BRANCHED POLYAMINO ACID BACTERIOSTATIC AGENT AND APPLICATION THEREOF

Abstract

The present invention provides a branched poly(amino acid) antimicrobial agent, comprising a branched poly(amino acid); the branched poly(amino acid) is obtained by the homopolymerization of one amino acid unit, or is obtained by the copolymerization of two or more amino acid units; the amino acid unit has a structure shown by Formula I. The present invention uses the amino acid as raw material, is non-toxic, has no side effects, and is a green and environmentally friendly new antimicrobial agent, and accessible to the users. The branched structure of the poly(amino acid) results in that such material has many active functional groups, may be further modified, has good biocompatibility, and will not develop drug resistance during the long-term use of this antimicrobial agent.

##STR00001##

Claims

1. A branched poly(amino acid) antimicrobial agent, comprising a branched poly(amino acid); the branched poly(amino acid) is obtained by the homopolymerization of one amino acid unit, or by the copolymerization of two or more amino acid units; the amino acid unit has a structure of Formula I or salts thereof: ##STR00010## wherein, a, b, c, d, e, and f are independently an integer of 06, and 1a+b+c+d+e+f20, preferably a+b+c+d+e+f10; T.sub.1, T.sub.2, T.sub.3, T.sub.4, T.sub.5, and T.sub.6 are independently selected from the group consisting of hydrogen, hydroxyl, mercapto, amino, carboxyl, C1C18 alkyl and derivatives thereof, C6C30 aryl and derivatives thereof, C3C8 cycloalkyl and derivatives thereof, C2C8 alkenyl and derivatives thereof, C2C8 alkynyl and derivatives thereof, C1C8 alkoxy and derivatives thereof, C1C8 alkylthio and derivatives thereof, carboxylic acid and derivatives thereof, amine and derivatives thereof, nitrogen-containing heterocyclic group and derivatives thereof, oxygen-containing heterocyclic group and derivatives thereof, or sulfur-containing heterocyclic group and derivatives thereof.

2. The antimicrobial agent according to claim 1, wherein the branched poly(amino acid) is obtained by the homopolymerization of one amino acid unit, wherein at least one of T.sub.1, T.sub.2, T.sub.3, T.sub.4, T.sub.5, and T.sub.6 of the amino acid unit is selected from the group consisting of hydroxyl, amino, mercapto, carboxyl, C2C8 alkenyl and derivatives thereof, C2C8 alkynyl and derivatives thereof, C1C8 alkoxy and derivatives thereof, C1C8 alkylthio and derivatives thereof, carboxylic acid and derivatives thereof, amine and derivatives thereof, nitrogen-containing heterocyclic group and derivatives thereof, oxygen-containing heterocyclic group and derivatives thereof, or sulfur-containing heterocyclic group and derivatives thereof.

3. The antimicrobial agent according to claim 1, wherein the branched poly(amino acid) is obtained by the copolymerization of two or more amino acid units, wherein at least one of T.sub.1, T.sub.2, T.sub.3, T.sub.4, T.sub.5, and T.sub.6 of at least one amino acid unit is selected from the group consisting of hydroxyl, amino, mercapto, carboxyl, C2C8 alkenyl and derivatives thereof, C2C8 alkynyl and derivatives thereof, C1C8 alkoxy and derivatives thereof, C1C8 alkylthio and derivatives thereof, carboxylic acid and derivatives thereof, amine and derivatives thereof, nitrogen-containing heterocyclic group and derivatives thereof, oxygen-containing heterocyclic group and derivatives thereof, or sulfur-containing heterocyclic group and derivatives thereof.

4. The antimicrobial agent according to claim 1, wherein T.sub.1, T.sub.2, T.sub.3, T.sub.4, T.sub.5, and T.sub.6 are independently selected from the group consisting of any one of the following structures: ##STR00011## or salts thereof, ##STR00012## or salts thereof, ##STR00013## wherein, g is an integer of 0 to 10; wherein, xx, yy, and zz are independently selected from the group consisting of hydrogen, C1C18 alkyl, C6C30 aryl, C3C18 cycloalkyl, carbonyl derivatives; hh is independently selected from the group consisting of hydrogen, hydroxyl, amino, halogen elements, C1C18 alkyl, C6C30 aryl, C3C18 cycloalkyl, amine and derivatives thereof, alkoxy derivatives, alkylthio derivatives; ii, jj, and kk are independently selected from the group consisting of hydrogen, C1C18 alkyl, C6C30 aryl, C3C18 cycloalkyl, alkoxy and derivatives thereof; oo, pp, and qq are independently selected from the group consisting of hydrogen, carboxyl, hydroxyl, amino, C1C18 alkyl, C6C30 aryl, C3C18 cycloalkyl, halogens, amine and derivatives thereof, alkoxy derivatives, carbonyl derivatives; rr and tt are independently selected from the group consisting of hydrogen, C1C18 alkyl, C6C30 aryl, C3C18 cycloalkyl, alkylthio derivatives, alkoxy derivatives, carbonyl derivatives; and uu is independently selected from the group consisting of one or more of the structures represented by the following formulae: C.sub.nH.sub.2n3-hT.sub.h (n is an integer of 0 to 10), C.sub.nH.sub.2n3-hT.sub.h (n is an integer of 2 to 10), C.sub.n3-hT.sub.h (n is an integer of 2 to 10), C.sub.nH.sub.2nhT.sub.h (n is an integer of 6 to 18), wherein, h is an integer of 0 to 3, and T is independently selected from any one or more of halogen elements.

5. The antimicrobial agent according to claim 1, wherein T.sub.1, T.sub.2, T.sub.3, T.sub.4, T.sub.5, and T.sub.6 are independently selected from the group consisting of any one of the following structures: ##STR00014##

6. The antimicrobial agent according to claim 1, wherein the branched poly(amino acid) is obtained by the homopolymerization of one amino acid unit, and the amino acid has a functionality of 3.

7. The antimicrobial agent according to claim 6, wherein the amino acid unit is glutamic acid, lysine, arginine, ornithine, histidine, aspartic acid, tryptophan, serine, citrulline, tyrosine, cysteine, asparagine, glutamine, or threonine, preferably the amino acid unit is lysine, arginine, ornithine, or histidine.

8. The antimicrobial agent according to claim 1, wherein the copolymerization unit of the branched poly(amino acid) contains at least one or more amino acid having a functionality of 3, wherein the amino acid unit having a functionality of 3 accounts for of total amino acid units, 0<100%.

9. The antimicrobial agent according to claim 8, wherein the amino acid unit having a functionality of 3 is one or more of glutamic acid, lysine, ornithine, arginine, histidine, asparagine, glutamine, serine, tryptophan, aspartic acid, citrulline, threonine, tyrosine, and cysteine; preferably the amino acid unit includes at least one or more of lysine, ornithine, arginine, and histidine.

10. The antimicrobial agent according to claim 1, wherein the poly(amino acid) is subjected to any one or more of the following modifications: I. modifying the amino or the amino group in the amides into the following groups: ##STR00015## II. modifying the hydroxyl group into OR.sub.1 or OC(=O)R.sub.2; III. modifying the mercapto group into SR.sub.3; IV. modifying the carboxyl group into C(=O)NHR.sub.4 or C(=O)OR.sub.5; V. modifying the guanidine group into the group as shown by Formula V-1; VI. modifying the NH in the nitrogen-containing heterocyclyl into NR.sub.6; ##STR00016## wherein, X, Y, Z, and Q are independently selected from the group consisting of hydrogen, C1C18 alkyl and derivatives thereof, C6C30 aryl and derivatives thereof, C3C18 cycloalkyl and derivatives thereof, C2C18 alkenyl and derivatives thereof, C2C18 alkynyl and derivatives thereof, C1C18 alkoxy and derivatives thereof, carboxylic acid and derivatives thereof, amine and derivatives thereof, nitrogen-containing heterocyclic group and derivatives thereof, oxygen-containing heterocyclic group and derivatives thereof, or sulfur-containing heterocyclic group and derivatives thereof; R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6 are independently selected from the group consisting of H, C1C18 alkyl and derivatives thereof, C6C30 aryl and derivatives thereof, C3C18 cycloalkyl and derivatives thereof, C2C18 alkenyl and derivatives thereof, C2C18 alkynyl and derivatives thereof, C1C18 alkoxy and derivatives thereof, carboxylic acid and derivatives thereof, amino and derivatives thereof, nitrogen-containing heterocyclic group and derivatives thereof, oxygen-containing heterocyclic group and derivatives thereof, or sulfur-containing heterocyclic group and derivatives thereof; and R.sub.1, R.sub.3, R.sub.5, and R.sub.6 are not H.

11. The antimicrobial agent according to claim 10, wherein X, Y, Z, and Q are independently selected from the group consisting of hydrogen, C1C3 alkyl, C6C8 aryl, C3C6 cycloalkyl, C1C3 alkoxy, C205 nitrogen-containing heterocyclic group, C2C5 oxygen-containing heterocyclic group, or C2C5 sulfur-containing heterocyclic group; and R.sub.1, R.sub.2, and R3 are independently selected from the group consisting of C1C3 alkyl, C6C8 aryl, C3C6 cycloalkyl, C1C3 alkoxy, C2C5 nitrogen-containing heterocyclic group, C2C5 oxygen-containing heterocyclic group, or C2C5 sulfur-containing heterocyclic group.

12. The antimicrobial agent according to claim 1, wherein the branched poly(amino acid) has a number-average molecular weight in the range of 500 g/mol-500,000 g/mol.

13. The antimicrobial agent according to claim 1, further comprising an adjuvant.

14. The antimicrobial agent according to claim 1, wherein the antimicrobial agent is used in one or more forms selected from the group consisting of solid, solution, suspension, emulsion, hydrogel, oleogel, or aerosol, being coated or grafted onto the solid surface, or being blended with other materials.

15. The antimicrobial agent according to claim 1, for use in one or more of bacteria, virus, fungus, actinomyces, rickettsia, mycoplasma, chlamydia, and spirochete.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0078] FIG. 1 is the .sup.1H NMR spectrum of the branched polylysine prepared in Example 2 of the present invention;

[0079] FIG. 2 is the .sup.1H NMR spectrum of the branched polyarginine prepared in Example 10 of the present invention; and

[0080] FIG. 3 is the .sup.1H NMR spectrum of the branched poly(amino acid) prepared in Example 25 of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0081] In order to further illustrate the present invention, the branched poly(amino acid) antimicrobial agent provided by the present invention and its use will be described in detail below in conjunction with Examples. Unless otherwise specified, the reaction raw materials used in the following Examples are all commercially available products, and purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., Sigma-Aldrich Chemical Reagent Co., Ltd., J&K Bailingwei Technology Co., Ltd., Shanghai McLean Biochemical Technology Co., Ltd. or Sinopharm Group Chemical Reagent Co., Ltd.

[0082] In the following Examples, the molecular weight of the polymer is measured by gel-permeation chromatograph (GPC), and the specific measurement method is as follows: the molecular weight (MO of the polymer and the distribution thereof (PDI=M.sub.w/M.sub.n) are measured by Waters 2414 gel-permeation chromatograph system equipped with Waters 2414 interference refraction detector (mobile phase: 0.2M acetic acid/0.1M sodium acetate, flow rate: 0.6 mL/min, temperature: 35 C., standards: polyethylene glycol).

Example 1

[0083] 100 grams of arginine was added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 180 C. under stirring and heating for 4 hours. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with methanol and precipitated in diethyl ether, to give 82.7 grams of hyperbranched polyarginine as light yellow solid powder. GPC characterization: M.sub.n=2200 g/mol, PDI=1.91.

Example 2

[0084] 91.32 grams of lysine hydrochloride and 28.05 grams of KOH were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 250 C. under stirring and heating for 1 minute. After the stop of heating, the polymer was dissolved with methanol and filtered to remove salts, concentrated, then precipitated in diethyl ether, to give 84 grams of hyperbranched polylysine as light yellow solid powder. GPC characterization: M.sub.n=1100 g/mol, PDI=1.81.

[0085] FIG. 1 shows the .sup.1H NMR spectrum of the synthesized branched poly(amino acid).

Example 3

[0086] 50 grams of serine and 50 mL of n-hexanol were added to a 500 mL round bottom flask, and a water separator was connected. Under nitrogen atmosphere, the reaction was conducted at 190 C. under stirring and heating for 10 hours. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with ethanol and precipitated in diethyl ether, to give 28.5 grams of hyperbranched polyserine as light yellow solid powder. GPC characterization: M.sub.n=7800 g/mol, PDI=1.71.

Example 4

[0087] 91.32 grams of lysine hydrochloride, 28.05 grams of KOH, and 10 mg of antimony trioxide were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 180 C. under stirring and heating for 96 hours. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with methanol and filtered to remove salts, concentrated, then precipitated in diethyl ether, to give 55.5 grams of hyperbranched polylysine as light yellow solid powder. GPC characterization: M.sub.n=500000 g/mol, PDI=2.36.

Example 5

[0088] 80 grams of lysine, 20 grams of lysine hydrochloride, and 6.14 g KOH were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 100 C. under stirring and heating for 10 hours. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with ethanol and filtered to remove salts, concentrated, then precipitated in diethyl ether, to give 68.5 grams of hyperbranched polylysine as brown solid powder. GPC characterization: M.sub.n=800 g/mol, PDI=1.66.

Example 6

[0089] 50 grams of cysteine and 100 mL of DMF were added to a 500 mL round bottom flask, and a water separator was connected. Under nitrogen atmosphere, the reaction was conducted at 180 C. under stirring and heating for 10 hours. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with methanol and precipitated in diethyl ether, to give 33.5 grams of hyperbranched polycysteine as yellow solid powder. GPC characterization: M.sub.n=1900 g/mol, PDI=2.07.

Example 7

[0090] 50 grams of glutamic acid and 100 mL of ethylene glycol were added to a 500 mL round bottom flask, and a water separator was connected. Under nitrogen atmosphere, the reaction was conducted at 200 C. under stirring and heating for 1 minute. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with methanol and precipitated in diethyl ether, to give 31.5 grams of hyperbranched polyglutamic acid as pale yellow solid powder. GPC characterization: M.sub.n=2100 g/mol, PDI=1.86.

Example 8

[0091] 50 grams of arginine and 100 mL of ethylene glycol were added to a 500 mL round bottom flask, and a water separator was connected. Under nitrogen atmosphere, the reaction was conducted at 150 C. under stirring and heating for 96 hours. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with methanol and precipitated in ethyl acetate, to give 34.5 grams of hyperbranched polyarginine as light yellow solid powder. GPC characterization: M.sub.n=1800 g/mol, PDI=2.18.

Example 9

[0092] 100 grams of lysine and 0.1 grams of phosphoric acid were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 100 C. under stirring and heating for 96 hours. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with methanol and precipitated in diethyl ether, to give 78.5 grams of hyperbranched polylysine as tawny solid powder. GPC characterization: M.sub.n=6800 g/mol, PDI=1.97.

Example 10

[0093] 100 grams of arginine and 200 grams of ethylene glycol were added to a 500 mL round bottom flask, and a water separator was connected. N.sub.2 was bubbled for 30 min The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 170 C. under stirring and heating for 8 hours. After the stop of heating, the reaction system was cooled to room temperature. The ethylene glycol was separated, and the polymer was precipitated in diethyl ether, to give 71.2 grams of hyperbranched polyarginine as light yellow solid powder. GPC characterization: M.sub.n=3100 g/mol, PDI=1.78.

[0094] FIG. 2 shows the .sup.1H NMR spectrum of the synthesized branched poly(amino acid).

Example 11

[0095] 100 grams of histidine and 200 grams of ethylene glycol were added to a 500 mL round bottom flask, and a water separator was connected. N.sub.2 was bubbled for 30 min The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 180 C. under stirring and heating for 24 hours. After the stop of heating, the reaction system was cooled to room temperature. The ethylene glycol was separated, and the polymer was washed with diethyl ether for 5 times, to give 71.2 grams of hyperbranched polyhistidine as yellow solid powder. GPC characterization: M.sub.n=1500 g/mol, PDI=1.71.

Example 12

[0096] 100 grams of ornithine and 50 g of water were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 150 C. under stirring and heating for 5 hours. After the stop of heating, the polymer was grounded to give 87 grams of hyperbranched polylysine as brown solid powder. GPC characterization: M.sub.n=3400 g/mol, PDI=1.77.

Example 13

[0097] 2 g of hyperbranched polyarginine of Example 10 and 0.2 g of silver nitrate were dissolved into 5 mL of water, stirred and uniformly dispersed, then freeze-dried, to give 2.18 g of a mixture of hyperbranched polyarginine with silver ions.

Example 14

[0098] 100 grams of lysine and 50 g of water were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 180 C. under stirring and heating for 5 hours. After the stop of heating, the polymer was grounded to give 87 grams of hyperbranched polylysine as brown solid powder. GPC characterization: M.sub.n=2400 g/mol, PDI=1.77.

Example 15

[0099] 2 g of hyperbranched polylysine of Example 14 and 0.2 g of chitosan were dissolved into 5 mL of water, stirred and uniformly dispersed, then freeze-dried, to give 2.18 g of a mixture of hyperbranched polylysine and chitosan.

Example 16

[0100] 100 grams of citrulline and 50 g of water were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 190 C. under stirring and heating for 5 hours. After the stop of heating, the polymer was dissolved with water and dialyzed by secondary water, to give 57 grams of hyperbranched polycitrulline as light yellow solid powder. GPC characterization: M.sub.n=5700 g/mol, PDI=1.27.

Example 17

[0101] 100 grams of (2S,3R,4S)--(carboxylcyclopropyl) glycine and 50 g of water were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 180 C. under stirring and heating for 5 hours. After the stop of heating, the polymer was grounded to give 86.4 grams of hyperbranched poly(amino acid) as brown solid powder. GPC characterization: M.sub.n=7500 g/mol, PDI=1.87.

Example 18

[0102] 100 grams of 5-amino-2-hydrazinopentanoic acid (CAS: 60733-16-6) and 50 g of water were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 180 C. under stirring and heating for 5 hours. After the stop of heating, the polymer was grounded to give 83.4 grams of hyperbranched poly(amino acid) as brown solid powder. GPC characterization: M.sub.n=8400 g/mol, PDI=1.94.

Example 19

[0103] 100 grams of 4-amino-3-hydroxylbutanoic acid and 50 g of water were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 180 C. under stirring and heating for 5 hours. After the stop of heating, the polymer was fractionated by gel column to give 78.4 grams of hyperbranched poly(amino acid) as a brown solid powder. GPC characterization: M.sub.n=7300 g/mol, PDI=1.48.

Example 20

[0104] 36 mg of hyperbranched poly(amino acid)s prepared in Examples 1-19 were respectively weighted and dissolved into 3 mL of sterile PBS, to give 12 mg/mL of stock solution. The antimicrobial activity of the hyperbranched poly(amino acid)-based antimicrobial agents was measured in accordance with the following method, and a part of experiment results were shown in Table 1.

[0105] The various strains used in the following Examples were purchased from the National Institute for the Control of Biological Products.

[0106] The antimicrobial activity of the hyperbranched poly(amino acid)-based antimicrobial agents was tested through the 96-well plate method, and -polylysine synthesized by fermentation was used as the control to evaluate the antibacterial capacity of the resulting hyperbranched poly(amino acid)-based antimicrobial agents. The minimum inhibitory concentration (MIC) is defined as the lowest polymer concentration that inhibits microbial growth by 90% compared to the control group.

[0107] A small amount of strains were picked from the agar slant medium with the inoculating ring to the ordinary MH medium, incubated at 37 C. overnight to recover the strains and achieve exponential growth. The microbial solution was diluted so that the concentration of the microbial solution was 10.sup.6 CFU/mL. In each well, 175 pL of microbial solution and 25 L of polymer solutions at different concentrations were added. The 96-well plate was incubated at 37 C. for 20 hours, and the OD.sub.600 value was measured by the microplate reader.

TABLE-US-00001 TABLE 1 Comparisons of MIC values of different antimicrobial polymers against different microbes MIC values of different antimicrobial polymers against different microbes (ng/mL) Ex. Ex. Ex. Ex. Ex. Ex. Ex. Ex. Ex. Ex. Ex. - Strain Name 1 5 9 10 11 12 13 16 17 18 19 polylysine Gram- Escherichia 24 47 24 24 47 24 24 12 24 12 12 47 negative coli bacteria ATCC8739 Pseudomonas 24 24 12 12 47 24 12 12 12 12 24 47 aeruginosa ATCC9207 Klebsiella 24 47 47 24 24 24 12 12 24 24 24 24 pneumoniae ATCC700603 Salmonella 24 24 24 24 24 12 12 24 47 6 12 47 paratyphi B CMCC50094 Acinetobacter 24 24 24 24 24 24 12 12 12 6 6 24 baumannii ATCC19606 Gram- Staphylococcus 12 12 12 12 24 12 24 12 6 24 12 24 positive aureus bacteria ATCC 25923 Methicillin- 24 24 24 24 47 24 47 47 24 24 24 47 resistant Staphylococcus aureus ATCC 43300 Bacillus 12 12 12 12 24 12 24 6 47 12 12 24 subtilis ATCC 6633 Micrococcus 12 12 12 6 24 12 24 12 24 12 12 24 luteus ATCC10240 Bacillus 12 12 12 12 47 6 12 12 24 12 24 24 pumilus ATCC700814 Fungi Candida 24 24 24 12 96 24 12 12 12 6 12 47 albicans ATCC10231 Saccharomyces 24 47 47 24 47 24 12 12 47 12 24 47 cerevisiae ATCC 9763

Example 21

[0108] 36 mg of hyperbranched poly(amino acid)s prepared in Examples 1-19 were weighted respectively and dissolved into 3 mL of sterile PBS, to give 12 mg/mL of stock solution. The in vitro hemolytic activity of the hyperbranched poly(amino acid)-based antimicrobial agents was measured in accordance with the following method, and a part of experiment results were shown in Table 2.

[0109] The in vitro hemolytic activity of the hyperbranched poly(amino acid)-based antimicrobial agents was tested by a 96-well plate method, and -polylysine synthesized by fermentation was used as the control to evaluate the in vitro hemolytic activity of the resulting hyperbranched poly(amino acid)-based antimicrobial agents.

[0110] Preparation of 2% (v/v) erythrocyte suspension: 2 mL of fresh healthy human blood was taken and dilute with 10 mL of endotoxin-free PBS buffer solution. The blood was transferred to a triangular flask with glass beads and shaken for 10 minutes, or stirred with a glass rod, to remove the fibrin and make it into deflbrinated blood. The deflbrinated blood was centrifuged at 1000-1500 r/min at 20 C. for 1015 minutes, and the supernatant was removed. The precipitated erythrocyte was then washed with the PBS buffer solution for 4 times in accordance with the above method, until the supernatant does not appear red. The obtained erythrocyte was formulated into a 2% suspension with physiological saline for subsequent experiments.

[0111] The polymer was diluted with the PBS buffer solution to prepare solutions of different concentrations and added to 96-well plates. The PBS buffer solution alone was used as the negative control, and 0.2% Triton-X-100 dissolved in water was used as the positive control for in vitro hemolytic activity test. The washed erythrocyte (2% v/v, 50 L) was added to the 96-well plate, mixed thoroughly and incubated. The absorption at 540 nm was measured with a microplate reader.

TABLE-US-00002 TABLE 2 Test results for the in vitro hemolytic activities of different antimicrobial polymers Concentration of the Hemolysis ratio (%) antimicrobial polymer Ex. Ex. Ex. Ex. Ex. Ex. Ex. Ex. Ex. Ex. Ex. (g/mL) 1 5 9 10 11 12 13 16 17 18 19 -polylysine 3 0 0 0 0 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 0 0 0 0 0 12 0 0 0 0 0 0 0 0 0 0 0 0 24 0 0 0 0 0 0 0 0 0 0 0 0 47 0 0 0 0 0 0 0 5 0 0 0 0 94 0 0 0 0 1 0 0 8 0 0 0 0 188 0 0 0 0 3 0 0 12 0 0 0 0 375 0 1 0 0 10 0 0 19 0 0 0 0 750 0 2 1 0 25 0 1 32 0 0 0 0 1500 1 3 2 1 45 0 1 54 1 0 0 0

Example 22

[0112] The Example was used to test the in vivo acute toxicity of the hyperbranched poly(amino acid)-based antimicrobial agents in animals, and -polylysine synthesized by fermentation was used as the control to evaluate the in vivo acute toxicity of the resulting hyperbranched poly(amino acid)-based antimicrobial agents in animals.

[0113] One hundred mice (Balb/C mice, purchased from Jilin University), half male and half female, weighing 213 g, were used. The hyperbranched poly(amino acid)-based antimicrobial agents prepared in Examples 1-19 were taken at a dose of 1 mg/mL respectively and intramuscularly injected into mice once a day for 15 consecutive days to observe the toxic reaction of mice. The experimental results showed that after intramuscular injection for 21 consecutive days, except for few mice with reduced vitality, the rest had no significant abnormal reactions, and all mice survived. It was demonstrated that the resulting hyperbranched poly(amino acid)-based antimicrobial agents have lower toxicity in vivo.

Example 23

[0114] 80 grams of arginine and 20 grams of alanine were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted under stirring and heating at 160 C. for 5 hours. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with ethanol and precipitated in diethyl ether, to give 78.7 grams of hyperbranched poly(amino acid) as light yellow solid powder. GPC characterization: M.sub.n=3100 g/mol, PDI=1.76.

Example 24

[0115] 50 grams of ornithine, 50 grams of leucine, and 10 mg of scandium trifluoromethylsulfonate were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 180 C. under stirring and heating for 2 hours. After the stop of heating, the polymer was dissolved with methanol and precipitated in diethyl ether, to give 81.5 grams of hyperbranched poly(amino acid) as dark yellow solid powder. GPC characterization: M.sub.n=500 g/mol, PDI=1.82.

Example 25

[0116] 91.32 grams of lysine hydrochloride, 28.05 grams of KOH, and 20 grams of alanine were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 180 C. under stirring and heating for 10 hours. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with methanol and precipitated in diethyl ether, to give 75.2 grams of branched poly(amino acid) as light yellow solid powder. GPC characterization: M.sub.n=14100 g/mol, PDI=2.72.

[0117] FIG. 3 shows the .sup.1H NMR spectrum of the synthesized branched poly(amino acid).

Example 26

[0118] 90 grams of histidine, 10 grams of phenylalanine, and 10 mg of ferric trichloride were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 180 C. under stirring and heating for 2 hours. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with water and precipitated in tetrahydrofuran, to give 79.5 grams of hyperbranched poly(amino acid) as light yellow solid powder. GPC characterization: M.sub.n=1100 g/mol, PDI=1.62.

Example 27

[0119] 80 grams of ornithine was first added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 190 C. under stirring and heating for 4 hours. Then 20 grams of 6-aminohexanoic acid was added to the reaction system and reacted for another 4 hours. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with methanol and precipitated in diethyl ether, to give 82.3 grams of hyperbranched poly(amino acid) having a core-shell structure, as light yellow solid powder. GPC characterization: M.sub.n=11100 g/mol, PDI=1.94.

Example 28

[0120] 91.32 grams of lysine hydrochloride, 20 grams of NaOH, and 20 grams of alanine were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 180 C. under stirring and heating for 36 hours. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with methanol and precipitated in diethyl ether, to give 74.8 grams of hyperbranched poly(amino acid) as light yellow solid powder. GPC characterization: M.sub.n=81100 g/mol, PDI=3.98.

Example 29

[0121] 91.32 grams of lysine hydrochloride, 20 grams of NaOH, and 20 grams of alanine were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 100 C. under stirring and heating for 96 hours. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with methanol and precipitated in diethyl ether, to give 72.8 grams of hyperbranched poly(amino acid) as dark yellow solid powder. GPC characterization: M.sub.n=481100 g/mol, PDI=2.21.

Example 30

[0122] 91.32 grams of lysine hydrochloride and 20 grams of NaOH were first added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 250 C. under stirring and heating for 0.5 h. Then 20 grams of alanine was added to the reaction system and reacted for another 0.5 h. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with methanol and precipitated in diethyl ether, to give 73.1 grams of hyperbranched poly(amino acid) having a core-shell structure, as light yellow solid powder. GPC characterization: M.sub.n=1200 g/mol, PDI=1.51.

Example 31

[0123] 50 grams of lysine and 50 grams of arginine were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 180 C. under stirring and heating for 4 hours. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with methanol and precipitated in ethyl acetate, to give 80.1 grams of branched poly(amino acid) as light yellow solid powder. GPC characterization: M.sub.n=3200 g/mol, PDI=1.73.

Example 32

[0124] 80 grams of (25,3R,45)--(carboxylcyclopropyl)glycine and 20 grams of alanine were added to a 500 mL round bottom flask and 80 g of secondary water was added for dissolution, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 180 C. under stirring and heating for 5 hours. After the stop of heating, the polymer was ground, to give 86.4 grams of hyperbranched poly(amino acid) as brown solid powder. GPC characterization: M.sub.n=5000g/mol, PDI=1.88.

Example 33

[0125] 80 grams of 5-amino-2-hydrazinopentanoic acid (CAS:60733-16-6) and 20 grams of alanine were added to a 500 mL round bottom flask and 80 g of secondary water was added for dissolution, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 160 C. under stirring and heating for 10 hours. After the stop of heating, the polymer was ground, to give 84.5 grams of hyperbranched poly(amino acid) as brown solid powder. GPC characterization: M.sub.n=8700g/mol, PDI=1.92.

Example 34

[0126] 20 grams of phenylalanine and 10 grams of alanine were first added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 100 C. under stirring and heating for 30 hours. Then 70 grams of lysine hydrochloride and 20 g of KOH were added to the reaction system and reacted for another 70 hours. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with methanol and precipitated in diethyl ether, to give 68.5 grams of hyperbranched poly(amino acid) having a core-shell structure, as light yellow solid powder. GPC characterization: M.sub.n=6200 g/mol, PDI=1.88.

Example 35

[0127] 80 grams of 4-amino-3-hydroxylbutanoic acid and 20 grams of glycine were added to a 500 mL round bottom flask and 80 g of secondary water was added for dissolution, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 170 C. under stirring and heating for 12 hours. After the stop of heating, the polymer was dissolved in water and precipitated in tetrahydrofuran, to give 84.5 grams of hyperbranched poly(amino acid) as brown solid powder. GPC characterization: M.sub.n=13700g/mol, PDI=1.72.

Example 36

[0128] 2 g of hyperbranched poly(amino acid) of Example 28 and 2 g of polyhexamethylene biguanidine were dissolved into 5 mL of water, stirred and uniformly dispersed, then freeze-dried, to give 4 g of a mixture of hyperbranched poly(amino acid) with polyhexamethylene biguanidine.

Example 37

[0129] 2 g of hyperbranched poly(amino acid) of Example 24 and 2 g of polyhexamethylene biguanidine were dissolved into 5 mL of water, stirred and uniformly dispersed, then freeze-dried, to give 4 g of a mixture of hyperbranched poly(amino acid) with polyhexamethylene biguanidine.

Example 38

[0130] 36 mg of hyperbranched poly(amino acid)s prepared in Examples 23-37 were weighted respectively and dissolved into 3 mL of sterile PBS, to give 12 mg/mL of stock solution. The antimicrobial activity of the hyperbranched poly(amino acid)-based antimicrobial agents was measured in accordance with the following method, and the experiment results were shown in Tables 3-1 and 3-2. The various strains used in the following examples were purchased from the National Institute for the Control of Biological Products.

[0131] The antimicrobial activity of the hyperbranched poly(amino acid)-based antimicrobial agents was tested by a 96-well plate method, and -polylysine synthesized by fermentation was used as the control to evaluate the antimicrobial capacity of the resulting hyperbranched poly(amino acid)-based antimicrobial agents. The minimum inhibitory concentration (MIC) is defined as the lowest polymer concentration that inhibits microbial growth by 90% compared to the control group.

[0132] A small amount of strains were picked from the agar slant medium with the inoculating ring to the ordinary M-H medium, incubated at 37 C. overnight to recover the strains and achieve exponential growth. The microbial solution was diluted so that the concentration of the microbial solution was 10.sup.6 CFU/mL. In each well, 175 L of microbial solution and 25 L of polymer solutions at different concentrations were added. The 96-well plate was incubated at 37 C. for 20 hours, and the OD.sub.600 value was measured by the microplate reader.

TABLE-US-00003 TABLE 3-1 Comparisons of MIC values of different antimicrobial polymers against different microbes MIC values of different antimicrobial polymers against different microbes (g/mL) Ex. Ex. Ex. Ex. Ex. Ex. Ex. Ex. Ex. Strain Name 23 24 25 26 27 28 29 30 31 -polylysine Gram- Escherichia 24 24 24 24 47 47 47 24 24 47 negative coli bacteria ATCC8739 Pseudomonas 24 12 12 12 24 24 47 12 24 47 aeruginosa ATCC9207 Klebsiella 24 24 24 24 24 47 96 24 24 24 pneumoniae ATCC700603 Salmonella 24 24 24 24 24 24 47 47 24 47 paratyphi B CMCC50094 Acinetobacter 24 24 24 24 47 24 47 24 24 24 baumannii ATCC19606 Gram- Staphylococcus 6 12 12 12 24 12 47 12 12 24 positive aureus bacteria ATCC 25923 Methicillin- 12 24 24 24 47 24 96 24 24 47 resistant Staphylococcus aureus ATCC 43300 Bacillus subtilis 6 12 12 12 47 12 47 12 6 24 ATCC 6633 Micrococcus 12 6 6 6 24 12 47 6 12 24 luteus ATCC10240 Bacillus 6 12 12 12 24 12 47 12 6 24 pumilus ATCC700814 Fungi Candida 24 12 12 12 47 24 96 12 24 47 albicans ATCC10231 Saccharomyces 24 24 24 24 47 47 96 24 24 47 cerevisiae ATCC 9763

TABLE-US-00004 TABLE 3-2 Comparisons of MIC values of different antimicrobial polymers against different microbes MIC values of different antimicrobial polymers against different microbes (g/mL) Ex. Ex. Ex. Ex. Ex. Ex. - Strain Name 32 33 34 35 36 37 polylysine Gram- Escherichia coli 24 24 24 24 24 24 47 negative ATCC8739 bacteria Pseudomonas 12 12 24 12 12 24 47 aeruginosa ATCC9207 Klebsiella 24 12 24 12 12 12 24 pneumoniae ATCC700603 Salmonella 47 12 47 24 12 24 47 paratyphi B CMCC50094 Acinetobacter 24 12 24 12 12 24 24 baumannii ATCC19606 Gram- Staphylococcus 12 6 12 6 6 12 24 positive aureus bacteria ATCC 25923 Methicillin- 24 24 47 12 12 24 47 resistant Staphylococcus aureus ATCC 43300 Bacillus subtilis 12 24 24 12 12 12 24 ATCC 6633 Micrococcus 6 24 24 24 12 12 24 luteus ATCC10240 Bacillus pumilus 12 12 24 12 12 24 24 ATCC700814 Fungi Candida 12 12 47 24 24 24 47 albicans ATCC10231 Saccharomyces 24 12 47 24 24 24 47 cerevisiae ATCC 9763

Example 39

[0133] 36 mg of hyperbranched poly(amino acid)s prepared in Examples 23-37 were weighted respectively and dissolved into 3 mL of sterile PBS, to give 12 mg/mL of stock solution. The in vitro antihemolytic activity of the hyperbranched poly(amino acid)-based antimicrobial agents was measured in accordance with the following method, and the experiment results were shown in Tables 4-1 and 4-2.

[0134] The in vitro hemolytic activity of the hyperbranched poly(amino acid)-based antimicrobial agents was tested through the 96-well plate method, and -polylysine synthesized by fermentation was used as the control to evaluate the in vitro hemolytic activity of the resulting hyperbranched poly(amino acid)-based antimicrobial agents.

[0135] Preparation of 2% (v/v) erythrocyte suspension: 2 mL of fresh healthy human blood was taken and dilute with 10 mL of endotoxin-free PBS buffer solution. The blood was transferred to a triangular flask with glass beads and shaken for 10 minutes, or stirred with a glass rod, to remove the fibrin and make it into deflbrinated blood. The deflbrinated blood was centrifuged at 20 C. at 10001500 r/min for 1015 minutes, and the supernatant was removed. The precipitated erythrocyte was then washed with the PBS buffer solution for 4 times in accordance with the above method, until the supernatant does not appear red. The obtained erythrocyte was formulated into a 2% suspension with physiological saline for subsequent experiments.

[0136] The polymer was diluted with the PBS buffer solution to prepare solutions of different concentrations and added to 96-well plates. The PBS buffer solution alone was used as the negative control, and 0.2% Triton-X-100 dissolved in water was used as the positive control for in vitro hemolytic activity test. The washed erythrocyte (2% v/v, 50 L) was added to the 96-well plate, mixed thoroughly and incubated. The absorption at 540 nm was measured with a microplate reader.

TABLE-US-00005 TABLE 4-1 Test results for the in vitro hemolytic activities of different antimicrobial polymers Concentration of the antimicrobial Hemolysis ratio (%) polymer Ex. Ex. Ex. Ex. Ex. Ex. Ex. - (g/mL) 23 24 25 26 27 28 29 polylysine 3 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 0 12 0 0 0 0 0 0 0 0 24 0 0 0 0 0 0 0 0 47 0 1 0 1 1 0 1 0 94 1 4 0 4 4 0 4 0 188 3 7 0 7 7 0 7 0 375 6 12 2 12 12 0 12 0 750 8 20 6 20 20 1 20 0 1500 15 24 9 24 24 3 24 0

TABLE-US-00006 TABLE 4-2 Test results for the in vitro hemolytic activities of different antimicrobial polymers Concentration of the Hemolysis ratio (%) antimicrobial polymer Ex. Ex. Ex. Ex. Ex. Ex. Ex. Ex. - (g/mL) 30 31 32 33 34 35 36 37 polylysine 3 0 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 0 0 12 0 0 0 0 0 0 0 0 0 24 0 0 0 0 0 0 0 0 0 47 0 0 0 0 2 0 0 0 0 94 0 0 0 1 3 0 0 0 0 188 2 1 1 3 8 0 0 0 0 375 5 3 6 11 11 0 0 0 0 750 8 5 9 14 12 1 1 1 0 1500 13 10 12 20 22 11 2 3 0

Example 40

[0137] The Example was used to test the in vivo acute toxicity of the hyperbranched poly(amino acid)-based antimicrobial agents in animals, and -polylysine synthesized by fermentation was used as the control to evaluate the in vivo acute toxicity of the resulting hyperbranched poly(amino acid)-based antimicrobial agents in animals.

[0138] One hundred mice (Balb/C mice, purchased from Jilin University), half male and half female, weighing 213 g, were used. The hyperbranched poly(amino acid)-based antimicrobial agents prepared in Examples 23-37 were taken at a dose of 1 mg/mL respectively and intramuscularly injected into mice once a day for 15 consecutive days to observe the toxic reaction of mice. The experimental results showed that after intramuscular injection for 15 consecutive days, except for few mice with reduced vitality, the rest had no significant abnormal reactions, and all mice survived. It was demonstrated that the resulting hyperbranched poly(amino acid)-based antimicrobial agents have lower toxicity in vivo.

Example 41

[0139] 100 grams of ornithine was added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 180 C. under stirring and heating for 4 hours. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with methanol and precipitated in diethyl ether, to give 82.7 grams of hyperbranched polyornithine.

Example 42

[0140] 2 g of hyperbranched polyornithine of Example 41 was dissolved under heating in 20 mL of N,N-dimethyl formamide (DMF), and 5 g of iodomethane was added. The reaction was conducted at 80 C. under stirring for 24 hours. After the stop of heating, it was cooled to room temperature, and precipitated in ethyl acetate, to give 2.4 g of quaternary ammonium salt-modified hyperbranched polyornithine.

Example 43

[0141] 91.32 grams of lysine hydrochloride and 20 grams of NaOH were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 180 C. under stirring and heating for 6 hours. After the stop of heating, the polymer was dissolved with methanol and precipitated in diethyl ether, to give 72.8 grams of hyperbranched polylysine.

Example 44

[0142] 2 g of hyperbranched polylysine of Example 43 was dissolved in 5 mL of methanol, and acetyl chloride was slowly dropwise added at 0 C. The system was warmed to room temperature and reacted for another 12 hours, then precipitated in ethyl acetate, to give 2.3 g of acetyl-modified hyperbranched polylysine.

Example 45

[0143] 2 g of hyperbranched polyornithine of Example 41 was dissolved in 5 mL of methanol, and 4.8 g of methylisothiourea hemisulphate and 5 mL of triethylamine were added. The reaction was conducted at 60 C. for 12 hours. After the stop of heating, it was cooled to room temperature and precipitated in ethyl acetate, to give 2.1 g of guanidino-modified hyperbranched polyornithine.

Example 46

[0144] 2 g of -polylysine were dissolved into 5 mL of water, and 5.1 g of 1H-pyrazole-1-carboxamidine hydrochloride and 5 mL of triethylamine were added. The reaction was conducted at 60 C. for 12 hours. After the stop of heating, it was cooled to room temperature and precipitated in ethyl acetate, to give 2.2 g of guanidino-modified -polylysine.

Example 47

[0145] 5.6 g of N-benzyloxycarbonyllysine was weighted into a 250 mL three necked flask, and 100 mL of tetrahydrofuran was added, stirred and dispersed. 2.5 g of triphosgene was carefully weighted and dissolved into 30 mL tetrahydrofuran, and slowly dropwise added to the reaction system under the protection of nitrogen. The system was refluxed under stirring for 3 hours until the solution was completely clear. After the reaction was completed, a large number of n-hexane was added such that the crude product was precipitated. The crude product was recrystallized twice using the tetrahydrofuran-n-hexane system, and dried under vacuum to give 4.96 g of product (yield: 88.6%). The NCA ring-opening of the lysine was initiated by using DMF as solvent and n-butylamine as initiator, to give -polylysine.

Example 48

[0146] 2 g of a-polylysine of Example 47 were dissolved into 5 mL of water, and 5.1 g of 1H-pyrazole-1-carboxamidine hydrochloride and 5 mL of triethylamine were added. The reaction was conducted at 60 C. for 12 hours. After the stop of heating, it was cooled to room temperature and precipitated in ethyl acetate, to give 2.3 g of guanidino-modified -polylysine.

Example 49

[0147] 80 grams of arginine and 20 grams of serine were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 180 C. under stirring and heating for 4 hours. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with methanol and precipitated in diethyl ether, to give 81.2 grams of branched poly(amino acid).

Example 50

[0148] 2 g of poly(amino acid) of Example 49 was dissolved into 20 mL methanol and 0.5 g of p-toluenesulfonic acid was added, and the system was heated under reflux for 10 hours. The polymer was settled in diethyl ether, to give 2.2 g of ether group-modified poly(amino acid).

Example 51

[0149] 80 grams of arginine and 20 grams of glutamic acid were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 180 C. under stirring and heating for 4 hours. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with methanol and precipitated in diethyl ether, to give 80.2 grams of branched poly(amino acid).

Example 52

[0150] 2 g of poly(amino acid) of Example 51 was dissolved in 20 mL of dry methanol, and 0.9 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 0.1 g of 4-imethylaminopyridine (DMAP) were added. The system was stirred at room temperature for 10 hours. The polymer was precipitated in diethyl ether, to give 2.1 g of methyl ester-modified poly(amino acid).

Example 53

[0151] 80 grams of ornithine and 20 grams of cysteine were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 180 C. under stirring and heating for 4 hours. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with methanol and precipitated in diethyl ether, to give 80.2 grams of branched poly(amino acid).

Example 54

[0152] 2 g of poly(amino acid) of Example 53 was dissolved in 20 mL of dry DMF. The argon gas was introduced for 30 min to remove the oxygen. 0.9 g of propynol and 0.1 g DMAP were added. The system was stirred at room temperature for 10 min. The reaction mixture was reacted at room temperature under the irradiation of ultraviolet light (365nm) for 120 min. Then, the polymer was precipitated in diethyl ether, to give 2.1 g of hydroxyl-modified poly(amino acid).

Example 55

[0153] 80 grams of histidine and 20 g of serine were added to a 500 mL round bottom flask, and a water separator was connected. The nitrogen purge was conducted for three times (each for more than 10 minutes) so as to finally maintain nitrogen atmosphere. The reaction was conducted at 180 C. under stirring and heating for 4 hours. After the stop of heating, the reaction system was cooled to room temperature. The polymer was dissolved with methanol and precipitated in diethyl ether, to give 80.2 grams of branched poly(amino acid).

Example 56

[0154] 2 g of poly(amino acid) of Example 55 was dissolved into 20 mL methanol and 0.5 g of p-toluenesulfonic acid was added, and the system was heated under reflux for 10 hours. The polymer was precipitated in diethyl ether, to give 2.2 g of ether group-modified poly(amino acid).

Example 57

[0155] 36 mg of poly(amino acid)s prepared in Examples 41-56 were respectively weighted and dissolved into 3 mL of sterile PBS, to give 12 mg/mL of stock solution. The antimicrobial activity of the poly(amino acid)-based antimicrobial agents was measured in accordance with the following method, and a part of experiment results were shown in Tables 5-1 and 5-2.

[0156] The various strains used in the following examples were purchased from the National Institute for the Control of Biological Products.

[0157] The antimicrobial activity of the hyperbranched poly(amino acid)-based antimicrobial agents was tested through the 96-well plate method, and -polylysine (M.sub.n=4000 g/mol) synthesized by fermentation was used as the control to evaluate the antimicrobial capacity of the resulting poly(amino acid)-based antimicrobial agents. The minimum inhibitory concentration (MIC) is defined as the lowest polymer concentration that inhibits microbial growth by 90% compared to the control group.

[0158] A small amount of strains were picked from the agar slant medium with the inoculating ring to the ordinary M-H medium, incubated at 37 C. overnight to recover the strains and achieve exponential growth. The microbial solution was diluted so that the concentration of the microbial solution was 10.sup.6 CFU/mL. In each well, 175 L of microbial solution and 25 L of polymer solutions at different concentrations were added. The 96-well plate was incubated at 37 C. for 20 hours, and the OD.sub.600 value was measured by the microplate reader.

TABLE-US-00007 TABLE 5-1 Comparisons of MIC values of different antimicrobial polymers against different microbes MIC values of different antimicrobial polymers against different microbes(pg/mL) Ex. Ex. Ex. Ex. Ex. Ex. - Strain Name 41 42 43 44 45 46 polylysine Gram- Escherichia coli 47 24 47 24 12 24 47 negative ATCC8739 bacteria Pseudomonas 47 24 47 24 12 12 47 aeruginosa ATCC9207 Klebsiella 47 24 96 47 47 24 94 pneumoniae ATCC700603 Salmonella 24 12 24 12 24 6 47 paratyphi B CMCC50094 Acinetobacter 24 12 24 12 12 12 24 baumannii ATCC19606 Gram- Staphylococcus 24 12 24 12 12 12 24 positive aureus bacteria ATCC 25923 Methicillin- 24 6 24 24 6 12 47 resistant Staphylococcus aureus ATCC 43300 Bacillus subtilis 24 6 24 12 12 12 24 ATCC 6633 Micrococcus 47 12 24 24 12 6 24 luteus ATCC10240 Bacillus pumilus 12 6 47 12 12 12 24 ATCC700814 Fungi Candida 24 12 24 12 12 12 47 albicans ATCC10231 Saccharomyces 24 12 24 12 12 24 47 cerevisiae ATCC 9763

TABLE-US-00008 TABLE 5-2 Comparisons of MIC values of different antimicrobial polymers against different microbes MIC values of different antimicrobial polymers against different microbes(g/mL) Ex. Ex. Ex. Ex. Ex. - - Strain Name 47 48 50 52 54 polylysine polylysine Gram- Escherichia coli 47 24 24 24 12 47 47 negative ATCC8739 bacteria Pseudomonas 94 12 24 12 12 47 94 aeruginosa ATCC9207 Klebsiella 94 12 24 24 12 94 94 pneumoniae ATCC700603 Salmonella 47 24 24 12 24 47 47 paratyphi B CMCC50094 Acinetobacter 47 24 47 12 12 24 47 baumannii ATCC19606 Gram- Staphylococcus 94 24 12 24 6 24 94 positive aureus bacteria ATCC 25923 Methicillin- 94 12 24 47 12 47 94 resistant Staphylococcus aureus ATCC 43300 Bacillus subtilis 24 6 6 24 6 24 24 ATCC 6633 Micrococcus 47 24 12 24 6 24 48 luteus ATCC10240 Bacillus 24 12 6 12 12 24 24 pumilus ATCC700814 Fungi Candida 24 12 24 12 12 47 24 albicans ATCC10231 Saccharomyces 47 24 24 12 12 47 47 cerevisiae ATCC 9763

Example 58

[0159] 36 mg of poly(amino acid)s prepared in Examples 41-56 were weighted respectively and dissolved into 3 mL of sterile PBS, to give 12 mg/mL of stock solution. The in vitro hemolytic activity of the poly(amino acid)-based antimicrobial agents was measured in accordance with the following method, and a part of experiment results were shown in Tables 6-1 and 6-2.

[0160] The in vitro hemolytic activity of the poly(amino acid)-based antimicrobial agents was tested by a 96-well plate method, and -polylysine synthesized by fermentation was used as the control to evaluate the in vitro hemolytic activity of the resulting poly(amino acid)-based antimicrobial agents.

[0161] Preparation of 2% (v/v) erythrocyte suspension: 2 mL of fresh healthy human blood was taken and dilute with 10 mL of endotoxin-free PBS buffer solution. The blood was transferred to a triangular flask with glass beads and shaken for 10 minutes, or stirred with a glass rod, to remove the fibrin and make it into deflbrinated blood. The deflbrinated blood was centrifuged at 20 C. at 10001500 r/min for 1015 minutes, and the supernatant was removed. The precipitated erythrocyte was then washed with the PBS buffer solution for 4 times in accordance with the above method, until the supernatant does not appear red. The obtained erythrocyte was formulated into a 2% suspension with physiological saline for subsequent experiments.

[0162] The polymer was diluted with the PBS buffer solution to prepare solutions of different concentrations and added to 96-well plates. The PBS buffer solution alone was used as the negative control, and 0.2% Triton-X-100 dissolved in water was used as the positive control for in vitro hemolytic activity test. The washed erythrocyte (2% v/v, 50 L) was added to the 96-well plate, mixed well and incubated. The absorption at 540 nm was measured with a microplate reader.

TABLE-US-00009 TABLE 6-1 Test results for the in vitro hemolytic activities of different antimicrobial polymers Concentration of Hemolysis ratio (%) the antimicrobial Ex. Ex. Ex. Ex. Ex. - - polymer (g/mL) 41 42 43 44 45 polylysine polylysine 3 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 12 0 0 0 0 0 0 0 24 0 0 0 0 0 0 0 47 0 0 0 0 0 0 0 94 0 0 0 0 0 0 0 188 0 0 0 0 0 0 0 375 0 1 0 0 0 0 1 750 1 4 1 0 1 2 3 1500 3 6 2 1 2 3 9

TABLE-US-00010 TABLE 6-2 Test results for the in vitro hemolytic activities of different antimicrobial polymers Concentration of the Hemolysis ratio (%) antimicrobial polymer Ex. Ex. Ex. Ex. Ex. - - (g/mL) 46 48 50 52 54 polylysine polylysine 3 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 12 0 0 0 0 0 0 0 24 0 0 0 0 0 0 0 47 0 0 0 0 5 0 0 94 0 0 0 0 8 0 0 188 0 0 0 0 12 0 0 375 0 0 0 0 19 0 1 750 0 2 0 1 32 2 3 1500 1 4 0 1 54 3 9

Example 59

[0163] The Example was used to test the in vivo acute toxicity of the poly(amino acid)-based antimicrobial agents in animals, and -polylysine synthesized by fermentation was used as the control to evaluate the in vivo acute toxicity of the resulting poly(amino acid)-based antimicrobial agents in animals.

[0164] One hundred mice (Balb/C mice, purchased from Jilin University), half male and half female, weighing 213 g, were used. The poly(amino acid)-based antimicrobial agents prepared in Examples 41-56 were taken at a dose of 1 mg/mL respectively and intramuscularly injected into mice once a day for 15 consecutive days to observe the toxic reaction of mice. The experimental results showed that after intramuscular injection for 15 consecutive days, except for few mice with reduced vitality, the rest had no significant abnormal reactions, and all mice survived. It was demonstrated that the resulting poly(amino acid)-based antimicrobial agents have lower toxicity in vivo.

Example 60

[0165] 50 mL of styrene, 50 mL of chloromethylstyrene, and azodiisobutyronitrile (both styrene and chloromethylstyrene passed through a section of neutral alumina column to remove the polymerization inhibitor) were added to a three-necked bottle equipped with the nitrogen port, stirrer, and thermometer, stirred, and N.sub.2 was bubbled for 30 min. The nitrogen atmosphere was maintained, and the reaction was conducted at 70 C. for 24 hours. After the reaction was completed, the system was precipitated in ethanol, to give chloromethylated polystyrene.

[0166] 5 g of chloromethylated polystyrene, 0.5 g of hyperbranched polylysine prepared in Example 14, and 30 mL of distilled water were added to the reaction bottle. After bubbling N2 for 30 min, the bottle was sealed and the reaction was conducted at 120 C. under nitrogen atmosphere for 10 hours. The solid product was filtered and rinsed with a large amount of distilled water, to give hyperbranched polylysine-modified polystyrene (HBPL-PS). The HBPL-PS was pressed at high temperature into a sample film of 2 cm1.5 cm for antimicrobial test.

[0167] By using Escherichia coli and Staphylococcus aureus, we studied the antimicrobial adhesion performances of the material. First, Escherichia coli (ATCC8739) and Staphylococcus aureus (ATCC25923) were cultured in LB and TSB medium at 37 C. for 24 hours, respectively. Then, the medium containing bacteria was centrifuged at 2700 rmp for 10 min. The supernatant was removed, and resuspended to a concentration of 110.sup.8 cells/mL. The sample membrane was transferred to a 48-well plate, and 1 mL of bacterial suspension was added respectively to adhere for 1 h and then the medium was aspirated. After that, Escherichia coli (using 0.5% (wt/vol) glucose medium) and Staphylococcus aureus (using TSB medium) was continued with static culture at 37 C. for 4 hours. The sample film adhered with bacteria was put in 1 mL of fresh sterile PBS solution and ultrasonically cleaned for 1 min to achieve the desorption of bacteria from the surface. The PBS solution containing bacteria was diluted by a certain fold, and applied to the solid medium. After culturing at 37 C. overnight, the bacterial colonies formed on the surface of the solid medium was counted. Compared with polystyrene (PS) as the control group, the bacterial adhesion of Escherichia coli and Staphylococcus aureus on the surface of HBPL-PS decreased by 95.5% and 98.8%, respectively.