NANOPARTICLES FOR TRANSFECTION

Abstract

This invention is directed to nanoparticles for delivery of nucleic acids to target cells of interest for transfection and expression. The nanoparticles typically include a complex of a cationic peptide bound to a protective hydrophilic polymer through a chelator. The nucleic acid is held to the complex by ionic interactions with the cationic peptide. The chelator is adapted to allow release of the hydrophilic polymer in a time frame suitable to facilitate transfection with the nanoparticle at the target cell surface.

Claims

1-36. (canceled)

37. Nanoparticles for transfection of a cell with a nucleic acid, the nanoparticles comprising: an unnatural cationic peptide comprising a majority of at least two different amino acids selected from the group consisting of: histidine (H) and at least one of: 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine, and lysine (K); and a nucleic acid associated with the cationic peptide through ionic interactions; and wherein the nucleic acid is a functional nucleic acid capable of encoding an active gene useful for gene therapy.

38. Nanoparticles of claim 37, the nanoparticles further comprising: an unnatural hydrophilic polymer bonded to a chelator coordinated to a metal ion and wherein the cationic peptide coordinates to the metal ion.

39. Nanoparticles of claim 37, provided in a physiologically acceptable buffer such as PBS, HEPES, saline, lactated ringers, or ultrapure water.

40. Nanoparticles of claim 37, wherein cationic peptide and the nucleic acid charge:charge ratio is balanced.

41. Nanoparticles of claim 38, wherein the molar ratio of the unnatural hydrophilic polymer bonded to a chelator coordinated to a metal ion and the cationic peptide is greater than 50:1.

42. Nanoparticles of claim 38, wherein the hydrophilic polymer forms a protective layer around the cationic peptide-nucleic acid nanoparticle core and/or wherein the hydrophilic polymer stabilizes the nanoparticles as demonstrated by resistance to agglomeration in a high ionic strength environment, substantially no aggregation in 50 mM NaCl for at least 3 hours.

43. Nanoparticles of claim 37, wherein the nucleic acid is an expression vector expressing functional peptides selected from CFTR, A1AT, sickle cell hemoglobin, hexosaminidase A (Tay-Sachs disease), or phenylalanine hydroxylase (phenylketonuria) or a CFTR sequence having at least 90% identity to a functional CFTR gene or comprises an A1AT sequence having at least 90% identity to a functional A1AT gene.

44. Nanoparticles of claim 37, adapted for topical delivery on a mucus membrane, intranasal, intrabronchial, intramuscular, subdermal, intraocular, trans-dermal, topical, on an ocular surface, intrathecal, or synovial surface.

45. Nanoparticles of claim 37, wherein the nanoparticles has an average diameter ranging from about 50 nm to about 250 nm.

46. Nanoparticles of claim 38, wherein the hydrophilic polymer is PEG or mPEG, wherein the PEG or mPEG is linear or branched.

47. Nanoparticles of claim 37, wherein the chelator moiety is selected from the group consisting of: an iminodiacetic acid (IDA), an ethylenediamine, ethylenediaminetetraacetic acid (EDTA), egtazic acid (EGTA), carboxylmethylaspartate (CMA), dimercaptopropanol, and nitrilotriacetic acid (NTA).

48. Nanoparticles of claim 38, wherein the metal ion is selected from the group consisting of: Ca.sup.2+, Zn.sup.2+, Mg.sup.2+, Ni.sup.2+, Cu.sup.2+, Fe.sup.2+, Fe.sup.3+ and Co.sup.2+.

49. Nanoparticles of claim 38, wherein the hydrophilic polymer is adapted to be releasably bound to the cationic peptide and wherein a half-life of a chelation bond between the hydrophilic polymer and cationic peptide in serum at 37° C. is adapted to be between 5 minutes and 8 hours.

50. Nanoparticles of claim 37, wherein the cationic peptide has at least 90% identity with any of the following peptides: TABLE-US-00003 No. Names Sequence I HHHHNHHHHKKK(KHKHHKHHKHHKHHKHHKHH).sub.4 II HK KHKHKHKHKGKHKHKHKHK III H2K KHKHKHKHKGKHKHKHKHK IV H2K2b K(KHKHHKHHKHHKHHKHHKHK).sub.2 V H2K3b KK(KHKHHKHHKHHKHHKHHKHK).sub.3 VI H2K4b KKK(KHKHHKHHKHHKHHKHHKHK).sub.4 see note 3 VII H3K4b KKK(KHHHKHHHHKHHHKHHHK).sub.4 H3K8b See Fig. 11 (+RGD) VIII H2K4bT KKK(KHKHHKHHKHHKHHKHHKHK).sub.4T 2070 See note 2 IX H3K4BT KKK(KHHHKHHHKHHHKHHHK).sub.4T X 2595 (H-Orn-His-Orn-His-His-Orn-His-His- Orn-His-His-Orn-His-His-Orn-H His-Orn-His-Orn).sub.4-Lys-Lys-Lys-His- His-His-His-Asn-His-His-His-His OH XI 2596 (H-Lys-His-Lys-His-Lys-His-Lys-His- 1:1  Lys-His-Lys-His-Lys-His-Lys-H Lys:His Lys-His-His-Lys).sub.4-Lys-Lys-Lys-His- His-His-His-Asn-His-His-His-His- OH XII 2597 (H-Lys-His-Lys-His-His-Lys-His-Lys- 9:11  His-His-Lys-His-Lys-His-His-Ly Lys:His His-Lys-His-Lys).sub.4-Lys-Lys-Lys-His- His-His-His-Asn-His-His-His-His- OH indicates data missing or illegible when filed

51. Nanoparticles of claim 37, wherein the nanoparticle further comprises an extracellular targeting ligand.

52. A method of manufacturing nanoparticles according to claim 38, comprising the steps of: (i) combining the nucleic acid and the cationic peptide to form nucleic acid bearing nanoparticles; (ii) adding to the nanoparticles in solution, the hydrophilic polymer functionalized with a chelating group chelated to the chelatable metal ion.

53. The method of claim 52, further comprising the step of controlling the solution pH to vary the nanoparticles average particle diameter.

54. A method of treating and/or alleviating the symptoms of one or more of cystic fibrosis, lung disease and liver disease, comprising the step of administering to a subject in need thereof, a therapeutically effective amount of the nanoparticles as defined in claim 37.

55. A non-viral vector for transfection of a bronchial cell with nucleic acid encoding a CFTR sequence having at least 90% identity to a functional CFTR gene, the vector comprising nanoparticles having an average particle diameter of from about 50 nm to about 250 nm, and including: unnatural branched cationic peptides comprising a majority of at least two different amino acids selected from the group consisting of: histidine (H) and at least one of: 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine, and lysine (K); and a plasmid DNA associated with the cationic peptide through ionic interactions; and a PEG or mPEG polymer bonded to a chelator coordinated to a Zn.sup.2+ metal ion and wherein the cationic peptide also coordinates to the Zn.sup.2+ metal ion, and wherein the DNA is a plasmid DNA or a mRNA capable of encoding the CFTR sequence.

56. The non-viral vector of claim 55, wherein the plasmid DNA is pGM160, pGM169, pCF1-CFTR, pGM151, pd1GL3-RL, pBAL, pBACH, pUMVC-nt-β-gal, pcDNA3.1 WT-CFTR, pEGFP WT-CFTR, or luciferase plasmid DNA.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0069] FIG. 1 is a schematic diagram of alternate defects that may lead to cystic fibrosis disease.

[0070] FIG. 2 is a chart showing transfection of epithelial cells with nanoparticles having releasably bound hydrophilic polymer. Nanoparticle mediated DNA transfection in BEAS-2B cells (48 hr) Human bronchial epithelial (BEAS-2B) cells were transfected with luciferase DNA formulated in Lipofectamine-2000 (Lipo) or in the forms of nanoparticle without PEG (LG15HKD) or with PEG (LG15HKD-p50) in the presence of 10% FBS (left) or absence of FBS (right) for 4 hour. The transfection was conducted with serial dilution of DNA concentration in triplicated wells. The transfection medium was replaced with regular culture medium and cells were incubated for 48 hours. The transfected cells were lysed and luciferase activity in each well was measured, and normalized against that in cells transfected with 0.05 μg DNA-lipofectamine-2000. Data were represented as the average value from two independent experiments.

[0071] FIG. 3 is a chart showing transfection efficiency using nanoparticles with and without PEG, and with and without FBS in the culture media. Nano-particle mediated DNA transfection in BEAS-2B cells (72 hr) Human bronchial epithelial (BEAS-2B) cells were transfected with luciferase DNA formulated in Lipofectamine-2000 (Lipo) or in the forms of nano-particle without PEG (LG15HKD) or with PEG (LG15HKD-p50) in the presence of 10% FBS (left) or absence of FBS (right) for 4 hours. The transfection was conducted with serial dilution of DNA concentration in triplicated wells. The transfection medium was replaced with regular culture medium and cells were incubated for 72 hours. The transfected cells were lysed and luciferase activity in each well was measured, and normalized against that in cells transfected with 0.05 μg DNA-lipofectamine-2000.

[0072] FIG. 4 is a chart showing transfection efficiency five days after transfection. Human bronchial epithelial (BEAS-2B) cells were transfected with luciferase DNA formulated in Lipofectamine-2000 (Lipo) or in the forms of nanoparticle without PEG (LG15HKD) or with PEG (LG15HKD-p50) in the presence of 10% FBS (left) or absence of FBS (right) for 4 hour. The transfection was conducted with serial dilution of DNA concentration in triplicated wells. The transfection medium was replaced with regular culture medium and cells were incubated for 5 days. The transfected cells were lysed and luciferase activity in each well was measured, and normalized against that in cells transfected with 0.05 μg DNA-lipofectamine-2000.

[0073] FIG. 5 is a chart showing the results of nanoparticle mediated DNA transfection in BEAS-2B cells (48 hr). Human bronchil epithelial (BEAS-2B) cells were transfected with luciferase DNA formulated in Trans-Hi (0.025 ug/well) or in the forms of nanoparticle without PEG or with PEG in the presence or absence of 10% FBS for 5 hour. The transfection was conducted with serial dilution of DNA concentration in triplicated wells. The transfection medium was replaced with regular culture medium at 5 hr post transfection and cells were incubated for 48 hours. The transfected cells were lysed and luciferase activity in each well was measured. Data were represented as the average value from two independent experiments (except F1 one which from only one study).

[0074] FIG. 6 is a chart showing the results of nanoparticle mediated DNA transfection in BEAS-2B cells (48 hr). Human bronchil epithelial (BEAS-2B) cells were transfected with luciferase DNA formulated in Trans-Hi (0.025 ug/well) or in the forms of nanoparticle without PEG or with PEG in the presence or absence of 10% FBS for 5 hour. The transfection was conducted with serial dilution of DNA concentration in triplicated wells. The transfection medium was replaced with regular culture medium at 5hr post transfection and cells were incubated for 48 hours. The transfected cells were lysed and luciferase activity in each well was measured.

[0075] Data were represented as the average value from two independent experiments (except F1 one which from only one study).

[0076] FIG. 7 is a chart showing FACS results in frames a, b and c. Frame a shows a negative control population of cells with no transfection. Frame b shows transfection results for cells treated with pGFP using PEGylated nanoparticles (5 μg DNA/well). Frame c shows cells treated with pGFP/Lipofectamine. M1 represents the population of transfected cells compared to the non-transfected cells represented in Frame a. Frame b shows that the nanoparticle formulation effected transfection in 43.8% of the cells.

[0077] FIG. 8 is a chart showing the results of a FACS assay at 72 hours, in terms of the percentage of cells transfected.

[0078] FIG. 9 is a chart showing the results of a GFP FACS assay at 72 hours, measuring the degree of GFP activity.

[0079] FIG. 10 is a schematic drawing of an exemplary cationic peptide and a self-assembly stage of nanoparticle production.

[0080] FIG. 11 is schematic diagram of the 8 branched H3K8b(+RGD) where R=HHHKHHHKHHHK—HHH. The three solid circles connected by a solid line represent the 3-lysine core and K represents the lysine with which the two-terminal branches are conjugated (see Leng at al., Drug News Perspect 20(2), March 2007, pg. 77-86 (‘Mixon’), the content of which is hereby incorporated by reference, particularly Table II and FIG. 6C therein).

DETAILED DESCRIPTION

[0081] The present inventions are directed to certain nanoparticles adapted to transfect cells, and methods of their manufacture and use. The nanoparticles generally comprise a capsule complex and a nucleic acid encoding a bioactive peptide. The complex typically comprises a hydrophilic polymer associated with and/or bound to a cationic peptide to capture, protect, and deliver the nucleic acid. The nanoparticles can be delivered to target cells for transfection by methods of administration including, e.g., localized topical application or an injection. Methods of manufacture include, e.g., Fmoc fabrication of the cationic peptide on a solid support, covalent binding of a chelator to the hydrophilic polymer, charging of the chelator with a divalent metal cation, and (reversibly) binding the hydrophilic polymer to the cationic peptide by interaction with the chelated divalent metal cation.

[0082] A number of methods and compositions are discussed in the Summary of the Invention and further details are provided herein and in the Examples section. As would be readily appreciated by the skilled person, the disclosures can be read in combination.

[0083] As explained above, preferred nanoparticles may comprise a cationic peptide (e.g., rich in H and K) component which associates with a nucleic acid, a chelator moiety bonded to hydrophilic polymer (e.g., PEG) and a metal ion to which the cationic peptide and the chelator coordinate. Combining the nucleic acid and the cationic peptide forms nanoparticles. Combining the nanoparticles with a metal complex of the metal ion and hydrophilic functionalized chelator results in the formation of a shell of the hydrophilic polymer around the nanoparticles.

[0084] The nanoparticles useful for transfection of cells generally include a nucleic acid, preferably plasmid DNA or mRNA, for transfection which is associated with cationic peptide. Preferred nanoparticles are insulated or covered in a shell of protective hydrophilic polymer. The hydrophilic polymer functions in providing stability to the nanoparticles (in vivo, in vitro, and/or in storage and/or administration e.g., by nebulisation) by forming the protective shell around the nucleic acid and cationic peptide, aids in migration through biologic fluids and matrices, and improving pharmacokinetics. The hydrophilic polymer includes a chelator which allows it to bind to the metal cation. The cationic peptide provides features (e.g., positive charges) that interact to bind the nucleic acid cargo and histidines that interact to bind with the chelated metal cation. The nanoparticle is designed to carry the nucleic acid to a cell surface in an efficient fashion, e.g., penetrating viscous body fluids. Particularly, preferred nanoparticles are small, e.g., in a range of about 100 nm diameter allowing diffusion through pores of viscoelastic biofluid polymers (e.g., mucus). Diffusion of the nanoparticles is also aided by the hydrophilic polymer which has little affinity for polymers found in many biofluids. The hydrophilic polymer can be adapted to be releasable from the cationic peptide, aiding in transfection on reaching the target cell.

[0085] The nucleic acid cargo in the nanoparticles is surrounded by the protective hydrophilic polymer shell. Preferably, the hydrophilic polymer is adapted to provide increased product stability in storage, reduced aggregation, reduced capture or interference by body fluids, and enhanced diffusion characteristics in body fluids. It is preferred the hydrophilic polymer be hypo-allergenic and not immuno-stimulating. Typically, the hydrophilic polymer is negatively charged or presents a polar surface. In many cases, the hydrophilic polymer is not a natural polymer, e.g., not a naturally occurring carbohydrate, nucleic acid, or peptide.

[0086] In certain embodiments, the hydrophilic polymer is a polyethylene glycol (PEG) molecule. For example, the hydrophilic polymer can be PEG or methoxypolyethylene glycol (mPEG). The PEG can be linear or branched. The molecular weight can range from less than 500 to more than 40,000, from 1000 to 25,000, from 2000 to 15,000, or about 10,000.

[0087] In many embodiments, the nanoparticles can be directed to target cells by the means of administration, e.g., physically in an organ or tissue compartment. However, the nanoparticles can be even more specifically directed by features providing specific affinity interactions between the nanoparticle and the target cell surface. For example, the hydrophilic polymer and/or cationic peptide can have a ligand (e.g., extracellular targeting ligand) directly or indirectly attached, e.g., covalently or non-covalently. The ligand can be configured to bind to a target cell receptor, preferably a receptor relatively abundant (or found only) on the target cell of interest. In certain embodiments, the ligand can be bound, e.g., at a free end of the hydrophilic polymer. Preferably, the hydrophilic polymer populating the outside of the nanoparticle includes a first hydrophilic polymer type linked to the extracellular binding ligand and a second hydrophilic polymer type that does not comprise an extracellular binding ligand. It can be preferred that the first hydrophilic polymer type be longer than the second type. It can be preferred that the second type be somewhat more releasable (shorter half-life) than the first type. In practice, the nanoparticles can bind to a specific target cell through the specific ligand. This can happen while the nanoparticle is still fully populated with a complete coat of the hydrophilic polymers, or after much of the hydrophilic polymers have been released from the nanoparticle. The presence of the ligand binding feature can allow the nanoparticle to loiter at the cell surface until enough of the hydrophilic polymer is released for transfection to proceed

[0088] The hydrophilic polymer is bound to the cationic peptide through a metal ion jointly coordinating to the chelator associated with the hydrophilic polymer and the amino acid residues of the cationic polymer. The chelator is typically associated with a chain end of the hydrophilic polymer via a covalent bond for example. For example, the hydrophilic polymer can covalently bind to a chelator moiety via reaction between suitably reactive functional groups on both entities. The chelator can coordinate with and capture a metal, e.g., leaving other coordination sites to further interact with suitable groups associated with the cationic peptide. Alternately, a chelator can also be associated (e.g. covalently bonded) with the cationic peptide. Any suitable chelator can be used to provide the bond between the hydrophilic polymer and cationic peptide. Exemplary chelators include, e.g., an iminodiacetic acid (IDA), an ethylenediamine, EGTA, dimercaptopropanol, NTA, DPTA, citrate, an oxalate, a tartrate, and the like. Typically, the chelator in the present nanoparticles is an IDA, EDTA, or NTA.

[0089] Any suitable metal ion can be used to interact with the chelator on the hydrophilic polymer and with coordinating groups on the cationic peptide. The metal ions are preferably di-cations or tri-cations. For example, the metal ions can be Ca.sup.+2, Zn.sup.+2, Mg.sup.+2, Ni.sup.+2, Cu.sup.+2, Cd.sup.+2, Fe.sup.+2, Fe.sup.+3, and Co.sup.+2 . Preferably, the chelated metal ion in the present nanoparticles is a Zn.sup.+2, Fe.sup.+2, Fe.sup.3+, Mg.sup.+2, or Ca.sup.+2 or combinations thereof.

[0090] The cationic peptide is configured to interact with the negatively charged nucleic acid to form nanoparticles and also to coordinate with the chelated metal ion, e.g., associated with the hydrophilic polymer. The cationic peptide will have a net positive charge at a pH of use, typically pH 5 to pH 8, or about pH 7.4. The cationic peptide typically features, or has a contiguous region of at least 10 amino acids of, for example including mostly or exclusively positively charged amino acids depending on the pH of the local environment. For example, preferred cationic peptides range in composition from about 10 to about 70 amino acids, from about 15 to about 50, or about 30 amino acids (in the entire peptide, or in a cationic region of the peptide). Preferred cationic peptides include all or a section of from 100% to about 80% positively charged amino acid residues in a section at least 12 amino acids long. In more preferred embodiments, the cationic peptide comprises about 30 to about 50 consecutive amino acids with at least 90% having a positive charge under physiological conditions. In some embodiments, preferred cationic peptides include a majority of H, and one other amino acid selected from the following group: K, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, and ornithine, can also be included in the cationic peptides of the invention in some embodiments. In more preferred embodiments, at least 90% of the cationic peptide consists of H and K residues; preferably more H residues than K residues (e.g., about 1/3 K residues and about 2/3 H residues). For example, the cationic peptide can include a cationic region abundant in positively charged amino acids. Other amino acids such as arginine (R), asparagine (N) or tyrosine (Y) can also be included in varying amounts. Amino acid analogues, such as histidine (H), 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine, can also be included in the cationic peptides of the invention in some embodiments. The cationic peptides are linear or branched. In most applications, there can be benefits to using branched peptides. For example packaging and delivery of the nucleic acid can often be improved using branched cationic peptides. The cationic peptide can include 2, 3, 4, 5 branches or more. The cationic peptides are usually prepared synthetically. This usually involves sequential amino acid synthesis on a solid support, e.g., using Fmoc/t-Boc chemistries.

[0091] The chelation bond is preferably adapted to render the hydrophilic polymer releasable in an appropriate time frame under conditions of the nanoparticle administration. For example, where the nanoparticles are administered in the presence of a viscous body fluid, the hydrophilic polymer is designed to stay bound long enough for delivery to a membrane surface of a cell targeted for transfection. Generally, under physiological conditions of ionic strength, temperature, and pH, the hydrophilic polymer and its attachment to the metal ion through the chelator functionality should have a half-life from 5 minutes to about 8 hours or more, from 10 minutes to 4 hours, from 30 minutes to 3 hours, or about 2 hours. The optimal half-life would of course depend on, e.g., the distance the nanoparticles must travel between the point of administration and the target cells, the viscosity of the relevant body fluid, and the pore size of any matrix or membrane the nanoparticles must traverse. The half-life of the chelation bond between the hydrophilic polymer, cationic peptide and the metal ion centre can be influenced by, e.g., the choice of chelator, metal ion, and cationic peptide sequence. For example, whereas a Zn.sup.+2 ion strongly interacts forming longer half-life bonds, the half-life of the bond can be moderated using another ion, such as Ca.sup.+2 which tends to form a more labile coordination bond with the chelator. Where the chelator is of a type coordinating at three sites (tridentate), the half-life can be reduced by electing a chelator coordinating at 2 sites (bidentate). Where the cationic peptide binds strongly with a peptide rich in histidine, the half-life can be reduced by reducing the number or percent H in the region interacting with the metal ion. Each of these techniques can be used in combination. The reverse of the operation can strengthen the chelation and extend the half-life. In some cases the environment around the chelation can affect the half-life. For example, where feasible, the chelation bond half-life can be influenced by the pH, ionic strength, or presence of competing ions in the local environment.

[0092] The nanoparticle includes nucleic acids of interest, or a nucleic acid encoding a peptide of interest. For example the nucleic acid can be a biologically active RNA, particularly mRNA or DNA preferably encoding a therapeutic peptide. In certain preferred embodiments, the nucleic acid cargo of the nanoparticle can be a DNA (e.g., plasmid) encoding a peptide, e.g., repairing a defect in a cell. For example, the plasmid may be an expression vector expressing functional peptides, such as cystic fibrosis transmembrane conductance regulator (CFTR), sickle cell hemoglobin, hexosaminidase A (Tay-Sachs disease), phenylalanine hydroxylase (phenylketonuria), and the like.

[0093] The assembled nanoparticle can have characteristics that aid in delivery to the surface of cells. The nanoparticle can be configured to have a desired charge, hydrophobicity, size, antigenicity, stability, nucleic acid capacity, and the like. In many cases, the nanoparticle has a size well suited to penetration of biologic fluids and membranes. For example, the nanoparticle can be adapted to effectively diffuse through many biological fluids, such as CSF, cell membranes, connective tissue, synovial fluid, mucus, interstitial fluid, clot, vitreous humour, and the like. In many cases, depending on the target cell environment, adequate penetration can be achieved with an assembled nanoparticle ranging in size from less than 50 nm, or from about 50 nm to about 500 nm, preferably from about 75 nm to about 200 nm, more preferably from about 90 nm to 150 nm, most preferably from about 90 nm to 110 nm. In certain preferred embodiments, the average diameter is about 110 nm or 120 nm. The nanoparticle capacity for nucleic acid cargo/payload can be changed by adjustment of the cationic peptide. This can also affect the size of the nanoparticle. The nucleic acid carrying capacity of the nanoparticle can be generally increased by provision of a longer cationic peptide sequence and/or by provision of branch points in the cationic peptide. The outside surface of the nanoparticle can be made less prone to aggregation, have less affinity to biologic fluid matrices, and be less immunogenic, by choice of the hydrophilic polymer. In a preferred embodiment, PEG and PEG-containing copolymers can be the hydrophilic polymer of the nanoparticles. The PEG can form a protective layer around the nanoparticles and disguise the particles against active and passive immune detection. The protective layer around the cationic peptide-nucleic acid may also stabilize the nanoparticles from agglomeration in a high ionic strength environments, for example, in one embodiment, can prevent aggregation in 50 mM NaCl for at least 3 hours.

Methods of Transfection with Nanoparticles

[0094] The nanoparticles can be manufactured, e.g., by bonding a chelator to a hydrophilic polymer, introducing an appropriate metal ion to the chelator to form a metal complex, then combining a cationic peptide associated with a nucleic acid in the form of nanoparticles to form the hydrophilic polymer coated nanoparticles. In other words, the nanoparticles can be manufactured, e.g., by treating a cationic peptide-nucleic acid nanoparticle complex with a pre-assembled hydrophilic polymer covalently linked to a chelator in the form of a pre-formed metal chelate. The nanoparticles of the invention can be stored in a liquid, frozen, freeze-dried, or dried powder formulation before use. The nanoparticles can be administered to a patient in any suitable fashion, e.g., topical, inhalation, or injection.

[0095] The formulated nanoparticles can be administered to the intended cells directly or indirectly. In preferred embodiments, the nanoparticles are physically deposited on the cells or within a short diffusion distance from the cells. Depending on the target cells, it may be beneficial to target the nanoparticles using affinity molecules. For example, the nanoparticle can include a ligand (bound anywhere in the complex) specific to any target cell surface feature. This, e.g., in combination with the physical localization of the nanoparticle on administration, can enhance transfection efficiency in the desired cells.

[0096] In one aspect of the invention, the nanoparticle is administered to a mucus membrane. For example, the formulated nanoparticles can be inhaled into the lungs to treat cystic fibrosis by introduction of a functional CFTR gene. The formulation can be inhaled as dry powder particles or as an aerosol of liquid droplets, e.g., of a particle size (e.g., about 3 microns, about 1 micron, or less) which can reach the lower reaches of the air passages and alveoli.

[0097] In other instances, the nanoparticles can be applied to the intended cells topically (e.g., in a salve) or injected directly into the tissue comprising the intended cells. For example, the nanoparticles can be injected as a liquid suspension through a needle or catheter to a mucus membrane, intranasal, intrabronchial, intramuscular, intraocular, subdermal, trans-dermal, topical, on an ocular surface, intrathecal, the urinary bladder, or synovial surface

EXAMPLES

[0098] The following examples are offered to illustrate, but not to limit the claimed invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Example 1—Nanoparticle Overview

[0099] Preparation of the subject nanoparticles is accomplished on combination of the nucleic acid, e.g., plasmid DNA (pDNA) with cationic peptide e.g., HK polymer. Due to electrostatic interactions occurring between the negatively charged nucleic acid and the cationic peptide, nanoparticles comprising cationic peptide associated with the nucleic acid spontaneously form. By manipulating the formulation conditions, nanoparticles with a desired diameter, for example, nanoparticles with diameters less than 100 nanometers, can be reproducibly produced. Furthermore, additional of the stabilizing hydrophilic polymers described, e.g. via PEGylation by grafting PEG-chelator to the surface of the nanoparticles provides added stability with no significant impact on overall nanoparticle size.

Example 2—Development of the Nanoparticle Formulation Method

[0100] The inventors established protocols to: a) Prepare sub 140 nm diameter nanoparticles comprising cationic peptide (HK rich peptide designated 2070 in Table 1 above) and pDNA to form HKD nanoparticles (HKplasmidDNA particles); b) PEGylate nanoparticles post formation to form HKDp particles (HKplasmidDNA-PEGylated; c) Test nanoparticle stability in PBS and NaCl. Only PEGylated HKD particles (HKDp) resisted size increase over time and with increasing ionic strength; d) Test the pH range for optimal HKD particle formation.

[0101] Preparation of nanoparticles was studied at pH 5 to 8 in 0.5 pH value increments. Particle size and DNA encapsulation was monitored. Optimal particle sizes were achieved between pH 5.0-7.0. Optimal DNA encapsulation was achieved between pH 5.5-7.0. pH 6.5+/−0.5 was selected in order to ensure robust particle formation close to physiological pH.

[0102] In cell culture media, HKD particles aggregated within a few minutes. HKDp particles were relatively more stable. In cell culture media with serum, there was an initial size increase to greater than 100 nm with stabilization under 200 nm up to 96 hrs. In cell culture with no serum, HKDp particle diameters remained unchanged during the first 40 minutes. However, after 24 hours, the particle sizes increased more rapidly than noted for particles in serum. Non-PEGylated nanoparticles are determined not stable in PBS, and NaCl solutions while PEGylated nanoparticles show prolonged stability over time and with incrementally increasing ionic strength. Longer stability of the nanoparticle formulations on storage at 2-8° C. requires study.

[0103] Nanoparticles (PEGylated and non-PEGylated) were successfully fluorescently labelled using PicoGreen, Propidium Iodide, and Alexa 488 labeling reagents. Labeled particles with diameters less than 100 nm could not be detected using optical microscopes. Very few particles in the range of 150-200 nm and a few more particles greater than 200 nm could be observed. With these observations, it is important to recognize that particles form in a size distribution with larger particles making up a minority of the overall formulation.

[0104] Overall, the inventors have developed reproducible methodologies for preparation of HKD particles under 80 nm, and HKDp particles under 100 nm. Along with HKD and HKDp formulation development, the inventors have developed a sensitive and robust Agarose Gel method for monitoring pDNA encapsulation qualitatively, and/or particle stability at various salt concentrations. Also, there was evidence that PEG disassociated from the nanoparticle after 48 to 72 hours, as the inventors had designed it to do.

Example 3—Transfections with Nano-Particles

[0105] The inventors' nanoparticles are active in mediating DNA transfection in BEAS-2B cells. In three transfection studies conducted 1-week apart, the nanoparticles demonstrated comparable activities in effecting DNA transfection in BEAS-2B cells. In general, at 48 hours, the nanoparticles (LG15HKD)(LG15 is a luciferase plasmidDNA) and LG15HKD-p50 (pegylated nanoparticle) yielded lower luciferase expression than achieved by Lipofectamine-mediated transfection. It is estimated that 5 to 10-fold more DNA is required for the nanoparticles to achieve similar or higher levels of transfection compared to Lipofectamine (see, FIGS. 2 and 3). Overall the LG15HKD was more effective for transfection compared to LGHKD-p50. However, the difference may not be significant (see, FIGS. 2, 3, and 4). The nanoparticles of the invention are well tolerated by BEAS-2B cells with no observed significant cell count reduction as a function of nanoparticle concentration. The low cellular toxicity of the nanoparticles is further confirmed by the linear curve of DNA levels as indicated by luciferase activity (see, FIGS. 2, 3, and 4). This reflects a dose like response.

[0106] The nanoparticles mediated DNA transfection with greater efficiency compared to Lipofectamine-mediated at 72 hours (FIG. 2). In contrast, at 120 hours post transfection, transfection efficiency of the nanoparticles was lower compared to Lipofectamine-mediated transfection (FIG. 3). This observation suggests that the nanoparticle transfection may be via a pathway which differs from Lipofeactamine-mediated transfection. It may also be the result of a higher DNA concentration per well.

[0107] Repeat experiments using different formulations yield consistent results showing strong transfection. Formulations comprising each of the 4 below peptides and PEG were tested against Luc transfection facilitated by the Trans-Hi transfection agent (similar to lipfectamine). Testing was executed at 3 concentrations (0.1 ug/well, 0.5 ug/well and 1.0 ug/well) in a 96 well plate format. Each peptide formulation was prepared both with and without PEG, and tested accordingly. Generally the formulation with the even number comprises PEG grafted to the nanoparticles (F2 ,F6, F10 and F14). F1 and F2 are reproductions of the original formulations. Formulations—F1: 2070/Luc non-PEG, 50 ug/mL; F2: 2070/Luc-PEG, 47.62 ug/mL; F5: 2595/Luc non-PEG, 50 ug/mL; F6: 2595/Luc-PEG, 48 ug/mL; F9: 2596/Luc non-PEG, 50 ug/mL; F9: 2596/Luc-PEG, 48 ug/mL; F13: 2597/Luc non-PEG, 50 ug/mL; F14: 2597/Luc-PEG, 48 ug/mL; F17: Luc in Hepes, 50 ug/mL, whereby 2070 is (original peptide HK, 2:3 Lys:His in Table 1 above), 2595 is the ornithine peptide X above in Table 1, 2596 is 1:1 Lys:His is peptide XI in Table 1 above, and 2597 is 9:11 Lys:His, peptide XII in Table 1 above.

TABLE-US-00002 Average Particle Peptide Diameter (via ZETA Pals Code Cationic Peptide Sequence particle size analysis) 2070 (H-Lys-His-Lys-His-His-Lys-His-His-Lys- 58.5 nm (luciferase, non-PEG) HK His-His-Lys-His-His-Lys-His-His-Lys-His- 66.0 nm (GFP, non-PEG) 2:3 Lys:His Lys)4-Lys-Lys-Lys-His-His-His-His-Asn- 94.5 nm (luciferase, PEG) His-His-His-His-OH 127.4 nm (GFP, PEG) 2595 (H-Orn-His-Orn-His-His-Orn-His-His-Orn- 65.9 nm (luciferase, non-PEG) 2:3 Orn:His His-His-Orn-His-His-Orn-His-His-Orn-His- 73.7 nm (GFP, non-PEG) Orn).sub.4-Lys-Lys-Lys-His-His-His-His-Asn- 70.9 nm (luciferase, PEG) His-His-His-His-OH 101.3 nm (GFP, PEG) 2596 (H-Lys-His-Lys-His-Lys-His-Lys-His-Lys- 64.3 nm (luciferase, non-PEG) HK His-Lys-His-Lys-His-Lys-His-Lys-His-His- 82.1 nm (GFP, non-PEG) 1:1 Lys:His Lys).sub.4-Lys-Lys-Lys-His-His-His-His-Asn-His- 77.4 nm (luciferase, PEG) His-His-His-OH 124.1 nm (GFP, PEG) 2597 (H-Lys-His-Lys-His-His-Lys-His-Lys-His- 51.3 nm (luciferase, non-PEG) HK His-Lys-His-Lys-His-His-Lys-His-Lys-His- 54.7 nm (GFP, non-PEG) 9:11 Lys:His Lys).sub.4-Lys-Lys-Lys-His-His-His-His-Asn-His-  86.0 nm (luciferase, PEG) His-His-His-OH 90.9 nm (GFP, PEG)

[0108] Some observations: with the original formulations, consistency with earlier experiments is demonstrated. The PEG formulations perform better than those without. Serum appears to aid transfection in the higher concentration but not so much in the lower concentrations. Transfection is lower in absolute terms at 72 hours but much better in comparative terms to the Trans Hi-Luc formulation—consistent with consideration that it takes time for the PEG to dissociate prior to transfection. It is possible that the PEG dissociated more rapidly for F2, F6, and F10 compared to F14. Weaker transfection for non-PEGylated formulations may be related to inherent nanoparticle instability in the cell culture media. For the most effective formulation to date, transfection efficiency may be up to approx. 700 percent better compared to the Trans-Hi formulation (noting different concentrations). Most important is that Luc without a transfection agent produced no signal. There is an apparent dose-response correlating well concentration to transfection.

Example 4—Nano-Particle Stability

[0109] The nanoparticle formulations are stable for mid-term to long-term storage. The nanoparticles (LG15HKD and LGHKD-p50), stored at 4° C., retained activity during the 3 week period of the 3 transfection studies. However, there is a possibility that these formulations may become less effective in the presence of 10% FBS during the transfection process. In the first transfection study conducted 5 days after nanoparticle preparation, nanoparticles were ineffective for transfection in the presence of 10% FBS. However, in the later transfection studies (11 days and 18 days after nanoparticle preparation), both LG15HKD and LGHKD-p50 remained effective for transfection in the presence of 10% FBS. These results were comparable to parallel studies run in the absence of FBS.

Example 4—Fluorescently Activated Cell Sorting

[0110] In this experiment, using sophisticated lasers, all of the cells in each well in a plate of wells were counted to see how many cells are transfected. This is a more precise way of measuring the efficacy of the nanoparticles and is an important assay for optimization for maximum stability, size and transfection efficiency. The results indicate transfection in up to 43.8% of cells which, if replicated in a human lung, would deliver a clinically meaningful result. The inventors then conducted a similar experiment using variants of the peptide to show the pathway to optimization. The nanoparticles show GFP activity up to 32 times higher than the control particles, being naked DNA. FIGS. 8 and 9 show the result of transfection using the same variants of the peptides as were used in FIGS. 5 and 6 above. As can be seen the transfection rate improved to up to 48% of cells. In the case of GFP activity, the nanoparticles perform up to 32 times better than the control, being naked DNA.

Example 5—Protocol for Preparing Peptide/DNA Nanoparticles & PEGylated Nanoparticles

[0111] The DNA was diluted to 50-100 ug/mL in 5mM HEPES pH 7.4. A peptide solution at a concentration of 100-180 ug/mL in 5 mM HEPES was prepared. A 250 uL volume of the DNA solution was transferred into a 2 mL Eppendorf tube. Using a 100 uL peptide, an equal volume of the peptide solution was titrated, at an appropriate rate to avoid aggregation, into the DNA solution while vortexing. The solution slowly turns translucent and no visible particles should occur. The particle size is measured using a dynamic light scattering (DLS) instrument (such as the Brookhaven ZetaPALS). The particle size should be below 100 nm, preferably below 80 nm. The nanoparticles were filtered through a 0.22 um sterile filter. The formulation was stored in a refrigerator (2-8 deg C.) until desired for use and/or for PEGylation. Where PEGylated peptide/DNA nanoparticles are required, a PEG-Zn solution was prepared in a separate tube at a concentration of about 400 mg/mL. 25-100 uL of the PEG solution was slowly added to 500 uL of the peptide/DNA nanoparticle preparation, depending on the degree of PEG-coating required. The particle size was measured by DLS. The particle size should be about 100-140 nm depending on the amount of PEG added. The formulation was store in a refrigerator (2-8 deg C.). Above describes a range for the peptide/DNA ratio which can be used in one embodiment. In a preferred example, the ratios used for nanoparticle preparation were: 100 ug/mL DNA and 100 ug/mL peptide mixed at equal volume. PEGylation: 50 uL of 440 mg/mL PEG-IDA-Zn into 500 uL nanoparticles as prepared above.

[0112] While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and/or other document were individually indicated to be incorporated by reference for all purposes