MAMC-MEDIATED BIOMIMETIC NANOPARTICLES (Machine-translation by Google Translate, not legally binding

专利摘要:
Biomimetic magnetic nanoparticles comprising MamC. The present invention provides magnetite comprising superparamagnetic biomimetic nanoparticles, which can be manufactured by a scalable process. Furthermore, these nanoparticles have promising properties, since, if functionalized, they can become drug transporters or contrast agents for clinical imaging. They can be used in clinical settings also to purge bone marrow as well as molecule separators and/or in environmental applications as biosensors. These nanoparticles, coupled with a drug, can be encapsulated in liposomes, obtaining magnetoliposomes, which can be functionalized for use in the targeted administration/release of drugs. In addition, mixtures of magnetoliposomes (both functionalized and non-functionalized with a targeting agent) and functionalized biomimetic magnetic nanoparticles or liposomes containing mixtures of functionalized BMNPs and MNPs can be used to combine different treatments such as targeted administration/release of drugs and hyperthermia. (Machine-translation by Google Translate, not legally binding)
公开号:ES2758400A1
申请号:ES201831064
申请日:2018-11-02
公开日:2020-05-05
发明作者:López Concepción Jiménez；Tercedor Carmen Valverde；Navarro Ana Peigneux；Ruz Ylenia María Jabalera；Maria Prat；Francesca Oltolina
申请人:Universidad de Granada；
IPC主号:

专利说明:

[0001]
[0002]
[0003]
[0004] TECHNICAL SECTOR
[0005]
[0006] The present invention deals with compositions that integrate biomimetic magnetic nanoparticles. The magnetic nanoparticles can be used as a medicine, in particular as a medicine for treatment of diseases with an associated marker that can be recognized, such as cancer.
[0007]
[0008] STATE OF THE ART
[0009]
[0010] Nanotechnology, and in particular the production of magnetic nanoparticles, generates millions of dollars every year in the United States. Nanotechnology application is based on the fact that these can be easily manipulated by applying an external magnetic field and, therefore, can be directed towards the target site by means of an external controller (Arakaki et al., 2014; Prozorov et al ., 2013). To date, these particles are used in numerous applications ranging from ferrofluids and magnetic storage to the clinical / scientific area such as the detection of nucleotide polymorphism (Maruyama et al., 2004, 2007; Matsunaga et al., 2007), separation cell (Matsunaga et al., 2007), DNA isolation and purification (Ota et al., 2006), magnetic resonance imaging contrast agent (Lisy et al., 2007), early diagnosis, drug transporter for chemotherapy Targeted (Sun et al., 2008) and hyperthermic treatments against cancer, understanding therefore the thermal damage that induces the rotation of the localized nanoparticles (Alphandéry et al., 2011).
[0011]
[0012] For these applications, especially in the clinic, one of the most important requirements is that the MNPs used as nanotransporters respond as efficiently as possible to the external magnetic field that is applied to guide said nanotransporter to the target site (Prozorov et al., 2013) . This response depends on the magnetic moment per particle, which, for superparamagnetic and crystalline stoichiometric magnetic magnetic nanoparticles, actually depends on the size of the MNP. For magnetite, the ideal size of the nanoparticle to integrate a single magnetic domain is between 30 and 120 nm. Most of the superparamagnetic nanoparticles already on the market are small (<30nm) and therefore their magnetic moment could be increased if larger MNPs were used. Above 120 nm, the magnetic nanoparticles are multidomains and their magnetic moment is not stable and depends on how these nanoparticles are oriented. Size is also important when hyperthermia treatments come into play. In fact, the heating power generated per unit mass of particle caused by the application of an external alternating magnetic field is directly related to the amount of iron in the MNP, and this must be as high as possible to maintain the applied magnetic field within clinically accepted ranges and a low dose of MNPs. Furthermore, the magnetic nanoparticles to be used in the clinic must also be biocompatible and do not carry any risk associated with the doses that need to be applied. Another important requirement of MNPs is that they expose functional groups on their surface that allow drug functionalization / release based on external stimuli such as changes in the pH of the environment. To this end, most commercial MNPs are coated with compounds such as polyethylene glycol and organic acids. This procedure not only introduces additional steps in the process of producing the MNPs (obviously increasing the preparation time and total costs), but also this coating can shield the magnetic heart and interfere with the magnetic response of the nanoparticle against a applied external magnetic field (response that is already suboptimal due to its small size). Therefore, most commercial MNPs have problems that need to be addressed in order for these MNPs to be efficient nanotransporters.
[0013]
[0014] Magnetite (Fe2 + and Fe3 + oxide, Fe ³ O ⁴ ) is a mineral found in numerous environments, from igneous and metamorphic rocks, to all kinds of sediments, both terrestrial and extraterrestrial (Thomas-Keprta et al., 2000) . It has also been found in higher organisms, such as those with migratory behavior and chitons. Inorganically, magnetite can be formed as a primary phase from a solution containing Fe2 + and / or Fe3 + (coprecipitation and oxide-reduction method) to which the pH is raised by adding chemical compounds (Arató et al. , 2005; Perez-Gonzalez et al., 2010; Prozorov et al., 2007; Schwertmann and Cornell, 2000). This is the most widely used method of producing magnetic nanoparticles. It is relatively easy to make, nanoparticles are produced at room temperature, and large amounts of material can be obtained per batch. The main disadvantage is that these nanoparticles are usually They are small (<30 nm) and therefore have a small magnetic moment per particle, which increases the doses to be used, which can pose a risk.
[0015]
[0016] Magnetites can also be formed by transforming precursors, usually at high temperatures (Jimenez-Lopez et al., 2012). An advantage is that well crystallized cubic magnetites can be formed. However, a major disadvantage is that this protocol is very expensive and it is difficult to control the size of the nanoparticles. The magnetite nanoparticles obtained by this process are usually either very small (<30nm) or multi-domain (> 120nm).
[0017]
[0018] Magnetite can also be formed biologically, either through an induced biomineralization (BIM) or controlled biomineralization (BCM) process (Bazylinski and Frankel, 2004). The formation of BIM magnetite is the result of the metabolic activity of organisms and subsequent chemical reactions that occur mediated by metabolic products. Minerals originating from BIM are indistinguishable from those formed inorganically under these conditions (Perez-Gonzalez et al., 2000). These BIM nanoparticles are not commonly used in nanotechnology applications.
[0019]
[0020] In contrast, magnetite nanoparticles made up of magnetotactic bacteria are the result of an exquisitely genetically controlled biomineralization (BCM) process, making these particles the ideal magnetic nanoparticle. They are ideal because they have very specific characteristics such as perfect crystal structures, high chemical purity, non-equilibrium morphologies and a narrow size distribution (Bazylinski and Frankel, 2004), which makes these crystals a unique magnetic domain and have predictable and stable magnetic properties (Amemiya et al., 2007; Prozorov et al., 2013). Furthermore, another advantage is that they are biocompatible. Therefore, there is a great demand for these nanoparticles, especially in the clinic.
[0021]
[0022] However, these nanoparticles cannot be commercialized because the culture of the magnetotactic bacteria cannot be scaled because they grow very slowly and have very demanding nutritional requirements. To solve this problem, different alternatives are being explored with the aim of improving the production yields of magnetosome-like nanoparticles. Basically three strategies are followed. The first is to try to transform non-bothersome non-magnetotactic microorganisms into magnetotactic bacteria. In this sense, the work of Kolinko et al. (2014). These authors demonstrated, for the first time, that gene insertion specificities of Magnetospirillum gryphiswaldense in Rhodospirilllum rubrum caused the latter to produce heterologous magnetosomes. They also showed that when genes involved in the production of these heterologous magnetosomes were silenced in Rhodospirillum , the changes that occurred were comparable to those in M. gryphiswaldense. With this important work, these authors opened the door to scaling heterologous magnetosomes into microorganisms that are easier to grow. However, while promising, mutant stability and scaling viability of these heterologous magnetosomes has yet to be demonstrated and the authors did not explore these aspects.
[0023]
[0024] The second alternative is to use recombinant DNA technology to silence or overexpress genes of interest in specific strains. In this sense, there is already a patent US2010 / 0292495 A1 that aims to obtain recombinant magnetite nanoparticles by controlling the expression of mamG, mamF, mamD, and mamC genes in magnetotactic bacteria.
[0025]
[0026] Finally the third alternative is the biomimetic approach, that is, learning from nature, which can inspire new strategies to produce advanced functional materials. Thus, in order to chemically produce magnetosome-like crystals whose production can be scaled, some magnetosome proteins, both expressed and recombinant complete proteins and synthetic peptides, have been tested in in vitro magnetite production experiments . Thanks to the mediation of these proteins, magnetite nanoparticles have been obtained with different magnetic properties to those of inorganic chemical precipitation. In this sense, most of the work done so far has been the in vitro obtaining of magnetite nanoparticles using the entire Mms6 protein from various Magnetospirillum species (Arakaki et al., 2010, 2014; Amemiya et al., .2007 ; Prozorov et al., 2007; Galloway et al., 2012; Bird et al., 2016). Most of the magnetic nanoparticles obtained through this protein are superparamagnetic, with a size of around 20 nm. Therefore, commercial nanoparticles that are already on the market do not improve much. Larger nanoparticles are achieved with the Mms6 using the biomoulding technique (Galloway et al., 2012b; Bird et al., 2016), but the magnetic properties of these nanoparticles have not been studied and their scaling has not been addressed either, but it is most likely difficult and quite expensive. The MmsF protein of Magnetospirilum magneticum AMB-1 is another potential candidate to mediate in vitro formation of biomimetic magnetite nanoparticles of sizes between 80-90 nm (Rawlings et al., 2014). These Nanoparticles could be magnetic single domain, but the magnetic properties of these nanoparticles have not been studied and have not been characterized enough to determine if these nanoparticles could be useful in nanotechnology. Anyway, all these experiments have been done using a single recombinant protein in the aqueous solution in which the magnetite is formed.
[0027]
[0028] Biomimetic nanoparticles have also been produced using chimeras constructed with synthetic peptides from magnetosome proteins bound to fusion proteins (Nudelman et al., 2016 and 2018). Some of these peptides have already been patented (WO 2017153996). When using MamC loop peptides, nanoparticles larger than those produced in the presence of other peptides or in the absence of peptides are obtained. However, the size distribution of the nanoparticles, and therefore their heterogeneity, was greater than that of the nanoparticles produced in the presence of the entire MamC protein expressed as recombinant.
[0029]
[0030] Prof. Jimenez-Lopez's research team has made important advances in the production of biomimetic nanoparticles. In fact, it is the only group that works with MamC of Magnetococcus marínus MC-1, which is the most abundant protein of the magnetosome in most magnetotactic bacteria. Furthermore, this group is the only one that has expressed and purified the three proteins that control the size and morphology of MC-1 magnetosomes (MamC, Mms6 and Mms7) as recombinant proteins. MamC of Magnetococcus marínus MC-1 has proven to be a strong candidate to produce new biomimetic nanoparticles (BMNPs). These are higher (~ 40 nm) than most commercial MNPs (<30 nm) and have (1) higher blocking temperature, while they are superparamagnetic at room temperature, (2) a slower increase in magnetization with temperature and (3) Verwey's transition temperature. All these characteristics are compatible with nanoparticles with greater structure and with a greater magnetic moment per particle compared to other inorganic nanoparticles and / or to most nanoparticles mediated by Mms6. These characteristics make them behave as if they were non-magnetic at room temperature in the absence of an external magnetic field, which prevents agglomeration, while they respond efficiently when an external magnetic field is applied, therefore increasing the efficiency of the magnetic guide. Furthermore, previous results of the group showed that MamC confers new surface properties to the BMNPs, in particles, an isoelectric point (iep) at a pH of 4.4. This is important because the BMNPs are negatively charged at physiological pH and can be functionalized with molecules, such as doxorubicin (DOXO), which are positively charged at that pH, through electrostatic interactions. Furthermore, when the pH decreases (something that occurs naturally in the tumor microenvironment), that electrostatic interaction weakens and the molecule is released from the BMNP. These BMNPs are cytocompatible and biocompatible. The properties of the resulting nanoparticles depend on the type of protein introduced into the solution before the formation of said nanoparticle and / or the relative concentration of the different protein (s) used.
[0031]
[0032] However, the process of formation of magnetite biomimetic nanoparticles using MamC results in the formation of siderite [Valverde-Tercedor et al., 2015 (magnetite> 90% siderite <10%)] or goethite [Lopez-Moreno et al., 2017 (magnetite 85% goethite 15%)] as an unwanted product. Once produced, and due to their nanometric size, these mineral phases (siderite and / or goetite) are very difficult, if not impossible, to remove from the mixture. Therefore, a method is needed to produce only magnetite and not other phases. Furthermore, it is necessary to develop a composition that integrates a substantially pure mineral phase of magnetite nanoparticles (> 95%) where the resulting nanoparticles are superparamagnetic, magnetic single domain and have surface properties that allow functionalization with different molecules without the need for treatment. after the production processes.
[0033]
[0034] Nanoparticles can be seen as efficient drug nanotransporters especially when they are functionalized with molecules (antibodies, ligands, cellular surface receptor aptamers) capable of recognizing certain markers linked to a disease. This makes it possible to reach large local amounts of the drug and low systemic exposure, therefore reducing the toxicity of the treatment, increasing its efficacy (Brigger et al., 2001; De Jong and Borm, 2008; Singh and Lillard, 2009).
[0035]
[0036] DESCRIPTION OF THE FIGURES
[0037]
[0038] Figure 1 : CLUSTAL O (1.2.1) multiple alignment of Mms6 sequences in different magnetotactic bacteria.
[0039]
[0040] Figure 2 : SDS-PAGE gel from purified MamC (lane 3) and Mms6 (lane 5). Lysates of E. coli TOP10 before the Purification of MamC (lane 2) and Mms6 (lane 4). Lane 1, molecular weight marker (KDa).
[0041] Figure 3 : Magnetite crystals synthesized in the presence of MamC (10 ^ g / mL) (BMNPs): (A) TEM images, (B) Crystal size distribution. Detail: Modeling of BMNPs from HRTEM data using SHAPE v7.3 Magnetite crystals synthesized in the absence of protein (inorganic magnetite: MNPs): (C) TEM images, (D) Crystal size distribution.
[0042]
[0043] Figure 4 : HRTEM images of inorganic magnetite nanoparticles (A and B) and magnetic nanoparticles produced in the presence of MamC (C, D and E). Dotted lines represent crystal faces and lines represent crystallographic directions.
[0044]
[0045] Figure 5 : HRTEM images of magnetic nanoparticles produced in the presence of Mms6 (A and B) and magnetite nanoparticles produced in the presence of MamC and Mms6 (C and D). Dotted lines represent crystal faces and lines represent crystallographic directions.
[0046]
[0047] Figure 6 : (A) Potential □ of MNPs and BMNPs, (B) Thermogravimetric analysis of MNPs and BMNPs, (C) Hysteresis cycle of BMNPs and MNPs at 300 K, (D) ZFC-W and FC-C of MNPs and BMNPs. Blocking temperature (T ^b ) and irreversibility temperature (Tirr) are indicated in the figure for each sample.
[0048]
[0049] Figure 7: Doxorubicin (DOXO) adsorption isotherm (A: kinetics; B: dose-dependent adsorption. Saturation is reached at 1 mmol DOXO per gram of nanoparticles) and monoclonal antibody DO-24 (C) in nanoparticles magnetite.
[0050]
[0051] Figure 8 : DOXO desorption profile.
[0052]
[0053] Figure 9: Effect of non-functionalized biomimetic nanoparticles and ternary nanoparticles (functionalized with Doxorubicin [DOXO] and with monoclonal antibody DO-24) on the viability of human tumor cells expressing the Met, Met / HGF-R + receptor GTL-16, and which do not express the met receptor, Met / HGF-R-Huh7. Non-functionalized nanoparticles reduce cell viability only up to 0-95%. Doxo [^ g / ml] indicates the DOXO concentration in each sample and the data expresses cell viability compared to a (untreated) control at the same time interval. At DOXO concentrations of 10 ^ g / ml, ternary nanoparticles were significantly much more toxic to GTL-16 than to Huh7 compared to nanoparticles without functionalize. Cell viability was measured by MTT test after three days of treatment.
[0054]
[0055] Figure 10 : Real-time toxicity of non-functional biomimetic nanoparticles, binary nanoparticles MNPs (DOXO-MNPs, -) and ternary nanoparticles (- ■ -) on Met / HGF-R + GTL-16 and Met / HGF-R-Huh7 cells . The presence of DO-24 mAbs significantly increases the toxicity of ternary nanoparticles compared to that of binary nanoparticles in GTL-16 cells, while no differences were observed in those that did not express the Met receptor (Huh7). These tests were done with XCELLIGENCE.
[0056]
[0057] Figure 11 : Histological analysis of different organs of female BALB / c mice that had been injected intravenously with biomimetic nanoparticles (10 ^ g / g mouse). Mice were sacrificed at different times after injection (1h, 4h, 1d, 7d, and 60d) and organs were processed by Prussian blue and Hematoxylin-Eosin staining. Small amounts of iron were detected in the lungs, which decreased after 60 days. In the spleen, which normally contains iron, iron levels were increased at the time of the injection, which then decreased during the following days, then recovered to normal levels at 60 d. In the other organs (brain, heart, liver and kidneys) a slight increase in iron was detected during the first day, which decreased later. Biomimetic nanoparticles are highly biocompatible in vivo.
[0058]
[0059] BRIEF DESCRIPTION OF THE INVENTION
[0060]
[0061] The present invention relates to a method of producing a composition that is substantially a pure mineral phase of superparamagnetic biomimetic magnetite (BMNPs,> 95% of total solid) that encompasses the following steps: ( a ) preparing a carbonate solution, ( b ) add FeCh to the carbonate solution, ( c ) add MamC and, optionally, Mms6 to the solution obtained in step (b), ( d ) incubate the solution obtained in step (c) for at least 30 minutes, ( e ) adding Fe (ClO ^{4) 2} to the solution obtained in step (d), and ( f ) adjusting the pH of the solution obtained in step (e) to 9 using a base; This method is carried out at 25 ° C and 1 pressure atmosphere and all the solutions are previously deoxygenated. In this aforementioned method, the concentrations of the protein reservoirs must be in the following range: [MamC] 2-5 mg / mL, [Mms6] and [Mms7]> 1 mg / mL.
[0062] Furthermore, the present invention provides a composition that encompasses: (i) a substantially pure mineral phase of superparamagnetic magnetite, (ii) MamC, and (iii) optionally, Mms6; where at least components (i) and (ii) form superparamagnetic magnetic nanoparticles containing up to 5 wt% MamC (MamC-mediated BMNPs are then composed of -95 wt% magnetite and -5 wt% MamC), with an average particle size between 30-120 nm, isoelectric point of -4.4, surface area of ~ 90 m2 / g, blocking temperature of -145 K, and irreversibility temperature of -292 K. Furthermore, the present invention provides a formulation to make magnetoliposomes encompassing: (i) the composition of the present invention, (ii) a liposome-forming agent, and (iii) optionally superparamagnetic inorganic magnetites.
[0063]
[0064] The present invention also provides a pharmaceutical composition encompassing the composition of the present invention or the formulation of the magnetoliposomes of the present invention and a pharmaceutically acceptable carrier and / or diluent. The composition of the present invention, the formulation of the magnetoliposomes, or the pharmaceutical composition can be used as a medicine. In particular, the composition of the present invention, the formulation of the magnetoliposomes, or the pharmaceutical composition can be used in cancer treatments.
[0065]
[0066] In closing, the present invention also provides the use of the composition of the present invention, the formulation of the magnetoliposomes, or the pharmaceutical composition of the present invention for the preparation of contrast agents for image-based clinical techniques. The present invention also provides the use of the composition of the present invention for (i) nucleic acid isolation; (ii) as a molecular separator; (iii) as biosensors.
[0067]
[0068] DETAILED DESCRIPTION OF THE INVENTION
[0069]
[0070] Definitions
[0071]
[0072] The terms "treatment" and "therapy" used herein refer to a set of hygienic, pharmacological, surgical and / or physical protocols that are used with the intention of curing and / or alleviating disease and / or symptoms with the objective of improving the state of health. The terms "treatment" and "therapy" include preventive and curative methods, since both are intended to maintain or restore the health of an individual or a animal. Regardless of the origin of the symptoms, illness or disability, the administration of an appropriate medication to alleviate or cure a health problem should be interpreted as a form of therapy treatment in the context of this report.
[0073]
[0074] The term "effective therapeutic amount" refers to the amount of compound in a composition or formulation that has a therapeutic effect that is capable of treating the disease.
[0075]
[0076] As used herein, "pharmaceutically acceptable carrier " or "pharmaceutically acceptable diluent" refers to any and all solvents, dispersed media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption retarders compatible with pharmaceutical administration. The use of these media and agents as pharmacologically active substances is well known. Acceptable carriers, excipients, or stabilizers are not toxic to the subject at the dose and concentration employed and, without limiting the scope of the present invention, include: buffering agents; preservatives; co-solvents; antioxidants, including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (eg, Znprotein complexes); biodegradable polymers such as polyesters; salt-forming counterions such as sodium, polyhydric sugar alcohols, amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid and threonine; organic sugars or alcoholic sugars such as lactitol, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (eg inositol), polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [alpha] -monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human albumin serum, bovine albumin serum, gelatin, or other immunoglobulins; and hydrophilic polymers like polyvinylpyrrolidone. Other pharmaceutically acceptable carriers, excipients, or stabilizers such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) may also be included in the pharmaceutical composition described herein, provided that they do not adversely affect the characteristics of the composition. pharmaceutical.
[0077]
[0078] The term "therapeutic agent" refers to any substance that can be used to treat and / or prevent disease when used in therapeutically effective amounts. The therapeutic agent can be a small chemical molecule (such as, for example, doxorubicin, antihistamine, etc.), or biological (for example, therapeutic protein) and / or nucleic acid (for example, siRNA, gRNA for CRISPR / Cas9, etc. .).
[0079] The term "self-therapeutic agent" refers to any drug that can be used to treat or prevent cancer. Non-limiting examples include: Actinomycin. All-trans retinoic acid, Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamida, Cytarabine, Daunorubicin, Doxifluridine, Doxorubicin, Epirubicin, Doxorubicin, Epirubicin, Epirubin Irinotecan, Mechloretamine, Mercaptopurine, Methotrexate, Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Thioguanine, Topotecan, Valrubicin, Vemurafenib, Vinblastine, Vincristine, Vindesine and Vinorelbine.
[0080]
[0081] The term "cancer" refers to a group of diseases that can be defined as abnormal, benign or malignant new growth of tissue that does not have a physiological function and that arises from an uncontrolled and usually rapid cell proliferation and that has the potential to invade or spread to other parts of the body. The magnetoliposome composition and formulation of the present invention can specifically recognize cancer cells through functionalization with a signaling substance and can lead antiproliferative agents to solid tumors or hematological cancers. Some non-limiting examples include: acute granulocytic leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, adenocarcinoma, adrenal cancer, anaplastic astrocytoma, angiosarcoma, cancer of the appendix, astrocytoma, basal cell carcinoma, B-cell lymphoma, cancer of the bile ducts, bladder cancer, bone cancer, bone marrow cancer, intestinal cancer, brain cancer, brain stem glioma, brain tumor, breast cancer, carcinoid tumors, cervical cancer, cholangiocarcinoma, chondrosarcoma, chronic lymphocytic leukemia, myelogenous leukemia chronic, colon cancer, colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, endometrial cancer, ependymoma, epithelioid sarcoma, esophageal cancer, Ewing's sarcoma, cancer of extrahepatic bile ducts, cancer of eye, fallopian tube cancer, fibrosarcoma, gallbladder cancer, gastric cancer, cancer gastrointestinal cer, gastrointestinal carcinoid cancer, gastrointestinal stromal tumors, germ cell tumor, glioblastoma multiforme, glioma, hairy cell leukemia, head and neck cancer, hemangioendothelioma, hodgkin lymphoma, hypopharyngeal carcinoma, infiltrating ductal carcinoma, infiltrating lobular carcinoma , inflammatory breast cancer, intestinal cancer, intrahepatic bile duct cancer, invasive / infiltrating breast cancer, islet cell cancer, jaw cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, leptomeningeal metastases, leukemia, lip cancer, liposarcoma, liver cancer, lobular carcinoma in situ, low-grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesenchymal chondrosarcoma, mesenchymal, mesothelioma, metastatic breast cancer, metastatic melanoma , metastatic squamous neck cancer, mixed gliomas, mouth cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, cancer of the nasal cavity, nasopharyngeal cancer, neck cancer, neuroblastoma, neuroendocrine tumors, non-Hodgkin lymphoma, non-small cell lung cancer, oat cell cancer, eye cancer, eye melanoma, oligodendroglioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteogenic sarcoma, osteosarcoma, ovarian cancer, epithelial ovarian cancer, tumor of ovarian germ cells, ovarian primary peritoneal carcinoma, ovarian sexual cord stromal tumor, pancreatic cancer, cancer Papillary cynoma, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal region tumor, pituitary gland cancer, lymphoma Primary central nervous system, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvic cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, bone sarcoma, soft tissue sarcoma, uterine sarcoma, breast cancer, cancer of skin, small cell lung cancer, small bowel cancer, spinal cancer, spinal cord cancer, spinal tumor, squamous cell cancer, stomach cancer, synovial sarcoma, T-cell lymphoma, testicular cancer, throat cancer, thymoma / thymic carcinoma, thyroid cancer, tongue cancer, tonsil cancer, transitional cell cancer, triple negative breast cancer, tubal cancer, ca tubular rcinoma, urethral cancer, uterine adenocarcinoma, uterine cancer, vaginal cancer, and vulvar cancer.
[0082]
[0083] The term “substantially pure mineral phase” refers to a mineral phase that consists mostly of a single type of mineral (> 95%). In this case, a substantially pure mineral phase of magnetite means that the magnetite crystals do not contain siderite. The substantially pure mineral phase of magnetite may contain small amounts of goetite (<5%) if the pH rises above 9 during the production process.
[0084]
[0085] The term "supemaramagnetism" refers to a form of magnetism that appears in small ferromagnetic or ferrimagnetic nanoparticles. Below a certain size, the magnetic nanoparticles cannot maintain the static walls of the different magnetic domains, behaving like a giant spin of magnetic momentum.
[0086] A superparamagnetic particle can be free (thermally balanced) or blocked (unbalanced). The blocking temperature (T ^b ) was determined as the one at which the maximum magnetization occurs in the ZFC curves, while the irreversibility temperature (Tirr) is that just below the blocking of the superparamagnetic nanoparticles that are no longer thermally balanced.
[0087]
[0088] The term "signaling substance" refers to any molecule that can specifically bind to another given substance. Non-limiting examples include antibodies, antibodies, aptamers, etc. In a preferential situation the signaling substance is a monoclonal antibody.
[0089]
[0090] The term "inorganic magnetite nanoparticles" or MNPs refers to any magnetite nanoparticle that is obtained or can be obtained by chemical synthesis methods in the absence of any biological agent and / or product.
[0091]
[0092] The term "MamC" refers to a complete protein that is derived from the mamC gene (NCBI Database, accession number ABK44766.1, protein accession Mmc1_2265). The term "MamC" also includes functional fragments and variants of the protein derived from the mamC gene that could be expressed in biological systems or synthesized. MamC functional fragments are previously described in (Nudelman et al., 2016; Nudelman et al., 2018; patent WO 2017153996). In particular, the functional fragments that contain the region of the MamM-region of interaction (MamC-MIL) (Nudelman et al., 2016). The functional fragments can be linked to another protein like MBP.
[0093]
[0094] The term "Mms &' refers to a complete protein that is derived from the mms6 gene (NCBI Database, accession number ABK44766.1, protein accession Mmc1_2275). The term "Mms6" also includes functional fragments and variants of the protein derived from the mms6 gene that could be expressed in biological systems or synthesized.
[0095]
[0096] The term "MmsT ' refers to a complete protein that is derived from the mms7 gene (UniProtKB reference number Q2W8R9). The term "MmsT also includes functional fragments and variants of the protein derived from the mms7 gene that could be expressed in biological systems or synthesized.
[0097]
[0098] The term "functional variant" refers to any variant or mutant that has a certain percentage (degree) of homology with the protein and that maintains the function of the protein. In a preferred situation, a functional variant has a degree of homology with the protein greater than 75%. Preferably degrees of homology of 80, 85, 90, 95, 98 or 99% with the original protein. More preferably a degree of homology of 95% with the original protein.
[0099]
[0100] The degree of identity between protein sequences can be determined by conventional methods. For example, using standard algorithms for sequence alignment, known in the area as BLAST (Altschul et al. 1990 J Mol Biol. 215 (3): 403-10) or CLUSTAL O (1.2.1). In a preferential situation the degree of homology is determined using BLAST or CLUSTAL O.
[0101]
[0102] Method
[0103]
[0104] First, the present invention provides a method of producing a substantially pure superparamagnetic magnetite mineral phase composition comprising the following steps: (a) preparing a carbonate solution, (b) adding FeCh to the carbonate solution, ( c) add MamC and, optionally, Mms6 to the solution obtained in step (b), (d) incubate the solution obtained in step (c) for at least 30 minutes, (e) add Fe (ClO ^{4) 2} to the solution obtained in step (d), and (f) adjusting the pH of the solution obtained in step (e) to pH 9 using a base; the method is performed at 25 ° C and 1 pressure atmosphere. All the solutions used are previously deoxygenated. For a better result, the method of the present invention is carried out under anoxic conditions, that is, less than 40 ppb of oxygen in the solution.
[0105]
[0106] The present invention provides a sequence of steps, which are essential to produce a composition that is free of any detectable level of siderite. The sequence of steps provided allows the MamC protein, which is highly hydrophobic, to remain folded and functional while maintaining the solution supersaturated for the magnetite such that the precipitation of the magnetite is kinetically enhanced relative to that of the siderite. Therefore, obtaining the correct sequence requires a careful balance of these three aspects.
[0107]
[0108] Under the best conditions, Mms7 is also added to the solution obtained in step (b) in step (c) of the method.
[0109] Under the best conditions, the concentration of the protein (s) added in step (c) should be at least 2 mg / ml. Under the best conditions, the concentration of the MamC solution that is added to the solution in step (c) is 2-5 mg / mL. Higher concentrations can induce aggregation and thus prevent or reduce the efficacy of MamC mediation in magnetite biomineralization. Lower concentrations will result in very small (<5nm) magnetite crystals due to the prevalence of the effect of the TRIS buffer in the biomineralization process. This embodiment does not refer to the final concentration of the protein solution (s). This condition refers to the concentration of the stock solution that is added to the solution obtained after step (b).
[0110]
[0111] Under the best conditions, the solution is incubated for at least 1 hour in step (d).
[0112]
[0113] Under the best conditions, the carbonate solution comprises NaHCO ³ and / or Na2CO3 and, optionally, the base is NaOH.
[0114]
[0115] Under the best conditions, the final concentration of the solution obtained in step (f) is 3.5 mM NaHCO3, 3.5 mM Na2CO3, 2.78 mM Fe (ClO ⁴ ) ² , 5.56 mM of FeCh and a variable amount of MamC and, optionally, Mms6. Preferably, the final concentration of MamC is 10 ^ g / ml and, if Mms6 is included, the final concentration of MamC is at least 5 ^ g / ml and the final concentration of Mms6 is 10 ^ g / ml.
[0116]
[0117] Composition
[0118]
[0119] Secondly, the present invention provides a composition obtained or obtainable through the methods of the present invention. Third, the present invention provides a composition comprising: (i) a substantially pure mineral phase of superparamagnetic magnetite, (ii) the MamC protein and (iii) optionally, the Mms6 protein; wherein, at least components (i) and (ii) form superparamagnetic magnetic nanoparticles containing up to 5 wt% MamC (MamC-mediated BMNPs are then composed of -95 wt% magnetite and -5 wt% MamC), with an average particle size between 30-120 nm, isoelectric point of ~ 4.4, surface area of ~ 90 m2 / g, blocking temperature of -145 K and irreversibility temperature of -292 K.
[0120]
[0121] Under best conditions, all of the following aspects of the present invention are applicable to the second and third aspects of the present invention. It is evident that the second and the third aspect of the present invention can refer to the same composition.
[0122] Under the best conditions, there are no detectable levels of siderite in the composition and goetite levels are <5% of the total solid.
[0123]
[0124] Under the best conditions, the average particle size of the magnetic nanoparticles is 30-50 nm. Preferably, the average particle size of the magnetic nanoparticles is 30-40 nm. Under the best conditions, the average particle size is determined by Transmission Electron Microscopy. In the examples of the present invention, sizes were obtained by measuring the size of more than 1000 crystals in each Transmission Electron Microscopy image.
[0125]
[0126] Under the best conditions, the wt% of MamC in the BMNPs is 2-20 wt%. Preferably, the wt% of MamC in the BMNPs is 2-10 wt%. Under the best conditions, the wt% of MamC in the BMNPs are determined by thermogravimetric analysis. In the examples of the present invention, the wt% of MamC in the BMNPs is 5 wt%.
[0127]
[0128] The composition may further comprise other proteins involved in magnetite formation in bacterial magnetosomes and / or other proteins with acidic domains capable of binding iron and / or those with a structure such that they could function as a template for nucleation and growth of magnetite . Non-limiting examples of such proteins include Mms6, Mms7, MmsF / MamF and their homologous proteins in different magnetotactic bacteria. Under the best conditions, the composition further comprises Mms7.
[0129]
[0130] Under the best conditions, the isoelectric point of the BMNPs is 3-7. Preferably, the isoelectric point of the BMNPs is 3-5. Under the best conditions, the isoelectric point of the BMNPs is calculated from measurements of electrophoretic mobility. In the examples of the present invention, the isoelectric point of the BMNPs is 4.4.
[0131]
[0132] Under the best conditions, the specific surface area of the BMNPs is 30-120 m2 / g. Preferably, the specific surface area of the BMNPs is 50-100 m2 / g. Under the best conditions, the specific surface area of the BMNPs is determined from BET. In the examples of the present invention, the specific surface area of the BMNPs is 97 m2 / g.
[0133]
[0134] Nanoparticles formed using the MamC protein exhibit high magnetization per particle at room temperature that is equal to or greater than that of the nanoparticles obtained by inorganic methods or by the unique use of the Mms6 protein. Therefore, under the best conditions, the magnetization of the BMNPs is 40-70 emu / g at 300 K when an external magnetic field of 500 Oe is applied. Preferably, the magnetization of the BMNPs is 55-65 emu / g at 300 K when an external magnetic field of 500 Oe is applied. Optimally, the magnetization of the BMNPs is 55 emu / g (61 emu / g when the amount of MamC in the crystal is taken into account) at 300 K when an external magnetic field of 500 Oe is applied.
[0135]
[0136] Under the best conditions, the blocking temperature of the BMNPs is at least 100 K when an external magnetic field of 500 Oe is applied. Preferably, the blocking temperature is at least 120 K when an external magnetic field of 500 Oe is applied. Under optimal conditions, the blocking temperature is at least 130 K when an external magnetic field of 500 Oe is applied.
[0137]
[0138] Under the best conditions, the irreversibility temperature of the BMNPs is at least 200 K when an external magnetic field of 500 Oe is applied. Preferably, the irreversibility temperature is at least 250 K when an external magnetic field of 500 Oe is applied. Under optimal conditions, the irreversibility temperature is at least 280 K when an external magnetic field of 500 Oe is applied.
[0139]
[0140] An advantage of biomimetic nanoparticles compared to inorganic nanoparticles is the fact that proteins change surface properties due to MamC adsorbing and / or incorporating up to 5 wt% on the crystal surface (Figure 6B). In particular, for example, MamC-magnetites are not charged at a pH of about 4 (Figure 6A). Therefore, they are heavily loaded at a physiological pH (pH = 7.4) and can adsorb high amounts of polar drugs. Said adsorption is stable at physiological pH and an insignificant release of drug occurs at this pH. However, when the functionalized nanoparticle is exposed to acidic pH values (such as those that exist in the tumor microenvironment or within the cell lysosome), the particle discharges and releases the drug. In contrast, inorganic magnetite nanoparticles (MNPs) are uncharged at a pH of about 7.4 and are therefore neutral or lightly charged at physiological pH. Therefore, low adsorption and high drug release are expected at physiological pH values. To avoid this, the nanoparticle must be covered by a molecular coating that allows stable functionalization. This step is not necessary in the biomimetic nanoparticles that are the subject of this patent.
[0141] In the best conditions, the magnetic nanoparticles are functionalized with a therapeutic agent. The therapeutic agent can be any agent that has a therapeutic effect when administered in a therapeutically effective amount. Preferably, the therapeutic agent is a chemotherapeutic agent or a nucleic acid molecule. Nucleic acid molecules include both RNA and DNA. The RNA can be a siRNA, miRNA or gRNA (gRNA for use in CRISPR / Cas9 gene editing approaches).
[0142]
[0143] Under the best conditions, the magnetic nanoparticles were functionalized with a chemotherapeutic agent. Under the best conditions, the chemotherapeutic agent is polar. Preferably, the chemotherapeutic agent is doxorubicin.
[0144]
[0145] In the best conditions, the magnetic nanoparticles are functionalized with a signaling substance. Preferably, the signaling substance is a ligand for a growth factor receptor (such as a growth factor), an antibody, or an aptamer. More preferably, the signaling substance is a monoclonal antibody.
[0146]
[0147] Under the best conditions, the magnetic nanoparticles are functionalized with a chemotherapeutic agent and a signaling substance. Preferably, the chemotherapeutic agent is doxorubicin and the signaling substance is a monoclonal antibody.
[0148]
[0149] Under the best conditions, the composition consists of: (i) a substantially pure mineral phase of superparamagnetic magnetite, (ii) MamC and (iii) optionally, Mms6 and / or Mms7; wherein, at least components (i) and (ii) form superparamagnetic magnetic nanoparticles containing up to 5 wt% MamC (MamC-mediated BMNPs are then composed of -95 wt% magnetite and -5 wt% MamC), with an average particle size between 30-120 nm, isoelectric point of -4.4, surface area of ~ 90 m2 / g, blocking temperature of -145 K and irreversibility temperature of -292 K.
[0150]
[0151] Under the best conditions, the composition does not derive, is neither obtained nor can be obtained from a magnetosome. More preferably, the composition is not obtained from a magnetosome. To clarify, in this condition, although the proteins used in the composition can be derived from a magnetosome, BMNPs are obtained using the in vitro precipitation approach described in the present invention. In other words, superparamagnetic magnetic nanoparticles are superparamagnetic biomimetic magnetic nanoparticles (BMNPs).
[0152] Under the best conditions, superparamagnetic magnetic nanoparticles are superparamagnetic biomimetic magnetic nanoparticles.
[0153]
[0154] The term "magnetosome" refers to both natural magnetosomes present in magnetotactic bacteria and recombinant magnetosomes or magnetosome-like structures that are produced by a host that does not normally contain magnetosomes or magnetosome-like structures.
[0155]
[0156] Magnetoliposome formulation
[0157]
[0158] In a fourth aspect, the present invention provides a magnetoliposome formulation which comprises: (i) the composition of the present invention, (ii) a liposome-forming agent; and (iii) optionally, superparamagnetic magnetite inorganic nanoparticles (MNPs).
[0159]
[0160] The forming agent involved in the formulation of magnetoliposomes is preferably a hydrogenated, partially hydrogenated, or non-hydrogenated phospholipid. The phospholipid used can be or comprise, for example: phosphatidylcholine, phosphatidylserine and phosphatidyl-inositol. The most typical phospholipid is phosphatidylcholine, which can be synthesized or isolated from a wide variety of natural sources. In addition to phosphatidylcholine, there are other phospholipids that can also be used in the formulation, either as forming agents or as additional components. These phospholipids are: Diacetyl Phosphate (DCP), Dimiritoylphosphatidylcholine (DMCP), Dimiristoyl Phosphatidylglycerol (DMPG), Dioleoyl Phosphatidylcholine (DOPc), Dipalmitoyl Phosphatidylcholine (DPPC), Dipalmitoyl Phosphatidylglycerin (DPPC), Dipalmitoyl Phosphatidylglycolin (DPPC), Dipalmitoyl Phosphatidylglycerol (DPPC), Dipalmitoyl Phosphatidylglycerin (DPPC), Dipalmitoyl Phosphatidylglycerin (DPPC), Dipalmitoyl Phosphatidylglycerin (DPPC) , whereby the lipid involved in the formulation can be hydrogenated, partially hydrogenated or non-hydrogenated. Magnetoliposomes can be formed using conventional auxiliary lipids by techniques known to the person skilled in the art, such as those described in patent application ES2231037-A1.
[0161]
[0162] Although liposomes have been used for coating inorganic magnetite nanoparticles, encapsulation of biomimetic nanoparticles in liposomes has not previously been performed. Because the surface properties of the two particles are very different, the stabilization process of both types of particles prior to encapsulation in the liposome is very different. Encapsulating the nanoparticles without prior stabilization could result in the agglomeration of said particles. Agglomerated nanoparticles would not be useful for use in nanotechnology applications.
[0163] Therefore, the process for obtaining magnetoliposomes that involves the use of biomimetic magnetite nanoparticles is not obvious or simple.
[0164]
[0165] Superparamagnetic nanoparticles such as inorganic magnetite nanoparticles (MNPs) can be especially useful for use as contrast agents in magnetic resonance imaging, as well as for use in hyperthermia treatments resulting from increased temperature caused by rotation of magnetic nanoparticles, which is induced by an alternating magnetic field or by radiation. Therefore, a magnetoliposome presenting larger and smaller MNPs can take advantage of both types of nanoparticles. In this regard, in a preferred embodiment of the invention, the magnetoliposome formulation further exhibits MNPs.
[0166]
[0167] In a preferred embodiment of the invention, the magnetoliposomes of the magnetoliposome formulation are functionalized with therapeutic agents and / or with a targeting substance. Magnetoliposomes can be functionalized by any common method known in the art. For example, magnetoliposomes can be functionalized by the methods described by Torchilin et al., 2001. The therapeutic agent can be any agent that exhibits a therapeutic effect when administered in a therapeutically effective amount. In a preferred embodiment of the invention, the therapeutic agent is a chemotherapeutic agent and the targeting substance is an antibody. More preferably, the antibody is a monoclonal antibody.
[0168]
[0169] Pharmaceutical composition
[0170]
[0171] In a fifth aspect, the present invention provides a pharmaceutical composition which comprises the composition of the present invention or the magnetoliposome formulation of the present invention and a pharmaceutically acceptable carrier and / or solvent.
[0172]
[0173] In a preferred embodiment of the invention, the pharmaceutical composition may comprise one or more solutions, which are suitable for intravenous, intra-arterial, intramuscular and / or subcutaneous administration. In another embodiment, the pharmaceutical composition may comprise one or more solutions, which are suitable for sublingual, buccal and / or inhalation routes of administration. In an alternative embodiment, the pharmaceutical composition may comprise one or more aerosols, which are suitable for administration by inhalation.
[0174] In a preferred embodiment of the invention, the pharmaceutical composition may comprise one or more creams and / or ointments, which are suitable for topical administration. In a preferred embodiment of the invention, the pharmaceutical composition may comprise one or more suppositories which are suitable for rectal or vaginal administration. In this embodiment, the composition can be used with the aim of achieving a loco-regional effect.
[0175]
[0176] Treatment of diseases such as cancer
[0177]
[0178] In a sixth aspect, the present invention provides the composition of the present invention, the magnetoliposome formulation of the present invention, or the pharmaceutical composition of the present invention for use as a medicament. In a seventh aspect, the present invention provides the composition of the present invention, the magnetoliposome formulation of the present invention, or the pharmaceutical composition of the present invention for use in the treatment of cancer.
[0179]
[0180] In a preferred embodiment, the cancer is selected from the group consisting of acute granulocytic leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, adenocarcinoma, adrenal cancer, anaplastic astrocytoma, angiosarcoma, cancer of the appendix, astrocytoma, basal cell carcinoma, cell lymphoma B, bile duct cancer, bladder cancer, bone cancer, bone marrow cancer, intestinal cancer, brain cancer, brain stem glioma, brain tumor, breast cancer, carcinoid tumors, cervical cancer, cholangiocarcinoma, chondrosarcoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, endometrial cancer, ependymoma, epithelioid sarcoma, cancer of the esophagus, Ewing sarcoma, cancer of the extrahepatic bile ducts, eye cancer, cancer of the fallopian tubes, fibrosarcoma, cancer of the Gallbladder, Gastric Cancer, Gastrointestinal Cancer, Gastrointestinal Carcinoid Cancer, Gastrointestinal Stromal Tumors, Germ Cell Tumor, Glioblastoma Multiforme, Glioma, Hairy Cell Leukemia, Hemangioendothelioma, Hodgkin Lymphoma, Hypopharyngeal Cancer, Carcinoma infiltrating ductal, infiltrating lobular carcinoma, inflammatory breast cancer, intestinal cancer, intrahepatic bile duct cancer, invasive / infiltrating breast cancer, islet cell cancer, jaw cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer , leiomyosarcoma, leptomeningeal metastases, leukemia, lip cancer, liposarcoma, liver cancer, lobular carcinoma in situ, low-grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesenchymal chondrosarcoma, mesenchymal, mesothelioma, metastatic breast cancer, metastatic squamous neck cancer, mixed gliomas , mouth cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, cancer of the nasal cavity, nasopharyngeal cancer, neck cancer, neuroblastoma, neuroendocrine tumors, non-small cell lung cancer , oat cell cancer, eye cancer, eye melanoma, oligodendroglioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteogenic sarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian primary peritoneal carcinoma , stromal tumor of the ovarian sexual cord, pancreatic cancer, papillary carcinoma, paranasal sinus cancer, can Cer of parathyroid, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal region tumor, pituitary gland cancer, primary lymphoma of the central nervous system, cancer of the prostate, rectal cancer, renal cell carcinoma, renal pelvic cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, bone sarcoma, soft tissue sarcoma, uterine sarcoma, breast cancer, skin cancer, small cell lung cancer, cancer small intestine, spinal cancer, spinal cancer, spinal cord cancer, spinal tumor, squamous cell cancer, stomach cancer, synovial sarcoma, T-cell lymphoma, testicular cancer, throat cancer, thymoma / thymic carcinoma , thyroid cancer, tongue cancer, tonsil cancer, transitional cell cancer, triple negative breast cancer, tubal cancer, tubular carcinoma, urethral cancer, adenocarcinom a uterine, uterine cancer, vaginal cancer, acute lymphoblastic leukemia, acute myeloblastic leukemia, thyomas, transitional cell bladder cancer, Wilms tumor, Waldenstrom macroglobulinemia, and vulva cancer. Preferably, the cancer is selected from the group consisting of acute lymphoblastic leukemia, acute myeloblastic leukemia, bone sarcoma, breast cancer, endometrial cancer, gastric cancer, head and neck cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, liver cancer , kidney cancer, multiple myeloma, neuroblastoma, ovarian cancer, small cell lung cancer, soft tissue sarcoma, thyomas, thyroid cancer, transitional cell bladder cancer, uterine sarcoma, Wilms tumor, and Waldenstrom macroglobulinemia.
[0181]
[0182] In a preferred embodiment, the present invention provides a composition comprising: (i) a substantially pure mineral phase of superparamagnetic biomimetic magnetite, (ii) MamC, and (iii) optionally, Mms6; where at least the Components (i) and (ii) form superparamagnetic magnetic nanoparticles containing up to 5 wt% MamC (MamC-mediated BMNPs are then composed of -95 wt% magnetite and -5 wt% MamC), with an average particle size between 30-120 nm, isoelectric point of -4.4, surface area of ~ 90 m2 / g, blocking temperature of -145 K and irreversibility temperature of -292 K and where the magnetic nanoparticles are functionalized with a chemotherapeutic agent; and a pharmaceutically acceptable carrier and / or diluent. Preferably, the chemotherapeutic agent is doxorubicin and the cancer is selected from the group consisting of acute lymphoblastic leukemia, acute myeloblastic leukemia, bone sarcoma, breast cancer, endometrial cancer, gastric cancer, head and neck cancer, Hodgkin lymphoma, non-lymphoma. Hodgkin, liver cancer, kidney cancer, multiple myeloma, neuroblastoma, ovarian cancer, small cell lung cancer, soft tissue sarcoma, thyomas, thyroid cancer, transitional cell bladder cancer, uterine sarcoma, Wilms tumor, and macroglobulinemia of Waldenstrom.
[0183]
[0184] In a preferred embodiment, the treatment of cancer involves the use of a hyperthermia treatment. In such treatment, an alternating magnetic field or radiation is used to rotate the magnetic nanoparticles. Then the magnetic nanoparticles increase the ambient temperature in response to the energy generated through the rotations. If magnetite nanoparticles are located in cancer cells, they can cause cancer cells to die due to increased heat.
[0185]
[0186] Applications
[0187]
[0188] In an eighth aspect, the present invention provides the use of the composition of the present invention, the magnetoliposome formulation of the present invention, or the pharmaceutical composition of the present invention for the preparation of a contrast agent for use in techniques imaging clinics such as magnetic resonance imaging. In a ninth aspect, the present invention provides the use of the composition of the present invention for the isolation and / or purification of nucleic acids.
[0189]
[0190] Other uses of the composition of the present invention, of the magnetoliposome formulation of the present invention or of the pharmaceutical composition of the present invention are also envisaged, which also form part of the invention. These uses include the use of the composition of the present invention, of the magnetoliposome formulation of the present invention, or of the pharmaceutical composition of the present invention as a molecular separator (for example, by functionalizing the nanoparticles with antibodies to capture a specific molecule and then separate said molecule by using a magnetic force) and using the composition of the present invention, the magnetoliposome formulation of the present invention, or the pharmaceutical composition of the present invention as a biosensor. The biosensor can be used for use in a clinical or environmental setting.
[0191]
[0192] Examples
[0193]
[0194] Example 1: Multiple alignment of MamC and Mms6 sequences
[0195] A multiple alignment of the MamC and Mms6 sequences of MC-1 with other homologous proteins from other magnetotactic bacteria showed some similarities in their C-terminal domain and also in the loop within the two transmembrane domains. This loop is rich in acidic amino acids (aspartate and glutamate) and in amino acids that contain hydroxyl groups (tyrosine, threonine, and serine) that can bind metal cations. (Figures 1 and 2).
[0196]
[0197] Example 2: Cloning and Expression of MamC and Mms6
[0198] MamC cloning, expression and purification was carried out as described in Valverde-Tercedor et al. 2015. In summary, the mamC gene (NCBI Database, gene accession ABK44766.1, protein accession Mmc1_2265) was amplified by polymerase chain reaction and cloned into a pTrcHis-TOPO vector (Life Technologies: Invitrogen, Grand Island, NY) to that the recombinant MamC protein was expressed with an N-terminal hexahistidine tail. The recombinant vector was transformed into a TOP10 strain of Escherichia coli (Life Technologies: Invitrogen) and verified by dideoxynucleotide sequencing using an ABI model 3100 sequencer (Life Technologies: Applied Biosystems).
[0199]
[0200] For expression and purification of the MamC protein, transformed E. coli TOP10 cells were incubated at 37 ° C and expression of the protein was induced with isopropyl pD-1-thiogalactopyranoside (IPTG). Cells were harvested by centrifugation, resuspended in buffer A (20mM sodium phosphate buffer, 500mM NaCl, 6M guanidine, pH 8.0) and disrupted by sonication. The soluble fraction was separated by centrifugation and loaded onto a HiTrap chelating HP column (GE Healthcare) previously equilibrated with buffer B (20 mM sodium phosphate buffer, 500 mM NaCl, 8 M urea, pH 8.0) using a ÁKTA Prime Plus FPLC system (GE Healthcare). The column was then washed with buffer B, followed by buffer B adjusted to pH 6. Finally, the protein was eluted with buffer B adjusted to pH 4. The eluate was dialyzed overnight at 4 ° C against buffer C (Tris 50 buffer). mM, 150 mM NaCl, 6M urea, pH 8.5). To reduce the concentration of urea, the dialysis buffer was diluted step-by-step 1: 2 (four times) with fresh urea-free buffer C (called buffer D) and dialyzed for another 2-4 h after each dilution step except for the last dialyzed step overnight.
[0201]
[0202] The mms6 gene (NCBI Database, gene accession ABK44776.1, protein accession Mmc1_2275) was amplified by polymerase chain reaction using the specific primers: f6 (SEQ ID NO: 1, 5'-ATGCCTGTTGCTGTACCAAATAAAGC-3 ') and r6 ( SEQ ID NO: 2, 5'-TCAGCTAATGGCCTCTTCCAATTC-3 '). As in the case of mamC, the amplified mms6 gene was cloned into a pTrcHis-TOPO vector. The recombinant vector was also used to transform a TOP10 strain of Eschenchia coli and was verified by dideoxynucleotide sequencing.
[0203]
[0204] Expression and purification of the Mms6 protein was carried out following the same protocol as described above for the purification of MamC, but using 1mM IPTG instead. Cells were harvested by centrifugation (4508 g, 10 min, 4 ° C), resuspended in 20 mM sodium phosphate buffer (pH 7.4) supplemented with 0.5 mg / ml lysozyme and 5% sodium lauroyl sarcosinate. % (sarcosil) and were broken by sonication. The soluble fraction was separated by centrifugation (15151 g, 40 min, 4 ° C) and loaded onto a HiTrap chelating HP column (GE Healthcare) using an ÁKTA Prime Plus FPLC system (GE Healthcare). The column was pre-equilibrated with 20mM Sodium Phosphate Buffer (pH 7.4) supplemented with 20mM Imidazole and TRITON X-100 at 1.3x Critical Micellar Concentration (CMC) to reduce protein aggregation and improve the protein stability. Elution of Mms6 (2 ml / min) was performed by applying a continuous imidazole gradient of 20 to 500 mM. Fractions were collected and analyzed by 12% SDS-PAGE electrophoresis. Fractions containing Mms6 protein were subjected to an additional chromatographic step on a C4 hydrophobicity column (Jupiter® 5 ^ m C4300 Á, LC 150 x 4.6 mm column) using an HPLC system (Agilent 1100) to remove minor contaminants, E. coli proteins and nucleic acids. In this case, the elution of the Mms6 protein (0.5 ml / min) was produced by applying a gradient of continuous organic solvent (trifluoroacetic acid and acetonitrile) in water due to the high hydrophobicity of Mms6. The purity of the Mms6 protein was tested by 12% Coomassie stained SDS-PAGE. Protein concentration was determined using a Bradford protein assay (Bradford, 1976) and using a NanoDrop 2000 UV-Vis spectrophotometer (Thermo Scientific), using the corresponding molar extinction coefficient at 280 nm (17085 M-1 cm-1) .
[0205] As a control experiment, TOP10 competent cells were also transformed with pTrcHis-TOPO that did not contain the genes of interest. The MamC and Mms6 purification protocol was followed with these transformed bacteria and their corresponding elution fractions were used for magnetite precipitation experiments (control).
[0206]
[0207] Figure 1 is an SDS-PAGE gel of the purified MamC and Mms6 proteins.
[0208]
[0209] Example 3: Biomineralization of magnetite
[0210] Deoxygenated solutions of NaHCO ³ / Na ² CO ³ (0.15 M / 0.15 M), FeCh (1 M), Fe (ClO ⁴ ) ² (0.5 M) and NaOH (5 M) were prepared using water Milli-Q oxygen free deoxygenated (ultrapure) or "Type 1" as defined by various authorities, eg. ex. ISO 3696) according to the following procedure:
[0211]
[0212] (1) To prepare anaerobic solutions:
[0213] to. Boil Milli-Q water in an Erlenmeyer flask in the presence of bubbling rocks until large bubbles form and escape the solution. b. Once boiled, place the flask in an ice bath and immediately cover it and let it bubble with N ² O ² free for one hour / L.
[0214] c. Once bubbled, place the water inside the anaerobic Coy Chamber and prepare the solutions inside.
[0215] (2) Prepare anaerobic protein solutions from a protein stock solution with a protein concentration greater than 2 mg / ml:
[0216] to. Cover the protein solution with a rubber septum and sparged with N ² O ² free.
[0217] b. Once bubbled, place the solution into the anaerobic Coy Chamber.
[0218] (3) To prepare the final mixture for magnetite precipitation (preferably 60 ml in 100 ml glass bottles):
[0219] to. Add the relevant volume of NaHCO ³ and Na ² CO ³ solutions to ensure a final concentration in the reaction mixture of 3.5 mM each,
[0220] b. Add the relevant total volume of Milli-Q water.
[0221] c. Add the relevant volume of the FeCh solution to ensure a final concentration in the reaction mixture of 5.56 mM.
[0222] d. Add protein (s) if necessary.
[0223] and. Let it react for an hour.
[0224] F. Add the relevant volume of Fe (ClO ^{4) 2} solution to ensure a final concentration in the reaction mixture of 2.78 mM.
[0225] g. While stirring, add drops of the NaOH solution to raise the pH value to a pH of 9.
[0226] h. Close the bottles with a rubber septum and seal them and let them react within the Coy anaerobic chamber for 30 days.
[0227]
[0228] The COY chamber was filled with 4% H ² in N ² to avoid possible oxidation. Magnetite precipitation was carried out in experiments under uncontrolled conditions and maintained at 25 ° C and 1 atm of total pressure following the protocol described by the authors inside the anaerobic chamber. The final reaction mixture from which the magnetite precipitated contained NaHCO33.5mM / Na2CO33.5mM, Fe 2.78mM (ClO ^{4) 2} and FeCh 5.56mM, and had a pH = 9. MamC was added and / or Mms6 to this reaction mixture at concentrations ranging from 0 to 10 | jg / ml. Specifically, nineteen magnetite co-precipitation experiments were carried out under the following conditions (three repeats per condition): (1) sixteen experiments performed by adding MamC and Mms6 to the reaction solution at protein concentrations of 0.2.5, 5, 10 jg / ml and MamC / Mms6 and Mms6 / MamC ratios ranging from 0 to 4, here are called experiments carrying MamC, Mms6, MamC-Mms6; (2) an experiment performed by adding purified "contaminating" proteins from transformed cells with the "empty" pTrcHis-TOPO to the reaction mixture, referred to herein as the empty vector experiment; (3) two experiments carried out by adding to the reaction mixture the buffer in which each of the proteins is stored (50 mM Tris and 150 mM NaCl (here called the MamC-buffer experiment) and 1.3 CMC TRITON X100 in water (here called the Mms6 buffer experiment), (4) an inorganic experiment in which no protein and / or buffer was added to the reaction mixture.
[0229]
[0230] Each experiment was allowed to advance into the anaerobic chamber for 30 days, after which the precipitated product was collected. The solids were concentrated in tubes with a magnet and the supernatant (which appeared completely transparent) was discarded. The precipitates were then washed with oxygen-free deoxygenated Milli-Q water twice and a final wash with absolute ethanol (5 ml in each reaction) was performed. Between washes, each reaction flask was shaken vigorously for several seconds, the precipitate was magnetically concentrated, and the liquid was removed. After the last wash with ethanol, the precipitate was concentrated in 1-2 ml of ethanol, hermetically sealed and stored at -20 ° C until analyzed.
[0231] Example 4: Identification of precipitates
[0232] The dust samples from the precipitates were analyzed with an Xpert Pro X-ray diffractometer (PANalytical, The Netherlands) using Cu Ka radiation, with the scan range adjusted from 20 to 60 ° to 20 (0.01 ° / step ; 3 s per step). The identification of the precipitates was carried out using the XPowder software (Martín Ramos, 2004).
[0233]
[0234] The solids formed in all the biomineralization experiments (with and without the proteins) were identified as magnetite using X-ray powder diffraction (XRD). The solids did not contain detectable levels of siderite.
[0235]
[0236] Example 5: Size and morphology of the particles
[0237] The morphology and size of the magnetite nanoparticles collected in these experiments were studied by transmission electron microscopy (TEM) using a Carl Zeiss SMT LIBRA 120 PLUS microscope. Magnetic nanoparticles were embedded in Embed 812 resin. Ultrathin sections (50-70nm) were prepared using a Reichert Ultracut S microtome (Leica Microsystems GmbH, Wetzlar, Germany) after which the sections were deposited on copper grids. The size of the crystals was measured using the ImageJ 1.47 program, and the size distribution curves were determined from those measurements using Origin pro 9. To ensure the reproducibility of the results, the particle sizes were measured in multiple micrographs with an excess of 1000 nanoparticles measured in each experiment. Furthermore, the statistical significance of the results obtained was tested using the Tukey test with a fixed value of a <0.05. High resolution TEM (HRTEM) was also performed using an FEI TITAN G2 80-300. The selected area electron diffraction patterns (SAED) were collected using a 10 ^ m aperture. D-spacings were measured using HRTEM images and crystallographic direction was determined using magnetite data on the RRUFF Project website (http://rruff.info/ams/amcsd.php).
[0238]
[0239] TEM analysis of the magnetite particles produced in the MamC-buffer experiment, the Mms6 buffer experiment, the empty vector experiment, and the Mms6-mediated magnetites show crystal sizes similar (16 ± 6 nm) to those of the magnetites collected from the inorganic control experiment. On the other hand, no differences in morphology were observed either, since all the particles were poorly faceted. Therefore, the potential effect on crystal size and / or morphology observed in magnetites Collected from the remaining experiments should be attributed solely to the proteins involved.
[0240]
[0241] TEM images of Mms6-mediated magnetites show differences in size and shape with respect to inorganic control experiments as a function of Mms6 concentration in solution. At an Mms6 concentration of 2.5 jg / mL, non-faceted crystals of 17 ± 7 nm were formed. However, at Mms6 concentrations of 5 and 10 jg / mL, magnetite crystals had more uniform polyhedral morphologies with well-faceted faces and were slightly larger (23 ± 9 and 22 ± 8 nm, respectively) compared to magnetites. obtained from the inorganic control (MNPs, Figure 3). The size and shape of the MamC-mediated magnetite particles (BMNPs) also depended on protein concentration. The BMNPs crystals formed in the presence of 2.5 µg / ml and 5 jg / ml of MamC were rounded and had sizes of 20 ± 6 nm and 22 ± 7 nm, respectively. At 10 jg / ml MamC, the magnetite crystals showed well-developed crystal faces with two-dimensional rhombic, rectangular and square morphologies and sizes of 37 ± 12 nm (Figure 3).
[0242]
[0243] When both MamC and Mms6 were present in the reaction solution, cumulative effects of both proteins were observed, since the magnetite crystals collected from these experiments showed better faceted morphologies and / or larger sizes compared, not only with crystals from the experiment of inorganic control, but also to crystals collected from experiments in which only one of the proteins was present. The effect of the protein mixture on the magnetite crystals depended on the concentration of the two proteins and the protein ratio. At low concentrations of Mms6 (2.5 jg / mL), the size of the crystals increased with the concentration of MamC to [MamC] = 5 jg / mL. This trend is identical to that observed in the highest concentration of Mms6 (10 jg / ml). However, at [Mms6] = 5 jg / mL no change in crystal size was observed regardless of the MamC concentration in the solution. The same result is observed when the size of the crystals is plotted against the MamC / Mms6 ratio, where the largest average crystal size (31 ± 10 nm) is obtained at a MamC / Mms6 ratio equal to 0.64 and a concentration of MamC of 5 jg / mL (0.28 jM) and an Mms6 concentration of 10 jg / mL (0.44 jM) (Figure 3). Interestingly, at higher MamC / Mms6 ratios, the crystal size decreased and this decrease was found to be statistically significant.
[0244] The HRTEM images show that the crystals obtained from the inorganic control experiments have a square and some rhombic 2-D shapes delimited by the faces (111) (Figure 4). Furthermore, some crystals showed rounded corners corresponding to the face (110) of the crystal (Figure 4). MamC-mediated nanoparticles expressed the glass face (111) with the rounded corners corresponding to the nascent glass faces (110) and (311) (Figure 4). In this case, the crystals seemed elongated along the direction [111]. The crystals obtained in the presence of the Mms6 protein showed rhomboidal, rectangular and hexagonal shapes delimited by the face (111) of the crystal and rounded corners that correspond to the faces (311), (110) and (400) of the crystal (Figure 5) . These crystals were elongated along the direction [111]. The nanoparticles obtained at 5 µg / ml of MamC and 10 jg / ml of Mms6 had shapes and corners that were more defined than those observed in the nanoparticles obtained when only one of the proteins was present. In particular, the crystals from this experiment showed rhomboidal, rectangular, and hexagonal shapes bounded by the faces (111) of the crystal and elongated along the direction [111]. The well-defined corners observed correspond to the faces (110), (311) and (400) of the crystal (Figure 5).
[0245]
[0246] Example 6: Magnetic measurements
[0247] Magnetization measurements were performed using a Quantum Design Superconducting Quantum Interference Device (SQUID) 5T Magnetic Property Measurement System (MPMS). Under gentle argon flow, 1.6 mg of MNPs and 1.01 mg of BMNPs were placed in a double-walled polycarbonate capsule. Hysteresis cycles were determined for each type of nanoparticles at 5 K and 300 K.
[0248]
[0249] Zero-field cooling (ZFC-W) and field cooling (FC-C) measurements were performed using a 5T superconducting quantum interference (SQUID) magnetometer (Quantum Design MPMS XL, USA). Under a smooth flow of argon, a different amount of each sample powder was placed in a double-walled polycarbonate capsule. The samples were immediately cooled in an applied field from zero to 5 K to maintain randomized magnetization of the nanocrystals, after which a 500 Oe magnetic field was applied. To allow comparison between nanoparticles synthesized differently, the M (T) curves were normalized by the quantity of each sample analyzed and by the magnetization value at 300 K.
[0250]
[0251] The nanoparticles synthesized in the inorganic control experiment exhibit the lowest blocking temperature (T ^b ~ 50 K) that is characteristic of nanoparticles small and little crystalline (Figure 6). Mms6-mediated nanoparticles show similar magnetization curves (Figure 6) while MamC-mediated nanoparticles exhibit a higher T ^b (~ 140 K), consistent with their larger size. The nanoparticles obtained at 5 ^ g / ml of MamC and 10 ^ g / ml of Mms6 show the highest TB (Tb ~ 300 K) with the slowest increase in magnetization, characteristic of particles with high crystallinity and a high magnetic moment due to particle.
[0252]
[0253] Both MNPs and BMNPS show remaining magnetization at 5 K in the absence of an external field, but not at 300 K (Figure 6C), confirming that both particles are superparamagnetic and have a blocking temperature <300 K. According to the data obtained, the magnetization saturation value (Ms) for BMNPs is 55 emu / g, while for MNPs it is 66 emu / g (Figure 6C). The difference in magnetization saturation between BMNPs and MNPs is not so high, taking into account the dilution effect due to the coating, so the reduction in the Ms value for the BMNPs could be due to the incorporation of MamC. In fact, taking into account that the percentage of MamC incorporated according to the TGA data (9.4% in BMNPs and 4.5% in MNPs; Figure 6B), the corrected values of Ms for BMNPs and MNPs should be respectively 55 / 1-0.094) = 61 emu / g and 66 / (1-0.045) = 69 emu / g, indicating that they are identical within the experimental error range.
[0254]
[0255] Furthermore, the blocking temperatures (TB) and the irreversibility temperatures (Tirr) of biomimetic particles and MNPs are also different. The lowest TB (103 K) and the Tirr (274 K) correspond to the MNPs (Figure 6D), followed by Mms6-BMNPs and MamC-BMNPs (Figure 6D). While the highest TB (260 K) and Tirr (296 K) correspond to the Mms6-MamC-BMNPs complex. The slowest magnetization and the highest TB values correspond to particles with a higher magnetic moment per particle. Furthermore, the smaller differences between TB and Tirr indicate less polydispersity.
[0256]
[0257] Example 7: Specific surface area measurements
[0258] Powder samples were analyzed to obtain 77 K nitrogen adsorption isotherms on a TriStar 3000 kit (Micromeritics). About 50 mg of sample were degassed at 100 ° C for 4 h before analysis using a degasser (VacPrep 061, Micrometrics). The specific surface area (SSA) of the samples was determined using the BET method [27]. The SSA determined by BET is 97 ± 2 m2 / g.
[0259] Example 8: Electrophoretic mobility measurements and thermogravimetric analyzes Electrophoretic mobility was measured in inorganic magnetites (MNPs) and BMNPs. Suspensions of each type of nanoparticle were prepared in 10 mL of oxygen-free NaClO ⁴ (10 mM). 200 ^ L aliquots from each of the above suspensions were inoculated into eleven tubes containing oxygen-free NaClO ⁴ (10mM), with the final volume of each tube being 10 mL. The pH of each tube was adjusted by adding oxygen-free HCl (0.1 M) or oxygen-free NaOH (0.1 M) until a pH in the range 2 to 11 was achieved, depending on the sample. Samples were sonicated for 2 minutes before measurements. Nine replicates were made for each measurement.
[0260]
[0261] Thermogravimetric analyzes (TGA) were made in D10 mg of solid, heating the sample in an aluminum cell under N ² atmosphere, at a speed of 20 ° C min-1 until a final temperature of 950 ° C.
[0262]
[0263] The graphs of Z potential versus pH (Figure 6A) reveal significant differences between the measured values for MNPs and BMNPs. Both are positively charged at low low pH values and negatively charged at high pH values, but differ at the isoelectric point (iep). While this iep is 7.0 for MNPs, this value is 4.4 for BMNPs. These data suggest that MamC is strongly adhered (or perhaps incorporated) to the crystal. This observation is confirmed by TGA analyzes (Figure 6B). The total weight% (wt%) lost by the BMNPs is 9.4, while by the MNPs it is 4.5, which indicates that the BMNPs are composed of 95.1 wt% of magnetite and 4.9 wt% of MamC. Thus, MamC seems to have an important role in controlling not only the nanoparticle size distribution but also their surface properties.
[0264]
[0265] Example 9: Magnetoliposomes
[0266] (1) Biomimetic magnetoliposomes
[0267] Magnetite nanoparticles tend to aggregate due to their magnetic properties, and additional treatment is necessary to prevent such aggregation before magnetoliposomes are produced. With this objective, the biomimetic nanoparticles were incubated in 5 mL of 100 mM glutamate for 12 hours. The concentration of the nanoparticles was 4.5 mg / mL. The particles were then washed 3 times with water to remove the glutamate. After washing, the particles were concentrated using a magnet and the supernatant was discarded. As mentioned, this procedure was repeated three times. Then the particles were re-dispersed in 1.67 mL of water ([Magnetite nanoparticles] «24 mg / mL]. This suspension was filtered through a filter with a pore size of 0.22 ^ m.
[0268]
[0269] Biomimetic magnetoliposomes were synthesized using the film hydration method. To obtain the thin layer of lipid film, phosphatidylcholine (PC) was dissolved in 8 mL of chloroform ([PC] = 1.25 mg / mL) forming a homogeneous suspension. The solvent was evaporated using a rotavapor (Büchi, Rotavapor-R) under a vacuum stream at 400 rpm and 37 ° C. Furthermore, in order to remove any remaining chloroform, the sample was under a vacuum current for 90 minutes. Then, the thin layer of lipid film was hydrated and dispersed with the ferrofluid suspension ([PC] «6 mg / mL). To ensure complete dispersion, the mixture was stirred for 2 hours at 180-200 rpm. After this, the magnetoliposome suspension was stored at 4 ° C for 24 hours. Finally, the unilamellar magnetoliposomes were obtained by the extrusion method. Specifically, the magnetoliposome solution was passed 5 times through a 200 and 100 nm polycarbonate membrane (Whatman), respectively, with the aid of an extruder (Avanti Polar Lipids) at 45 ° C.
[0270]
[0271] (2) Inorganic Magnetoliposomes
[0272] Inorganic nanoparticles, like biomimetic nanoparticles, tend to add. However, the treatment applied to disaggregate them is different from that followed for biomimetic nanoparticles, due to the different surface properties of both particles. Because of this, the inorganic nanoparticles were incubated in 5 mL of 2M citrate. The rest of the protocol used to obtain inorganic magnetoliposomes was identical to that followed for obtaining biomimetic magnetoliposomes.
[0273]
[0274] Example 10: Functionalization of particles with doxorubicin and DO-24
[0275] The nanoparticles tested were those obtained using 10 ^ g / mL of the MamC protein. The resulting precipitates were concentrated in tubes with a magnet and the supernatant was discarded. The precipitates were then washed sequentially with oxygen-free Milli-Q water three times, with a 0.5% SDS solution and with oxygen-free water again. Finally, the precipitates were resuspended in HEPES buffer with saline (0.01 M HEPES, pH 7.2, 0.15 M NaCl) and sterilized by autoclaving them at 121 ° C for 21 minutes.
[0276] The magnetite nanoparticles were functionalized with DOXO and with the purified DO-24 monoclonal antibody (mAb), which recognizes the human Met / HGF receptor ectodomain, which is considered a tumor marker, being overexpressed in numerous cancers, as already described with minor modifications (Iafisco et al., 2010; Iafisco et al., 2013; Oltolina et al., 2015). Briefly, couplings were performed by mixing 2 mg of magnetite nanoparticles with 1 mg / mL DOXO dissolved in water or with the mAb dissolved in the HEPES buffer with saline (binary magnetite nanoparticles) or with the mAb followed by DOXO (nanoparticles ternary magnetite) inside hermetically sealed cans to prevent magnetite oxidation. Adsorption kinetics experiments were carried out at 25 ° C with stirring (200 rpm) for different time periods up to 24 hours. At the end of each incubation period, the mixtures were washed 3 times to separate the particles from the supernatants using a magnet. The amounts of DOXO and mAb adsorbed were evaluated by UV-Vis spectroscopy (A = 490 and 280 nm, respectively), calculating the differences between the concentrations of the molecules in the solution before and after adsorption on the magnetite nanoparticles (the mentioned supernatant). The functionalized nanoparticles were resuspended in the HEPES buffered saline and stored at 4 ° C until use.
[0277]
[0278] In the case of the magnetite nanoparticles conjugated with both motifs (DOXO and mAb) (the so-called ternary nanoparticles), the same protocols were applied sequentially, each followed by extensive washes. DO-24 mAb was coupled first and DOXO was coupled in the next step, based on previous experiments (Iafisco et al., 2013).
[0279]
[0280] Example 11: Cytocompatibility and cytotoxicity of binary and ternary nanoparticles Cell cultures: the GTL-16 cell line, derived from poorly differentiated human gastric carcinoma expressing Met, and the Huh-7 cell line, derived from well differentiated hepatocellular carcinoma that is negative for Met, they were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (FCS), 50 U / ml penicillin and 50 ^ g / ml streptomycin. Cells were transplanted when they reached 80-90% confluence.
[0281]
[0282] Cells (approximately 12 * 103 GTL-16 / well or 6 * 103 Huh-7 / well) were incubated in 96-well plates for 24 hr; Then 100 ^ L of the different concentrations of the different magnetite nanoparticles, functionalized or non-functionalized, were added to each microwell. After 3 days of incubation, cell viability was evaluated by the 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazole bromide colorimetric assay. (MTT), as described in Oltolina et al., 2015. [Briefly, 20 | jL of MTT solution (5 mg mL-1 in PBS solution) was added to each well. The plate was incubated at 37 ° C for 2 h and then the supernatants were carefully aspirated. Then 125 ^jL of isopropanol 0.2 N HCl was added to dissolve the formed formazan crystals. An aliquot of 100 µL was then carefully taken from each well and its optical density measured in a multiwell reader (2030 Multilabel Reader Victor ™ X4, PerkinElmer) at 570 nm. The viability of parallel cultures of untreated cells was taken as 100% viability and the values obtained in the cells subjected to the different treatments referred to this value. Experiments were performed 3-5 times using 3 replicates for each sample. In some experiments, predefined or equimolar amounts of soluble DOXO were used.
[0283]
[0284] For experiments with the XCELLIGENCE® instrument the cells, approximately 12 x 103 GTL-16 / well or 6 x 103 HuH-7 / well, were seeded in appropriate multi-well plates for 24 hr. From this moment on, the impedance was controlled (time 0 of the experiment) and 100 ^ L of the different functionalized magnetite nanoparticles were added to each microwell. Equimolar amounts of DOXO, either soluble or adsorbed on the nanoparticles, were also used.
[0285]
[0286] When the binary and ternary magnetite nanoparticles, both DOXO transporters, were incubated for 3 days in the cells, they behaved similarly, that is, they exerted the same level of toxicity, with respect to the untreated reference samples or samples treated with soluble DOXO, except in the case where 10 ^ g / ml DOXO were used in GTL-16 cells (Figure 9). In this case, the mAb-functionalized ternary magnetite nanoparticles were significantly more toxic than the binary magnetite nanoparticles that carried only DOXO; no such difference was observed in the case of Huh7, which do not express the target receptor of the mAb. In all cases, both types of magnetite nanoparticles showed dose-dependent toxicity, and the toxicity exerted by DOXO on magnetite nanoparticles was less than that exerted by soluble DOXO, according to what the research team has already reported. and others for other types of functionalized nanoparticles. Therefore, the specificity of the toxicity was restricted to a narrow dose of ternary magnetite nanoparticles. Since only final-time data is obtained in the MTT experiments, experiments were performed that monitored the kinetics of the cytotoxic response of the magnetite nanoparticles in real time using the XCELLIGENCE apparatus. In this case, in the experiments performed with GTL-16 cells at doses of 100 jg / ml DOXO, the mAb functionalized ternary magnetite nanoparticles were significantly more toxic than binary magnetite nanoparticles that only transport DOXO and as effective as soluble DOXO up to 48 h. On the third day, all three DOXO treatments, either soluble or bound to both types of magnetite nanoparticles, induced the same level of toxicity, in line with data from the MTT assay. When the same experiments were performed on Huh7 cells, no significant difference was observed between the effects exerted by the two types of functionalized magnetite nanoparticles, which behaved similarly and were only slightly less toxic than soluble DOXO. Taken together, these data indicate that functionalization with the mAb provides specificity to the magnetite nanoparticles, giving them the ability to target complementary antigen expressing cells.
[0287]
[0288] Example 12: Biocompatibility and distribution of nanoparticles in vivo
[0289] Female BALB / c mice were injected into the tail vein with magnetite nanoparticles (10 µg nanoparticles / g mouse weight) diluted in a final volume of 100 µl of sterile PBS. Animals were monitored every other day for up to 1 month. Mice were subdivided into 5 groups, differentiated at the time point of euthanasia (from 1 hour to 2 months). For each group consisting of 3 mice, an untreated control mouse was also used. His organs were collected, fixed, paraffin-embedded and processed for histological analysis. Serial sections were stained with Prussian blue and hematoxylin-eosin (Sigma Aldrich) and underwent histological evaluation by an independent pathologist not informed of the identity of the samples. All procedures were carried out in accordance with the Directive of the European Community for the Care and the Italian Laws on animal experimentation (Law by Decree 116/92).
[0290]
[0291] All mice injected with the magnetite nanoparticles (10 jg / g mouse weight) were observed to be alive and in good shape for at least 60 days. The brain, heart, lung, spleen, liver and kidney sections prepared from the animals 1, 4 h, 1, 7, 60 days after the injection do not show any morphological alteration compared to those of the control mice (Figure eleven). Few magnetite nanoparticles were detected, mainly as aggregates in the lungs. Furthermore, in the case of the spleen, which in the untreated control animals was already positive for Prussian blue staining, this staining was undetectable 4 h and 1 day after the injection of the magnetite nanoparticles, but was detectable at least 1 week later, if not earlier. Taken together, these data confirm minimal / low toxicity of magnetite nanoparticles up to 10 | jg / g mouse weight.
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权利要求:
Claims (18)
[1]
1. A composition containing:
(i) a pure mineral phase of superparamagnetic biomimetic magnetite;
(ii) MamC; and
(iii) optionally, Mms6;
where, at least components (i) and (ii) form superparamagnetic magnetic nanoparticles containing up to 5 wt% MamC (MamC-mediated BMNPs are then composed of -95 wt% magnetite and -5 wt% MamC), with an average particle size between 30-120 nm, isoelectric point of -4.4, surface area of ~ 90 m2 / g, blocking temperature of -145 K and irreversibility temperature of -292 K.
[2]
2. The composition according to clause 1, in which there is no siderite in the composition at detectable levels and <5% goetite.
[3]
3. The composition according to any of the preceding clauses, where the average particle size is 30-50 nm.
[4]
4. The composition according to any of the preceding clauses, where the biomimetic magnetic nanoparticles are functionalized with a therapeutic agent, preferably with a chemotherapeutic agent, more preferably doxorubicin.
[5]
5. The composition according to any of the preceding clauses, where the biomimetic magnetic nanoparticles are functionalized with a signaling substance, preferably with a monoclonal antibody.
[6]
6. A formulation to make magnetoliposomes that includes:
(i) the composition in accordance with any of clauses 1-5;
(ii) an agent that forms liposomes; and
(iii) optionally superparamagnetic inorganic magnetites (MNPs).
[7]
7. The magnetoliposome formulation according to clause 6, where the liposomes are functionalized with a therapeutic agent and / or with a signaling substance.
[8]
8. A pharmaceutical composition encompassing the composition of the present invention or the formulation of the magnetoliposomes of the present invention and a pharmaceutically acceptable carrier and / or diluent.
[9]
9. The composition according to any of clauses 1-5, the formulation of the magnetoliposomes according to any of clauses 6-7 or the pharmaceutical composition according to clause 8 for use as a medicine.
[10]
10. The composition according to any of clauses 1-5, the formulation of the magnetoliposomes according to any of clauses 6-7 or the pharmaceutical composition according to clause 8 for use in cancer treatments.
[11]
11. The composition, the formulation of the magnetoliposomes or the pharmaceutical composition for use according to clause 10, where the cancer is selected is selected from the group consisting of acute lymphocytic leukemia, acute myelogenous leukemia, bone cancer, breast cancer , cervical cancer, gastric cancer, brain and neck tumor, Hodgkin lymphoma, non-Hodgkin lymphoma, liver cancer, kidney cancer, multiple myeloma, neuroblastoma, ovarian cancer, non-small cell lung cancer, sarcoma of soft tissues, thymomas, thyroid cancer, transitional cell bladder cancer, Wilms tumor, Waldenstrom macroglobulinemia.
[12]
12. A method of producing a substantially pure mineral phase composition of superparamagnetic biomimetic magnetite comprising the following steps:
(a) prepare a carbonate solution;
(b) adding FeCh to the carbonate solution;
(c) add MamC and, optionally, Mms6 to the solution obtained in step (b); (d) incubating the solution obtained in step (c) for at least 30 minutes;
(e) add Fe (ClO ^{4) 2} to the solution obtained in step (d) and
(f) adjusting the pH of the solution obtained in step (e) to pH 9 using a base;
Where the method is performed at 25 ° C and 1 pressure atmosphere and all the solutions used are previously deoxygenated.
[13]
13. The method according to clause 12, where the carbonated solution consists of NaHCO ³ and Na ² CO ³ and, optionally, the base is NaOH.
[14]
14. The method according to clause 13, where the final concentration of the solution obtained in step (f) is 3.5 mM NaHCO ³ , 3.5 mM Na ² CO ³ , 2.78 mM Fe (ClO ⁴ ) ² , 5.56 mM FeCl ³ and a variable amount of MamC and, optionally, Mms6.
[15]
15. The method according to clause 13, where the concentration of the protein reservoirs in solution are [MamC] = 2-5 mg / mg, [Mms6] and [Mms7]> 1 mg / mL.
[16]
16. The use of the composition according to any of clauses 1-5, the formulation of the magnetoliposomes according to any of clauses 6-7 or the pharmaceutical composition according to clause 8 for the preparation of a Contrast for clinical imaging techniques, preferably magnetic resonance imaging techniques.
[17]
17. The use of the composition according to any of clauses 1-5 for the isolation and purification of nucleic acids.
[18]
18. The use of the composition according to any of clauses 1-5 as a biosensor or as a molecular separator.

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同族专利:

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公开号 | 公开日

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ES2758400B2|2021-01-20|

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WO2020089505A1|2020-05-07|

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EP3875433A1|2021-09-08|

引用文献:

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优先权:

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ES201831064A|ES2758400B2|2018-11-02|2018-11-02|BIOMIMETIC NANOPARTICLES MEDIATED BY MAMC|ES201831064A| ES2758400B2|2018-11-02|2018-11-02|BIOMIMETIC NANOPARTICLES MEDIATED BY MAMC|

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EP19877759.1A| EP3875433A1|2018-11-02|2019-11-04|Mamc-mediated biomimetic nanoparticles|

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PCT/ES2019/070747| WO2020089505A1|2018-11-02|2019-11-04|Mamc-mediated biomimetic nanoparticles|