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WO2024256488A1 - Procédé de production continue de nanoparticules de ferrite fonctionnalisées par un polymère - Google Patents

Procédé de production continue de nanoparticules de ferrite fonctionnalisées par un polymère Download PDF

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Publication number
WO2024256488A1
WO2024256488A1 PCT/EP2024/066274 EP2024066274W WO2024256488A1 WO 2024256488 A1 WO2024256488 A1 WO 2024256488A1 EP 2024066274 W EP2024066274 W EP 2024066274W WO 2024256488 A1 WO2024256488 A1 WO 2024256488A1
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Prior art keywords
solution
linker
thermo
mnps
reaction
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Teresa Pellegrino
John Santigie CONTEH
Thanh Binh MAI
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DIMES Dipartimento di Medicina Sperimentale Universita degli Studi di Genova
Fondazione Istituto Italiano di Tecnologia
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DIMES Dipartimento di Medicina Sperimentale Universita degli Studi di Genova
Fondazione Istituto Italiano di Tecnologia
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    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
    • H01F1/00—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
    • H01F1/0036—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties showing low dimensional magnetism, i.e. spin rearrangements due to a restriction of dimensions, e.g. showing giant magnetoresistivity
    • H01F1/0045—Zero dimensional, e.g. nanoparticles, soft nanoparticles for medical/biological use
    • H01F1/0054—Coated nanoparticles, e.g. nanoparticles coated with organic surfactant
    • 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
    • A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y25/00—Nanomagnetism, e.g. magnetoimpedance, anisotropic magnetoresistance, giant magnetoresistance or tunneling magnetoresistance

Definitions

  • the present invention relates to a continuous process for the production of ferrite nanoparticles having the surface functionalized with thermo-responsive polymeric moieties.
  • MNPs magnetic nanoparticles
  • Ms magnetic resonance and particle imaging
  • AMF alternating magnetic field
  • Ms magnetic resonance and particle imaging
  • MHT magnetic hyperthermia
  • MNPs can also be used as nanocarriers for targeted combined MHT and drug delivery applications.
  • the hydrophobic surface of the MNP can be engineered to bind hydrophilic and biocompatible stimuli sensitive (pH, heat, etc.) polymers containing covalently linked or encapsulated drug molecules.
  • hydrophilic and biocompatible stimuli sensitive (pH, heat, etc.) polymers containing covalently linked or encapsulated drug molecules are often preferred.
  • TR-polymers thermoresponsive polymers
  • the MNPs heat is used not only to directly damage cancer cells, but also to trigger deformations/cleavage on the grafted TR-polymer shell, which thereby enable the release of the linked drug only upon heat application.
  • MNPs are embedded in a lipidic bilayer in the form of a vesicle enclosing a drug; upon exposure to an alternating magnetic field, the MNPs heat up and cause deformation and permeability change of the lipidic bilayer, and as a consequence the release of the drug present in the vesicle cavity.
  • the process of this document is based on a nanoemulsion encapsulation technique; the present inventors have noted that this process has several drawbacks, namely, poor size distributions, big size of iron oxide nanoparticles (> 100 nm), clustering phenomena which result in multiple NPs encapsulated in a polymer shell (that can reduce their heating performance under applied alternating magnetic field), or the need to use a micro-fluidic device to aid the emulsion formation, which is however expensive and difficult to microfabricate and leads to limited flowrates of reagents and as a consequence low throughput.
  • Patent application WO 2013/150496 A1 describes the production of ferrite nanocrystals (meaning iron oxides and mixed oxides of Fe-Mn or Fe-Co) in cubic shape; despite their potential excellent properties, the NPs obtained according to this document have not found wide application because they are not soluble in aqueous media due to the coverage of their surface with a layer of hydrophobic ligands; besides, they tend to form aggregates, making their functionalization with a polymeric coating difficult.
  • Patent application WO 2018/138677 A1 describes a method for the production of magnetic nanoparticles coated with a thermo- or pH-responsive polymer, comprising providing a solution including magnetic nanoparticles functionalized at their surface with a polymerization initiator, and causing the radical polymerization of a monomer or co-monomers forming a thermo-polymer or pH-responsive copolymer on the surface of the magnetic nanoparticles.
  • the particles produced with this method have suitable properties for the intended application, in particular in the field of nanomedicine, and the therapeutic efficacy of the produced MNPs was demonstrated through in vitro and in vivo experiments done on skin and colorectal cancers in murine model at clinical MHT conditions (11 KA/m and 110 kHz).
  • WO 2018/138677 A1 also describes the relevance of the lower critical solution temperature (also referred to as LOST) of the polymeric shell of the MNPs: this is the temperature threshold that, when overcome, causes the contraction of the polymeric molecules of the shell, and thus a change in the capability of the shell to shield the inner part of the MNPs; when MNPs are loaded with a drug during their production, the heating of the magnetic core during hyperthermia treatment causes thus the shrinking of the shell and the release of the drug at the site reached by the MNPs. It is thus necessary that the LCST is above the physiological temperature (37 °C) to prevent premature leakage of the drug from the MNPs.
  • the LCST varies depending on several parameters, among which the specific monomers the polymer shell is made of and its production process.
  • Patent application WO 2020/222133 A1 discloses a method for producing ferrite nanocrystals, which comprises the steps of providing a solution comprising a fatty acid, an aliphatic amine and an alcoholic solvent; adding to the solution at least one organometallic precursor compound comprising a metal selected from Fe, Mn, Co or Zn and an aromatic organic molecule obtaining a reaction mixture; transferring the reaction mixture to a sealed reactor, filling the reactor at a percentage between 20 and 70 vol.%; and heating the sealed reactor to a temperature between 160-240 °C for at least 3 hours.
  • This process yields nanoparticles of cubic shape, having excellent properties in view of the intended application in nanomedicine, but again is a batch process suffering of the limits of low productivity indicated above for discontinuous processes. Continuous processes are well-known in the chemical industry.
  • Patent applications US 2018/0339914 A1 and US 2021/0261431 A1 describe a continuous, in-flow process for producing metallic nanoparticles.
  • a first flow stream of an aqueous salt solution comprising ions of a transition metal and a branched polymeric template that is not a dendrimer; providing a second flow stream comprising a reducing agent to reduce the ions of the transition metal to form metallic nanoparticles within the branched polymeric template; and separating the metallic nanoparticles from the branched polymeric template (using various possible purification methods).
  • the process of these documents is about the in-situ synthesis of metallic nanocrystals in the presence of a preformed polymer and has no reference to the production of metallic oxide nanoparticles, nor to their surface modification with polymers for drug delivery and bio applications.
  • thermo-responsive polymer a continuous process for the production of magnetic nanoparticles comprising a ferrite core functionalized on its surface with a thermo-responsive polymer, comprising the steps of: a) preparing a solution of nanoparticles of: i) a ferrite pre-functionalized with an atom transfer radical polymerization initiator linker; ii) a mixture of at least two thermo-responsive oligomer molecules; iii) a catalyst of the reaction between said initiator linker and said thermo-responsive oligomer molecules; and iv) a radical reaction initiator; the solvent of the solution being selected from tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, acetonitrile, methyl cyanide, methanol, ethanol, water, ethylene glycol, and mixtures thereof; b) degassing the solution of step a); c) feeding, as a continuous flow, the degassed solution obtained in step b)
  • FIG. 1 shows a schematic representation of a preferred embodiment of apparatus for carrying out the process of the invention
  • - Fig. 2 reproduces FT-IR spectra collected on magnetic nanoparticles produced with the process of the invention and precursors of the same;
  • TEM transmission electron microscope
  • - Fig. 4 is a graph showing the particle size distribution of the magnetic nanoparticles produced with the process of the invention.
  • Fig. 5 is a graph showing the dependence on temperature of the particle size of the magnetic nanoparticles produced with the process of the invention.
  • - Fig. 6 is a graph showing the values of specific absorption rates (SAR) of magnetic nanoparticles produced with the process of the invention, measured at two values of magnetic field frequency;
  • Fig. 7 is a graph showing the increase of temperature, in three consecutive cycles, of magnetic nanoparticles produced with the process of the invention when irradiated with a magnetic field;
  • - Fig. 8 is a graph confirming the non-cytotoxicity of the magnetic nanoparticles produced with the process of the invention.
  • - Fig. 9 reproduces a picture showing the results of five tests of preparation of suspensions, one test carried out according to the invention and four tests carried out in comparative conditions.
  • MNPs that stands for magnetic nanoparticles
  • the abbreviation MNPs refers to the final product of the process, that is, the complete particles with a polymer shell encasing the magnetic material core.
  • the inventors have devised a process that allows to produce magnetic nanoparticles consisting of a ferrite core functionalized on its surface with a thermo- responsive polymer, in a continuous way and thus with an increased rate compared to the batch processes of the prior art.
  • the inventors have tried to adopt in a continuous process and apparatus the conditions developed in the past (WO 2018/138677 A1 and WO 2020/222133 A1 ), changing one parameter/condition from the prior art at a time, but these attempts had no success.
  • the present invention derives from the realization that to achieve an effective continuous process it was necessary to change simultaneously various parameters/conditions compared to those adopted in the cited prior art documents.
  • the inventors have also noted that the continuous process of the invention affords said magnetic nanoparticles with a higher degree of homogeneity as to particle size and properties.
  • the first passage of the inventions, a) consists in preparing a solution of precursors of the MNPs of the invention.
  • This solvent useful for the preparation of the solution is selected from tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetonitrile (ACN), methyl cyanide (MeCN), methanol (MeOH), ethanol (EtOH), water, ethylene glycol and mixtures thereof.
  • THF tetrahydrofuran
  • DMSO dimethyl sulfoxide
  • DMF dimethylformamide
  • ACN acetonitrile
  • MeCN methyl cyanide
  • MeOH methanol
  • EtOH ethanol
  • the first precursor of the final MNPs are pre-produced nanoparticles of a ferrite on the surface of which are present molecules of a compound adapted for chemically binding to oligomer and/or polymer compounds.
  • Ferrites are a class of magnetic compounds consisting of a mixture of iron oxides and optionally of other metals selected among Fe Mn, Co and Zn, having a high degree of magnetic response.
  • Preferred ferrites are iron (III) oxide (Fe2Os) and magnetite (FesCM).
  • nanoparticles of ferrite, and in particular the preferred ferrites of Fe2Os and FesCM are preferably produced in the shape of nanocubes; in fact, previous works of research groups of one of the present applicants have demonstrated that these nanocubes have superior magnetic heat properties in terms of specific absorption rates (SAR) compared to other nanoparticles shapes, deriving the specific cubic shape and the control of size and monodispersion (i.e. narrow size distribution) afforded.
  • SAR specific absorption rates
  • the starting ferrite nanocrystals, that form the core of the final nanoparticles may be produced according to any method known in the literature.
  • these can be produced following the procedure described in WO 2013/150496 A1 and have preferably a transmission electron microscopy TEM edge-size between about 10 and 35 nm.
  • linker molecules having a moiety that electrostatically binds (through Van der Walls force, or similar coordination mechanisms) on the surface of the metal oxide core, and a moiety adapted to react with the oligomers or polymers precursors of the thermoresponsive polymeric shell of the final MNPs.
  • the linker may be selected among phosphonic acid (PO(OH)2) based linkers, linkers with a terminal functional group such as COOH, OH- and NH2 and, preferably, catechol derivatives; catechol derivatives are compounds having a 1 ,2-dihydroxybenzene moiety modified by a radical that bears a functionality capable to react with a functional group in said oligomers or polymers precursors.
  • a preferred class of catechols for use in the present invention are the derivatives of dopamine, that is, 4-(2-aminoethyl)benzen-1 ,2-diol.
  • This precursor that is, the linker-coated ferrite nanocrystals, is present in solution at a concentration between 0.2 and 4.0 g/L of iron.
  • This range is higher than the precursor/solvent weight range taught by the available prior art (WO 2018/138677 A1 ) and is the first parameter that must be modified along with other parameters described below to obtain a successful process.
  • the second precursor is a mixture of thermo-responsive oligomer molecules.
  • Thermo-responsive oligomer molecules useful for the objects of the invention are N- isopropylacrylamide (NIPAM) derivatives, methacrylate derivatives such as methyl methacrylate (MMA) and butyl methacrylate, acrylamide derivatives and, preferably, ethylene glycol methyl ether methacrylate derivatives.
  • NIPAM N- isopropylacrylamide
  • methacrylate derivatives such as methyl methacrylate (MMA) and butyl methacrylate
  • acrylamide derivatives and, preferably, ethylene glycol methyl ether methacrylate derivatives.
  • the preferred oligomers of ethylene glycol methyl ether methacrylate are in particular diethylene glycol methyl ether methacrylate (DEGM EMA) and oligoethylene glycol methyl ether methacrylate (OEGMEMA) monomers with up to 15 ethylene glycol derived moiety units.
  • the DEGMEMA/OEGMEMA molar ratio useful for the objects of the invention is in the range 50/50 to 75/25; this means that useful ratios are the two ends of the range and the intermediate ratios 55/45, 60/40, 65/35 and 70/30.
  • This ratio is the second parameter that must be simultaneously changed compared to the teachings of WO 2018/138677 A1 to achieve a successful process; the reported ratios are calculated on the ethylene glycol monomeric units present in the oligomers, that are 2 in DEGMEMA and 3 to 15 in OEGMEMA.
  • the inventors have found that adjusting the molar ratio of DEGMEMA/OEGMEMA in the range 50/50 to 75/25 allows producing a stable aqueous suspension of MNPs, while adopting higher DEGMEMA/OEGMEMA ratios, such as the ratio 81/19 of WO 2018/138677 A1 (see Example 3 in connection with the information that the used OEGMEMA has a molecular weight of 500 Da), results in the precipitation of the MNPs.
  • DEGMEMA/OEGMEMA it is possible to tune the LOST of the polymer from 25 to 60 °C.
  • the two oligomers were present in the starting solution in volumes of 0.83 mL of DEGMEMA and 0.7 mL of OEGMEMA.
  • the third precursor is a catalyst of the polymerization reaction of the oligomers described above.
  • This catalyst may be selected among amines, imines, or copper (l/ll) halide based catalysts; examples of amines/imines are tris(2-pyridylmethyl)amine (TPMA), pentamethyldiethylenetriamine (PMDETA), hexamethyltriethylenetetramine (HMTETA), pyridineimine and 2,2'-bipyridine or their combinations; copper based catalysts may be CuBr, CuBr2, or the preferred catalyst for this step, namely, copper tris(2-dimethylaminoethyl)amine bromide, generally indicated with the abbreviated formula [Cu(MeeTREN)Br]Br.
  • the catalyst is added to the solution with a concentration between 0.05 and 0.10 M.
  • the fourth precursor is a radical reaction initiator, that may be the same molecule as the linker present on the surface of the ferrite nanocrystals.
  • Initiators useful for the objects of the invention are alkyl or aryl halides and pseudohalide derivatives; preferred initiators are ethyl a-bromophenylacetate, phenylethyl bromide, methyl 2- bromopropionate, methyl 2-bromoisobutyrate, ethyl chloroisobutyrate, and ethyl a- bromoisobutyrate;
  • a particularly preferred radical reaction initiator is 2-bromo-N-[2- (3,4-dihydroxy-phenyl)-ethyl]-butyramide, a derivative of dopamine, also referred to in the following as DOPA-BiBA.
  • This component is conveniently added to the solution at a concentration between 7 x 10’ 4 and 1 x 10’ 3 M.
  • the amount of initiator is the third parameter that must be changed, compared to the teachings of WO 2018/138677 A1 , to achieve an effective continuous process: the inventors have in fact observed that, adopting the conditions of the cited prior art in the continuous process, the result was again the precipitation of the MNPs; in the present invention, the amount of initiator is increased of about 30% compared to the conditions described in WO 2018/138677 A1 .
  • the solution is preferably prepared in separate steps, by preparing partial solutions that are then mixed in the amounts and ratios required to obtain the concentrations reported above for the different precursors.
  • first partial solution in one of the cited solvents
  • This first partial solution is degassed from oxidizing gases, for instance through sonication and nitrogen purging for about 15 minutes before mixing with a second partial solution.
  • the second partial solution contains instead the catalyst.
  • the second step of the process, b) the solution containing all the precursors (obtained for instance by mixing the two partial solutions above) is degassed for removing possible oxidizing gases. This passage takes place as indicated above for the first partial solution.
  • the degassed solution obtained in step b) is continuously fed at a first end of a reactor consisting in an elongated chamber with at least one wall transparent to UV radiation and irradiated with UV causing the polymerization of the thermo-responsive oligomer molecules.
  • the reactor chamber Before feeding the reactant solution, the reactor chamber is preferably flushed for some minutes (e.g., 15 minutes) with the same solvent, previously degassed in the same way as described above, as the solvent of the reactant solution.
  • This operation has the function of cleaning the reaction chamber and avoiding the presence of any impurities that could contaminate the final product or interfere with the polymerization reaction.
  • the wavelength range of the UV radiation useful for the objects of the invention is 315-578 nm, preferably about 365 nm.
  • the inventors have found that optimal reaction efficiency is obtained with a residence time of the solution in the reactor chamber between 30 and 150 min.
  • the flowrate of the solution fed to the reaction chamber is regulated so as to obtain a residence time of the solution in the chamber in the range above, depending on the length of the chamber.
  • the reaction temperature is between 5 and 25 °C.
  • the magnetic nanoparticles coated with a polymeric shell (MNPs), useful in theranostic applications are collected at a second end of the reactor.
  • MNPs polymeric shell
  • the apparatus comprises four main elements, that are: i) an elongated reaction chamber with at least one wall transparent to UV radiation; ii) one or more UV lamps for irradiating the solution in the reaction chamber; iii) means connected to a first end of the elongated reaction chamber for pushing the degassed solution of step b) of the process into the chamber; and iv) a tank connected to a second end of the elongated reaction chamber for collecting the magnetic nanoparticles produced with the process.
  • the means for pushing the solution into the reaction chamber can be a pump or a syringe with automated control (namely, a syringe pump), for regulating the flowrate of the solution to a value such that the residence time of the solution inside the reaction chamber is between 30 and 150 min, that is the range of duration of the reaction.
  • a pump or a syringe with automated control namely, a syringe pump
  • the automated means for pushing the solution into the chamber are preferably capable of delivering very low flowrates; commercial pumps or syringe pumps are available to this end with flowrates of 0.05 mL/min.
  • the reaction chamber may have any geometry that ensures a residence time of the reacting mixture inside the chamber at the selected flowrate.
  • the chamber may be, for instance, in the form of a channel obtained in a chip and closed with a flat slab or a tubular coil of a material transparent to UV.
  • the wall of the reactor chamber facing the UV lamp(s) must be transparent to this radiation.
  • Preferred materials for the production of this transparent wall are perfluoroalkoxy alkanes (PFA) and polytetrafluoroethylene (PTFE).
  • the thickness of the wall of the chamber has maximum value of 1 mm in the direction perpendicular to the incident UV radiation; this condition ensures that the radiation extinction due to absorbance in the first layer of solution (that is, the layers closer to the UV lamp(s)) is low, and the radiation that reaches the layers of solution farther from the UV source is still intense enough to cause the polymerization reaction.
  • the elongated reaction chamber is preferably in the form of a tube having a convoluted shape, for instance in the form of a serpentine or, preferably, helically wound around a UV lamp; this latter configuration maximizes the surface of the reaction chamber directly exposed to the UV source, thus reaching the maximum possible efficiency of the apparatus.
  • the inner diameter of said chamber is preferably in the range 0.1 -7.0 mm, more preferably of about 0.75 mm, while the wall of the reaction chamber has preferably a thickness in the range of 0.1 up to 1 mm, more preferably of about 0.85 mm.
  • Fig. 1 is a schematic representation of a preferred configuration of an apparatus of the kind described above.
  • the apparatus, 10, comprises a means 11 for pushing the degassed solution of step b) of the process into the reaction chamber 12.
  • the means 11 is represented in the form of a syringe pump, while the reaction chamber is represented as a UV- transparent tubing, that in this preferred embodiment is coiled around a UV lamp 13; at the end of the reaction chamber opposite to the end connected to the syringe 11 , there is the tank 14 for collecting the MNPs produced in the chamber 12.
  • the apparatus also comprises a second coil wrapped around the system described above, in which a refrigerant fluid is circulated, to keep the system and the reacting mixture in the desired temperature range.
  • TEM JEOL JEM 1400Plus electron microscope. This instrument is equipped with a LaBe crystal thermionic electron source having an acceleration voltage of 120 kV, and an Orius CCD Camera purchased from Gatan Company, USA.
  • FT-IR Vertex 70 (Bruker) having an ATR Golden Gate accessory.
  • This example refers to the preparation of magnetic nanoparticles according to the continuous process of the invention.
  • the apparatus depicted in Fig. 1 was used; the reaction chamber had a total inner volume of 3.1 mL.
  • DOPA-BiBA Iron oxide
  • ATRP photo-atom transfer radical polymerization
  • a syringe loaded with the reaction solvent medium (10% v/v THF/DMSO) was mounted onto the syringe pump and charged into the photo-tubular reactor (with switched off UV-lamps), to flush the tube. This step takes about 15 min and ensures that the lamps remain cool before the polymerization starts.
  • the stock solution containing reactants and catalyst was then loaded into a 24 mL plastic syringe and mounted onto the syringe pump, replacing the one bearing the blank solvent.
  • the UV-lamps were switched on, and the flow rate of the pump operated at a flow rate of 0.07 mL/min, which is equivalent to an observed reaction time of 50 minutes.
  • the obtained crude product was rinsed three times to precipitate the TR-cubes from the reaction mixture, by addition of 25.0 mL diethyl ether followed by centrifugation and 10.0 mL THF dissolution of the black gel-like pellet collected at the bottom of the falcon tube after removal of the solvent.
  • the MNPs were dried over nitrogen for 1 h before re-dispersing them in 10.0 mL Milli-Q water.
  • the result of the procedure is shown in Fig. 9: the obtained product is the transparent suspension in the first test tube from right in the figure. Then the excess free polymer molecules were removed by centrifugal filtration (1500 rpm, 10 min) done twice and the sample concentrated to about 200 pL before being dispersed in saline for further characterizations.
  • FT-IR Fourier-transform infrared
  • the process described above was repeated three times.
  • the yield of the process calculated as ratio of grams of iron in the recovered product over the grams of iron used in the process and averaged over the three runs, resulted 72.8 ⁇ 3.8%.
  • the product obtained in this Example is specimen 1 .
  • Example 1 For comparative purposes, the batch procedure of WO 2020/222133 A1 was followed, using the same starting materials and solutions of Example 1. The only difference was that, due to higher efficiency and kinetics of the continuous process of Example 1 , the reaction time was increased to 150 minutes. The result of the test is shown in Fig. 9 and is represented by the first test tube on the left in the picture; the MNPs are essentially completely precipitated and separated from the limpid solution above.
  • Example 1 For comparative purposes, the procedure of Example 1 was repeated adopting the process parameters of WO 2020/222133 A1 , namely, a DEGMEMA/OEGMEMA molar ratio equal to 81/19, higher than that of the present invention.
  • the result of the test is shown in Fig. 9 and is represented by the second test tube from right in the picture; the MNPs are essentially completely precipitated and separated from the limpid solution above.
  • This example refers to the morphological and polydispersity characterization of the MNPs produced according to the invention.
  • MNPs of specimen 1 were observed to be easily water dispersible and characterized by TEM; the obtained TEM picture is reproduced in Fig. 3, the inset representing a magnification of part of the picture.
  • the MNPs show a clear cubic shape, with size of about 20 nm.
  • MNPs of specimen 1 were deposited on the TEM grid with a 1 % uranyl acetate solution, that made the polymer shell on single nanocubes more visible: in these conditions, the polymer shell has a darker contrast.
  • the hydrodynamic size (Dh) of the MNPs of specimen 1 was further determined using dynamic light scattering (DLS) technique.
  • the measurements were conducted at 25 °C using a Malvern ZetasizerNano series instrument and the scattered light was detected at an angle of 173°.
  • 500 pL of MNPs sample in saline was pipetted into a disposable cuvette and triplicate readings were made following an equilibration time of
  • LCST lower critical solution temperature
  • the DLS technique was employed to measure the change in hydrodynamic size (Dh) of MNPs in solution as a function of temperature.
  • the test was carried out on specimen 1 produced according to the invention. The results of the test are reproduced in the graph in Fig. 5: as is clear from the graph, the size of the MNPs remains essentially unchanged for temperatures below about 41 -42 °C, and the LCST is of about 43 °C.
  • Example 1 The procedure of Example 1 was repeated adopting process parameters derived from WO 2020/222133 A1 ; in particular, 1 .6 mg of free DOPA-BiBA initiator (lower than in Example 1 ) was used.
  • the result of the test is shown in Fig. 9 and is represented by the second test tube from left in the picture; the MNPs are essentially completely precipitated and separated from the limpid solution above.
  • Example 1 The procedure of Example 1 was repeated adopting process parameters derived from WO 2020/222133 A1 ; in particular, a weight ratio between the sum of reactants and the solvent equal to 11 .2 was used.
  • the result of the test is shown in Fig. 9 and is represented by the central test tube in the picture; as can be seen in the figure, the MNPs are not dispersible in water.
  • the magnetic heating losses of a part of specimen 1 was first performed by calorimetric measurements to determine the specific absorption rates (SAR) values, which measures the heating efficiency of the magnetic nanoparticles.
  • SAR is defined as the power absorbed per mass of the heat mediator (expressed here as Watt per gram of iron, W/g Fe).
  • MHT field conditions were used (frequency: 111 and 182 kHz; field: 24 kA/m) on a sample of specimen 1 dissolved in saline solution (0.9% NaCI).
  • the SAR value of the MNPs of the invention is about 150 and 260 W/g Fe at frequency of 111 and 182 kHz, respectively (Fig. 6). These values are completely comparable to those reported for MNPs prepared by in-batch synthesis.
  • the sample of specimen 1 was subjected to three cycles of magnetic hyperthermia of 30 minutes each, and the temperature was recorded during the hyperthermia cycle using an optical fiber probe while switching on and off the AMF.
  • the sample of specimen 1 was diluted at 2.5 g/L [Fe] in saline solution and exposed to a clinically accepted MHT condition (field intensity of 15 kA/m, frequency 182 kHz). The results of the test are reproduced in Fig. 7. Within 30 minutes when the AMF was on, a therapeutic temperature of 46 °C was reached in few minutes and it was maintained constant until the MHT was switched off.
  • the MHT cycle could be repeated for three times in a row with just 5 minutes in between without losing the ability of the MNPs of specimen 1 to reach the 46 °C temperature under the same AMF conditions. This suggests that the MNPs of the invention are stable and do not aggregate during heating, which would lead to a reduced or compromised heating performance.
  • This example is directed to verifying the biocompatibility of the MNPs prepared according to the invention; this is a necessary condition for the nanoparticles to be admitted as a possible clinical candidate to mediate cancer treatment.
  • example 1 invention
  • example 2 comparatives the comparison between the results obtained in example 1 (invention) and example 2 (comparison) shows a yield of the process of the invention higher of about 40%, obtained with a duration of the process that is one third, compared to the yield and duration of the known in-batch process.

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Abstract

L'invention concerne un procédé continu pour la production de nanoparticules de ferrite dont la surface est fonctionnalisée avec des fractions polymères thermosensibles, utiles dans des applications thérapeutiques et diagnostiques dans une thérapie anticancéreuse. L'invention concerne également un appareil destiné à réaliser le procédé continu.
PCT/EP2024/066274 2023-06-14 2024-06-12 Procédé de production continue de nanoparticules de ferrite fonctionnalisées par un polymère Pending WO2024256488A1 (fr)

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Citations (5)

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WO2011147926A2 (fr) 2010-05-26 2011-12-01 Erik Reimhult Structures de membranes à réaction magnétique
WO2013150496A1 (fr) 2012-04-06 2013-10-10 Fondazione Istituto Italiano Di Tecnologia Procédé de préparation des nanocristaux de ferrite
WO2018138677A1 (fr) 2017-01-27 2018-08-02 Fondazione Istituto Italiano Di Tecnologia Procédé de synthèse de nanoparticules magnétiques sensibles à un stimulus
US20180339914A1 (en) 2017-05-23 2018-11-29 Tuskegee University High-throughput synthesis of metallic nanoparticles
WO2020222133A1 (fr) 2019-04-30 2020-11-05 Fondazione Istituto Italiano Di Tecnologia Méthode de préparation à l'échelle du gramme de nanocristaux cubiques de ferrite pour des applications biomédicales

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011147926A2 (fr) 2010-05-26 2011-12-01 Erik Reimhult Structures de membranes à réaction magnétique
WO2013150496A1 (fr) 2012-04-06 2013-10-10 Fondazione Istituto Italiano Di Tecnologia Procédé de préparation des nanocristaux de ferrite
WO2018138677A1 (fr) 2017-01-27 2018-08-02 Fondazione Istituto Italiano Di Tecnologia Procédé de synthèse de nanoparticules magnétiques sensibles à un stimulus
US20180339914A1 (en) 2017-05-23 2018-11-29 Tuskegee University High-throughput synthesis of metallic nanoparticles
US20210261431A1 (en) 2017-05-23 2021-08-26 Tuskegee University High-throughput synthesis of metallic nanoparticles
WO2020222133A1 (fr) 2019-04-30 2020-11-05 Fondazione Istituto Italiano Di Tecnologia Méthode de préparation à l'échelle du gramme de nanocristaux cubiques de ferrite pour des applications biomédicales

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