WO2023238060A1 - Nanocomposite injectable à base d'hydrogel pour imagerie chirurgicale - Google Patents

Nanocomposite injectable à base d'hydrogel pour imagerie chirurgicale Download PDF

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WO2023238060A1
WO2023238060A1 PCT/IB2023/055879 IB2023055879W WO2023238060A1 WO 2023238060 A1 WO2023238060 A1 WO 2023238060A1 IB 2023055879 W IB2023055879 W IB 2023055879W WO 2023238060 A1 WO2023238060 A1 WO 2023238060A1
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nanocomposite according
hydrogel
injectable
injectable nanocomposite
icg
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PCT/IB2023/055879
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English (en)
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Chiara CRESSONI
Adolfo SPEGHINI
Alessandro Antonelli
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Universita' Degli Studi Di Verona
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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
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • 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
    • A61K49/0073—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 semi-solid, gel, hydrogel, ointment
    • 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
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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

Definitions

  • the present invention applies to diagnostics in the field of imaging aimed at intraoperative navigation during surgery, in particular mini-invasive surgery; in addition, it can apply to magnetic resonance diagnostics and focal therapies .
  • target lesion The precise knowledge of the anatomical site in which a certain disease resides ( "target lesion” ) is an indispensable prerequisite for care , because it allows respecting the healthy tissues not involved while completely treating the disease during surgical interventions , especially in the field of oncology .
  • the target lesion is not visible to the naked eye because the tissue features prevent the possibility of distinguishing it from healthy tissues or because the neoplasm resides in a deep - not superficial - anatomical site in the af fected organ .
  • This obliges clinicians to mentally reproduce the location of the tumor lesion based on what is understood during the preoperative investigations ; such a mode is subj ective and therefore suf fers from limited precision and prevents the unambiguous sharing of information among the surgical team .
  • Image-guided surgery or fluorescence-guided surgery are two in vivo imaging techniques, which already assist the surgeon daily in many procedures (e.g., identification of the sentinel lymph node) providing him with the information necessary to perform some surgical procedures (e.g., removal of axillary lymph nodes in breast cancer, inguinal lymph nodes in penile cancer, etc.) .
  • the marking occurs using the natural dissemination pathways of the fluorescent marker, injected into a superficial site (e.g., breast, penis) so that it can flow to the site of the surgical target.
  • the ICG use methods described above cannot be used to mark tumor lesions (e.g., prostate cancer) , as there are no superficial access points and vascular structures which allow the dye to selectively reach the tumor site.
  • tumor lesions e.g., prostate cancer
  • the ICG introduced by direct injection into a vascularized tissue would have the disadvantage of a short retention time, a typical problem of molecules in solution, which undergo rapid dissemination within the surrounding tissues, causing loss of signal intensity and hindering a correct and precise localization .
  • thermosensitive hybrid thermosensitive hydrogels for the intravaginal release of anti- HIV compounds .
  • Kankala Ranjith Kumar “Nanoarchitectured two-dimensional layered double hydroxides-based nanocomposites for biomedical applications", Advanced drug delivery reviews Elsvier, Amsterdam, NL, vol. 186, 12 April 2022) describes hybrid nanocomposites with LDH and organic polymers for drug delivery applications.
  • the inventors of the present patent application have developed an inj ectable nanocomposite for application as a tissue marker in optical and magnetic imaging techniques .
  • Such a nanostructured material is mainly applied to image-guided surgery, forming an innovative and promising field of clinical research .
  • the hydrogel-based inj ectable nanocompos ite is described for medical use in imaging diagnosis .
  • the inj ectable nanocomposite o f the invention is described for therapeutic medical use , possibly in combination with diagnosis .
  • Figure 1 shows a depiction of the injectable composite of the invention nanoLDH-ICG@hydrogel .
  • Figure 2 shows X-ray diffraction patterns showing diffraction signals of nanoLDH powder samples consisting of only Mg-Al-OH (above) , nanoLDH consisting of Mg-Al-Gd- OH (center) and nanoLDH bound to Indocyanine Green dye (below) .
  • Figure 3 shows Raman spectra of nanoLDH powder samples at the excitation wavelength of 532 nm.
  • Figure 4 shows TEM images of nanoLDH Mg-Al-OH (a) , nanoLDH Mg-Al-Gd-OH (b) , nanoLDH-ICG (c) samples captured at 80 kV.
  • Figure 5 shows the results of the DLS analysis of nanoLDH samples in aqueous dispersion.
  • the graphs show the hydrodynamic diameter (left) and zeta potential (right) dimensions of the nanoLDH nanoparticles of Mg-Al and Mg- Al-Gd and the nanoLDH-ICG complex.
  • Figure 6 shows the absorption spectrum in the UV-Vis- NIR range of an aqueous solution of Indocyanine Green (left) and the values obtained from the calculation of the loading efficiency for variable ratios of nanoLDH: ICG, indicated by the values shown at the top of the bars of the histogram (right) .
  • Figure 7 shows (a) image captured by Optical Imager exciting at 745 nm and capturing at 820 nm of hydrogel samples in which serial dilutions of nanoLDH-ICG have been added (sample 1 is the most diluted, sample 6 is the most concentrated, sample 7 is only hydrogel without nanoLDH- ICG) and (b) related numerical representation of the radiance extrapolated from the ROIs directly from the image .
  • Figure 8 shows an example of MRI imaging on one of the mice with tumor, monitored pre-inj ection (a) and after 1 h from injection (b) by MRI tomography (Bruker, Biospin) for small animals.
  • Figure 9 shows Optical Imager IVIS spectrum acquisition on 2 pre-in ection naked mice (a) and 2 hours post-injection subcutaneously (b) of the nanoLDH- ICG@hydrogel composite.
  • Figure 10 shows the comparison between the in vivo 01 acquisitions of the three experimental groups (hydrogel, ICG and Nanocomposite) , at different times.
  • Figure 11 shows: a) in vivo optical imaging of the behavior of the positive control compared to the nanocomposite, at different times, using the bandpass emission filter at 820 nm; b) trend of the signal dissemination over time for the control group and the nanocomposite, obtained from the statistical analysis of the data.
  • Figure 12 shows: a) magnetic resonance imaging of two animals captured before and after injection of the hydrogel (above) and nanocomposite (below) . b) The TR signal for muscle tissue with respect to the hydrogel (above) or nanocomposite nanoLDH-ICG@hydrogel (below) .
  • Figure 13 shows ex vivo imaging tests using a bovine liver, simulating acquisition during a surgical procedure through the Da Vinci X robotic system (shown above) for fluorescence-guided surgery.
  • the nanocomposite of the invention comprises : a) metal hydroxide nanoparticles, b) a functional agent, c) a hydrogel.
  • said nanoparticles (a) comprise divalent and trivalent metal hydroxides according to the following structure:
  • M 2+ is the divalent metal ion
  • M' 3+ is the trivalent metal ion
  • the divalent metal (M 2+ ) and the trivalent metal (M 3+ ) are present together in a ratio of 2: 1-4:1 and preferably 2:1 or 3:1 (mol /mol ) .
  • M 2+ is for example magnesium, zinc or calcium.
  • M 3+ is for example aluminum.
  • a n is for example the nitrate, chloride or carbonate ion.
  • the particles of the invention comprise layered hydroxides according to a structure referred to as the Layered Double Hydroxides (LDH) , comprising cationic layers of divalent and trivalent metal atoms.
  • LDH Layered Double Hydroxides
  • a percentage of about 1-10% (mol/mol) divalent metal ions can be replaced by gadolinium, in order to impart paramagnetic properties to the particles and allow use in magnetic resonance imaging (MRI) .
  • MRI magnetic resonance imaging
  • yttrium (III) can be employed.
  • lanthanide ions for example Europium (III)
  • the functional agent (b) this can be: a dye, a therapeutic agent.
  • die means both a substance which emits radiation in the visible spectrum and a substance which, when excited, emits radiation from the optical region of the ultraviolet, visible or near-infrared spectrum, which can be detected by appropriate techniques.
  • a dye is preferably Indocyanine Green (trade name: Cardiogreen or ICG) , with near-infrared fluorescent properties.
  • ICG can be detected by virtue of an absorption peak at 780 nm and an emission between 800-900 nm (in what is known as "first biological window”) .
  • Other dyes can be chosen from the group of anionic dyes, one of which is fluorescein.
  • a therapeutic agent can instead be drugs, such as ibuprofen or paracetamol, or macromolecules such as for example nucleic acids (RNA or DNA) , oligopeptides and peptides, to activate or inactivate biological functions or to act as substrates for the production of proteins with activities of interest.
  • drugs such as ibuprofen or paracetamol
  • macromolecules such as for example nucleic acids (RNA or DNA) , oligopeptides and peptides, to activate or inactivate biological functions or to act as substrates for the production of proteins with activities of interest.
  • the functional agent can be dual, i.e., have both dye and therapeutic agent functions, or it can be envisaged that two different functional agents, each having dye and/or therapeutic function, are included.
  • Indocyanine Green (or ICG) is capable of producing, in an aerobic environment, reactive oxygen species (ROS) once excited to a certain radiation power and for a certain time; this can be exploited to lead to the degradation of specific molecules or to cause the death of cells linked to disease situations, such as cancer cells .
  • ROS reactive oxygen species
  • the functional agent (s) can be released slowly, for example over the course of long-term therapies, together with the display of the injection site by imaging techniques.
  • the functional agent described above can further comprise a composite sensitive to certain characteristic conditions of the injection site (pH, presence of particular ions, etc.) .
  • the functional agent can be employed not only non-specif ically (not interacting with any biological structure) but also specifically, to detect certain conditions of interest linked for example to certain diseases (e.g., the more acidic pH in tumors) .
  • hydrogel (c) this is a biocompatible and thermo-responsive hydrogel.
  • the hydrogel of the invention preferably has a sol-gel transition passing from the temperature of 4°C to room temperature.
  • such a hydrogel comprises hydrophilic polar organic functional groups, which allow a certain dispersion in water.
  • block copolymers derived from (PEG) or hydrogels based on 1-2% chitosan, preferably 15 kDa for example in a solution of 0.1 M HC1 and glycerophosphate can be employed.
  • step A) of particle preparation proceeds with step A) of particle preparation .
  • the solution of step Al) comprises divalent (M 2+ ) and trivalent (M 3+ ) metal salts.
  • the divalent metal (M 2+ ) and the trivalent metal (M 3+ ) are present together in a ratio of 2: 1-4:1 and preferably 2:1 or 3:1 (mol /mol ) .
  • M 2+ is for example magnesium, zinc or calcium.
  • M 3+ is for example aluminum.
  • Salts of the above metals are preferably hydrated nitrates, for example: Mg(NC>3)2 ’ H2O and Al (NO3) 3 -IbO .
  • solution A of step Al) is an alcoholic solution of ethanol or methanol.
  • step A2 an alcoholic solution of a base is prepared .
  • a sodium hydroxide solution can be prepared, preferably with the same alcohol as solution A.
  • Each of solutions A and B is prepared at room temperature, in a closed container and until complete dissolution .
  • step A3) solution A is added to solution B.
  • such an addition is conducted dropwise and under vigorous magnetic stirring.
  • the mixture is kept under constant vigorous stirring for a period of 20-60 minutes, and preferably 40 minutes (20 to 60 minutes) .
  • step A4 the hydroxide particles formed are collected by centrifugation.
  • a centrifugation can be carried out for 10 minutes at 7000 rpm.
  • step A4 the supernatant is removed and the precipitate is re-dispersed in 20 ml of alcohol using a vortex.
  • Ethanol or methanol can be used for this purpose.
  • Step A5) of heat treatment comprises treating the obtained dispersion, placed in a special glass vial, to a heat treatment.
  • Such a heat treatment can be conducted at 100°C for a time of 1-8 hours.
  • step A5) can be conducted in a microwave reactor for about 15 minutes at 100°C.
  • the method proceeds with a further washing of the particles.
  • the dispersion can be centrifuged for a period of 10 minutes at 7000 rpm adding deionized water, after which the supernatant is removed and the precipitate is dispersed in deionized water using the vortex. [0095] This procedure can be repeated another 2/3/4 times to eliminate the alcohol and excess reagents.
  • the nanoparticles thus obtained can be stored for months in a refrigerator in the form of a gelatinous pellet and weighed if necessary for dispersion in an aqueous environment .
  • step Al) can comprise the addition of a gadolinium (ITT) salt.
  • the gadolinium (ITT) salt are 1-10% mol with respect to the divalent ion (M 2+ ) added to the reaction mixture.
  • the preparation of the nanocomposite of the invention comprises the further step B) to obtain a particle preparation and functional agent.
  • Said step B) comprises the steps of:
  • step B3 adding the dispersion of step Bl) to the functional agent solution and stirring.
  • step Bl) is preferably obtained in deionized water.
  • the functional agent of step B2) can be a dye, as described above .
  • step B3 an appropriate ratio of particles to functional agent is employed in step B3) .
  • the ratio of particles to dye is about 10:1- 100:1 by weight .
  • step B3) is conducted under appropriate light and temperature conditions .
  • step B3 is conducted at room temperature, in the dark and under vigorous magnetic stirring.
  • step B3 The mixture obtained from step B3) is stirred vigorously in the dark for 4-6 hours, at the end of which the dispersion is centrifuged at 7000 rpm for 10 minutes, washing with water 3/4 times to eliminate the excess functional agent, for example a dye.
  • the pellet is stored in a refrigerator at 4°C and in the dark and is stable for months.
  • the preparation of the hydrogel is obtained in a step C) .
  • a hydrogel buffer solution can be prepared, for example with phosphate buffered saline (PBS) .
  • PBS phosphate buffered saline
  • the hydrogel (c) this is a biocompatible hydrogel .
  • the hydrogel is a thermo-responsive hydrogel.
  • such a hydrogel comprises hydrophilic polar organic functional groups, which allow the dispersion in water.
  • block copolymers derived from polyethylene glycol (PEG) , or hydrogels based on 1-2% chitosan, preferably 15 kDa for example in a solution of 0.1 M HC1 and glycerophosphate can be employed.
  • step DI an amount of particles obtained with step B3) of about 25-200 mg of particles per ml of hydrogel solution is dispersed in the hydrogel solution obtained with step C) .
  • the final hydrogel comprises about 20-40% (w/w) and preferably 25-30% (w/w) polymer.
  • hydrogel-based injectable nanocomposite according to the present invention is described for medical use.
  • the hydrogel-based injectable nanocomposite is described for diagnostic medical use.
  • the present invention applies to diagnostic imaging.
  • said imaging is multifunctional.
  • optical imaging (01)
  • magnetic resonance imaging MRI
  • fluorescence fluorescence
  • CT computerized axial tomography
  • the injectable nanocomposite of the present invention is described for use as a contrast agent.
  • the imaging techniques that can be employed comprise optical imaging (01) , magnetic resonance imaging (MRI) , fluorescence, computed axial tomography (CT) techniques.
  • the nanocomposite described is inj ected and then displayed with the appropriate technique .
  • the described nanocomposite is used as an auxiliary agent in surgery (known as image-guided surgery, IGS ) .
  • the use of the composite for the described purposes comprises the inj ection thereof before starting the surgical procedure .
  • Such an inj ection is at a human or animal body district , at which the surgery must be carried out , in order to provide and maintain the indication of the site of said district over time .
  • Such a correspondence should be understood as a spatial relationship between the inj ection site and the operative intervention site , for example where the inj ection cannot be carried out exactly at the intervention site .
  • the composite can be inj ected from 1 minute to 3 days before surgery or within a time compatible with needs , which can be for example from 1-24 hours , or within 1 hour or 30 minutes from surgery or 15 minutes before surgery .
  • the nanocomposite is described for therapeutic medical use , possibly in combination with diagnosis or use as a contrast agent.
  • Nanoparticles are prepared by coprecipitation of precursor salts, followed by heat treatment in microwave reactor (Monowave-400, Anton Paar) .
  • the dispersion is placed in a special glass vial and the heat treatment is carried out in the microwave reactor for 15 minutes at 100 ° C .
  • the dispersion is centri fuged ( 10 minutes at 7000 rpm) adding deioni zed water, after which the supernatant is removed and the precipitate is dispersed in deioni zed water using the vortex .
  • This procedure is repeated another 2 / 3/ 4 times to eliminate all the ethanol ( or methanol ) and excess reagents .
  • the nanoparticles thus obtained can be stored for months in a refrigerator in gelatinous pellet form and weighed i f necessary for dispersion in an aqueous environment .
  • Nanoparticles are prepared by the same method described above based on co-precipitation of precursor salts , followed by microwave reactor heat treatment (Monowave-400 , Anton Paar ) .
  • nanoLDH-ICG nanocomposite 200 mg of nanoLDH are weighed and dissolved in 5 ml of deionized water .
  • the LDH dispersion is quickly added to the ICG solution, at room temperature, in the dark and under vigorous magnetic stirring.
  • the pellet is stored in a refrigerator at 4°C and in the dark and is stable for months.
  • a solution of PBS (phosphate buffered saline) at pH 7.4 is prepared by dissolving 0.01 M phosphate buffer, 0.0027 M potassium chloride (KC1) and 0.137 M sodium chloride in 200 ml of deionized water in a vessel and then cooled by immersing it in a second vessel containing ice.
  • PBS phosphate buffered saline
  • nanoLDH-ICG@hydrogel composite 25 mg of nanoLDH-ICG are weighed per ml of hydrogel (other options: 50 mg/ml, 100 mg/ml, 200 mg/ml nanoparticles) and are incorporated by stirring with magnetic anchor and dispersing them by an ultrasonic bath. [00166] Characterization and results
  • wavelengths of 488 nm, 514 nm, 633 nm and 785 nm can be employed.
  • Figure 3 shows the Raman spectrum of the vibrational signals corresponding to the structure of the nanoLDH centered at 554, 724, 1049, 1068 and 1380 cur 1 ( these are only possible alternative values ) .
  • the signal around 1050 cur 1 in this speci fic example can be attributed to the presence of the ion NOs- in the interplanar space .
  • Figure 4 shows that the dimensions of the nanoLDH nanoparticles are between 10 nm and 300 nm and the morphology appears in large spherical lines , appearing as a few nanometer needles seen from the side .
  • the hydrodynamic diameter and zeta potential of the nanoparticles are measured using the Dynamic Light Scattering ( DLS ) technique , in our case operated by the Malvern Zetasi zer Nano ZS instrument , provided with a HeNe laser at 633 nm .
  • DLS Dynamic Light Scattering
  • the technique measures the si ze of the nanoparticles surrounded by the solvent molecules and the Stern layer, moreover the value of the net surface charge of the nanoparticles in solution is measured by applying a voltage .
  • Figure 5 shows that the nanoparticles in aqueous dispersion have a hydrodynamic diameter between 30 and 300 nm, while the zeta potential for all the nanoparticles ranges from +20 to +40 mV.
  • the absorption spectrum of the sample in aqueous dispersion is measured by a UV-Vis spectrophotometer, in our case the Cary60 instrument (Agilent) was used.
  • the absorption spectra from 300 nm to 1000 nm of the aqueous dispersions of nanoLDH-ICG and ICG alone, as well as of the supernatant obtained after the nanoLDH-ICG composite formation procedure, were captured. From the absorbance measurement of the supernatant it is possible to calculate, using Lambert-Beer's law, the concentration of the solution and, from here, the mass of ICG in the supernatant. In fact, this measurement allows calculating the loading efficiency, which indicates the goodness of the preparation of the nanoLDH-ICG composite, by means of the formula:
  • FIG. 6 shows the loading efficiency (LE) which is calculated by determining the concentration of ICG by measuring the absorption spectrum of solutions at known volume. In particular, the weight (or moles) of the loaded ICG is calculated by subtraction (initial ICG - ICG in the supernatant) .
  • the figure shows an absorption spectrum example of an ICG solution in the UV-Vis-NIR range, with the characteristic maximum absorption centered around 780 nm.
  • An example of results of the loading efficiency calculation obtained by the measurement of supernatants deriving from washes by means of centrifugation, following the adsorption of a fixed amount of nanoLDH with variable ratios of ICG, is also reported. Using 10:1 to 100:1 ratios of nanoLDH: ICG, LE values greater than 90% are obtained.
  • the emission can also be captured by centering the window at 800 nm or 810 nm, or using a filter which cuts before 850 nm and captures the rest of the spectrum in the NIR.
  • Multimodal in vivo imaging After capturing images in pre-acquisition 01 and MRI , the animals were anestheti zed by inhalation of isoflurane and a volume of 50 pl of nanoLDH- ICG@hydrogel nanocomposite was inj ected subcutaneously at a temperature below 4 ° C ( kept on ice until the moment of inj ection) . Immediately afterwards, fluorescence images were captured using the Optical Imager IVIS spectrum, exciting Indocyanine Green at 745 nm and capturing a 10 nm window centered at 820 nm . Then, still under anesthesia, the images are captured using a magnetic resonance tomograph (Bruker Biospin) .
  • Figure 8 shows a transverse slice corresponding to a scan of the magnetic resonance tomography and, in particular, of the portion corresponding to the inj ection site (highlighted in white ) .
  • the presence of a contrast is apparent ( lighter color ) in the highlighted portion, due to the presence of the nanoLDH- ICG@hydrogel nanocomposite .
  • NC nanocomposite
  • MRI magnetic imaging
  • the main innovation of this composite is the ability to non-speci f ically mark the inj ection site and to remain in this site for at least a few hours , without spreading rapidly into the surrounding environment .
  • the abi lity to act as a dual contrast agent , optical ( near-infrared) and magnetic, leveraging technologies commonly employed in clinical practice lays the foundation for the application thereof as an inj ectable marking tool for image-guided surgery .
  • nude atypical mice as the animal model for the study, parameters such as signal intensity, persistence time in the body, tendency to disseminate around the inj ection site and clearance of the composite were evaluated .
  • a preliminary as sessment of signal persistence over time is necessary to obtain information on the behavior of the nanomaterial in the body and on the time required for the elimination thereof.
  • Figure 10 shows the images captured by optical imaging (01) of the animals injected with the composite, compared with those administered with the positive control (only ICG in solution) and the negative control (only hydrogel) , captured using the emission filter at 820 nm.
  • Figure 11 shows a sequence representative of the behavior of the nanoLDH-ICG@hydrogel nanocomposite, with respect to the ICG dye in aqueous solution, in which a different dissemination in the tissues surrounding the injection point is highlighted both from a qualitative and quantitative point of view.
  • Magnetic resonance imaging was then evaluated in vivo , capturing the image of the animal pre-inj ection, inj ecting the nanocomposite into the desired sub-cutaneous position, and capturing the measurement within 30 minutes of inj ection .
  • the nanoLDH- ICG@hydrogel nanocomposite shows a clearer contrast (positive contrast ) in the Ti-weighted images , circumscribed to the nanocomposite inj ection site .
  • Figure 13 thus shows the images taken during the surgical simulation, showing two subcutaneous inj ections of the nanoLDH- ICG@hydrogel on a portion of bovine liver and the image taken from the Da Vinci system display screen ( acquisition window 700- 900 nm) .
  • This experiment allowed testing the ability of the nanocomposite to be a tissue marker which can be displayed with an imaging system normally used in clinical practice , with an important positive impact on human health .
  • the advantages of fered by the present invention will be apparent to those skilled in the art .
  • the composites of the present invention allow a preoperative marking of the tissue of interest in surgical operations such as prostatectomy or mastectomy, with excellent precision and locali zation .
  • the present invention allows developing biocompatible, safe , non-toxic and ef ficient agents which are stable and persistent at the inj ection site for a suf ficient period of time to perform a surgical procedure guided by target lesion marking .
  • the nanostructured system of the present invention has the potential typical of new nanostructured systems with applications in biomedicine and for bioimaging, which have demonstrated safety, low toxicity and ef ficacy in animal models , allowing the development of new biotechnological companies operating in the field of nanotechnologies or of interest to companies already existing and operating in the field of biomedical diagnostics and imaging .
  • nanocomposites described lend themselves to further implementation by incorporating therapeutic compounds , forming the basis of a theranostic product which, in addition to diagnosis , can convey the sitespeci fic pharmacological treatment .
  • the product can be stored under normal conditions even for a long time .

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  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Nanotechnology (AREA)
  • Medicinal Chemistry (AREA)
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Abstract

La présente demande de brevet décrit un nanocomposite injectable à base d'un hydrogel thermosensible pour marquer des sites anatomiques à des fins chirurgicales.
PCT/IB2023/055879 2022-06-08 2023-06-07 Nanocomposite injectable à base d'hydrogel pour imagerie chirurgicale WO2023238060A1 (fr)

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IT102022000012128A IT202200012128A1 (it) 2022-06-08 2022-06-08 Nanocomposto iniettabile a base di un idrogel per imaging chirurgico

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Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
BEHZADI NIA SAMAN ET AL: "Carboxymethylcellulose/layered double hydroxides bio-nanocomposite hydrogel: A controlled amoxicillin nanocarrier for colonic bacterial infections treatment", INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, vol. 155, 1 July 2020 (2020-07-01), NL, pages 1401 - 1409, XP093014762, ISSN: 0141-8130, DOI: 10.1016/j.ijbiomac.2019.11.115 *
GUAN SHANYUE ET AL: "An NIR-sensitive layered supramolecular nanovehicle for combined dual-modal imaging and synergistic therapy", NANOSCALE, vol. 9, no. 29, 1 January 2017 (2017-01-01), United Kingdom, pages 10367 - 10374, XP093014514, ISSN: 2040-3364, DOI: 10.1039/C7NR01771E *
KANKALA RANJITH KUMAR: "Nanoarchitectured two-dimensional layered double hydroxides-based nanocomposites for biomedical applications", ADVANCED DRUG DELIVERY REVIEWS, ELSEVIER, AMSTERDAM , NL, vol. 186, 12 April 2022 (2022-04-12), XP087088734, ISSN: 0169-409X, [retrieved on 20220412], DOI: 10.1016/J.ADDR.2022.114270 *
NARENDRA K. SINGH ET AL: "Nanostructure controlled sustained delivery of human growth hormone using injectable, biodegradable, pH/temperature responsive nanobiohybrid hydrogel", NANOSCALE, vol. 7, no. 7, 1 January 2015 (2015-01-01), United Kingdom, pages 3043 - 3054, XP055468201, ISSN: 2040-3364, DOI: 10.1039/C4NR05897F *
NATH JAYASHREE ET AL: "Synthesis of carboxymethyl cellulose-g-poly(acrylic acid)/LDH hydrogel for in vitro controlled release of vitamin B 12", APPLIED CLAY SCIENCE, vol. 155, 1 April 2018 (2018-04-01), AMSTERDAM, NL, pages 65 - 73, XP093014745, ISSN: 0169-1317, DOI: 10.1016/j.clay.2018.01.004 *
TIAN WENXUE ET AL: "RETRACTED ARTICLE: LDH hybrid thermosensitive hydrogel for intravaginal delivery of anti-HIV drugs", ARTIFICIAL CELLS, NANOMEDICINE AND BIOTECHNOLOGY, vol. 47, no. 1, 10 April 2019 (2019-04-10), US, pages 1234 - 1240, XP093014814, ISSN: 2169-1401, DOI: 10.1080/21691401.2019.1596935 *
WANG SHUO ET AL: "A remarkable thermosensitive hydrogel cross-linked by two inorganic nanoparticles with opposite charges", JOURNAL OF COLLOID AND INTERFACE SCIENCE, vol. 538, 1 March 2019 (2019-03-01), US, pages 530 - 540, XP093014737, ISSN: 0021-9797, DOI: 10.1016/j.jcis.2018.12.012 *
WEI PEI-RU ET AL: "Synthesis of chitosan-coated near-infrared layered double hydroxide nanoparticles for in vivo optical imaging", JOURNAL OF MATERIALS CHEMISTRY, vol. 22, no. 12, 1 January 2012 (2012-01-01), GB, pages 5503, XP093014561, ISSN: 0959-9428, DOI: 10.1039/c2jm16447g *
YU XINYING ET AL: "Alginate-chitosan coated layered double hydroxide nanocomposites for enhanced oral vaccine delivery", JOURNAL OF COLLOID AND INTERFACE SCIENCE, ACADEMIC PRESS,INC, US, vol. 556, 9 August 2019 (2019-08-09), pages 258 - 265, XP085859484, ISSN: 0021-9797, [retrieved on 20190809], DOI: 10.1016/J.JCIS.2019.08.027 *

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