REVERSIBLE IMMOBILIZATION AND/OR CONTROLLED RELASE OF NUCLEIC ACID CONTAINING NANOPARTICLES BY (BIODEGRADABLE) POLYMER COATINGS

Abstract

The present invention relates to nanoparticles comprising nucleic acids coated with a (biodegradable) polymer for reversible immobilization and/or controlled release of the nucleic acid comprising nanoparticles. Furthermore, the present invention is directed to medical or diagnostic devices, particularly stents and implants coated by a (biodegradable) polymer with the nucleic acid comprising nanoparticles for reversible immobilization and/or controlled release. Furthermore, the present invention is directed to the use of these nanoparticles coated with a (biodegradable) polymer and to the use of medical devices and implants coated by the (biodegradable) polymer with these nucleic acid comprising nanoparticles in the prophylactic or therapeutic treatment of diseases, particularly in the prevention or treatment of restenosis, calicification, foreign body reaction, or inflammation. Additionally, the present invention is directed to a method of preparing these nucleic acid comprising nanoparticles coated with a (biodegradable) polymer and to a method for coating nucleic acid comprising nanoparticles by a (biodegradable) polymer on medical or diagnostic devices.

Claims

1.-35. (canceled)

36. A method for preparing a coated nanoparticle, the method comprising the following steps: a) providing a nanoparticle comprising a complex of a nucleic acid and a polymeric carrier molecule according to generic formula (I):
L-P.sup.1—S—[S—P.sup.2—S].sub.n—S—P.sup.3-L wherein, P.sup.1 and P.sup.3 are different or identical to each other and represent a linear or branched hydrophilic polymer chain, the linear or branched hydrophilic polymer chain selected independent from each other from polyethylene glycol (PEG), poly-N-(2-hydroxypropyl)methacrylamide, poly-2-(methacryloyloxy)ethyl phosphorylcholines, poly(hydroxyalkyl L-asparagine), poly(2-(methacryloyloxy)ethyl phosphorylcholine), hydroxyethylstarch or poly(hydroxyalkyl L-glutamine), wherein the hydrophilic polymer chain exhibits a molecular weight of 1 kDa to 100 kDa, P.sup.2 is a cationic or polycationic polypeptide, having a length of about 3 to about 100 amino acids, and comprising at least 2 cysteine residues; —S—S— is a (reversible) disulfide bond, wherein one of the sulfur positions of each of the disulfide bonds is provided by the at least 2 cysteine residues of the polypeptide P.sup.2; L is an optional ligand, which may be present or not, and may be selected independent from the other from RGD, Transferrin, Folate, a signal peptide or signal sequence, a localization signal or sequence, a nuclear localization signal or sequence (NLS), an antibody, a cell penetrating peptide, TAT, a ligand of a receptor, cytokines, hormones, growth factors, small molecules, carbohydrates, mannose, galactose, synthetic ligands, small molecule agonists, inhibitors or antagonists of receptors, or RGD peptidomimetic analogues; and n is an integer, selected from a range of 1 to 50; and b) contacting the nanoparticle of a) with a biodegradable polymer in an organic solvent containing solution.

37. The method of claim 36, wherein contacting is further defined as mixing.

38. The method of claim 36, further comprising removing the organic solvent and/or any other solvent in the organic solvent containing solution.

39. The method of claim 37, wherein removing comprises drying.

40. The method of claim 36, wherein the biodegradable polymer is a PLGA polymer.

41. The method of claim 40, wherein the PLGA polymer is defined by an average molecular weight in the range of 4 kDa to 210 kDa.

42. The method of claim 40, wherein the PLGA polymer is defined by an average molecular weight in the range of 10 kDa to 110 kDa.

43. The method of claim 40, wherein the proportion of lactic acid in the PLGA polymer is greater than 50%.

44. The method of claim 36, wherein the linear or branched hydrophilic polymer chain is PEG.

45. The method of claim 36, wherein preparing the polymeric carrier of step (a) comprising the following steps: a) providing at least one cationic or polycationic polypeptide comprising at least two cysteine residues as component P.sup.2, and optionally at least one further component (AA).sub.x, wherein x is an integer selected from a range of 1 to 100, and wherein (AA).sub.x comprises at least two cysteine residues, mixing these components to mild oxidation conditions, and thereby condensing and thus polymerizing these components with each other via disulfide bonds in a polymerization condensation or polycondensation to obtain a repetitive component H—[S—P.sup.2—S].sub.n—H or H{[S—P.sup.2—S].sub.a[S-(AA).sub.x-S].sub.b}H; H{[S—P.sup.2—S].sub.a[S-(AA).sub.x-S].sub.b}H; b) providing a hydrophilic polymer P.sup.1 and/or P.sup.3 optionally modified with a ligand L and/or an amino acid component (AA).sub.x as defined according to claim 1; and c) mixing the hydrophilic polymer P.sup.1 and/or P.sup.3 according to step b) with the repetitive component H—[S—P.sup.2—S].sub.n—H or H{[S—P.sup.2—S].sub.a[S-(AA).sub.x-S].sub.b}H obtained according to step a) in a ratio of about 2:1, and thereby typically terminating the polymerization condensation or polycondensation reaction and obtaining the polymeric carrier molecule of claim 1.

46. The method of claim 44, further comprising purifying the polymeric carrier molecule obtained according to step c).

47. The method of claim 44, further comprising complexing the nucleic acid to the polymeric carrier of step c) to obtain the nanoparticle.

48. The method of claim 47, wherein the nucleic acid is a DNA, a coding mRNA, a siRNA or an immunostimulatory RNA (isRNA).

49. The method of claim 47, wherein the nucleic acid encodes a therapeutically active polypeptide, tumor antigen, pathogenic antigen, animal antigen, viral antigen, protozoal antigen, bacterial antigen, allergic antigen, autoimmune antigen, allergen, antibody, immunostimulatory protein or an antigen-specific T-cell receptor.

50. The method of claim 47, further comprising lyophilizing the nanoparticle and reconstituting the nanoparticle in an organic solvent containing solution prior to contacting the nanoparticle with the biodegradable polymer.

51. The method of claim 36, wherein the organic solvent containing solution comprises acetone, ethanol and/or THF.

52. The method of claim 36, wherein the organic solvent containing solution comprises 80% to 95% organic solvent.

53. The method of claim 36, wherein the polymeric carrier molecule additionally comprises an amino acid component (AA).sub.x, wherein x is an integer selected from a range of about 1 to 100.

54. The method of claim 36, wherein component P.sup.2 of the polymeric carrier is selected from a polypeptide comprising the formula (IIb):
Cys{(Arg).sub.l;(Lys).sub.m;(His).sub.n;(Orn).sub.o;(Xaa).sub.x}Cys,  (formula IIb) wherein l+m+n+o+x=8-16, and l, m, n or o are independently any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, provided that the overall content of Arg, Lys, His and Orn represents at least 10% of all amino acids of the polypeptide; and Xaa may be any amino acid selected from native or non-native amino acids except of Arg, Lys, His or Orn; and x may be any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, provided, that the overall content of Xaa does not exceed 90% of all amino acids of the polypeptide.

55. The method of claim 36, wherein component P.sup.2 of the polymeric carrier comprises at least 3 Arg amino acids.

Description

FIGURES

[0292] The following Figures are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

[0293] FIG. 1: Expression of Luciferase in medium supernatant of endothelial cells. 100,000 EA.hy 926 cells were cultured on cover slips coated by PLGA with mRNA containing nanoparticles. Different PLGAs were used. The supernatant of the cells was used to measure Luciferase expression after 0, 6 hours, 24 hours, 48 hours and 72 hours and 6 days. After each measure point cells were washed excessively with PBS for several times to remove luciferase. The RLU of the luciferase assay was always added to the previous measurement. The control was a cell cultured coverslips without coating.

[0294] FIG. 2: Schematic diagram of the application of metallic plates coated with mRNA containing nanoparticles by PLGA in porcine vein grafts. MRNA containing nanoparticles were coated by PLGA on phynox plates. Therefore mRNA comprising nanoparticles were together with PLGA pipetted in stainless steel plates (phynos plates). The solution was again dried over night. Freshly excised porcine vein of the jugularis were cut longitudinal. The veins were placed into one 6-well with 2 ml endothelial medium (Vasculife, endothelial growth medium). Afterwards the coated phynox plate was laid with the coated side on the endothel of the vein.

[0295] FIG. 3: Expression of Luciferase in medium supernatant after 24 hours of transfection of porcine vein grafts with immobilized Luciferase mRNA containing nanoparticles (20 μg/plate) coated on phynox plates with the help of Lactel-PLGA (40 μg/plate). The procedure corresponds to FIG. 2. The supernatant of the medium was measured as described in Example 5.

[0296] FIG. 4: Expression of Luciferase in medium supernatant after 24 hours of transfection of porcine vein grafts with immobilized Luciferase mRNA containing nanoparticles (20 μg/plate) coated on phynox plates with the help of Lactel-PLGA (40 μg/plate). In this experiment the mRNA containing nanoparticles were prepared in different solutions; mannose containing solution or H.sub.2O.

[0297] FIG. 5: shows the mRNA sequence encoding Gaussia luciferase (SEQ ID NO: 128). [0298] The mRNA sequence contains following sequence elements: [0299] the coding sequence encoding Gaussia luciferase; [0300] stabilizing sequences derived from alpha-globin-3′-UTR (muag (mutated alpha-globin-3′-UTR)); [0301] 70× adenosine at the 3′-terminal end (poly-A-tail); [0302] 30× cytosine at the 3′-terminal end (poly-C-tail).

[0303] FIG. 6: shows the corresponding DNA sequence encoding Gaussia luciferase (SEQ ID NO: 129).

EXAMPLES

[0304] The following examples are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto. [0305] 1. Preparation of DNA and mRNA Constructs Encoding Gaussia luciferase (Gaussia) [0306] For the present examples DNA sequences, encoding Gaussia luciferase, were prepared and used for subsequent in vitro transcription reactions. [0307] According to a first preparation, the DNA sequence was prepared, which corresponds to the Gaussia luciferase coding sequence. Additionally sequences derived from alpha-globin-3′-UTR (muag (mutated alpha-globin-3′-UTR)), a histone stem-loop sequence, a stretch of 70× adenosine at the 3′-terminal end (poly-A-tail) and a stretch of 30× cytosine at the 3′-terminal end (poly-C-tail) were introduced 3′ of the coding sequence. [0308] The sequence contains following sequence elements: [0309] the coding sequence encoding Gaussia luciferase; [0310] stabilizing sequences derived from alpha-globin-3′-UTR (muag (mutated alpha-globin-3′-UTR)); [0311] 70× adenosine at the 3′-terminal end (poly-A-tail); [0312] 30× cytosine at the 3′-terminal end (poly-C-tail). [0313] 2. In Vitro Transcription: [0314] The respective DNA plasmid prepared according to Example 1 was transcribed in vitro using T7-Polymerase. Subsequently the mRNA was purified using PureMessenger® (CureVac, Tübingen, Germany). [0315] In SEQ ID NO: 129 (see FIG. 6) the sequence of the DNA corresponding to the mRNA is shown. [0316] 3. Reagents: [0317] Peptides: The peptides used in the present experiments were as follows: [0318] PB83: HO-PEG.sub.5000-S—(S-CHHHHHHRRRRHHHHHHC-S-).sub.7-S-PEG.sub.5000-OH [0319] 4. Synthesis of the Polymeric Carrier: [0320] The condensation reaction was performed with the calculated amount of peptide (component P.sup.2) which is dissolved in a mixture of a buffered aqueous solution at pH 8.5 with an optional additive of 5% (v/v) Dimethylsulfoxide (DMSO) (which are mild oxidation conditions and therefore allow the establishment of an equilibrium) and stirred for 18 h at ambient temperature. Afterwards the calculated amount of a thiol group containing PEG derivative (alpha-Methoxy-omega-mercapto poly(ethylene glycol)) (component P.sup.1) (dissolved in water) is added and the resulting solution is stirred for another 18 h. Subsequent lyophilisation and purification yield the desired polymer. The ratio between PEG component P.sup.1 to peptide component P.sup.2 defines the chain length of the P.sup.2 polymer. [0321] The condensation reaction in this reaction environment is reversible, therefore the chain length of the polymer is determined by the amount of the monothiol compound which terminates the polymerisation reaction. In summary the length of the polymer chain is determined by the ratio of oligo-peptide and monothiol component. This reaction is supported by the chosen mild oxidation conditions. With more stringent oxidation conditions (30% DMSO) the generation of high molecular (long chain) polymers is induced.

[0322] 4.1. 1. Step: Exemplary Polymerization Reaction: [0323] n HS-CHHHRRRHHHC-SH.fwdarw.H-(-S-CHHHRRRRHHHC-S).sub.n-H

[0324] 4.2. 2. Step: Exemplary Stop Reaction: [0325] H-(-S-CHHHRRRRHHHC-S).sub.n-H+2 PEG-SH.fwdarw.PEG-S-(S-CHHHRRRRHHHC-S).sub.n-S-PEG

[0326] 4.3. Exemplary Synthesis Reaction:

[0327] Step 1) [0328] 5×HS-CHHHRRRHHHC-SH.fwdarw.H-(S-CHHHRRRRHHHC-S).sub.5-H

[0329] Step 2) [0330] H-(S-CHHHRRRRHHHC-S).sub.5-H+2×PEG.sub.5000-SH [0331] .fwdarw.PEG.sub.5000-S-(S-CHHHRRRRHHHC-S).sub.5—S-PEG.sub.5000 [0332] To achieve a polymer length of 5 (n=5), a molar ratio of peptide:PEG of 5:2 was used. [0333] Some variations of the synthesis reaction were done to show the effect of the PEGchains and the effects of the reversible attachment of the PEG chains.

[0334] 4.4. Synthesis Reaction for Polymeric Carriers without PEG Chains: [0335] The reaction conditions are the same as mentioned above, but the step of the addition of a sulfhydryl containing PEG derivative is not performed/skipped.

[0336] 4.5. Synthesis Reaction for Irreversible Attached PEG Chains: [0337] The reaction conditions are the same as mentioned above, but instead of a sulfhydryl containing PEG derivative a maleimide containing PEG derivative is utilized. The maleimide moiety reacts rapidly with free sulfhydryl groups forming a covalent bond. Therefore the termination of the polymerization is not under the dynamic equilibria conditions as for sulfhydryl containing PEG derivatives but under irreversible conditions which results in a “frozen” polymerization pattern of high polydiversity and not the defined reaction products of the dynamic equilibria reaction. [0338] 5. Complexation of RNA: [0339] The mRNA construct defined above in Example 1 and prepared according to Example 2, were complexed for the purposes of the present invention with the polymeric carrier, preferably as defined in Example 4. Therefore, 4 μg RNA coding for Gaussia luciferase according to SEQ ID NO: 128 were mixed in molar ratios as indicated with the polymeric carrier (according to formula I) thereby forming a complex. Afterwards the resulting solution was adjusted with water to a final volume of 50 μl and incubated for 30 minutes at room temperature. [0340] Following ratio of polymeric carrier/RNA were used in the experiments:

TABLE-US-00005 Präfix Polymer Ratio Cationic AS N/P PB83 HO-PEG.sub.5000-S—(S—CHHHHHHRRRRHHHHHHC—S-).sub.7-S-PEG.sub.5000-OH 250 28 2.8 [0341] Ratio=molar ratio of RNA:peptide [0342] cationic AS=cationic amino acids, which are positively charged at a physiological pH (i.e. not histidine (H) but e.g. arginine (R)) [0343] N/P=is the ratio of basic nitrogen atoms in the polymeric carrier to phosphate residues in the nucleic acid, considering that nitrogen atoms confer to positive charges and phosphate of the phosphate backbone of the nucleic acid confers to the negative charge. Histidine residues are counted neutral, because complex formation is done at physiological pH, therefore the imidazole residue is uncharged. [0344] N/P is calculated by the following formula:

[00001]$N/P=\frac{p.Math..Math.\mathrm{mol}.Math.\left[\mathrm{RNA}\right]\star \mathrm{ratio}\star \mathrm{cationic}.Math..Math.\mathrm{AS}}{\mathrm{.Math.g}.Math..Math.\mathrm{RNA}\star 3\star 1000}$ [0345] For the calculations mRNA coding for Gaussia luciferase according to SEQ ID NO: 128 was applied, which has a molecular weight of kDa. Therefore 1 μg mRNA according to SEQ ID NO: 128 confers to pmol mRNA according to SEQ ID NO: 128 [0346] 6. Coating of the Polymeric Carrier Cargo Complexes with the Biodegradable Polymer PLGA:

[0347] Different PLGAs Used in the Experiments: [0348] 1. Lactel, PLGA, no indication of the molecular weight ratio 50:50, ester terminated [0349] 2. Sigma Aldrich, Resomer RG 752 H, catno. 719919, Poly(DL-lactide-co-glycolide) acid terminated, (75:25), MW 4000-15000 [0350] 3. Sigma Aldrich, Resomer RG 502 H, catno. 719897, Poly(DL-lactide-co-glycolide) acid terminated, (50:50), MW 7000-17000 [0351] 4. Sigma Aldrich, Resomer RG 504 H, catno. 719900, Poly(DL-lactide-co-glycolide) acid terminated, (50:50), MW 38000-54000
100 μl of the solution comprising the mRNA containing nanoparticles prepared according to example 5 (basal medium, 18 μg mRNA containing nanoparticles and 7 μl Interferin) were mixed with 100 μl of 0.05 PLGA solution (solved in 100% acetone). [0352] 7. Transfection of Endothelial Cells:

[0353] The solution as prepared according to example 6 was pipetted in duplets on coverslips (Thermanox Plastic Coverslips with 15 mm diameter) in one or in a second repeat to prevent solution from floating away from the cover slip. The solution on the coverslips was dried overnight at RT under sterile conditions and corresponds to 18 μg mRNA containing nanoparticles coated with 50 μg PLGA on one slide.

[0354] At the next day EA.hy 926 cells which are immortalized endothelial cells were cultured on the coverslips with 1 ml medium (DMEM high glucose, supplemented with L-Glutamine and antibiotics). Around 100,000 cells were cultured on one coverslip.

[0355] The measurement of secreted luciferase was measured 6 h, 24 h, 48 h, 72 h and 6 days after culturing. After each measurement cells were washed excessively with PBS to remove luciferase enzyme. For each measurement 20 μl of cell supernatant was pipetted into one well of a 96-well plate. The measurement buffer was consisting of PBS without Ca and Mg, supplemented with 5 mM NaCl and 0.04 mg Coelenterazien. With the Mithras LB 940 (Berthold) 100 μl of the measurement buffer was injected to the 20 μl supernatant. The luminescence detection (RLU) was performed for 10 sec by the Mithras apparatus. For calculating the luciferase expression the RLU of each measurement was added to the previous one. [0356] 8. Expression of Luciferase Ex Vivo:

[0357] 8.1. Coating of Phynoxplates (Stainless Steal Plates):

[0358] Nanoparticles containing mRNA coding for Gaussia Luciferase were formulated at a concentration of 0.1 g/l. 200 μl of this solution was mixed with an equal volume solution containing 40 μg PLGA in acetone. The coating solution was applied to the plates and after drying at RT over night the plates were ready for use in transfection studies.

[0359] 8.2. Transfection of Vein Grafts:

[0360] Freshly prepared lumen tissue of Vena jugularis externa (pig) were incubated in 6 well plates in 2 ml medium. The coated metal plates were placed on top of the tissue (endothelium), therefore enabling direct contact of the PLGA matrix to the cells. After 24 h the amount of expressed Gaussia luciferase was quantified in the supernatant (see Example 7). [0361] 11. Results:

[0362] 11.1. Expression of Luciferase in EA.hy926 Cells: [0363] The results show that all cells growing on coverslips with PLGA immobilized mRNA containing nanoparticles secrete luciferase, compared to control cells growing on slips without mRNA containing nanoparticles coding for Gaussia luciferase. Furthermore, cells grown on the PLGA resomer 752 with a ratio of 75:25 show the highest luciferase expression compared to the 50:50 resomers. The RLU of each measurement point was added to the previous one, resulting in a curve that shows a burst release between 0-24 h, by the dissociation of the mRNA out of the PLGA coating. Afterwards the expression of luciferase increases slow, which indicates the hydrolyzation of the PLGA.

[0364] 11.5. Expression of Luciferase Ex Vivo: [0365] Expression of luciferase in porcine vein grafts was determined 24 hours after application of metallic plates coated by PLGA with polymeric carrier cargo complexes comprising 20 μg mRNA coding for Gaussia luciferase. The secreted luciferase was again measured in the surrounding endothelial cell medium. As can be seen in FIG. 3, PLGA coating of the metallic plates with the mRNA containing nanoparticles leads to a high luciferase expression in the porcine vein grafts. [0366] Furthermore, expression of luciferase in porcine vein grafts 24 hours after application of metallic plates coated by PLGA with polymeric carrier cargo complexes is independent of the polymeric carrier medium which was containing mannose or H2O as shown in FIG. 4.