More particularly, the present invention relates to a cationic highly branched polyaminoester which can carry genetic materials into cells at high efficiency and is of low cytotoxicity. Also, the present invention is concerned with a method for preparing such a cationic highly branched polyaminoester.

BACKGROUND ART Highly branched polymers such as dendrimers or hyperbranched polymers have attracted extensive attention owing to their novel uses based on their characteristic three-dimensional structures. Highly branched polymers, whose chemistry was first discussed by Flory, P. J. (J. Am. Chem. Soc. , 1952,74, 2718; Principles of Polymer Chemistry, Cornell University Press, 1953, pp. 361-70), can be prepared by polymerizing Ax-R-By-type monomers. Nowadays, highly branched polymers find numerous applications in various fields, including combinatorial chemistry, surface coating, catalysts, gene delivery, drug delivery, etc.

Highly branched polymers are usually synthesized from the monomers in the form of AX-R-By by one-step polymerization. The synthesized polymers with branch structures have surface functional groups which can be modified by surface functionalization reaction as needed.

Emerging as a potent curative means, gene therapy is under active study.

Usually, gene therapy needs a gene delivery carrier which can effectively deliver a gene of interest into the inside of cells. As such, virus, liposomes, cationic lipids and cationic polymers are currently in use.

Various forms of cationic polymers are found as exemplified by poly-L- lysine, poly (ethyleneimine), poly (2-dimethylamino) ethylmethacrylate (pDMAEMA), starburst PAMAM dendrimer, poly (N-ethyl-4- vinylpyridiumbromide) (PVP), poly (4-hydroxy-L-proline ester) (PHP ester), and poly [a- (4-aminobutyl)-L-glycolic acid] (PAGA). Among them, PHP ester and PAGA exhibit very low level of cytotoxicity, since their backbones are linked via an ester bond which is biodegradable.

Over the other gene delivery systems (virus, liposomes, cationic lipids), cationic polymers have the advantages because they can be mass-produced, cause immune responses in a low level, and are easy to control for optimal gene delivery.

However, conventional gene delivery systems based on cationic polymers have much margin for improvement in cytotoxicity and gene delivery efficiency.

DISCLOSURE OF THE INVENTION Leading to the present invention, the thorough and intensive research into gene carriers, conducted by the present inventors, resulted in finding that a highly branched polyaminoester based on a unit represented by Ax-N-By (N is a tertiary amine; x and y are integers; and A and B are functional groups which can form an ester group) can be improved in gene delivery capacity when it is provided with primary, secondary, tertiary or quaternary amines on its surface or the internal tertiary amine is further aminated to a quaternary amine.

Therefore, it is an object of the present invention to overcome the above problems encountered in prior arts and to provide a cationic polymer which is excellent in terms of gene delivery into cells.

It is another object of the present invention to provide a cationic polymer which is suitable for use in gene therapy.

It is a further object of the present invention to provide a method for preparing a cationic polymer for use in gene delivery.

BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which: Fig. 1 is a diagram showing the synthesis of a precursor molecule of the present invention.

Fig. 2 is a diagram showing the synthesis of a cationic hyperbranched polyaminoester having primary amines on its surface.

Fig. 3 is a diagram showing the synthesis of a cationic hyperbranched polyaminoester having quaternary amines at the interior of its structure.

Fig. 4 is a photograph taken after an electrophoresis experiment, showing the formation of a cationic hyperbranched polyaminoester/DNA complex.

Fig. 5 is a histogram showing the transfection efficiencies obtained by use of transfectants Polymer 5 and PAGA, each being associated with DNA in a weight ratio of 15 or 30 (polymer/DNA).

Fig. 6 is a graph showing the cytotoxicity of various transfectants.

Fig. 7 is a diagram showing the synthesis of a network-type cationic polyaminoester with primary amines on its surface.

Fig. 8 is photographs showing electrophoresis results after polyplexes of a network-type cationic polyaminoester withprimary amines on the surface and DNA with weight ratios 2 (a), 5 (b), 10 (c) and 20 (d) are run.

Fig. 9 is a diagram showing the determination of configuration and dimension of a polyplex (B) in which a network-type cationic polyaminoester with primary amines on its surface is associated with a plasmid DNA (A), by use of an atomic force microscope.

Fig. 10 is a histogram showing the transfection efficiency of a network-type cationic polyaminoester with primary amines on the surface, along with the transfection efficiency of other transfectants, in which numerals in parentheses mean weight ratios between transfectants and DNA.

Fig. 11 is a graph showing the cytotoxicity of a network-type cationic polyaminoester and other transfectants in 239-cells and HepG2 cells.

Fig. 12 is histograms showing transfection efficiency ratios of various transfectants in the absence of and in the presence of chloroquine or nigericin, showing the excellent endosome buffering effect of a network-type cationic polyaminoester.

BEST MODES FOR CARRYING OUT THE INVENTION The present invention pertains to cationic highly branched polyaminoester based on the following precursor polymers: (1) Precursor Polymers (a) Precursor polymer 1 A precursor useful in the synthesis of the cationic highly branched polyaminoester of the present invention may be polymerized to a molecular weight of 500-20,000, 000 from the unit molecule represented by the following chemical formula 1: [X (R') p] qRON [ (R2). Y] 2 wherein, m and p each are an integer of 0 or 1 ; q is an integer of 1 to 3; N stands for a nitrogen atom ; R°, Ru, and R2 are independently selected among aliphatic and aromatic hydrocarbons and derivatives thereof, containing 0-20 carbon atoms ; and X and Y are functional groups which can form an ester bond therebetween (e. g. , X = alcohol group, Y = carboxyl group, or X = carboxyl group, Y = alcohol group).

The precursor polymer 1 has a network-type structure when q is 2 or 3.

(b) Precursor polymer 2

A precursor for synthesizing the cationic highly branched polyaminoester of the present invention may be obtained by copolymerizing the unit molecule of the chemical formula 1 with the core-forming molecule represented by the following chemical formula 2 to a molecular weight of 1,000-50, 000,000 (precursor polymer 2a). Alternatively, a polymer polymerized from the unite molecule (the precursor polymer 1) may be reacted with the core-forming molecule to synthesize a precursor polymer with a molecular weight of 1,000-50, 000,000 (precursor polymer 2b).

Preferably, the core-forming molecule is linked to the unit molecule via an ester bond or a peptide bond.

The core-forming molecule is represented by the following chemical formula 2: R' [ (R') aZ] b [2] wherein, a is an integer of 0 or 1; R5 is a nitrogen element or a carbon atom; b is an integer of 2 or 3 when R5 is a nitrogen atom, or an integer of 2 to 4 when R is a carbon atom; R6 is selected from among aliphatic hydrocarbons, aromatic hydrocarbons, and derivatives thereof, preferably containing 0 to 20 carbon atoms; Z is selected from among alcohol moieties, amine moieties, carboxyl moieties and alkyl ester moieties.

(2) Cationic highly branched polyaminoester The cationic highly branched polyaminoesters of the present invention are synthesized by subjecting the precursor polymer 1 or 2 to surface amine- functionalization reaction or internal quaternary amination. The term"surface amine-functionalization reaction"as used herein means the coupling of an external functional group (e. g. alcohol, carboxyl, or alkylester) on the surface of a polymer to an amine group via an ester linkage or a peptide linkage.

Synthesis of Cationic highly branched Polyaminoesters By Surface Amine- Functionalization Reaction The cationic highly branched polyaminoesters of the present invention are synthesized by the amination of a part of or all of the functional groups present on the surface of the precursor polymer 1 or 2 to one selected from among the moieties represented by the following chemical formulas 3 to 6: - (O) OR7N (R8) c (R9) d [3] -OC (O) R7N (R8) c (R9) d [4] -C (O) NHR7N (R8) c (R9) d [5] -OR'N (R'),

wherein, R7 is selected from among aliphatic hydrocarbons, aromatic hydrocarbons, and derivatives thereof, preferably containing 0 to 20 carbon atoms; R8 and R9 are independently a hydrogen atom, or are selected from among aliphatic hydrocarbons, aromatic hydrocarbons and derivatives thereof, preferably containing 1 to 20 carbon atoms; and c and d each are an integer of 1 or 2 with the proviso that the sum of c and d is 2 or 3.

In the chemical formulas 3 and 4, the precursor polymer is coupled with the surface amine moieties via ester linkages, while peptide bonds are intercalated between the precursor polymer and the surface amine moieties in the chemical formulas 5 and 6.

Synthesized by the surface amine-functionalization reaction, the cationic highly branched polyaminoesters of the present invention may be exemplified as follows: Example 1. Where the sum of c and d is 2 and both R8 and R9 are hydrogen atoms, the cationic highly branched polyaminoesters have primary amines on their surfaces.

Example 2. Where the sum of c and d is 2 and both R8 and R9 are alkyl groups, the cationic highly branched polyaminoesters have tertiary amines on their surfaces.

Example 3. Where the sum of c and d is 3 and both R8 and R9 are alkyl groups, the cationic highly branched polyaminoesters have quaternary amines on their surfaces.

Positively charged in aqueous solutions, the amine groups on the surface of the polymer thus obtained can be associated with negative charged DNA through electrostatic attraction to form a polymer/gene complex (polyplex). For gene therapy, a foreign gene of interest must be transfected into cells (nuclei) to express a protein which directly or indirectly exerts a therapeutic effect. Without any external aid, it is virtually impossible for DNA itself to enter cells. That is, transfection is difficult to conduct with DNA alone. The reason is that because cellular membranes is negatively charged on the whole, electric repulsion occurs between cellular membranes and DNA with negative charges, making it difficult for DNA to pass through the cellular membranes. By contrast, the polyplex in which DNA is associated with the cationic highly branched polymer is electrically neutral or positive on the whole, so that it can easily penetrate into cells. Therefore, the cationic highly branched polymer of the present invention is suitable for use as a gene delivery vector.

Synthesis of Cationic Highly branched Polyaminoesters By Internal Quaternary Amination Cationic highly branched polyaminoesters of the present invention may be obtained by reacting the tertiary amine groups existing inside of the precursor polymer 1 or 2 with an alkyl halide represented by the following chemical formula: R10x wherein X is a halogen atom selected from among chlorine, bromine and iodine and Rl° is selected from aliphatic hydrocarbons, aromatic hydrocarbons and derivatives thereof, preferably containing 1 to 20 carbon atoms.

Positively charged in aqueous solutions, the polymer with internal quaternary amine groups is electrostatically attracted to negatively charged DNA to form a polyplex.

To extend the time for which the cationic highly branched polyaminoester/gene complex survives in blood streams, hydrophilic polymer chains may be linked to the cationic highly branched polyaminoesters of the present invention (by for example graft polymerization). Examples of the hydrophilic polymer chains suitable for use in the extension of the cationic highly branched polyaminoesters include polyethylene glycol (PEG), polylactic acid, polyglycolic acid, polyvinylpyrrolidone, polymethyloxazoline, and polyethyloxazoline with preference for polyethylene glycol (PEG). PEG, synthesized by the polymerization of ethylene glycol, is an amphipathic compound concurrently comprising both hydrophobic and hydrophilic moieties. The PEG tail reduces the imunogenicity of the cationic highly branched polyaminoester/gene complex.

To play the role of a gene delivery carrier so as to implement gene therapy, the cationic highly branched polyaminoester of the present invention is associated with a genetic material of interest. The genetic material useful in the present invention may comprise single-or double-strand DNA, RNA, PNA (peptide nucleic acid), plasmids, and fragments thereof.

Instead of being directly associated with the cationic highly branched polyaminoester of the present invention, a gene of interest may be contained in a plasmid which is then associated with the carrier. Examples of RNA, associable with the cationic highly branched polyaminoester of the present invention, include mRNA, tRNA, rRNA, antisense RNA sequences complementary to cellular target DNA or RNA sequences, and rybozymes.

Genes of interest to be transfected into cells may encode various hormones, histocompatible antigens, cell-adhering proteins, cytokines, various antibodies, cell receptors, intracellular or extracellular enzymes, and fragments thereof. DNA fragments useful in the present invention may comprise gene

expression regulators, such as promoters, enhancers, silencers, operators, terminators, attenuators, and other expression controllers.

In addition, specific markers or ligands may be linked to the cationic highly branched polyaminoesters of the present invention in order for the carriers to adhere specifically to target cells. Suitable for use in this purpose are various antibodies, transferrins, biotin, folic acid, low-density lipoproteins (LDL), carbohydrates, for example, monosaccharides such as mannose, glucose and galactose, and diaccharides such as lactose. The markers or ligands react specifically with corresponding receptors existing on the surface of cells, playing a role in identifying cells. The identifiers for certain cells, if necessary, can be added to the carrier of the present invention by known methods in the art, and a detail description for the methods is omitted.

Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.

EXAMPLE 1: Synthesis of Precursor Polymer 2a To a solution of ethanol amine (5 ml, 82 mmol) in methanol was added excess methyl acrylate (148 ml, 1.64 mol) with stirring, and the resulting solution was reacted at 35 °C for 24 hours. The evaporation of the solvent gave a unit 1 shown in Fig. 1 (yield 99 %). Then, the unit 1 (3g, 12.9 mmol) and a core-forming molecule 3 (7.7 mg, 64.3 pmol) shown in Fig. 1 were mixed together with the catalyst Al (O'Pr) 3 (27 mg, 1 mol%) in a reactor and stirred under vacuum. The reactants were heated to 120 °C over 2 hours and maintained thereat for 10 hours.

Then, the temperature was increased to 140 °C and maintained thereat for 10 hours, followed by precipitating the product in diethyl ether to give a precursor polymer 4 shown in Fig. 1 (Yield 67 %). The above reaction procedure is illustrated in Fig. 1.

Characteristics of unit 1 :'H NMR (300 MHz, CDC13) 2.47 (t, 4H,- CH2CH2COO-), 2.59 (t, 2H, HOCH2CH2N-), 2.79 (t, 4H,-CH2CH2COO-), 3.59 (t, 2H, HOCH2CH2N-), 3.68 (s, 6H,-CH3). i3C NMR (75 MHz, CDCl3) 32.50 (- CH2CH2COO-), 49.14 (-CH2CH2COO-), 51.48 (-CH3), 55.88 (HOCH2CH2N-), 59.03 (HOCH2CH2N-), 172.87 (-COO-). MS (MALDI) Calc'd for CloHlyNOs (M+H) + 234.273 ; found 234.298.

Characteristics of precursor polymer 4 :'H NMR (300 MHz, CDC13) 2.45 (br t,-CH2CH2COO-), 2.71 (br t,-COOCH2CH2N-), 2.82 (br t,-CH2CH2COO-), 3.66 (br s,-CH3), 4.10 (br t,-COOCH2CH2N-).'3C NMR (150 MHz, INVGATE,

CDCI3) 32.65-32. 80 (-CH2CH2COO-), 49. 64-49. 83 (-CH2CH2COO-), 51. 58 (- CH3), 51.96-52. 11 (-COOCH2CH2N-), 62.43 (-COOCH2CH2N-), 172.20-172. 76 (ester carbonyl carbons). Mw =42,500 (by GPC, relative to PAMAM standard), PDI (polydispersity index=1.43 EXAMPLE 2: Synthesis of Cationic Hyperbranched Polyaminoester with Primary Amines on Surface In a polymer reactor, the precursor polymer 4 (1.53 g) was reacted with N- cbz-ethanolamine (1.47 g) at 140 °C under vacuum for 5 hours in the presence of the catalyst Al (OPr) 3 (25 mg), followed by precipitation in diethyl ether to produce a polymer which was associated with N-cbz-ethanolamine on its surface (Yield 67 %).

From the polymer, the protecting cbz group was removed by hydrogenolysis in the presence of a palladium catalyst (10 % Pd/C) to allow a precursor polymer 5, a cationic hyperbranched polyaminoester which has primary amines on its surface, (Yield 62 %). The above reaction procedure is illustrated in Fig. 2.

Characteristics of precursor polymer 5:'H NMR (300 MHz, DMSO-d6) 2.34 (br,-CH2CH2COO-), 2.70 (br,-COOCH2CH2N-), 2.88 (br,-CH2CH2COO-), 3.22 (br,-OCH2CH2NH2), 3.62 (br,-CH3), 4.07 (br,-COOCH2CH2-).

EXAMPLE 3: Synthesis of Cationic Hyperbranched Polyaminoester With Internal Quaternary Amines The tertiary amines existing inside of the precursor polymer 4 was further aminated to quaternary ones. To this end, methyl iodide (2 ml) was reacted with the precursor polymer 4 (1 g). After completion of the amination, the evaporation of the solvent left a precursor polymer 6, a cationic hyperbranched polyaminoester with internal quaternary amines (Yield 99 %). The above reaction procedure is illustrated in Fig. 3.

Characteristics of precursor polymer 6 :'H NMR (300 MHz, D20) 2.73-3. 25 (br m,- CH2CH2COO-,-CH2CH2COO-), 3.73 (s,-COOCH3), 3.77 (s, -NCH3), 3.87 (br, - COOCH2CH2N-), 4.34-4. 69 (br m,-COOCH2CH2-).

EXAMPLE 4: Formation of Cationic Hyperbranched Polyaminoester/Gene Complex A cationic hyperbranched polyaminoester (polymer 5 of Fig. 2) was dissolved at various concentrations in a buffer (Hepes 25 mM, NaCl 150 mM, pH

7.4) and mixed with an aqueous plasmid solution. After the mixture was allowed to stand for 1 hour to form complexes, they were run on a 0.7 % agarose gel under an electric field. After electrophoresis at 100 volts for 1 hour, the mobility of the plasmids was examined by visualizing the formation of the complexes with ethidium bromide under a UV beam, as shown in Fig. 4.

EXAMPLE 5: Transfection with Cationic Hyperbranched polyaminoester/Gene Complex 293 cells (embryonic human kidney cells) aliquoted on 96-well plates were cultured in a minimal essential medium (MEM) supplemented with 10 % fetal bovine serum (FBS) under a 5 % C02 atmosphere in an incubator. Separately, the polymer 5 was mixed with a plasmid (pGL3-Control vector, Promega, U. S. A.) which can express a gene encoding a luciferase, to form a polyplex which was then transfected into the cultured cells at 37 °C for 4 hours. The polymer 5 was found to exhibit at least 10-fold higher gene delivery efficiency than the preexisting biodegradable cationic polymer PAGA (Lim, Y. et al. , J. Am. Chem. Soc. , 2000), as shown in Fig. 5. The higher transfection efficiency is believed to result from the three-dimensional structure and internal tertiary amines of the cationic hyperbranched polyaminoester, as explained by the following transfection mechanism. When passing through a cellular membrane, the polyplex is enveloped by a vesicle, so-called endosome. In order to express the gene which the cationic hyperbranched polyaminoester carries, the polyplex must free itself from the endosome and reach the nucleus. Whereas they are hardly cationized at pH 7.4, tertiary amines present inside of the polymer become cationic at an acidic pH (about pH 5-6). The cationized tertiary amines repel each other so that the polymers swell in three-dimensional direction to break the endosome (endosome buffering effect).

In other words, the high transfection efficiency of the cationic hyperbranched polyaminoester of the present invention can be explained by the endosome buffering effect.

EXAMPLE 6: Alleviation of Cytotoxicity of Cationic Hyperbranched Polyaminoester 293 cells were cultured on 96-well plates containing a MEM medium supplemented with 10 % FBS and then treated with the polymer 5, and other cationic polymers (PAGA, PAMAM and PEI) for 24 hours. Afterwards, treatment was carried out with 3- [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) for 2 hours and then with an extraction buffer (a solution of 20 % w/v SDS

in 50 % DMF, pH 4.7) for 24 hours. Absorbance was measured at 570 nm. The results are given in Fig. 6.

As shown in Fig. 6, the cationic hyperbranched polyaminoester (polymer 5) were found to be of much lower cytotoxicity than PEI, which is a hardly-degradable cationic polymer, and PAMAM. The low cytotoxicity of the cationic hyperbranched polyaminoester is believed to be attributed to the fact that the backbone of the polymer is composed of ester bonds which are readily decomposed in water.

EXAMPLE 7: Synthesis of Network-Type Cationic Polyaminoester (n-PAE) With primary Amines On Surface To methyl acrylate (150 ml, 1.67 mol) was added a solution of tris (hydroxymethyl) aminomethane (5 g, 41 mmol) in methanol (250 ml) at 45 °C, and the mixture was stirred at 45 °C for 24 hours. Methanol and surplus methacrylate were evaporated to afford the unit molecule 7 (12 g, 41 mmol) shown in Fig. 7 as a yellow oil. Yield 99 %.

For polycondensation, the unit molecule 7 (5 g) in a vial was placed a silicon bath. A polycondensation reaction was carried out under a constant flux of argon at 170 °C to which the reaction temperature was increased at a rate of 10 °C/min. After 5 hours, there was obtained a precursor polymer 8 of a network-type structure, which is in a solid state at room temperature.

A solution of the precursor polymer 8 (0.73 g) and Fmoc-6-aminohexane (Fmoc-eAhx, 0.91 g, 2.58 mmol) in DMF (7 ml) was treated with DCC (1.06 g, 5.15 mmol), DMAP (0.16 g, 1.29 mmol), and PTSA. (0.23 g, 1.2 mmol) at room temperature. After 15 hours at room temperature, the reaction mixture was filtered to remove DCU. The polymer thus produced was precipitated by immersing the reaction mixture in excess water three times, while removing DMAP and PTSA.

After being harvested, the precipitate was dissolved in CHC13 and added with excess EtOAc/ether (1 : 1) three times to remove remaining DCC and Fmoc-eAhx while affording the polymer as a viscose semi-solid phase.

The obtained polymer (0.5 g) in which the polymer 8 was coupled with Fmoc-eAhx was treated with a solution of 20 % (v/v) piperidine in DMF to remove the protecting group Fmoc-eAhx. After the deprotecting reaction was conducted for 5 min, the resulting mixture was dropwise added to excess EtOAc/ether (1: 1) to afford n-PAE (0.21 g) as a white solid. This precipitation was repeated twice further.

Characteristics of unit molecule 7 :'H NMR (300 MHz, DMSO-d6) 2.39 (t, 4H, CH2CH2COO), 2.75 (t, 4H, CH2CH2COO), 3.28 (s, 6H, HOCH2), 3.57 (s, 6H, CH3).

13C NMR (75 MHz, DMSO-d6) 35.41 (CH2CH2COO), 36.93 (CH2CH2COO), 51.41 (CH3), 59.98 (quaternary carbon), 61.05 (HOCH2), 173.16 (ester carbonyl carbons).

MS (MALDI) Calc'd for C12H23NO7 (M+H) + 294.325 ; found 294.323.

Characteristics of unit molecule 8 :'H NMR (300 MHz, DMSO-d6) 1.41-2. 77 (br m, CH2CH2COO and CH2CH2COO), 3.24-3. 42 (br m, HOCH2), 3.50-3. 57 (br m, CH3), 4.01-4. 10 (br m, COOCH2-). 13C NMR (75 MHz, DMSO-d6) 33.63-35. 83 (CH2CH2COO), 36.61-37. 79 (CH2CH2COO), 51.60 (CH3), 59.32, 62.62 (quaternary carbons), 60.86, 61.44, 64.27 (HOCH2), 165.13-173. 53 (ester carbonyl carbons).

Characteristics of n-PAE (polymer 9) :'H NMR (300 MHz, DMSO-d6) 1.24-1. 47 (br m, CH2 (CH2) 3CH2COO), 2.27 (br, CH2 (CH2) 3CH2COO), 2.66 (br, CH2 (CH2) 3CH2COO), 3.21-3. 40 (br m, HOCH2), 3.51-3. 55 (br m, CH3), 3.85-4. 15 (br m, COOCH2).

EXAMPLE 8: Formation of network-type cationic polyaminoester having primary amines on its surface/gene complex.

A network-type cationic polyaminoester (polymer 9) was dissolved at various concentrations in a buffer (Hepes 25 mM, NaCl 150 mM, pH 7.4) and mixed with an aqueous plasmid solution. After the mixture was allowed to stand for 1 hour to form complexes, they were run on a 0.7 % agarose gel under an electric field. After electrophoresis at 100 volts for 1 hour, the mobility of the plasmids was examined by visualizing the formation of the complexes with ethidium bromide under a UV beam.

Fig. 8 contains photographs showing electrophoresis results after polyplexes with weight ratios of n-PAE/DNA of 2 (a), 5 (b), 10 (c) and 20 (d) are run as a supercoiled form (Form I), a nicked circular form (Form II), and a linear form (Form III).

EXAMPLE 9: Determination of Configuration and Dimension of n-PAE (Polymer 9) /Gene Complex Using Atomic Force Microscope (AFM) To take DNA images, a plasmid (pGL3-control vector) was dissolved at a concentration of 1 pg/ml in Hepes-Mg (25 mM Hepes, 10 mM MgCl2, pH 7.6) buffer. Two ul of the DNA solution was applied onto a mica substrate which had just been cleaved. After the absorption of the DNA solution into the mica substrate for 2 min, the mica substrate was washed with 1 ml of distilled water and immediately dried with N2 gas. A polyplex was prepared by mixing a solution of the plasmid in water (5 ug/ml) with one volume of a polymer solution. Two PI of the polyplex was applied onto a mica substrate which had just been cleaved, and

dried for 2 min, followed by the removal of surplus liquid through a filter. Prior to image formation, the solution was dried at room temperature.

The AFM was operated with the aid of a nanoscope nia (Digital Instruments, Santa Babara, CA) equipped with an E scanner. All AFM images were taken in a typical ambient tapping mode at a scanning speed of about 5 Hz with 512x512 pixels.

From the AFM photographs, it was seen that a polyplex consisting of n- PAE and DNA was formed and most plasmid DNAs were in supercoil or nicked circular forms, as shown in Fig. 9A. AFM images of the n-PAE/DNA polyplex demonstrated that all plasmid molecules were associated with n-PAE to form polyplexes which were circular with heterogeneity, as shown in Fig. 9B. The polyplex was made of polymer/DNA with a weight ratio of 5.

EXAMPLE 10: Transfection with n-PAE (Polymer 9) /Gene Complex 293 cells (embryonic human kidney cells) or HepG2 cells (human liver cells) aliquoated to suitable volumes on 96-well plates were cultured in an MEM (minimal essential medium) supplemented with 10 % FBS (fetal bovine serum) at 37 °C under a 5 % CO2 atmosphere. n-PAE and a plasmid harboring a gene encoding a luciferase (pGL3-control vector, Promega, U. S. A. ) were mixed to give a polyplex which was then transfected into the cultured cells at 37 °C for 4 hours. The transfection efficiency (TE) was determined by the activity of the luciferase expressed in the cells.

As shown in Fig. 10, n-PAE is excellent in terms of TE as it performs equally to PEI, known as the most efficient transfectant ever developed. The TE of n-PAE was measured to be 106-and 318-fold higher in 293 cells and HepG2 cells, respectively, than that of PAGA, demonstrating its excellent gene delivery capacity.

EXAMPLE 11 : Cytotoxicity of n-PAE (Polymer 9) in 293 cell and HepG2 cell 293 cells or HepG2 cells were grown in an MEM supplemented with 10 % FBS on 96-well plates and treated with n-PAE, polymer 8, PEI or PAGA for 4 hours, with 3- [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) for 2 hours, and with an extraction buffer (a solution of 20 w/v SDS in 50 % DMF, pH 4.7) for 24 hours, followed by measuring absorbance at 570 nm Fig. 11 shows relative cell viability, based on the absorbance at 570 nm, of 293 cells and HepG2 cells treated with n-PAE (0), polymer 8 (D), PEI (0) and PAGA (V). As shown in the curves of Fig. 11, n-PAE is of low cytotoxicity.

When treated with as high as 200 gg/rnl of n-PAE, 293-cells and HepG2 cells

exhibited relative cell viability of 93 % and 87 %, respectively. However, PEI was found to be highly toxic as most cells treated with PEI did not survive.

EXAMPLE 12: Endosome Buffering Activity of n-PAE (Polymer 9) Cells were treated with the polyplex in the same manner as in Example 10, with the modification of adding 50 uM of chloroquine or 5 M of nigericin together.

Chloroquine is known to accumulate in endosomal compartment, buffer endosome acidification, and induce osmotic swelling of the endosome, which eventually results in endosome destabilization and release of internalized polyplex. Nigericin is a carboxylic ionophore that mediates exchange of monovalent cations through the membrane and is known to be an inhibitor of endosomal acidification. The endosome buffering effects of the polyplex are shown in Fig. 12.

When chloroquine was used, as seen in the histograms of Fig. 12, TE of PEI and n-PAE were slightly decreased and increased, respectively, while PAGA was highly increased in TE (899 times). Because PEI and n-PAE themselves act as endosome buffers, they are not affected by the endosome buffer chloroquine. In contrast, because PAGA has no endosome buffering activity, its TE is greatly improved when it is aided by the endosome buffer chloroquine. PEI and n-PAE shows much higher TE in the absence of than in the presence of nigericin whose activity is contrary to that of chloroquine. On the other hand, PAGA showed reverse results. Taken together, the data of Fig. 12 demonstrates that the high gene delivery capacity of n-PAE is attributed to its endosome buffering activity.

INDUSTRIAL APPLICABILITY The cationic highly branched polyaminoesters of the present invention, as described hereinbefore, are associated with negatively charged DNA by electrostatic attraction to form complexes (polyplexes) which can effectively pass through cellular membranes, thereby introducing DNA into cells. In addition, the cationic highly branched polyaminoesters of the present invention are superior in terms of transfection efficiency owing to their endosome buffering activity, in addition to being of low toxicity owing to its biodegradability.

The present invention has been described in an illustrative manner, and it is to be understood that the terminology used is intended to be in the nature of description rather than of limitation. Many modifications and variations of the present invention are possible in light of the above teachings. Therefore, it is to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.