pH by increasing the hydrodynamic volume as the pH increases.
  • the carboxyl groups of the polymeric network are completely deprotonated at physiological pH (7.4), allowing greater electrostatic repulsion and swelling of the nanosystem thanks to the recall of water from the external environment.
  • weakly acidic pH 5.5
  • they are partially protonated, causing the expulsion of water, the formation of hydrophobic domains and the collapse of the structure.
  • the result is that the volume of the nanosystem decreases approximately 11 times at a weakly acidic pH, typical of the tumor microenvironment ( Figure 4b).
  • the nanosystem also responds to temperature variations, decreasing its volume by about 3 times when heated from 37°C to 45°C ( Figure 4a)
  • the variation in the swelling degree of the nanosystem as a function of the temperature and pH of the microenvironment causes significant changes in the fluorescence intensity and contrast properties in MRI of the nanosystem. More particularly, the quantum yield (QY) of the nanosystem, measured by comparing the emission of a solution of CDs in water at pH 5.5 or 7.4 with absorption equal to 0.2 and a reference solution of 6G rhodamine at pH 13 with the same absorption (Julien Laverdant et al., Experimental Determination of the Fluorescence
  • Quantum Yield of Semiconductor Nanocrystals ranges from 2% at pH 5.5 to 11.7% at pH 7.4, highlighting the possibility of using this nanosystem as a pH sensor ( Figure 5a).
  • the diameter variation of the nanosystem caused by heating is at the basis of the phenomenon of fluorescence quenching observed shifting from 43 to 25°C, where the QY shifts from 19 to 13%, underlining that the nanosystem can also be used as a temperature sensor ( Figure 5b).
  • the same can be used to obtain information on the local temperature following the heating of the nanosystem with a diode laser with a wavelength between 750 and 900 nm.
  • the nanosystem object of the present invention can be formulated both as an injectable preparation and as tablets, capsules or a preparation for topical use.
  • formulations for parenteral use both intramuscular and bolus
  • isotonizing agents such as sodium chloride or glucose, pH 7.4 phosphate buffer and the appropriate quantity of nanosystem dispersed at the colloidal level.
  • the lyophilized formulation can be obtained together with the quantity of water for injectable preparations useful for reconstituting the colloidal suspension.
  • a suitable antimicrobial for multidose preparations it is appropriate to introduce a suitable antimicrobial to be chosen from sodium metabisulphite, phenol, cresol, methyl p-hydroxybenzoate, benzyl alcohol or other similar.
  • the nanosystem can also be formulated as capsules and tablets for oral administration. In the case of tablets, the appropriate amount of nanosystem can be diluted in a suitable diluent powder such as calcium carbonate or microcrystalline cellulose, glidants (0.5-3%) such as magnesium stearate or talc, disaggregating agents such as corn starch (5-10%) and super-disintegrants such as croscarmellose sodium (1 -2%).
  • the lyophilized nanosystem can be added to a diluent such as corn starch in the presence of a gliding agent such as talc (1 -4%) and the solid formulation can be used to fill the hard gelatin capsules.
  • the nanosystem can also be formulated as soft capsules containing a gel or sol in which the nanosystem is dispersed. In the latter case, the nanosystem is dispersed in purified water at the appropriate concentration and gelling agents such as hydroxyethylcellulose (1 - 5%) or carbopol (1 -4%) are added in the presence of a suitable base such as sodium hydroxide; the resulting gel can be used as a filling of soft gelatin-based capsules.
  • the nanosystem can be formulated as an emulsion, suspension or gel for topical applications, such as in the case of skin cancer.
  • the nanosystem object of the present invention is versatile and can be used in the medical field for different types of applications, both in the diagnostic and in the therapeutic field. More particularly, the nanosystem can be used as a contrast agent in MRI for the evaluation of cancerous lesions and for monitoring the progress of the disease, both following a therapeutic treatment and during patient follow-up.
  • the injectable formulation should be injected to the patient during the MRI scan to allow the acquisition of MRI sequences with a contrast different from the normal one that allows the visualization of lesions and anatomical-pathological details not easily visible in the absence of a contrast agent.
  • a second application area is the possibility of using the nanosystem as a photothermal agent for the photothermal ablation of solid tumors.
  • the nanosystem can be injected and then the tumor selectively eradicated by applying an infrared laser (700-1100 nm). Tumor overheating occurs selectively because nanosystems are able to recognize tumor cells and usually accumulate in tumor tissues.
  • the physician decides where to direct the laser source via an optical fiber and therefore the intervention is by its nature highly selective.
  • it is possible to combine photothermal and diagnostic action by operating a photothermal ablation guided by MR images.
  • the physician decides where to apply the laser source in a non- invasive way through the images produced online by an MRI device for clinical use.
  • SPIONs can be used to increase the production of toxic reactive oxygen species (ROS) in tumor cells (C. Janko et al. Functionalized Superparamagnetic Iron Oxide Nanoparticles (SPIONs) as Platform for the Targeted Multimodal Tumor Therapy. Front Oncol . 2019; 9:59).
  • ROS toxic reactive oxygen species
  • SPIONs Functionalized Superparamagnetic Iron Oxide Nanoparticles
  • the hyperthermia that can be achieved thanks to the use of SPIONs can allow to increase the efficacy of radiotherapy in poorly perfused tumor tissues (Chatterjee DK et al., Nanoparticle- mediated hyperthermia in cancer therapy. Ther Deliv. (2011 ) 2: 1001-14).
  • the nanosystem can incorporate anticancer drugs to release them at the site of action following its heating by means of an infrared optical fiber (Scialabba et al., Highly Homogeneous Biotinylated Carbon Nanodots: Red- Emitting Nanoheaters as Theranostic Agents toward Precision Cancer Drug, ACS Applied Materials & Interfaces, 2019 Jun 5; 11 (22): 19854-19866).
  • the nanosystem object of the invention has distinctive features not present in the literature and which are highlighted here:
  • the nanosystem object of the present invention is considered a new chemical entity, consisting of a biopolymer (based on functionalized hyaluronic acid) crosslinked through covalent bonds with carbon-based crosslinkers (CDs) and iron oxide nanoparticles (SPIONs), which can be used in diagnostics and photothermal therapy.
  • a biopolymer based on functionalized hyaluronic acid
  • CDs carbon-based crosslinkers
  • SPIONs iron oxide nanoparticles
  • the red fluorescence intensity and pH sensitivity, the ability to transform near infrared (NIR) light into heat, and the magnetic resonance contrast properties of the nanosystem are not the mere sum of the individual properties of the single components, but they are more pronounced (the QY of CDs alone is 4% and is not pH dependent, as reported in the literature (Scialabba C. et al. ACS Applied Material & Inter. 2019), and the QY of CDS-N3 alone is 2%).
  • the contrast properties in magnetic resonance imaging (MRI) of the nanosystem allow to measure pH changes in the range 7.4-5 allowing to monitor a pH range quite comparable to that observed in the tumor microenvironment during the healing process (Arig Bennett Hashim et al., NMR Biomed. 2011 ; 24: 582-591).
  • MRI magnetic resonance imaging
  • Calibration is a method within the reach of the skilled in the art and can be carried out by making measurements of the intensity of fluorescence and/or RM signal as the pH varies and then plotting the data in a spreadsheet. At this point, having a correlation coefficient between fluorescence or RM signal and pH, the pH can be measured from any measurement of fluorescence and/or RM. 4.
  • the nanosystem allows for the combination of MR imaging and MR guided NIR-induced photothermal ablation. In principle, there are already tools that allow you to do MRI Guided Photothermal Therapy.
  • Medtronic's Visualase Thermal Therapy System https://www.medtronic.com/en- qb/oper3 ⁇ 4torj-s3 ⁇ 4nit3 ⁇ 4ri/product$/neuroiogical/i3 ⁇ 4ser- ablation/visualase.htmj
  • This system can be used following the systemic or topical administration of the nanosystem and after verifying the effective biodistribution in the tumor site.
  • instruments that are already widely used, such as: 1 ) MRI for clinical use, 2) guided optical fiber with catheter to arrive laparoscopically up to the tumor site.
  • the optical fiber can be used by applying it directly to the tumor site.
  • the use of an endoscope is necessary.
  • the nanosystem object of the present invention is to be understood as a nanocomposite structure of nanometric dimensions (80-180 nm) ( «100 nm) comprising or consisting of an amphiphilic polymeric matrix, a charge of super-paramagnetic nanoparticles (SPIONs), with magnetic targeting and contrast properties in magnetic resonance (MRI), and a charge of fluorescent carbon nanoparticles (CDs), with photothermal and photoluminescence (FL) properties, covalently interconnected.
  • SPIONs super-paramagnetic nanoparticles
  • MRI magnetic targeting and contrast properties in magnetic resonance
  • CDs fluorescent carbon nanoparticles
  • FL photothermal and photoluminescence
  • the nanosystem has been rationalized to group in a single three- dimensional structure of nanometric dimensions, contrast and molecular sensing properties in MRI and FL, useful for the identification and anatomopathological characterization of tumor masses in a non-invasive way, and photothermal and release properties controlled and selective (by selective we mean that it is released locally in the tumor mass and therefore avoiding the phenomena of toxicity in tissues not to be treated and highly perfused organs).
  • the trigger for the release is the excitation with a laser from 700 to 900 nm, that can be performed on demand by the physician only at the site of action of drugs for the eradication of tumor masses guided by images. Therefore, the nanosystem of the invention can be used in therapy, in particular for the treatment of tumors, lymph nodes and related metastases and relapses.
  • the nanosystem object of the invention is made up of:
  • a pH/thermo-sensitive polymeric matrix based on a semi-synthetic amphiphilic polymer derivative, obtained through a chemical crosslinking process POLYMER NETWORK.
  • the Polymer Network has a dual function, as well as being chosen for its high biocompatibility and because it is typically recognized by tumor cells that overexpress the CD44 receptor (active targeting) (George Mattheolabakis et al., Hyaluronic acid targeting of CD44 for cancer therapy: from receptor biology to nanodrug, J Drug Target 2015; 23 (7-8): 605-18), is able to modify its three-dimensional conformation depending on external stimuli such as: pH, temperature, ionic strength, etc..
  • SPIONs super-paramagnetic nanoparticles
  • Polymeric Network acts as a crosslinker and contrast agent in magnetic resonance (MRI), obtained through surface coupling with common activating agents (carbonyl diimidazole, 1 -ethyl-3(3-dimethylaminopropyl) carbodiimide, N- hydroxysuccinimide and others similar, which are activating groups (leaving groups) which then will not form part of the structure;
  • CDs carbon-based nanoparticles
  • FLI fluorescence imaging
  • photothermal agent for image-guided phototherapy, obtained through solvolitic and surface coupling through common activating agents (carbonyl diimidazole, EDC, NHS, and related reagents);
  • the magnetic and fluorescence properties of the nanostructures present in the nanosystem are strongly influenced by the average distance between them. More in detail, the fluorescence of the CDs is prevented when it is at an average distance from the SPIONs below 0.8 nm while it progressively increases above this distance. For instance, conjugating the SPIONs directly to the CDs via a PEG2000 spacer, typically 2.5 nm in diameter, turns off the fluorescence. Therefore, any variation of the microenvironment in which the particles of the nanosystem of the invention are dispersed that changes this parameter, modifies its contrast properties both in FL and in MRI.
  • the Polymeric Network makes it possible to respond to variations in pH and temperature, determining a significant variation in hydrodynamic volume which, as a final consequence, determines a variation in the average particle-particle bond distances (above we quantified the variation in hydrodynamic volume, it is not possible to make precise estimates of the particle-to-particle distance in the medium) thus inducing detectable modifications of the contrast properties.
  • the nanosystem can be used as a multimodal pH sensor to detect changes in the tumor microenvironment in response to a specific therapeutic treatment, thus allowing the personalization of the therapy based on the individual responses of each single patient.
  • the pH of the tumor mass tends to increase (from pH 6 to pH 7.4) in response to an optimal therapeutic effect.
  • CDs in the hybrid nanosystem also confers photothermal properties, allowing the transformation of NIR light into local heat, which can be exploited for the thermal eradication of tumor masses or for the on-demand delivery of anticancer drugs directly in the site of action.
  • This property is also strongly influenced by the pH of the microenvironment in which the superstructure is located, improving the photothermal performance in more typically tumor microenvironments (pH 5.5). This guarantees greater selectivity of action and fewer unwanted side effects.
  • SPIONs allow to obtain MR images of small areas and to monitor the pH of the medium in which they are dispersed, on the other hand it allows magnetic targeting following the application of an applied external static magnetic field in the tumor mass, thus increasing the bioavailability of the nanodrug at the site of action.
  • the combination of fluorescence and magnetic resonance signals could be used for selective photothermal ablation of tumor mass and metastases and relapses using image-guided therapeutic procedures.
  • the extemporaneous identification of the tumor masses through magnetic resonance and fluorescence can be exploited to eradicate the tumor masses during the therapeutic treatment by applying an infrared laser, which is able to heat the nanodrug present inside the mass to be treated by exploiting its photothermal properties.
  • the super-structure object of the present invention is also able to respond to thermal stimuli, emitting more photons as the temperature increases.
  • the temperature increase can therefore be monitored using fluorescence imaging techniques. Therefore, this property, together with the contrast and photothermal properties, can be exploited in image-guided applications of solid tumors photothermal ablation as a sensor to reach desired temperatures at the site of action.
  • the distinctive feature of the nanosystem object of the present invention is that the combination of the contrast properties in RM and FL, of the photothermal and magnetic properties listed above is not present in any of the single isolated components that make up the nanosystem itself. To date, according to the inventors, a nanosystem with similar characteristics is not known.
  • the device proposed in this invention can allow to identify changes in the pH of the tumor microenvironment both with fluorescence imaging techniques and with magnetic resonance imaging in different acquisition modalities.
  • the advantage of the present invention is to have a nanosystem capable of 1) accumulating magnetically and actively in the action site, 2) acting as a contrast and sensing agent in magnetic resonance and fluorescence, 3) causing the death of tumor cells photothermally, 4) releasing large quantities of anticancer drugs in situ and as needed.
  • the components that make up the present invention are all bio- eliminable as they are or after degradation.
  • Other similar devices such as colloidal gold nanoparticles or carbon nanotubes, as well as not having all the functions described here, are instead designed in such a way as to allow only the photothermal eradication of the tumor mass (without magnetic accumulation, without multimodal pH/thermo sensitive imaging).
  • these nanoparticles are not biodegradable, as they are chemically inert, and are not bioeliminable due to their size above the renal excretion cut-off (> 5 nm), effectively constituting a limit in biomedical applications.
  • the innovation represented by the nanosystem object of the present invention lies in the possibility of inducing the release of cytotoxic heat in situ and possibly of anticancer drugs and, at the same time, of verifying their therapeutic efficacy instrumentally in a non-invasive way and in real time.
  • This treatment could be easily conducted in vivo in any surgically accessible or highly perfused site, without inducing adverse effects due to off-target treatment.
  • the nanosystem proposed in the present invention represents a significant advancement of the state of the art, considering that it allows to integrate the properties of CDs in a versatile and multifunctional nanosystem that allows to respond to small variations in pH and temperature in such a way as to monitor the variations of the microenvironment in which they are dispersed.
  • CDs and SPIONs suitably functionalized on the surface, and we used them as building blocks of a stimulus-sensitive superstructure capable of modifying the optical and magnetic properties by virtue of the pH in which it is. In this way it is possible to instrumentally verify (in a non-invasive way), both by means of MRI and fluorescence, the pH of the medium in which they are.
  • the molecular architecture of the super-structures obtained is designed to amplify the sensitivity of the CDs towards the chemical environment in which they are, in particular in response to variations in pH and temperature, and at the same time to combine the optical properties of the CDs with the magnetic ones of the SPIONs.
  • innovation consists of having a single nanocomposite and multicomponent nanosystem equipped with a multiplicity of functions that can be used in oncology, both for therapeutic and diagnostic objects.
  • image-guided therapeutic procedures ITT
  • ITT image-guided therapeutic procedures
  • MRgFUS Magnetic Resonance-guided Focused Ultrasound Surgery
  • the device of the invention can be used with high versatility in identifying personalized therapies, monitoring the therapeutic effect of the selected treatment for the single patient and evaluating in real time and in a non-invasive way the pH changes of the tumor microenvironment typically found in the healing process.
  • the device proposed in the present invention falls within at least three fundamental segments of anticancer therapy: - the diagnostic one, being an excellent contrast medium in magnetic resonance and fluorescence imaging,
  • the surgical one constituting the present invention a tool that can be used in image-guided laser-induced surgical resection, and
  • anticancer drugs doxorubicin, danaurobicin, dacarbazine, irinotecan, topotecan, paclitaxel, docetaxel, dexamethasone, sorafenib, imatinib, gefitinib, sirolimus, nutlina, mechloretamine, cyclophosphamide, cisplatin, carboplatin, methotrexate, fluorouracil, capecitabine, gemcitabine, asparaginase, axitinib, bosutinib, cabazibitaxel, cetuximab, cyclophosphamide, dactinomycin, dasatinib, interleukin-2, interferon alfa-2, lapatinib, lomustine
  • anticancer drugs doxorubicin, danaurobicin, dacarbazine, irinotecan, topotecan, paclit
  • those incorporated in the nanosystem that constitutes the present invention have unique properties: they can modify the T2 values by virtue of appreciable variations of the microenvironment that surrounds them, constituting in fact a potential tool for a prognostic evaluation of the chemotherapeutic treatment.
  • the innovation lies in the fact that the incorporation of SPIONs in a polymeric matrix sensitive to external stimuli, of which it is an integral part through covalent bonds, determines modifications of the particle-particle distances depending on the surrounding microenvironment, thus influencing the surface phenomena of both electromagnetic and electronic nature (fluorescence).
  • the nanosystem object of the present invention represents a therapeutic and diagnostic tool with considerably more advanced performances when compared with the current therapeutic and diagnostic agents present in the clinic. In fact, it is designed to be exploited as a multimodal therapeutic and diagnostic agent, the potential of which can be exploited even only partially and adapted to the specific application and patient.
  • This nanosystem offers the possibility of extending the market to highly personalized precision therapies, imagining being able to produce the same nanosystem loaded with a combination of specific anticancer drugs for the single patient as needed.
  • the nanosystem can be used on biological material excised from the patient's body. In this way, it is possible to request the nanosystem that is able to release a specific combination of chemotherapeutic agents, so as to maximize the therapeutic effect.
  • This kind of highly personalized treatments which could be optionally supported in countries with insurance coverage, constitute a growing and potentially profitable market.
  • the HA-EDA-C18-C5 derivative was synthesized using a similar protocol previously described (F.S. Palumbo et al., RSC Adv. 2015,
  • NPBC NPBC selected in such a way as to obtain a ratio of 4-NPBC mol/mol of FIA-TBA repeating units equal to 0.7, respectively, was solubilized in 12 ml of anhydrous dimethyl sulfoxide; this solution was added drop by drop to the HA-TBA solution at a temperature of 40°C under constant stirring and the reaction was maintained for 4 hours. After this time, the reaction was brought to 60°C and appropriate quantities of octadecylamine (C-is) in such a way as to obtain a ratio of C-is mol/mol 4-NPBC equal to 0.5 were added and left to react for 4 hours.
  • C-is octadecylamine
  • the solid was washed repeatedly using the hot solvent mixture and finally washed with 80:20 ethanol/water.
  • the solid thus obtained was characterized by 1 H NMR in D20/THF-d8 66:24, comparing the integrals of the peaks at d 0.99 (Chb of group C-ie) and of those at d
  • Table 1 shows as an example the amount of molar functionalization obtained in Cs, C-ie and ethylenediamine groups linked to hyaluronic acid, for a typical reaction. Table 1 The obtained compound had the appearance and consistency of a gel.
  • CDs with a diameter of 1.5 nm and emitting in red were synthesized as previously described (Scialabba et al., Highly Homogeneous Biotinylated Carbon Nanodots: Red-Emitting
  • SPIONS-N3 were prepared using commercially available SPIONs with a diameter of about 20 nm and containing surface carboxyl groups. Similarly, to what was expected for the preparation of CDs- N3, 10 mg of SPIONs-COOH were dispersed in 4 ml of phosphate buffer at pH 6.4 and functionalization was performed using the same protocol described above for CDS-N3. The functionalization of the surface was always confirmed by IR spectroscopy, highlighted by the presence of the vibration at 2100 cm 1 , typical of azides. Also in this case, as evidenced by the AFM analyses, the dimensions of the SPIONS-N3 are completely superimposable to the starting nanoparticles (21.2 ⁇ 1.1 nm).
  • the evaluation of the functionalization degree in azide groups was evaluated by potentiometric titration, by titrating a dispersion of SPIONS-N3 in water with a standard solution of sodium hydroxide 0.025 M and by comparing the base meq necessary to reach the titration end point with those necessary to titrate a dispersion of SPIONs-COOH.
  • the depletion of carboxylic groups due to functionalization with azide groups by amide bond was evaluated by difference and found to be between 0.25 and 2.10 meq/mg of SPIONS-N3.
  • the final nanosystem was obtained by chemical crosslinking of the HA-EDA-C18-C5 derivative in the presence of the two crosslinking agents SPIONS-N3 and CDS-N3.
  • 20 mg of HA-EDA-C18- C5 were solubilized in 20 ml of ultrapure water and 4 ml of THF at
  • the reaction was also confirmed by differential scanning thermal analysis (DSC), which highlights the disappearance of the typical endothermic decomposition peak of FIA-EDA-C18-C5 at 350°C following crosslinking with nanoparticulate fillers. Furthermore, a net endothermic peak is highlighted at about 200°C, similar to the fusion of crystalline domains present in the nanosystem following the crosslinking process.
  • the quantity of SPIONs incorporated in the nanosystem was calculated by the alizarin red assay, showing the presence of 2.2% w/w of elemental iron, compatible with 0.046 mg of SPIONs per mg of nanosystem (99.8% yield).
  • nano-sized nanosystems were confirmed by HR TEM (Figure 3a), AFM (Figure 3b) and DLS ( Figure 3c) and Z- potential measurements. Following the formation of the nanosystem, a significant increase in the Z-potential is observed, from -17 to -34 mV, compatible with the formation of a nanostructure consisting of a core of negative nanoparticles covered by a hyaluronic acid shell.
  • the DLS analysis shown in Figure 3c shows nanoparticles which in pH 6 water have dimensions of about 95 nm; while the AFM and HR TEM images shown in Figure 3a-b highlight nanosystems of about 90 nm characterized by the presence of a polymeric shell that covers nanoparticles with structural characteristics completely superimposable to the starting SPIONs and CDs.
  • the presence of well separated nanoparticles with d-spacing of 4,852 A and others of about 1 .5 nm with d-spacing of about 2,252 A is clearly visible (Figure 3a).
  • the resulting nanosystem is in the form of a nanogel.
  • the size distribution of the nanosystem was evaluated in different mediums at varying temperature and pH.
  • a nanosystem dispersion in pH 7.4 PBS at a concentration of 0.1 mg/ml was placed in a cuvette for DLS measurements and the size distribution was obtained at different temperatures by DLS analysis, using a Malvern Zetasizer Nano ZS with 173° scattering angle.
  • the sample was equilibrated at each temperature for 120 seconds.
  • the average volume of the nanoparticles was calculated by approximating it to the volume of a perfect sphere and using the Z-average as the diameter. As can be seen in Figure 4a, the volume of the particles increases approximately three times, passing from 25 to 45°C, underlining the marked dependence on the temperature of the dispersing medium.
  • the ability of the nanosystem to transform light stimuli of NIR frequency into heat was evaluated by varying the pH and using a diode laser with a wavelength of 810. More in detail, a dispersion of a nanosystem of concentration equal to 0.25 mg/ml was used or in 0.15 mM pH 7.4 phosphate buffer or in 0.15 mM pH 6.4 phosphate buffer or in 0.15 mM pH 5.5 acetate buffer. 0.2 ml of the dispersion was placed in a 96-well multiwell and each well was laser treated with a power of 5 W/cm 2 for up to 200 seconds. The temperature was recorded both by means of a thermal imager placed above the wells and by means of an infrared optical sensor. Water was used as a negative control.
  • the emission spectra of an aqueous solution of the nanogels of Example 1 were recorded with a JASCO FP-6500 spectrofluorimeter with a spectral resolution of 3 nm.
  • the solution was placed in a 1 cm cuvette.
  • the pH of the solution was checked by means of different buffer solutions with controlled pH of 5.5, 6.2 and 7.5.
  • Magnetic resonance imaging (MRI) acquisitions were made using a Philips Achieva 1 .5T magnetic field clinical scanner (with head coil) used for patient diagnostic examinations.
  • the reconstruction matrix on the plane is 256 c 256 and the dimensions of the voxels are 1.0x1.0x2.5 mm 3 .
  • the regions of interest were considered (ROI positioned in the central area of the sample and characterized by a cubic shape with a volume of 27 voxels) within each sample and the average value and the corresponding standard deviation of the voxel signal in the ROI.
  • the trend of the RM signal was obtained as a function of the inversion time Tl.
  • the Ti relaxation time values for the various ROIs present within the various samples were extracted from these data by means of an appropriate numerical fitting procedure with an exponential saturation function.
  • the Ti value tends to increase with the pH value.
  • the same data can be reported as a function of the SPIONs concentration as the pH varies. It is observed that as the concentration of SPIONs increases, the Ti relaxation times decrease. The effect is greater the greater the concentration of
  • the transverse relaxation process is much faster than in water samples.
  • the samples with the greatest relaxation times are those of distilled water and free SPIONs in solution at concentrations of 0.25 and 0.5 mg/ml.
  • nanosystem samples have transverse relaxation times of about an order of magnitude lower (between 250 and 500 ms). Therefore, the effect of the nanosystem is remarkable and acts as a negative contrast medium because it reduces the intensity of the T2-weighted signal in the regions where it is concentrated.
  • the transverse relaxation time T2 increases with the pH.
  • ELI Contrast Measurements in Fluorescence Imaging Using Confocal Microscopy
  • the ability of the nanosystem to penetrate the tumor mass and generate detailed images of the tumor microenvironment was assessed by confocal microscopy, using the red autofluorescence of the nanosystem due to the presence of CDs and marking the cells’ nuclei that make up the organoid with DAPI.
  • Three-dimensional organoids consisting of a core of MDA-MB-231 breast cancer cells and a shell of HDFa human fibroblasts incubated with the nanosystem at a concentration of 0.25 mg/ml were used for the tests. After incubation, the organoids were washed three times with DPBS and placed on a slide for confocal. The nuclei were labeled with DAPI for 10 minutes before and the organoids were rewashed 5 times with
  • Three-dimensional breast cancer organoids were subsequently obtained as described above. These were incubated with the nanosystem in DMEM for 48 hours at a concentration of 0.25 mg/ml. After this time, the culture medium was replaced with fresh DMEM and the organoids were treated with an 810 nm laser for 300 seconds. Cell vitality was assessed by transferring the treated organoid to a 96-well multiwell and by MTS test. Vitality was reported by comparing results with untreated organoids. The experiment was repeated by applying more treatment cycles (up to three) to demonstrate that it is possible to increase the power of the treatment as the hyperthermia cycles applied increase. In Figure 8 it is observed that after 300 seconds and a cycle of phototherapy we witness the almost complete death of the entire organoid.

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Abstract

L'invention concerne un nanosystème pour le diagnostic, le traitement guidé par image de tumeurs et la surveillance du microenvironnement tumoral. Le nanosystème est un agent de contraste comprenant une enveloppe polymère à base d'un nanogel d'acide hyaluronique, des nanoparticules d'oxyde de fer super-paramagnétique (SPION) et des nanoparticules de carbone (CD).
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CN119923563A (zh) * 2022-09-13 2025-05-02 核心量子有限公司 聚合物复合物纳米材料封装系统
CN115487319A (zh) * 2022-09-16 2022-12-20 吉林大学 一种靶向肿瘤相关巨噬细胞的光磁双模态纳米粒子的制备方法及其应用
CN115721629A (zh) * 2022-10-31 2023-03-03 西安交通大学 多西他赛与恩杂鲁胺联合用药pH响应型铁-铜磁性纳米载药系统及其制备和应用
CN115721629B (zh) * 2022-10-31 2024-02-20 西安交通大学 多西他赛与恩杂鲁胺联合用药pH响应型铁-铜磁性纳米载药系统及其制备和应用
CN116726260A (zh) * 2023-04-27 2023-09-12 郑州大学 一种涂层及带有涂层的血管植入材料、制备方法和应用
CN116836700A (zh) * 2023-06-29 2023-10-03 辽宁大学 一种透明质酸修饰红光碳点HA-R-CDs的制备方法及其在肺癌细胞靶向成像中的应用
CN116836700B (zh) * 2023-06-29 2024-05-31 辽宁大学 一种透明质酸修饰红光碳点HA-R-CDs的制备方法及其在肺癌细胞靶向成像中的应用

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