Poly(Beta-Amino Ester)s With Additives for Drug Delivery

Abstract

Disclosed are nanoparticles comprising an end-modified poly(β-amino ester) and an additive that is a sugar or sugar derivative, such as a sugar, a sugar alcohol or chitosan. The nanoparticles may be used in any field where polymers have been found useful, including in medical fields, particularly in drug delivery. The polymers are useful in delivering a polynucleotide such as DNA, RNA or siRNA, a small molecule or a protein. Also disclosed are compositions comprising said nanoparticles and an active agent, methods for preparing said nanoparticles, said nanoparticles and compositions for use in medicine, and in vitro methods using said nanoparticles and compositions.

Claims

1. A nanoparticle comprising an end-modified poly(β-amino ester) and 1 to 35 weight percent, relative to the end-modified poly(β-amino ester), of a sugar or sugar alcohol.

2. A nanoparticle according to claim 1, comprising 2 to 15 weight percent, relative to the end-modified poly(β-amino ester), of the sugar or sugar alcohol.

3. A nanoparticle according to claim 1 wherein the nanoparticle has a coating comprising a sugar or sugar alcohol.

4. A nanoparticle according to claim 3, comprising 1 to 18 weight percent, relative to the end-modified poly(β-amino ester), of the sugar or sugar alcohol.

5. A nanoparticle according to any one of claims 1 to 4, wherein the sugar or sugar alcohol is a sugar alcohol having the general formula HOCH.sub.2(CHOH).sub.nCH.sub.2OH wherein n is from 3 to 4

6. A nanoparticle according to claim 5, wherein the sugar alcohol is mannitol.

7. A nanoparticle comprising an end-modified poly(β-amino ester) and chitosan, or a pharmaceutically acceptable salt thereof, at 0.15 to 3.0 weight percent relative to the end-modified poly(β-amino ester).

8. A nanoparticle according to any preceding claim, wherein each end modification of the end-modified poly(β-amino ester) is independently selected from an oligopeptide and R.sub.y; wherein R.sub.y is selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, heteroalkyl, heterocycloalkyl, acyl, aryl or heteroaryl.

9. A nanoparticle according to any preceding claim, wherein the end-modified poly(β-amino ester) is a polymer of formula I: ##STR00021## wherein each L.sub.1 and L.sub.2 is independently selected from the group consisting of ##STR00022## O, S, NR.sub.x and a bond; wherein R.sub.x is independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, heteroalkyl, heterocycloalkyl, acyl, aryl or heteroaryl; L.sub.3 is independently selected from the group consisting of alkylene, alkenylene, heteroalkylene, heteroalkenylene, arylene, heteroarylene and ##STR00023## wherein T.sub.1 is ##STR00024## and T.sub.2 is selected from H, alkyl or ##STR00025## wherein L.sub.T is independently selected from the group consisting of: ##STR00026## O, S, NR.sub.x and a bond; wherein R.sub.x is independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, heteroalkyl, heterocycloalkyl, acyl, aryl or heteroaryl; L.sub.4 is selected from the group consisting of ##STR00027## L.sub.5 is independently selected from the group consisting of alkylene, alkenylene, heteroalkylene, heteroalkenylene, arylene or heteroarylene; R.sub.1, R.sub.2 and R.sub.T, where present, are independently selected from an oligopeptide and R.sub.y; wherein R.sub.y is selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, heteroalkyl, heterocycloalkyl, acyl, aryl or heteroaryl each R3 is independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, heteroalkyl, heterocycloalkyl, acyl, aryl or heteroaryl; and n is an integer from 5 to 10,000; or a pharmaceutically acceptable salt thereof.

10. The nanoparticle of claim 9, wherein R.sub.1 and R.sub.2 are both oligopeptides.

11. The nanoparticle of claim 9 or 10, wherein n is from 1 to 20.

12. The nanoparticle of any of claims 8 to 11, wherein R.sub.y is independently selected from a group consisting of hydrogen, —(CH.sub.2).sub.mNH.sub.2, —(CH.sub.2).sub.mNHMe, —(CH.sub.2).sub.mOH, —(CH.sub.2).sub.mCH.sub.3, —(CH.sub.2).sub.2(OCH.sub.2CH.sub.2).sub.mNH.sub.2, —(CH.sub.2).sub.2(OCH.sub.2CH.sub.2).sub.mOH or —(CH.sub.2).sub.2(OCH.sub.2CH.sub.2).sub.mCH.sub.3 wherein m is an integer from 1 to 20.

13. The nanoparticle according to any preceding claim, further comprising an active agent.

14. A composition comprising at least one nanoparticle of any preceding claim.

15. A method for preparing nanoparticles comprising the steps of (i) preparing an end-modified poly(β-amino ester) in the presence of a sugar or sugar alcohol and (ii) preparing nanoparticles from the product of step (i).

16. The method according to claim 15, wherein the method comprises the steps of (i-a) reacting the acrylate terminated intermediate of Formula II with compounds of formulae R.sub.1L.sub.1H and R.sub.2L.sub.2H in the presence of a sugar or sugar alcohol and (ii) preparing nanoparticles from the product of step (i).

17. The method according to claim 15 or 16, wherein the sugar or sugar alcohol is present at 1 to 35 weight percent relative to the end-modified poly(β-amino ester).

18. A method for preparing nanoparticles comprising the steps of (i) preparing nanoparticles from an end-modified poly(β-amino ester) and (ii) contacting the nanoparticles with a sugar or sugar alcohol.

19. The method according to claim 18, wherein the sugar or sugar alcohol is present at 1 to 35 weight percent relative to the end-modified poly(β-amino ester).

20. A method according to any one of claims 15 to 19, wherein the sugar alcohol is mannitol.

21. A method for preparing nanoparticles comprising the steps of (i) mixing an end-modified poly(β-amino ester) with chitosan and (ii) preparing nanoparticles from the product of step (i).

22. A method according to claim 21, wherein the chitosan is present at 0.15 to 3.0 weight percent relative to the end-modified poly(β-amino ester).

23. A method according to any one of claims 15 to 22 wherein the nanoparticles are prepared in the presence of an active agent.

24. A nanoparticle produced by the method of any of claims 15 to 23.

25. A nanoparticle according to any one of claim 1 to 13 or 24 or a composition according to claim 14 for use in medicine.

26. An in vitro method of inhibiting gene expression comprising contacting one or more cells or a tissue with a nanoparticle according to any one of claim 1 to 13 or 24 or a composition according to claim 14.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0170] FIG. 1 shows the mean diameter and zeta potential of nanoparticles according to the first aspect of the invention formulated with mannitol, trehalose and sucrose at various weight percents.

[0171] FIG. 2 shows the mean diameter and zeta potential of nanoparticles according to the third aspect of the invention coated with chitosan at various weight percents.

[0172] FIG. 3 shows a gel retardation assay of R/DNA coated with different molecular weights of chitosan at various weight percents.

[0173] FIG. 4 shows the mean diameter and zeta potential of nanoparticles according to the second aspect of the invention coated with mannitol, trehalose and sucrose at various weight percents.

[0174] FIG. 5 shows the mean size of nanoparticles in the presence of buffers of differing ionic strength and/or prepared by different procedures.

[0175] FIG. 6 shows the change in particle size over time for nanoparticles with differing amounts of chitosan.

[0176] FIG. 7 shows a second experiment monitoring the change in particle size over time for nanoparticles with differing amounts of chitosan.

[0177] FIG. 8 shows the change in particle size over time for nanoparticles with differing amounts of chitosan having different molecular weights.

[0178] FIG. 9 shows the change in count rate (in kilo counts per second) over time for nanoparticles with differing amounts of chitosan.

[0179] FIG. 10 shows the mean diameter and zeta potential of R/DNA nanoparticles coated with different molecular weights of chitosan at various weight percents.

[0180] FIG. 11 the effect on stability of coating nanoparticles with differing amounts of mannitol.

[0181] FIG. 12 shows the effect on stability of coating nanoparticles with mannitol, trehalose or sucrose.

[0182] FIG. 13 shows the change in particle size over time for nanoparticles coated with specific amounts of mannitol, trehalose and sucrose

[0183] FIG. 14 shows the change in particle size over time for three of the nanoparticles described in FIG. 12.

[0184] FIG. 15 shows the change in particle size over time for nanoparticles formulated with 20% mannitol or 20% sucrose.

[0185] FIG. 16 shows the change in particle size over time for nanoparticles coated with mannitol, formulated with mannitol, or without mannitol.

[0186] FIG. 17 shows the change in particle size over time for nanoparticles formulated with 20 weight percent mannitol.

[0187] FIG. 18 shows GFP expression in NRK-52e cells as analysed by flow cytometry, 48 hours after transfection with nanoparticles having differing amounts of chitosan.

[0188] FIG. 19 shows GFP expression in NRK-52e cells as analysed by fluorescence microscopy, 48 hours after transfection with nanoparticles having differing amounts of chitosan.

[0189] FIG. 20 shows GFP expression in NRK-52e cells as analysed by flow cytometry, 48 hours after transfection with nanoparticles having differing mixtures of PBAEs with or without chitosan.

[0190] FIG. 21 shows GFP expression in NRK-52e cells as analysed by fluorescence microscopy, 48 hours after transfection with nanoparticles having differing mixtures of PBAEs with or without chitosan.

[0191] FIG. 22 shows transfection efficacy of complexes with or without coating agents in COS-7 cells. (A) Fluorescent images of GFP expression in COS-7 cells: (i) RD; (ii) R/CS0.17; (iii) R/CSM0.17. (B) Percentage of GFP positive cells multiplied by the GeoMean fluorescence of the positive population. (C) GFP expression was determined after 48 h by flow cytometry.

[0192] FIG. 23 shows GFP expression in COS-7 cells after transfection with MntR20 nanoparticles prepared using different incubation conditions and durations.

[0193] FIG. 24 shows GFP expression in COS-7 cells after transfection with nanoparticles according the invention with different weight percents of additives.

[0194] FIG. 25 shows viability of cells after transfection with PBAE/DNA complexes modified with sugar or sugar alcohol.

[0195] The invention is further illustrated by the following examples. It will be appreciated that the examples are for illustrative purposes only and are not intended to limit the invention as described above. Modification of detail may be made without departing from the scope of the invention.

EXAMPLES

Materials and Methods

[0196] Reagents and solvents were obtained from Sigma-Aldrich and Panreac and used as received unless otherwise stated. H-Cys-Arg-Arg-Arg-NH2.4HCl (CR3.4HCl) was obtained from GL Biochem Ltd. (Shanghai, China). Chitosan with molecular weight 22 kg/mol and deacetylation degree 85% was purchased from Fluka. Chitosan with molecular weight 20-50 kg/mol and deacetylation degree 85% was purchased from Creative PEGWorks. Chitosan with molecular weight 60 kg/mol and deacetylation degree 60% was purchased from Sigma Aldrich. Chitosan with molecular weight 60-120 kg/mol and deacetylation degree 60% was purchased from Sigma Aldrich. Oligopeptides were obtained from GL Biochem Ltd. (Shanghai, China). Plasmid encoding green fluorescent protein (pmaxGFP, 3486 bp) was obtained from Amaxa (Gaithersburg, Md., USA). Cell lines were obtained from ATCC (Manassas, Va.) and maintained at 37° C. in 5% CO2 atmosphere in complete DMEM, containing 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.1 mM MEM Non-Essential Amino Acids (NEAA), 2 mM L-glutamine obtained from Gibco. All reagents were analytical grade and used without further purification.

[0197] Unless otherwise noted, the mean diameter, zeta potential and polydispersity of nanoparticles were determined, diluted in PBS to a final concentration of 0.25 mg/ml, by DLS using a Zetasizer nano zs90 (Malvern Instruments) at 25° C. Each experiment was carried out in triplicate and the mean result was reported.

[0198] .sup.1H-NMR spectra were acquired at 25° C. on a Varian NMR instrument operating at 400 MHz with samples dissolved in either deuterated dimethyl sulfoxide (d6-DMSO) or deuterated methanol (CD.sub.3OD) and using tetramethylsilane (TMS) as internal reference. 8-10 mg of sample dissolved in 1 ml of solvent was used for .sup.1H-NMR.

[0199] IR spectra were obtained using a Nicolet Magna 560 (Thermo Fisher Scientific, Waltham, Mass.) with a KBr beamsplitter, using methanol as solvent in evaporated film.

[0200] Molecular weight was determined by gel permeation chromatogram carried out at 35° C. with a refraction-index detector with 0.05 M tetrahydrofuran (THF) as mobile phase using a GPC KF-603 column with a flow rate of 0.5 ml/min. Chromatograms were calibrated against polystyrene monodisperse standards.

Example 1: Synthesis of PBAE Polymers

[0201] Acrylate-terminated poly(β-amino ester) C32 was obtained by mixing 1,4-butanediol diacrylate (8.81 g, 40 mmol) and 5-amino-1-pentanol (3.44 g, 33 mmol) in Teflon-lined screw cap vials. The mixture was left under stirring at 90° C. for 24 h. Then it was cooled to room temperature to form a transparent yellow viscous solid, C32, and was stored at −20° C. used for each experiment.

[0202] Weight-average molecular weight 2100 g/mol.

[0203] Number-average molecular weight 1320 g/mol.

[0204] .sup.1H NMR of C32 (d6-DMSO): δ (ppm) 1.2-1.4 (m, —NCH.sub.2(CH.sub.2).sub.3CH.sub.2OH), 1.6 (br, —N(CH.sub.2).sub.2COOCH.sub.2CH.sub.2— and CH.sub.2CHCOOCH.sub.2CH.sub.2—), 2.3-2.4 (m, —COOCH.sub.2CH.sub.2N— and —NCH.sub.2(CH.sub.2).sub.4OH), 2.6 (m, —COOCH.sub.2CH.sub.2N—), 3.4 (br, —N(CH.sub.2).sub.4CH.sub.2OH), 4.0 (br, —N(CH.sub.2).sub.2COOCH.sub.2CH.sub.2—), 4.1 (m, CH.sub.2CHCOOCH.sub.2CH.sub.2—), 4.3 (br, —N(CH.sub.2).sub.5OH), 5.9 (m, CH.sub.2CHCOO CH.sub.2CH.sub.2—), 6.1-6.2 (m, CH.sub.2CHCOOCH.sub.2CH.sub.2—), 6.3-6.4 (m, CH.sub.2CHCOO CH.sub.2CH.sub.2—).

Example 2: Synthesis of PBAEs End Modified with Oligopeptides

[0205] In general, oligopeptide-modified PBAEs may be obtained as follows: acrylate-terminated polymer C32 or C32SS and either amine- or thiol-terminated oligopeptide (for example, HS-Cys-Arg-Arg-Arg (CR3), H.sub.2N-Arg-Arg-Arg (R3) or HS-Cys-Glu-Glu-Glu (CE3)—other oligopeptides are indicated by similar abbreviations using the standard one-letter code) were mixed at 1:2.5 molar ratio in DMSO. The mixture was stirred overnight at room temperature and the resulting polymer may be obtained by precipitation in diethyl ether:acetone (3:1). The polymers may then be dissolved at 100 mg/ml in DMSO and stored at −20° C. until further use.

[0206] (a) In a typical example, for the R polymer, C32 (150 mg, 0.07 mmol), CR3.4HCl (115 mg, 0.18 mmol) and DMSO (3 ml) were placed in Teflon-lined screw cap vials and stirred at room temperature for 24 h. End-modified polymer, R3C-C32-CR3 (R) was purified by precipitation in diethyl ether/acetone (7:3 v/v) for twice and dried under vacuum. Dried polymers were finally dissolved at 100 mg/ml in DMSO and stored at −20° C. until further use.

[0207] The chemical structure of the oligopeptide-modified PBAEs was confirmed in .sup.1H-NMR spectroscopy by the disappearance of acrylate signals and the presence of signals typically associated with amino acid moieties.

[0208] .sup.1H-NMR of R (400 MHz, CD.sub.3OD, TMS) (ppm): δ=4.43-4.34 (br, NH.sub.2—C(═O)—CH—NH—C(═O)—CH—NH—C(═O)—CH—NH—C(═O)—CH—CH.sub.2—), 4.17 (t, CH.sub.2—CH.sub.2—O), 3.57 (t, CH.sub.2—CH.sub.2—OH), 3.22 (br, NH.sub.2—C(═NH)—NH—CH.sub.2—, OH—(CH.sub.2).sub.4—CH.sub.2—N—), 2.83 (dd, —CH.sub.2—S—CH.sub.2), 2.75 (m, CH.sub.2—CH.sub.2—N—), 2.48 (br, —N—CH.sub.2—CH.sub.2—C(═O)—O), 1.90 (m, NH.sub.2—C(═NH)—NH—(CH.sub.2).sub.2—CH.sub.2—CH—), 1.73 (br, —O—CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—O), 1.69 (m, NH.sub.2—C(═NH)—NH—CH.sub.2—CH.sub.2—CH.sub.2—), 1.56 (br, —CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—OH), 1.39 (br, —N—(CH.sub.2).sub.2—CH.sub.2—(CH.sub.2).sub.2—OH).

##STR00018##

[0209] IR (evaporated film): ν=721, 801, 834, 951, 1029, 1133 (C—O), 1201, 1421, 1466, 1542, 1672 (C═O, from peptide amide), 1731 (C═O, from ester), 2858, 2941, 3182, 3343 (N—H, O—H) cm.sup.−1

[0210] .sup.1H-NMR (400 MHz, CD.sub.3OD, TMS) (ppm): δ=4.41-4.33 (br, NH.sub.2—C(═O)—CH—NH—C(═O)—CH—NH—C(═O)—CH—NH—C(═O)—CH—CH.sub.2—, 4.11 (t, CH.sub.2—CH.sub.2—O), 3.55 (t, CH.sub.2—CH.sub.2—OH), 3.22 (br, NH.sub.2—C(═NH)—NH—CH.sub.2—, OH—(CH.sub.2).sub.4—CH.sub.2—N—), 3.04 (t, CH.sub.2—CH.sub.2—N—), 2.82 (dd, —CH.sub.2—S—CH.sub.2), 2.48 (br, —N—CH.sub.2—CH.sub.2—C(═O)—O), 1.90 (m, NH.sub.2—C(═NH)—NH—(CH.sub.2).sub.2—CH.sub.2—CH—), 1.73 (br, —O—CH.sub.2—CH2-CH2-CH.sub.2—O), 1.69 (m, NH.sub.2—C(═NH)—NH—CH.sub.2—CH.sub.2—CH.sub.2—), 1.56 (br, —CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—OH), 1.39 (br, —N—(CH.sub.2).sub.2—CH.sub.2—(CH.sub.2).sub.2—OH).

[0211] (b) in a further example, tri-lysine modified oligopeptides (K3C-C32-CK3) was prepared by mixing a solution of intermediate C32 in DMSO (2 ml) with the corresponding solution of oligopeptide (Cys-Lys-Lys-Lys (CK3) in DMSO (1 ml) in an appropriate molar ratio, 1:2 respectively. The mixture was stirred overnight at room temperature, then precipitated in diethyl ether/acetone (3:1).

[0212] IR (evaporated film): ν=721, 799, 834, 1040, 1132, 1179 (C—O), 1201, 1397, 1459, 1541, 1675 (C═O, from peptide amide), 1732 (C═O, from ester), 2861, 2940, 3348 (N—H, O—H) cm.sup.−1

[0213] .sup.1H-NMR (400 MHz, CD.sub.3OD, TMS) (ppm): δ=4.38-4.29 (br, NH.sub.2—(CH.sub.2).sub.4—CH—), 4.13 (t, CH.sub.2—CH.sub.2—O—), 3.73 (br, NH.sub.2—CH—CH.sub.2—S—), 3.55 (t, CH.sub.2—CH.sub.2—OH), 2.94 (br, CH.sub.2—CH.sub.2—N—, NH.sub.2—CH.sub.2—(CH.sub.2).sub.3—CH—), 2.81 (dd, —CH.sub.2—S—CH.sub.2), 2.57 (br, —N—CH.sub.2—CH.sub.2—C(═O)—O), 1.85 (m, NH.sub.2—(CH.sub.2).sub.3—CH.sub.2—CH—), 1.74 (br, —O—CH.sub.2-CH2-CH2-CH.sub.2—O), 1.68 (m, NH.sub.2—CH.sub.2—CH.sub.2—(CH.sub.2).sub.2—CH—), 1.54 (br, —CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—OH), 1.37 (br, —N—(CH.sub.2).sub.2—CH.sub.2—(CH.sub.2).sub.2—OH).

[0214] (c) Tri-histidine modified oligopeptides (H3C-C32-CH3) were prepared according to the same protocol as K3C-C32-CK3 and characterized as follows:

[0215] IR (evaporated film): ν=720, 799, 832, 1040, 1132, 1201, 1335, 1403, 1467, 1539, 1674 (C═O, from peptide amide), 1731 (C═O, from ester), 2865, 2941, 3336 (N—H, O—H) cm.sup.−1

[0216] .sup.1H-NMR (400 MHz, CD.sub.3OD, TMS) (ppm): 6=8.0-7.0 (br —N(═CH)—NH—C(═CH)—) 4.61-4.36 (br, —CH2-CH—), 4.16 (t, CH2-CH2-O—), 3.55 (t, CH2-CH2-OH), 3.18 (t, CH2-CH.sub.2—N—, 3.06 (dd, —CH2-CH—), 2.88 (br, OH—(CH.sub.2).sub.4—CH.sub.2—N—), 2.82 (dd, —CH.sub.2—S—CH.sub.2—), 2.72 (br, —N—CH.sub.2—CH.sub.2—C(═O)—O), 1.75 (br, —O—CH.sub.2-CH2-CH2-CH.sub.2—O), 1.65 (m, NH.sub.2—CH.sub.2—CH.sub.2—(CH.sub.2).sub.2—CH—), 1.58 (br, —CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.2—OH), 1.40 (br, —N—(CH.sub.2).sub.2—CH.sub.2—(CH.sub.2).sub.2—OH).

Example 3: Synthesis of PBAEs with Asymmetric End Modifications

[0217] In general, asymmetric oligopeptide-modified PBAEs were obtained as follows: Acrylate-terminated polymer C32 (or C32SS) and either amine- or thiol-terminated oligopeptide (for example, CR3, R3 or CE3) were mixed at 1:1 molar ratio in DMSO. The mixture was stirred overnight at room temperature. Equimolar amount of a second amine- or thiol-terminated oligopeptide, or of a primary amine, was added and the mixture was stirred overnight at room temperature. The resulting asymmetric PBAE polymers were obtained by precipitation in diethyl ether/acetone (3:1). The following synthetic procedure to obtain asymmetric end-modified B3-C32-CR3 PBAEs is shown as an example: a solution of intermediate C32 (0.15 g, 0.075 mmol) in DMSO (2 ml) was mixed with the corresponding solution of oligopeptide Cys-Arg-Arg-Arg (CR3; 0.055 g, 0.075 mmol) in DMSO (1 ml) and was stirred overnight at room temperature. Subsequently, 2-methyl-1,5-pentanediamine (0.017 g, 0.02 ml, 0.15 mmol) was added in the mixture for 4h at room temperature in DMSO. A mixture of asymmetric end-modified polymer B3-C32-CR3 with B3-C32-B3 and R3C-C32-CR3 was obtained by precipitation overnight in diethyl ether/acetone (3:1). The mixture may be used without further purification or the asymmetric end-modified polymer B3-C32-CR3 may be separated from the mixture by standard methods.

##STR00019## ##STR00020##

Example 4: Library of Compounds

[0218] A library of different oligopeptide end-modified PBAEs was synthesized by adding primary amines to diacrylates followed by end-modification. According to Formula I, the oligopeptide end-modified PBAEs shown in Table 1 were synthesized.

TABLE-US-00001 TABLE 1 Library of oligopeptide end-modified PBAEs Polymer L.sub.3 L.sub.4 HL.sub.1-R.sub.1 HL.sub.2-R.sub.2 B3 -CH.sub.2-(CH.sub.2).sub.2-CH2- >N-(CH.sub.2).sub.5-OH NH.sub.2-CH.sub.2-(CH.sub.2).sub.2-CH(CH.sub.3)-CH.sub.2-NH.sub.2 H.sub.2N-CH.sub.2-(CH.sub.2).sub.2-CH(CH.sub.3)-CH.sub.2-NH.sub.2 R3-C32-R3 (R) -CH.sub.2-(CH.sub.2).sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH H.sub.2N-Arg-Arg-Arg H.sub.2N-Arg-Arg-Arg K3-C32-K3 -CH.sub.2-(CH.sub.2).sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH NH.sub.2-Lys-Lys-Lys H.sub.2N-Lys-Lys-Lys H3-C32-H3 -CH.sub.2-(CH.sub.2).sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH NH.sub.2-His-His-His NH.sub.2-His-His-His R3C-C32-CR3 -CH.sub.2-(CH.sub.2).sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH HS-Cys-Arg-Arg-Arg HS-Cys-Arg-Arg-Arg K3C-C32-CK3 -CH.sub.2-(CH.sub.2).sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH HS-Cys-Lys-Lys-Lys HS-Cys-Lys-Lys-Lys H3C-C32-CH3 -CH.sub.2-(CH.sub.2).sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH HS-Cys-His-His-His HS-Cys-His-His-His B3-C32-R3 -CH.sub.2-(CH.sub.2).sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH H.sub.2N-CH.sub.2-(CH.sub.2).sub.2-CH(CH.sub.3)-CH.sub.2-NH.sub.2 H.sub.2N-Arg-Arg-Arg B3-C32-CR3 -CH.sub.2-(CH.sub.2).sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH H.sub.2N-CH.sub.2-(CH.sub.2).sub.2-CH(CH.sub.3)-CH.sub.2-NH.sub.2 HS-Cys-Arg-Arg-Arg B3-C32-CK3 -CH.sub.2-(CH.sub.2).sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH H.sub.2N-CH.sub.2-(CH.sub.2).sub.2-CH(CH.sub.3)-CH.sub.2-NH.sub.2 HS-Cys-Lys-Lys-Lys B3-C32-CH3 -CH.sub.2-(CH.sub.2).sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH H.sub.2N-CH.sub.2-(CH.sub.2).sub.2-CH(CH.sub.3)-CH.sub.2-NH.sub.2 HS-Cys-His-His-His R3C-C32-CK3 -CH.sub.2-(CH.sub.2).sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH HS-Cys-Arg-Arg-Arg HS-Cys-Lys-Lys-Lys R3C-C32-CH3 -CH.sub.2-(CH.sub.2).sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH HS-Cys-Arg-Arg-Arg HS-Cys-His-His-His K3C-C32-CH3 -CH.sub.2-(CH.sub.2).sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH HS-Cys-Lys-Lys-Lys HS-Cys-His-His-His B3-C32SS-B3 -CH.sub.2-CH.sub.2-S-S-CH.sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH H.sub.2N-CH.sub.2-(CH.sub.2).sub.2-CH(CH.sub.3)-CH.sub.2-NH.sub.2 H.sub.2N-CH.sub.2-(CH.sub.2).sub.2-CH(CH.sub.3)-CH.sub.2-NH.sub.2 R3C-C32SS-CR3 -CH.sub.2-CH.sub.2-S-S-CH.sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH HS-Cys-Arg-Arg-Arg HS-Cys-Arg-Arg-Arg K3C-C32SS-CK3 -CH.sub.2-CH.sub.2-S-S-CH.sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH HS-Cys-Lys-Lys-Lys HS-Cys-Lys-Lys-Lys H3C-C32SS-CH3 -CH.sub.2-CH.sub.2-S-S-CH.sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH HS-Cys-His-His-His HS-Cys-His-His-His B3-C32SS-CR3 -CH.sub.2-CH.sub.2-S-S-CH.sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH H.sub.2N-CH.sub.2-(CH.sub.2).sub.2-CH(CH.sub.3)-CH.sub.2-NH.sub.2 HS-Cys-Arg-Arg-Arg B3-C32SS-CK3 -CH.sub.2-CH.sub.2-S-S-CH.sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH H.sub.2N-CH.sub.2-(CH.sub.2).sub.2-CH(CH.sub.3)-CH.sub.2-NH.sub.2 HS-Cys-Lys-Lys-Lys B3-C32SS-CH3 -CH.sub.2-CH.sub.2-S-S-CH.sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH H.sub.2N-CH.sub.2-(CH.sub.2).sub.2-CH(CH.sub.3)-CH.sub.2-NH.sub.2 HS-Cys-His-His-His R3C-C32SS-CK3 -CH.sub.2-CH.sub.2-S-S-CH.sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH HS-Cys-Arg-Arg-Arg HS-Cys-Lys-Lys-Lys R3C-C32SS-CH3 -CH.sub.2-CH.sub.2-S-S-CH.sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH HS-Cys-Arg-Arg-Arg HS-Cys-His-His-His K3C-C32SS-CH3 -CH.sub.2-CH.sub.2-S-S-CH.sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH HS-Cys-Lys-Lys-Lys HS-Cys-His-His-His D3C-C32-CD3 -CH.sub.2-(CH.sub.2).sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH HS-Cys-Asp-Asp-Asp HS-Cys-Asp-Asp-Asp E3C-C32-CE3 -CH.sub.2-(CH.sub.2).sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH HS-Cys-Glu-Glu-Glu HS-Cys-Glu-Glu-Glu D3C-C32-CE3 -CH.sub.2-(CH.sub.2).sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH HS-Cys-Asp-Asp-Asp HS-Cys-Glu-Glu-Glu E3C-C32SS-CD3 -CH.sub.2-CH.sub.2-S-S-CH.sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH HS-Cys-Asp-Asp-Asp HS-Cys-Asp-Asp-Asp E3C-C32SS-CE3 -CH.sub.2-CH.sub.2-S-S-CH.sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH HS-Cys-Glu-Glu-Glu HS-Cys-Glu-Glu-Glu D3C-C32SS-CE3 -CH.sub.2-CH.sub.2-S-S-CH.sub.2-CH.sub.2- >N-(CH.sub.2).sub.5-OH HS-Cys-Asp-Asp-Asp HS-Cys-Glu-Glu-Glu

Example 5: Preparation of MntR, TreR and SucR

[0219] In a typical example of the first aspect of the invention, the R polymer was prepared in the presence of mannitol, trehalose or sucrose to form MntR, TreR or SucR, respectively. The procedure was as follows: C32 (75 mg, 0.035 mmol), CR3.4HCl (55 mg, 0.09 mmol) and DMSO (1.5 ml) were placed in Teflon-lined screw cap vials. Then, 10, 20, or 30% (w/w) of the sugar or sugar alcohol were added in each vial and stirred at room temperature for 24 h. The product was purified by precipitation in diethyl ether/acetone (7:3 v/v) for twice and dried under vacuum.

Example 6: Formation of Complexes of Polymer with DNA

[0220] Nanoparticles were formulated by mixing polymer and pGFP (plasmid green fluorescent protein) in a weight ratio of 50:1. For example, pGFP was diluted to 60 μg/ml in a final concentration of ˜5 mM sodium acetate (NaOAc) buffer at pH 5.5 and PBAE stock solution (100 μg/μl) in DMSO was diluted in the same buffer. 100 μl of diluted PBAE solution (3 μg/μl) was added to 100 μl of pGFP, and mixed with vortex for a few seconds and incubated at 37° C. for 30 min.

Example 7: Further Method for Formation of Complexes of Polymer with DNA

[0221] pGFP stock solution was diluted to 60 μg/ml in a final concentration of 11-12 mM of NaAc buffer (pH 5.2). Polymer stock solutions (for example R, MntR, TreR or SucR; 100 mg/ml in DMSO) were diluted in the same buffer. 100 μl of diluted pGFP was added into 100 μl of PBAE solutions (3 mg/ml), and mixed with vortex for a few seconds and incubated at room temperature for 10 min.

[0222] The mean diameter and zeta potential of nanoparticles of pDNA with polymer formed in the presence of 10, 20 or 30% of mannitol, trehalose or sucrose are shown in FIG. 1. MntR polymers were formulated with DNA at a weight ratio of 50:1. MntR/pDNA formed with 20 weight percent mannitol showed the smallest particle size (120.7±1.1 nm) and the highest zeta potential (22.5±1.1 mV). TreR polymers were formulated with DNA at a weight ratio of 100:1, which was general ratio of formulation PBAE/DNA complexes. Increasing the weight percent of trehalose from 10 to 30 decreased the mean size of TreR/pDNA nanoparticles from 202 to 154 nm and negligibly changed their surface charge. SucR:DNA ratio was 50:1. SucR/pDNA formed with 20 weight percent sucrose showed better results than that with 10 or 30 weight percent sucrose. However, the mean size and zeta potential of SucR/pDNA nanoparticles were larger and lower, respectively, than the other sugar-based nanoparticles.

Example 8: Formation of Complexes of Polymer Plus Chitosan with DNA

[0223] 20 mg of chitosan was suspended in 0.5% acetic acid (AcOH, 10 ml) and left overnight under stirring at room temperature. The chitosan stock solution (2 mg/ml) was adjusted at pH 5 with 0.1 M NaOH and filtered through 0.22 μm. The chitosan (60 kg/mol and deacetylation degree 60%) stock solution was diluted with 25 mM NaOAc buffer in a proportion ranging from 0.17 to 2.75 weight percent relative to the weight of PBAE when mixed with the PBAE solution. 50 μl of diluted chitosan solution was then added to 50 μl of diluted PBAE solution, and mixed with vortex. 100 μl of GFP (60 μg/ml in the same buffer) was then added to the mixture solution, mixed slightly with vortex and incubated at (a) 37° C. for 30 min or (b) room temperature for 10 min.

[0224] The particle size and zeta potential of R/DNA nanoparticles without or with a coating of 0.17, 0.34, 0.69, 1.38, and 2.75 weight percent chitosan prepared using incubation conditions (a) above were measured and the results are shown in FIG. 2. The mean particle size and zeta potential of R/pDNA as a positive control was 143.2 nm and 23.1 mV, respectively. The R/pDNA nanoparticles coated with chitosan ranged from 126 to 162 nm in the mean diameter and from 12 to 14 mV in zeta potential. There were small differences in size observed between nanoparticles coated with or without chitosan. However, zeta potential values show a significant decrease with the small amount of chitosan. This indicates that chitosan was perfectly formulated on the surface of the complexes.

Example 9: Formation of Complexes of Polymer Plus Chitosan with DNA

[0225] In a separate experiment, further complexes were prepared according to the method of Example 8(b), using chitosan having (a) molecular weight 22 kg/mol, (b) molecular weight 60-120 kg/mol or (c) molecular weight 20-50 kDa in a proportion ranging from 0.17 to 2.67 weight percent relative to the weight of PBAE when mixed with the PBAE solution. Polymers and GFP were in a weight ratio of 50:1.

[0226] The formation of complexes was confirmed by agarose gel electrophoresis with ethidium bromide, as shown in FIG. 3. In comparison with the mobility of the naked pDNA, the movement of DNA was completely retarded in the test compositions, indicating that addition of chitosan did not affect the formation of PBAE/DNA complexes.

Example 10: Formation of Sugar-Coated PBAE/DNA Complexes

[0227] Complexes of R and pDNA prepared according to the method of Example 7 were coated with 10, 20, 30 and 40 weight percent of mannitol, trehalose or sucrose by incubation at RT for 10 min with a solution containing the relevant amount of mannitol, trehalose or sucrose. Resulting complexes are denoted, for example, R/Mnt10%.

[0228] The mean diameter of the resulting sugar-coated nanoparticles was found to be in a range of 130-154 nm with a positive zeta potential of about 19-22 mV, which was slightly decreased compare to the nanoparticles uncoated with sugar (FIG. 4). In the case of nanoparticles coated with mannitol, increasing the amount of mannitol from 10 to 40% increased the mean size from 130 to 150 nm. However, the size of R/pDNA coated with 5% mannitol was larger than that coated with 10% (data not shown). In the case of nanoparticles coated with trehalose or sucrose, the mean particle size and zeta potential of nanoparticles were not dependent on the amount of sugar. R/Mnt10%, 30% R/Tre30%, and R/Suc30% had the smallest diameter.

Example 11: Factors Affecting Stability

[0229] The effect of different factors on the stability of nanoparticles was investigated. (i) Preparation according to Example 6 (i.e. incubation at 37° C. for 30 min) with a final concentration of ˜5 mM sodium acetate; (ii) preparation according to Example 6 but with a final concentration of 11-12 mM sodium acetate and (iii) Example 7 (i.e. incubation at RT for 10 min) with a final concentration of 11-12 mM sodium acetate. The stability of the nanoparticles, diluted in PBS to a final concentration of 0.25 mg/ml, was determined. While PBAE stock solution can be obtained at a constant concentration of 100 mg/ml, DNA stock solutions can be obtained at various concentrations and this may influence the various size and zeta potential of complexes.

[0230] The results are shown in FIG. 5 (indicated values are mean±SD of at least three experiments). The complexes at a final concentration of 11-12 mM of sodium acetate buffer were slightly smaller and much more stable than those in 5 mM of sodium acetate buffer. These results suggest that the final concentration of sodium acetate buffer affects the ionic strength of the complexes, and preparation of nanoparticles in the presence of buffers at 11-12 mM significantly increases stability. Further, the duration and temperature of incubation did not have an effect on the size or the stability of the nanoparticles over the 30 min measurement time frame. Accordingly, it is noted that the stability of complexes are not affected by the incubation conditions, but by the concentration of sodium acetate buffer (ionic strength).

Example 12: Stability of Particles Coated with Chitosan

[0231] Nanoparticles comprising polymer R in combination with differing amounts of chitosan prepared according to Example 8(a) were incubated for 8 h in phosphate-buffered saline and were analysed by DLS every 5 min in order to monitor changes in the size of the nanoparticles. The results are shown in FIG. 6. Nanoparticles having 0.17 or 0.35 weight percent chitosan were seen to be particularly resistant to agglomeration.

[0232] The stability over 4 h of R/DNA nanoparticles prepared according to Example 8(b) with a coating of 0.17, 0.34, 0.69, 1.38, and 2.75 weight percent chitosan was measured. The results are shown in FIG. 7. Notably, the mean particle size of R/pDNA coated with 0.69% of chitosan was still less than 400 nm at 4 h, indicating that 0.69 weight percent of chitosan may be an optimal content to sustain the formulation of the nanoparticles.

[0233] The stability over 4 h of R/DNA nanoparticles prepared according to Example 9(a) and Example 9(b) with a coating of 0.17, 0.33, 0.67, 1.33, and 2.67 weight percent chitosan was measured. The results are shown in FIGS. 8(a) and 8(b) respectively.

[0234] NRK-52e cells using nanoparticles comprising R and differing amounts of chitosan prepared according to Example 8(a) as carriers of pGFP at 0.6 μg/well were also analysed by fluorescence microscopy every 5 min over 8 h in order to monitor changes in the degradation profile of the nanoparticles over time. The results are shown in FIG. 9. Nanoparticles with higher amounts of chitosan showed a slower decrease in the count rate, suggesting that they were resistant to degradation.

[0235] The stability over 4 h of R/DNA nanoparticles prepared according to Example 9 with a coating of 0.17, 0.33, 0.67, 1.33, and 2.67 weight percent chitosan was measured. The results are shown in FIG. 10.

Example 13: Stability of Nanoparticles Coated with Mannitol

[0236] R/Mnt complexes prepared according to Example 10 were analysed by DLS every hour in order to monitor changes in the diameter of the nanoparticles.

[0237] The results are shown in FIG. 11 (indicated values are mean±SD of at least three experiments). The mean diameter for each type of nanoparticles is in the range 140-150 nm at t=0 and increases at each hourly measurement. It can be seen that nanoparticles coated with 10 weight percent mannitol are more stable than those without mannitol, whereas those coated with 5 or 20 weight percent mannitol are less stable than those without mannitol.

Example 14: Stability of Nanoparticles Coated with Mannitol, Sucrose or Trehalose

[0238] Complexes coated with 10 weight percent mannitol, sucrose or trehalose prepared according to Example 10 were analysed by DLS every hour in order to monitor changes in the diameter of the nanoparticles. The results are shown in FIG. 12 (indicated values are mean±SD of at least three experiments). It can be seen that nanoparticles coated with 10 weight percent mannitol are more stable than those coated with 10 weight percent sucrose or 10 weight percent trehalose.

[0239] In a separate experiment, nanoparticles coated with 10 to 40% of mannitol, trehalose or sucrose were incubated for 4 h in PBS at pH7.4 and analysed by DLS every hour in order to observe changes in the size of complexes. The results are shown in FIG. 13 (indicated values are mean±SD of at least three experiments). There highest stability for each coating material was observed with 10% of mannitol (R/Mnt10%), 30% of both sucrose (R/Suc30%) and trehalose (R/Tre30%).

[0240] The results for R/Mnt10%, R/Tre30% and R/Suc30% are shown in FIG. 14. The mean size of both R/Mnt10% and R/Tre30% was less than 800 nm within 4h. On the other hand, the size of R/Suc30% rapidly increased compare to that of the others. These results support that the stability of nanoparticles was dependent on the amount of sugar, and 10 weight percent of mannitol, or 30 weight percent of trehalose or sucrose are optimal weight contents for improving the stability of complexes. The zeta potential of all nanoparticles slightly decreased within 4 h (data not shown).

Example 15: Stability of Nanoparticles Formed from MntR or with Mannitol Coating

[0241] The stability over 10 h of complexes of MntR20 and DNA and of SucR20 in PBS at pH7.4 was measured by DLS every hour. The results are shown in FIG. 15 (indicated values are mean±SD of at least three experiments). There was minimal increase in the particle size of MntR20 and SucR20 within 4 h in PBS. The particle size of the complexes increased with decreasing zeta potential (data not shown). All results indicate that the presence of additives during polymerization results in complexes having high stability.

Example 16: Stability of Nanoparticles Formed from MntR or with Mannitol Coating

[0242] Nanoparticles comprising pGFP were prepared from R polymer according to Example 7 either (i) without mannitol, (ii) with coating of mannitol at 10 weight percent, (iii) polymerized with mannitol at 10 weight percent. The properties of these nanoparticles are shown in Table 2. There were no significant differences between the zeta potentials of these nanoparticles.

TABLE-US-00002 TABLE 2 Properties of pGFP nanoparticle Nanoparticle Mean diameter zeta potential preparation (nm) Polydispersity (mV) (i) R 143.2 ± 2.4 0.184 ± 0.044 23 ± 3.1 (ii) R/Mnt10 139.7 ± 2.0 0.198 ± 0.014 24.1 ± 2.5   (iii) MntR10 137.6 ± 2.4 0.135 ± 0.025 24 ± 3.0

[0243] The nanoparticles were diluted in phosphate buffer pH 7.4 and analysed by DLS every hour in order to monitor changes in the diameter of the nanoparticles. The results are shown in FIG. 16 (indicated values are mean±SD of at least three experiments). As expected from Example 14, the nanoparticles coated with 10 weight percent mannitol were more stable (less prone to aggregation) than the nanoparticles without mannitol. The nanoparticles having 10 weight percent mannitol added prior to the polymerization step are significantly more stable even than those coated with 10 weight percent mannitol: after 8 hours the mean diameter was still <200 nm.

[0244] The stability of MntR20% nanoparticles over time was also measured. The results are shown in FIG. 17. The mean diameter of MntR20/pDNA was 600.4±17.6 nm within 7 h, indicating that MntR20/pDNA nanoparticles significantly increased the stability of the nanoparticles. The particle size of nanoparticles increased with decreasing zeta potential, indicating the weak binding between DNA and polymers (data not shown).

Example 17: Effect of Chitosan on Transfection Efficiency in NRK-52e Cells

[0245] NRK-52e cells were seeded in 96-well plates at 10,000 cells/well and incubated overnight to roughly 80% confluence. The cells were then transfected with the following: [0246] Nanoparticles comprising R [0247] Nanoparticles comprising R and coatings of differing amounts of chitosan [0248] Chitosan alone [0249] GeneJuice (positive control) [0250] No treatment (negative control)

[0251] In each case GFP expression was evaluated 48 h after transfection by fluorescence microscopy and quantified by flow cytometry based on a sample of 2000-5000 cells. The properties of transfected cells that were measured were relative size, relative granularity or internal complexity, and relative fluorescence intensity. Finally, data were analysed by BD LSRFortessa cell analyser software.

[0252] Results of flow cytometry are shown in FIG. 18 and results of fluorescence microscopy are shown in FIG. 19. It can be seen that all the polymer formulations achieved higher transfection efficiency than positive control and chitosan alone. Further, when the weight percent of chitosan was increased the transfection efficiency was at a level similar to that of the polymer alone, but with further increase in the weight percent of chitosan, transfection efficiency was reduced. In particular, chitosan was found to result in the highest transfection efficiency when present at 0.17 to 0.35 weight percent.

[0253] The effect of the choice of end modification of the PBAE was also investigated. The following nanoparticles were prepared and tested according to the protocol above: [0254] Nanoparticles comprising R. [0255] Nanoparticles comprising R and H3C-C32-CH3 at 1:1 weight ratio, with or without inclusion of chitosan at 0.35 weight percent. [0256] Nanoparticles comprising R and D3C-C32-CD3 at 7:3 weight ratio (use of oppositely charged polymers results in increased stability as a result of electrostatic interactions), with or without inclusion of chitosan at 0.35 weight percent. [0257] Chitosan alone. [0258] GeneJuice (positive control). [0259] No treatment (negative control).

[0260] Results of flow cytometry are shown in FIG. 20 and results of fluorescence microscopy are shown in FIG. 21. The lower GFP expression after transfection with nanoparticles comprising chitosan, in spite of transfection levels in the same range, seems to be a result of the increased stability of these nanoparticles, which leads to decreased release of pGFP within transfected cells.

Example 18: Effect of Chitosan on Transfection Efficiency in COS-7 Cells

[0261] COS-7 cells were transfected with pGFP plasmid DNA using nanoparticles prepared according to Example 9(b) and Example 9(c). Non-coated R complexes were used as a positive control to compare the influence of chitosan on the transfection efficiency because R/DNA complexes were reported to provide higher gene expression in cell-type-specific manner and better cellular viability compared to other end-modified PBAEs and commercial transfection agents (Segovia et al., 2014). Cells without any treatment were included as a negative control (NC)

[0262] The pGFP expression was determined by flow cytometry analysis at 48 h post-transfection. Results of flow cytometry and fluorescence microscopy are illustrated in FIG. 22 (indicated values in b and c are mean±SD of at least three experiments). In FIG. 22c, GFP expression was determined after 48 h by flow cytometry and bars represent percentage of cells positively transfected and the normalized total gene expression.

[0263] In the case of a coating of chitosan 60-120 kDa, the transfection efficiency of R/DNA coated with chitosan 60-120 kDa decreased with increasing the amount of CSM. The lower GFP expression after transfection with complexes comprising CSM may be a result of the increased size and the decreased zeta potential of these complexes, leading to reduced release of GFP within transfected cells.

[0264] Although GFP expression levels slightly decreased with increasing the amount of a coating of chitosan 20-50 kDa, all such complexes except that coated with 2.67% chitosan showed high transfection efficiency (≧75%). Interestingly, higher gene expression was maintained for complexes coated with 0.67 wt % of chitosan 20-50 kDa, which also showed the smallest size and the highest stability among the chitosan coated complexes.

Example 19: Effect of Sugar, Sugar Alcohol or Chitosan on In Vitro Transfection

[0265] Cellular transfection was carried out using pDNA plasmid in COS-7 cells. Cells were seeded on 96-well plated at 10,000 cells/well and incubated overnight prior to performing the transfection experiments. PBAE:pDNA complexes were prepared as described above (PBAE:DNA=wt:wt, 50:1). Complexes were diluted in serum-free DMEM medium and added to cells at a final plasmid concentration of 0.3 μg pDNA/well. Briefly, 33 μl of PBAE/DNA complexes were diluted into 450 μl of serum-free DMEM medium and cells were washed once with PBS. Then, 150 μl of the resulting solutions were added to each well, achieving a final concentration of 0.3 μg pDNA/well. Cells were incubated for 3 h at 37° C. in 5% CO.sub.2 atmosphere. Subsequently, cells were washed once with PBS, and complete DMEM medium was added. Cells were harvested after 48 h and analysed for GFP expression by flow cytometry (BD LSRFortessa cell analyzer). GFP expression was compared against a negative control (untreated cells), and both GeneJuice (Merck KGaA, Germany) and unmodified R/pDNA as a positive control.

[0266] Transfection conditions for GeneJuice control were optimized in order to achieve maximal transfection efficiency. Different GeneJuice:pGFP ratios were evaluated and 3:1 ratio (v:w) was found to be optimal. Briefly, 300 μl serum-free DMEM medium and 2.88 μl of GeneJuice were mixed vigorously with vortex and incubated at room temperature for 5 min. Then, 15 μl of a plasmid solution at a concentration of 0.06 μg/μl in NaAc buffer was added into the mixture, mixed gently by pipetting and incubated at room temperature for 15 min. Finally, 100 μl of the resulting solution was added to each well, achieving a final concentration of 0.3 μg/well DNA dose.

[0267] The influence of incubation conditions on transfection efficiency was investigated using MntR20/pDNA, which was shown above to have the highest stability, and R/pDNA obtained under different incubation conditions as shown in FIG. 23. The transfection efficiency of R/pDNA significantly increased when nanoparticles were prepared at RT. However, incubation time resulted in a negligible change in transfection efficiency.

[0268] The transfection efficiencies of the nanoparticles modified with chitosan, sugar or sugar alcohol as described in the examples above were measured and the results, in comparison with nanoparticles formed from R, GeneJuice (positive control) and pGFP alone are shown in FIG. 24.

[0269] In the case of R/pDNA coated with chitosan, the transfection efficiency decreased as the amount of chitosan was increased. R/pDNA coated with 0.69 weight percent chitosan, which showed the highest stability among those with chitosan, exhibited similar transfection efficiency compared to GeneJuice. However, the transfection efficiency of R/pDNA coated with 1.38 and 2.75 weight percent of chitosan was significantly reduced. This is believed to be because the transfection efficiency of nanoparticles formulated with chitosan is dependent on the pH of the medium due to its pKa, and efficiency is dramatically decreased at a transfection medium pH of 7.4 (Mao 2010).

[0270] Overall high transfection efficiency was obtained from R/pDNA coated with sugar, especially with mannitol. R/pDNA coated with 5 and 10 weight percent of mannitol or 30 weight percent sucrose showed higher transfection efficiency compared with uncoated nanoparticles. The lowest expression efficiencies were seen using 20 weight percent mannitol and 10 weight percent trehalose. These results confirm that the transfection efficiency of nanoparticles is dependent on either weight percent or type of sugar, and mannitol can promote higher both stability and transfection of nanoparticles than those of trehalose or sucrose.

[0271] MntR nanoparticles showed much higher transfection efficiency than coated nanoparticles. Moreover, MntR showed higher expression efficiency than TreR or SucR. Surprisingly, MntR20 significantly decreased the transfection efficiency. This corresponds to the results obtained from nanoparticles coated with 20 weight percent mannitol.

[0272] Very low transfection efficiency was observed with the TreR-based nanoparticles, which may be a result of the lower plasmid dosage of 0.075 μg/well (0.3 μg/well) that was used to avoid cytotoxicity resulting from the high TreR:DNA ratio of 100:1 that was needed for formulated nanoparticles.

[0273] The lowest transfection efficiency was detected for MntR20, TreR30, and SucR20, which showed the best formulation and the highest stability among their series. These results are in agreement with the results obtained by Vuorimaa et al., who reported that for tight DNA binders further increase in binding affinity is expected to decrease transfection due to the impaired DNA release in the cells.

Example 20: Effect of Complexes on Cell Viability

[0274] Sugar and sugar alcohol modified complexes were tested for their effect on cell viability of transfected cells. Complexes were formulated at a 50:1 polymer:DNA weight ratio and tested using a plasmid dosage of 0.3 μg/well, except TreR/DNA complexes which were formulated at a 100:1 weight ratio of polymer to DNA was tested using a 0.075 μg DNA/well. As shown in FIG. 25, no large decrease in cell viability was observed for any of the modified complexes compared with the unmodified R complexes, which presented cell viability of ˜81%. These results indicate a good safety profile for the sugar or sugar alcohol modified PBAE/DNA complexes.