HK1236412A1 - Functionalized nanoparticles for intracellular delivery of biologically active molecules - Google Patents

Abstract

Functionalized biocompatible nanoparticles capable of penetrating through a mammalian cell membrane and delivering intracellularly a plurality of bioactive molecules for modulating a cellular function are disclosed herein The functionalized biocompatible nanoparticles comprise: a central nanoparticle ranging in size from about 5 to about 50 nm and having a polymer coating thereon, a plurality of functional groups covalently attached to the polymer coating, wherein the plurality of bioactive molecules are attached to the plurality of the functional groups, and wherein the plurality of bioactive molecules include at least a peptide and a protein, and wherein the peptide is capable of penetrating through the mammalian cell membrane and entering into the cell, and wherein the protein is capable of providing a new functionality within the cell. The protein may be a transcription factor selected from the group consisting of Oct4, Sox2, Nanog, Lin28, cMyc, and Klf4.

Description

Functionalized nanoparticles for intracellular delivery of biologically active molecules

The application is a divisional application of Chinese application with the application number of 201280063870.2, the international application date of 2012, 10 and 22 and the invention name of 'functionalized nano-particles for intracellular delivery of bioactive molecules'.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application having application number 61/550,213, filed 2011, 10/21, which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates generally to organic synthesis and nanobiotechnology, and more particularly, to functionalized nanoparticles for delivering bioactive molecules into cells to modulate cellular function and methods related thereto.

Background

The ability of cells to normally proliferate, migrate and differentiate into various cell types is critical to embryogenesis and the function of mature cells, including but not limited to hematopoietic cells and/or cells of the cardiovascular system in various genetic or acquired diseases. The functional capacity of stem cells and/or further differentiated specialized cell types varies in various pathological conditions but can be normalized by intracellular introduction of bioactive components. For example, abnormal cell function, such as Impaired survival and/or differentiation of bone marrow stem/progenitor cells into neutrophils, is observed in patients with periodic neutropenia or severe congenital neutropenia, who may have severe life-threatening infections and may develop acute myelogenous leukemia or other malignancies [ Aprikyan et al, infected Survival of bovine myelogenous inflammatory prognosticator cells in cyclic neutropenia ], Blood, 97, 147 (2001); GoranCarlsson et al, Kostmann syndrome: severer genetic neutral associated with defective expression of Bcl-2, constitutive mitogenic release of cytochrome C, and anaerobic apoptosis of myelogenous spongioblasts (Costmann syndrome: severe congenital neutropenia associated with defective expression of Bcl-2 protein, constitutive mitochondrial release of cytochrome C, and excessive apoptosis of myeloid progenitor cells), Blood, 103, 3355(2004) ]. Genetic or acquired diseases, such as severe congenital neutropenia or bart syndrome, are caused by various genetic mutations and subsequent neutropenia, myocardial infarction and/or heart failure due to insufficient manufacture and function of The patient's blood and/or cardiomyocytes [ Makaryan et al, The cellular and molecular mechanisms for neutropenia in bart syndrome ], Eur J Haematol, 88: 195-. The severe congenital neutropenia phenotype may be caused by various substitutions, deletions, insertions or truncation Mutations of the neutrophil elastase gene, HAX1 gene or the Wiskott-Aldrich syndrome protein gene [ Dale et al, Mutations in the gene encoding neutrophile elastase in genetic and cycloneutropenia in congenital and periodic neutropenia ], Blood, 96:2317-2322 (2000); devirindedt et al, Constitutent activating mutation WASP mice X-linked segment convergent neutropenia (constitutive activating mutation in WASP results in X-linked severe congenital neutropenia), Nat Genet.27:313-7 (2001); klein et al, HAXl specificity mice autoimmunal receiveable Severe genetic neutropenia (Kostmann disease) (HAXl deficiency causes severe congenital neutropenia with autosomal recessive inheritance (Costmann disease)), Nat Genet, 39:86-92 (2007)).

Other genetic diseases, such as barter's syndrome, which is a multi-system stem cell disease that may be caused by loss-of-function mutations in the mitochondrial TAZ gene, are associated with neutropenia (decreased blood neutrophil levels), may lead to recurrent severe and sometimes life-threatening fatal infections and/or myocardial infarction that may lead to heart failure that can be addressed by heart transplantation. In most cases, the mutant gene products associated with pathogenesis and development of inherited or acquired human disease affect different intracellular events (intracellular events) that result in abnormal cellular function and a particular disease phenotype.

Treatment of these patients with granulocyte colony stimulating factor (G-CSF) induces a conformational change in the G-CSF receptor molecule located on the cell surface, which in turn triggers a series of intracellular events that ultimately restore neutrophil production to near normal levels and improve the quality of life of the patient [ Welte and Dale, Pathophysiology and treatment of severe chronic neutropenia, ann. hematol.72,158(1996) ]. However, patients treated with G-CSF may evolve into leukemia [ Aprikyan et al, Cellular and molecular abortions in secondary genetic predisposition to leukemia (Cellular and molecular abnormalities in severe congenital neutropenia are liable to induce leukemia), Exp Hematol.31,372 (2003); philip Rosenberg et al, neutrophile elastas mutations and risk of leukaemia in severe genetic neutropenia (risk of Neutrophil elastase mutations and leukemia in severe congenital neutropenia), Br J Haematol.140,210 (2008); peter Newburger et al, Cyclic Neutropenia and Severe genetic Neutropenia in Patients with a Shared Elane Mutation and Paternal Haplotype: evaluation for Phosphotype depletion by modification Genes (consensus ELANE Mutation and Evidence of Severe congenital Neutropenia in Patients of parent Haplotype: Evidence of Phenotype Determination by modifier Genes), Peditar. blood Cancer,55,314(2010), which is the reason for exploring new alternative methods.

Different bioactive molecules can be delivered intracellularly by using distinct functionalized nanoparticles, thereby enabling more effective influencing and modulation of intracellular events. These bioactive molecules can normalize cellular function or eliminate unwanted cells as desired. However, the cell membrane acts as an effective barrier to protect the cascade of intracellular events from exogenous stimuli.

Thus, there is a need in the art for novel bioactive molecules that are capable of penetrating cell membranes and effecting intracellular events of interest. The present invention fulfills these needs and provides further related advantages.

Disclosure of Invention

In some embodiments, the present invention relates to a functionalization method for attaching proteins and/or peptides to biocompatible nanoparticles to modulate cellular function. In some embodiments, the present invention relates to the functionalized biocompatible nanoparticles themselves.

In one embodiment, a functionalized biocompatible nanoparticle capable of penetrating a mammalian cell membrane and delivering intracellularly a plurality of bioactive molecules for modulating cell function, comprising: a central nanoparticle having a size range of 5-50nm and having a polymer coating thereon; a plurality of functional groups covalently attached to the polymer coating, wherein the plurality of bioactive molecules are attached to the plurality of functional groups, and wherein the plurality of bioactive molecules comprise at least a peptide and a protein, and wherein the peptide is capable of penetrating a mammalian cell membrane and entering a cell, and wherein the protein is capable of providing a new function within the cell.

The central nanoparticle may comprise iron and be magnetic. The peptides of the invention may be linked to proteins (on the opposite side to the nanoparticles). The peptide and protein may each be attached to the nanoparticle by one or more intervening linker molecules. In some embodiments, the peptide may include 5-9 basic amino acids, while in other embodiments, the peptide includes more than 9 basic amino acids. The protein may be a transcription factor selected from the group consisting of Oct4, Sox2, Nanog, Lin28, cMyc, and Klf 4.

In another aspect, the invention relates to a method of altering cellular function within a mammalian cell. The novel method comprises administering an effective amount of functionalized biocompatible nanoparticles to a cell and altering cellular function within the cell. Alterations in cell function involve alterations in physicochemical properties of the cell, alterations in proliferative properties of the cell, alterations in viability of the cell, or alterations in morphological phenotypic properties of the cell. Changes in cell function are related to the ability of the cell to acquire new cell types, including stem cells or more specialized cell types.

The above and other aspects of the present invention will become more apparent by referring to the following detailed description and the accompanying drawings. However, it should be understood that various changes, alterations, and substitutions may be made to the embodiments disclosed in this specification without departing from the spirit and scope of the invention. Finally, various references cited in this specification are expressly incorporated herein by reference.

Drawings

FIG. 1 illustrates a multi-step functionalization scheme for nanoparticles based on simultaneous attachment of peptide and protein molecules to the nanoparticles according to embodiments of the present invention.

FIG. 2A shows the reaction of amine group-containing nanoparticles with equimolar ratios of long chain LC1-SPDP and iodoacetic acid nanoparticles according to an embodiment of the present invention.

FIG. 2B illustrates the reduction of disulfide bonds of a PDP to provide free SH group nanoparticles, according to an embodiment of the present invention.

Fig. 2C shows the reaction of long chain LC1-SMCC with lysine groups of protein nanoparticles according to an embodiment of the invention.

Figure 2D illustrates the reaction of multifunctional nanoparticles according to embodiments of the present invention with a protein that has been reacted with SMCC and that comprises terminal-reactive maleimide-based nanoparticles.

FIG. 2E shows the reaction of the amino group of the peptide with LC 2-SMCC. This reaction is followed by a reaction with mercaptoethanol to convert the terminal maleimide to alcohol nanoparticles according to embodiments of the present invention.

Figure 2F shows the reaction of functional beads (and attached proteins) with modified peptides at the free carboxyl terminus on nanoparticles according to embodiments of the present invention.

FIG. 3A shows the reaction of amine group-containing nanoparticles with LC1-SPDP nanoparticles according to an embodiment of the present invention.

FIG. 3B illustrates the reduction of disulfide bonds of a PDP to provide free SH group nanoparticles, according to an embodiment of the present invention.

Fig. 3C shows the reaction of long chain LC2-SMCC with lysine groups of protein nanoparticles according to an embodiment of the invention.

Figure 3D illustrates the reaction of multifunctional nanoparticles according to embodiments of the present invention with a protein that has been reacted with SMCC and that contains terminal-reactive maleimide-based nanoparticles.

The above and other aspects of the present invention will become apparent to those skilled in the art upon reference to the following detailed description when taken in conjunction with the accompanying drawings.

Detailed Description

For intracellular delivery of bioactive molecules, the inventors of the present invention propose a general approach based on cell membrane penetrating nanoparticles with covalently linked bioactive molecules. To this end, the inventors propose herein a new functionalization method which ensures covalent attachment of proteins and peptides to the nanoparticles. The modified cell permeable nanoparticles of the present invention provide a general mechanism for intracellular delivery of bioactive molecules for modulating cellular function and/or normalization of cellular function.

The ability of cells to normally proliferate, migrate and differentiate into various cell types is critical to embryonic development and the function of mature cells, including but not limited to hematopoietic cells and stem/progenitor cells of the cardiovascular system and further differentiated cells in various genetic or acquired diseases. This functional capacity of stem cells and/or further differentiated specialized cell types can be altered in a variety of pathological conditions resulting from abnormal changes in intracellular events, but can be normalized by intracellular introduction of bioactive components. For example, impaired survival and/or ability to differentiate into neutrophils of human bone marrow progenitor cells, normalized by the cell membrane-penetrating small molecule inhibitor of neutrophil elastase, are observed in patients with severe life-threatening infections and possibly developing leukemia with periodic neutropenia or severe congenital neutropenia, which interfere with abnormal intracellular events and apparently restore the normal phenotype. However, such small molecules are largely ineffective against disease-causing target mutation products, which is why alternative effective cell membrane penetration means are required for intracellular delivery of bioactive molecules capable of modulating cellular function.

The methods disclosed in the present specification utilize biocompatible nanoparticles, including, for example, superparamagnetic iron oxide particles similar to those described previously in the scientific literature. This type of nanoparticle can be used in a clinical setting for magnetic resonance imaging of bone marrow cells, lymph nodes, spleen and liver [ see, e.g., Shen et al, monocystalline iron oxiranecellular complexes (MIONs)); physiochemical properties, magn. Harisinghani et al, MR lymphagingraphying ultra small superparamagnetic iron oxide in tissues with primary and cathodic malignneces (MR lymphangiography with ultra small superparamagnetic iron oxide for patients with primary abdominal and pelvic malignancies), am.J.Roentgenol.172,1347(1999)]. These magnetic iron oxide nanoparticles comprise a cross-linked dextran coated core of around 5nm and have a total particle size of about 45 nm. Importantly, these cell membrane permeable Tat-derivatives comprising cross-linking have been demonstratedNanoparticles of peptides, in amounts up to 30pg per cell of superparamagnetic iron oxide nanoparticles, are efficiently internalized into hematopoietic and neural progenitors [ Lewis et al, Tat peptide-derived magnetic nanoparticles in vivo tracking and recovery of progenitors ], nat Biotechnol.18,410(2000)]. Furthermore, insertion of the nanoparticles did not affect bone marrow-derived CD34⁺Proliferation and differentiation characteristics of progenitor cells or cell viability [ MaiteLewin et al, nat. Biotechnol.18,410(2000)]. These nanoparticles can be used to track labeled cells in vivo.

The labeled cells retain their ability to differentiate and can also be detected in a tissue sample using magnetic resonance imaging. The inventors herein propose a novel nanoparticle-based approach that is functionalized to carry peptides and proteins that can serve as excellent carriers to deliver biologically active molecules intracellularly for cellular reprogramming solutions to target intracellular events and to modulate cellular function and properties.

Overview of nanoparticle-peptide/protein conjugates:

nanoparticles are based on iron or other materials with biocompatible coatings (e.g. dextran polysaccharides) having X/Y functional groups with linkers of different lengths attached, which in turn are covalently linked to proteins and/or peptides (or other small molecules) through their X/Y functional groups.

Functional groups useful for crosslinking include:

-NH₂(e.g., lysine, α -NH)₂)；

-SH，

-COOH，

-NH-C(NH)(NH₂)，

A carbohydrate in a mixture of at least one carbohydrate,

-a hydroxyl group (OH),

-linkage by photochemical reaction of the azide group on the linker.

The crosslinking reagent comprises:

SMCC [4- (N-maleimido-methyl) cyclohexane-1-carboxylic acid succinimidyl ester ] can also be sulfo-SMCC, the sulfosuccinimidyl derivative being used to crosslink amino and thiol groups.

LC-SMCC (long chain SMCC). Or sulfo-LC-SMCC.

SPDP [ N-succinimidyl-3- (pyridyldithio) -propionate ] may also be sulfo-SPDP. Reaction with an amine provides a sulfhydryl group.

LC-SPDP (Long chain SPDP). Or sulfo-LC-SPDP.

EDC [ 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride]For reacting-COOH groups with-NH₂A group-linking agent.

Sm (peg) n, where n ═ 1,2,3,4 … … 24 ethylene glycol mono-positions. Also sulfo-SM (PEG) n derivatives.

Spdp (peg) n, wherein n ═ 1,2,3,4 … … 12 ethylene glycol units. Also sulfo-SPDP (PEG) n derivatives are possible.

PEG molecules containing both carboxyl and amino groups.

PEG molecules containing carboxyl and thiol groups.

Blocking and blocking agents include:

citraconic anhydride-specific for NH

Ethylmaleimide-specificity for SH

Mercaptoethanol-specific for maleimide

In view of the above, the present inventors treated biocompatible nanoparticles to produce functional amines on the surface, which in turn were used to chemically bond proteins and short peptides.

In case a protein, such as green fluorescent protein or transcription factor, is attached to a superparamagnetic or alternatively a nanoparticle, the following method can be used: superparamagnetic beads comprising an amino function externally are commercially available from different manufacturers. They may range in size from 20 to 50nm, with 10 per ml¹⁵-10²⁰Each nanoparticle having more than 10 amine groups. The nanoparticles were placed into the appropriate reaction buffer (0.1M phosphate buffer, ph7.2) through Amicon centrifugal filtration units (micro-columns) using molecules with a molecular weight cutoff of 10,000 daltons. Typically requiring about four washes to ensure a proper buffer system. The nanoparticles were removed from the filter unit (via a low speed rotating flip column/filter unit) as recommended by the manufacturer.

SMCC (from Thermo Fisher) was dissolved in Dimethylformamide (DMF) from ACROS (sealed vial and anhydrous) at a concentration of 1 mg/ml. The samples were sealed and used almost immediately.

Mu.l of the solution was added to the nanoparticles to a volume of 200. mu.l. This provided a large excess of SMCC over the available amine groups present and allowed the reaction to proceed for 1 hour. Excess SM and DMF can be removed using Amicon centrifugal filtration columns with a molecular weight cutoff of 3000 daltons. Typically 5 volume changes are required to ensure proper buffer exchange. It is important that excess SMCC be removed at this stage.

Any peptidyl molecule, such as the commercially available Green Fluorescent Protein (GFP) or purified recombinant green fluorescent protein or other protein, is added to a solution containing a quantity of ethylene glycol and frozen at-30 ℃. A PBS solution containing 3. mu.g of protein in 14. mu.l, 10. mu.l of freshly prepared DTT (dithiothreitol, Cleland's reagent) was added with vigorous stirring. Since proteins usually contain more than one cysteine, it is prone to cross-link different GFP molecules. Thus, excess DTT reduces the disulfide bonds and releases green fluorescent protein. The reaction was allowed to proceed at 4 ℃ for 2 hours, and then excess reagent was removed by an Amicon centrifugal filtration unit with a molecular weight cut-off of 3000.

The activated nanoparticles and protein solution were mixed and allowed to react for 2 hours, after which unreacted protein was removed by an Amicon centrifugal filtration device with the appropriate molecular weight cut-off (50,000 daltons in this case with GFP). Samples were stored at-80 ℃. Instead of using Amicon spin filter columns, small spin columns containing fixed size filter modules, such as Bio Rad P columns, can also be used. They are all size exclusion columns. It should also be noted that SMCC is also commercially available as a sulfo derivative (sulfo-SMCC) to make it more soluble in water. DMSO may also be used as a solvent carrier for the labeling reagent in place of DMF, and it should be anhydrous.

All other crosslinking agents can be used in a similar manner. SPDP is also administered to the protein/appropriate peptide in the same manner as SMCC. It is readily soluble in DMF. The disulfide bond is cleaved by reacting with DTT for 1 hour or more than 1 hour. After removing the by-products and unreacted materials, they were purified by centrifugation using Amicon filtration column with a molecular weight cut-off of 3,000.

Other more directly controllable means of labeling nanoparticles with peptides and proteins is the use of two different bifunctional coupling agents. The reaction sequence is somewhat analogous to that of FIG. 1. Iodoacetic acid is used to introduce a selected number of "carboxyl" groups to the nanoparticle surface.

The LC-SMCC containing peptide was treated with amino mercaptoethanol. This enables bonding via a thiol group and provides a free amino group. This amino group is then coupled to the carboxyl group on the nanoparticle by using EDC. EDC is known as 1-ethyl-3 [ 3-dimethylaminopropyl ] carbodiimide hydrochloride. This coupling step is performed at the end of the reaction scheme.

Figure 1 shows a general depiction of a magnetic nanoparticle-protein/peptide adduct. The magnetic nanoparticles are coated with a polysaccharide and then functionalized. The surface of which may be substituted with amines. But may be changed/modified into any other functional form. The extender/linker physically links the two units together.

A wide variety of functional groups can be used to chemically attach the nanoparticles to the protein via a cross-linking reaction. Variations in available functional groups allow a large number of proteins/peptides to be attached to the nanoparticle, one step at a time.

Similarly, various crosslinking reagents or reaction catalysts can be used to crosslink the nanoparticles with the proteins/peptides by the heterobifunctional reagents. It should also be noted that these crosslinking agents have different lengths. For example, many contain LC symbols representing extenders or "long chains". The pegylated compounds may also be of different lengths. Thus linkers of different lengths may be added to the nanoparticle and provide different linker lengths for larger molecules (e.g. proteins) and small molecules (e.g. peptides).

In general, different proteins may contain the same functional groups, and thus it is difficult to label nanoparticles with various proteins. Since a reagent for changing the functional group is present, the functional group of the protein can be changed, and selectivity can be obtained in stages without being interfered by other proteins. This requires alteration of functional groups on the protein.

Various reagents can be used to alter the protein so that different chemistries can be used to attach the protein by the desired functional group. For example, a compound (such as SPDP) can be used to convert an amine to a thiol, which will then react with the maleimide moiety.

In stepwise attachment of proteins to beads (nanoparticles), residues and reactive groups of previously attached proteins may interfere with coupling chemistry. Thus, permanent or reversible capping agents may be used to block these active moieties from interference by the agents that will be used to attach the second or third protein to the nanoparticle.

A large number of different end-capping compounds can be used to block unreacted moieties. Because the end-capping compound may also interfere with protein activity, care is taken to use. Typically, this is used when a second chemical linking step is necessary and the functional group may interfere.

To show that proteins can be attached to beads (nanoparticles) using the above mentioned chemistry, the synthesis of magnetic nanoparticles is given, which contain green fluorescent protein derived from jellyfish. LCC-SMCC was used in this synthesis scheme.

The N-hydroxysuccinimide chemically reacts with free amine groups on the nanoparticles to form chemical bonds. Maleimide end groups are provided which are capable of reacting with GFP. GFP is known to have two cysteines, and cysteines from different GFP molecules react to form disulfide bonds. To eliminate this interference, the molecule is first reduced with Cleland's reagent.

The protein was purified and then reacted with beads containing LC-maleimide groups. The reaction was allowed to proceed for 1 hour and purified on an Amicon spin filter (50K molecular weight cut-off). The photographs were taken with a fluorescence electron microscope.

Various types of functional groups may be disposed on the nanoparticles. Allowing the linkage of three or more different proteins.

First with amines on the surface.

The Telot's reagent can be used to convert some of these amines to sulfhydryl groups. In addition thereto, iodoacetic acid may be used to convert part of the amine to carboxylic acid.

For both proteins and peptides, the amine is converted to functional groups with different linker lengths as described in detail below. It will serve as a universal group to link proteins and peptides.

FIG. 1 shows a schematic representation of nanoparticle functionalization, attachment of peptides and proteins to nanoparticles.

The synthesis and coating were performed as follows: NHS-LC-SPDP, commercially available by Thermo Fisher, is a long chain extender with a bifunctional coupling agent specific for amines at both ends and with disulfide bonds that can be converted to sulfides.

The extender has an N-hydroxysuccinimide ester at one end and a pyridyldithio group at the other end. This dithio group can be reduced to produce a mercapto group. The NHS-LC-SPDP is reacted with the nanoparticle and the reaction can be cleared from the non-incorporated NHS-LC-SPD. The coupled nanoparticles are then reduced as shown in figure 1.

Production of coupled proteins: biologically active proteins purified by affinity chromatography columns contain free-amine groups from carboxy-terminal lysine residues that are added to facilitate attachment to the nanoparticles. NHS-LC-SMCC was used as a bifunctional coupling agent. This molecule has an LC1 chain extender. An N-hydroxysuccinimide reagent with amine specificity at one end. The other end contains a maleimide group, specifically a thiol group. Once the material is attached to the protein, it is separated from the reaction mixture and the maleimide-coupled protein will be added to the thiol-containing nanoparticle. The resulting material was isolated by gel filtration.

Peptide coupling to nanoparticles: in this case, the peptide also contains a carboxy-terminal lysine, which functions as a matrix for NHS ester-LC-maleimide coupling. This molecule has an LC2 chain extender. All schemes are similar to those described above for proteins.

In the most preferred embodiment, the membrane-permeable peptide and protein will be mixed in different ratios to achieve the maximum number of molecules attached to the nanoparticle. According to previously published studies, surface-bound cell-permeable peptides having 3-4 molecules per nanoparticle are sufficient for efficient intracellular delivery of superparamagnetic nanoparticles.

The use of an LC 2-extension arm provides an important means to increase the amount of peptidyl molecular bonding. The use of different concentrations of NHS-LC-SPDP increases the number of peptide and protein molecules anchored to the nanoparticle surface, thus enabling more efficient penetration and more reliable cell reprogramming activity.

Peptides and proteins attached to the nanoparticles: this can be achieved using the flow shown in fig. 1. In this case, SMCC-tagged proteins and peptides were added to the beads in proportion and allowed to react.

Another more directly controllable means of labeling nanoparticles with peptides and proteins is to use two different bifunctional coupling reagents (fig. 2A-F). The reaction sequence is somewhat similar to that of FIG. 1, with some modifications described below.

Iodoacetic acid is used to introduce a selected number of "carboxyl" groups to the nanoparticle surface. This is done in step I (see FIGS. 2A-F, Steps (I-VII)).

The NH-LC-SMCC containing peptide was treated with aminoethanol. This enables bonding via a thiol group and provides a free amino group. This amino group is then coupled 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 at the end of the reaction scheme.

In another aspect, the invention also relates to methods of delivering bioactive molecules attached to functionalized nanoparticles for modulating intracellular activity. For example, human cells, fibroblasts, or other types of cells commercially available or available using standard or modified experimental procedures are first plated under sterile conditions onto a solid surface with or without a cell adhesion substrate (feeder cells, gelatin, matrigel, fibronectin, etc.). Plated cells are cultured in combination with specific factors for a period of time to allow cell division/proliferation or to maintain acceptable cell viability. Examples are serum and/or various growth factors, which can be removed or renewed later and the culture continued. Plating the plated cells in the presence of functionalized biocompatible cell-permeable nanoparticles having biologically active molecules attached thereto in the presence or absence of a magnetic field using the various methods described herein. The use of a magnet in the case of superparamagnetic nanoparticles allows for a significant increase in the contact surface area between the cell and the nanoparticle, thereby further enhancing the penetration of the functionalized nanoparticles through the cell membrane. The cell population is repeatedly treated with functionalized nanoparticles as necessary to deliver the bioactive molecule intracellularly.

The cells are suspended in culture medium and the unincorporated nanoparticles are removed by centrifugation or cell separation, leaving the cells present as colonies. The colony cells are then resuspended and cultured in fresh medium for an additional suitable period of time. The cells may be harvested by multiple cycles of isolation, resuspension and re-culture until the specific bioactive molecule delivered intracellularly is observed to cause a consequent biological effect.

One use of the invention is to screen for a compound (or compounds) that is effective for reprogramming cells. The compounds are attached to the nanoparticles as desired cell populations using one or more of the methods disclosed in the specification, cultured for an appropriate time, and then any modulation produced by the compounds is determined. The method can comprise the following steps: initiation of cell reprogramming and generation of pluripotent stem cells, differentiation or transdifferentiation of cells into further or differently specialized cell types, examination of cytotoxicity, metabolic changes or effects on contractile activity, and the like.

Another use of the invention is to generate specialized cells as a drug or in a delivery device for treatment of the human or animal body. This enables the clinician to administer cells into or around damaged tissue (whether heart, muscle, liver, etc.) either transvascularly or directly into the muscle or organ wall, thereby implanting specialized cells, controlling damage, and participating in muscle tissue regeneration and specialized functional recovery of the tissue.

One use of the invention involves nanoparticles functionalized with other proteins (such as Oct4 and Sox2 transcription factors) to ensure reprogramming or production of cells with well-preserved genomic stem cells or further differentiated cell types.

Another use of the invention is to screen for a compound (or compounds) that is effective for reprogramming cells. The compounds are attached to the nanoparticles as desired cell populations using the various methods disclosed in the specification, cultured for an appropriate time, and then assayed for any modulation produced by the compounds. The method can comprise the following steps: initiation of cell reprogramming and generation of pluripotent stem cells, differentiation or transdifferentiation of cells into further or differently specialized cell types, examination of cytotoxicity, metabolic changes or effects on contractile activity, and the like.

The invention also has use in the generation of specialized cells for use as a medicament or in a delivery device for the treatment of the human or animal body. This enables the clinician to administer cells into or around damaged tissue (whether heart, muscle, liver, etc.), either through the vasculature or directly into the muscle or organ wall, to implant specialized cells, control damage, and participate in the regeneration of muscle tissue and restoration of specialized function of the tissue.

By way of further illustration, and not limitation, the following examples disclose other aspects of the invention.

Examples

Example 1

GFP was linked to superparamagnetic particles (attached to the amine groups of the beads) using LC-SMM as a cross-linker, which were then directly coupled to the thiol group on GFP. LC-SMCC (from Thermo Fisher) was dissolved in Dimethylformamide (DMF) from ACROS (sealed vial and anhydrous) at a concentration of 1 mg/. mu.l. The samples were sealed and used almost immediately.

Mu.l of the solution was added to the nanoparticles to a volume of 200. mu.l. This provided a large excess of SMCC over the available amine groups present and allowed the reaction to proceed for 1 hour. Excess SMCC and DMF can be removed with an Amicon spin filter with a molecular cut-off of 3000 daltons. Typically 5 volume changes are required to ensure proper buffer exchange. It is important that excess SMCC be removed at this stage.

Any peptidyl molecule, such as the commercially available Green Fluorescent Protein (GFP) or purified recombinant green fluorescent protein or other protein, is added to a solution containing a quantity of ethylene glycol and frozen at-30 ℃. A PBS solution containing 3. mu.g of protein in 14. mu.l, 10. mu.l of freshly prepared DTT (dithiothreitol, Cleland's reagent) was added with vigorous stirring. Since proteins usually contain more than one cysteine, it is prone to cross-link different GFP molecules. Thus, excess DTT reduces the disulfide bonds and releases green fluorescent protein. The reaction was allowed to proceed at 4 ℃ for 2 hours, and then excess reagent was removed by an Amicon centrifugal filtration unit with a molecular weight cut-off of 3,000.

The activated nanoparticles and protein solution were mixed and allowed to react for 2 hours, after which unreacted protein was removed by an Amicon centrifugal filtration unit with the appropriate molecular weight cut-off (50,000 daltons in this case with GFP). Samples were stored at-80 ℃. It should also be noted that a sulfo derivative of SMCC (sulfo-SMCC), which is more soluble in water, may also be used. DMSO may also be used as a solvent carrier for the labeling reagent in place of DMF, and it should be anhydrous.

Example 2

In this method, the amino group of lysine is used to perform a coupling reaction with a thiol group on a bead. Beads that had just been equilibrated with 0.1M phosphate buffer, pH7.2, were used in these studies. LC-SPDP was freshly prepared at 1mg/ml (in DMF). Mu.l of SPDP solution were added to the bead suspension with vigorous stirring and allowed to react for 1 hour. Next, unreacted material was removed by centrifugation and the nanoparticles were washed with phosphate buffer using an Amicon spin filter with a molecular weight cut-off of 10K. The disulfide bond of SPDP was cleaved using claimant's reagent, 1mg was added to the solution, and allowed to react for 1 hour. The by-products and unreacted claimant reagent were removed by Amicon rotary filter with a molecular weight cut-off of 10K.

In the above reaction, GFP was blocked using N-ethylmaleimide. Excess ethylmaleimide was added to the GFP solution. The reaction was run at room temperature for 1 hour with an Amicon rotary filter with a molecular weight cut-off of 3K to remove unwanted material. Then, GFP was reacted with excess SMCC for 1 hour. Subsequently, GFP was purified using spin columns and then reacted with PDP-nanoparticles. The reaction was run for 1 hour and the final product was purified on an Amicon spin filter with a molecular weight cut-off of 50K.

Example 3

The standard test methods described will be obtained commercially or used [ Moretti et al, Mouse and human induced pluripotent stem cells as a source for pluripotent Isllcardiovascular progenitor cells). FASEB J.24:700(2010)]The obtained human fibroblasts were plated under sterile conditions at a density of 150,000 cells on a solid surface in a 6-well plate with or without feeder cells pre-plated at a density of 150,000-200,000. Feeder cells can be obtained commercially or by standard laboratory methods. The plated cells are cultured in combination with specific factors for a period of time to allow the cells to divide/proliferate or maintain an acceptable viability of the cells in serum-containing media, so that they can be removed or regenerated later, and in a humidified incubator (5% CO)₂And environment O₂) The culture is continued under aseptic conditions.

The cells collected at the bottom of the conical tube or the plated cells were treated with 50 μ l of a suspension containing functionalized biocompatible cell-permeable nanoparticles to which bioactive molecules were attached in the presence or absence of a magnetic field using the various methods disclosed in this specification.

The use of a magnetic field in the case of superparamagnetic nanoparticles allows a significant increase in the contact surface area between the cell and the nanoparticle, thus ensuring a further enhancement of the penetration of the functionalized nanoparticles through the cell membrane. Importantly, several protein-based drugs (PEG-GCSF, Amgen, CA; PEG-Interferon, Schering-Plough/Merck, NJ) for PEG-mediated protection similar to PEG-linked poly (ethylene glycol) PEG increase the size of the polypeptide and mask the surface of the protein, thereby reducing degradation of the protein by proteolytic enzymes and resulting in longer term stability of the protein molecules used. Repeated treatment of cell populations with functionalized nanoparticles as needed for intracellular delivery of bioactive molecules

Cells were suspended in culture medium and unincorporated nanoparticles were removed by centrifugation at about 1200 Xg for 10 minutes, leaving the cells present as colonies. The colony cells are then resuspended, washed using a similar procedure, and re-cultured in fresh medium for an appropriate period of time. Cells can be harvested by multiple cycles of isolation, resuspension, and re-cultivation in culture medium until the observed intracellular delivery of a particular bioactive molecule causes a consequent biological effect.

In the specific example using green fluorescent protein, the cell-permeable nanoparticles deliver the protein into the cell and new green fluorescence is obtained by the target cell. This newly acquired capability allows for subsequent sorting, and separation of cells with intracellular delivered proteins with high homogenization, which can further be used for a variety of applications. Importantly, the protein-linked cell-permeable functionalized nanoparticles do not integrate in any way into the cell genome, thereby ensuring that each cell with new (in this case fluorescent) properties maintains an intact genome and retains the integrity of the cellular DNA.

The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the above-described embodiments should be regarded as illustrative rather than restrictive of the invention described in this specification. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (13)

1. A functionalized biocompatible nanoparticle capable of penetrating a mammalian cell membrane and delivering intracellularly a biologically active molecule for modulating cell function, comprising:

a central nanoparticle having a polymer coating thereon,

a plurality of functional groups covalently attached to the central nanoparticle or polymer coating, and a plurality of bioactive molecules attached to the plurality of functional groups, wherein the plurality of bioactive molecules comprises one or more cell penetrating peptides for penetrating the mammalian cell membrane and one or more proteins for modulating the cellular function of the cell,

wherein one or more of said peptides are attached to said nanoparticle independently of each of said proteins.

2. The functionalized biocompatible nanoparticle of claim 1 wherein each of the peptides is attached to the nanoparticle by a linker molecule.

3. The functionalized biocompatible nanoparticle of claim 2 wherein each of the peptide molecules and each of the protein molecules are each attached to the nanoparticle by one or more intervening linker molecules.

4. The functionalized biocompatible nanoparticle of claim 1 wherein the peptide comprises 5-9 basic amino acids.

5. The functionalized biocompatible nanoparticle of claim 1 wherein the peptide comprises 9 or more basic amino acids.

6. The functionalized biocompatible nanoparticle of claim 4 wherein the protein is a transcription factor.

7. The functionalized biocompatible nanoparticle of claim 6 wherein the transcription factor is selected from the group consisting of Oct4, Sox2, Nanog, Lin28, cMyc, and Klf 4.

8. A method of altering cellular function within a mammalian cell comprising administering an effective amount of the functionalized biocompatible nanoparticle of claim 1 to the cell and altering cellular function within the cell.

9. The method of claim 8, wherein said alteration of cellular function is related to one or more of said cell physicochemical properties, said cell proliferative properties, said cell viability, said cell morphological phenotypic properties, or an alteration of a cell's ability to acquire for the production of a new cell type, said new cell type comprising a stem cell or a more specialized cell type.

10. The functionalized biocompatible nanoparticle of claim 4 wherein the linker length linking each of the peptide molecules to the nanoparticle is different from the linker length linking the protein to the nanoparticle.

11. The functionalized biocompatible nanoparticle of claim 1 wherein the nanoparticle comprises iron.

12. The functionalized biocompatible nanoparticle of claim 16 wherein the central nanoparticle ranges in size from 5-50 nm.

13. A method of screening for a compound effective for cell reprogramming, the method comprising:

attaching a desired compound to the nanoparticle of any one of claims 1-7;

introducing the compound and the attached nanoparticle into a desired cell population in vitro;

culturing the population of cells for an effective period of time; and

determining the modulation elicited by the compound on one or more cells of the cell population.