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    • 专利摘要:
    Polymers for gene therapy. The present invention relates to a polymer, more specifically a polyphosphazene polymer, which is useful for gene therapy. The invention also relates to a complex comprising the polymer, to the preparation process and its uses. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2809348A1
    申请号:ES202031074
    申请日:2020-10-27
    公开日:2021-03-03
    发明作者:Mazas Carla Maria Garcia；Noemi Csaba；Fuentes Marcos Garcia
    申请人:Universidade de Santiago de Compostela；
    IPC主号:
    专利说明:

    [0001] Polymers for gene therapy
    [0003] Technical sector
    [0004] The present invention relates to a polymer, more specifically a polyphosphazene polymer that is useful for gene therapy. The invention also relates to a complex comprising the polymer, to the preparation process and its uses.
    [0006] Background
    [0008] In gene therapy, in addition to other biomedical areas, new biodegradable materials are required and polymers play a critical role in this field. Compared to natural polymers, synthetic biodegradable polymers are generally better defined and easier to modulate with respect to their mechanical and degradation properties. Polyesters such as polylactide (PLA), poly (lactide-co-glycolide) (PLGA) or polycaprolactone (PCL) are some of the most widely used synthetic polymers as biomaterials, but more recently, other families of polymers have begun to be investigated, such as polyphosphazenes ( Hsu WH, Csaba N, Alexander C, Garcia-Fuentes M. Polyphosphazenes for the delivery of biopharmaceuticals. J. Appl. Polym. Sci. 2019, 137 ( 25)).
    [0010] Polyphosphazenes are a relatively new family of polymers based on a nitrogen-phosphorous backbone where side groups can be inserted.
    [0012] The first known polyphosphazene, published by Stokes in 1897, was obtained by heating (250 ° C) of a mixture of PCl 5 and NH 4 CL Due to its insolubility and lack of knowledge about its chemical structure, at first it was called "inorganic rubber". In 1956, Allcock and collaborators carried out the first stable synthesis of polyphosphazenes, at which point this type of materials began to be of greater interest for use in biomedical applications. The scheme developed by Allcock remains the basis of the more general procedure for linear synthesis of polyphosphazenes: the precursor, poly (dichlorophosphazene) (PDCP), is prepared in the first step, and then the final polymer is formed by nucleophilic substitution of the desired side chains.
    [0014] Since the synthetic route of polyphosphazenes is very versatile, it is possible to add many different functionalities that completely modify the physicochemical and biological properties of polymers. In this way, polyphosphazenes can be designed incorporating a series of organic side groups that make these materials biodegradable and highly biocompatible ( Hsu WH, Sanchez-Gomez P, Gomez-Ibarlucea E, Ivanov DP, Rahman R, Grabowska AM, Csaba N, Alexander C, Garcia-Fuentes M Structure-Optimized Interpolymer Polyphosphazene Complexes for Effective Gene Delivery against Glioblastoma. Adv. Ther. 2018, 2 ( 3)). These particularities of polyphosphazenes have stimulated growing interest, particularly concentrated in some areas such as tissue engineering, gene delivery, protein delivery and vaccination. Particularly critical to this interest is the biodegradability of most poly (organophosphazenes) and the chemical flexibility of these materials. Although other types of polymers can also be chemically diverse, engineers find the special synthesis pathway of polyphosphazenes attractive, where a precursor is modified to adapt the structure of the material to meet the requirements of specific applications. This synthesis route simplifies the adaptation of existing technologies to new areas of interest ( Hsu WH, Csaba N, Alexander C, Garcia-Fuentes M. Polyphosphazenes for the delivery of biopharmaceuticals. J. Appl. Polym. Sci. 2019, 137 ( 25)).
    [0015] Even so, it is still necessary to have polyphosphazene derivatives that allow better transfection and are biologically safe. And even more, that they can effectively load biological molecules to improve their transfection.
    [0017] Brief description of the invention
    [0019] The inventors have designed and prepared different polyphosphazenes that are suitable for the transfection of biological molecules.
    [0021] Thus, in a first aspect the invention is directed to a polyphosphazene comprising:
    [0023] - at least one hydrocarbon chain (A) of between 6 and 24 links, where between 1 and 4 links are heteroatoms independently selected from O, N and S, and the end of the chain is a linear C 1 -C 6 alkyl group or branched, optionally the hydrocarbon chain has between 1 and 3 substituents independently selected from hydroxyl and thiol; Y,
    [0024] - at least one hydrocarbon chain (B) of between 6 and 24 links, where between 1 and 4 links are heteroatoms independently selected from O, N and S, and the terminal group of the chain is a group of formula -NH 2 .
    [0025] Furthermore, the polyphosphazenes of the invention are capable of complexing with biological molecules. And thus, a second aspect of the invention is directed to a complex comprising a polyphosphazene of the first aspect of the invention and a biologically active molecule.
    [0026] In other aspects, the invention relates to the use of the polyphosphazene or the complex of the invention, to a pharmaceutical composition, to a kit and method of preparing the same.
    [0028] Description of the figures
    [0030] Figure 1. Synthesis and modification scheme of the polyphosphazene precursor by thiol-ene chemistry.
    [0032] Figure 2. Cellular metabolic activity of the commercial line corresponding to human glioblastoma (U87MG) and that has been treated with different concentrations of nanocomplexes (ratio 8.0.1 and 8.4.1) at 48 hours after treatment. Concentrations are expressed in plasmid µg to compare the toxicities of formulations with the same loading capacity.
    [0034] Figure 3. Transfection in two-dimensional cultures of the human glioblastoma cell line (U87MG) measured by luminescence 48 hours after transfection and after the addition of the luciferin substrate. Results are expressed with respect to lipofectamine luminescence (transfection control).
    [0036] Figure 4. Clonogenicity assay in two glioblastoma cell lines (U251 and U87MG). The clonogenic capacity of the cells was studied after incubation for 48 h with the different treatments, the data are expressed in number of colony-forming units per plate (untreated cells were assumed to be 100%); Tz: temozolomide; NCs: nanocomplexes; BMP4: Bone morphogenic protein (p <0.05).
    [0038] Figure 5. In vivo efficacy of the formulation alone and in combination with a chemotherapeutic agent. A. Evolution of tumor volume. B. Evolution of the volume of the tumor corrected for its volume before the administration of the treatment. C. Evolution in the weight of the mice. D. Survival percentage of the mice taking into account that the animals with necrosis in the tumor or a size greater than 1500mm3, were sacrificed in order to reduce the suffering of the animal. The red arrows illustrate the four administrations on consecutive days. Treatment group legend: Control (gray circle), White nanocomplexes (gray triangle), Protein-BMP4 Temozolomide (inverted black triangle), Temozolomide (gray square), BMP4 nanocomplexes (black diamond), BMP4 nanocomplexes Temozolomide (circle black).
    [0040] Figure 6. Photographs of the dissected tumors after euthanasia of the mice. NCs: Nanocomplexes; BMP4: Bone Morphogenic Protein-4; Tz: Temozolomide.
    [0042] Figure 7. Representations of the expression of genes involved in the development of tumors, the expression was analyzed by real-time PCR (RT-PCR). NCs: Nanocomplexes; BMP4: Bone Morphogenic Protein-4; Tz: Temozolomide.
    [0044] Detailed description of the invention
    [0045] As discussed above, in a first aspect the invention is directed to a polyphosphazene comprising:
    [0047] - at least one hydrocarbon chain (A) of between 6 and 24 links, where between 1 and 4 links are heteroatoms independently selected from O, N and S, and the end of the chain is a linear C 1 -C 6 alkyl group or branched, optionally the hydrocarbon chain has between 1 and 3 substituents independently selected from hydroxyl and thiol; Y,
    [0048] - at least one hydrocarbon chain (B) of between 6 and 24 links, where between 1 and 4 links are heteroatoms independently selected from O, N and S, and the terminal group of the chain is a group of formula -NH 2 .
    [0050] Each monomeric unit of polyphosphazene can be represented by the following formula:
    [0055] So that the phosphorus atom is linked to a type (A) chain and to a type (B) chain. In a particular embodiment, the polyphosphazenes of the invention have between 150 and 600 monomeric units. In a more particular embodiment, the polyphosphazenes of the invention have between 200 and 500 monomeric units.
    [0056] For the present invention, by "hydrocarbon chain of between 6 and 24 links" is understood a hydrocarbon that has between 6 and 24 carbon atoms in its backbone and that can be linear or branched. Between 1 and 4 carbon atoms of these hydrocarbon chains may have been substituted by heteroatoms. Thus, in hydrocarbon chains there may be 1, 2, 3 or 4 heteroatoms that are forming part of the chain, occupying a link in it. Said heteroatoms are selected from between O, N and S. Preferably, the hydrocarbon chains have between 6 and 16 links. Preferably, 2 or 3 links of the hydrocarbon chains are heteroatoms independently selected from N and S.
    [0058] For the present invention, "substituent" is understood as a set of atoms that have a chemical function, that is, a functional group, which is attached to the hydrocarbon chain in one of its positions, but is not part of it.
    [0060] For the present invention, the term "end of the chain" is understood to be the final part of the hydrocarbon chain (A), the beginning of that end of the chain being the link following the heteroatom immediately before the end, or what is the same, the heteroatom located in the link farthest from the phosphorus atom.
    [0062] For the present invention, by "terminal group" is understood a set of atoms that have a chemical function, that is, a functional group, which occupies the position of the end of the hydrocarbon chain.
    [0064] The polyphosphazenes of the invention are characterized in that they comprise a hydrocarbon chain (A) whose alkyl group at the end of the chain is a group with an aliphatic and hydrophobic character. The presence of hydrophobic groups in the polymer has the advantage that it improves its diffusion through cell membranes.
    [0066] In a particular embodiment, the hydrocarbon chain (A) has the following structure:
    [0070] where m can have the value 0, 1 or 2,
    [0072] n can have the value 1, 2 or 3,
    [0074] q can have the value 0 or 1,
    [0075] each Xi, X 2 and X 3 are independently selected from NH, S and O atoms,
    [0076] Alk is a straight or branched C 1 -C 6 alkyl, optionally having a substituent selected from a hydroxyl or thiol group. Alq is what we have previously called the end of the chain. Preferably methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl and tert-butyl.
    [0078] In a particular embodiment, these hydrocarbon chains (A) have a heteroatom in position 1 of the chain, more particularly that hetero atom is N. In another particular embodiment, the hydrocarbon chains (A) have a heteroatom in position 5, more particularly that heteroatom is S. In another particular embodiment, the hydrocarbon chains (A) have two heteroatoms in the chains, in position 1 an N and in position 5 an S.
    [0080] In a particular embodiment, the hydrocarbon chains (A) of the polyphosphazenes of the invention are selected from the group consisting of:
    [0082]
    [0085] The polyphosphazenes of the invention are also characterized in that they comprise a hydrocarbon chain (B) with a terminal group -NH 2 . This terminal group has the advantage of increasing the carrying capacity of proteins or nucleic acids through electrostatic interaction.
    [0087] In a particular embodiment, these hydrocarbon chains (B) have a heteroatom in position 1 of the chain, more particularly that hetero atom is N. In another particular embodiment, the hydrocarbon chains (B) have a heteroatom in position 5, more particularly that heteroatom is S. In another particular embodiment, the hydrocarbon chains (B) have two heteroatoms in the chains, in position 1 an N and in position 5 an S.
    [0089] The presence of the hydrocarbon chains (A) and (B) with the technical characteristics set forth above, in the polyphosphazenes of the invention, favors the loading capacity and the internalization of biologically active molecules, and the final effect obtained is that a transfection much more efficient.
    [0090] Furthermore, the polyphosphazenes of the invention form complexes with biologically active molecules. Thus, in a second aspect the invention is directed to a complex comprising a polyphosphazene, as described above, and a biologically active molecule.
    [0092] In a particular embodiment, the complexes of the invention have a particle size between 50 nm and 300 nm, preferably between 80 nm and 200 nm. In a particular embodiment, the complexes of the invention have a surface charge of between 20 mV and 50 mV.
    [0094] In a particular embodiment, the biologically active molecule is selected from among protein and polynucleotide. In a more particular embodiment, the biologically active molecule is a plasmid encoding BMP-4.
    [0096] Furthermore, the complexes of the invention may also incorporate an anionic polyphosphazene.
    [0098] For the present invention, "anionic polyphosphazene" is understood to be polyphosphazene having anionic groups, for example carboxylic groups.
    [0100] In a particular embodiment, the anionic polyphosphazene comprises a hydrocarbon chain of between 6 and 24 links, where between 1 and 4 links are heteroatoms independently selected from O, N and S, and at least one carboxylic group.
    [0102] The complexes of the invention that further comprise anionic polyphosphazenes have the advantage of being less toxic as demonstrated in Example 3.1. and figure 2. Furthermore, these complexes have a higher transfection capacity, as demonstrated in example 4 and figure 3. Being the one with the highest transfection capacity PPZ-Aliphatic.
    [0103] In a preferred embodiment, the invention is directed to a complex comprising a polyphosphazene of the invention, an anionic polyphosphazene and a biologically active molecule. In a more preferred embodiment, the invention is directed to a complex comprising a polyphosphazene of the invention whose type (A) chain has the formula 1.
    [0105] In a particular embodiment, the complexes of the invention also comprise a chemotherapeutic molecule. In a more particular embodiment, the chemotherapeutic molecule is temozolomide.
    [0106] In another aspect, the invention is directed to a pharmaceutical composition comprising the complex of the invention and pharmaceutically acceptable excipients.
    [0108] In another aspect, the invention is directed to the polyphosphazenes of the invention or the pharmaceutical composition of the invention for use in medicine.
    [0110] In a particular embodiment, the invention is directed to the polyphosphazenes of the invention or to the pharmaceutical composition of the invention for use in gene therapy or oncology, or for use in the treatment of brain tumors, preferably glioblastomas.
    [0112] In another aspect the invention is directed to a kit comprising at least two containers where one of the containers comprises a complex of the invention, and the other container comprises a chemotherapeutic molecule, together with instructions for its use in the treatment of a disease for sequential or simultaneous administration of both ingredients.
    [0114] In another embodiment the invention is directed to the use of the complex of the invention in the elaboration of vectors for the transport of biologically active molecules.
    [0116] Materials and methods
    [0117] Aluminum Chloride (99.99%), Anhydrous Tetrahydrofuran (THF), Cysteamine (Cis), Chloroform-deuterated (99.96 atom% D contains 0.03% TMS), Deuterium oxide (99.9 atom% D, contains 0.05% acid weight 3- ( trimethylsilyl) propionic-2,2,3,3-d4), Heparin as sodium salt (from porcine intestinal mucosa), HEPES (> 99.5%), Hexachlorocyclotriphosphazene (99%), Potassium chloride (BioXtra> 99%), Triethylamine (TEA), Tris-Acetate-EDTA buffer solution (10x), 1-mercapto-2-propanol (MP), 2-butylamino) ethanethiol (BET), 2- (dimethylamino) ethanethiol hydrochloride (DMAES), 2, 2,2-Trifluoroethanol (TFE), 2,2-dimethoxy-2-phenylacetophenone (DMPA), 2-methyl-1-propanethiol (MPT), 6-mercaptohexanoic acid (6MHA), Temozolomide, Xylazine all of them were purchased from Sigma -Aldrich. Ketamine for anesthesia was ordered from Pfizer. Ethanol, Crystal Violet, and Glacial Acetic Acid were purchased from Merk. DNAse / RNAse-free water (Invitrogen), Dulbecco's Modified Eagle's Medium (DMEM) (Gibco), OptiMEM (Gibco), Minimal Essential Eagle's Medium (EMEM), Fetal Calf Serum (FBS) (Gibco), Penicillin-Streptomycin for medium Culture (Gibco), Dialysis Membrane (7kDa pore size), Lipofectamine 2000 transfectant (Life Technologies), nucleic acid stain SYBR® Gold (Life Technologies) were purchased from Thermo Fisher. The Bio-Rad protein determination test was purchased from BioRad (CA, USA), Test for Luciferase determination Roche (Germany) MTS BioVision Cell Proliferation Determination Kit (USA), Alamar Blue Promega (Spain ). Bone morphogenic protein 4 (BMP-4) was purchased from Preprotech (UK). The plasmid expressing BMP4 is from Sino Biological Inc. (Germany).
    [0119] Example 1. Preparation of cationic polyphosphazenes and anionic polyphosphazene.
    [0121] 1.1. Synthesis of the poly (allylamino-phosphazene) precursor
    [0122] The synthesis of the precursor was carried out following the protocol developed by Dr. Wei-Hsin Hsu (Hsu, W.-H., Sánchez-Gómez, P., Gomez-Ibarlucea, E., Ivanov, DP, Rahman, R., Grabowska, AM, Garcia-Fuentes, M. (2019). Structure-Optimized Interpolymer Polyphosphazene Complexes for Effective Gene Delivery against Glioblastoma. Advanced Therapeutics, 2 (3), 1800126. https://doi.org/10.1002/adtp.201800126 ). In a previously dry flask, 14.4 mmol of hexachlorocyclophosphazene was mixed with 7.5% (w / w) aluminum chloride (catalyst) under an inert nitrogen atmosphere and heated at 240-250 ° C for 3 hours, the aluminum chloride produced the polymerization after the opening of the ring and the elimination of part of the chlorides giving as product dichlorophosphazene. The product was then cooled to 120 ° C and solubilized in diglyme to minimize cross-linking and prevent product solidification and was centrifuged to remove aluminum chloride (-10 ° C, 7000G, 5 min). The supernatant was transferred to a flask with THF, TEA (3 equivalents / chlorine) and allylamine (3 equivalents / chlorine), the reaction was kept in an ice bath for 24h and then 24h at room temperature. The resulting product was filtered to remove TEA hydrochloride and precipitated with water, then centrifuged (4 ° C, 7000G, 10 min) and the precipitate was collected and dried under vacuum overnight (Figure 1). Allylphosphazene (APPZ) was characterized by phosphorus, proton and DOSY magnetic resonance imaging.
    [0124] 1.2. Modification of the precursor by thiol-ene chemistry
    [0125] The side chains of the precursor were modified by thiol-ene chemistry to introduce different radicals, the thiol group of the new compounds reacts with the allyl group of the precursor obtaining five different polyphosphazenes.
    [0127] The product obtained in the previous point was dissolved in trifluoroethanol (TFE) and mixed with the desired substituent (s) in the following ratio: 3 equivalents / allyl group.
    [0128] The substituents used were: 1-mercapto-2-propanol (to obtain polyphosphazene PPZ-Hydroxyl), 2- (butylamine) ethanethiol (to obtain polyphosphazene PPZ-Amine2), 2-methyl-1-propanethiol (to obtain polyphosphazene PPZ-Aliphatic). The mixture was sparged with nitrogen to achieve an inert atmosphere and 2,2-dimethoxy-2-phenylacetophenone (DMAES) catalyst (0.05 equivalents / allyl group) was added. The reaction took place for 3h with magnetic stirring and UV irradiation (X = 365 nm) (Figure 1). The product was then dialyzed (membrane pore size 7 kDa) against 2 mM HCl for 24 hours and 48 hours against water. The dialysate was lyophilized and the compounds were characterized by phosphorus and proton NMR, COZY and HSQC.
    [0130] The polyphosphazene PPZ-Aminol was also synthesized to be able to compare it with the polyphosphazenes of the invention. This polyphosphazene was prepared as above and using cysteamine as a reagent to react with the allylphosphazene.
    [0132] An anionic polyphosphazene (PPZ-anionic) was also synthesized using 6-mercaptohexanoic acid as a substituent, using a ratio of 3 equivalents of mercaptohexanoic acid / allyl group to react with the allylphosphazene, following the instructions previously described.
    [0134] The percentages of substitution of each substituent in the heteropolymers is 50% for the PPZ-Aliphatic. However, for PPZ-Amine2 the substitution is 68% of the cysteamine radical and 32% of the 2- (butylamine) ethanethiol radical and in the case of PPZ-Hydroxyl, the substitution was 33% of the cysteamine radical group and of the 66% of the 1-mercapto-2-propanol radical.
    [0136] To determine the molar mass of the polymers, they were dissolved in 10mM sodium chloride at a concentration of 5mg / mL and measured by field-flow fractionation asymmetric flow (AF4) using AF2000 MultiFlow coupled to a multi-angle light scattering detector ( MALS) (Postnova, Germany). The detector was calibrated with a bovine serum albumin monomer standard (66 kDa) and quality control was performed every day by pullulan (48.8 kDa).
    [0138] Table 1 Molar mass of the polymer and distribution measured by AF4.
    [0140]
    [0143] Mw: weight average molecular weight; Mn: number average molecular weight; 0: Polydispersity index.
    [0145] Example 2. Formation of nanocomplexes
    [0146] Example 2.1. Formation of the nanocomplex with a plasmid
    [0147] The nanocomplexes were prepared by ionic complexation, using a model plasmid encoding enhanced green fluorescent protein (eGFP) and luciferase (Luc). The polymers were dissolved in 10 mM HEPES (pH 5.5) and the plasmid in water. The formation of the complexes takes place by ionic complexation when the plasmid, or the mixture of the plasmid with the anionic polymer, is added to the cationic polymer solution with magnetic stirring. Different charge ratios have been prepared, in the case of nanocomplexes containing only cationic polymer, the ratios are based on the number of positively charged amines of the cationic polymer (N) and negatively charged phosphates of the plasmid (F), in the case of nanocomplexes that also contain anionic polymer, the relationships were established between the number of positively charged amines of the cationic polymer (N), the number of negatively charged carboxyl groups of the anionic polymer (C) and the number of negatively charged phosphate groups of cDNA (F ), therefore N / F and N / C / F, respectively. The nanocomplexes were prepared for a final concentration of encapsulated plasmid of 25 µg / mL, in order to be able to compare the formulations with each other, all of them having the same amount of plasmid.
    [0149] Characterization of the size, surface charge and concentration of nanocomplexes
    [0151] The characterization of the nanocomplexes, in terms of size, was performed using Dynamic Light Scattering (DLS) and the Zeta potential was determined by laser doppler anemometry using a Nanosizer ZS instrument (Malvern, UK). Each analysis was performed in triplicate at 25 ° C, with a backscatter angle of 173 °. Nanocomplex concentrations were determined by nanocomplex follow-up analysis using a Nanosight NS300 system (Malvern Instruments, Worcestershire, UK Bonded) equipped with a laser measuring at X = 488 nm, after diluting the samples 1: 400 in 10 mM HEPES. In the case of the zeta potential, measurements were made with a 1:10 dilution in 1 mM KCl.
    [0153] Table 2 Characterization of cationic nanocomplexes by size and surface charge. nm: nanometers; PDI: Polydispersion; mV: millivolts.
    [0155]
    [0158] The polymer: plasmid ratio affects the size and surface charge of nanocomplexes. when the proportion of cationic polymers increases, the surface charge of the nanocomplexes increases slightly, being positive in all cases, with respect to size we can observe a small variation although all the prototypes showed a size between 100-150nm, which makes them suitable as vectors in gene therapy.
    [0160] Table 3 Characterization of cationic and anionic nanocomplexes by size and surface charge, and concentration. nm: nanometers; PDI: Polydispersion; mV: millivolts.
    [0162]
    [0163]
    [0166] The size of all the nanocomplexes formed is between 100 and 150 nm, the surface charge between 30 and 40 mV and it was observed that the concentration of nanocomplexes that associate cationic and anionic polymers is between 2 and 3 times greater than those containing only cationic polymers.
    [0168] Morphological analysis was performed using field emission scanning electron microscopy (FESEM) with energy dispersive X-ray spectroscopy (Zeiss Gemini Ultra Plus, Germany) and using scanning transmission electron microscopy (STEM) and immersion lens detectors. (InLens) for observation of the sample. For sample preparation, 10 ^ L of the nanocomplexes were placed on a copper grid with carbon films and the excess was removed. Then the same volume of phosphotungtic acid (2%) was added and washed twice with water. Once dry, the sample was observed through STEM detectors and immersion lenses (InLens).
    [0170] In all cases the particles are spherical and those containing anionic polymer show a higher intensity, which may be indicative of a higher particle density compared to the cationic ones.
    [0172] Efficiency of nucleic acid association in nanocomplexes
    [0174] The binding efficiency of the nanocomplexes was determined by agarose gel delay assay. The samples were loaded onto the agarose gel (1% w / v in 1x Tris-EDTA buffer). Each well contained 0.33 µg of cDNA, free plasmid was used as a control. For sample visualization and ease of loading, all samples contained 1x SYBR® Gold for nucleic acid staining and loading buffer (30% glycerol and 0.25% bromophenol blue). The dissociation assay was performed by incubating samples with an excess of an anionic competitor (20: 1 w / w heparin: cDNA) for 1 hour at 37 ° C.
    [0176] It was shown that the nucleic acid does not bind irreversibly to the vehicle and can be released into the physiological environment and perform its function.
    [0178] Example 2.2. Formation of the nanocomplex with a protein
    [0179] The nanocomplexes were prepared by ionic complexation, using a model protein, heparin. The polymers were dissolved in 10 mM HEPES (pH 5.5) and the protein in water. The formation of the complexes takes place by ionic complexation when the protein, or the mixture of the protein with the anionic polymer, is added to the cationic polymer solution with magnetic stirring.
    [0181] The size, polydispersity (PDI) and charge of the prepared nanocomplexes were measured.
    [0182] Table 4. Characterization of cationic and anionic nanocomplexes by size and surface charge. nm: nanometers; PDI: Polydispersion; mV: millivolts.
    [0187] Example 3. Toxicity studies
    [0189] 3.1. 2D in vitro toxicity
    [0191] All in vitro assays were performed on U87MG human glioblastoma cells grown in DMEM medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and penicillin / streptomycin (P / S) at 1% (Gibco, USA) incubating them at 37 ° C, an atmosphere saturated with humidity and 5% CO 2 . In all of them the negative control is medium and the positive control is Triton 0.1%.
    [0193] For the 2D toxicity assay, 8,000 cells / well were seeded in a 96-well plate and incubated 24 hours prior to treatment to allow adherence to the bottom of the well. Then, the nanocomplexes were incubated in supplemented medium for 4 hours at different reference concentrations to the plasmid concentration (0.1-2 pg / cm2). Afterwards, they were washed with PBS, fresh medium was added and they were allowed to grow for an additional 48h. The cytotoxicity evaluation was carried out by adding 10 µl of MTS per well (BioVision, USA) and the absorbance was measured after 3 h of incubation in a plate reader at 495 nm.
    [0194] Figure 2 shows that in all cases there was a decrease in the toxicity of nanocomplexes containing the anionic polymer compared to those containing only cationic polymer.
    [0196] 3.2. In vitro 3D toxicity
    [0198] For the 3D toxicity assay, first the neurospheres were formed, 500 cells / well were seeded in a 96-well ultra low adherence plate, the cells were centrifuged for 20 minutes at 200 rcf. After 3 days, the nanocomplexes were incubated for 12 h at a concentration of nanocomplexes relative to 2 pg plasmid / ml, after 12 h the nanocomplexes were replaced with fresh medium and the neurospheres were incubated for a further 72 h. We evaluated two parameters: neurosphere size at 0, 24, 48 and 72 h and cytotoxicity at 72 h using the resazurin reduction metabolic assay (CellTiter-Blue®, Promega, USA U U). For the metabolic assay, 40 µl of reagent was added per well and incubated for 4 h. Fluorescence was evaluated by a plate reader at excitation wavelength 539 nm and emission wavelength 620 nm.
    [0200] Similar growth and morphology was observed in the untreated neurospheres (viability control adding fresh culture medium) and those treated with the formulations, while a disintegration and decrease in size was observed in the neurospheres treated with newt (toxic control ). On the other hand, a metabolic test was also carried out with alamar blue, which has shown that the formulations are not toxic at that concentration, data consistent with those obtained in the study of the evolution of the growth of neurospheres.
    [0202] Example 4. In vitro transfection studies
    [0204] For the transfection assay, the glioblastoma cell line U87MG was used, 56,000 cells / well were seeded in a 24 multi-well plate in DMEM medium supplemented with 10% FBS and 1% P / S. After 24 h, nanocomplexes were added at a concentration of 0.5 pg plasmid / cm2 in OptiMEM medium (Gibco, USA) and incubated for 4 h. They were then washed and replaced with fresh DMEM medium and the cells were incubated for 48 h. To measure transfection, the commercial Luciferase Reporter Gene Assay kit (Roche, Germany) was used. The cells were washed twice with PBS and 100 µl of lysis buffer was added, after 5 minutes the lysate was centrifuged. 50 µl of the supernatant was placed on a white plate and, using an automatic injector, 25 µl of luciferin from the commercial kit was added immediately before each luminescence measurement on a Mithras LB940 luminometer (Berthold, Germany). The results were corrected for protein by the Bio-Rad Protein Assay (BioRad, USA), 40 µl of the reagent was added to the sample and the absorbance was measured at 595 nm.
    [0205] In figure 3 (the results are expressed as percentage of lipofectamine luminescence) we can see that 48 hours after treatment, the prototypes containing the anionic polymer have shown a marked increase in transfection compared to the cationic prototypes and the free plasmid. . The luminescence obtained after transfection with PPZ-Aliphatic combined with PPZ-Anionic was superior even compared to the commercial transfectant, lipofectamine.
    [0207] Example 5. Acute toxicity studies in zebra fish embryos (TEF)
    [0209] For this test, the OECD regulation has been followed, freshly fertilized zebrafish eggs were selected around 2-3 hours after fertilization, specifically they are selected in the blastomer stages of 16-32 cells. Ten viable fertilized eggs were placed in osmosis water per group, one per well. The embryos were incubated with different concentrations of the polymers that form the nanocomplexes at 26 ± 1 ° C for 96 h and toxicity was observed every 24 hours until the end of the test. Observations made to determine toxicity include detection of embryo clotting, lack of somite formation, detachment of the tail, lack of heartbeat (after 48 h), and edema in the embryo.
    [0211] The formulations were diluted in reverse osmosis water at different concentrations, to compare the toxicity of the nanocomplexes. The embryos were incubated for 96 hours and their evolution was studied every 24 hours until the end of the study. The mortality of the negative control was less than 10% and the hatching rate was greater than 80%. To express the toxicity results, the parameters listed in the following table were calculated; Lethal concentration necessary to kill 50% of the population (LC50); No Observed Effect Concentration (NOEC); Minimum observed effect concentration (LOEC).
    [0213] In all cases, the LC50 is higher in the prototypes that contain the anionic polymer, and the results also coincide with the trend found in vitro. The results showed that the aliphatic polymer is the least toxic, but it must be considered that for the same plasmid loading, the concentration of the aliphatic polymer is almost twice that of PPZ-Aminal or PPZAmin2, due to the number of amine groups per monomer.
    [0218] Table 5. In vivo toxicity determined by zebra fish embryo acute toxicity test (FET). The results are expressed in mg of cationic polymer / mL of osmosis water. LC50: lethal concentration necessary to kill 50% of the population; NOEC: No Observed Effect Concentration; LOEC: lowest observed effect concentration.
    [0220] Example 6. Preparation of nanocomplexes with plasmid encoding BMP-4
    [0222] The nanocomplexes were prepared by ionic complexation using the cationic PPZ-Aliphatic polymer associated with PPZ-Anionic, in this case the nanocomplexes were prepared for a final concentration of encapsulated plasmid of 83.3 ^ g of plasmid / mL, which corresponds to a concentration theoretical nanocomplexes of 0.967 mg / mL. In this case, the plasmid encoding bone morphogenic protein 4 (BMP4) was used as nucleic acid and nanocomplexes were used as target nanocomplexes associating a non-therapeutic plasmid, pEGFP-Luc used in the optimization of the formulation.
    [0224] The size of the nanocomplexes was determined by dynamic light scattering (DLS) using a Nanosizer ZS instrument (Malvern, UK) after 1:10 dilution in 1 mM KCl. Each analysis was performed in triplicate at 25 ° C, with a backscatter angle of 173 °.
    [0227] The nucleic acid complexation efficiency was evaluated using the same technique described in example 2, and no changes were observed in the association / dissociation of the nucleic acid, which shows that the particles can be prepared more concentrated without affecting their physicochemical properties.
    [0229] Example 7. Clonogenicity test
    [0231] Two human glioblastoma cell lines (U87MG and U251) have been used in this experiment. Both cell lines were grown in supplemented EMEM medium, 105 cells / well were seeded in a 12-well plate. After 24 hours, the treatment was added, the cells were treated with seven different treatments: Medium, BMP4 protein, white nanocomplexes (NCs-white), nanocomplexes with BMP-4 (NCs-BMP4) prepared in example 6, Temozolamine ( Tz), BMP4-Tz protein combination and NCs-BMP4 Tz combination. The concentration was 23.2 pg of nanocomplexes / ml, 30 ng of BMP4 protein / ml and 2.4 pg of temozolamide / ml. Two days later, 500 cells / well were reseeded in a 6-well plate and grown for an additional 12 days.
    [0233] The cells were stained with a 50% ethanol, 5% acetic acid and 0.5% crystal violet preparation, washed with water twice, and the number of colonies per well was counted.
    [0235] In Figure 4, it is observed that in both cell lines the BMP4 nanocomplexes reduce the clonogenicity of tumor cells compared to the direct administration of the protein or the white nanocomplexes, in addition, their association with temozolomide generates a synergistic effect which is observed when only the chemotherapeutic agent is used.
    [0236] Example 8. In vivo efficacy in mouse glioblastoma xenograft model
    [0237] This experiment was approved by the animal care ethics committee of the Université Catholique de Louvain (2019 / UCL / MD004) and was carried out in accordance with the Belgian National Guidelines in accordance with the European Directive. The animals had free access to water and food at all times.
    [0239] Eight-week-old female NMRI nude mice (Janvier, France) were anesthetized intraperitoneally with 150 ^ L of a 10 mg / ml solution of ketamine (Pfizer, USA) and 1 mg / ml of xylazine (Sigma , USA) Two million fresh U87MG cells were administered subcutaneously in the flank. The tumor was grown ten days before the administration of the treatment.
    [0240] When the tumor size was around 35mm3, the mice, randomized into six groups, were anesthetized and the treatment was administered intratumorally except for temozolamide, which was administered intraperitoneally, four doses in four days in a row. Group distribution: Group 1: Control, saline (n = 7); Group 2: temozolomide (n = 6); Group 3: white nanocomplexes (n = 6); Group 4: BMP4 protein Tz (n = 6); Group 5: nanocomplexes-BMP4 prepared in example 6 (n = 7) and Group 6: nanocomplexes-BMP4 prepared in example 6 Tz (n = 7). The doses administered were 5pg of temozolomide / g mouse intraperitoneally, 1.54 pg of nanocomplexes of BMP4 / g mouse and 2 ng of BMP4 protein / g mouse intratumorally.
    [0242] After the administration of the different treatments, the tumor size and the body weight of the animals were measured every 2 days. Tumor size was measured by digital caliper and volume was calculated according to the following formula V = L x A x H, where L is the length, A is the width, and H is the height of the tumor. The relative volume of the tumor was calculated as V / Vo (Vo is the volume of the tumor before the first administration). Mice were considered dead and were euthanized when tumor volume was greater than 1500 mm3, appearance of necrosis or ulcers,> 20% weight loss, or presence of signs of distress.
    [0244] Given that there is always some heterogenicity in the size of the tumors, the mice were distributed keeping the groups comparable, even so the results were also corrected with respect to the initial tumor volume to reduce the variables due to tumor heterogenicity.
    [0246] Once the tumors were dissected, they were photographed and the expression of some RNAs implicated in cancer was analyzed. For RNA extraction, the tumors were homogenized in 1 ml of TRI-Reagent (ThermoFisher) using the GentleMACS Dissociator (Miltenyi Biotec, Germany), the homogenized tumor was mixed with 200 µl of chloroform / ml of TRI-Reagent and the samples were centrifuged, the aqueous phase was transferred to an eppendorf containing 500 µl of isopropanol / mL of TRI-Reagent and cooled to -20 ° C. The sample was centrifuged and the supernatant was decanted, the pellet was washed with ethanol, allowed to dry at room temperature and resuspended in water. RNA concentration was determined by NanoDrop 2000 (ThermoFisher). RNA samples were reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher). Real-time PCR (RT-PCR) was performed using the NZYSpeedy qPCR Green Master Mix kit (2x) (NZYTech, Portugal). Both PCRs were carried out using the Mastercycler Nexus (Eppendorf, Germany). GAPDH served as a gene to normalize gene expression. The primer sequences for the genes of interest are shown in the following table.
    [0248]
    [0251] It is observed that the combination of the nanocomplexes that encode for BMP4 in combination with temozolomide, slow down the evolution of the tumor significantly compared to the rest of the groups, we can also observe that the nanocomplexes-BMP4 by themselves did not reduce the tumor size of significantly compared to the control group (Figure 5a).
    [0253] When the results are corrected taking into account the initial volume of the tumor, figure 5b, the same trend is observed as in the previous graph.
    [0255] Regarding weight, no variations in weight due to toxicity were observed between the different treatments.
    [0257] Finally, an increase in survival was observed in the animals in the group combining BMP4-nanocomplexes with Tz compared to the control group and the group treated only with Tz (Figure 5.c). These results were also observed after dissection of the tumors at the end of the trial (Figure 6).
    [0259] Finally, the expression of some genes of interest in cancer was analyzed, in figure 7 we can see that after the administration of the therapeutic nanocomplexes, the expression of BMP4 is more than a thousand times higher compared to the rest of the groups. The Expression of two overexpressed genes in tumor stem cells (Sox2 and Nanog) in both cases treatment with therapeutic nanocomplexes decreased the expression, in addition, an additional effect on Nanog was observed when BMP4 nanocomplexes were associated with Tz. Finally, the expression of a gene involved in resistance to chemotherapeutic agents (MDR) and therefore causing chemotherapy to be less effective was analyzed. After treatment with temozolomide, this gene is overexpressed, but the association with nanocomplexes reduce their expression to basal levels.
    权利要求:
    Claims (18)
    [1]
    1. Polyphosphazene comprising:
    - at least one hydrocarbon chain (A) of between 6 and 24 links, where between 1 and 4 links are heteroatoms independently selected from O, N and S, and the end of the chain is a linear C 1 -C 6 alkyl group or branched, optionally the hydrocarbon chain has between 1 and 3 substituents independently selected from hydroxyl and thiol; Y
    - at least one hydrocarbon chain (B) of between 6 and 24 links, where between 1 and 4 links are heteroatoms independently selected from O, N and S, and the terminal group of the chain is a group of formula -NH 2 .
    [2]
    2. Polyphosphazene according to claim 1, wherein the hydrocarbon chains have between 6 and 16 links.
    [3]
    3. Polyphosphazene according to any of claims 1-2, wherein 2 or 3 links of the hydrocarbon chains are heteroatoms independently selected from N and S.
    [4]
    4. Polyphosphazene according to any of the preceding claims, wherein the hydrocarbon chain (A) has the following structure:

    [5]
    5. Polyphosphazene according to claim 1, wherein the hydrocarbon chain (A) is selected from the group consisting of:

    [6]
    6. Complex comprising a polyphosphazene, according to any of the preceding claims, and a biologically active molecule.
    [7]
    7. Complex according to claim 6, wherein the biologically active molecule is selected from protein and polynucleotide.
    [8]
    8. Complex according to claim 6, wherein the biologically active molecule is a plasmid encoding BMP-4.
    [9]
    A complex according to any of claims 6-8, further comprising an anionic polyphosphazene.
    [10]
    10. Complex according to claim 9, wherein the anionic polyphosphazene comprises a hydrocarbon chain of between 6 and 24 links, where between 1 and 4 links are heteroatoms independently selected from O, N and S, and at least one carboxylic group.
    [11]
    A complex according to any of claims 6-10, further comprising a chemotherapeutic molecule.
    [12]
    12. Complex according to claim 11, wherein the chemotherapeutic molecule is temozolamide.
    [13]
    13. Pharmaceutical composition comprising the complex according to any of claims 6-1, and pharmaceutically acceptable excipients.
    [14]
    14. Complex according to any of claims 6-12, or pharmaceutical composition according to claim 13, for use in medicine.
    [15]
    15. Complex according to any of claims 6-12, or pharmaceutical composition according to claim 13, for use in gene therapy or oncology.
    [16]
    16. Complex according to any of claims 6-12, or pharmaceutical composition according to claim 13, for use in the treatment of brain tumors, preferably glioblastomas.
    [17]
    17. Kit comprising at least two containers where one of the containers comprises a complex according to any of claims 6-12, and the other container comprises a chemotherapeutic molecule, together with instructions for its use in treating a disease for sequential administration or simultaneous of both ingredients.
    [18]
    18. Use of the complex according to any of claims 6-12, in the preparation of vectors for the transport of biologically active molecules.
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2017191345A1|2016-05-02|2017-11-09|Universidade De Santiago De Compostela|Biodegradable scaffold comprising messenger rna|
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