The present invention relates to methods for producing oxide ferrimagnetics with spinel structure and iron oxide nanoparticles by soft mechanochemical synthesis using inorganic salt hydrates, oxide ferrimagnetics with spinel structure and iron oxide nanoparticles obtainable by the methods, stable and biocompatible aqueous colloidal systems comprising oxide ferrimagnetics with spinel structure and iron oxide nanoparticles, carriers comprising oxide ferrimagnetics with spinel structure and iron oxide nanoparticles, and uses thereof in medicine.
Magnetic resonance (MR) imaging (MRI) is a diagnostic method that enables tissue differentiation on the basis of different relaxation times. Contrast agents alter the relaxation times and are used to enhance the visualization of properties correlated with patient anatomy and physiology. The change in relaxation times depends on the contrast agent concentration as well as on magnetic field strength. Two types of contrast agents are known: T1 contrast agents that shorten spin-lattice relaxation time of the nearby protons and T2 contrast agents, which enhance spin-spin relaxation to darken the contrast media-containing structures. Contrast agent specificity is a desired property for enhancing signal-to-noise ratio at a site of interest and providing functional information through imaging. Natural biodistribution of contrast agents depends upon the size, charge, surface chemistry and administration route. Contrast agents may concentrate at healthy tissue or lesion sites and increase the contrast between the normal tissue and the lesion. In order to increase contrast, it is necessary to concentrate the agents at the site of interest and increase relativity. In addition, it is also desirable to increase the uptake of the agents by diseased cells in relation to healthy cells. Till now, superparamagnetic nanoparticles are used for MRI negative contrast, of which superparamagnetic iron oxide (SPIO) is the representative example.
WO 2008/127031 discloses magnetic resonance imaging contrast agents that comprise zinc-containing magnetic metal oxide nanoparticles. Optimized nanoparticles are proposed for conjugation with a bioactive material such as proteins, antibodies, and chemical materials. The proposed methods in WO 2008/127031 thus have limitations in accessibility of the materials used for such conjugation and careful analyses of their efficiency upon binding should be always performed.
The commercially available standard SPIOs, such as SHU 555A (Resovist®, Bayer HealthCare AG), are extremely strong enhancers of proton relaxation, but have very short useful half-lives after intravenous administration, as they are rapidly cleared from the blood within minutes and accumulate in the reticuloendothelial cells of the liver and spleen.
The properties and the further use of magnetic nanoparticles depends on the method by which they were obtained. The standard one step method of the preparation of magnetic nanoparticles by the method of co-precipitation was described before (Lopez et al., 2010; Morais et al., 2006). In contrast to that approach, the milling of reagents in the planetary mill will result in the ultrasmall sized nanoparticles with unique properties described in the current invention. Moreover, the use of saline crystal hydrates (proposed in the current invention) instead of the anhydrous salts that have been used in the work of Naiden et al., (Naiden et al., 2008), will change the solid-phase mechanism to a soft mechanochemical synthesis in aqueous media, resulting in a significantly increased reaction rate.
The main limiting factor for using of superparamagnetic nanoparticles in vivo is their low colloidal stability. To prevent nanoparticles aggregation, different methods by creating an electrostatic double layer have been developed. Mainly, those methods are based on the use of polymer surfactants functioning as a steric stabilizer, such as dimercaptosuccinic acid (DMSA) (Morais et al., 2004), polysaccharide polymer (dextran or dextran derivatives), starch, polyvinyl alcohol (PVA), polyethylene oxide (PEO), polyethylene glycol (PEG) or by modifying the isoelectric point with a citrate or silica coating (Bacri et al., 1990; Cornell, 1991). The most commonly used iron oxide nanoparticles are dextran coated, and are physiologically well tolerated (Babincova and Machova, 1999). There coating of magnetic nanoparticles by citric acid which form anionic monolayer on the particle surface was proposed by Morais et al., (Morais et al., 2006). However, the use of acid in state of buffers on the base of citrate salts could decrease the bioavailability of nanoparticles solution, particularly in the in vivo systems. Thus, to enable higher bioavailability of nanoparticles, a multiplex buffer containing HEPES, as a main component, and physiological pH (7.4) was developed in the current invention. Moreover, the salinity of this multiplex stabilizing buffer can be changed by variable concentration of saline component (NaCl). WO2009/002569 describes a procedure of effective polyurethane coupling of nanoparticles. Particles loaded and stable were obtained by this method. However, the described procedure is making impossible the coupling to the composite of additional components except nanoparticles. It can be critical point in use of such nanoparticles in more complicated systems, for example targeted delivery.
Another way is a coupling of nanoparticles by hydrophobic environment e.g polystyrene as described in WO2006/061835 or oleic acid (Lopez et al., 2010). Hydrophobic monolayer covered nanoparticles formed nanocrystals are stable and suspendable in non-polar and polar solvents. However, using nanoparticles thus stabilized in carrier systems, e.g. liposomes, there is possibility of incorporation of hydrophobic nanocrystals into the liposome bilayer which can destroy the liposome structure.
Therefore, there is a need for novel paramagnetic nanoparticles, in particular iron oxide and ferrite nanoparticles having improved MR contrast properties and which offer the possibility of their targeted delivery, for use in the field of medicine, in particular diagnostics and treatment. There is also a need for stabilized formulations of iron oxide and ferrite nanoparticles as a prerequisite for use in medicine and/or for use as starting material for carriers comprising iron oxide nanoparticles, in particular ferriliposomes. In particular, there is a need for iron oxide nanoparticles with high colloidal stability in aqueous media as well as biocompatibility.
Compounds used for MRI contrast should be of a nanosize, stably dispersed both in aqueous media and in vivo environments and exhibit excellent MR contrast effects. Superparamagnetic nanoparticles currently suggested for the MRI have nanonsize and are stable for injection to the bloodstream what is very important to prevent thrombosis and blood vessels embolism. However, the use of such superparamagnetic nanoparticles for the high performance MRI applications is limited by their contrast properties.
In one embodiment, the present invention relates to a method for preparing oxide ferrimagnetics with spinel structures nanoparticles of ultra small size (below 30 nm) and high specific surface area (50-200 m2/g) according to formula (I):
MxFe3-xO4 (I)
wherein M is selected from Fe, Cu, Co, Ni, Mg and Mn, in particular M is Fe, and wherein 0≦x≦1, preferably 0.05≦x≦1,
comprising the following steps:
In a preferred embodiment, a salt crystal hydrate selected from FeCl3 6H2O; CoCl2.6H2O, CuCl2.2H2O is used etc.
The method of the invention surprisingly results in nanoparticles of ultrasmall size (less than about 50 nm, preferably less than about 30 nm) and a high specific surface area.
In a preferred embodiment, the oxide ferrimagnetics with spinel structure nanoparticles thus obtained are characterized by a diameter of less than about 50 nm, more preferably less than about 30 nm, even more preferably less than about 15 nm.
Such nanoparticles are particles of ultrasmall size.
In a preferred embodiment, the oxide ferrimagnetics with spinel structure nanoparticles thus obtained are characterized by specific surface area of about 50 to about 200 m2/g more preferably of about 100 to about 160 m2/g.
Such nanoparticles are characterized by a high specific surface area.
In a more preferred embodiment, the oxide ferrimagnetics with spinel structure nanoparticles thus obtained are characterized by
a) a diameter of less than about 50 nm, more preferably less than about 30 nm, even more preferably less than about 15 nm, and
b) specific surface area of about 50 to about 200 m2/g more preferably of about 100 to about 160 m2/g.
In a preferred embodiment, the diameter of the oxide ferrimagnetics with spinel structure nanoparticles thus obtained is at least about 1 nm, preferably at least about 3 nm.
In an especially preferred embodiment,
In a preferred embodiment, a salt crystal hydrate selected from FeCl3.6H2O; CoCl2.6H2O and CuCl2.2H2O is used.
In a further preferred embodiment, the crystal salt hydrate FeSO4.7H2O is used.
In a particularly preferred embodiment,
(i) the crystal salt hydrate FeSO4.7H2O, and
(ii) a crystal salt hydrate selected from FeCl3.6H2O; CoCl2.6H2O and CuCl2.2H2O is used.
In a preferred embodiment, grinding and/or milling is performed in a planetary mill.
In another preferred embodiment, the weight ratio of metal salt hydrates in total to the inert diluent, in particular NaCl, is about 1 to about 10 to about 1:1.5, preferably about 3:8.
In another preferred embodiment, the atmosphere in the planetary mill is vacuum, air or inert gas, in particular Ar.
In another preferred embodiment, the ratio of the mass of iron balls to the mass of reaction mixture according to step (i) is about 1:1 to about 50:1, preferably about 20:1.
In a preferred embodiment, the oxide ferrimagnetics with spinel structure are selected from the group consisting of Fe3O4, CoFe2O4, CuFe2O4, MnFe2O4, MgFe2O4, and NiFe2O4.
In another embodiment, the present invention relates to a method for preparing iron oxide nanoparticles according to formula (II):
Fe3O4 (II)
comprising the following steps:
The reaction is as follows
FeSO4+2FeCl3+8NaOH→Fe3O4+Na2SO4+6NaCl+4H2O
The method of the invention surprisingly results in nanoparticles of ultrasmall size (less than about 50 nm, preferably less than about 30 nm) and a high specific surface area. Moreover, as explained below, the method of the invention surprisingly results in novel iron oxide nanoparticles which are were proven to be ultrasmall and spherical with narrow size distribution and could successfully be formulated in a stable colloidal system. Moreover, the particles exhibit several fold higher relaxivities than commercial MRI contrast agents, resulting in ultra-sensitive MRI detection (
In a preferred embodiment, the iron oxide nanoparticles thus obtained are characterized by a diameter of less than about 50 nm, more preferably less than about 30 nm, even more preferably less than about 15 nm
Such nanoparticles are particles of ultrasmall size.
In a preferred embodiment, the iron oxide nanoparticles thus obtained are characterized by specific surface area of about 50 to about 200 m2/g, more preferably of about 100 to about 160 m2/g.
Such nanoparticles are characterized by a high specific surface area.
In a more preferred embodiment, the iron oxide nanoparticles thus obtained are characterized by
In a preferred embodiment, the diameter of the iron oxide nanoparticles thus obtained is at least about 1 nm, preferably at least about 3 nm.
In one preferred embodiment, the salt crystal hydrate FeCl3.6H2O is used.
In a further preferred embodiment, the crystal salt hydrate FeSO4.7H2O is used.
In a particularly preferred embodiment, the crystal salt hydrates FeSO4.7H2O and
FeCl3.6H2O are used.
In another preferred embodiment, the ratio of the mass of iron balls to the mass of reaction mixture according to step (ii) is about 1:1 to about 50:1, preferably about 20:1.
In another preferred embodiment, the weight ratio of metal salt hydrates in total to the inert diluent, in particular NaCl, is about 1 to about 10 to about 1:1.5, preferably about 3:8.
The inert compound prevents heating of the reagent mixture.
In another preferred embodiment, the atmosphere in the planetary mill is vacuum, air or inert gas, in particular Ar.
In another preferred embodiment, mechanochemical synthesis in the planetary mill is performed in an MPV planetary mill.
In yet another preferred embodiment, mechanochemical synthesis in the planetary mill is performed in a planetary mill at about 30 g to about 100 g, preferably at about 60 g.
In another preferred embodiment, the washing of the nanoparticles according to the methods of the invention is performed by washing with distilled water.
In another preferred embodiment, the washing of the nanoparticles according to the methods of the invention is performed by washing on a filter.
In another preferred embodiment, the washing of the nanoparticles according to the methods of the invention is performed until the salts are completely removed from the filter.
In another embodiment, the present invention relates to an iron oxide nanoparticle or a oxide ferrimagnetics with spinel structure nanoparticle obtainable by any of the above methods of the invention.
In another embodiment, the present invention relates to an iron oxide nanoparticle or a oxide ferrimagnetics with spinel structure nanoparticle obtainable by any of the above methods of the invention.
In a preferred embodiment, the iron oxide nanoparticles and/or oxide ferrimagnetics with spinel structure nanoparticles obtainable by any of the above methods of the invention are characterized by a diameter of less than about 50 nm, more preferably less than about 30 nm, even more preferably less than about 15 nm.
Such nanoparticles are particles of ultrasmall size.
In a preferred embodiment, the diameter of the oxide ferrimagnetics with spinel structure nanoparticles and/or oxide ferrimagnetics with spinel structure nanoparticles obtainable by any of the above methods of the invention is at least about 1 nm, preferably at least about 3 nm.
In a preferred embodiment, the iron oxide nanoparticles and/or oxide ferrimagnetics with spinel structure nanoparticles obtainable by any of the above methods of the invention are characterized by specific surface area of about 50 to about 200 m2/g, more preferably of about 100 to about 160 m2/g.
Such nanoparticles are characterized by a high specific surface area.
In a more preferred embodiment, the iron oxide nanoparticles and/or oxide ferrimagnetics with spinel structure nanoparticles obtainable by any of the above methods of the invention are characterized by
In another embodiment, the present invention relates to a method for preparing oxide ferrimagnetics with spinel structures nanoparticles according to formula (III):
CoxFe3-xO4 (III)
comprising the following steps:
The reaction is as follows:
2FeCl3+CoCl2+Ca(OH)2+3Na2CO3=CoxFe3-xO4+CaCl2+6NaCl+3CO2↑+H2O, wherein 0.1≦x≦0.99, preferably 0.6≦x≦0.98.
In one preferred embodiment, the salt crystal hydrate FeCl3.6H2O, and the salt crystal hydrate CoCl2.6H2O is used.
In another preferred embodiment, the ratio of the mass of iron balls to the mass of reaction mixture according to step (ii) is about 1:1 to about 50:1, preferably about 20:1.
The conditions for accomplishment of a given technical effect of the invention, which is a production of non-stoichiometric oxide ferrimagnetic nanoparticles with spinel structure are the strict adherence to the weight ratios of the mass of reaction mixture to the mass of inert component, preferably of about 1:2 to 1:3, the mass of powder to the mass of balls, preferably about 20:1, and the time for performing of mechanochemical synthesis of 10÷60 min.
In another preferred embodiment, the atmosphere in the planetary mill is vacuum, air or inert gas, in particular Ar.
In another preferred embodiment, mechanochemical synthesis in the planetary mill is performed in an MPV planetary mil.
1
In yet another preferred embodiment, mechanochemical synthesis in the planetary mill is performed in a planetary mill at about 30 g to about 100 g, preferably at about 60 g.
In another preferred embodiment, the washing of the nanoparticles according to the methods of the invention is performed by washing with distilled water.
In another preferred embodiment, the washing of the nanoparticles according to the methods of the invention is performed by washing on a filter.
In another preferred embodiment, the washing of the nanoparticles according to the methods of the invention is performed until the salts are completely removed from the filter.
In another embodiment, the present invention relates to an oxide ferrimagnetics with spinel structure nanoparticles obtainable by the above described
method by formula: CoxFe3-xO4, wherein 0.1≦x≦0.99, preferably 0.6≦x≦0.98.
In another embodiment, the present invention relates to an oxide ferrimagnetics with spinel structure nanoparticles obtainable by the above described method of the invention with the size of nanoparticles below 50 nm, in particular below 15 nm and high specific surface area in the range of 50-200 m2/g, in particular 100-160 m2/g.
In another embodiment, the present invention relates to a method for preparing oxide ferrimagnetic with spinel structure nanoparticles according to formula (IV):
MnxFe3-xO4 (IV)
comprising the following steps:
The reaction is as follows:
2FeCl36H2+MnO2+6NaOH→MnxFe3-xO4+6NaCl+15H2O+1/2O2,
wherein 0.1≦x≦0.99, preferably 0.6≦x≦0.98.
In one preferred embodiment, only the salt crystal hydrate FeCl3.6H2O is used.
In another preferred embodiment, the ratio of the mass of iron balls to the mass of reaction mixture according to step (ii) is about 1:1 to about 50:1, preferably about 20:1.
In another preferred embodiment, the atmosphere in the planetary mill is vacuum, air or inert gas, in particular Ar.
In another preferred embodiment, mechanochemical synthesis in the planetary mill is performed in an MPV planetary mill.
In yet another preferred embodiment, mechanochemical synthesis in the planetary mill is performed in a planetary mill at about 30 g to about 100 g, preferably at about 60 g.
In another preferred embodiment, the washing of the nanoparticles according to the methods of the invention is performed by washing with distilled water.
In another preferred embodiment, the washing of the nanoparticles according to the methods of the invention is performed by washing on a filter.
In another preferred embodiment, the washing of the nanoparticles according to the methods of the invention is performed until the salts are completely removed from the filter.
In another embodiment, the present invention relates to an oxide ferrimagnetics with spinel structure nanoparticles obtainable by the above described method by formula: MnxFe3-xO4, wherein 0.1≦x≦0.99, preferably 0.6≦x≦0.98.
In another embodiment, the present invention relates to an oxide ferrimagnetics with spinel structure nanoparticles obtainable according the above described method of the invention to the methods of the invention with the size of nanoparticles below 50 nm, in particular below 15 nm and high specific surface area in the range of 50-200 m2/g, in particular 100-160 m2/g.
As shown in the Examples, the novel iron oxide nanoparticles of the invention were proven to be ultrasmall and spherical with narrow size distribution and high specific surface area and could successfully be formulated in a stable colloidal system. Moreover, the particles exhibit several fold higher relaxivities than commercial MRI contrast agents, resulting in ultra-sensitive MRI detection (
As shown in the Example 2 the obtaining of the oxide ferrimagnetic nanoparticles with spinel structure which chemical composition is CoxFe3-xO4, where 0.1≦x≦0.99 provides the end product of high contrast properties at T1T2-relaxation time (
In a preferred embodiment, the iron oxide nanoparticles or the oxide ferrimagnetics with spinel structure nanoparticles of the present invention have a diameter of about 1 to about 50 nm, in preferably of about 1 to about 30 nm, even more preferably of about 1 to 15 nm, in particular of about 3 to about 14 nm.
In another preferred embodiment, more than about 70% of the particles of the iron oxide nanoparticles or the oxide ferrimagnetics with spinel structure nanoparticles of the present invention have a diameter of less than about 8 nm. Preferably more than about 70%, more preferably more than about 80%, of the nanoparticles have a diameter of more than about 1 nm.
The novel iron oxide particles of the present invention were shown to exhibit such size distribution as shown in Example 1.
Thus, in another embodiment, the present invention relates to a population of iron oxide particles and/or oxide ferrimagnetics with spinel structure particles obtainable by above methods,
in particular wherein at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95% of the nanoparticles
In another preferred embodiment, the negative surface zeta potential of the iron oxide nanoparticles or the oxide ferrimagnetics with spinel structure nanoparticles of the present invention is about 20 to about 30 mV, in particular about 28 mV, at pH 7.4 and 37° C.
The novel iron oxide particles of the present invention were shown to exhibit such zeta potential as shown in the Examples.
In another embodiment, the method for preparing iron oxide nanoparticles or oxide ferrimagnetics with spinel structure nanoparticles of the invention, further comprises the step of suspending the iron oxide nanoparticles or oxide ferrimagnetics with spinel structure nanoparticles in a biocompatible saline solution.
In a further embodiment, the present invention relates to a suspension of iron oxide nanoparticles of the present invention or oxide ferrimagnetics with spinel structure nanoparticles of the present invention, obtainable the method
In a further embodiment, the present invention relates to a method for preparing a biocompatible aqueous colloidal system comprising iron oxide nanoparticles or oxide ferrimagnetics with spinel structure nanoparticles comprising the following steps:
In a preferred embodiment of the present invention, the iron oxide nanoparticles or oxide ferrimagnetics with spinel structure nanoparticles of the present invention are used in step (i).
It was surprisingly shown, that stable colloidal systems could be generated, which do not result in agglomeration as a prerequisite for use in medicine. This could be shown in Example 3.
In another preferred embodiment, iron oxide nanoparticles may be used for in step (i) of the method. In particular, Feridex®, ferucarbotran, SHU 555C, or the zinc-containing particles disclosed in WO 2008/127031 may be used.
In another preferred embodiment of the present invention, the biocompatible saline solution comprises at least one buffering compound, in particular at least one buffering compound selected from citrate, HEPES ADA, Bicine, MES and Tris, in particular citrate and HEPES, in particular at a total concentration of buffering compounds of about 5 to about 100 mM, even more preferably of about 10 to about 70 mM.
In another preferred embodiment of the present invention, the biocompatible saline solution comprises about 50 to about 500 mM NaCl, preferably about 80 to about 400 mM NaCl, more preferably about 100 to about 350 mM NaCl, even more preferably about 108 mM NaCl.
In another preferred embodiment of the present invention, the biocompatible saline solution has a pH of about 4.0 to about 10.0, preferably has a pH of about 5.5 to about 9.0, more preferably has a pH of about 6.5 to about 8.5, even more preferably has a pH of about 7.4.
In another preferred embodiment of the present invention, the biocompatible saline solution comprises, in particular consists of 20 mM sodium citrate, 108 mM NaCl, and 10 mM HEPES, and wherein the pH of the biocompatible saline solution is about 7.4, which is equal to physiological pH.
In another preferred embodiment of the present invention, the biocompatible saline solution is sterile.
In another embodiment of the present invention, the invention relates to a biocompatible aqueous colloidal system comprising iron oxide nanoparticles or oxide ferrimagnetics with spinel structure nanoparticles obtainable by the methods of the invention for preparing a biocompatible aqueous colloidal system.
In a further embodiment, the present invention relates to a method for preparing ferriliposomes comprising:
In a preferred embodiment, step (iii) comprises:
In a further embodiment, the present invention relates to a ferriliposome
The ferriliposomes of the present invention are surprisingly useful for targeted delivery in vivo and in vitro as shown in Examples 11, 13, 18, 19 and 24 as well as sections “Ferriliposome delivered JPM-565 inhibits growth of mammary tumour lesions” and “Efficacy of ferriliposomes as an MRI-visible drug delivery system in vivo” in the Examples. Moreover, the ferriliposomes are shown to be non-toxic (Example 17).
In a preferred embodiment, the method for preparing ferriliposomes of the invention, or the ferriliposome of the invention are characterized by:
As could be shown in
As shown in the Examples, the ferriliposomes according to the present invention are magnet-sensitive and can target both the tumour and its microenvironment. The nanosized ferriliposomes display outstanding magnetic resonance T2 contrast properties (r2=573-1286 s−1 mM−1), both in vitro and in vivo in transgenic tumour bearing mice, enabling drug delivery to be monitored noninvasively. Successful tumour microenvironment targeting and uptake of a probe administered by ferriliposomes were visualized in vivo. Targeted delivery of an inhibitor of tumour promoting proteases to the mouse mammary tumour and its microenvironment substantially reduced tumour size compared to systemic delivery of the same drug.
In a preferred embodiment, the ferriliposome of the present invention further comprises at least one therapeutically active agent and/or at least one diagnostically active agent and/or at least one agent allowing targeting of the ferriliposome, preferably
As shown in the Examples, doxorubicin and a Cathepsin-inhibitor could successfully be targeted to the peri-tumoral region of mouse breast cancer, resulting in significant tumour growth reduction without any adverse effect (see Examples, section “Ferriliposome delivered JPM-565 inhibits growth of mammary tumour lesions”, Examples 9 and 15).
In one embodiment, the present invention relates to a ferriliposome of the present invention, or an iron oxide nanoparticle of the present invention or a oxide ferrimagnetics with spinel structure nanoparticle of the present invention, for use in medicine, in particular
In another embodiment, the present invention relates to a ferriliposome of the present invention, or an iron oxide nanoparticle of the present invention or an oxide ferrimagnetics with spinel structure nanoparticle of the present invention, for the preparation of a medicament and/or diagnostic, in particular
In another embodiment, the present invention relates to a method of diagnosing and/or treating a disease, a preferably a neoplastic, a neuronal and/or an inflammatory disease, comprising administering to a patient in need thereof a diagnostically an/or therapeutically effective amount of a ferriliposome of the present invention, or an iron oxide nanoparticle of the present invention or an oxide ferrimagnetics with spinel structure nanoparticle of the present invention. In a preferred embodiment, the patient is a mammal, in particular a human.
In a preferred embodiment, the method further comprises applying a magnetic field by a magnet, in particular applying the magnetic field locally at the site to be diagnosed and/or treated. Typically the magnetic field is in the range of about 0.3 to about 4.5 Tesla, in particular in the range of about 1.0 to about 3.5 Tesla.
The present invention further provides iron oxide and oxide ferrimagnetics with spinel structure nanoparticles as well as ferriliposomes which allow targeted delivery of the nanoparticles and ferriliposomes respectively, to a site of interest using a magnetic field. Thereby, the iron oxide and oxide ferrimagnetics with spinel structure nanoparticles as well as ferriliposomes a magnetic resonance imaging represent (MRI)-visible drug delivery systems. The iron oxide and oxide ferrimagnetics with spinel structure nanoparticles as well as ferriliposomes are useful for determining the distribution of drugs using MRI, as well as organ-, tissue-, and/or site-specific drug delivery.
The oxide ferrimagnetics with spinel structure nanoparticles and iron oxide nanoparticles may be formulated and administered as a pharmaceutical composition.
The present invention therefore also relates to a pharmaceutical composition comprising at least one ferriliposome of the present invention, or iron oxide nanoparticle of the present invention or an oxide ferrimagnetic with spinel structure nanoparticle of the present invention or biocompatible aqueous colloidal system of the present invention.
The pharmaceutical composition of the present invention comprises therapeutically and/or diagnostically effective amounts oxide ferrimagnetics with spinel structure nanoparticles and/or iron oxide nanoparticles and/or ferriliposomes of the invention and generally an acceptable pharmaceutical carrier, diluent or excipient, e.g. sterile water, physiological saline, bacteriostatic saline, i.e. saline containing about 0.9% mg/ml benzyl alcohol, phosphate-buffered saline, Hank's solution, Ringer's-lactate, lactose, dextrose, sucrose, trehalose, sorbitol, mannitol, and the like. In a preferred embodiment, a formulation in a biocompatible saline solution according to the present invention, optionally comprising further excipients and additives, may be used. In another preferred embodiment, a biocompatible aqueous colloidal system according to the present invention, optionally comprising further excipients and additives may be used. The composition is generally a colloid, dispersion or suspension. It can be administered systemically, intravenously, orally, subcutaneously, intramuscularly, pulmonary, by inhalation and/or through sustained release administrations. Preferably, the composition is administered systemically, in particular intravenously.
The term “therapeutically effective amount” generally means the quantity of a therapeutically active agent, where applicable, which results in the desired therapeutic effect. A typical dosage range is from about 0.01 μg to about 1000 mg per application.
The term “diagnostically effective amount” generally means the quantity of a oxide ferrimagnetics with spinel structure nanoparticle and/or iron oxide nanoparticle and/or ferriliposome of the invention which results in the desired diagnostic effect without causing unacceptable side-effects. A typical dosage range is from about 0.01 μg to about 1000 mg per application.
Generally, the administration of the oxide ferrimagnetics with spinel structure nanoparticle and/or iron oxide nanoparticle and/or ferriliposome and/or pharmaceutical composition to a patient is one made one or several times, for example one or several times per day, or one or several times a week, or even during longer time periods as the case may be. For diagnostic purposes, a single administration may be sufficient.
In a further embodiment, the present invention relates to a carrier comprising at least one iron oxide nanoparticle and/or oxide ferrimagnetics with spinel structure nanoparticle of the present invention, or at least one iron oxide nanoparticle and/or oxide ferrimagnetics with spinel structure nanoparticle of the suspension of the present invention, or at least one iron oxide nanoparticle and/or oxide ferrimagnetics with spinel structure nanoparticle of the biocompatible aqueous colloidal system of the present invention, preferably wherein the carrier is selected from a nanotube, a liposome, a lipoplex, a polymersome, a micell, a nanogel, a mesoporous silica nanoparticle, a dendrimer, and a nanoshell, in particular the carrier is a liposome.
In a further embodiment, the present invention relates to a kit comprising:
In one embodiment, the iron oxide nanoparticles can be used as T2 MR contrast agents and/or negative MR contrast agents. The negative T2 effect is very strong, as the MR signal diminishes in the vicinity of the presence of the contrast agent on T2-weighted MR images.
In one embodiment, the oxide nanoparticles facilitate an increased contrast in the MRI images and/or an increased signal. In a preferred embodiment, the increased signal can be translated to shorter acquisition times, and/or higher spatial resolution and/or a reduction in dose of the contrast agent.
In another embodiment, the oxide ferrimagnetics nanoparticles with spinel structure according to the formula CoxFe3-xO4 can be used as T1 and T2 contrast agents.
“About” according to the present invention is understood as meaning the experimental error range, in particular ±5% or ±10%.
“Nanoparticle” according to the present invention is understood as particle with a diameter in at least one dimension exceeding at least about 1 nm, preferably at least about 10 nm, more preferably at least about 20 nm. In a preferred embodiment, a nanoparticle has a diameter in at least two dimensions, preferably in three dimensions exceeding at least about 1 nm, preferably at least about 10 nm, more preferably at least about 20 nm, as determined by dynamic light scattering, as for example described in the Examples. In a preferred embodiment, a nanoparticle is spherical. In a further preferred embodiment, a nanoparticle is less than about 1 μm, preferably less than 150 nm in diameter in at least one dimension, preferably in at least two dimensions, preferably in three dimensions.
“Iron oxide nanoparticle” according to the present invention is understood as nanoparticles consisting of at least about 80%, preferably at least about 90%, more preferably at least about 95%, even more preferably at least about 99% iron oxide. In particular, iron oxide is understood as Fe3O4 or FeO.Fe2O3.
A “spinel” according to the present invention is understood as compounds of the formula [AyB2-y]O4, wherein A is a divalent metal, preferably Fe2+, Cu, Co, Ni, Mg or Mn, in particular A is Fe2+, and wherein B is a 3- or 4-valent metal, for example Al, Fe3+, V, Cr, Ti, in particular B is Fe3+, and wherein y is 0 or 1.
“Oxide ferrimagnetics with spinel structure” according to the present invention are understood as spinel of the formula M2+Fe3+2O4 or MIIO.Fe2O3, wherein M is a divalent metal, preferably Fe2+, Cu, Co, Ni or Mn, in particular Fe2+. Suitable oxide ferrimagnetics with spinel structures comprise for example Fe3O4, CuFe2O4, NiFe2O4, MgFe2O4 and CoFe2O4, preferably Fe3O4 and nonstoichiometric oxide ferrimagnetics with spinel structure comprise for example CoxFe3-xO4 and MnxFe3-xO4, wherein 0.1≦x≦0.99, preferably 0.6≦x≦0.98.
A carrier according to the present invention is understood as a chemical entity with a length in at least one dimension exceeding at least about 1 nm, preferably at least about 10 nm, more preferably at least about 100 nm, and which allows covalent or non-covalent binding of further moieties. In a preferred embodiment of the present invention, the carrier is selected from a nanotube, a liposome, a lipoplex, a polymersome, a micell, a nanogel, a mesoporous silica nanoparticle, a dendrimer, and a nanoshell, in particular the carrier is a liposome. In the case of a liposome, the nanoparticles and, where applicable, a further therapeutic and/or diagnostic moiety may be encapsulated within the liposome, or, where applicable, the further therapeutic and/or diagnostic moiety may integrated in the bilayer of the liposome. The generation of liposomes may be performed by methods known to a skilled person. In particular, the liposomes may be generated by extrusion as described in Example 4, or by sonification. In other embodiment, like e.g. dendrimers, the nanoparticles of the present invention, and, where applicable, the therapeutic and/or diagnostic moiety are bound covalently to the dendrimer, preferably via a linker. The synthesis and use of a nanotube, a liposome, a lipoplex, a polymersome, a micell, a nanogel, a mesoporous silica nanoparticle, a dendrimer, and a nanoshell are for example described in (Orive et al., 2009) A “liposome” according to the present invention is understood as vesicle made of a lipid bilayer. A liposome may comprise one or more lipids, in particular phospholipids, more preferably phosphatidylcholine. A liposome may comprise further lipids, preferably PEGylated lipids, even more preferably. In a preferred embodiment, the lipid may be present. Such lipids are described in Example 4. A liposome may be unilamellar or multilamellar. In case of multilamellar liposomes, it may comprise 2, 3, 4, 5 or more lamelles.
“Ferriliposome” according to the present invention is understood as liposome comprising one or more iron oxide nanoparticles and/or oxide ferrimagnetics with spinel structure nanoparticles of the invention. In a preferred embodiment, the nanoparticle(s) are located in the hollow space of the liposome. A ferriliposome may comprise further diagnostically and/or therapeutically active agents as described above. Also, a ferriliposome may be coated with an optionally functionalized polymer. For example, a ferriliposome may be coated with dextran or functionalized dextran. Ferriliposomes coated with Alexa Fluor-conjugated dextran are described in Example 4. The ferriliposomes typically have a diameter of about 20 nm to about 1 μm, preferably of about 50 nm to about 500 nm, more preferably of about 80 nm to about 200 nm, in particular preferably of about 90 nm to about 110 nm. A ferriliposome may be unilamellar or multilamellar. In case of multilamellar ferriliposomes, it may comprise 2, 3, 4, 5 or more lamelles.
A “therapeutic moiety” according to the present invention is a chemical moiety, which is capable of exhibiting a therapeutic effect when administered to a patient in need thereof in an effective amount. The therapeutic moiety is preferably selected from:
a chemotherapeutic agent,
a radioactive agent,
a protease inhibitor, in particular a cathepsin inhibitor, more preferably JPM-565,
an apoptosis-inducing agent, and
an anti-inflammatory agent,
It is understood, that the selection of the therapeutic moiety depends on the disease to be treated.
A “diagnostic moiety” according to the present invention is a chemical moiety, which can be detected. Preferably, the chemical moiety can be detected in vitro, ex vivo, or in vivo, preferably in vivo. The diagnostic moiety is preferably selected from:
a radioactive agent,
a paramagnetic agent,
a PET-imagable agent,
an MRI-imagable agent,
a fluorophore, in particular Alexa Fluor,
a chromophore,
a phosphorescing agent,
a chemiluminescent agent, and
a bioluminescent agent.
“Biocompatible” according to the present invention is understood as a solution that will not induce any undesirable local or systemic response in the animal, in particular human, to which it is administered. In a preferred embodiment, the administration is systemic, in particular intravenous.
The iron oxide particles according to the present invention were prepared by mechanochemical synthesis using saline crystal hydrates (see Example 1). The use of saline crystal hydrates instead of conventional methods utilizing anhydrous salts changes the solid phase mechanism to soft mechanochemical synthesis in aqueous media, surprisingly resulting in a significantly increased reaction rate. Furthermore, this modification surprisingly resulted in ultrasmall spherical particles of 3-14 nm in diameter (>70% less than 8 nm) (
The main limiting factor in using nanoparticles, in particular iron oxide nanoparticles in vivo is their low colloidal stability. The method of the present invention for preparing a biocompatible aqueous colloidal system prevents their agglomeration, leading to a more narrow nanoclusters particle size distribution (
The suspension of the present invention surprisingly exhibits high colloidal stability under physiological conditions as well as at various pH values and ionic strengths (Example 1,
Stabilized iron oxide nanoparticles were encapsulated in sterically stabilized polyethylene glycol (PEG) coated liposomes (PEGylated; Stealth liposomes), forming ferriliposomes of about 100 nm diameter. Modification of the liposome surface with PEG is known to greatly reduce the opsonization of liposomes and their subsequent clearance by the reticuloendothelial (mononuclear-phagocyte) system, resulting in a substantially prolonged circulation half-life. This was confirmed in a cellular experiment (
The oxide ferrimagnetics with spinel structure nanoparticles according to formula Co0.84Fe2.16O4 were prepared by mechanochemical synthesis using saline crystal hydrates (see Example 2) with size distribution of nanoparticles (
The size of spinel oxide ferrimagnetic nanoparticles in a suspension (aqueous colloidal system) was determined using a dynamic light scattering (Dynamic Light Scattering Detector PD 2000 DLS Plus). Ion concentration was determined by flame-spectroscopy using a Varian Spectr AA 110 atomic absorption spectrometer.
Characterization of MR Contrast Properties of Iron Oxide Nanoparticles In Vitro
The MR contrast properties of the stabilized nanoparticle suspension in vitro have been evaluated in MRI agarose phantoms (MRAP), simulating the tumour tissue, using 1% agarose, with T2≈80 ms, which are similar to those of tumour tissues. MRAPs containing iron oxide nanoparticles with nanoclusters mean hydrodynamic diameter of 39 nm and 57 nm, respectively, were screened for the MRI contrast properties. The longitudinal (T1) and transverse (T2) relaxation times were measured at different iron oxide nanoparticle concentrations, and r1 and r2 relaxivities were determined to be 12 s−1 mM−1 and 573 s−1 mM−1 for the 39 nm nanoparticles, and 31 s−1 mM−1 and 1286 s−1 mM−1 for the 57 nm iron oxide nanoparticles. Comparison with the commercially available SPIO nanoparticles Feridex® (Bayer HealthCare Pharmaceuticals) and the standard gadolinium-based T1 contrast agent Magnevist® (Bayer HealthCare Pharmaceuticals) revealed that iron oxide nanoparticles exhibited several fold higher relaxivities than commercial MRI contrast agents, resulting in ultra-sensitive MRI detection (
To verify the effectiveness of the iron oxide nanoparticles as positive T1 and negative T2 contrast agents, T1-weighted and T2-weighted images of the control phantom and MRAPs with 0.017 mM of the 39 nm and 0.17 mM of the 57 nm iron oxide nanoparticles were obtained (
To demonstrate the targeting effectiveness of the magnetic nanoparticles suspension and their MM contrast properties upon internalization into the phantom, two techniques for iron oxide nanoparticle delivery into the MRAP were used: (i) direct injection of the iron oxide nanoparticle solution with an 0.3 mm needle into the centre of MRAP (sample 2 in
Characterization of MR contrast properties of cobalt oxide ferrimagnetic nanoparticles with spinel structure according to formula CO0.84 Fe2.16O4 in vitro
The longitudinal (T1) and transverse (T2) relaxation times were measured at different concentrations of cobalt oxide ferrimagnetics nanoparticles with spinel structure in 1% agarose at room temperature for the frequency 1H MP νH=100 MHz (
Also, the loss of signal was observed in the sample with the concentration of nanoparticles of 3.4 mm. The signal intensity for the concentration of 0.034 mM was comparable to that for 1% agarose. The results have demonstrated the possibility of using nanoparticles as contrast agents in selecting a desired concentration. The properties of oxide ferrimagnetics nanoparticles with spinel structure according to formula CO0.84 Fe2.16O4 as T1 and T2 contrast agents have also been demonstrated using a sample containing 1% agarose, which is locally added with a solution of 0.34 mM nanoparticles. The nanoparticles were injected into the middle of the agarose phantom (
Efficacy of ferriliposomes as an MRI-visible drug delivery system in vivo
To establish the efficacy of the prepared ferriliposomes for in vivo applications a genetically engineered mouse model of human breast cancer (MMTV-PyMT) was employed resulting in a widespread transformation of the mammary epithelium and in the development of multifocal mammary adenocarcinomas. Initially, ferriliposomes were demonstrated to be non-cytotoxic in mouse embryonic fibroblasts (MEFs) and primary mouse tumour cells (see
Having shown that the system is suitable for in vivo applications, ferriliposomes were injected intraperitoneally into a MMTV-PyMT tumour bearing mouse, under a magnetic field applied for 1 hour to the first left inguinal mammary tumour. iron oxide nanoparticles delivered by ferriliposomes were detected as a dark area on the T2-weighted MR images, 1 and 48 hours post injection (
The efficacy of ferriliposomes as an MRI-visible drug delivery system for oxide ferrimagnetic nanoparticles with spinel structure according to formula Co0.84 Fe2.16O4 in vivo is shown in
Ferriliposome Delivered JPM-565 Inhibits Growth of Mammary Tumour Lesions
The initial testing of the ferriliposome system for the targeted drug delivery was performed with a standard cancer chemotherapy drug, doxorubicin. Even a single dose treatment with doxorubicin targeted by ferriliposomes resulted in a 90% reduction of tumour volume two weeks after administration, compared with 60% decrease obtained by the standard doxorubicin administration (
Although intraperitoneal administration of therapeutic agents is an important adjunct to surgery and systemic chemotherapy of cancer in selected patients 44 it was evaluated the effectiveness of intravenous administration of the delivery system for targeting a tumour and its microenvironment in MMTV-PyMT transgenic female mouse. The fluorescence of ferriliposome cargo (Alexa Fluor 555™) was found to be co-localized both with the stroma (
The iron oxide nanoparticles synthesized are preferably ferrimagnetic (magnetic ferrite spinel, magnetite, Fe3O4) and are also called FMIO (ferrimagnetic iron oxide nanoparticles). Ferrimagnetic iron oxide (iron oxide) nanoparticles (magnetite, Fe3O4) were manufactured by modified and optimised mechano-chemical synthesis. The standard mechanochemical synthesis of iron oxide nanoparticles is for example described in Naiden et al., 2003. However, according to the present invention, saline crystal hydrates are used for the first time for generating iron oxide nanoparticles. In particular, the salt crystal hydrates FeSO4. 7 H2O and FeCl3. 6H2O were used. In order to prevent heating of the reagent mixture during activation, we additionally introduced sodium chloride as an inert component in the ratio 1:2. The mechanochemical synthesis was performed in an MPV planetary mill at 60 g acceleration and the weight ratio of the powder (i.e. the reaction mixture comprising the two salt crystal hydrates and NaOH) and balls was 1:20. The reaction in the planetary mill was performed for 30 min. The obtained product was washed on a filter with distilled water until the salts were completely removed. The electron microscopy and size distribution of iron oxide nanoparticles are shown in
The oxide ferrimagnetics nanoparticles with spinel structure were obtained by mechanochemical synthesis using iron and cobalt chlorides as basic reagents in the presence of sodium chloride as inert component according to the following reaction:
2FeCl3+CoCl2+Ca(OH)2+3Na2CO3=CoxFe3-xO4+CaCl2+6NaCl+3CO2↑+H2O
where 0.1≦x≦0.99.
The value of 0.6≦x≦0.98 is preferable.
The starting materials used for the synthesis were FeCl3, CoCl2 in the form of salt crystal hydrates. For the purpose of changing the ratio of cobalt and iron in the end product and preventing the heating of the mixture of reagents and the aggregation of nanoparticles sodium chloride as additional inert component was injected. The mixture was sealed in a hardened steel drums with steel balls with a diameter of 4-5 mm. Mechanochemical synthesis was carried out in a MPV planetary mill with the acceleration of 55-60 g.
The conditions for accomplishment of a given technical effect of the invention are the strict adherence to the weight ratios of the mass of reaction mixture to the mass of inert component of 1:(1÷4) and the mass of powder to the mass of balls equal to 1:20, and the time for performing of mechanochemical synthesis of 10÷60 min. The product obtained through a heat treatment at 100° C. for 0.5÷1:00 (or without treatment) was washed in the filter with distilled water until free of salts and dried at room temperature, and then, if necessary, sonicated and centrifuged (UZDN-2T and <<Bekman J2-21″).
Phase composition, morphology, dispersion, and structural parameters of nanoparticles were determined by X-ray diffraction (XRD) using the Schimadzu XRD-6000 device with CuKα-radiation and by transmission electron microscopy (TEM) using the EM-125 device. The specific surface area (S) was determined by the method of thermal desorption of nitrogen (‘SORBI’ N 4.1) and the chemical composition was analyzed by X-ray fluorescence analysis (XRF) using Schimadzu XRD-1800 device and by inductively coupled plasma-atomic emission spectrometry (ICP-AES) using iCAP-6300 Duo, Thermo Scientific spectrometer. The data of X-ray structure analysis were processed using the full-profile analysis program POWDER CELL 2.5. The average diameter of particles was calculated from the values of specific surface area and particle density.
In the study of magnetic properties of ferrite spinel according invention used methods for analyzing the temperature dependence of initial magnetic permeability at a frequency of 10 kHz, and the magnetization curves and their derivatives are obtained in pulsed magnetic fields up to 3 T by the method described in (V. U. Kreslin, E. P. Naiden/PTE, 2001, No. 5, p. 63).
The investigation of the end products of mechanochemical synthesis showed that the powder consists of nanosized spherical particles with a diameter of 3-20 nm which are loosely coupled with each other (
Table II. Effect of activation time on the chemical and phase composition of oxide ferrimagnetic nanoparticles with spinel structure (CoxFe3-xO4) (acceleration is 60 g, the mass of the balls: the mass of the reacting mixture=20:1, the mass of NaCl: the mass of the reacting mixture=2:1 and FeCl3.6H2O, CoCl2.6H2O are reagents).
The specific surface area of the resulting nanoparticles of cobalt spinel ferrite was 113 m2/g and the specific saturation magnetization was G.22-25 cm3/g.
The increase in the content of inert diluent from 1:2 to 1: (3 or 4) for the time of mechanochemical synthesis of 30 min results in the product of a nearly stoichiometric composition (Co0.99Fe2.01O4) instead of the product with the chemical formula CO0.84Fe2.16O4 (Table III).
With further increase in the content of inert component in the reaction mixture to 5:1 the yield of the end product significantly reduced. In this regard, the lower boundary value of the ratio of mass of reaction mixture to the mass of inert component was assumed to be 1:3.
The yield of the final product having chemical composition CoxFe3-xO4 was also determined by the conditions of mechanochemical synthesis. For the mass ratio of balls or sodium chloride to the mass of the reaction mixture of 20:1 and 2:1 respectively, the time of mechanical activation of 5 min. or less, and low conversion degree the yield of the end product was very low (35%) respectively (Table II).
With further increase in the duration of mechanical activation to 10 min the yield of the end product increased to 60% (lower value), and after treatment for 25÷40 min the yield was 94-96% (upper value). As a result, the value of the ratio of the mass of reaction mixture to the mass of inert component 1:2 was taken as the upper limit value. With increasing time of mechanical treatment the time of mechanical activation of 60 min was determined by a decrease in yield of the end product to 85%, other conditions being the same. The further increase in time of the mechanochemical process did not influence significantly the final product yield.
Thus, it is advisable to carry out the process of mechanical activation in the range of following parameters: the ratio of the mass of sodium chloride to the mass of the reaction mixture of (2:1)÷(3:1) and the time of mechanical activation of 10÷60 min. The heat treatment of the product of mechanochemical activation at 100±20° C. during 0.5÷1 h helps to ensure a final product having chemical composition CoxFe3-xO4, where 0.1≦x≦0.99 and which reveals high contrast properties at T1 and T2 relaxation times as shown below.
The investigation of the final products of mechanochemical synthesis shows that the manganese cubic spinel ferrite powder consists of nanosized spherical particles with a diameter range of 5-19 nm.
Depending on the synthesis conditions, the final products could contain to 90-99% vol. of spinel phase and the rest is mostly β-FeOOH and hematite.
As X-ray fluorescence analysis reveals that the chemical composition of nanosized manganese cubic spinel ferrite powder obtained at a ratio of the mass of reaction mixture to the mass of NaCl inert component 1:2 and at the time of the mechanochemical synthesis of 30-40 min has chemical formula MnxFe3-xO4, where x=0.50-0.96 and differs noticeably from stoichiometric composition. The increase in the content of inert diluent to 1:4 allows the obtaining of powders of nearly stoichiometric composition (Table IV).
The magnetic properties of nanosized powders of cubic spinel ferrites whose specific magnetization is 20-30 Gs·cm3/g determine their ability to be controlled by magnet.
Iron oxide nanoparticles of Example 1 were suspended in a stabilizing buffer (20 mM sodium citrate buffer pH 7.4, containing 108 mM NaCl, 10 mM HEPES), sonicated with an ultrasonic disintegrator at 20 kHz, 3 min (Ultrasonic disintegrator, Branson) and centrifuged at 500 g for 3 min to separate the remaining undisrupted agglomerates. The resulting stable colloidal dispersion of non-aggregating nanoparticle clusters was characterized using flame atomic absorption spectrometry on a Varian SpectrAA 110 atomic absorption spectrometer (Varian, Mulgrave, Australia), dynamic light scattering (DLS) using a PDDLS/BatchPlus System (Precision Detectors), and field emission gun scanning electron microscopy (FEG-SEM) using an FESEM SUPRA 35 VP (Carl Zeiss) equipped with energy dispersive spectroscopy Inca 400 (Oxford Instruments). The zeta potential of iron oxide nanoparticles was measured by PALS Zeta Potential Analyzer Ver. 3.19 at pH 7.4 and 37° C. The result is shown in
Iron oxide nanoparticles were suspended in a stabilizing buffer (20 mM sodium citrate buffer pH 7.4, containing 108 mM NaCl, 10 mM HEPES). The resulting agglomerates were disrupted with an ultrasonic disintegrator (Branson), followed by separation of the remaining agglomerates by centrifugation at 500 g for 3 min (Eppendorf Centrifuge 5417C, Eppendorf). The nanoparticle drying step used in the initial procedure (Naiden, et al., 2003) was omitted. The colloidal stability of iron oxide nanoparticles was tested by increasing the ionic strength of the solution (NaCl concentration from 108 mM to 324 mM), and at two different pH values (pH 5.5, pH 9.0) of the solution. The colloidal stability was assessed by measurements of iron oxide cluster average sizes in the resulting suspensions by dynamic light scattering (DLS), using a PDDLS/BatchPlus System (Precision Detectors). It could be shown, that also stable suspensions could be obtained using these buffers.
A suspension of nanoparticles of CO0.84 Fe2.16O4 in a stabilizing buffer was obtained (pH=7) via ultrasonic disintegration (Bandelin). For the preparation of nanosuspension, for example, 100 mg of nanoparticles were dissolved in a stabilizing buffer (20 mM sodium citrate, 108 mM NaCl, 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazinethan sulfonic acid) and sonificated (20 kHz, 50 V) for 5 min. In the course of sonification the large aggregates of nanoparticles were broken and covered with macromolecules of sodium citrate to prevent a reverse aggregation. Unbroken particles were precipitated in the gravitational field of 500 g.
Iron oxide nanoparticles loaded liposomes (ferriliposomes) were prepared from 95% L-a-phosphatidylcholine (Avanti Lipids) and 5% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (Avanti Lipids) with a total lipid concentration of 2.75 mM. Organic solvent was evaporated in an Eppendorf Concentrator 5301 (Eppendorf), resulting in formation of dry lipid films. Their subsequent hydration with iron oxide nanoparticles in 20 mM citrate buffer pH 7.4, containing 108 mM NaCl and 10 mM HEPES, led to multilamellar vesicles containing nanoparticles. The multilamellar vesicles were extruded by mini-extruder containing a polycarbonate membrane, pore size 100 nm (Avanti Lipids), in order to generate nanosized unilamellar bilayer liposomes forming ferriliposomes. Non-encapsulated nanoparticles were removed by gel filtration on Sephadex™ G-25 M PD-10 columns (GE Healthcare). Ferriliposomes were separated magnetically from empty liposomes on a Dynal MPC-S magnetic separator (Dynal) and resuspended in stabilizing buffer. The morphology and size of the ferriliposomes was followed by atomic force microscopy (images were obtained with a Nanoscope III Multimode scanning probe microscope (Digital Instruments) operated in tapping mode) and DLS.
For fluorescence studies, ferriliposomes were functionalized with Alexa Fluor 546™-labelled dextran (Invitrogen) or non-conjugated Alexa Fluor 555™ (Invitrogen). Alexa Fluor 546™ or Alexa Fluor 555™ were suspended (100 μg/ml) in iron oxide nanoparticles containing the stabilizing buffer, and encapsulated in PEGylated liposomes as described. The fluorescent ferriliposomes were separated from non-encapsulated Alexa Fluor dye by gel filtration on a Sephadex™ G-25 M column (GE Healthcare).
Liposomes (ferriliposomes) loaded with oxide ferrimagnetic nanoparticles with spinel structure according to formula: Co0.84 Fe2.16O4 were prepared. An aliquot of lipids (2.6 mM of phosphatidylcholine and 0.2 mM of 0.2-distearol-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (Avanti Polar Lipids Inc.), dissolved in chloroform was placed in a vacuum for the evaporation of the solvent with the formation of dry lipid films. Dry films were hydrated by adding of stabilized nanoparticles (3.4 mM).
The dispersion was emulsified by sonication in an ultrasonic bath for 5 min. The size of liposomes was determined using a dynamic light scattering (Dynamic Light Scattering Detector PD 2000 DLS Plus). Liposomal spheroids with a diameter of 90-110 nm were visualized by atomic force microscopy (AFM).
Female FVB/N and FVB/N-TgN(MMTVPyVT)634Mu1 mice were used in accordance with protocols approved by the Veterinary Administration of the Republic of Slovenia (VARS) and the government Ethical Committee. Procedures for animal care and use were in accordance with the “PHS Policy on Human Care and Use of Laboratory Animals” and the “Guide for the Care and Use of Laboratory Animals” (NIH publication 86-23, 1996). In order to generate tumours for the treatment study, primary MMTV-PyMT tumour cells were obtained from 14 week old MMTV-PyMT transgenic mice as described 36, culture-expanded, suspended in 200 μl serum free Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen), and 5×105 cells were injected into the left inguinal mammary gland of the recipient mouse (FVB/N mouse strain).
All MR experiments were performed on a TecMag Apollo MRI spectrometer with a superconducting 2.35 T horizontal bore magnet (Oxford Instruments) using a 25 mm saddle-shaped Bruker RF coil. Spin-lattice and spin-spin relaxation times (T1 and T2) were measured for different concentrations of iron oxide nanoparticles in 1% agarose at room temperature, using inversion recovery and spin-echo techniques, respectively. The longitudinal (rt) and transverse (r2) relaxivities were calculated from ri=(1/Ti−1/Ti0)/c, where c is the concentration of iron oxide nanoparticles in mM, Ti is the relaxation time at concentration c, Ti0 the relaxation time of 1% agarose, and i=1, 2 for T1 and T2. 2D MR images were taken using a standard multi-echo pulse sequence with an echo time (TE) of 8.5 ms and a repetition time (TR) of 400 ms for T1 MR images, and with TE=60 ms and TR=2000 ms for T2-weighted MR images. The field of view was 40 mm with an in-plane resolution of 156 μm and a slice thickness of 1 mm. Ferriliposomes were detected ex vivo by taking T2-weighted MR images before and after injection of 50 μl ferriliposome solution (3.4 mM nanoparticles) into one of the tumours. For in vivo detection, an external magnet of 0.33 T (diameter 4.5 mm) was glued to the right inguinal mammary gland of 12 weeks old mouse by cyanoacrylate and 200 μl of ferriliposomes (3.4 mM nanoparticles) were intraperitoneally injected. The magnet was removed 1 hour after ablution with acetone. T2-weighted MR images were taken before injection, 1 hour post-injection and 48 hours post-injection of ferriliposomes. During imaging, the mouse was anaesthetized by subcutaneous injection of ketamine/xylazine/acepromazine (50/10/1.0 mg/kg).
Primary MMTVPyMT cells were isolated and cultured as described 36. Mouse embryonic fibroblasts (MEFs) were generated from 12.5 days post-coitum mouse embryos of FVB/N mice; only low passage number cells (<4 passages total) were used for experiments. All primary cells were maintained in DMEM supplemented with 10% fetal bovine serum (Sigma), 2 mM L-glutamine (Invitrogen), 100 units of penicillin and 100 μg/ml streptomycin (Invitrogen). Cultured cells were maintained at 37° C. in a humidified 5% CO2 atmosphere. For fluorescence microscopy studies, primary MMTV-PyMT tumour cells and fibroblasts were cultured with 500 μl of Alexa Fluor 555™-functionalized ferriliposomes in normal culture medium on Lab-Tek™ Chamber Slides (Nunc). After incubation with nanoparticles for 3 hours, cells were washed with PBS, stained with Hoechst 33342 (Fluka) and examined with an Olympus fluorescence microscope (Olympus IX 81) with Imaging Software for Life Science Microscopy Cellf.
Female FVB/N-TgN(MMTVPyVT)634Mul mice (PyMTtg/+) developing multifocal adenocarcinomas were crossed with the FVB/N mouse strain expressing firefly luciferase under the control of the β-actin promoter (FVB.luctg/+)30. Resulting double transgenic mice (FVB.luctg/+;PyMTtg/+) develops breast tumours with simultaneous expression of luciferase through the whole body. For in vivo control of ferriliposomes distribution and content release, ferriliposomes were functionalized with D-luciferin (Sigma) by suspending in nanoparticles containing stabilizing buffer (2.5 mg/ml), followed by encapsulation in PEGylated liposomes. 400 μl of ferriliposomes loaded with D-luciferin (30 mg/kg) were intraperitoneally administered to the 10 weeks old FVB.luctg/+;PyMTtg/+ mouse and a magnet was attached to the 1st right pectoral mammary tumour. In the control experiment magnet was omitted. 24 hours after ferriliposomes administration magnet was detached and mice were imaged non-invasively by IVIS® Imaging System (integration time 5 minutes, IVIS® 100 Series). During the scan mice were kept under gaseous anaesthesia (5% isofluorane) and at 37° C. Due to the luciferase present in all cells, D-luciferin release and its subsequent conversion by luciferase resulted in emission of a bioluminescent signal that could be imaged with an IVIS® Imaging System.
The dosing regimen for JPM-565 treatment was determined based on previous reports and studies on RIP1-Tag2 and MMTV-PyMT mouse models 33-35, 45. JPM-565 had no discernable toxic side effects in the animal trials 33, 45. The regular injections, followed by magnetic targeting, were started when tumours reached a volume of 125 mm3. JPM-565 was dissolved in iron oxide nanoparticles containing stabilizing buffer and then encapsulated into PEGylated liposomes. The ferriliposomes loaded with JPM-565 were administered at a dose of 100 mg/kg every second day in 10 intraperitoneal injections (JPM+FL, n=9). Magnets of 0.33 T (diameter 4.5 mm) were attached to the tumour before the first injection and removed 24 hours after the last injection as described above. Control groups were treated with stabilizing buffer (Control, n=7), ferriliposomes with magnetic targeting (FLt, n=5), JPM-565 in nanoparticle stabilizing buffer (JPM, n=7) and ferriliposomes containing JPM-565 without magnetic targeting (JPM+FL, n=7). The horizontal and vertical tumour diameters were measured by digital calliper every second day until the end of treatment and volume was calculated using the formula V=(a×b2)π/6 where a and b are the longer and shorter diameter of the tumour. On the next day after the last injection mice were sacrificed and the excised tumour volumes calculated.
For the in vivo study, 200 μl of Alexa Fluor 546™ functionalized ferriliposomes were daily injected intraperitoneally to the orthotopic allograft breast cancer mouse model for 3 days. Alexa Fluor 555™ functionalized ferriliposomes were daily injected intravenously into the PyMT transgenic breast cancer mouse model for 2 days. A magnetic field was applied to the tumour for 12 hours immediately after each injection. On the next day after the last injection mice were sacrificed and the corresponding tumours were resected, fixed in 10% formalin overnight, dehydrated using Shandon Tissue Processor (Shandon Citadel 1000) and moulded with paraffin (Microm EC 350 Paraffin Embedding Station) or cryopreserved by snap freezing in liquid nitrogen. Paraffin sections were cut with 5 μm thickness, mounted with anti-fade media containing DAPI (Prolong@ Gold antifade reagent with DAPI, Invitrogen) and visualized as described above.
Histological measurement of proliferation by Ki67 staining, and tumour vascularisation rate by CD31, were performed on frozen tissue slides. For data assessment, 10 fields per tumour were randomly selected using a 40× objective and quantified using TissueQuest software (TissueGnostics). Rabbit anti-mouse E-cadherin (Abeam; 1:100 dilution) and secondary antibody goat anti-rabbit Alexa Fluor™ 488 (Invitrogen; 1:100 dilution) were used for immunodetection of cell-adhesion protein E-cadherin on cryopreserved tumour sections. Rat anti-mouse monoclonal FITC conjugated CD206 (1:100; AbD Serotec) were used for the detection of tumour associated macrophages on cryopreserved tumour sections. Samples were co-stained with Hoechst 33342 (5 μg/ml, Fluka) and mounted in ProLong® Gold antifade reagent (Invitrogen) and examined with an Olympus fluorescence microscope (Olympus IX 81) with Imaging Software for Life Science Microscopy Cellf.
Quantitative data are presented as means plus/minus standard error. The differences of the JPM-565 treatment effect were compared using Student's t-test. When P-values were 0.05 or less, differences were considered statistically significant.
THP-1 monocytic cell line was grown at 37° C. in a humidified air atmosphere with 5% CO2. Cells were cultured in the RPMI 1640 medium, supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 10 mM HEPES buffer. THP-1 cells were differentiated into macrophages by the addition of 30 μl of 10 μM phorbol 12-myristate 13-acetate (PMA) for 24 hours. Cells were then incubated with 100 μl aliquots of Alexa Fluor 546™ functionalized PEGylated and non-PEGylated liposomes in Dulbecco's Modified Eagle Medium (DMEM) on 96-well optical bottom plate (Nunc, USA) for 15 minutes. In the next step, cells were washed 3 times with the phosphate buffer, pH 7.4, and examined in a 96-well Saphire (TECAN, Austria) plate reader at excitation and emission wavelengths of 561 nm and 572 nm, respectively.
Mouse embryonic fibroblasts (MEFs) and primary MMTV-PyMT cells were maintained in DMEM supplemented with 10% fetal bovine serum (Sigma), 2 mM L-glutamine (Invitrogen), 100 units of penicillin and 100 μg/ml streptomycin (Invitrogen) at 37° C. in a humidified 5% CO2 atmosphere. For in vitro toxicity assay cells were incubated with ferriliposomes containing 3.4 mM iron oxide nanoparticles, or 3.4 mM and 55 mM iron oxide nanoparticles, in phosphate buffer, pH 7.4, for 24 hours at 37° C. In the control experiment nanoparticles were omitted. Exposure to phosphatidylserine and DNA fragmentation were measured by labelling cells with Annexin V-PE in the presence of propidium iodide according to the manufacturer's instructions. Cells were then subjected to flow cytometry (FACS) analysis using FACScalibur flow cytometer (Becton Dickinson, USA) and CellQuest software.
Treatment study was performed using the newly developed orthotopic allograft mouse breast cancer model using congenic immunocompetent mice (described in the manuscript).
Doxorubicin hydrochloride (Sigma) was dissolved in ferromagnetic iron oxide nanoparticles containing stabilizing buffer and then encapsulated into PEGylated liposomes. Three weeks after transplantations of primary cells (average tumour volume 75 mm3) ferriliposomes loaded with doxorubicin were administered at a dose of 15 mg/kg (Dox+FLt, n=4). Magnets of 0.33 T (diameter 4.5 mm) were glued to the tumour by cyanoacrylate before injection and removed by acetone 24 hours after administration. Control group was treated with systemic administration of doxorubicin without any targeting (Dox, n=4). The horizontal and vertical tumour diameters were measured by digital calliper twice per week and volumes were calculated using the formula V=(a×b2)π/6 where a and b are the longer and shorter diameter of the tumour, respectively.
Primary MMTV-PyMT tumour cells, obtained from 14 week old MMTV-PyMT transgenic mice, were culture-expanded, suspended in 200 μl serum free Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen) and injected (5×105 cells) into the left inguinal mammary gland of the recipient female FVB/N mouse expressing firefly luciferase under the control of the β-actin promoter (FVB.luctg/+). For in vivo control of ferriliposomes distribution and content release, ferriliposomes were functionalized with D-luciferin (Sigma) by suspending in nanoparticles-containing stabilizing buffer (2.5 mg/ml), followed by encapsulation in PEGylated liposomes. 400 μl of ferriliposomes loaded with D-luciferin (30 mg/kg) were intraperitoneally administered to the FVB.luctg/+ mouse bearing transplanted tumour and a magnet was attached to the left inguinal mammary gland tumour. 24 hours after ferriliposomes administration magnet was detached and mice were imaged non-invasively by IVIS® Imaging System (integration time 5 minutes, IVIS® 100 Series). During the scan mice were kept under gaseous anaesthesia (5% isofluorane) and at 37° C. After whole body imaging, mice were sacrificed and the organs were harvested and imaged with an IVIS® Imaging System (2 minutes, IVIS® 100 Series). Due to the luciferase presence in all the cells, D-luciferin release and its subsequent conversion by luciferase resulted in emission of a bioluminescent signal that could be imaged with an IVIS® Imaging System.
To clarify the pathway of ferriliposome clearance we employed transgenic mice expressing luciferase through the whole body and ferriliposomes loaded with the luciferase substrate, D-luciferin (Sigma), administered by intraperitoneal or intravenous injection and magnetic targeting. Mice were scanned non-invasively by IVIS® Imaging System (integration time 5 minutes, IVIS® 100 Series for i.p., IVIS Spectrum for i.v.). During the scan mice were kept under gaseous anaesthesia (5% isofluorane), at 37° C. The luminescence signal was clearly detected in both cases from the urinary tract of mice, providing evidence that the nanoparticles were eliminated by renal clearance, very similarly in both approaches of ferriliposome administration.
Female rats were treated with 500 mg/kg of iron oxide nanoparticles (n=16) and stabilizing buffer (n=8) as control. Mice were sacrificed at day 7 and 12 after injection. Blood was collected and serum separation performed by centrifugation in Li-heparin 0.6 ml flasks (Fuji Photo Film Co., Ltd. Life Science Products Division). Biochemical parameters of creatinine, urea nitrogen, alanine transaminase, aspartate transaminase, creatine phosphokinase and total bilirubin in blood were analyzed by biochemical analyzer Fujifilm DRI CHEM 3500i (Fuji Photo Film Co., Ltd. Life Science Products Division). The kidneys, spleen, liver, heart and lung tissues were collected and fixed in 10% neutral formalin. Organs were dehydrated and maintained in paraffin blocks. 5 μm paraffin sections were stained by hematoxylin and eosin for histo-pathological analysis. iron oxide nanoparticles were detected in animal tissues by Prussian blue staining with carmine (Sigma).
Frozen tissues of primary tumours, lungs, kidneys, pancreas and liver were disrupted in 200 μl of 0.1 M TRIS buffer (5 mM EDTA, 200 mM sodium chloride, 0.2% SDS, pH 8.5) using an Ultrathurrax (IKA, Staufen, Germany) and centrifugation at 1000 g for 10 min. Cysteine cathepsin activity was determined by hydrolysis of the general cathepsin substrate Z-Phe-Arg-4-methyl-coumarin-7-amide (Z-Phe-Arg-AMC, 25 μM; Bachem, Bubendorf, Switzerland) in 0.1 M phosphate buffer, pH 6.0, containing 1 mM EDTA, 0.1% (v/v) PEG and 1 mM dithiothreitol. Kinetics of substrate hydrolysis was monitored continuously during 10 min by spectrofluorometry at excitation and emission wavelengths of 370 and 460 nm, respectively.
Total RNA was isolated from the tumour samples and mRNA was reverse transcribed into cDNA. RTQ-PCR was performed by detection of SYBR-green dye DNA-intercalation in the newly formed PCR-products, using the Mx3005PTM Real-Time PCR System (Agilent, Stratagene Products). The relative amount of target gene expression was normalized to the β-actin transcripts. Primers: b-actin.1: 5′-ACC CAG GCA TTG CTG ACA GG-3′ (SEQ ID No. 1); b-actin.2: 5′-GGA CAG TGA GGC CAG GAT GG-3′ (SEQ ID No. 2); Ctsb.1: 5′-TGC GTT CGG TGA GGA CAT AG-3′ (SEQ ID No. 3); Ctsb.2: 5′-CGG GCA GTT GGA CCA TTG-3′ (SEQ ID No. 4); Ctsl.1: 5′-GCA CGG CTT TTC CAT GGA-3′ (SEQ ID No. 5); Ctsl.2: 5′-CCA CCT GCC TGA ATT CCT CA-3′ (SEQ ID No. 6); Ctsx.1: 5′-TAT GCC AGC GTC ACC AGG AAC-3′ (SEQ ID No. 7); Ctsx.2: 5′-CCT CTT GAT GTT GAT TCG GTC TGC-3′ (SEQ ID No. 8); Ctsh.1: 5′-CAT GGC TGC AAA GGA GGT CT-3′ (SEQ ID No. 9); Ctsh.2: 5′-CTG TCT TCT TCC ATG ATG CCC-3′ (SEQ ID No. 10).
For evaluation of the peritoneum clearance pathway for ferriliposomes, 200 μl of Alexa Fluor 555™-functionalized ferriliposomes were daily injected intraperitoneally into the PyMT transgenic breast cancer mouse for 3 days. Magnetic field was applied to the tumour for 12 hours immediately after each injection. 24 hours after the last injection mice were sacrificed and renal lymph nodes were resected, fixed in 10% formalin overnight, dehydrated using Shandon Tissue Processor (Shandon Citadel 1000) and moulded with paraffin (Microm EC 350 Paraffin Embedding Station). 5 μm paraffin sections were cut, mounted with anti-fade media containing DAPI (Prolong@ Gold antifade reagent with DAPI, Invitrogen) and visualized as described above.
Number | Date | Country | Kind |
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2011132913 | Aug 2011 | RU | national |
PCT/RU2011/000574 | Aug 2011 | RU | national |
This application is a Continuation application of International Patent Application PCT/RU2012/000632, filed on Aug. 3, 2012, which in turn claims priority to Russian Patent Applications No. RU 2011132913, filed Aug. 4, 2011 and International Patent Application PCT/RU2011/000574, filed Aug. 4, 2011, all of which are incorporated herein by reference in their entirety.
Number | Date | Country | |
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Parent | PCT/RU2012/000632 | Aug 2012 | US |
Child | 14172401 | US |