Carbonate polymer containing a functional group of disulfide five-membered ring in the side chain and application thereof
10072122 ยท 2018-09-11
Assignee
Inventors
Cpc classification
A61K47/34
HUMAN NECESSITIES
C08G63/64
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
A61K47/34
HUMAN NECESSITIES
Abstract
Apparatus relates to a carbonate polymer containing a functional group of disulfide five-membered ring in the side chain and application thereof. The polymer can be prepared from cyclic carbonate monomer containing a disulfide five-membered ring functional group through the activity controllable ring-opening polymerization. For polymer, molecular weight is controlled, molecular weight distribution is narrowed and does not require the protection and deprotection procedures. Polymer prepared from the carbonate monomer through the ring-opening polymerization has biodegradability, can be used for controlling drugs release system, and can be used to prepare tumor-targeted nano-drug carrier which is sensitive to reduction and is reversible cross-linking, can support long circulation in the body, in high concentration of cancers cells can rapidly release cross-linking in the cancer cells, to release drugs, to kill cancer cells with high efficiency and specificity. Biodegradable polymer has a good application value in the tissue engineering and bio-chip coating.
Claims
1. A carbonate polymer containing a functional group of disulfide five-membered ring in the side chain, which contains a cyclic carbonate monomer unit containing a disulfide five-membered ring functional group, wherein the chemical structure of the said carbonate polymer containing a functional group of disulfide five-membered ring in the side chain is one of the following formulas: ##STR00022## wherein x, y, and n are repeating units, R1 is selected from one of the following groups: ##STR00023## in the formula, k=20-250, R4 is selected from one of the following groups: ##STR00024## R2 is selected from one of the following groups: ##STR00025## R3 is selected from one of the following groups: ##STR00026## in the formula, a=2, 3 or 4; b=20-250; and the molecular weight of said carbonate polymer containing a functional group of disulfide five-membered ring in the side chain is 800-100,000 Da.
2. The carbonate polymer containing a functional group of disulfide five-membered ring in the side chain according to claim 1, wherein the number of units of the cyclic carbonate monomer containing a disulfide five-membered ring functional group in the molecular chain of said carbonate polymer containing a functional group of disulfide five-membered ring in the side chain is from 4 to 50.
3. A method for preparing a drug-controlled release carrier, the method comprising utilizing the carbonate polymer of claim 1, the molecular weight of said carbonate polymer being 3,000-70,000 Da.
4. A method for preparing a scaffold material for a biologic tissue engineering, the method comprising utilizing the carbonate polymer of claim 1, the molecular weight of said carbonate polymer being 5,000-100,000 Da.
5. A method for preparing a biochip coating, the method comprising utilizing the carbonate polymer of claim 1, the molecular weight of said carbonate polymer being 800-10,000 Da.
6. A method for preparing a drug-controlled release carrier, the method comprising utilizing the carbonate polymer of claim 2, the molecular weight of said carbonate polymer being 3,000-70,000 Da.
7. A method for preparing a scaffold material for a biologic tissue engineering, the method comprising utilizing the carbonate polymer of claim 2, the molecular weight of said carbonate polymer being 5,000-100,000 Da.
8. A method for preparing a biochip coating, the method comprising utilizing the carbonate polymer of claim 2, the molecular weight of said carbonate polymer being 800-10,000 Da.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
Description of Drawings
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EXAMPLES FOR THE INVENTION
Detailed Description of the Embodiments
(22) The present invention will further be described in detail below with reference to examples and figs:
Example 1 Synthesis of the Cyclic Carbonate Monomer Containing a Disulfide Five-Membered Ring Functional Group (CDC)
(23) ##STR00010##
(24) 1. The sodium hydrosulfide monohydrate (28.25 g, 381.7 mmol) was dissolved in 400 mL N,N-Dimethylformamide (DMF), then heated at 50 C. until dissolution completely; dibromo-ne neopentyl glycol (20 g, 76.4 mmol) was added dropwise, then reaction was carried out for 48 h. The solvent of DMF was removed by distillation under reduced pressure to the reactants, then was diluted with 200 mL distilled water and extracted four times with 250 mL ethyl acetate, and finally the organic phase was rotary evaporated to give compound A as a yellow viscous, Yield: 70%;
(25) 2. The compound A was dissolved in 400 mL tetrahydrofuran solution, oxidized in the air for 24 h, when the sulfydryl between molecules was oxidized to disulfide bond, to give compound B, Yield: >98%;
(26) 3. Under nitrogen atmosphere, the compound B (11.7 g, 70.5 mmol) was dissolved in dry tetrahydrofuran (150 mL), stirring until completely dissolved. Then cool to 0 C., and the ethyl chloroformate (15.65 mL, 119.8 mmol) was added, then Et.sub.3N (22.83 mL, 120.0 mmol) was dropwise added. When the addig was completed, the reaction was carried out in an ice-water bath for 4 h. When the reaction is completed, the reaction mixture was filtered to removal the Et.sub.3N.HCl and the filtrate mixture was concentrated by rotational evaporation and recrystallized from diethyl ether for many times to give a yellow crystal, that is the cyclic carbonate monomer containing a disulfide five-membered ring functional group (CDC), Yield: 64%.
Example 2 Synthesis of PEG5k-b-PCDC2.8k Double Block Copolymer Containing a Functional Group of Disulfide Five-Membered Ring in the Side Chain
(27) Under a nitrogen atmosphere, 0.3 g (1.56 mmol) CDC monomer, 2 mL dichloromethane were added into a sealing reactor, then 0.5 g (0.1 mmol) polyethylene glycol which molecular weight is 5000 and 1 mL zinc bis[bis(trimethylsilyl)amide] solution (0.1 mol/L) in dichloromethane as the catalyst were added, then the reactor was sealed and moved out of the glovebox, and placed in an oil-bath at 40 C., the reaction was carried out for 1 day, then the reaction was terminated by adding glacial acetic acid, the reaction mixture was precipitated in cold ethyl ether and filtered and dried under vacuum to give product PEG5k-b-PCDC2.8k.
(28) .sup.11H NMR (400 MHz, CDCl.sub.3): 3.08 (s, CCH.sub.2), 3.30 (m, OCH.sub.3), 4.05 (s, CH.sub.2OCOCHCH.sub.2), 4.07 (s, OCH.sub.2CCH.sub.2O), 4.31 (m, CCH.sub.2).
(29) ##STR00011##
in the formula, m=113.6, n=14.6.
Example 3 Synthesis of PEG5k-P (CDC2.5k-Co-CL3.9k) Double Block Copolymer Containing a Disulfide Five-Membered Ring in the Side Chain
(30) Under a nitrogen atmosphere, 0.28 g (1.46 mmol) CDC monomer and 0.4 g (3.51 mmol) caprolactone (-CL) were dissolved in 3 mL dichloromethane, then added into a sealing reactor, then 0.5 g (0.1 mmol) polyethylene glycol which molecular weight is 5000 and 0.1 mol/L of zinc bis[bis(trimethylsilyl)amide] solution (0.1 mol/L) in dichloromethane as the catalyst were added, then the reactor was sealed and moved out of the glovebox, and placed in an oil-bath at 40 C., the reaction was carried out for 1 day, then the reaction was terminated by adding glacial acetic acid, the reaction mixture was precipitated in cold ethyl ether and filtered and dried under vacuum to give product PEG5k-P(CDC2.5k-co-CL3.9k). The molecular weight of GPC: 14.0 kDa, molecular weight distribution: 1.56.
(31) ##STR00012##
in the formula, m=113.6, x=34.2, y=13.0, n=47.2.
(32)
Example 4 Synthesis of PEG5k-P(CDC3.8k-co-CL14k) Double Block Copolymer Containing a Disulfide Five-Membered Ring in the Side Chain
(33) Under a nitrogen atmosphere, 0.5 g (2.6 mmol) CDC monomer and 1.5 g (13.2 mmol) caprolactone (-CL) were dissolved in 10 mL dichloromethane, then added into a sealing reactor, then 0.5 g (0.1 mmol) polyethylene glycol which molecular weight is 5000 and 1 mL of zinc bis[bis(trimethylsilyl)amide] solution (0.1 mol/L) in dichloromethane as the catalyst were added, then the reactor was sealed and moved out of the glovebox, and placed in an oil-bath at 40 C., the reaction was carried out for 1 day, then the reaction was terminated by adding glacial acetic acid, the reaction mixture was precipitated in cold ethyl ether and filtered and dried under vacuum to give product PEG5k-P(CDC3.8k-co-CL14k). The molecular weight of GPC: 30.6 kDa, molecular weight distribution: 1.34.
(34) ##STR00013##
in the formula, m=113.6, x=122.8, y=19.8, n=142.
Example 5 Synthesis of PEG1.9k-P(CDC3.9k-co-CL3.8k) Double Block Copolymer Containing a Disulfide Five-Membered Ring in the Side Chain
(35) Under a nitrogen atmosphere, 0.4 g (2.1 mmol) CDC monomer and 0.4 g (3.51 mmol) caprolactone (-CL) were dissolved in 3 mL dichloromethane, then added into a sealing reactor, then 0.4 g (0.21 mmol) polyethylene glycol which molecular weight is 1900 and 1 mL of zinc bis[bis(trimethylsilyl)amide] solution (0.1 mol/L) in dichloromethane as the catalyst were added, then the reactor was sealed and moved out of the glovebox, and placed in an oil-bath at 40 C., the reaction was carried out for 1 day, then the reaction was terminated by adding glacial acetic acid, the reaction mixture was precipitated in cold ethyl ether and filtered and dried under vacuum to give product PEG1.9k-P(CDC3.9k-co-CL3.8k). The molecular weight of GPC: 0.96 kDa, molecular weight distribution: 1.35.
(36) ##STR00014##
in the formula, m=43.2, x=33.3, y=20.3, n=53.6.
Example 6 Synthesis of Alk-PCDC2.8k Homopolymer Containing a Functional Group of Disulfide Five-Membered Ring in the Side Chain
(37) Under a nitrogen atmosphere, 0.3 g (1.6 mmol) CDC monomer was dissolved in 1 mL dichloromethane, then added into a sealing reactor, then 1 mmol/L of refined propargyl alcohol and 1 mL zinc bis[bis(trimethylsilyl)amide] solution (0.1 mol/L) in dichloromethane as the catalyst were added, then the reactor was sealed and moved out of the glovebox, and placed in an oil-bath at 40 C., the reaction was carried out for 1 day, then the reaction was terminated by adding glacial acetic acid, the reaction mixture was precipitated in cold ethyl ether and filtered and dried under vacuum to give product Alk-PCDC2.8k.
Example 7 Synthesis of iPr-P(CDC0.8k-Co-CL92k) Carbonate Polymer Containing a Disulfide Five-Membered Ring in the Side Chain
(38) Under a nitrogen atmosphere, 0.1 g (0.52 mmol) CDC monomer and 10 g (87.7 mmol) -CL caprolactone (CL) were dissolved in 10 mL dichloromethane, then added into a sealing reactor, then 6 mg (0.1 mmol) isopropyl alcohol and 1 mL of zinc bis[bis(trimethylsilyl)amide] solution (0.1 mol/L) in dichloromethane as the catalyst were added, then the reactor was sealed and moved out of the glovebox, and placed in an oil-bath at 40 C., the reaction was carried out for 2 days, then the reaction was terminated by adding glacial acetic acid, the reaction mixture was precipitated in cold ethyl ether and filtered and dried under vacuum to give product iPr-P(CDC-co-CL)(0.8k-92k). The molecular weight of GPC: 102.3 kDa, molecular weight distribution: 1.36.
(39) ##STR00015##
in the formula, x=4.2, y=80.7, n=84.9.
Example 8 Synthesis of PEG5k-PCDC1.0k-PCL3.2k Triple Block Copolymer Containing a Disulfide Five-Membered Ring in the Side Chain
(40) Under a nitrogen atmosphere, 0.12 g (1.5 mmol) CDC monomer was dissolved in 2 mL dichloromethane, then added into a sealing reactor, then 0.5 g (0.31 mmol) polyethylene glycol which molecular weight is 5000 and 1 mL of zinc bis[bis(trimethylsilyl)amide] solution (0.1 mol/L) in dichloromethane as the catalyst were added, then the reactor was sealed and moved out of the glovebox, and placed in an oil-bath at 40 C., the reaction was carried out for 1 day, then under a nitrogen atmosphere 0.35 g (0.31 mmol) caprolactone (-CL) was added in the glovebox, the reaction was carried out for 1 day, then the reaction was terminated by adding glacial acetic acid, the reaction mixture was precipitated in cold ethyl ether and filtered and dried under vacuum to give product triple block copolymer PEG5k-PCDC1.0k-PCL3.2k. The molecular weight of GPC: 10.4 kDa, molecular weight distribution: 1.45.
(41) .sup.1H NMR (400 MHz, CDCl.sub.3): 1.40 (m, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2), 1.65 (m, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2), 2.30 (t, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2), 3.08 (s, CCH.sub.2), 3.30 (m, OCH.sub.3), 4.03 (t, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2O), 4.05 (s, CH.sub.2OCOCHCH.sub.2), 4.07 (s, OCH.sub.2CCH.sub.2O), 4.31 (m, CCH.sub.2).
Example 9 Synthesis of PEG5k-P(CDC3.2k-Co-TMBPEC3.5k) Double Block Copolymer Containing a Disulfide Five-Membered Ring in the Side Chain
(42) Under a nitrogen atmosphere, 0.4 g (2.1 mmol) CDC monomer and 0.4 g (1.2 mmol) 2,4,6-trimethoxy benzylidenepentaery thritol carbonate (TMBPEC) were dissolved in 5 mL dichloromethane, then added into a sealing reactor, then 0.5 g (0.1 mmol) polyethylene glycol which molecular weight is 5000 and 1 mL of zinc bis[bis(trimethylsilyl)amide] solution (0.1 mol/L) in dichloromethane as the catalyst were added, then the reactor was sealed and moved out of the glovebox, and placed in an oil-bath at 40 C., the reaction was carried out for 1 day, then the reaction was terminated by adding glacial acetic acid, the reaction mixture was precipitated in cold ethyl ether and filtered and dried under vacuum to give product PEG5k-P(CDC3.2k-co-TMBPEC3.5k). The molecular weight of GPC: 12.4 kDa, molecular weight distribution: 1.47.
(43) ##STR00016##
in the formula, m=113.6, x=16.7, y=10.2, n=26.9.
Example 10 Synthesis of PEG1.9k-PCL1.8k-PCDC0.7k Triple Block Copolymer Containing a Functional Group of Disulfide Five-Membered Ring in the Side Chain
(44) Under a nitrogen atmosphere, 0.2 g (1.76 mmol) caprolactone (-CL) was dissolved in 2 mL dichloromethane, then added into a sealing reactor, then 0.19 g (0.1 mmol) polyethylene glycol which molecular weight is 1900 and 1 mL of zinc bis[bis(trimethylsilyl)amide] solution (0.1 mol/L) in dichloromethane as the catalyst were added, then the reactor was sealed and moved out of the glovebox, and placed in an oil-bath at 40 C., the reaction was carried out for 1 day, then under a nitrogen atmosphere 80 mg (0.42 mmol) CDC monomer was added in the glovebox, the reaction was carried out for 1 day, then the reaction was terminated by adding glacial acetic acid, the reaction mixture was precipitated in cold ethyl ether and filtered and dried under vacuum to give product triple block copolymer PEG1.9k-PCL1.8k-PCDC0.7k. The molecular weight of GPC: 0.64 kDa, molecular weight distribution: 1.32.
(45) .sup.1H NMR (400 MHz, CDCl.sub.3): 1.40 (m, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2), 1.65 (m, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2), 2.30 (t, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2), 3.08 (s, CCH.sub.2), 3.30 (m, OCH.sub.3), 4.03 (t, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2O), 4.05 (s, CH.sub.2OCOCHCH.sub.2), 4.07 (s, OCH.sub.2CCH.sub.2O), 4.31 (m, CCH.sub.2).
Example 11 Synthesis of PEG5k-P(CDC4.9k-Co-TMC19k) Double Block Copolymer Containing a Disulfide Five-Membered Ring in the Side Chain
(46) Under a nitrogen atmosphere, 0.1 g (0.52 mmol) CDC monomer and 0.4 g (3.85 mmol) trimethylene cyclic carbonate (TMC) were dissolved in 3 mL dichloromethane, then added into a sealing reactor, then 0.1 g (0.02 mmol) polyethylene glycol which molecular weight is 5000 and 0.1 mol/L of zinc bis[bis(trimethylsilyl)amide] solution (0.1 mol/L) in dichloromethane as the catalyst were added, then the reactor was sealed and moved out of the glovebox, and placed in an oil-bath at 40 C., the reaction was carried out for 1 day, then the reaction was terminated by adding glacial acetic acid, the reaction mixture was precipitated in cold ethyl ether and filtered and dried under vacuum to give product PEG5k-P(CDC4.9k-co-TMC19.0k). The molecular weight of GPC: 34.5 kDa, molecular weight distribution: 1.48.
(47) .sup.1H NMR (400 MHz, CDCl.sub.3): 2.08 (t, COCH.sub.2CH.sub.2CH.sub.2O), 3.08 (s, CCH.sub.2), 3.30 (m, OCH.sub.3), 3.65 (t, OCH.sub.2 CH.sub.2O), 4.28 (t, COCH.sub.2CH.sub.2CH.sub.2O), 4.31 (m, CCH.sub.2).
(48) ##STR00017##
in the formula, m=113.6, x=25.5, y=186.3, n=211.8.
Example 12 Synthesis of iRGD-PEG6k-P(CDC4.8k-Co-TMC19.2k) Double Block Targeted Copolymer with iRGD Polypeptide Containing a Disulfide Five-Membered Ring in the Side Chain
(49) Polymer iRGD-PEG6k-P(CDC4.8k-co-TMC19.2k) was synthesized through two steps, the synthesis of polymer functionalized by maleimide Mal-PEG6k-P(CDC4.8k-co-TMC19.2k) as the first step, which as the same with example 11 except that the mPEG of molecular weight 5000 was replaced by Mal-PEG of molecular weight 6000 Da, which as the initiator for polymerization. .sup.1H NMR (400 MHz, CDCl.sub.3): 2.08 (t, COCH.sub.2CH.sub.2CH.sub.2O), 3.08 (s, CCH.sub.2), 3.30 (m, OCH.sub.3), 3.65 (t, OCH.sub.2 CH.sub.2O), 4.28 (t, COCH.sub.2CH.sub.2CH.sub.2O), 4.31 (m, CCH.sub.2), 6.70 (s, Mal). The molecular weight of GPC: 38.6 kDa, molecular weight distribution: 1.42.
(50) ##STR00018##
in the formula, m=136.4, x=24.8, y=188.4, n=213.2.
(51) The michael addition reaction between iRGD polypeptide and the polymer as got above as the second step. Polymer Mal-PEG6k-P(CDC4.8k-co-TMC19.2k) was dissolved in DMF, then became the nanoparticles with the dropwise adding of PB buffer solution, then the organic solvent was removed by dialysis, then two times the molar weight of iRGD was added, the reaction was carried out at 30 C. for 2 days, then the free iRGD which was not bonded was removed by dialysis, freeze-dried to give the final product iRGD-PEG6k-P(CDC4.8k-co-TMC19.2k). The grafting ratio of iRGD was 92%, by the analysis of nuclear magnetic and BCA protein kit.
Example 13 Synthesis of cRGD-PEG6k-P(CDC4.6k-Co-TMC18.6k) Double Block Targeted Copolymer with cRGD Polypeptide Containing a Disulfide Five-Membered Ring in the Side Chain
(52) There were two steps for the synthesis of polymer cRGD-PEG6k-P(CDC4.6k-co-TMC18.6k), which was similar as the example 12, the synthesis of polymer functionalized by N-hydroxysuccinimide NHS-PEG6k-P(CDC4.6k-co-TMC18.6k) as the first step, which as the same with example 11 except that the mPEG which molecular weight is 5000 Da was replaced by NHS-PEG which molecular weight is 6000 Da, which as the initiator for polymerization. .sup.1H NMR (400 MHz, CDCl.sub.3): 2.08 (t, COCH.sub.2CH.sub.2CH.sub.2O), 3.08 (s, CCH.sub.2), 3.30 (m, OCH.sub.3), 3.65 (t, OCH.sub.2 CH.sub.2O), 4.28 (t, COCH.sub.2CH.sub.2CH.sub.2O), 4.31 (m, CCH.sub.2), 2.3 (s, NHS). The molecular weight of GPC: 37.6 kDa, molecular weight distribution: 1.38.
(53) ##STR00019##
in the formula, m=136.4, x=24.0, y=178.8, n=202.8.
(54) The amide reaction for bonding cRGD polypeptide and the polymer as got above as the second step. Polymer said above was dissolved in DMF, then two times the molar weight of cRGD was added, the reaction was carried out at 30 C. for 2 days, then the free cRGD which was not bonded was removed by dialysis, freeze-dried to give the final product cRGD-PEG6k-P(CDC4.6k-co-TMC18.6k). The grafting ratio of cRGD was 88%, by the analysis of nuclear magnetic and BCA protein kit.
Example 14 Synthesis of PEG5k-PLA7.8k-PCDC1.7k Triple Block Copolymer Containing a Disulfide Five-Membered Ring in the Side Chain
(55) Under a nitrogen atmosphere, 0.45 g (3.13 mmol) lactide (LA) was dissolved in 3 mL dichloromethane, then added into a sealing reactor, then 0.25 g (0.05 mmol) polyethylene glycol which molecular weight is 5000 and 1 mL of zinc bis[bis(trimethylsilyl)amide] solution (0.1 mol/L) in dichloromethane as the catalyst were added, then the reactor was sealed and moved out of the glovebox, and placed in an oil-bath at 40 C., the reaction was carried out for 1 day, then under a nitrogen atmosphere 100 mg (0.52 mmol) CDC monomer was added in the glovebox, the reaction was carried out for 1 day, then the reaction was terminated by adding glacial acetic acid, the reaction mixture was precipitated in cold ethyl ether and filtered and dried under vacuum to give product triple block copolymer PEG5k-PLA7.8k-PCDC1.7k. The molecular weight of GPC: 16.8 kDa, molecular weight distribution: 1.47.
(56) .sup.1H NMR (400 MHz, CDCl.sub.3): 1.59 (m, COCH (CH.sub.3) O), 3.08 (s, CCH.sub.2), 3.30 (m, OCH.sub.3), 3.65 (m, OCH.sub.2CH.sub.2O), 4.07 (s, OCH.sub.2CCH.sub.2O), 5.07 (m, COCH (CH.sub.3).
(57) ##STR00020##
in the formula, m=113.6, x=122.2, y=8.9, n=131.1.
Example 15 Synthesis of P(CDC-Co-CL)(6.21k)-PEG(0.5k)-P(CDC-Co-CL)(6.21k) Triple Block Copolymer Containing a Disulfide Five-Membered Ring in the Side Chain
(58) Under a nitrogen atmosphere, 1.5 g (13.2 mmol) -CL and 0.0625 g (0.325 mmol) CDC were dissolved in 8 mL dichloromethane, then added into a sealing reactor, then 0.05 g (0.01 mmol) PEG500 and 1 mL of zinc bis[bis(trimethylsilyl)amide] solution (0.1 mol/L) in dichloromethane as the catalyst were added, the reaction was carried out for 1 day, then the reaction was terminated by adding glacial acetic acid, the reaction mixture was precipitated in cold ethyl ether and filtered and dried under vacuum to give product triple block copolymer P(CDC-co-CL)(6.21k)-PEG(0.5k)-P(CDC-co-CL)(6.21k). The molecular weight of GPC: 14.6 kDa, molecular weight distribution: 1.38.
(59)
(60) ##STR00021##
in the formula, m=11.4, x=6.3, y=43.9, n=51.2.
(61) It was known from the above results that, ring-opening homopolymerization or copolymerization of CDC was controllable and the correct molecular weights with the expected and the molecular weight distribution of polymer was narrowed, by the characterization of the polymers.
Example 16 Preparation of the Polymeric Micelle Nanoparticles PEG5k-b-PCDC2.8k
(62) Dialysis was used for the preparation of the polymeric micelle nanoparticles. 200 L of DMF solution of PEG5k-b-PCDC2.8k (2 mg/mL) was dropwise added into 800 L phosphate buffer (10 mM, pH7.4, PB), then the obtained solution was put in the dialysis bag (MWCO 3500 Da) and dialyzed against PB (10 mM, pH7.4) for one night and change water five times. The size of obtained micelles nanoparticles was 173 nm by dynamic light scattering particle size analyzer (DLS), and the particle size distribution was narrow, which was showed in
Example 17 Cross-Linking, Decross-Linking and Cytotoxicity of the Polymeric Micelle Nanoparticles PEG5k-b-PCDC2.8k
(63) To clearing the air, nitrogen was bubbled into water of micelle nanoparticles for 20 mins, and 10 L secondary water of dithiothreitol (DTT)(0.007 mg, 4.6710.sup.5 mmol, lipoic acid group moles 10%) was added into nanoparticles solution (1 mL, 0.25 mg/mL, 3.2110.sup.5 mmol) which in the a sealing reactor, the reaction was carried out at room temperature for 1 day with stirring. The measured size of particles was 150 nm after dialysis for 1 day, which was 15% smaller than the size of not cross-linking particles. The particle size and particle size distribution was not change almost when the concentration of cross-linking nanoparticles diluted below the CMC; it was stability in physiological environment, thus it can be seen that the stability of nanoparticles was largely improved by disulfide cross-linking, which was showed in
(64) Disulfide bond was ruptured under the action of reducing agent such as glutathione (GSH) or DTT. Under a nitrogen atmosphere and at 37 C., nitrogen was bubbled into solution of cross-linking nanoparticles for 10 mins, then GSH was added which final concentration was 10 mM in the solution of cross-linking nanoparticles. The change of the decross-linking size of nanoparticles was follow-up by DLS, which was showed in
(65) The toxicity of cross-linked nanoparticle was assayed by MTT. Cell as MCF-7 (human breast cancer cell) and Raw 264.7 (mouse macrophages) were used. HeLa or Raw 264.7 cells were plated in a 96-well plate by 110.sup.4 cells/mL, 100 L per well. Culture of different concentration of micelle nanoparticles was added as the experiment group and wells containing only cells and wells containing only culture medium were other setted (quadrupled parallel hole) after cells adherence. Take out the 96-well plate after the cells were cultured for 24 h, 10 L MTT (5.0 mg/mL) was added. The cells were cultured for another 4 h, then 150 L Crystal Violet from DMSO dissolution was added, and the absorbance (A) at 492 nm was measured using a microplate reader. The cell viability was determined by comparing the absorbance with control wells containing only culture medium.
(66)
in the formula, A.sub.T was the absorbance of experiment group at 492 nm, A.sub.C was the absorbance of control group at 492 nm. Concentration of polymer was 0.1, 0.2, 0.3, 0.4 or 0.5 mg/mL.
Example 18 Loaded Drug, In Vitro Release and Cytotoxicity of the Cross-Linked Nanoparticle PEG5k-b-PCDC2.8k
(67) Doxorubicin (DOX) was used as a drug, and the whole operation was proceed under dark condition. Desalting the hydrochloride of doxorubicin hydrochloride as the first step, which was proceed as: 1.2 mg (0.002 mmol) DOX was dissolved in 225 L DMSO, then 0.58 mL (m=0.419 mg, 0.004 mmol) triethylamine was added and stirred for 12 h, then absorbed the above procedure. The concentration of DMSO solution of DOX was 5.0 mg/mL. PEG5k-b-PCDC2.8k was dissolved in DMF, which was mixed with the DMSO solution of DOX according to the predetermined quality ratio for the drug and polymer, then quadrupled secondary water was added slowly with stirring, then takeed a dialysis in water.
(68) The cross-linking for nanoparticle loaded drug was the same as the cross-linking method in Example 17. 100 L the cross-linked micelle nanoparticle loaded drug was freeze-dried and was dissolved in 3.0 mL DMSO, drug loading efficiency was calculated according to the fluorescence spectroscopy and the standard curve of DOX.
(69) Drug loading content (DLC) and drug loading efficiency (DLE) were calculated according to the following formula:
(70) Drug loading content (wt. %)=(weight of loaded drug/weight of polymer)100% Drug loading efficiency (%)=(weight of loaded drug/weight of drug in feed)100%
(71) The loading result of PEG5k-b-PCDC2.8k micelle nanoparticle to DOX was in table 1, showed the efficient loading effect.
(72) TABLE-US-00001 TABLE 1 drug loading content and drug loading efficiency of cross-linked polymer nanoparticle loaded Doxorubicin feed ratio DLC DLE size size polymer (wt. %) (wt. %) (%) (nm) distribution PEG5k-b-PCDC2.8k 5 4.0 83.3 150.3 0.17 10 7.4 80.0 162.1 0.22 15 9.1 68.2 173.2 0.19
(73) The in vitro release studies of DOX were conducted in a thermostatic shaker (200 rpm) at 37 C., double duplicate sample per group. The cross-linked micelle nanoparticle loaded DOX released in the PB (10 mM, pH 7.4) of 10 mM GSH which mimicked the intracellular reducing environment as the first group; and the cross-linked micelle nanoparticle loaded DOX released in the PB (10 mM, pH 7.4) as the second group. The concentration of micelle nanoparticle loaded drug was 25 mg/L, and 0.5 mL of release media dialysis against 25 mL of dialysis solvent per tube in the dialysis bag (MWCO: 12,000-14,000). At desired time intervals, 5 mL of release media was taken out and replenished with an equal volume of fresh media of 5 mL. The concentration of drug in the solution was determined using fluorometry EDINBURGH FLS920.
(74) The toxicity of cross-linked nanoparticle loaded DOX of PEG5k-b-PCDC2.8k to mouse macrophages Raw 264.7 and human breast cancer cell MCF-7 was assayed by MTT, and decross-linked micelle nanoparticle loaded drug and free drug as the control group. Take Raw 264.7 cell as an example, Raw 264.7 cells were plated in a 96-well plate by 110.sup.4 cells/mL, 100 L per well. After cells adherence, Fresh culture containing 0.01, 0.1, 1, 5, 10, 50 and 100 g/mL solution of cross-linked nanoparticle loaded DOX and free DOX were added as experiment group. Take out the 96-well plate after the cells were cultured for 48 h in incubator, 10 L MTT (5.0 mg/mL) was added. The cells were cultured for another 4 h, then 150 L crystal violet from DMSO dissolution was added, and the absorbance (A) at 492 nm was measured using a microplate reader. The cell viability was determined by comparing the absorbance with control wells containing only culture medium.
(75)
Example 19 Preparation of the Cross-Linked Polymeric Vesicle Nanoparticles PEG5k-P(CDC4.9k-Co-TMC19k) and its Biocompatibility and the Toxicity of the Cross-Linked Vesicle Loaded Drug to MCF-7, U87MG and A549
(76) The same as example 16, the polymer PEG5k-P (CDC4.9k-co-TMC19k) can become nanoparticle and it has the structure of vesicle by TEM and CLSM, which shown in
(77) DOX.HCl was loaded by pH-gradients method, and hydrophilic DOX was loaded because the different pH among inside and outside for the vesicle. The cross-linked vesicle loaded drug was prepared with the different proportion of drug inventory from 10% to 30%, then the free drug which was not loaded was dislodged by dialysis, and the size of cross-linked vesicle was 105 to 124 nm by DLS, and the particle size distribution which from 0.10 to 0.15 was narrow, and the efficiency of loading hydrophilic DOX was high (63% to 77%).
Example 20 the Blood Circulation in Mice of the Cross-Linked Nanoparticle PEG5k-b-PCDC2.8k Loaded Drug
(78) Aged 4 to 6 weeks old, weight 18 to 20 g C57BL/6 mice (Shanghai Institutes for Biological Sciences Laboratory Animal Center) were collected. They were evenly divided into groups after weighting. Mice were intravenously injected with nanoparticle loaded drug and free drug, and DOX was 10 mg/kg. 10 L of blood was taken at different time points of 0, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h, and the weight of blood was calculated with the method of balance. The blood samples upon withdrawing were immediately dissolved in 100 L 1% triton and 500 L of extraction solution DMF (containing 1M HCl and 20 mM DTT). Taken the supernatant by centrifugation (20000 rpm, 20 min). The DOX level in the supernatant of each time was determined by fluorometry.
(79)
Example 21 Biodistribution of the Cross-Linked Nanoparticle PEG5k-b-PCDC2.8k Loaded Drug to Mice Bearing Melanoma Tumors
(80) Aged 4 to 6 weeks old, weight 18 to 20 g C57BL/6 mice (Shanghai Institutes for Biological Sciences Laboratory Animal Center) were collected. They were evenly divided into groups after weighting. B16 melanoma tumor of 110.sup.6 cells were generated by subcutaneous injection into the hind flank of the mice. Mice were intravenously injected with nanoparticle loaded drug and DOX (DOX was 10 mg/kg) when the tumor volume reached 100-200 mm.sup.3 about two weeks. The mice were sacrificed at 6, 12 or 24 h and the tumor and organs of heart, liver, spleen, lung and kidney were collected, wet weighed, then 500 L 1% triton was added, homogenized and extracted by 900 L DMF solution (containing 1 mM HCl and 20 mM DTT). Taken the supernatant by centrifugation (20000 rpm, 20 min). The DOX level at each time was determined by fluorescence measurement.
(81)
Example 22 Therapeutic Efficacy of the Cross-Linked Nanoparticle PEG5k-b-PCDC2.8k Loaded Drug to Mice Bearing Melanoma Tumors
(82) Aged 4 to 6 weeks old, weight 18 to 20 g C57BL/6 mice (Shanghai Institutes for Biological Sciences Laboratory Animal Center) were collected. They were evenly divided into groups after weighting. B16 melanoma tumor of 110.sup.6 cells were generated by subcutaneous injection into the hind flank of the mice. Mice were intravenously injected with nanoparticle loaded drug and DOX at 0, 2, 4, 6.8 day when the tumor volume reached 30-50 mm.sup.3 about one week, where the DOX proportion of nanoparticle loaded drug was 10, 20, 30 mg/kg and the DOX was 10 mg/kg. The tumor size was measured every day using calipers from 0 to 15 day. The tumor size was calculated by V=(L*W*H)/2 (L was length of tumor, W was width of tumor, H was thickness of tumor). Continue to observe the survival of mice until 46 day.
(83)
Example 23 the Blood Circulation of the Targeting Cross-Linked Vesicle Loaded Drug cRGD-PEG6k-P(CDC4.6k-co-TMC18.6k)/PEG5k-P(CDC4.9k-co-TMC19k)
(84) Aged 4 to 6 weeks old, weight 18 to 20 g Balb/C mice (Shanghai Institutes for Biological Sciences Laboratory Animal Center) were collected. They were evenly divided into groups after weighting. The vesicle was formed with different ratio of cRGD-PEG6k-P(CDC4.6k-co-TMC18.6k) and PEG5k-P(CDC4.9k-co-TMC19k). The results from experiment showed that the size of cross-linked vesicle was 100 nm and the particle size distribution was 0.10 when proportion of cRGD was 20% which had the best targeting. Mice were intravenously injected with targeting vesicle loaded drug cRGD20/CLPs, vesicle loaded drug CLPs, targeting decross-linked vesicle cRGD20/PEG-PTMC and DOX.HCl as control (the DOX was 10 mg/kg). 10 L of blood was taken at different time points of 0, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h, and the weight of blood was calculated with the method of balance. The blood samples upon withdrawing were immediately dissolved in 100 L 1% triton and 500 L of extraction solution DMF (containing 1M HCl and 20 mM DTT). Taken the supernatant by centrifugation (20000 rpm, 20 min). The DOX level in the supernatant of each time was determined by fluorometry.
(85)
Example 24 Biodistribution of the Targeting Cross-Linked Vesicle Loaded Drug cRGD-PEG6k-P(CDC4.6k-co-TMC18.6k)/PEG5k-P(CDC4.9k-co-TMC19k) to Mice Bearing Human Glioma Cells
(86) Aged 4 to 6 weeks old, weight 18 to 20 g Balb/C mice (Shanghai Institutes for Biological Sciences Laboratory Animal Center) were collected. They were evenly divided into groups after weighting. U87MG human glioma cells of 510.sup.6 cells were generated by subcutaneous injection into the hind flank of the mice. Mice were intravenously injected with cRGD20/CLPs, CLPs and DOX.HCl (DOX was 10 mg/kg) when the tumor volume reached 100 to 200 mm.sup.3 about 3 to 4 weeks. The mice were sacrificed at 4 h and the tumor and organs of heart, liver, spleen, lung and kidney were collected, wet weighed, then 500 L 1% triton was added, homogenized and extracted by 900 L DMF solution (containing 1 mM HCl and 20 mM DTT). Taken the supernatant by centrifugation (20000 rpm, 20 min). The DOX level at each time was determined by fluorescence measurement.
(87)
Example 25 the Application Therapeutic Efficacy of the cRGD-PEG6k-P(CDC4.6k-co-TMC18.6k)/PEG5k-P(CDC4.9k-co-TMC19k) Targeting Cross-Linked Vesicle Loaded Drug to Mice Bearing Human Glioma Cells
(88) Aged 4 to 6 weeks old, weight 18 to 20 g Balb/C mice (Shanghai Institutes for Biological Sciences Laboratory Animal Center) were collected. They were evenly divided into groups after weighting. U87MG human glioma cells of 510.sup.6 cells were generated by subcutaneous injection into the hind flank of the mice. Mice were intravenously injected with cRGD20/CLPs, CLPs, targeting decross-linked nanoparticle vesicle (cRGD20/PEG-PTMC), DOX.HCl and PBSat 0, 4, 8.12 day when the tumor volume reached 30 to 50 mm.sup.3 about two weeks, where the DOX proportion was 10 mg/kg. The tumor size was measured every two days using calipers from 0 to 18 day. The tumor size was calculated by V=(L*W*H)/2 (L was length of tumor, W was width of tumor, H was thickness of tumor). Continue to observe the survival of mice until 45 day.
(89)
Example 26 Therapeutic Efficacy of iRGD-PEG6k-P(CDC4.8k-co-TMC19.2k)/PEG5k-P(CDC4.9k-co-TMC19k) Targeting Cross-Linked Vesicles Loaded Drug to Mice Bearing Melanoma Tumors
(90) The vesicle was formed with different ratio of iRGD-PEG6k-P(CDC4.8k-co-TMC19.2k) and PEG5k-P(CDC4.9k-co-TMC19k). The size of vesicle was 110 nm and the particle size distribution was 0.12 when proportion of iRGD (internalizing RGD) polymer was 0, 25% and 50%. The function of iRGD was targeting tumor cell and mediated penetrate tumor cell and histamine, and a certain amount of free iRGD enhanced the function of nanoparticle to penetrate tumor histamine. A pH-gradient method was used to load DOX.HCl which had an efficiency of 60 to 80%.
(91) Aged 4 to 6 weeks old, weight 18 to 20 g C57BL/6 mice (Shanghai Institutes for Biological Sciences Laboratory Animal Center) were collected. They were evenly divided into groups after weighting. B16 melanoma tumor of 110.sup.6 cells were generated by subcutaneous injection into the hind flank of the mice. Mice were intravenously injected with polymer cross-linked vesicles loaded drug which containing 0, 25%, 50% or 100% iRGD, DOX.HCl and PBS at 0, 3, 6, 9, 12 day when the tumor volume reached 30 to 50 mm.sup.3 about one week, where the DOX.HCl was 10 mg/kg. The tumor size was measured every day using calipers from 0 to 20 day. The tumor size was calculated by V=(L*W*H)/2 (L was length of tumor, W was width of tumor, H was thickness of tumor). Continue to observe the survival of mice until 46 day.
(92)
Example 27 the Blood Circulation, Biodistribution and Inhibit Tumor Growth of the cNGQ-PEG6k-P(CDC4.8k-co-TMC19.2k)/PEG5k-P(CDC4.9k-co-TMC19k) Targeting Cross-Linked Vesicle Loaded Drug to Mice Bearing Lung Cancer Cells
(93) The synthesis of polymer cNGQ-PEG6k-P(CDC4.8k-co-TMC19.2k) was similar as the example 13, that was the synthesis of NHS-PEG6k-P(CDC4.8k-co-TMC19.2k) as the first step. The amide reaction for bonding cNGQ polypeptide and the polymer as got above as the second step. The grafting ratio of cRGD was 87%, by the analysis of nuclear magnetic and BCA protein kit. The vesicle was formed with different ratio of cNGQ-PEG6k-P(CDC4.8k-co-TMC19.2k)/PEG5k-P(CDC4.9k-co-TMC19k). A pH-gradient method was used to load DOX.HCl which had an efficiency of 60 to 80%. The results from experiment of in vitro experiment for cells showed the best targeting when proportion of cNGQ was 20% in the vesicle. Half-lives of eliminate in mice of the targeting cross-linked vesicle loaded drug (cNGQ20/CLPs) which was prepared from the vesicle with the proportion of cNGQ was 20% was 4.78 h. The lung cancer model was build subcutaneous the mice by injection the cNGQ20/CLPs modification by near-infrared molecular into the hind flank of the mice as the same as example 24. Results of in vivo imaging confirmed that the concentration of cNGQ20/CLPs to cancer was happened quickly and the fluorescence of cNGQ20/CLPs kept strongly in the tumor site after 48 h. Results of biodistribution confirmed that accumulation of cNGQ20/CLPs was 9 ID %/g in tumor site at 8 h which higher than accumulation of cRGD20/CLPs, CLPs and DOX.HCl, even other viscera.
(94) The A549 lung cancer model and orthotopic A549 lung tumor with bioluminescence model which can observe tumor growth by bioluminescence from the in vivo imaging were acquired by injecting subcutaneous the mice. After injection the drug into the hind flank of the mice at 0, 4, 8 and 12 day, bioluminescence from the in vivo imaging shown the less fluorescence of lung of the mice treated by cNGQ20/CLPs. It confirmed that cNGQ20/CLPs can target to lung cancer and inhibit tumor growth.
Example 28 Nano-Gold Rods Modified on the Surface of PEG5k-PLGA7.8k-PCDC1.7k Loaded DOX and Drug Release by NIR
(95) Synthesis of nano-gold rods modified of PEG5k-PLGA7.8k-PCDC1.7k triple block copolymer nanoparticle: the polymer solution of DMSO (2 mL, 5 mg/mL) was added dropwise into dispersion of nano-gold rods (5 mL, 0.1 mg/mL) under vigorous stirring, then stirred 4 h. The free polymer was removed by centrifugation twice and disperse into phosphate buffer. The yield of polymer modified of nano-gold rods was got by TGA and it was 80% charge to the free polymer (the feeding polymer was 100%).
(96) Loading drug of nano-gold rods modified of polymer: DMSO containing 10%, 20% or 30% DOX was added dropwise into the above said nano-gold rods modified of polymer solution, then stirred for 0.5 h and incubated for 12 h. Free micromolecule was removed by dialysis against phosphate buffer of pH7.4 for 12 h. Loading efficiency of DOX was from 70 to 90% by fluorescence, which says nano-gold rods modified of polymer can load drug efficiently.
(97) Drug release of nano-gold rods modified of polymer by NIR: nano-gold rods modified of polymer were dispersed in 10 mL phosphate buffer and irradiated by infrared light of 0.2 W/cm.sup.2 and 808 nm for 5 min every hour. 500 L solution was taken out at given time and centrifugation, then the release of DOX was got by the fluorescence of supernatant. The release for nano-gold rods modified of polymer after irradiating was 92%, which faster than non irradiating group (18% only). So the nano-gold rods modified of polymer can be used for the release by nearfrared.
Example 29 Polymer PEG1.9k-PCDC0.8k was Used for the Surface of Surface Plasmon Resonance (SPR) Sensor
(98) The gold surface of the SPR sensor was previously treated with aqua regia, then washed with ethanol and dried, and then added to a THF solution of triblock polymer PEG1.9k-PCDC0.8k (1 mL, 5 mg/mL). After reacting for 24 h under slow shaking, the sensor chip was taken out and washed three times. The surface density of PEG1.9k modified on the sensor gold plate was 20 nmol/cm.sup.2 by XPS, ellipsometer and SPR detection. Compared with the traditional chip, the sensor chip modified by polymer can reduce the non-specific adsorption, improve the stability of measurement and so on, and can be widely used in biomedicine and so on.
Example 30 Cross-Linked Polymer P(CDC0.8k-Co-CL92k) as a Biodegradable Scaffold Material
(99) The polymer P(CDC0.8k-co-CL92k) was dissolved in chloroform (40 mg/mL) and film formation on a 11 cm.sup.2 glass plate (scaffold material). The solvent was removed completely in a vacuum oven for 48 h. The disulfide five-membered ring to cross-linking by heating 10 minutes at 40 C. of heat gun, then soaked in saline for two weeks, it was still intact on the glass plate, however, membrane of PCL as the control group has been off, as