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    • 专利摘要:
    functionalized biocompatible nanoparticle and method for changing cellular functionality within a mammalian cell the present invention relates to disclosed functionalized biocompatible nanoparticles capable of penetrating through a mammalian cell membrane and administering various bioactive molecules intracellularly to modulate a cellular function. the functionalized biocompatible nanoparticles comprise: a central nanoparticle with size from about 5 to about 50 nm and with a polymeric coating on it, and several functional groups covalently linked to the polymeric coating, in which the various bioactive molecules are linked to the various functional groups , where the various bioactive molecules include at least one peptide and one protein, where the peptide is able to penetrate through a mammalian cell membrane and enter the cell, and where the protein is capable of providing new functionality within of the cell. the protein may be a transcription factor selected from the group consisting of oct4, sox2, nanog, lin28, cmyc and klf4.
    公开号:BR112014009753B1
    申请号:R112014009753-4
    申请日:2012-10-22
    公开日:2020-09-15
    发明作者:Andranik Andrew Aprikyan；Kilian Dill
    申请人:Stemgenics, Inc；
    IPC主号:
    专利说明:

    DESCRIPTIVE REPORT REMISSIVE REFERENCE TO RELATED ORDERS
    [0001] This Application claims the priority benefit of U.S. Provisional Application No. 61 / 550,213, filed on October 21, 2011, which is incorporated by reference into this document in its entirety for all purposes. TECHNICAL FIELD
    [0002] In general terms, the present invention refers to oiled assesse organic synthesis and nanobiotechnology and, more specifically, functionalized nanoparticles for the administration of bioactive molecules to cells for the modulation of cellular function, as well as methods related to them . BACKGROUND OF THE INVENTION. The ability of cells to proliferate, migrate and differentiate from various cell types is often critical to embryogenesis and the function of mature cells, including, but not limited to, cells in the hematopoietic and / or cardiovascular systems in a variety of hereditary or acquired. This functional capacity of stem cells and / or of more differentiated specialized cell types is altered in several pathological conditions, but can be normalized when intracellular introduction of biologically active components. For example, abnormal cell functions, such as impaired survival and / or impaired differentiation of stem cells / bone marrow progenitor cells to neutrophils, are seen in patients with severe cyclic or congenital neutropenia, who may experience severe fatal infections and develop leukemia acute myeloid or other malignancies [Aprikyan et al, “Impaired survival of bone marrow hematopoietic progenitor cells in cyclic neutropenia”, Blood, 97, 147 (2001); Goran Carlsson et al, “Kostmann syndrome: severe congenital neutropenia associated with defective expression of Bcl-2, constitutive mitochondrial release of cytochrome C, and excessive apoptosis of myeloid progenitor cells”, Blood, 103, 3.355 (2004)]. Hereditary or acquired disorders, such as severe congenital neutropenia or Barth's syndrome, are triggered by several genetic mutations and result from the patient's deficient blood and / or cardiac cell production and function, leading subsequently to neutropenia, cardiomyopathy and / or heart failure [Makaryan et al., “The cellular and molecular mechanisms for neutropenia in Barth syndrome. Eur J Haematol ”, 88: 195 to 209 (2012)]. The phenotype of severe congenital neutropenia can be caused by different mutations by substitution, deletion, introduction or truncation in the neutrophil elastase gene, in the HAX1 gene or in the Wiskott-Aldrich Syndrome Protein gene [Dale et al, “Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia ”, Blood, 96: 2,317 to 2,322 (2000); Devriendt et al, “Constitutively activating mutation in WASP causes X-linked severe congenital neutropenia”, Nat Genet, 27: 313 to 317 (2001); Klein et al, “HAX1 deficiency causes autosomal recessive severe congenital neutropenia (Kostmann diseasef, Nat Genet, 39: 86 to 92 (2007)].
    [0003] Other hereditary diseases, such as Barth syndrome, a multisystemic disorder of stem cells induced by mutations, presumably with loss of the functions of the mitochondrial TAZ gene, are associated with neutropenia (reduced levels of neutrophils in the blood), which can cause serious and sometimes fatal recurrent infections and / or cardiomyopathy, which can lead to heart failure, which could be resolved with heart transplantation. In most cases, mutant gene products, involved in the pathogenesis and development of inherited or acquired human diseases, cause distinct intracellular events that lead to abnormal cellular functions and the specific disease phenotype.
    [0004] Treatment of these patients with granulocyte colony stimulating factor (G-CSF) induces adaptive changes in the G-CSF receptor molecule located on the cell surface, which subsequently triggers a chain of intracellular events that ultimately , recover neutrophil production to a level close to normal and improve the patient's quality of life [Welte and Dale, “Pathophysiology and treatment of severe chronic neutropenia”, Ann. Hematol., 72, 158 (1996)]. Nevertheless, patients treated with G-CSF can develop leukemia [Aprikyan et al, “Cellular and molecular abnormalities in severe congenital neutropenia predisposing to leukemia”, Exp Hematol, 31, 372 (2003); Philip Rosenberg et al, “Neutrophil elastase mutations and risk of leukaemia in severe congenital neutropenia”, Br J Haematol, 140, 210 (2008); Peter Newburger et al, “Cyclic Neutropenia and Severe Congenital Neutropenia in Patients with a Shared ELANE Mutation and Paternal Haplotype: Evidence for Phenotype Determination by Modifying Genes”, Pediatr. Blood Cancer, 55, 314 (2010)], which is why innovative alternative approaches are being explored.
    [0005] Intracellular events can be influenced and regulated more efficiently when intracellular administration of different biologically active molecules using distinctly functionalized nanoparticles. These bioactive molecules can normalize cell function or eliminate unwanted cells when necessary. However, the cell membrane serves as an active barrier that prevents the cascade of intracellular events from being affected by exogenous stimuli.
    [0006] Therefore, there is a need in the art for new types of bioactive molecules capable of penetrating through cell membranes and carrying out the intracellular events of interest. The present invention satisfies these needs and brings even more related advantages. SUMMARY OF THE INVENTION
    [0007] The present invention, in some embodiments, relates to functionalization methods that consist of attaching proteins and / or peptides to biocompatible nanoparticles to modulate cellular functions. In some embodiments, the present invention relates to the functionalized biocompatible nanoparticles themselves.
    [0008] In one embodiment, a functionalized biocompatible nanoparticle capable of penetrating through a mammalian cell membrane and intracellularly administering several bioactive molecules to modulate a cell function comprises: a central nanoparticle with a size of 5 to 50 nm and with a polymeric coating on it, and several functional groups covalently linked to the polymeric coating, in which the various bioactive molecules are linked to the various functional groups, in which the various bioactive molecules include at least one peptide and one protein, in which the peptide is able to penetrate through of a mammalian cell membrane and enter the cell, and where the protein is capable of providing new functionality within the cell.
    [0009] The central nanoparticle can comprise iron and be magnetic. The peptides of the present invention can be linked to the protein (instead of linked to the nanoparticle). Each of the peptides and proteins can be linked to the nanoparticle by means of one or more interconnected binding molecules. The peptide can include five to nine basic amino acids in some embodiments, while in others, it includes nine or more basic amino acids. The protein can be a transcription factor such as, for example, a transcription factor selected from the group composed of Oct4, Sox2, Nanog, Lin28, cMyc and Klf4.
    [0010] In another aspect, the present invention relates to a method for changing cellular functionality within a mammalian cell. The innovative method comprises administering an effective amount of functionalized biocompatible nanoparticles to the cell and changing the cellular functionality within it. The change in cell functionality may involve a change in a cell's physical-chemical property, a change in the cell's proliferative property, a change in the cell's ability to survive or a change in the cell's morphological phenotypic property. Changing cellular functionality may involve an acquired cell's ability to produce a new cell type, including a stem cell or a more specialized cell type.
    [0011] These and other aspects of the present invention will appear better with reference to the detailed description and the accompanying drawings. It should be borne in mind, however, that various changes, changes and substitutions can be made to the specific embodiments disclosed in this document without diverging from its scope or essence. Finally, all the various references cited in this document are expressly incorporated into this document by reference. BRIEF DESCRIPTION OF THE DRAWINGS
    [0012] Figure 1 illustrates a scheme of functionalization of a nanoparticle in several stages based on the simultaneous binding of peptide and protein molecules to the nanoparticle according to an embodiment of the invention.
    [0013] Figure 2A illustrates a reaction of a nanoparticle containing amino groups with equimolar ratios of long chain LC1-SPDP and iodoacetic acid in the nanoparticle according to an embodiment of the present invention.
    [0014] Figure 2B illustrates a reduction in the disulfide bond of the PDP to produce a nanoparticle without SH group according to an embodiment of the present invention.
    [0015] Figure 2C illustrates a long-chain LC1-SMCC reaction with the lysine groups of a protein nanoparticle according to an embodiment of the present invention.
    [0016] Figure 2D illustrates a reaction of a multifunctional nanoparticle with the protein reacted with SMCC and contains a reactive maleimide terminal group nanoparticle in accordance with an embodiment of the present invention.
    [0017] Figure 2E illustrates a reaction of an amino group of a peptide with LC2-SMCC. The reaction is then followed by a reaction with mercaptoethanol to convert the terminal maleimide to an alcohol nanoparticle according to an embodiment of the present invention.
    [0018] Figure 2F illustrates a reaction of a functional bead (and linked protein) with a modified peptide to the carboxyl-free group on the nanoparticle according to an embodiment of the present invention.
    [0019] Figure 3A illustrates a reaction of a nanoparticle containing amine groups with a LC1-SPDP nanoparticle in accordance with an embodiment of the present invention.
    [0020] Figure 3B illustrates a reduction in the disulfide bond of the PDP to produce a nanoparticle without SH group according to an embodiment of the present invention.
    [0021] Figure 3C illustrates a long chain LC2-SMCC reaction with the lysine groups of a protein nanoparticle according to an embodiment of the present invention.
    [0022] Figure 3D illustrates a reaction of a multifunctional nanoparticle with the protein reacted with SMCC and contains a reactive terminal maleimide group nanoparticle in accordance with an embodiment of the present invention.
    [0023] These and other aspects of the present invention will appear better to those skilled in the art in the light of the detailed description below along with the accompanying drawings. DETAILED DESCRIPTION OF THE INVENTION
    [0024] In order to administer biologically active molecules intracellularly, the inventors of the present invention present a universal device based on nanoparticles penetrable in the cell membrane with biologically active molecules covalently attached to them. To this end, the inventors present, in this document, an innovative functionalization method that guarantees the covalent bonding of proteins and peptides to nanoparticles. The modified nanoparticles penetrable in the cell of the present invention provide a universal mechanism for the intracellular administration of biologically active molecules for the regulation and / or normalization of cellular function.
    [0025] The ability of cells to proliferate, migrate and differentiate from various types of cells is usually fundamental to embryogenesis and the function of mature cells, including, among others, stem cells / progenitor cells and the most differentiated cells from hematopoietic and / or cardiovascular systems in a variety of inherited or acquired diseases. This functional capacity of stem cells and / or of more differentiated specialized cell types is altered in various pathological conditions due to aberrant changes in intracellular events, but can be normalized when intracellular introduction of biologically active components. For example, impaired survival and impaired differentiation of human bone marrow progenitor cells into neutrophils seen in patients with cyclic or severe congenital neutropenia, who suffer from serious fatal infections and can develop leukemia, can be normalized by a small elastase inhibitory molecule. neutrophil penetrable in the cell membrane, which interferes with aberrant intracellular events and, apparently, restores the normal phenotype. Nevertheless, these small molecules specific for target mutant products causing the disorder are rarely available, which is why alternative efficient cell membrane penetration devices are needed for the cellular administration of biologically active molecules capable of modulating cellular function.
    [0026] The methods disclosed in this document use biocompatible nanoparticles, including, for example, superparamagnetic iron oxide particles similar to those previously described in the scientific literature. This type of nanoparticles can be used in clinical structures for magnetic resonance imaging of cells in the bone marrow, lymph nodes, spleen and liver [see, for example, Shen et al, “Monocrystalline iron oxide nanocompounds (MION ); physicochemical properties ”, Magn. Reson. Med., 29, 599 (1993); Harisinghani et. al, “MR lymphangiography using ultrasmall superparamagnetic iron oxide in patients with primary abdominal and pelvic malignancies”, Am. J. Roentgenol, 172, 1,347 (1999)]. These magnetic iron oxide nanoparticles contain a core of about 5 nm coated with cross-linked dextran and about 45 nm of the total particle size. Most importantly, these nanoparticles containing permeable reticulated Tat-derived peptides on the cell membrane have been shown to efficiently incorporate hematopoietic cells and neural progenitors in quantities of up to 30 pg of superparamagnetic iron nanoparticles per cell [Lewin et al, “Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells ”, Nat. Biotechnol., 18, 410 (2000)]. In addition, the incorporation of nanoparticles does not affect the proliferative characteristics or the differentiation characteristics of CD34 + primitive progenitor cells derived from bone marrow or cell viability [Maite Lewin et al, Nat. Biotechnol. 18, 410 (2000)]. These nanoparticles can be used for in vivo monitoring of the labeled cells.
    [0027] The labeled cells maintain their ability to differentiate and can also be detected in tissue samples using magnetic resonance imaging. In this document, innovative devices based on nanoparticles are presented, which are now functionalized to transport peptides and proteins that can serve as excellent vehicles for the intracellular administration of biologically active molecules in cellular reprogramming solutions to affect intracellular events and modulate functions and cellular properties.
    [0028] General Description of Nanoparticle-Peptide / Protein Conjugates:
    [0029] Nanoparticles based on iron or other material with a biocompatible coating (for example, dextran polysaccharide) with X / Y functional groups are bridges of various lengths, which, in turn, are covalently linked to proteins and / or peptides (or other small molecules) via their X / Y functional groups.
    [0030] Functional groups that can be used for crosslinking include:
    [0031] -NH2 (e.g., lysine, a— NH2),
    [0032] -SH,
    [0033] -COOH,
    [0034] -NH-C (NH) (NH2),
    [0035] carbohydrate,
    [0036] -hydroxyl (OH),
    [0037] - Photochemical connection of an azido group on the bridge.
    [0038] Crosslinking reagents may include:
    [0039] SMCC [succinimidyl-4- (N-maleimido-methyl) -cyclohexane-1-carboxylate]. Sulfo-SMCC, the sulfosuccinimidyl derivative for amino and thiol crosslinking groups, is also available.
    [0040] LC-SMCC (long chain SMCC). Also Sulfo-LC-SMCC.
    [0041] SPDP [N-succinimidyl-3- (pipridyldithium) -propionate]. Also Sulfo-SPDP. Reacts with amines and produces thiol groups.
    [0042] LC-SPDP (long chain SPDP). Also Sulfo-LC-SPDP.
    [0043] EDC [l-ethyl-3- (3-dimethylaminopropyl) -carbodimide hydrochloride]. Reagent used to link the -COOH group to the —NH2 group.
    [0044] SM (PEG) n, where n = 1,2, 3, 4, ... 24 glycol units. Also the derivative Sulfo-SM (PEG) n.
    [0045] SPDP (PEG) n, where n = 1, 2, 3, 4, ... 12 units of glycol. Also the derivative Sulfo-SPDP (PEG) n.
    [0046] PEG molecule containing carboxyl and amine groups.
    [0047] PEG molecule containing carboxyl and sulfhydryl groups.
    [0048] Capping and blocking reagents include:
    [0049] Citraconic anhydride; NH specific
    [0050] Ethyl-maleimide; SH specific
    [0051] Mercaptoethanol; specific for maleimide
    [0052] In light of the above, we treat biocompatible nanoparticles to produce functional amines on the surface, which, in turn, were used to chemically bind short proteins and peptides.
    [0053] In the case of binding proteins, for example, Fluorescent Green Protein or a transcription factor, to superparamagnetic or alternative nanoparticles, it is possible to adopt the following protocol: Superparamagnetic beads containing amino functional groups abroad can be purchased commercially from several manufacturers. Their size ranges from 20 to 50 nm, and they have 1015 to 1020 nanoparticles per ml with 10 or more amine groups per nanoparticle. The nanoparticles are placed in the correct reaction buffer (0.1 M phosphate buffer, pH 7.2) using an Amicon centrifugal filter unit (microcolumn) with a 10,000 dalton molecular cut. Generally, about 4 washes are required to ensure an adequate buffering system. The nanoparticles are removed from the filter unit according to the manufacturer's recommendations (inversion of the column / filter device by turning it at low speed).
    [0054] SMCC (from Thermo Fisher) is dissolved in dimethylformamide (DMF) obtained from ACROS (sealed and anhydrous ampoule) at a concentration of 1 mg / ml. The sample is sealed and used almost immediately.
    [0055] Ten (10) microliters of the solution are added to nanoparticles in a volume of 200 microliters. This provides a large excess of SMCC to the available amine groups present, and then the reaction is allowed to proceed for an hour. Excessive SM and DMF can be removed using an Amicon centrifugal filter column with a cut of 3000 daltons. Generally, five volume changes are required to ensure proper buffer change. It is important to remove excess SMCC at this stage.
    [0056] Any protein-based molecule, for example, Fluorescent Green Protein (GFP) available for sale, a purified recombinant GFP or other proteins, is added to the solution containing a certain amount of ethylene glycol to freeze at -30 ° C For 3 micrograms of the protein in 14 microliters, 10 microliters of a freshly prepared solution of DTT (dithiothreitol, Cleland's reagent) in PBS with vigorous vortex is added. Since proteins normally contain more than one cysteine, there is a tendency to cross-link different GFP molecules. Therefore, excessive DTT decreases the binding of dithiol and releases GFP. The reaction is allowed to continue for two hours at 4 ° C and then the excess reagent is removed using an Amicon centrifugal filter unit with a cut-off of 3,000 daltons molecular weight.
    [0057] The activated nanoparticles and protein solutions are combined and then allowed to react for two hours, after which the unreacted protein is removed by an Amicon centrifugal filter unit with a molecular weight cut appropriate (in the example with GFP, the cut is 50,000 daltons). The sample is stored at -80 ° C. Instead of using Amicon rotary filter columns, it is also possible to use small rotating columns containing solid size filter components, such as Bio Rad P columns. These are size exclusion columns. It should be borne in mind that SMCC can also be purchased as a sulfo-derivative (Sulfo-SMCC), making it more soluble in water. DMSO can also be replaced by DMF as a solvent vehicle for the labeling reagent; again, it must be anhydrous.
    [0058] All other crosslinking reagents can be applied in a similar way. SPDP is also applied to the applicable protein / peptide in the same way as SMCC. It is readily soluble in DME. Dithiol is cut by a reaction with DTT for an hour or more. After removing by-products and unreacted material, it is purified using an Amicon centrifugal filter column with a 3,000-dalton molecular weight cut.
    [0059] Another more direct and controlled way of labeling a nanoparticle with a peptide and protein would be to use two different bifunctional coupling reagents. The reaction sequence is somewhat similar to that in Figure 1. Iodoacetic acid is used to introduce a select number of “carboxyl” groups to the surface of the nanoparticle.
    [0060] The peptide containing LC-SMCC is treated with aminomercaptoethanol. This generates a bond through the sulfhydryl group and produces a free amino group. This amino group is then coupled to the carboxyl group on the nanoparticle using EDC. EDC is known as 1-ethyl-3 [3-dimethylaminopropyl] carbodimide hydrochloride. This coupling step is performed last in the reaction scheme.
    [0061] Figure 1 illustrates the general description of magnetic nanoparticles, protein / peptide adducts. A magnetic nanoparticle is coated with a polysaccharide and then functionalized. It can be acquired with amines on the surface. It can also be changed / transformed into any other functional formats. The extender / connector physically connects the two units to each other.
    [0062] Various functional groups can be used to chemically link the nanoparticle to the protein via cross-linking reactions. The variety of functional groups available allows several proteins / peptides to be attached to the nanoparticle, one at a time.
    [0063] Likewise, various cross-linking reagents or reactive catalysts can be used to cross-link nanoparticles to proteins / peptides by means of hetero-bifunctional reagents. It is also worth noting that these crosslinking reagents come in various lengths. For example, many contain the LC notation, which refers to extenders or "long chains". Pegylated compound is also available in various lengths. In this way, bridges of various lengths can be added to the nanoparticles and provide different binding lengths for larger molecules, such as proteins, and smaller molecules, such as peptides.
    [0064] Generally, different proteins can contain the same functional groups, which makes it difficult to mark the nanoparticle with the various proteins. There are reagents that allow you to change the functional groups; therefore, we can change the functional groups of the proteins, thus enabling us to selectively in a gradual manner without interference from other proteins. This requires changing the functional groups of the proteins.
    [0065] Various reagents can be used to alter proteins so that different chemicals can be used to link proteins to similar functional groups. For example, a compound, such as SPDP, can be used to convert an amine to sulfhydryl, which is then receptive to reaction with a maleimide terminal group.
    [0066] By attaching proteins to the bead (nanoparticle) gradually, generally residual and active groups of previously bound proteins can interfere with coupling chemicals. For this reason, permanent or reversible capping reagents can be used to block these terminal groups active against interference from reagents about to be used to attach a second or third protein to the nanoparticle.
    [0067] Several different capping compounds can be used to block unreacted end groups. Capping compounds must be used judiciously as they can also interfere with protein activity. They are used most often when a second chemical bonding step is needed and this functional group can interfere.
    [0068] To demonstrate that proteins can be attached to beads (nanoparticles) using the chemicals mentioned above, we performed the synthesis of magnetic nanopathicles, which contained Fluorescent Green Protein derived from jellyfish. The LCC-SMCC was used in this synthesis scheme.
    [0069] The N-hydroxysuccinimide is reacted chemically with the free amine groups on the nanoparticle to form a chemical bond. This produces a maleimide terminal group that can react with GFP. It is known that GPF has two cysteines and that cysteines from various GFP molecules can react to form disulfide bonds. To remove this interference, the molecule is first reduced with Cleland's reagent.
    [0070] The protein is purified and then allowed to react with beads containing the LC-maleimide group. The reaction is allowed to continue for 1 hour and then the reaction is purified on the Amicon rotary filter (cut 50,000 daltons). Images were taken with a fluorescence electron microscope.
    [0071] Various types of functional groups can be created on a nanoparticle. This allows the addition of three or more different proteins that will be linked.
    [0072] A first one starts with an amine on the surface.
    [0073] Traut's reagent can be used to convert some of these amines to sulfhydryl. In addition, iodoacetic acid can be used to convert some amines to carboxylic acid.
    [0074] In the case of both proteins and peptides, amines are converted into functional groups with different bridge lengths, as described in more detail below. This will serve as a generic group to link proteins and peptides.
    [0075] Figure 1 illustrates the schematic representation of the functionalization of a nanoparticle and the binding of peptides and proteins to that nanoparticle.
    [0076] The syntheses and coating are carried out as follows: NHS-LC-SPDP, available for sale by Thermo Fisher, is a long chain extender with bifunctional coupling reagents on both sides, which are specific for amines , and a disulfide, which can be converted to sulfide.
    [0077] One end has N-hydroxysuccinimide ester, while the other end of the extender contains a pyridyldithiol group. This dithiol group can be reduced to produce a sulfhydryl. The NHS-LC-SPDP is allowed to react with the nanoparticles, and the reaction of the unincorporated NHS-LC-SPD can be cleaned. The coupled nanoparticles are then reduced, as shown in Figure 1.
    [0078] Production of Coupled Proteins: Biologically active proteins purified using affinity columns contain a free epsilon-amine group resulting from the terminal carboxy-lysine residue added to facilitate binding with nanoparticles. NHS-LC-SMCC is used as a bifunctional coupling reagent. The molecule has an LC1 chain extender. One end has the amine-specific N-hydroxysuccinimide reagent. The other end contains the maleimide group, very specific for sulfhydryl groups. After the material is coupled to a protein and separated from the reaction mixture, the protein coupled to the maleimide will be added to the nanoparticles containing sulfhydryl. The resulting material is separated by gel filtration.
    [0079] Peptide Coupling to Nanoparticles: In this case, the peptide also contains a terminal carboxy-lysine that will serve as the basis for the NHS-LC-maleimide ester coupling. The molecule has an LC2 chain extender. All procedures are similar to those described above for the protein.
    [0080] During optimization, the permeable peptide and proteins in the membrane are mixed for different reasons to obtain the maximum number of molecules coupled to the nanoparticle. Based on previously published studies, 3 to 4 cell-penetrable peptide molecules attached to the surface by nanoparticles are sufficient for efficient intracellular administration of superparamagnetic nanoparticles.
    [0081] The use of the LC2-extensor arm provides an important means to increase the number of linked peptide-based molecules. The use of different concentrations of NHS-LC-SPDP allows a greater number of peptide and protein molecules anchored to the surface of nanoparticles and, therefore, more efficient penetration and, therefore, a more robust cellular reprogramming activity.
    [0082] Binding of Peptides and Proteins to a Nanoparticle: This can be accomplished by adopting the procedure illustrated in Figure 1. In this case, SMCC-labeled protein and peptide ratios are added to the beads and allowed to react.
    [0083] Another more direct and controlled way of labeling a nanoparticle with a peptide and a protein would be to use two different bifunctional coupling reagents (Figures 2A to 2F). The reaction sequence is somewhat similar to that in Figure 1, with some modifications described below.
    [0084] Iodoacetic acid is used to introduce a select number of "carboxyl" groups to the surface of the nanoparticles. This is done in step I, see Figures 2A to 2F, steps I to VII.
    [0085] The peptide containing NH-LC-SMCC is treated with aminoethanol. This generates a bond through the sulfhydryl group and produces a free amino group. This amino group is then linked to the carboxyl group on the nanoparticle using EDAC (EDC). EDAC is known as 1-ethyl-3 (3-dimethylaminopropyl) -carbodiimide hydrochloride. This coupling step is performed last in the reaction scheme.
    [0086] In another aspect, the present invention also relates to a method for administering bioactive molecules bound to functionalized nanoparticles for the modulation of intracellular activity. For example, human cells, fibroblasts or other types of cells available for sale or obtained using standard or modified experimental procedures are first laminated under sterile conditions on a solid surface with or without a substrate for the cells to adhere to (cells that feed , gelatin, martigel, fibronectin, etc.). Laminated cells are cultured for a period of time with a specific combination of factors that allows cell division / proliferation or the maintenance of acceptable cell viability. Examples include serum and / or various growth factors, which can later be removed or renewed and cultures continued. Laminated cells are cultured in the presence of permeable functionalized biocompatible nanoparticles in the cell with bioactive molecules attached to them using the various methods described in this document in the presence or absence of a magnetic field. The use of a magnet, in the case of superparamagnetic particles, produces an important increase in the area of the contact surface between cells and nanoparticles and, therefore, further reinforces the better penetration of functionalized nanoparticles through the cell membrane. When necessary, the cell population is treated repeatedly with the functionalized nanoparticles to administer the bioactive molecules intracellularly.
    [0087] The cells are suspended in a culture medium and the unincorporated nanoparticles are removed by centrifugation or cell separation, leaving the cells present in the form of clusters. The clustered cells are then resuspended and cultured in a fresh medium for an appropriate period of time. The cells can be subjected to several cycles of separation, resuspension and recultivation until the observation of a consequent biological effect triggered by the specific bioactive molecules administered intracellularly.
    [0088] One of the uses of the invention is to screen a compound (or compounds) for an effect on cell reprogramming. This involves combining the compound attached to the nanoparticle using one or more of the methods disclosed in this document to a cell population of interest, cultivating for an appropriate period of time, and then determining any modular effect resulting from the one or more compounds. This may include initiation of cell reprogramming and generation of pluripotent stem cells, cell differentiation or transdifferentiation into more specialized or different cell types, examination of cells for toxicity, metabolic changes or an effect on contractile activity, in addition to other functions.
    [0089] Another use of the invention consists in the formulation of specialized cells as a medicine or an administration device intended for the treatment of the body of a human or animal. This allows the doctor to administer the cells to the damaged tissue (heart, muscle, liver, etc.) through or around the vasculature or directly into the muscle or organ wall, thereby allowing specialized cells to graft, limit damage and participate in the remodeling of the tissue musculature and the restoration of specialized function.
    [0090] One of the uses of the present invention involves nanoparticles functionalized with other proteins, such as the transcription factors Oct4 and Sox2, in order to guarantee cell reprogramming and the generation of stem cells or cell types more differentiated with the preservation of the genome intact.
    [0091] Another use of the present invention is to screen a compound (or compounds) for an effect on cell reprogramming. This involves combining the nanoparticle-bound compound using the methods disclosed in this document to a cell population of interest, cultivating for an appropriate period of time, and then determining any modular effects resulting from the one or more compounds. This may include initiation of cell reprogramming and generation of pluripotent stem cells, cell differentiation or transdifferentiation into more specialized or different cell types, examination of cells for toxicity, metabolic changes or an effect on contractile activity, in addition to other functions.
    [0092] Yet another use of the present invention is the formulation of specialized cells as a medicine or in an administration device intended for the treatment of the body of a human or animal. This allows the doctor to administer the cells to the damaged tissue (heart, muscle, liver, etc.) through or around the vasculature or directly into the muscle or organ wall, thereby allowing specialized cells to graft, limit damage and participate in the reformulation of the tissue musculature and the restoration of specialized function.
    [0093] As a way of better illustrating the present invention, without however restricting it, the Examples below reveal other aspects of it.
    [0094] EXAMPLES
    [0095] Example 1
    [0096] GFP was attached to the superparamagnetic particle using LC-SMM as a crosslinker (linked to the amine groups of the beads), which was then coupled directly to the sulfhydryl groups in the GFP. SMCC (from Thermo Fisher) was dissolved in dimethylformamide (DMF) obtained from ACROS (sealed and anhydrous ampoule) at a concentration of 1 mg / ml. The sample was sealed and was used almost immediately.
    [0097] Ten (10) microliters of the solution were added to nanoparticles in a volume of 200 microliters. This provided a large excess of SMCC to the available amine groups present, and then the reaction was allowed to proceed for an hour. Excessive SMCC and DMF were removed using an Amicon rotary filter column with a cut of 3,000 daltons. Five volume changes were necessary to ensure an adequate buffer change. It was important to remove excess SMCC at this stage.
    [0098] Any protein-based molecule, for example, Fluorescent Green Protein (GFP) available for sale, a purified recombinant GFP or other proteins, was added to the solution containing a certain amount of ethylene glycol to freeze at -30 ° C For 3 micrograms of the protein in 14 microliters, 10 microliters of a freshly prepared solution of DTT (dithiothreitol, Cleland's reagent) in PBS with vigorous vortex was added. Since proteins normally contain more than one cysteine, there was a tendency to cross-link different GFP molecules. Therefore, excessive DTT decreased the dithiol binding and released GFP. The reaction was allowed to continue for two hours at 4 ° C and then the excess reagent was removed using an Amicon centrifugal filter unit with a molecular weight cut of 3,000 daltons.
    [0099] The activated nanoparticles and protein solutions were combined and then allowed to react for two hours, after which the unreacted protein was removed by an Amicon centrifugal filter unit with a molecular weight cut appropriate (in the example with GFP, the cut is 50,000 daltons). The sample was stored at -80 ° C. It should be kept in mind that a sulfo-derivative of SMCC (Sulfo-SMCC), which is more soluble in water, can be used. DMSO can also be replaced by DMF as a solvent vehicle for the labeling reagent; again, it must be anhydrous.
    [00100] Example 2
    [00101] In this method, the amino groups of lysine were used for the coupling reaction with sulfhydryl groups in the bead. Newly balanced beads with 0.1 M phosphate buffer with a pH of 7.2 were used in these studies. LC-SPDP at 1 mg / ml (in DMF) was freshly prepared. 10 microliters of SPDP solution was added to the bead suspension under vigorous vortexing, and then allowed to react for one hour. After that, the unreacted material was removed by centrifugation and the nanoparticles were washed with phosphate buffer using an Amicon rotary filter with a cut of 10,000 daltons. The disulfide binding of the SPDP was interrupted using Cleland's reagent; 1 mg was added to the solution and the reaction was allowed to proceed for one hour. By-products and unreacted Cleland's reagent were removed using an Amison rotary filter with a cutout of 10,000 daltons.
    [00102] While the above reaction continued, GFP was blocked using N-ethylmaleimide. Excessive ethylmaleimide was added to the GFP solution. The reaction proceeded for one hour at room temperature and unwanted materials were removed using an Amicon rotary filter with a cut of 3,000 daltons. The GFP was then allowed to react with the excessive SMCC for an hour. After that, the GFP was purified on a rotating column and then reacted with PDP-nanoparticles. The reaction proceeded for an hour and the final product was purified using an Amicon rotary filter with a cut of 50,000 daltons.
    [00103] Example 3
    [00104] Human fibroblasts available for sale or obtained using standard experimental procedures as described [Moretti et al, “Mouse and human induced pluripotent stem cells as a source for multipotent Isll cardiovascular progenitors”, FASEB J., 24: 700 (2010)] they are laminated at a density of 150,000 cells in sterile conditions on a solid surface with or without pre-laminated feeder cells at a density of 150,000 to 200,000 cells in six well plates. Feeder cells are obtained commercially or using standard laboratory procedures. Laminated cells are cultured for some time with a specific combination of factors that allows cell division / proliferation or the maintenance of acceptable cell viability in the culture medium containing serum, which can later be removed or renewed and the cultures continued under sterile conditions in an incubator moistened with 5% CO2 and ambient O2.
    [00105] Cells collected at the bottom of a conical tube or laminated cells are treated with 50 microliters of suspension containing permeable functionalized biocompatible nanoparticles in the cell with bioactive molecules attached to them using various methods disclosed in this document in the presence or absence of a magnetic field.
    [00106] The use of magnetic field, in the case of superparamagnetic particles, produces an important increase in the area of the contact surface between cells and nanoparticles and, thus, guarantees the best penetration of functionalized nanoparticles through the cell membrane. Importantly, similar to the protection mediated by polyethylene glycol (PEG) of various protein-based drugs (PEG-GCSF, Amgen, CA; PEG-Interferon, Schering-Plow / Merck, NJ) to which PEG is attached, nanoparticles used in conjunction with coupled peptides increase the size of the polypeptide and mask the surface of the protein, thereby decreasing protein degradation by proteolytic enzymes and resulting in greater stability of the protein molecules used. If necessary, a cell population is treated repeatedly with the functionalized nanoparticles to administer the bioactive molecules intracellularly.
    [00107] The cells are suspended in a culture medium and unincorporated nanoparticles are removed by centrifugation for 10 minutes at about 1,200 x g, leaving the cells present in the form of agglomerates in the granule. The agglomerated cells are then resuspended, washed again using a similar procedure and cultured in a fresh medium for an appropriate period of time. The cells can be subjected to several cycles of separation, resuspension and re-cultivation in a culture medium until the observation of a consequent biological effect activated by the specific bioactive molecules administered intracellularly.
    [00108] In this specific example with green fluorescent protein, the penetrable nanoparticles in the cell deliver proteins within the cells, which allows the acquisition of new green fluorescence by the target cells. This newly acquired property subsequently allows the classification and separation of cells with proteins administered intracellularly with a high degree of homogeneity that can be additionally used for various applications. Importantly, the use of permeable functionalized nanoparticles in the cell with proteins attached to them eliminates any integration into the cell genome, thus ensuring that any cell with new properties (in this case, fluorescence) keeps its genome intact and preserves the integrity of DNA cell phone.
    [00109] The present invention can be realized in other specific forms without deviating from its scope or its essential characteristics. The aforementioned embodiments should therefore be considered illustrative rather than limiting the invention described in this document. Therefore, the scope of the invention is defined by the appended Claims, instead of the previous description, and all modifications that fall within the meaning and equivalence limits of the Claims will be covered by it.
    权利要求:
    Claims (14)
    [0001]
    1. Functionalized Biocompatible Nanoparticle, to penetrate through a mammalian cell membrane, for the intracellular administration of a bioactive molecule to modulate a cellular function, said biocompatible nanoparticle being functionalized comprising: a central nanoparticle with one or more functional groups attached to it, a cell-penetrating peptide to penetrate through a membrane of said mammalian cell, characterized in that said cell-penetrating peptide is linked via a first linker molecule to a first functional group on said central nanoparticle, and a bioactive molecule to modulate a function of said mammalian cell, wherein said bioactive molecule is linked via a second binding molecule to a second functional group on said central nanoparticle; wherein said cell-penetrating peptide and said bioactive molecule are independently attached to said central nanoparticle by attaching said cell-penetrating peptide to said first binding molecule and attaching said bioactive molecule to said second binding molecule.
    [0002]
    2. Functionalized Biocompatible Nanoparticle, according to Claim 1, characterized in that said central nanoparticle has a size ranging from 5 to 50 nm.
    [0003]
    Functionalized Biocompatible Nanoparticle according to Claim 1, characterized in that said first functional group is the same as said second functional group.
    [0004]
    4. Functionalized Biocompatible Nanoparticle according to Claim 1, characterized in that said central nanoparticle further comprises polymeric coating.
    [0005]
    Functionalized Biocompatible Nanoparticle according to Claim 4, characterized in that one or both of said first functional group and said second functional group are attached to said polymeric coating on said central nanoparticle.
    [0006]
    6. Functionalized Biocompatible Nanoparticle according to Claim 1, characterized in that said central nanoparticle comprises iron.
    [0007]
    Functionalized Biocompatible Nanoparticle according to Claim 6, characterized in that said first binding molecule has a first length, wherein said second binding molecule has a second length and wherein said first length is greater than said second length.
    [0008]
    8. Functionalized Biocompatible Nanoparticle according to Claim 4, characterized in that said cell-penetrating peptide includes from five basic amino acids to nine basic amino acids.
    [0009]
    9. Functionalized Biocompatible Nanoparticle according to Claim 4, characterized in that said cell-penetrating peptide includes nine or more basic amino acids.
    [0010]
    10. Functionalized Biocompatible Nanoparticle, according to Claim 4, characterized in that said bioactive molecule is a transcription factor.
    [0011]
    11. Functionalized Biocompatible Nanoparticle, according to Claim 10, characterized in that said transcription factor is selected from the group consisting of Oct4, Sox2, Nanog, Lin28, cMyc and Klf4.
    [0012]
    12. Use of Functionalized Biocompatible Nanoparticles, characterized by the fact that it is for the manufacture of a drug to change a cellular functionality within a mammalian cell.
    [0013]
    13. Use of a Functionalized Biocompatible Nanoparticle, according to Claim 12, characterized in that the aforementioned change in said cellular functionality is selected from the group consisting of a change in a physical-chemical property, a proliferative property, the ability to survive, in the morphological phenotypic property and in the differentiation state of said cell.
    [0014]
    14. Use of a Functionalized Biocompatible Nanoparticle, according to Claim 13, characterized in that the change in cellular functionality involves an acquired ability of the cell to produce a new cell type, including a stem cell or a more differentiated cell type.
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    同族专利:
    公开号 | 公开日
    AU2020223737A1|2020-09-17|
    MX2014004778A|2014-10-17|
    EP2769217A4|2015-06-03|
    EP3400956A1|2018-11-14|
    RU2018135567A|2018-11-15|
    WO2013059831A1|2013-04-25|
    HK1201089A1|2015-08-21|
    EP2769217A1|2014-08-27|
    CN104094119A|2014-10-08|
    JP2014532628A|2014-12-08|
    SG11201401658SA|2014-07-30|
    MX367656B|2019-08-29|
    JP2017165781A|2017-09-21|
    KR20150001711A|2015-01-06|
    JP2018184485A|2018-11-22|
    CN106822868A|2017-06-13|
    KR20200040924A|2020-04-20|
    JP6560302B2|2019-08-14|
    AU2018203848A1|2018-06-21|
    US9675708B2|2017-06-13|
    BR112014009753A2|2017-04-25|
    IN2014DN03224A|2015-05-22|
    KR20190077124A|2019-07-02|
    US20140342004A1|2014-11-20|
    CA2853128C|2016-09-27|
    CA2938661A1|2013-04-25|
    AU2012325723A1|2014-05-15|
    RU2014120465A|2015-11-27|
    SG10201601746TA|2016-04-28|
    CA2853128A1|2013-04-25|
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    法律状态:
    2017-05-02| B15I| Others concerning applications: loss of priority|
    2017-07-11| B12F| Other appeals [chapter 12.6 patent gazette]|
    2019-03-12| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-11-05| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2020-04-07| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2020-09-15| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 22/10/2012, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
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    US201161550213P| true| 2011-10-21|2011-10-21|
    US61/550,213|2011-10-21|
    PCT/US2012/061391|WO2013059831A1|2011-10-21|2012-10-22|Functionalized nanoparticles for intracellular delivery of biologically active molecules|
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