BIOMIMETIC NON-IMMUNOGENIC NANOASSEMBLY FOR THE ANTITUMOR THERAPY

Abstract

Nanoassembly (1) for inducing apoptosis in cancer cells comprising: a core (2) comprising at least a nanoparticle of a nano structured and semiconductor metal oxide, said nanoparticle being monocrystalline or polycrystalline; a shell (3) formed by a double phospholipid layer and proteins derived from an extracellular biovesicole chosen between an exosome, an ectosome, a connectosome, an oncosome and an apoptotic body, and an oncosome, said core (2) being enclosed inside said shell (3); and a plurality of targeting molecules (4, 4, 4) of said cancer cells, preferably monoclonal antibodies (4, 4, 4), said molecules (4, 4, 4) being anchored to the external surface of said biovesicole.

Claims

1-23. (canceled)

24. A nanoassembly (1) for inducing apoptosis in cancer cells comprising: a core (2) comprising at least one nanoparticle of a nanostructured and semiconductor metal oxide, said nanoparticle being monocrystalline or polycrystalline; a shell (3) comprising a double phospholipid layer, proteins derived from at least one extracellular biovesicle, said core (2) being enclosed inside said shell (3); and a plurality of targeting molecules (4, 4, 4) of said cancer cells, said molecules (4, 4, 4) being anchored to the external surface of said at least one biovesicle, wherein said at least one biovesicle derived from cells of the same organism which said cancer cells belong to.

25. The nanoassembly (1) according to claim 24, wherein said targeting molecules (4, 4, 4) are monoclonal antibodies (4, 4, 4).

26. The nanoassembly (1) according to claim 24, wherein said nanoparticle is a sphere-shaped nanocrystal.

27. The nanoassembly (1) according to claim 24, wherein said nanoparticle is a nanocrystal in wurtzite phase.

28. The nanoassembly (1) according to claim 24, wherein said metal oxide is zinc oxide.

29. The nanoassembly (1) according to claim 24, wherein said biovesicle is chosen among an exosome, an ectosome, a connectosome, an oncosome and an apoptotic body.

30. The nanoassembly (1) according to claim 26, wherein the diameter of said at least one nanocrystal is comprised between 5 nm and 100 nm, and the diameter of said biovesicle is comprised between 30 nm and 300 nm.

31. The nanoassembly (1) according claim 24, wherein the core (2) of said nanoassembly (1) comprises a medicament.

32. The nanoassembly (1) according to claim 24, comprising a dye element having fluorescence properties.

33. The nanoassembly (1) according to claim 28, wherein the zinc oxide is conjugated or doped with an element which broadens the fluorescence radiation in the ultraviolet-visible spectrum of said zinc oxide.

34. The nanoassembly (1) according to claim 28, wherein the zinc oxide is conjugated or doped with an element which modifies the frequency spectrum of the fluorescence radiation of said zinc oxide.

35. The nanoassembly (1) according to claim 34, wherein said element, which modifies the spectrum, is adapted to broaden said spectrum up to comprise the frequencies in the infrared spectrum.

36. The nanoassembly (1) according to claim 28, wherein the zinc oxide is doped with a paramagnetic or diamagnetic element, said element being adapted to be detected by means of a nuclear magnetic resonance scanner.

37. A kit for using the nanoassembly (1) as a contrast medium according to claim 27, comprising: an injectable solution comprising said nanoassembly (1); means for emitting electromagnetic radiations in the ultraviolet spectrum by which radiating said nanoassembly (1); means for detecting the fluorescence radiation emitted by said nanoassembly (1) following the irradiation performed by said emitting means.

38. A method for manufacturing the nanoassembly (1) according to claim 28, comprising the steps of: synthesis of the zinc oxide; in vitro culture of a plurality of cancer cells; extraction of a plurality of biovesicles from said plurality of cancer cells, said biovesicles having a diameter comprised between 30 nm and 300 nm; coupling of the biovesicles with the zinc oxide; and separation of the biovesicles coupled to the zinc oxide, remained non-coupled biovesicles and remained non-coupled zinc oxide.

39. The method according to claim 38, wherein the zinc oxide synthesis is a wet-chemical, sol-gel, hydrothermal, or solvothermal synthesis.

40. The method according to claim 38, wherein the zinc oxide synthesis comprises the use of a microwave source.

41. The method according to claim 38, wherein the in vitro culture of cancer cells has a duration comprised between 24 and 48 hours.

42. The method according to claim 38, wherein the coupling of the biovesicles with the zinc oxide comprises the incubation of both in a solution obtained by mixing water and a phosphate-buffered saline with a volume ratio of 1:1.

43. The method according to claim 42, wherein the incubation is performed for a period comprised between 30 minutes and two hours and 30 minutes, at a temperature comprised between the room temperature and 37 C., in static conditions or under mechanical stirring generated by an orbital stirrer with a number of rotations comprised between 50 and 350 rpm.

44. The method according to claim 43, wherein said separation comprises the steps of: centrifugation with an acceleration of at least 10000 g for at least five minutes; and washing in a solution of water and a phosphate-buffered saline having a volume ratio of 1:1.

45. A medicament comprising the nanoassembly (1) according to claim 24, for use in antitumor therapy.

46. The medicament comprising the nanoassembly (1) according to claim 24, for use in the prevention of the immunologic response of an organism treated with an antitumor therapy.

Description

[0026] These and further objects of the present invention will be made clearer on reading the following detailed description of a preferred embodiment of the present invention, by way of example and not limitation of the more general claimed concepts, as well as by way of examples concerning experimental tests performed on the present invention.

[0027] The following description refers to the accompanying figures, wherein:

[0028] FIG. 1 is an exemplary diagram of the nanoassembly object of the present invention;

[0029] FIG. 2a is a scanning electron microscopy image of zinc oxide nanocrystals synthesized according to the method of the present invention;

[0030] FIG. 2b is a transmission electron microscopy image of zinc oxide nanocrystals synthesized according to the method of the present invention;

[0031] FIG. 2c is an image of the electron diffraction pattern in transmission electronic microscopy of zinc oxide nanocrystals synthesized according to the method of the present invention;

[0032] FIG. 2d shows a diffractogram obtained by X-ray diffraction (with radiation source Cu K alpha-=0.15418 nm) on the zinc oxide nanocrystals synthesized according to the method of the present invention;

[0033] FIG. 3a is a field emission scanning electron microscopy (FESEM) image of zinc oxide nanocrystals synthesized according to the method of the present invention;

[0034] FIG. 3b is a graph of the distribution of the diameters of the zinc oxide nanocrystals synthesized according to the method of the present invention, said nanocrystals being dispersed in an ethanol solution;

[0035] FIG. 4 summarizes the distribution graphs of the size of the nanocrystals synthesized according to the method of the present invention, said graphs being obtained by means of the Dynamic Light Scattering (DLS) technique. In particular, solid line curves refer to the ZnO nanocrystals, the dashed line curves relate to ZnO functionalized with amino-propyl groups (ZnONH.sub.2), the first line refers to nanocrystals dispersed in water (H.sub.2O), the second line to nanocrystals dispersed in methanol (MeOH), the third line to nanocrystals dispersed in ethanol (EtOH), and finally the fourth line refers to nanocrystals dispersed in ethylene glycol (EG);

[0036] FIG. 5a is a FESEM image of exosomes extracted according to the method of the present invention from epithelial oral cancer cells (KB type) in adhesion;

[0037] FIG. 5b is a graph, obtained by means of Nanoparticle Tracking Analysis (NTA) of the distribution of diameters of exosomes extracted according to the method of the present invention from cancer cells derived from an epithelial oral cancer (KB type) in adhesion;

[0038] FIG. 6a is a FESEM image of exosomes extracted according to the method of the present invention from type B lymphomatous cancer cells (Daudi type) in suspension;

[0039] FIG. 6b is a graph obtained by means of NTA analysis of the distribution of diameters of exosomes extracted with the method of the present invention from type B lymphomatous cancer cells (Daudi type);

[0040] FIG. 7 summarizes the distribution graphs of the size of the exosomes extracted by type KB cells, of the size of zinc oxide nanocrystals and complete nanoassemblies obtained according to the method of the present invention, said graphs being obtained by means of the Dynamic Light Scattering (DLS) technique. In particular, the solid line curve refers to the nanoassemblies, the dashed line curve with alternating points and dashes refers to exosomes, and the dashed line curve refers to zinc oxide crystals;

[0041] FIG. 8a shows the measurements of the concentrations of zinc, calcium and phosphate ions released by zinc oxide (ZnO), zinc oxide functionalized with amino-propyl groups (ZnONH.sub.2), and zinc oxide coated with a phospholipid layer, in simulated inorganic plasma (SBF, Simulated Body Fluid). The graphs are obtained by means of Inductive Coupled Plasma (ICP) techniques;

[0042] FIG. 8b shows the measurements of the concentrations of zinc, calcium and phosphate ions released by zinc oxide (ZnO), zinc oxide functionalized with amino-propyl groups (ZnONH.sub.2) and zinc oxide coated with a phospholipid layer, in a cell culture medium (EMEM). The graphs were obtained by inductively coupled plasma mass spectrometry (ICP-MS) (ICP);

[0043] FIG. 9 shows the fluorescence emission in the wavelength at approximately 400 nm and 550 nm, obtained by exciting the zinc oxide nanocrystals with a monochromatic source at 230 nm and 300 nm, respectively;

[0044] FIG. 10a shows the result of cell viability tests after 5 hours (H) of line KB cancer cell exposure to different concentrations of zinc oxide in a cell culture medium. The graph shows, in particular, the percentage of live cells with respect to the control according to the concentration of zinc oxide; and

[0045] FIG. 10b shows the result of cell viability tests after different exposure times (24, 48 and 72 hours (H)) of line KB cancer cells to different concentrations of zinc oxide in a cell culture medium. The graph shows, in particular, the percentage of live cells in comparison with respect the control as a function of the concentration of zinc oxide.

[0046] With reference to FIG. 1, in a preferred embodiment, the present invention regards a biomimetic nanoassembly (1) comprising: [0047] a core (2) comprising at least one zinc oxide nanocrystal, said nanocrystal being spherical-shaped and in wurtzite phase; [0048] a shell (3) formed by a double phospholipid layer and other molecules, including proteins, derived from an exosome, said core (2) being enclosed inside said shell (3); and [0049] a plurality of monoclonal antibodies (4, 4, 4), said antibodies (4, 4, 4) being anchored to the external membrane of said exosome.

[0050] The diameter of said at least one nanocrystal is comprised between 5 nm and 100 nm, and the diameter of said exosome is comprised between 30 nm and 300 nm.

[0051] The nanoassembly (1) of the present invention may optionally comprise also a compound having an anti-inflammatory action and/or a compound having a chemotherapeutic action as an adjuvant of the cytotoxic action of zinc oxide. In addition, the zinc oxide, per se capable of emitting a fluorescence radiation in a band astride the spectrum of ultraviolet and visible green light spectra when excited by ultraviolet radiations, can be doped with an element that amplifies this fluorescence radiation or with an element that changes the frequency spectrum of this fluorescence radiation. Actually, with a suitable doping, it is possible to broaden the spectrum to include the frequency of the infrared spectrum.

[0052] In such context, it is specified here that a kit for the use of the nanoassembly (1) as a contrast medium forms an object of the present invention, said kit comprising: [0053] an injectable solution comprising said nanoassembly (1); [0054] means for emitting electromagnetic radiations in the ultraviolet spectrum by which radiating said nanoassembly (1); [0055] means for detecting the fluorescence radiation emitted by said nanoassembly (1) following the irradiation performed by said emitting means.

[0056] The means for the detection of fluorescence radiation comprise a camera or an optoelectronic device or a spectrophotometer configured to detect radiations in the visible light spectrum and/or a camera or an optoelectronic device or a spectrophotometer configured to detect radiations in the infrared spectrum.

[0057] Also, a method for the production of the nanoassembly (1) described above is an object of the present patent application, said method comprising the steps of: [0058] synthesis of the zinc oxide; [0059] in vitro culture of a plurality of cancer cells for a period ranging between 24 and 48 hours; [0060] extraction of a plurality of biovesicles from said plurality of cancer cells, said biovesicles having a diameter comprised between 30 nm and 300 nm; [0061] coupling of the biovesicles with the zinc oxide; and [0062] separation of the biovesicles coupled to the zinc oxide, remained non-coupled biovesicles and remained non-coupled zinc oxide.

[0063] The synthesis of zinc oxide can be by wet-chemical synthesis, such as sol-gel or hydrothermal or solvothermal synthesis, or provide for the use of a microwave source, or an ultrasound source, or a solid-state synthesis of grinding or crushing with a mill. The coupling between biovesicles and zinc oxide provides for the incubation of both in a solution obtained by mixing water and a phosphate-buffered saline (PBS) with a volume ratio of one to one, for a period comprised between 30 minutes and two hours and thirty minutes, at a temperature comprised between the room temperature and 37 C., in static conditions or under mechanical stirring generated by an orbital stirrer with a number of rotations comprised between 50 and 350 rpm. At the separation between biovesicoles coupled to zinc oxide, the remained non-coupled biovesicoles and the remained non-coupled zinc oxide is carried out by means of centrifugation with an acceleration greater than 10000 g for at least five minutes, followed by a washing in a solution of water and phosphate-buffered saline with a volume ratio of one to one.

[0064] Finally, also a medicament or molecule comprising the nanoassembly (1), described above for use in antitumoral therapy and for use in the prevention of the immune response from the human organism treated with an antitumor therapy, is an object of the present invention. It is, therefore, possible to design and implement a treatment protocol, or a treatment method of cancers based on the administration of a drug comprising the nanoassembly (1) object of the present invention.

EXAMPLE 1

[0065] Synthesis of Zinc Oxide.

[0066] The zinc oxide nanocrystals were synthesized by means of the use of a microwave source. Thanks to this technology, it has been possible to obtain nanoparticles being spherical-shaped and monocrystalline in wurtzite phase with a homogeneous size distribution equal to approximately 20 nm, as shown in FIGS. 2a, 2b, 2c, 2d, 3a and 3b. The zinc oxide nanocrystals can remain dispersed in a colloidal manner (hydrodynamic diameter) in various solutions such as methanol (used in the synthesis), ethanol, water or polyethylene glycol, as shown in FIG. 4.

EXAMPLE 2

[0067] Pre-Treatment of Zinc Oxide to Prepare it for Subsequent Doping.

[0068] In order to subsequently bind a dye to ZnO and, for example, amplify its fluorescence radiation, a functionalization of its surface has been carried out with amino groups (NH.sub.2), that are reactive against specific dye molecules, which otherwise would not bind exclusively to the oxide structure. The reaction takes place, in particular, between the ZnO nanocrystals and a reagent called aminopropyl-trimethoxy silane (APTMS) pouring all the components in a pyrex glass flask under nitrogen atmosphere with an anhydrous solvent (e.g. ethanol), stirring at least at 200 rpm with magnetic stir bar, and bringing the whole to boil for a period ranging between 5 and 8 hours, with the addition of a cooling column in order to avoid the solvent evaporation.

EXAMPLE 3

[0069] Extraction of Exosomes from Cancer Cells.

[0070] The exosome vesicles were produced from cancer cells of epithelial oral cancer (KB type, FIGS. 5a, 5b) and lymphoma cells (Daudi type, FIGS. 6a, 6b). The cells are cultured in a complete culture medium (Eagle's Minimum Essential Medium, EMEM or Roswell Park Memorial Institute, RPMI-1640, complete with 10% foetal bovine serum) for 48 h, after which the medium is replaced with one compound of EMEM or RPMI-1640 and 10% foetal bovine serum, from which the bovine exosomes have been previously removed (to prevent contamination). After 24-48 hours, the exosomes are extracted directly from such cell culture medium by means of differential ultracentrifugation techniques.

[0071] The exosomes obtained have a size ranging from 30 to 100 nm and were visualized either by scanning electron microscopy (FIGS. 5a, 6a) or by the Nanoparticle Tracking Analysis (NTA) technique (FIGS. 5b, 6b).

EXAMPLE 4

[0072] Coupling Between Exosomes and Zinc Oxide Nanocrystals.

[0073] The coupling between exosomes and zinc oxide occurred by incubation of both in water and PBS (1:1 vol, V.sub.total 100 L) for certain time (1 h 30 min) at constant room temperature, and under stirring using a 250 rpm orbital shaker, followed by appropriate washings, centrifugation at 16870 g for 5 minutes, washing in a PBS:H.sub.2O=1:1 solution (V.sub.total 100 L), and purification processes to separate the exosomes and non-coupled ZnO from the final nanoassembly. The coupling process was then repeated on the supernatant portion (typically containing only exosomes) with a new aliquot of ZnO nanocrystals, followed by an appropriate washing.

EXAMPLE 5

[0074] Verification of Coupling Occurred Between Exosomes and Zinc Oxide Nanocrystals.

[0075] Verifications on the prepared materials (ZnO and exosomes), as well as on the coupling occurred into the nanoassembly, were conducted by means of various characterization techniques, including Dynamic Light Scattering (DLS, FIG. 7). Thanks to such technique, it was possible to see how the diameter of the particles obtained has a larger size than individual exosomes and ZnO nanocrystals as such.

[0076] In addition, by fluorescence microscopy (not shown in the Figure), after suitably marking nanocrystals with a dye emitting in the red wavelength, and exosomes with a dye emitting in the green wavelength, it was possible to acquire an image with the two different fluorescence channels (red and green) and superimpose the two images, evaluating the coupling rate between ZnO and the exosomes.

EXAMPLE 6

[0077] Coupling Between Monoclonal Antibodies and Nanoassembly Lipid Shell Obtained in Example 4.

[0078] The coupling between the monoclonal antibody and the lipid shell was performed by means of antibody fusion and diffusion/fusion (suitably bound to a bifunctional polyethylene glycol spacer and a lipid molecule) into the lipid exosomial membrane.

EXAMPLE 7

[0079] Verification of Radical Species.

[0080] The measurement of oxidizing radical species generated by ZnO alone, or by the nanoassembly as a whole, was performed after the immersion of a material sample in water or buffered saline solutions or cell culture media, after internalisation for at least 24 hours in cancer cells. Successively, a paramagnetic resonance electronic spectrometer was used, capable of detecting the presence of free radicals generated by using a suitable chemical trap (5,5-dimethyl-1-pyrroline N-oxide, DMPO) capable of keeping them stable for a time sufficient for the measurement.

[0081] The measurement of zinc ions released from only from ZnO alone or from the whole nanoassembly was, therefore, carried out with ICP (Inductive Coupled Plasma)-Mass techniques (FIGS. 8a and 8b) and colorimetric methods in fluorescence with probes that bind specifically to zinc ions either in a cellular or non-cellular environment.

EXAMPLE 8

[0082] Testing of the Imaging Properties of the Nanoassembly

[0083] Imaging properties of ZnO and the nanoassembly as a whole have been investigated and measured by means of spectroscopy and fluorescence microscopy, presenting an emission centred at 500-600 nm in wavelength (FIG. 9).

EXAMPLE 9

[0084] Cell Viability Tests after Exposure to Nanoassembly.

[0085] Cultured cells were exposed to the nanoassembly, and correct times of internalization were calibrated in cells (it resulted in the 62% of the cells of the KB line, which have internalized the nanoassembly after 5 hours, the 98% of cells which have internalized the nanoassembly after 24 hours), as well as the targeting specificity and the adequate doses of nanoassembly to be administered. Within the cell, but not externally, the nanoassembly loses its lipid coating and starts to develop radical species, and to dissolve into Zn.sup.2+ ions, both leading to cell death.

[0086] It has been found that, already at very low doses (15-20 g/ml) of zinc oxide nanocrystals in a cell culture medium, and already after just 5 hours after the administration, the cancer cells of the KB line die at a considerable rate (the survival rate of the cell population is lower than 40%), and such threshold decreases to 20% of survival rate for doses little higher than 25-30 g/ml (FIGS. 10a and 10b), and is reduced to zero for dosages exceeding 30 g/ml. The cell death was verified through various extensive testing of cell population counts, colorimetric (Trypan Blue and WST-1) as well as flow-cytometric methods, conventional in common biological practices.

EXAMPLE 10

[0087] Testing of the Nanoassembly Selectivity

[0088] The Anti-CD20 monoclonal antibody is used and coupled to the lipid shell of the nanoassembly. Such nanoassemblies are then cultured with leukemic cells (particularly, lymphomatous cancer cells of B, Daudi type) and, as a reference, with healthy B lymphocytes. It is demonstrated that the nanoassembly preferentiality and specific selectivity exist in binding by means of the Anti-CD20 antibody to the CD20-specific receptor on the Daudi cancer cell membrane. On the contrary, this selectivity does not exist when the nanoassembly with the AntiCD20 antibody is incubated with healthy B lymphocytes.