COMPOSITIONS AND METHODS FOR DELIVERING PHARMACEUTICALLY ACTIVE AGENTS

Abstract

The invention relates to modified lysines of the formula (I). The invention further relates to polypeptides comprising one or more modified lysine residues, pharmaceutical delivery systems comprising these polypeptides, pharmaceutical compositions containing them, and to their use in therapy.

##STR00001##

Claims

1. A modified lysine of formula (I): ##STR00028## wherein: A is a bond, C.sub.1-6alkylene, carbocyclyl or heterocyclyl; wherein said carbocyclyl or heterocyclyl may be optionally substituted on carbon by one or more R.sup.2; and wherein if said heterocyclyl contains an —NH— moiety that nitrogen may be optionally substituted by a group selected from R.sup.A; Q is a bond, carbocyclyl or heterocyclyl; wherein said carbocyclyl or heterocyclyl may be optionally substituted on carbon by one or more R.sup.3; and wherein if said heterocyclyl contains an —NH— moiety that nitrogen may be optionally substituted by a group selected from R.sup.B; Ring B is morpholinyl or thiomorpholinyl; wherein if said morpholinyl or thiomorpholinyl contains an —NH— moiety that nitrogen may be optionally substituted by a group selected from R.sup.C; R.sup.1, R.sup.2 and R.sup.3 are each independently selected from halo, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, carboxy, carbamoyl, mercapto, sulphamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulphinyl, ethylsulphinyl, mesyl, ethylsulphonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulphamoyl, N-ethylsulphamoyl, N,N-dimethylsulphamoyl, N,N-diethylsulphamoyl and N-methyl-N-ethylsulphamoyl; n is 0-4; R.sup.A, R.sup.B are R.sup.C are independently selected from methyl, ethyl, propyl, isopropyl, acetyl, mesyl, ethylsulphonyl, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, carbamoyl, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl and N-methyl-N-ethylcarbamoyl.

2. A modified lysine as claimed in claim 1 wherein A is a bond, C.sub.1-6alkylene or heterocyclyl.

3. A modified lysine as claimed in claim 1 or claim 2 wherein Q is a bond.

4. A modified lysine as claimed in any one of claims 1-3 wherein n is 0.

5. A modified lysine as claimed in any one of claims 1-4 wherein Ring B is morpholinyl.

6. A modified lysine as claimed in any one of claims 1-4 wherein Ring B is thiomorpholinyl.

7. A modified lysine as claimed in any one of claims 1-4 wherein: A is a bond, methylene or a pyridyl; Q is a bond; Ring B is morpholinyl or thiomorpholinyl; and n is 0.

8. A modified lysine as claimed in any one of claims 1-7 which is a modified lysine of formula (IB): ##STR00029##

9. A modified lysine as claimed in any one of claims 1-4, 7 or 8 selected from: 2-amino-6-{[6-(morpholin-4-yl)pyridine-3-carbonyl]amino}hexanoic acid; 2-amino-6-[(thiomorpholine-3-carbonyl)amino]hexanoic acid; and 2-amino-6-[2-(morpholin-4-yl)acetamido]hexanoic acid.

10. A polypeptide comprising one or more modified lysine residues as claimed in any one of claims 1-9.

11. A polypeptide as claimed in claim 10 comprising 20-50 amino acid residues.

12. A polypeptide as claimed in either claim 10 or claim 11 wherein fewer than 50% of the amino acid residues are modified lysine residues.

13. A polypeptide as claimed in any one of claims 10-12 wherein fewer than 50% of the amino acid residues are modified lysine residues and the remainder are unmodified lysine residues.

14. A polypeptide as claimed in any one of claims 10-13 further comprising a polyethylene glycol polymer.

15. A polypeptide as claimed in any one of claims 10-14 for use as a pharmaceutical delivery system.

16. A pharmaceutical composition which comprises a polypeptide as claimed in any one of claims 10-14 and a pharmaceutically active agent.

17. A pharmaceutical composition as claimed in claim 16 wherein the pharmaceutically active agent is genetic material.

18. A pharmaceutical composition as claimed in claim 17 wherein the genetic material is DNA.

19. A method of gene therapy in a warm-blooded animal, such as man, which comprises administering to said animal an effective amount of a pharmaceutical composition as claimed in any one of claims 16-18.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0283] FIG. 1A: Shows the results from Buffering Experiment 1a): Acid-Base Titration. The buffering properties in the pH range of 4.5-6.5 of the indicated polymers in solution are depicted in the graph.

[0284] FIG. 1B: Shows the results from Buffering Experiment 1b): Lysosomal Buffering. The buffering properties of the identified polymers in live cells is presented as the Mander's Overlap Coefficient (0-1): green signal (neutral pH) colocalized with red signal (acidic pH)/overall green signal. Mander's coefficient a measure from 0 to 1 that can roughly be defined as the ratio of complexes that acidified versus total complexes. If buffering happens, the cells stay green, if it does not, the cells become increasingly red.

[0285] FIG. 2A. Shows the results from Nanoparticle Stability Experiment 2a) Anionic Dissociation. Stability assessment of nanoparticles towards anionic displacement of pDNA by dextran sulfate (DS) quantified by DNA band intensities of nanoparticles exposed to different concentrations of DS and analysed via gel electrophoresis.

[0286] FIG. 2B. Shows the results from Nanoparticle Stability Experiment 2b) Nanoparticle stability in the bloodstream. Representative intravital ear-lobe PK images acquired at different time points post-tail vein injection of Cy5-pDNA nanoparticles where signal in the vascular structures indicated the NPs remain in circulation. Scalebars represent 100 μm.

[0287] FIG. 3A. Shows the results from Nanoparticle Transfections Experiment 3). Transfection of H1299 cells. Normalized luminescence after cells were treated with poly-L-lysine (PLL) nanoparticles encoding luciferase in OPTI-MEM.

[0288] FIG. 3B. Shows the results from Nanoparticle Transfections Experiment 3). Transfection of H1299 cell in Serum. Normalized luminescence after cells were treated with PLL nanoparticles encoding luciferase in media supplemented with 10% FBS and 100 nM chloroquine.

[0289] FIG. 4 Shows the results from Nanoparticle Transfections Experiment 3). C2C12 Myotube Transfection. Normalized luminescence after cells were treated with PLL nanoparticles encoding luciferase under the following conditions: (A) OPTI-MEM, (B), OPTI-MEM supplemented with 100 μM chloroquine, (C) media supplemented with FBS and (D) media supplemented with 100 μM chloroquine and 10% FBS.

[0290] FIG. 5 Shows the results from Intramuscular Transfection Experiment 5). Mouse Intramuscular Luciferase Expression. Luminescence in mouse hind limbs post intramuscular injection with 5 μg of complexed pDNA assessed via IVIS and IVIS Perkin Elmer software (n=8).

EXAMPLES

Abbreviations Used Herein

[0291] The following shorthand is used herein to denote a particular polymer or nanoparticie: [0292] “P” or “N”: “PLL” or “PEG-PLL” (modification) % modification

e.g. “P: PLL(M) 33” and “N: PEG-PLL(TM) 30”.

[0293] Key: [0294] P: Polymer; [0295] N: Nanoparticle; [0296] PLL: Poly-L-lysine is the polypeptide; [0297] PEG-PLL: The polypeptide comprises both a polyethylene glycol polymer and a poly-L-lysine polypeptide; [0298] Modification: is the modification at the ε-nitrogen of the lysine (as depicted in formula (I)) according to the following key “M”, “MN” or “TM”:

##STR00014## [0299] % Modification: refers to:

[00001]$\frac{\mathrm{modified}\epsilon -\mathrm{nitrogens}}{\mathrm{modified}\epsilon -\mathrm{nitrogens}+\mathrm{unmodified}\epsilon -\mathrm{nitrogens}}\times 100\%$

Method 1

[0300] Synthesis of 1-{[(morpholin-4-yl)acetyl]oxy}pyrrolidine-2.5-dione

##STR00015##

[0301] (Morpholin-4-yl)acetic acid (1 & 6.89 mmol) was dissolved in dichloromethane (DCM) (25 mL) and N-hydroxysuccinimide (NHS) (872 mg, 7.58 mmol) and N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.Math.HCl) (1.60 & 8.35 mmol) were added. The reaction was stirred at room temperature for 1 h before filtering through a 2″×3″ pad of silica gel. The pad was washed with DCM (3×25 mL) and the filtrate and washings were combined and concentrated to give 1-{[(morpholin-4-yl)acetyl]oxy}pyrrolidine-2,5-dione (1.3 & 78%) as white solid. .sup.1H NMR (300 MHz, CDCl.sub.3) δ 3.75 (d, J=3.6 Hz, 4H), 3.57 (s, 2H), 2.85 (s, 4H), 2.67 (d, J=4.2 Hz, 4H); MS (ESI) calc.:242.09, obs.:243.3 (M+1).

Method 2

[0302] Synthesis of 1-{[6-(morpholin-4-yl)pyridine-3-carbonyl]oxy}pvrrolidine-2,5-dione

##STR00016##

[0303] 6-(Morpholin-4-yl)pyridine-3-carboxylic acid (350 m& 1.68 mmol) was dissolved in DCM (25 mL) at room temperature with stirring. NHS (213 mg, 1.85 mmol) was added followed by EDC.Math.HCl ((418 mg, 2.18 mmol). The reaction mixture was stirred at room temperature for 1 h and then filtered through a 2″×3″ pad of silica gel. The pad was washed with DCM (3×25 mL) and ethyl acetate (25 mL). The filtrate and washings were combined and concentrated to give 1-{[6-(morpholin-4-yl)pyridine-3-carbonyl]oxy}pyrrolidine-2,5-dione (325 mg, 63%) as a white solid. .sup.1H NMR (300 MHz, CDCl.sub.3) δ 8.89 (s, 1H), 8.07 (dd, J=9.3 Hz, 1.5 Hz, 1H), 6.60 (d, J=9.3 Hz, 1H), 3.80 (d, J=4.2 Hz, 4H) 3.72 (d, J=4.2 Hz, 4H), 2.89 (s, 4H). MS (ESI) cak.: 305.1, obs.: 306.3 (M+1).

Method 3

[0304] Synthesis of tert-butyl 3-{[(2,5-dioxopyrrolidin-1-yl) oxy]carbonyl}thiomorpholine-4-carboxylate

##STR00017##

[0305] 4-(tert-Butoxycarbonyl)thiomorpholine-3-carboxylic acid (1 & 4.04 mmol) was dissolved in DCM (25 mL) at room temperature with stirring. NHS (511 m& 4.44 mmol) was added followed by addition of EDC.Math.HCl (1.01 & 5.25 mmol). The reaction mixture was stirred at room temperature for 1 h and then filtered through a 2″×3″ pad of silica gel. The pad was washed with DCM (3×25 mL) and the filtrate and washings were combined and concentrated to give tert-butyl 3-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}thiomorpholine-4-carboxylate (1.3 & 93.4%) as a white solid. .sup.1H NMR (300 MHz, CDCl3) δ 5.38 (br s, 1H), 4.43-4.22 (m, 1H), 3.46-3.21 (m, 1H), 3.19-3.10 (m, 1H), 3.06-2.97 (m, 1H), 2.85 (s, 4H), 2.81-2.62 (m, 1H), 2.61-2.42 (m, 1H), 1.47 (s, 9H). MS (ESI) calc.: 345.4 (M+1).

Method 4

[0306] Synthesis of N2-(((9H-fluoren-9-vllmethoxvlcarbonvll-N6-(6-morpholinonicotinovll-L-lysine

##STR00018##

[0307] Fluorenylmethyloxycarbonyl chloride L-Iysine (Fmoc-Lys-OH) (15.3 & 41.53 mmol, 1.2 eq) was dissolved in THF-water (1:1, 800 mL) under mechanical stirring at room temperature. A solution of the above prepared ester (Method 2) in DCM was added in one portion followed by DIPEA (10.73 g, 82.99 mmol, 2.4 eq). The reaction was stirred further at room temperature until consumption of the starting material (TLC, 2 h), then ethyl acetate (EtOAc) (250 mL) was added. The mixture was acidified with HQ (1M, 200 mL), poured into a separatory funnel, and the layers separated. The aqueous layer was extracted with EtOAc (2×250 mL). The organic layers were combined, washed with brine (200 mL), dried over anhydrous Na2504, filtered, and concentrated under vacuum. The crude product was obtained as light brown coloured oily residue which was dissolved in THF, adsorbed on silica gel and purified by flash chromatography over a column (7″×3″) of silica gel. The column was washed with 50% ethyl acetate in hexanes and 100% ethyl acetate to elute the product under vacuum suction. The fractions containing the required product were combined and concentrated under vacuum to provide N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(6-morpholinonicotinoyl)-L-lysine (12.6 g, 65%) as off-white coloured solid. .sup.1H NMR (500 MHz, CDCl3) δ 9.26 (br s, 1H), 8.62 (d, J=1.5 Hz, 1H), 7.93 (dd, J=2.5, 9 Hz, 1H), 7.71 (d, J=7.5 Hz, 2H), 7.53 (dd, J=4.5, 7.5 Hz, 2H), 7.35 (t, J=7.5 Hz, 2H), 7.23 (q, J=6.5 Hz,2H), 6.59 (t, J=5 Hz, 1H), 6.48 (d, J=9 Hz, 1H), 5.99 (d, J=8 Hz, 1H), 4.41 (dd, J=7.5, 12.5 Hz, 1H), 4.31 (dd, J=12, 18 Hz, 2H), 4.15 (d, J=7 Hz, 1H), 3.71 (t, =4.5 Hz, 4H), 3.56-3.32 (m, 6H), 1.98-1.87 (m, 1H), 1.86-1.75 (m, 1H), 1.71-1.57 (m, 2H), 1.56-1.39 (m, 2H) ppm; 13C NMR (125 MHz, CDCl3) δ 175.2, 166.6, 159.9, 156.6, 146.9, 144.1, 143.9, 141.4, 137.7, 127.9, 127.3, 125.3, 120.1, 119.5, 106.3, 67.2, 66.6, 53.8, 47.3, 45.3, 39.5, 32.0, 28.9, 22.4 ppm; MS (ESI) Exact mass cald. for C31H34N4O6 [M+H]+: 559.26, found: 559.35.

Example 1

[0308] Synthesis of (2S)-2-amino-6-{[6-(morpholin-4-yl)pyridine-3-carbonyl]amino}hexanoic acid (N6-(6-morpholinon icotinoyl)-L-lysine)

Method 1:

[0309] ##STR00019##

The Fmoc protecting group of N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(6-morpholinonicotinoyl)-L-lysine (Method 4) may be removed by standard procedures known in the art for example using 20% piperidine in DMF.

Method 2:

[0310] ##STR00020##

[0311] The modified lysine was dissolved in 700 μL of NMP (15 mg, 26.87 μmol. 300 μL piperidine was subsequently added to the solution (Piperidine:NMP=7:3; 1 mL) while stirring. The reaction was stirred at room temperature for 30 minutes. The modified lysine was subsequently precipitated and washed 3 times in cold diethyl ether (10 mL) using centrifugation (4000 g, 10 minutes, 4° C.). .sup.1H NMR (500 MHz, CDCl.sub.3) was used to confirm Fmoc removal 1H NMR (500 MHz, CDI3) δ11.89 (s, 1H), 8.85 (dd, 1H), 7.89 (dd, 1H), 7.34 (dd, 1H), 3.52-3.71 (m, 8H), 3.35 (t, 1H), 2.72-2.61 (t, 2H), 2.01-1.57 (m, 6H) ppm. MS (ESI) Exact mass cald. [M+H2O]+: 352.55, found: 352.04.

Method 5

[0312] Synthesis of N2-(((9H-fluoren-9-vllmethoxvlcarbonvll-N6-(4-(tert-butoxvcarbonvllthiomoroholine-3-carbonyl)-L-lysine

##STR00021##

[0313] Fmoc-L-Lys-OH (8.94 & 24.26 mmol, 1.2 eq) was dissolved in THE-water (1:1, 800 mL) under mechanical stirring at room temperature. A solution of the above prepared activated ester (Method 3) in DCM was added in one portion followed by DIPEA (6.27 & 48.53 mmol, 2.4 eq). The reaction was stirred further at room temperature until consumption of the starting material (TLC, 2 h), then EtOAc (250 mL) was added. The mixture was acidified with HCl (1 M, 200 mL), poured into a separatory funnel, and the layers separated. The aqueous layer was extracted with EtOAc (2×250 mL). The organic layers were combined, washed with brine (200 mL), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was obtained as light yellow coloured oily residue which was dissolved in DCM, adsorbed on silica gel and purified by flash chromatography over a column (7″×3″) of silica gel. The column was washed with 50%-70% ethyl acetate in hexanes to elute the product under vacuum suction. The fractions containing the required product were combined and concentrated under vacuum to provide N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(4-(tert-butoxycarbonyl)thiomorpholine-3-carbonyl)-L-lysine (9.1 & 75%) as off-white coloured solid. .sup.1H NMR (500 MHz, CDCl3)δ7.75 (d, J=7.5 Hz, 2H), 7.63-7.51 (m, 2H), 7.38 (t, J=7.5 Hz, 2H), 7.29 (t, J=7.5 Hz, 2H), 5.71 (dd, J=7.5, 23 Hz, 1H), 4.97 (br s, 1H), 4.57-4.23 (m, 4H), 4.20 (t, J=7 Hz, 1H), 3.51-3.18 (m, 3H), 3.17-2.96 (br s, 1H), 2.77 (d, J=12.5 Hz, 1H), 2.70-2.58 (m, 1H), 2.38 (d, J=12.5 Hz, 1H), 1.98-1.86 (m, 1H), 1.85-1.73 (m, 1H), 1.66-1.53 (m, 2H), 1.46 (br s, 12H) ppm; 13C NMR (125 MHz, CDCl3) δ 175.0, 156.4, 155.8, 143.9, 141.4, 127.9, 127.3, 125.3, 120.1, 67.3, 60.6, 53.8, 47.3, 39.2, 31.5, 29.1, 28.5, 26.7, 22.3 ppm; MS (ESI) Exact mass cald. for C.sub.31H.sub.39N.sub.3O.sub.7S [M+Na]+: 620.24, found: 620.35.

Example 2

[0314] Synthesis (2S1-2-amino-6-1(thiomorpholine-3-carbonvllaminolhexanoic acid (N6-(thiomorpholine-3-carbonyl)-L-lysine)

Method 1:

[0315] ##STR00022##

[0316] The Fmoc protecting group of N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(4-(tert-butoxycarbonyl)thiomorpholine-3-carbonyl)-L-lysine (Method 5) may be removed by standard procedures known in the art for example using 20% piperidine in DMF. Similarly, the Boc protecting group may be removed by standard procedures known in the art for example using 30% TFA in DCM.

Method 2:

[0317] ##STR00023##

[0318] The modified lysine was dissolved in 700 μL of NMP (15 mg, 25.12 μmol. 300 μL piperidine was subsequently added to the solution (Piperidine:NMP=7:3; 1 mL) while stirring. The reaction was stirred at room temperature for 30 minutes to remove the Fmoc protectant group. The modified lysine was subsequently precipitated and washed 3 times in cold diethyl ether (10 mL) using centrifugation (4000 g, 10 minutes, 4° C.). After air drying overnight, the product was dissolved in 50% TFA:DCM (1 mL) and stirred at room temperature for 15 minutes. The TFA:DCM solution was removed using a rotary evaporator and the precipitated and washed 2 times in cold ethyl ether Fmoc removal was confirmed using .sup.1H NMR: δ11.56-12.04 (1H, br), 7.34 (s, 1H), 3.65-3.76 (dd, 2H), 3.42 (t, J =7.3 Hz, 1H), 3.01-3.15 (m, 2H), 2.70-2.89 (m, 2H), 2.55-2.61 (t, J=5.6 Hz, 2H), 2.06-2.18 (m, 2H), 1.74-1.85 (m, 2H), 1.58-1.64 (q, 2H) 1.05 (1H, s) ppm and MS (ESI) Exact mass cald. [M+H2O]+: 279.22, found: 279.62.

Method 6

[0319] Synthesis of N2(1-{[(morpholin-4-yl)acetyl]oxy}pyrrolidine-2,5-dione)-L-lysine

##STR00024##

[0320] The following procedure could be employed to generate N2(1-{[(morpholin-4-yl)acetyl]oxy}pyrrolidine-2,5-dione)-L-lysine, Fmoc-L-Lys-OH 1.2 eq) may be dissolved in THE-water (1:1, 800 mL) under mechanical stirring at room temperature. A solution of the above prepared activated ester in DCM may be added in one portion followed by DIPEA (2.4 eq). The reaction may be stirred further at room temperature until consumption of the starting material (TLC, 2 h), then EtOAc (250 mL) may be added. The mixture may be acidified with HCl (1 M, 200 mL), poured into a separatory funnel, and the layers separated. The aqueous layer many be extracted with EtOAc (2×250 mL). The organic layers may be combined, washed with brine (200 mL), dried over anhydrous Na2504, filtered, and concentrated under vacuum. The crude product may be dissolved in DCM, adsorbed on silica gel and purified by flash chromatography over a column (7″×3″) of silica gel. The column may be washed with 50%-70% ethyl acetate in hexanes to elute the product under vacuum suction. The fractions containing the required product may be combined and concentrated under vacuum to provide N2-(N2(1-{[(morpholin-4-yl)acetyl]oxy}pyrrolidine-2,5-dione)-L-lysine.

Example 3

[0321] Synthesis of (2S)-2-amino-6-[2-(morpholin-4-yl)acetamido]hexanoic acid (N6-(2-morpholinoacetyl)-L-lysine)

Method 1:

[0322] ##STR00025##

[0323] The Fmoc protecting group of N2(1-{[(morpholin-4-yl)acetyl]oxy}pyrrolidine-2,5-dione)-L-lysine (Method 6) may be removed by standard procedures known in the art for example using 20% piperidine in DMF.

Method 2:

[0324] ##STR00026##

[0325] The modified lysine was dissolved in 700 μL of NMP (15 mg, 25.02 limo. 300 μL piperidine was subsequently added to the solution (Piperidine:NMP=7:3; 1 mL) while stirring. The reaction was stirred at room temperature for 30 minutes. The modified lysine was subsequently precipitated and washed 3 times in cold diethyl ether (10 mL) using centrifugation (4000 g, 10 minutes, 4° C.). Fmoc removal was confirmed using was confirmed using H-NMR: 1H NMR (500 MHz, CDI3) δ12.01 (br s, 1H), 3.62-3.74 (m, 4H), 3.40 (t, 1H), 3.29 (s, 2H), 3.10 (t, 1H), 2.59-2.70 (m, 4H), 1.88-2.01 (m, 2H), 1.58-1.65 (m, 2H), and 1.46-1.56 (q, 2H) and MS (ESI) Exact mass cald. [M+H2O]+: 273.17, found: 273.33.

Example 4

Poly(L-Lysine) and PEG-Poly(L-Lysine) Polypeptides

[0326] ##STR00027##

[0327] Modified PEG-PLL and PLL was prepared by reacting the NHS-esters prepared in Methods 1-3 with the corresponding PLL (MW 5000, Alamanda Polymers Inc., Huntsville Ala.) or PEG-PLL (MW 13000, Alamanda Polymers Inc., Huntsville Ala.). All polymers were supplied with a polymerisation initiator residue (referred to herein simply as PLL) or MeO-PEG-(CH.sub.2).sub.2—NH— group (referred to herein as PEG-PLL) at the terminal carboxy end of the PLL. PLL (20 mg, 2.5 limo) or PEG-PLL (20 mg, 1.5 μmot) was dissolved in freshly prepared 0.1 M sodium bicarbonate, pH 8.0 (4 mL). 40 mM of the compounds of Methods 1, 2 or 3 were prepared in dimethylacetamide (DMAC) (1.5 molar excess) and added dropwise to the polymer solution while stirring. A series of PLLs with different degrees of modification were prepared by controlling the molar feed ratio of the NHS-esters. Modification reactions were conducted at 12.5 to 50 molar equivalents NHS ester:polymer. The reaction was stirred at room temperature for 1 hour, and non-reacted groups were subsequently removed through dialysis in phosphate buffered saline (PBS) pH 7.4 and then water using a dialysis cassette with a molecular weight cut off (MWCO) of 3.5 kDa. For PLL(TM) and PEG-PLL(TM), the Boc protectant group on the intermediate products (*) was removed using a standard protocol used in prior art through incubation of the lyophilized product in 30% trifluoroacetic acid/DCM for 30 minutes. The degree of lysine modification was determined from the H-NMR spectra recorded in D20 by peak intensity ratio of β, y, and 6-methylene protons of Lys ((CH.sub.2).sub.3, δ=1.3-1.9 ppm) to the sum of peak intensities of methylene protons from morpholine rings of morpholine (M) (starting material Method 1) and morpholino-niacin groups (starting material Method 2) (MN) (CH.sub.2).sub.2, δ=3.86 and 2.58 ppm for morpholine and morpholino-niacin and (CH.sub.2).sub.2, δ=3.11 and 3.56 ppm for thiomorpholine (TM) (starting material Method 3), and the results are shown in the table below.

TABLE-US-00001 TABLE 1 Modified PLLs and PEG-PLLs Feed ratio (moles Polymer Polymer NHS:moles Modified Modified Unmodified # Abbreviation amine) lysines (%) lysines lysines 1 P: PLL(M) 24 0.25 24 12 38 2 P: PLL(M) 37 0.50 37 18.5 31.5 3 P: PLL(M) 53 1 53 26.5 23.5 4 P: PLL(MN) 24 0.25 24 12 38 5 P: PLL(MN) 32 0.50 32 16 34 6 P: PLL(MN) 51 1 51 25.5 24.5 7 P: PLL (TM) 21 0.25 21 10.5 39.5 8 P: PLL (TM) 39 0.50 39 16.5 33.5 9 P: PLL (TM) 50 1 50 25 25 10 P: PEG-PLL(M) 24 6.25 24 12 38 12 P: PEG-PLL(M) 35 0.50 35 17.5 32.5 13 P: PEG-PLL(M) 63 1 63 31.5 18.5 14 P: PEG-PLL(MN) 24 0.25 24 12 38 15 P: PEG-PLL(MN) 39 0.50 39 19.5 30.5 16 P: PEG-PLL(MN) 65 1 65 32.5 17.5 17 P: PEG-PLL (TM) 24 0.25 24 12 38 18 P: PEG-PLL (TM) 33 0.50 33 16.5 33.5 19 P: PEG-PLL (TM) 61 1 61 30.5 19.5

Example 5

[0328] Preparation and characterization of nanoparticles with genetic material

[0329] Nanoparticles were prepared by mixing polypeptide and nucleic acid solutions in a neutral buffer. Nanoparticles were assessed using standard techniques including dynamic light scattering (DLS), transmission electron microscopy (TEM), and ethidium bromide exclusion assays to confirm nanoparticle formation.

a) Nanooarticle Preparation

[0330] DNA (Gwiz Luciferase; Genlantis, San Diego, Calif.) (66.6 μg/mL) and polymer solutions were prepared in 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, pH 7.0. Polymer solutions were prepared with PLL (50 units, Alamanda Polymers, Inc., Huntsville Ala.), PEG (5K)-PLL (50 units) (Alamanda Polymers, Inc., Huntsville Ala.) and “P: PLL(M) 29”, “P: PLL(MN) 31”, “P: PLL(TM) 28”, “P: PEG-PLL(M) 33”, “P: PEG-PLL(MN) 31” and “P: PEG-PLL(TM) 30” (all prepared by a procedure analogous to Example 4). Polymer solutions also were prepared in HEPES so the ratio of amines (N) in the polymer to phosphates (P) in the DNA backbone (N:P ratio) would be between 0.5 and 10. DNA solutions were subsequently added drop-wise to polymer solutions while gently vortexing to ensure homogenous particles. DNA/polymer nanoparticles were allowed to form at room temperature for 30 minutes. The final concentration of DNA in the nanoparticles solution was 33.3 μg/mL. Initially, complexes were prepared a different N:P ratios (0.5-10) and DNA gel electrophoresis was used to determine the ratio required for nucleic acid condensation.

b) Dynamic Light Scattering (DLS)

[0331] DLS data was collected using a ZetaSizer Nano ZS instrument (Malvern Instruments Ltd., Worcester, UK) with a green laser (λ=532 nm) as the incident beam. All measurements were performed at 25° C. at a detection angle of 173′. Nanoparticle samples prepared as described above in 20 mM HEPES buffer (pH 7.4) at 33.3 μg of complexes pDNA/mL were loaded into a low volume ZEN2112 quartz cuvette (12.5 μL). The hydrodynamic diameter and polydispersity index (PDI) were derived using cumulant fit analysis. All measurements were conducted with automated attenuator adjustment and multiple scans (between 15-20) for accuracy. All data points represent the mean of three or more individually prepared samples.

TABLE-US-00002 TABLE 2 Summary of DNA nanoparticle properties Modified Hydrodynamic Zeta NP amines N/ diameter potential Polydispersity # Material (%).sup.a P.sup.f (nm).sup.b (mV).sup.c index.sup.b morphology.sup.d 1 N: PLL 0 0 3 90 ± 8 22 ± 1  0.34 ± 0.12 sphere 2 N: PLL(M) 29 29 5  95 ± 10 19 ± 3  0.36 ± 0.11 sphere 3 N: PLL(MN) 31 31 5 98 ± 8 20 ± 2  0.29 ± 0.08 sphere 4 N: PLL(TM) 28 28 5 91 ± 6 16 ± 3  0.29 ± 0.08 sphere 5 N: PEG-PLL 0 0 3 99 ± 8 1.7 ± 0.5 0.32 ± 0.09 mixed.sup.e 6 N: PEG-PLL(M) 33 33 5 100 ± 8  2.9 ± 0.4 0.29 ± 0.09 mixed.sup.e 7 N: PEG-PLL(MN) 31 31 5 106 ± 9  1.6 ± 0.7 0.34 ± 0.07 mixed.sup.e 8 N: PEG-PLL(TM) 30 30 5 103 ± 8  2.2 ± 0.4 0.31 ± 0.07 mixed.sup.e .sup.aDetermined by .sup.1H NMR .sup.bDetermined by dynamic light scattering .sup.cDetermined by electrophoretic light scattering/lase doppler electrophoresis .sup.dDetermined by transmission electron microscopy .sup.eToriods and rods .sup.fN/P = Molar amine to phosphate ratio

Results

Experiment 1

a) Buffering

[0332] Acid-base titrations of the polymer solutions were conducted to evaluate their buffering capacity/ability to maintain pH upon addition of acidic solution and intracellular lysosomal buffering studies were conducted to further assess the materials buffering capacity.

a) Acid-Base Titration

[0333] Polymer PEG (5K)-PLL (50 units) (Alamada Polymers), PEI (25K, Polysciences Inc), “P: PEG-PLL (M) 35” (Polymer 12), “P: PEG-PLL (MN) 39” (Polymer 15) and “P: PEG-PLL (TM) 33” (Polymer 18) solutions (3 mL) were prepared at 1 mg/mL in 150 mM NaCl and titrated to a pH of 11 by addition of 1 M NaOH. While stirring, polymer solutions were titrated to pH 4.5 with 0.1 M HCl at 5 μL increments. The buffering capacity was reported as the average volume (μL) needed to change the polymer solution pH by 0.5. The change in the protonation degree between extracellular neutral pH 7.4 and endosomal acidic pH 4.5 (Aa7.4-4.5) was calculated as the moles of HCl added to change the pH from 7.4 to 4.5 divided by the total moles of amine per solution. All titration experiments were performed in triplicate. The results are shown in FIG. 1A.

b) Lysosomal Buffering

[0334] Gwiz Luciferase (Genlantis, San Diego Calif.) was labelled with two fluorophores, a pH-insensitive green dye and a pH sensitive red dye to enable pH estimation by measuring the green:red fluorescence ratio colocalized in the same pixels. First, DNA was labelled using the MFP488 LabellT.Math.Nucelic Acid kit (Mirus Bio LLC, Madison Wis.) and then amine-functionalized with the Amine LabellT.Math.Nucelic Acid kit (Mirus Bio LLC, Madison Wis.) using the manufacturer's suggested protocol. Ethanol precipitation was used to removed non-reacted dye. The DNA was subsequently labelled with NHS ester pHRODO red (Thermo Fischer, Waltham, Mass.) and purified via ethanol precipitation. Spectrophotometry was used to confirm and quantify modification of the DNA with the different fluorophores (approximately 40 dyes per plasmid). MFP-488 and pHRODO red dual-labelled DNA was then used to prepare nanoparticles as described below. For cell studies, H1299 cells were seeded at 10,000 cells per well in 96-well plates and allowed to attach for 24 h in Roswell Park Memorial Institute (RPMI) media supplemented with 10% FBS and 1% P/S. Next, cells were washed twice with 100 μL of PBS and once with 100 μL of OPTI-MEM. DNA nanoparticles were prepared with PEI, PEG-PLL, “P: PEG-PLL (M) 35” (Polymer 12), “P: PEG-PLL (MN) 39” (Polymer (15) and “P: PEG-PLL (TM) 33” (Polymer 18)) by mixing DNA (66.6 μg/mL) and polymer solutions in 20 mM HEPES at an amine to phosphate ratio of 5 (final DNA concentration of 33.3 μg/mL) as described above. The NPs were then added to the cells at 0.1 μg of DNA per well in OPTI-MEM (1:10 dilution). After a 4-hour period, cells were treated with a Hoechst stain (5 μg/mL stock in PBS for 5 minutes) and imaged using fluorescent microscopy with the EVOS Cell Imaging System (Thermo Fischer, Waltham, Mass.). A scale bar approximating intracellular pH was generated by imaging fixed, permeabilized cells after nanoparticle treatment in buffers at acidic, neutral, and basic buffers. In this assay pHRODO red signal dramatically increases as pH decreases while MFP488 is pH insensitive, therefore acidic vesicles appear red or orange and neutral vesicles appear green. The results are shown in FIG. 1B.

Experiment 2

Nanoparticle Stability

[0335] Nanoparticle stability was assessed against anionic dissociation and in intravital pharma kinetic (PK) studies.

a) Anionic Dissociation

[0336] Nanoparticles were prepared as described above in Example 5 with PEG-PLL and Polymers 12, 15 and 19 at an N:P of 5 with 2 μg of Gwiz Luciferase DNA (Genlantis, San Diego, Calif.). 20 mM HEPES buffer and dextran sulphate (DS) (Sigma-Aldrich, 5 g/mL in 20 mM HEPES buffer) solution was added to each nanoparticle sample so the final pDNA concentration remained constant and the DS concentration varied between 0 and 200 mg/mL. After a 30-minute treatment period at 3TC, 15 μL of each formulation was added to a 2% electrophoresis gel (Thermo Fischer Scientific) with ethidium bromide and run for 10 minutes using the standard E- gel protocol. The results are shown in FIG. 2A.

b) Nanooarticle stability in the bloodstream

[0337] Nanoparticle stability in the bloodstream was determined by multi-photon confocal fluorescence microscopy imaging of blood vessels in the earlobes of mice. Nanoparticles solutions were prepared as described in Example 5 with PEG-PLL and Polymers 12, 15 and 19 using Cy5-labeled pDNA (20 dyes/plasmid) at 100 μg of DNA/mL in 5% trehalose solution (N:P=5). Balb/c mice were subsequently anesthetized with isoflurane in an induction chamber before being transferred to a nose cone located on the microscope stage. The ear of the mouse was then positioned and flattened using a custom slide holder and glass slide to enable imaging with a Leica SP8 DIVE multi-photon microscope. A 25×1.0 NA water immersion objective with an M32 back aperture was used for the imaging of the nanoparticles. The Cy5 dye was excited using the 1220 nM line of a Spectra-Physics X3 laser. Two non-descanned detectors of the DIVE system were used for imaging. One DIVE HyD detector was tuned to 605-615 nM for second harmonics imaging. The second detector was tuned to 635-775 nM for Cy5 emission detection. Second harmonics was used to locate a field of view with a vein and artery, after which mice were I.V. administered the nanoparticle formulations through a tail vein injection of 200 μL (20 μg of pDNA). Mice were imaged until the signal within the vessels equated that in the surrounding tissue or for a 2-hour period. Image) was used to generate maximum intensity projections of images collected over a is period for each specified time point. Each formulation was tested in a minimum of 3 mice. The results are shown in FIG. 2B.

Experiment 3

Nanoparticle Transfections

[0338] Nanoparticle stability was assessed against anionic dissociation and in circulation.

[0339] H1299 and C2C12 cells were seeded in 96-well plates at 10,000 cells/well in RPMI media or 20,000 cells/well in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin (P/S). Before transfection H1299 cells were allowed a recovery period of 241, while C2C12 myoblasts were differentiated into myotubes through incubation in DMEM supplemented with 2% horse serum for a minimum of 7 days. Subsequently, cells were washed twice with 100 μL of PBS and once with culture media or modified Eagle's Minimum Media (OPTI-MEM). Nanoparticles were prepared as described in Example 5 with PEI, PLL, “PLL (M) 37” (Polymer 2), “PLL (MN) 33” (Polymer 5), “PLL (TM) 39” (Polymer 8), PEG-PLL, “PEG-PLL (M) 35” (Polymer 12), “PEG-PLL (MN) 39” (Polymer 15), “PEG-PLL (TM) 33” (Polymer 18). Non-modified PEI, PLL and PEG-PLL nanoparticles were prepared with 1 μg of DNA at an N:P of 3 and the modified PLL nanoparticles at an N:P of 5. After complexation, NPs were added to the cells at 0.1 μg of DNA per well in either OPTI-MEM (1:10 dilution) or culture media. After a 16-hour period, the nanoparticle supplemented media was removed, and fresh culture media was added. Green fluorescent protein (GFP) expression was imaged using the Incucyte (Essen BioScience, Ann Arbor, Mich.) and luciferase/viability quantified using the standard protocol for the ONE-Glo™+ Tox Luciferase Reporter (Promega, Madison, Wis.) and PHERAstarFSX instrument (BMG Lab Tech, Cary, N.C.). The results are shown in FIGS. 3 and 4.

4) Toxicity

[0340] Material toxicity was assessed using live dead assays.

[0341] H1299 cells were seeded in 96-well plates (10000/well). After a 24 h recovery period, cells were treated with 0.1 to 2 μg of PEI (25K, Polyscience, Inc., Philadelphia Pa.), PLL (Alamanda Polymers, Inc., Huntsville Ala., 5 kDa), and P: PLL(M) 33, P: PLL(MN) 33, and P: PLL(TM) 33″ prepared in OPTI-MEM. After a 16-hour exposure, cells were analysed using a live/dead stain or metabolic assay. For the live/dead assay, 10 μL of Calcein AM (2 mM in DMSO) and 5 of propidium iodide (2 mM DMSO) stock solutions were dissolved in 5 mL of PBS. The cells were then washed with PBS twice and staining solution added (100 μL/well). After a 30-minute incubation period at 37° C., the cells were imaged using an IncuCyte plate reader (Sartorius). Alternatively, metabolic assays were performed using Celriter-Glo.Math.Luminescent Assay using the manufacturers standard protocol. Luminescence measurements were collected using a PHERAStar plate reader (BMG LabTech) and IC.sub.50 doses were derived by fitting the data in Microsoft excel.

TABLE-US-00003 TABLE 3 Toxicity/IC.sub.50 (μg/well) of polymer materials in vitro a Cell line tested material H1299.sup.a C2C12.sup.a P: PEI 0.43 0.82 P: PLL 0.21 0.33 P: PLL(M) 33 0.40 0.41 P: PLL(MN) 33 0.45 0.90 P: PLL(TM) 33 1.78 1.89

5) Intramuscular Transfection

[0342] Nanoparticle transfection efficiency was assessed in vivo through monitoring the expression of reporter protein luciferase after intramuscular injection.

[0343] Nude (nu/nu) mice were intramuscularly injected with 5 μg of DNA complexed with PEI, PEG-PLL or P: PEG-PLL (MN) 39 (Polymer (15)) in 50 μL of 5% trehalose solution per hind limb (n=2 per mouse). Expression was assessed daily/weekly using IVIS (Perkin Elmer) whereupon mice were administered 150 μL of Rediiect (30 mg/mL Luciferin-D) with an IP injection. Luminescence was collected in quadruplicate and presented as the mean+/−standard deviation. The results are shown in FIG. 5.