Polymeric nanoparticles, process for producing them and use thereof in nanomedicine

Abstract

The present invention relates to nanoparticles formed by a hollow polymeric wall, capable of transporting drugs and active substances, or contrast media, across cellular membranes and releasing them at sites of interest. The nanoparticles are formed by self-assembly of poly(isobutene-alt-maleic anhydride) functionalised with an aminosaccharide.

Description

[0001] POLYMERIC NANOPARTICLES, PROCESS FOR PRODUCING THEM AND USE THEREOF IN NANOMEDICINE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of nanomedicine, and in particular to carriers having nanoscale dimensions, capable of transporting drugs and active substances, or contrast media, through cellular membranes and releasing them at sites of interest.

[0004] BACKGROUND ART

[0005] Nanomedicine is a branch of medicine which exploits nanotechnologies in general. A particularly important field is that of nanoparticles, i.e. particles having dimensions typically less than 100 nm and in any case less than 1 pm, which can be built or functionalized so as to transport pharmacologically active molecules, or other species useful in diagnostics, to the treatment target cells, without involving healthy cells or organs, thereby reducing or eliminating the side effects of the treatments. An example of nanoparticles employed in the medical field are liposomes, essentially spherical structures consisting of a phospholipid bilayer defining a cavity, generally containing an aqueous solution in which an active substance is present; thereby the antitumor liposomal Doxorubicin is prepared, for example.

[0006] Other particles which find use in the medical field are polymersomes, analogous to liposomes but in which the surface of the particle consists of polymers.

[0007] Nanoparticles loaded or functionalized with active species are particularly employed in the field of bioimaging and in the treatment of tumors.

[0008] The related patent and non-patent literature is highly extensive, and only some examples of nanoparticles studied for use in the medical field are reported below.

[0009] Patent application WO 2011 / 147926 A2 describes liposomes, i.e. essentially spherical nanoparticles consisting of a phospholipid bilayer, in which drugs can be loaded, for example in an aqueous solution present in the cavity of the liposome. In this document, magnetic particles are present in the lipid bilayer, which can be activated from outside the body by destabilizing the bilayer itself and thus causing the release of the drug in the area of the body stimulated with magnetic fields.

[0010] Patent EP 3873535 Bl describes nanoparticles formed by self-assembly of the protein ferritin, in particular in the long-chain form (H-ferritin), the internal cavity of which can be loaded with transition metals, drugs, fluorescent molecules or contrast media. Ferritin nanoparticles are stable at pH values close to neutrality, while they are disassembled at acidic (2-3) or basic (10-12) pH; since the cytosol of tumor cells is more acidic than that of healthy cells, this feature of ferritin nanoparticles allows the release of the transported species only into the target cells.

[0011] Finally, the article “Rationally designed oxaliplatin-nanoparticle for enhanced antitumor efficacy”, A. Paraskar et al., Nanotechnology, 2012 Feb 24, 23(7):075103, describes nanoparticles formed from a polymer comprising monomer units of polyisobutene / maleic anhydride functionalized with glucosamine to open the maleic anhydride ring and allow the complexation of the platinum atom of the compound cyclohexanediamineplatinum(II) dihydroxide, obtained from the corresponding chloride. The thus-obtained functionalized and derivatized polymer self-assembles in the form of nanoparticles, which are described in the article as having more powerful antitumor effects, and with fewer side effects, compared to the reference compound oxaliplatin.

[0012] Similarly, US patent application 201218957A1 discloses a polymer comprising monomer units of poly(isobutene-maleic anhydride) functionalized with glucosamine to open the maleic anhydride ring and allow the complexation of the platinum atom of square planar Pt(II) complexes. The thus-obtained functionalized and derivatized polymer self-assembles in the form of nanoparticles, which are employed as carriers for platinum-based antitumor agents.

[0013] Despite the considerable milestones already achieved, the need remains in the field to have nanoparticles with improved features available, in particular of selectivity towards the cells reached, to allow releasing drugs or other useful species exclusively into tumor cells.

[0014] It is the object of the present invention to provide a new class of nanoparticles for the transport and selective release of contrast media, small molecules and / or drugs into tumor cells.

[0015] SUMMARY OF THE INVENTION In the first aspect thereof, the present invention relates to nanoparticles formed by selfassembly of the polymer poly(isobutene-aZz-maleic anhydride), in which a percentage between 1 and 25% of the maleic anhydride units are functionalized by reaction with an amino saccharide or with an aminosaccharide and N-(4-aminobutyl)guanidine, said nanoparticles containing in the internal cavity thereof one or more active compounds as imaging media or as antitumor agents.

[0016] In the second aspect thereof, the present invention relates to a process for preparing the nanoparticles loaded with active compounds as described above.

[0017] In the third aspect thereof, the present invention relates to a nanoparticle obtainable through the process of the present invention.

[0018] Finally, in the fourth aspect thereof, the present invention relates to the use of the nanoparticles in diagnostic and therapeutic methods.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Fig. 1 shows the IR spectra of the unfunctionalized polymer poly(isobutene-aZz-maleic anhydride), and of the same polymer functionalized with glucosamine at two different functionalization percentages.

[0021] Fig. 2 shows the IR spectra of the unfunctionalized polymer poly(isobutene-aZz-maleic anhydride), and of the same polymer functionalized with glucosamine and agmatine.

[0022] Fig. 3 shows the1H-NMR spectrum of the polymer poly(isobutene-aZz-maleic anhydride) functionalized with glucosamine at 12.5% of functionalized maleic anhydride units, in DMSO- de.

[0023] Fig. 4 shows a microphotograph obtained by transmission electron microscopy (TEM) of a sample of nanoparticles of the invention.

[0024] Fig. 5 depicts a size distribution histogram of the nanoparticles identified in the microphotograph in Fig. 4.

[0025] Figs. 6 and 7 show two size distribution charts of nanoparticles of the invention obtained with nanoparticle tracking analysis (NTA), for the polymer functionalized with glucosamine and for the polymer functionalized with glucosamine and agmatine, respectively. Fig. 8 shows a comparative size distribution chart of nanoparticles of the invention (functionalized at 12.5% and 25.0% of the maleic anhydride units) and not of the invention (functionalized at 50.0% and 75.0% of the maleic anhydride units), obtained by NTA.

[0026] Fig. 9 shows a comparative size distribution chart of nanoparticles not of the invention (functionalized at 50.0% and 75.0% of the maleic anhydride units) using a greater enlargement scale on the y axis compared to Fig. 8, obtained by NTA.

[0027] Fig. 10 shows a photograph of a dispersion sample of nanoparticles of the invention, functionalized at 12.5% of the maleic anhydride units, obtained by NTA.

[0028] Fig. 11 shows a photograph of a dispersion sample of nanoparticles of the invention, functionalized at 25.0% of the maleic anhydride units, obtained by NTA.

[0029] Fig. 12 shows the image of the agarose gel related to the qualitative assessment of the encapsulation of the siRNA in the nanoparticles.

[0030] Fig. 13 shows a chart of the results of a toxicity study conducted in vitro on the healthy fibroblast cell line, such as NIH-3T3, and on the glioblastoma tumor cell line, such as U87.

[0031] Fig. 14 shows a chart of the results, for cells not treated with labeled nanoparticles of the invention, of an uptake experiment of nanoparticles of the invention by tumor cells by flow cytometry.

[0032] Fig. 15 shows a chart of the results, for cells treated with labeled nanoparticles of the invention at a concentration equal to 0.5 pg / mL, of an uptake experiment of nanoparticles of the invention by tumor cells by flow cytometry.

[0033] Fig. 16 shows a chart of the results, for cells treated with labeled nanoparticles of the invention at a concentration equal to 5 pg / mL, of an uptake experiment of nanoparticles of the invention by tumor cells by flow cytometry.

[0034] Fig. 17 shows a chart of the results, for cells treated with labeled nanoparticles of the invention at a concentration equal to 25 pg / mL, of an uptake experiment of nanoparticles of the invention by tumor cells by flow cytometry.

[0035] Fig. 18 shows a chart of the results, for cells treated with labeled nanoparticles of the invention at a concentration equal to 50 pg / mL, of an uptake experiment of nanoparticles of the invention by tumor cells by flow cytometry.

[0036] Fig. 19 shows an image obtained with a confocal microscope in an uptake experiment of nanoparticles of the invention by tumor cells.

[0037] Fig. 20 shows an image obtained with a confocal microscope in an uptake experiment of nanoparticles of the invention by tumor cells.

[0038] Fig. 21 shows an image obtained with a confocal microscope in an uptake experiment of nanoparticles of the invention by tumor cells.

[0039] Fig. 22 shows the in vivo concentration of the nanoparticles of the invention in Balb / C mice as a function of time.

[0040] Fig. 23 shows the in vivo biodistribution of the nanoparticles of the invention in Balb / C mice.

[0041] Fig. 24 shows the in vivo biodistribution of the nanoparticles of the invention in NOD / SCID mice engrafted with tumor cells.

[0042] Figs. 25-27 show the ex vivo biodistribution of the nanoparticles of the invention in NOD / SCID mice engrafted with tumor cells.

[0043] DETAILED DESCRIPTION OF THE INVENTION

[0044] In the following description the following definitions are adopted:

[0045] - room temperature: it is meant a temperature between 22 and 27 °C;

[0046] - ambient pressure: it is meant a pressure in the range from 980 to 1030 hPa;

[0047] - under vacuum: it is meant a pressure less than ambient pressure;

[0048] - small molecule: it is meant a single molecule with a molecular weight of less than 1000 g / mol, in accordance with Macielag, M. J., 2012, “Chemical properties of antibacterials and their uniqueness” in Dougherty TJ, Pucci MJ (eds.). Antibiotic Discovery and Development, Springer, pp. 801-802. ISBN 978-1-4614-1400-1;

[0049] - in the formation of the polymer poly(isobutene-a / z-maleic anhydride), the carboncarbon double bond of maleic anhydride is engaged in the reaction with the double bond of isobutene, and it is no longer present in the final polymer which therefore exhibits succinic anhydride units; for simplicity and uniformity of nomenclature, however, these units will always be designated in the remainder of the description as maleic anhydride units, meaning the monomer from which they are derived; and

[0050] - the polymer poly(isobutene-aZz- maleic anhydride) will hereinafter also be referred to for brevity as the “base polymer”.

[0051] The present inventors have surprisingly observed that the polymer poly(isobutene-aZz- maleic anhydride), when functionalized with an aminosaccharide, or with an amino saccharide and N-(4-aminobutyl)guanidine, in an overall percentage between 1 and 25% of the units derived from maleic anhydride, exhibits optimal properties of capability of self-assembly in solution in the form of nanoparticles, as will be apparent from the experimental section.

[0052] The repeating unit of the base polymer, poly(isobutene-aZz-maleic anhydride), has the following structure: where n is between 20 and 60, preferably between 35 and 45.

[0053] Polymers of this type are commercially available; for example, a poly(isobutene-aZz- maleic anhydride) polymer with a weight-average molecular weight of about 6000 Da, equivalent to a mean value of n = 39, is available from Sigma-Aldrich with catalog number 531278.

[0054] For the purposes of the invention, the base polymer is functionalized with an amino saccharide and optionally is further functionalized with N-(4-aminobutyl)guanidine, obtaining a polymer having the following general formula: where R is a monosaccharide or disaccharide residue, R’ is the-(CH2)4-NH-C(NH)-NH2 residue of N-(4-aminobutyl)guanidine, n is between 20 and 60, x’ is between 0.01 and 0.25, z is a number between 0 and 0.24, the sum of x’ and z is a number between 0.01 and 0.25 and y = 1- (x’ + z).

[0055] In a preferred embodiment of the invention, the base polymer is functionalized with an aminosaccharide, i.e. a molecule derived from a saccharide (molecules also known as carbohydrates or sugars) in which a hydroxyl group is replaced with an amino group.

[0056] The functionalization occurs through the formation of an amide between the amino group of the amino saccharide and a carbonyl carbon of the maleic anhydride unit, according to the following scheme: where R is the aminosaccharide radical and x is a number between 0.01 and 0.25 and y is the complement to 1 of x (i.e., y = 1-x); the above reported formula of the functionalized polymer is a general formula, only indicative of the component units and of the quantitative ratio thereof, but does not imply the regular alternation of reacted and unreacted units (a situation which would not satisfy the condition that x be equal to at most 0.25).

[0057] The aminosaccharide can be a monosaccharide or a disaccharide. Glucosamine is preferred for the functionalization of the base polymer, in particular in the form 2-D-(+)- glucosamine, a compound of wide commercial availability having the structural formula reported below:

[0058] For the functionalization reaction of the base polymer, the amino saccharide is simply added to the polymer solution, in the desired stoichiometric ratio between maleic anhydride units and aminosaccharide, at room temperature in an anhydrous solvent; the reaction is conducted under a moisture-free inert atmosphere, and requires a few hours for completion. The reaction solvent can be any organic solvent in which the base polymer and the amino saccharide are soluble; preferred solvents are dimethylformamide (DMF) and tetrahydrofuran (THF).

[0059] In the case of glucosamine, which is commonly sold in the form of a salt thereof, typically sulfate or hydrochloride, a stoichiometric excess of base with respect to the salt is added to the solution to release the amino function, thus making it available for the reaction; a base useful for this purpose is triethylamine, which can be employed in a stoichiometric ratio between 1.1 and 1.5, preferably 1.2-1.3, with respect to glucosamine.

[0060] The reagents are employed at a concentration between 20 and 25 g / L for the base polymer, and between 3.5 and 4 g / L for the aminoglucoside.

[0061] Fig. 1 shows three IR spectra of the base polymer (upper line in the figure), of the polymer in which 12.5% of the maleic anhydride units are functionalized with glucosamine (intermediate line), and of the polymer in which 25% of the maleic anhydride units are functionalized with glucosamine (lower line). The presence of unreacted anhydride units is confirmed by characteristic bands at 1850 and 1770 cm'1(symmetric and asymmetric stretching). With increasing functionalization, the spectrum changes, showing the appearance of a broad band at 3400 cm'1, attributable to the stretching of the -OH groups of the sugar. Furthermore, the ring opening of the anhydrides, resulting from the functionalization, causes the appearance of bands at 1704 and 1563 cm'1assigned respectively to the stretching of the carboxylic acid C=O and to the stretching of the secondary amide. These spectra confirm the occurrence of the functionalization of the polymer under the reaction conditions described above.

[0062] Fig. 3 shows the1H-NMR spectrum of the polymer poly(isobutene-a / z-maleic anhydride) functionalized with glucosamine at 12.5% of functionalized maleic anhydride units, in DMSO- de. Said spectrum indicates the presence of an amide bond formed between glucosamine and the base polymer, highlighted by the presence of a broad multiplet signal in the region from 7.0 to 7.5 ppm.

[0063] In another preferred embodiment of the present invention, the base polymer is further functionalized with N-(4-aminobutyl)guanidine (also known by the common name “agmatine”), according to the following scheme: in which R is the amino saccharide radical, R’ is the -(CH2)4-NH-C(NH)-NH2 radical of agmatine, x’ is a number between 0.01 and 0.24, z is a number between 0.01 and 0.24, the sum of x’ and z is a number between 0.02 and 0.25, and y is the complement to 1 of x’ + z. Similarly to the previous case, the above formula of the polymer with double functionalization is a general formula which only indicates the type of units forming it and the quantitative ratios thereof, but does not imply a precise sequence of such units, which are distributed in a random manner along the polymer chain.

[0064] In the case of functionalization with both aminosaccharide and agmatine, the reaction can be conducted in two subsequent steps, i.e. first the functionalization with the aminosaccharide and then that with agmatine or vice versa, but preferably the reaction is carried out in a single operation. Agmatine is also generally sold and stored in the form of a salt thereof, typically dihydrochloride; therefore, also in this case the amino function must be released for the reaction, which is achieved with an amount of base (e.g., triethylamine) in a slight stoichiometric excess with respect to the amount of agmatine dihydrochloride; in the preferred case of simultaneous reaction of the base polymer with the aminoglycoside and agmatine, the amount of base to be employed for the release of the salts is the sum of the amounts required to release the two different reagents.

[0065] Fig. 2 shows the IR spectra of the base polymer (upper line in the figure), and of the polymer in which 6.25% of the maleic anhydride units are functionalized with glucosamine and 6.25% of the maleic anhydride units are functionalized with agmatine (lower line). Also in this case, the spectrum shows the appearance of a broad band at 3400 cm'1, attributable to the stretching of the sugar -OH groups and of the amino groups present in agmatine, again confirming the occurrence of the functionalization of the polymer under the reaction conditions described above.

[0066] The reaction between the base polymer and glucosamine is also described in the previously cited article by A. Paraskar et al. In that case, however, the carboxyl group and the carbonyl of the amide group which are formed by opening the ring of the maleic anhydride are employed in the complexation of the platinum atom of the compound oxaliplatin; in contrast, in the present invention these groups remain free and contribute to the balance of hydrophilic / hydrophobic properties of the functionalized polymer, allowing the self-assembly thereof into nanoparticles in aqueous solution.

[0067] The functionalized polymer thus obtained is employed for the preparation of nanoparticles loaded with active species; the loaded nanoparticles and the method for the production thereof form the first and the second aspect of the present invention, respectively, which being closely related are described together.

[0068] The loaded nanoparticles of the present invention are produced starting from a solution containing the polymer functionalized with the amino saccharide and optionally with agmatine and the active species to be incorporated into the nanoparticle in an organic solvent. The solvent is selected from solvents that are miscible with water; the preferred solvents include, for example, DMF, THF or dimethylsulfoxide (DMSO).

[0069] The functionalized polymer can be isolated and dried after the preparation thereof, for example for purification purposes from unreacted compounds, and re-dissolved in the selected solvent together with the active species. Preferably, however, when operating according to an alternative embodiment, the solution in which the nanoparticles are formed is the same solution resulting from the functionalization process of the base polymer; this alternative is preferred because it allows avoiding an intermediate step and simplifying the overall process; a final purification step, in any case necessary also after the production of the nanoparticles, also removes the residues of the previous step of functionalizing the base polymer.

[0070] Whether the solution in which the functionalized polymer has been produced is used, or whether the latter is separated from the initial solution and re-dissolved in solvent, the concentration of the functionalized polymer in the solution in which the nanoparticles are produced is between 23 and 30 g / L, while the active species is added to the solution at a concentration between 0.8 and 1.5 g / L. As said, the active species can be any molecule useful as a fluorescent marker for imaging diagnostics or having pharmacological activity, in particular cytotoxic activity against tumor cells. The active species can be selected for example from metal ions, inorganic colloidal nanoparticles, small molecules, oligonucleotides and / or peptides. Examples of molecules employed in imaging are Coumarin 6, manganese (II) phthalocyanine, pyrene, 1 -pyrenebutyric acid, Rhodamine 6G and indocyanine; example of molecules with pharmacological activity are oxaliplatin, doxorubicin, curcumin, cisplatin, paclitaxel, RNA fragments (siRNA, shRNA), DNA, therapeutic peptides. The polymer functionalized with only the aminoglycoside is suitable for the incorporation of individual molecules, while the polymer with dual functionalization with aminoglycoside and agmatine is more effective in the incorporation of RNA, DNA and therapeutic peptides.

[0071] Advantageously, the nanoparticles according to the present invention allow an effective solubilization of hydrophobic active species and a safe administration thereof, for example of paclitaxel, the known side effects of which are mainly due to the administration thereof into the bloodstream as a dispersion in castor oil.

[0072] The loading of the nanoparticle with platinum-based active species is also described in US patent application US 2012189571A1, cited above. In that case, however, the carboxyl group and the carbonyl of the amide group which form upon the opening of the maleic anhydride ring are employed in the complexation of the platinum atom, since the nanoparticle loaded with the platinum-based active species is formed with the active species in aqueous solution; in contrast, in the present invention, since the nanoparticle loaded with the platinumbased active species is formed with the polymer and the active species in organic solution, the platinum-based active species is embedded in the internal cavity of the nanoparticle, with the carboxyl group and the carbonyl of the amide group remaining free and contributing to the balance of hydrophilic / hydrophobic properties of the functionalized polymer, allowing the selfassembly thereof in the form of nanoparticles in aqueous solution.

[0073] The solution containing the functionalized polymer and the active species can be used in the preparation of the loaded nanoparticles of the invention according to two alternative modes.

[0074] According to the first possibility, the nanoparticles are produced in “batch” mode, i.e. discontinuously. Operating according to this mode, a sugar, preferably D-(+)-glucose at a concentration between 3.2 and 3.8 g / L is added as a thickening agent to the solution obtained as described above. The inventors have observed that the addition in solution of a thickening agent favors obtaining monodisperse and stable nanoparticles.

[0075] Ultrapure water is added to this solution by dripping under ultrasonic stirring; preferably the purified and deionized water known as MilliQ® is employed obtained by means of the automated systems produced and sold by Millipore Corporation.

[0076] The nanoparticles form spontaneously as a result of the balance of hydrophobic and hydrophilic properties of the functionalized polymer described above: with aminosaccharide functionalization percentages above 25%, the functionalized polymer is essentially hydrophilic, which does not allow the self-assembly of the polymer in the form of nanoparticles, as will be apparent from the experimental section.

[0077] The nanoparticles are obtained when the volume of added water is between 8 and 10 times the volume of the initial solution containing the functionalized polymer and the active species, a condition the implementation of which conventionally requires between 15 and 45 minutes.

[0078] In the second operating mode, the production of the nanoparticles is carried out continuously using microfluidic systems, which allow automating the production and increasing the hourly yield of product. The inventors verified this possibility by employing Dolomite Microfluidic and Sunshine systems, both sold by the company Alfatest of Cernusco sul Naviglio (MI); using these systems, the inventors produced nanoparticles by operating with a liquid flow rate between 1 and 12 mL / min and a water: organic solvent volume ratio between 1 and 10. By operating under microfluidic conditions and with microfluidic systems, the addition of a thickening agent is not required.

[0079] The final solution, either resulting from a “batch” process or microfluidic methods, is further filtered to recover the nanoparticles and to eliminate or minimize the amount of residual organic solvent.

[0080] The nanoparticles thus obtained, loaded with the active species of interest, exhibit good monodispersity, i.e. a monomodal size distribution within a narrow range, from 50 to 150 nm. Furthermore, analyses performed on samples of nanoparticles obtained with the method of the invention have shown that these do not exhibit aggregation phenomena, which could lead to the formation of particles of larger size and consequent difficulties, for example, in crossing cellular membranes.

[0081] Preferably, the nanoparticle according to the present invention has a dimensional mean value from 50 to 150 nm, more preferably from 75 to 100 nm, even more from 80 to 90 nm, as determined by at least one technique selected from the group consisting of nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS).

[0082] Analyses carried out by the inventors on the nanoparticles of the invention have shown that the loading of active species is generally no more than 17% by weight, with respect to the weight of the complete nanoparticle, a value corresponding to loadings indicatively between 30,000 and 80,000 molecules of active species per nanoparticle depending on the molecular weight.

[0083] The third aspect of the present invention relates to a nanoparticle obtainable through the process according to the present invention.

[0084] All the preferred aspects concerning the nanoparticle according to the present invention and the process according to the present invention also apply to the nanoparticle obtainable through the process according to the present invention and are not repeated here.

[0085] In a preferred embodiment of the invention, the active species is selected from the group consisting of oxaliplatin and cisplatin and said active species is encapsulated within the nanoparticle and is not complexed by the carboxyl and carbonyl groups of the amide group of the functionalized poly(isobutene-aZz-maleic anhydride) polymer.

[0086] Advantageously, since the nanoparticle loaded with the platinum-based active species is formed with the polymer and the active species in organic solution, the platinum-based active species is incorporated within the nanoparticle, with the carboxyl group and the carbonyl of the amide group remaining free and contributing to the balance of hydrophilic / hydrophobic properties of the functionalized polymer, allowing the self-assembly thereof in the form of nanoparticles in aqueous solution.

[0087] The fourth aspect of the present invention consists in the use of the above described nanoparticles in medical diagnostics and in the treatment of tumors.

[0088] The inventors have observed that functionalization of a well-defined fraction, between 0.01 and 0.25, of anhydride groups with amino saccharide molecules or with amino saccharide and agmatine, leads to a highly stable self-assembled nanostructure in aqueous solutions, even at high ionic strength, including serum and plasma. Furthermore, the presence of amino saccharide groups on the surface of the nanoparticles allows imparting thereto the properties of bioinvisibility (a feature referred to in the field by the term “stealth”) towards the cells of the immune system and specific tropism towards tumor cells which have high affinity for glucose and the analogs thereof.

[0089] The nanoparticles of the invention exhibit an excellent degree of biocompatibility both in vitro and in vivo, excellent colloidal stability and long-term stability during in vivo circulation, without premature release of the active species. Furthermore, the nanoparticles of the invention have the ability to cross the blood-brain barrier and have pH-dependent surface charge inversion features, capable of actively promoting endosomal escape and the release of drugs into the cytosol. This combination of features allows having a circulation of the nanoparticles in bodily fluids without elimination by the immune system, and the selective accumulation thereof in tumor cells, with rapid elimination of the nanoparticles in excess through the liver. Selectivity for tumor cells is due to the presence of glucose in the particles; indeed, the sugar avidity of tumors with respect to healthy cells is confirmed by the clinical use of glucose-based tracers for tumor imaging (e.g., glucose-F used for PET). The surface charge inversion of the nanoparticles during endosomal progression promotes endosomal escape and the release of the load at the cytosolic level into tumor cells.

[0090] The nanoparticles loaded with active species of the invention can find broad use in the field of nanomedicine to address the lack of effective therapies for certain forms of tumors, especially those lacking specific receptors directly targetable by antibodies with antitumor action.

[0091] Tests performed by the inventors to evaluate the biodistribution of the nanoparticles of the invention in an NSG (NOD scid gamma) mouse model subcutaneously injected with human (MDA-MB-231 cells) TNBC (triple-negative breast cancer) have confirmed the spontaneous tropism, with selective accumulation in the tumor. The isolation of the cells within the tumor tissue revealed that the nanoparticles had entered the cells and the related fluorescent signal was identified within the cellular cytoplasm.

[0092] The nanoparticles according to the present invention can be formulated in different manners, for example they can be administered in the form of tablets, capsules, granules, effervescent granules, pearls, drops, sticks, syrups and sachets or injectable solution.

[0093] The invention will be further illustrated by the following experimental section.

[0094] INSTRUMENTS, METHODS AND EXPERIMENTAL CONDITIONS

[0095] The NTA analyses were performed using an instrument NanoSight NS300 - Malvern.

[0096] The DLS analyses were performed using an instrument Zetasizer Nano ZS ZEN3600, Malvern

[0097] TEM micrographs were obtained using a Jeol JEM 2100Plus instrument by operating at an acceleration voltage of 200 kV and provided with a Gatan Rio9 9 MP CMOS (complementary metal oxide semiconductor) digital camera (Gatan, Inc., Pleasanton, CA, USA).

[0098] The IR analyses were performed with a Bruker Invenio®-S FTIR Spectrometer.

[0099] The1H-NMR analyses were performed using a Bruker Avance NEO 400 NMR.

[0100] The fluorescence emission measurements were obtained by means of FluoroMax-4 spectrofluorometer - HORIBA.

[0101] The absorbance was measured using a plate reader EnSight™ - PerkinElmer.

[0102] The analysis of the agarose gel was performed using an Amersham Imager (Cytiva).

[0103] The analyses to evaluate the in vitro uptake were performed by flow cytometry - Gallios Flow Cytrometer Beckman Coulter Inc., Brea, CA, USA. Using the FlowJo software (Tree Star, Ashland, OR, USA) for data analysis.

[0104] The fluorescence emission images pertaining to the in vitro experiments were acquired by means of a Nikon AIR confocal microscope.

[0105] The epifluorescence images were acquired by means of an IVIS Lumina imaging system (Perkin Elmer).

[0106] The compounds poly(isobutene-alt-maleic anhydride) (weight-average molecular weight -6000), triethylamine, anhydrous dimethylformamide (DMF), D-(+)-Glucose, 2-D-(+)- glucosamine hydrochloride, Manganese(II) phthalocyanine, Coumarin 6, pyrene, 1- pyrenebutyric acid, Rhodamine 6G, L-Glutamine were purchased from Sigma-Aldrich. Phosphate buffered saline (PBS), Dulbecco’s Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), Penicillin-Streptomycin were purchased from EuroClone. Ammonium-chlorine- potassium lysing buffer (ACK) and Hoechst dye were purchased from ThermoFisher. The water used in all processes was purified using the MilliQ® Millipore system.

[0107] EXAMPLE 1

[0108] This example relates to the functionalization of the polymer poly(isobutene-a / z-maleic anhydride) with an amino saccharide and to the production of the nanoparticles of the invention.

[0109] Operating under an inert, moisture-free atmosphere, 1.92 mg of 2-D-(+)-glucosamine hydrochloride were dissolved in 0.5 mL of anhydrous dimethylformamide; 1.08 mg of triethylamine were added to this solution, corresponding to a stoichiometric excess of 1.2 with respect to glucosamine. The solution was maintained at 25 °C for 30 minutes. 11 mg of poly(isobutene-alt-maleic anhydride) were then added to this solution, and the solution was maintained at 25 °C under gentle stirring for 12 hours (overnight). With the indicated amounts of reagents, a degree of functionalization of the polymer equal to about 12.5% was obtained. An IR spectrum of the thus obtained functionalized polymer was recorded, reproduced as an intermediate line in Fig. 1.

[0110] The obtained solution was used directly in the next nanoparticle formation step.

[0111] While continuing to operate under an inert and moisture-free atmosphere, 0.5 mg of the fluorophore compound Coumarin 6, and then 1.1 mL of a solution of D-(+)-glucose in anhydrous dimethylformamide (concentration 52.4 g / L) were added to the solution obtained in the previous step, and the system was left to stand for 30 minutes at room temperature.

[0112] 13.65 mL of MilliQ® water were then added to this solution over 30 minutes, dropwise and under ultrasonic stirring, thus obtaining a colloidal suspension of nanoparticles.

[0113] The solution was filtered by means of an Amicon Ultra 3 kDa MWCO centrifugal filter, using MilliQ® water at each washing, recovering 5-6 mg of nanoparticles of the invention.

[0114] To confirm the formation of the nanoparticles, the material recovered from the filter was examined by transmission electron microscopy (TEM), obtaining the photograph reproduced in Fig. 4, which indeed shows nanoparticles with essentially spherical morphology and similar size.

[0115] The size distribution was measured by nanoparticle tracking analysis (NTA), dynamic laser light scattering (DLS) and image analysis on the photograph obtained by TEM. Fig. 5 is a histogram of particle size distribution identified by TEM, while the chart obtained with the NTA measurement is reproduced in Fig. 6. Instrumental data analysis provided an average particle size of 86.1 ± 0.5 nm in the NTA measurement, 88.6 ± 1.4 nm in the DLS measurement, and 53.84 ± 7.72 nm in the TEM image analysis. These data are mutually consistent; the result obtained from the TEM measurement, lower than that of the other two measurements, is explained by the fact that this measurement was obtained on a very small fraction of particles (38 particles in total), while the NTA and DLS measurements provide an average result of all the particles in the sample.

[0116] EXAMPLE 2

[0117] The procedure of Example 1 was repeated using twice the amount of glucosamine hydrochloride (3.84 mg) obtaining a degree of functionalization equal to about 25%. An IR spectrum was recorded on the functionalized polymer thus obtained, reproduced as the lower line in Fig. 1.

[0118] EXAMPLE 3

[0119] Example 1 was repeated four times under identical conditions, with the only difference that in each test a different fluorophore compound was employed. The four fluorophores employed, and the encapsulation efficiency in the nanoparticles, are summarized in Table 1; the encapsulation efficiency was measured by interpolating the measurements on a previously constructed calibration line for each molecule in DMF by UV-Vis spectroscopy. For completeness, the table also reports the formula and the encapsulation efficiency of Coumarin 6 of Example 1.

[0120] Table 1

[0121] EXAMPLE 4

[0122] This example relates to the comparison between nanoparticles according to the present invention, obtained with a poly(isobutene-a / z-maleic anhydride) polymer functionalized with an aminosaccharide, at 12.5% and 25% of the maleic anhydride units, and nanoparticles according to the prior art, obtained with a poly(isobutene-alt-maleic anhydride) polymer functionalized with an aminosaccharide, at 50% and 75% of the maleic anhydride units.

[0123] The method for obtaining each nanoparticle is that reported in Example 1, modifying the molar ratios between poly(isobutene-aZz-maleic anhydride) and 2-D-(+)-glucosamine hydrochloride so as to obtain functionalizations equal to 12.5%, 25% (according to the present invention), 50% and 75% (according to the prior art) of the maleic anhydride units.

[0124] The nanoparticles thus obtained were subjected to NTA analysis. The analyses were carried out by diluting the samples obtained with functionalization equal to 12.5% and 25% with a dilution factor equal to 10000, while the samples obtained with functionalization equal to 50% and 75% were diluted with a dilution factor equal to 100.

[0125] Fig. 8 shows a comparison chart of particle size distribution of nanoparticles of the invention (functionalized to 12.5% and 25.0% of the maleic anhydride units) and not according to the invention (functionalized to 50.0% and 75.0% of the maleic anhydride units), obtained by NTA.

[0126] Fig. 9 shows a comparison chart of particle size distribution of nanoparticles not according to the invention (functionalized to 50.0% and 75.0% of the maleic anhydride units) using a greater enlargement scale on the y axis compared to Figs. 8-9, obtained by NTA. The detected signals are probably false positives.

[0127] Fig. 10 shows a photograph of a dispersion sample of nanoparticles of the invention, functionalized at 12.5% of the maleic anhydride units, obtained by NTA.

[0128] Fig. 11 shows a photograph of a dispersion sample of nanoparticles of the invention, functionalized at 25.0% of the maleic anhydride units, obtained by NTA.

[0129] The particles according to the present invention, obtained with a degree of functionalization less than 25% of maleic anhydride units, made a reliable analysis possible, since the number of particles per frame in the videos exceeds the minimum required value. Conversely, for nanoparticle samples according to the prior art, the number of particles per frame is less than 5, making the analysis of hydrodynamic distributions unreliable. Attempting to reproduce on samples obtained according to the procedures of the prior art images corresponding to Figs. 10-11, under the same operating and instrumental conditions, it was not possible to obtain the image of any nanoparticle.

[0130] As can be seen from the figures cited above, in particular Figs. 8 and 9, it is possible to observe a significant difference in the number of particles formed from the same amount of polymer. Such a difference underlines how the polymer becomes too hydrophilic to efficiently form nanoparticles at a high degree of functionalization. The presence of particles in the cases of functionalization equal to 50% and 75% of the maleic anhydride units is probably due to an incomplete and / or heterogeneous functionalization of the polymer during the reaction.

[0131] Finally, it is observed that the particles according to the present invention, with a lower degree of functionalization, show a better hydrodynamic distribution, which is a key factor for the therapeutic activity.

[0132] In conclusion, it has been demonstrated that the polymer poly(isobutene-aZz-maleic anhydride), when functionalized with an aminosaccharide, or with an amino saccharide and N- (4-aminobutyl)guanidine, in an overall percentage from 1 to 25% of the units derived from maleic anhydride, exhibits optimal properties of capability of self-assembly in solution as nanoparticles, unlike the prior art, represented by the article “Rationally designed oxaliplatin- nanoparticle for enhanced antitumor efficacy”, A. Paraskar el al., Nanotechnology, 2012 Feb 24, 23(7):075103 and by patent application US 2012189571A1.

[0133] EXAMPLE 5

[0134] This example relates to the dual functionalization of the polymer poly(isobutene-aZz- maleic anhydride) with an amino saccharide and with agmatine, and to the preparation of nanoparticles of the invention with the polymer thus functionalized.

[0135] Operating under an inert, moisture-free atmosphere, 10 mg of poly(isobutene-aZz-maleic anhydride) were dissolved in 333.33 pL of DMF obtaining a polymer concentration equal to 30 mg / mL. 0.825 mg of agmatine dihydrochloride (40 mg / mL) and 0.87 mg of glucosamine hydrochloride (40 mg / mL) were added to this first solution, in addition to triethylamine in a molar ratio of 1.2 with respect to the two salts to release the amino groups of the two reagents. The activation of the hydrochlorides required about 30 minutes; the system was further allowed to react at 25 °C overnight. 58.4 mg of D-(+)-glucose in 1.11 mL of DMF were then added to the solution. Polymer-only nanoparticles, not loaded with active species, were obtained by adding the organic solution dropwise to 10.86 mL of MilliQ® water over 30 minutes under ultrasound stirring. The recovered nanoparticles were transferred into Amicon Ultra filters with cellulose membranes with a 3 kDa cut-off and several washings by centrifuging with MilliQ® water (2220 RCF for 45 minutes) were carried out so as to remove the byproducts.

[0136] The size distribution was measured by NTA technique; the chart obtained with the measurement is reproduced in Fig. 7, and shows a nanoparticle size distribution comparable to that obtained in Example 1.

[0137] EXAMPLE 6

[0138] Example 5 was repeated, in this case however by dripping the DMF solution of functionalized polymer into an aqueous solution of siRNA.

[0139] An aqueous phase was prepared comprising 50 pL of 10 pM siRNA-Cy5 in 10 mM PBS buffer at pH 7.2; the siRNA used is labeled with the fluorescent molecule Cyanine5 (Cy5) to assess encapsulation by means of fluorescence emission.

[0140] 10 mg of DMF solution of functionalized polymer, prepared as described in Example 5, were added to the aqueous phase. Nanoprecipitation was carried out following the procedure of Example 5, including washings such as to remove non-encapsulated siRNA.

[0141] With the aim of qualitatively assessing the encapsulation of the siRNA in the nanoparticle of the invention, the nanoparticles were subjected to treatment with a 0.2% v / v Triton X-100 solution in TBE buffer at 37 °C for 20 minutes, so as to disrupt the colloidal solution into dispersed polymeric material. The samples treated with a 10% glycerol solution were then loaded onto a 1% agarose gel. The analysis of the gel was performed using an Amersham Imager (Cytiva). In well 1, non-encapsulated siRNA was loaded as a control, highlighting the presence of a band and a smear (Fig. 12). In wells 2 and 3, the TBE buffer and the Triton solution were loaded, respectively, demonstrating that the detected fluorescence exclusively derives from the genetic material. In wells 4 and 5, empty nanoparticles were loaded as a control, at two different concentrations. In wells 8 and 9, the same nanoparticles were loaded, but treated with the Triton solution. No fluorescence was detected in wells 4, 5, 8 and 9, as well as in the TBE buffer and in the Triton solution, confirming the absence of signal in the samples containing empty nanoparticles. In wells 6 and 7, nanoparticles containing encapsulated siRNA were loaded, where the smear observed in the control (well 1) became a well-defined band, showing that the genetic material is organized in a uniform structure capable of migrating through the gel in a regular manner. Finally in wells 10 and 11, nanoparticles containing siRNA treated with the Triton solution were loaded, highlighting the reappearance of the conventional smear of free siRNA, indicative of the destruction of the nanoparticles and the consequent release of the siRNA.

[0142] The results in Fig. 12 qualitatively demonstrate that the genetic material can be easily encapsulated within the nanoparticles.

[0143] EXAMPLE 7

[0144] This example relates to the toxicity study carried out in vitro on healthy fibroblast cell lines, such as NIH-3T3 and on glioblastoma tumor cell lines, such as U87.

[0145] U87 cells (human glioblastoma) were cultured in DMEM + 10% FBS (fetal bovine serum) 1% penicillin- streptomycin and L-glutamine, and seeded (5000 cells per well) into 96- well plates. NIH-3T3 cells (murine fibroblasts) were cultured in DMEM + 10% CS (calf serum) 1% penicillin- streptomycin and L-glutamine and seeded (4000 cells per well) into 96-well plates. On the following day, a medium change was performed, by introducing into the wells the medium containing different concentrations of nanoparticles (25 pg / mL, 50 pg / mL, 100 pg / mL), while in the control wells the medium without nanoparticles was inserted. After 24 and 48 hours of incubation at 37 °C, another medium change was performed, and the nonradioactive CellTiter 96® AQueous (MTS; Promega Corporation) cell proliferation assay was conducted. The absorbance was measured after 1, 2, 3 and 4 hours of incubation with the MTS reagent, using a plate reader (EnSight™, PerkinElmer) at 490 nm. The results were normalized with respect to the values of untreated cells and expressed as mean ± standard deviation, derived from three independent experiments conducted in quadruplicate. In Fig. 13 it is possible to appreciate that the nanoparticles of the invention do not show levels of toxicity in the tested cell lines, not even at high nanoparticle concentrations.

[0146] EXAMPLE 8

[0147] Once the non-toxicity of the nanoparticles in vitro had been established, the uptake of the nanoparticles labeled with coumarin 6G was assessed by flow cytometry (FACS) and through a confocal microscope.

[0148] The FACS analysis was performed on U87 cells (human glioblastoma). In particular, U87 cells were cultured with DMEM + 10% FBS (fetal bovine serum), 1% penicillin- streptomycin, and L-glutamine, and seeded (265,000 cells per well) in 6 well plates. After aspirating the medium, 1 mL / well of mixture is added (2 mixtures for each cell line, consisting of phenol red- free culture medium and nanoparticles at concentrations 0.5, 5, 25 and 50 pg / mL). It is incubated for 15 min at 37 °C and 2 washings with PBS are performed. After transferring the content of each well into tubes for FACS, the tubes are centrifuged at 300 g for 5 minutes and further washed with PBS. It is centrifuged again at 300 g for 5 minutes, the supernatant is removed, and the pellet is resuspended in PBS EDTA 2 mM.

[0149] In Figs. 14-18 it is possible to see the amount of uptake of the nanoparticles by the U87 tumor cells at the different concentrations of nanoparticles. The charts show the cell population density (U87) treated with labeled nanoparticles of the invention at concentrations of 0.5, 5, 25 and 50 pg / mL. The gates were set starting from untreated cells (Fig. 14) to show fluorescencepositive and fluorescence-negative cells.

[0150] Confocal microscopy imaging was, instead, performed on U87 (human glioblastoma), MDA-MB-231 (human breast carcinoma) and MET-1 (murine breast carcinoma) cells. After culturing the cell lines and incubating for 15 min at 37 °C at 2.5 pg / mL, the medium is removed and the cells are washed 2 times with prewarmed PBS IX. 350 pL of 4% PFA solution is added per well, and incubation is carried out for 20 minutes at room temperature. The cells are checked under a microscope to verify fixation. At this point the fixative is removed and the cells are washed 3 times with cold PBS IX. Blocking is performed with PBS IX + 2% BSA for 10 minutes at room temperature with stirring and washed 3 times with cold PBS. 100 pL of primary antibody (CD44 antibody, BioRad) diluted in PBS + 1% BSA are added and incubated for Ih at room temperature. After removing the unbound primary antibody, it is washed 2 times with PBS. Add the secondary antibody solution (Goat anti-Rat Alexa Fluor plus 647, ThermoFisher) and rinse 3 times with PBS IX in the dark. Stain the nuclei with the Hoechst solution (5 pg / mL) for 5 minutes at room temperature. Finally, wash with PBS IX and analyze under a Nikon confocal microscope (Al series).

[0151] Figs. 19-21 clearly show how the nanoparticles have a high capacity to diffuse into tumor cells.

[0152] EXAMPLE 9

[0153] After the in vitro experiments, the nanoparticles of the invention were evaluated in vivo, through analysis of the biodistribution in the organs of Balb / C mice.

[0154] 6 female Balb / C mice 10 weeks of age, of which 1 as control (injection of PBS only) and 5 for the test (intravenous injection into the caudal vein of 100 pg of nanoparticles labeled with Rhodamine B in 200 pL of PBS), were used to evaluate the biodistribution at different time intervals (20 min, 40 min and 60 min) (Fig. 22). At each time point, the plasma was collected, from which the PBMC were isolated. After 60 minutes, the mice were sacrificed and the explanted organs were analyzed with IVIS.

[0155] Fig. 23 shows the images obtained with IVIS in which the photon fluorescence emission intensity related to Rhodamine B detected in the organs of mice treated and not treated with nanoparticles is shown. Fig. 23, furthermore, shows a representative chart of the photon emission intensity related to Rhodamine B detected in the organs of mice treated with nanoparticles. From these data, a greater accumulation in the liver, the organ responsible for the clearance of materials, and a smaller amount in the kidneys, the organs responsible for excretion, are noted.

[0156] The in vivo biodistribution was also tested in NOD / SCID mice engrafted with MDA-MB- 231 Luc+ cells. For these experiments 5 week old NOD / SCID mice purchased from Inotiv were used. The animals were maintained in a fully equipped facility, housed in individual cages, fed ad libitum and monitored daily, in accordance with the guidelines of the Italian Ministry of Health.

[0157] 3.0 x 106MDA-MB-231 Luc+ cells were suspended in growth medium, mixed at a 1: 1 ratio with Cultrex and injected close to the mammary fat pad of the mice. After 56 days from implantation (with a tumor volume of 126-364 mm3), the mice (n = 4) were anesthetized with 2.5% isoflurane, and nanoparticles according to the present invention labeled with Rhodamine B (100 pg per mouse) were injected intravenously. An untreated mouse was used as a control. Five hours after the injection, retro-orbital blood samples (right side) were collected in EDTA- coated hematology tubes (BD Vacutainer) and centrifuged for 15 minutes at 1500 x g to recover the plasma. The fluorescence of the samples was further analyzed using the FluoroMax-4 spectrometer HORIBA Scientific and fitted to a calibration curve. Standard solutions were prepared by suspending known amounts of fluorescent nanoparticles in plasma and measuring the fluorescence intensity. After blood collection, the mice were sacrificed, and the major organs and tumor tissues were collected and analyzed.

[0158] Epifluorescence imaging was carried out by placing the dissected organs or the entire animal in an IVIS Lumina imaging system (Perkin Elmer) operating at 37 °C. Images were acquired with a 575-650 nm emission filter, while the excitation was scanned with 460, 500 and 535 nm bandpass filters. For the in vivo analysis, autofluorescence was removed by spectral unmixing and the images obtained were quantified by identifying an appropriate region of interest (ROI). For the ex vivo analysis, the ROI were analyzed in the images recorded with a 500 nm excitation filter.

[0159] Fig. 24 shows the in vivo biodistribution of the nanoparticles of the invention in NOD / SCID mice engrafted with tumor cells.

[0160] Figs. 25 and 27 provide images related to the epifluorescence (Epf) of tumors and organs excised from sacrificed mice bearing MDA-MB-231-L tumors, the mean fluorescence intensity thereof being calculated. Indeed, it emerged that in the analyzed mice an intense epifluorescence (Epf) signal was detected at the tumor, evidencing uptake of the nanoparticles by the tumor cells.

[0161] Furthermore, the dissected organs were, weighed, dissolved in PBS at 250 mg / mL and homogenized with the ball mill TissueLyser QIAGEN (2 cycles, 30 Hz). The fluorescence intensity of the samples was recorded by a FluoroMax-4 HORIBA Scientific spectrometer after subtraction of the blank (untreated tissue). The amount of nanoparticles per tissue was calculated using a calibration curve obtained by adding a known amount of nanoparticles to the untreated homogenized tissue.

[0162] In Fig. 26 it is possible to observe a representative chart of the mean epifluorescence (Epf) value (multiplied by 108) of organs and tumors harvested from the mice. It is noted that most of the nanoparticles are observed in the liver by virtue of the detoxification role thereof; nevertheless, high fluorescence values were significant at the tumor level, while they were highly reduced in the other organs. The high accumulation of the nanoparticles of the invention is favored by high selectivity for tumor cells by virtue of glucosamine as an active targeting molecule.

Claims

CLAIMS1. A nanoparticle formed by self-assembly of the polymer poly(isobutene-aZz-maleic anhydride) functionalized with an aminosaccharide, and optionally further functionalized with N-(4-aminobutyl)guanidine, in units derived from maleic anhydride, having thewhere R is a monosaccharide or disaccharide residue, R’ is the-(CH2)4-NH-C(NH)-NH2 residue of N-(4-aminobutyl)guanidine, n is between 20 and 60, x’ is between 0.01 and 0.25, z is a number between 0 and 0.24, the sum of x’ and z is a number between 0.01 and 0.25 and y = l-(x’ + z), said nanoparticle containing in the internal cavity one or more active compounds as imaging media or as antitumor agents.

2. The nanoparticle according to claim 1, wherein z is equal to 0.

3. The nanoparticle according to any one of claim 1 or 2, wherein n is between 35 and 45.

4. The nanoparticle according to any one of claims 1 to 3, wherein said aminosaccharide is2-D-(+)-glucosamine.

5. The nanoparticle according to any one of claims 1 to 4, wherein said nanoparticle has a dimensional mean value from 50 to 150 nm, preferably from 75 to 100 nm, more preferably from 80 to 90 nm, as determined through at least one technique selected from the group consisting of nanoparticle tracking analysis and dynamic light scattering.

6. The nanoparticle according to any one of claims 1 to 5, wherein said active compounds as imaging media or as antitumor agents are selected from metal ions, inorganic colloidal nanoparticles, small molecules, oligonucleotides and / or peptides.

7. The nanoparticle according to claim 6, wherein said active compounds as imaging mediaor as antitumor agents are selected from Coumarin 6, manganese (II) phthalocyanine, pyrene, 1 -pyrenebutyric acid, Rhodamine 6G, indocyanine, oxaliplatin, doxorubicin, curcumin, cisplatin, paclitaxel, RNA fragments, DNA and therapeutic peptides.

8. A process for preparing a nanoparticle of any one of claims 1 to 7 in a batch manner, comprising the following steps:- using a water-miscible organic solvent, preparing a solution containing a poly(isobutene-a / z-maleic anhydride) polymer functionalized in the units derived from maleic anhydride with an aminosaccharide and optionally further functionalized with N-(4-aminobutyl)guanidine according to claim 1, said solution also containing one or more active compounds as imaging media or as antitumor agents, wherein said functionalized poly(isobutene-a / z-maleic anhydride) polymer is present in the solution in a concentration of between 23 and 30 g / L and said one or more active compounds as imaging media or as antitumor agents are present in an overall concentration of between 0.8 and 1.5 g / L;- adding a sugar acting as a thickener to the solution prepared in the previous operation;- adding ultrapure water to the solution obtained in the previous operation, by dripping under ultrasonic stirring, in a volume between 8 and 10 times the volume of the solution obtained in the previous operation;- filtering the solution to recover the obtained nanoparticles.

9. A process for preparing a nanoparticle of any one of claims 1 to 7 in a continuous manner using a microfluidic system, said process comprising the following operations:- using a water-miscible organic solvent, preparing a solution containing a poly(isobutene-a / z-maleic anhydride) polymer functionalized in the units derived from maleic anhydride with an aminosaccharide and optionally further functionalized with N-(4-aminobutyl)guanidine according to claim 1, said solution also containing one or more active compounds as imaging media or as antitumor agents, wherein said functionalized poly(isobutene-a / z-maleic anhydride) polymer is present in the solution in a concentration of between 23 and 30 g / L and said one or more active compoundsas imaging media or as antitumor agents are present in an overall concentration of between 0.8 and 1.5 g / L;- mixing the solution obtained in the previous operation with ultrapure water, in a water: solution volume ratio of between 1 and 10, and with a liquid flow rate of between 1 and 12 mL / min;- filtering the solution to recover the obtained nanoparticles.

10. The process according to any one of claims 8 or 9, wherein said water-miscible organic solvent is selected from dimethylformamide, tetrahydrofuran and dimethyl sulfoxide.

11. The process according to claim 8, wherein said sugar acting as a thickener is D-(+)- glucose and is added in a concentration between 3.2 and 3.8 g / L.

12. A nanoparticle obtainable through the process according to any one of claims 8 to 11.

13. The nanoparticle according to claim 12, wherein the active species is selected from the group consisting of oxaliplatin and cisplatin and said active species is encapsulated within the nanoparticle and is not complexed by the carboxyl and carbonyl groups of the amide group of the functionalized poly(isobutene-a / z-maleic anhydride) polymer.

14. The nanoparticle according to any one of claims 1 to 7, 12 and 13 for medical use.

15. The nanoparticle according to claim 14, wherein said use is in medical diagnostics or in the treatment of tumors.

Images