CN103285400A - Acid sensitive polymer prodrug, nanoparticles of prodrug and application of nanoparticles - Google Patents
Acid sensitive polymer prodrug, nanoparticles of prodrug and application of nanoparticles Download PDFInfo
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Abstract
The invention discloses an acid sensitive polymer prodrug, nanoparticles of the prodrug and application of the nanoparticles. In the acid sensitive polymer prodrug, a polymer precursor is a vinyl ether-functionalized water-soluble A-B type two-block polymer; drug molecules are linked with the A-B type two-block polymer through an acetal bond; the acid sensitive polymer prodrug is self-assembled in water solution to form predrug micellar nanoparticles in which a polyethylene glycol hydrophilic chain segment serves as an outer surface and an anti-cancer drug bound to a polymer main chain through the acetal bond serves as a hydrophobic core. The pH sensitive prodrug and the nanoparticles of he prodrug are simple in preparation method, and good in tumor inhibitory effect; the nanoparticles also can efficiently encapsulate another hydrophobic anticancer drug; the kill capability to the cancer cell is enhanced; and the efficiency of endocytosis of the cell to the nanoparticles can be enhanced by coupling specific target molecules onto the surfaces of the nanoparticles. Thus, the inhibitory effect on the tumor cell is enhanced.
Description
Technical field
The present invention relates to a kind ofly have the polymeric prodrugs of sensitivity to acid, nanoparticle prepared therefrom and this nanoparticle as the application of pharmaceutical carrier, belong to the medical material field.
Background technology
The whole world has more than 700 ten thousand people to lose one's life because of cancer stricken every year, and this numeral rises year by year.Treatment for cancer is maximum test and the difficult problem that the 21st century mankind face.
Continuous development and progress along with science and technology, human continuous exploration to related sciences such as oncology, molecular biology, pharmacy and chemistry, feasible research to antitumor drug has had significant progress, and many effective cancer therapy drugs are designed to develop.But present most cancer therapy drug is micromolecule, has toxic and side effects greatly and the shortcoming of poorly water-soluble, thereby has greatly limited the application of cancer therapy drug in clinical.
For solve micromolecule cancer therapy drug water solublity low (hydrophobicity is strong), bioavailability low, easily be excreted and to problems such as the human body toxic and side effects are big, people study various polymeric prodrugs or pharmaceutical carrier and wait to change cancer therapy drug and enter the mode of human body and distribution in vivo.
Although polymeric prodrugs can improve the dissolubility of dewatering medicament to a great extent, but still there be, cell endocytic inefficient problem lower as drug loading in several water-soluble prodrugs that enter the clinical experiment stage that comprise of research at present, and medicine only depends on the degraded of polymer and slowly discharges, and makes cancerous cell be easy to generate drug resistance like this; The medicament contg height that the polymer nanocomposite pharmaceutical carrier can load usually, and by the enrichment in tumor cell of EPR effect, but have problems such as the body internal stability is poor, the easy premature disconnection of medicine.
People study employing and replace the only prodrug of end group bonding at a plurality of drug molecules of hydrophilic polymer side chain graft, it is that nuclear, hydrophilic polymer are the prodrug micelle nano grain of shell that but its self assembly forms with hydrophobic cancer therapy drug, and can link cancer therapy drug with the chemical bond (as acetal bonds, hydrazone key, ester bond, the amido link of peptide bond, phosphoric acid ester bond, acid-sensitive sense and the responsive cystine linkage etc. that reduces) of stimulating responsive, make Nano medication after arriving tumor locus, can discharge medicine fast, reach the purpose of oncotherapy.
In general, all the pH value than normal body fluid is low for the pH value of pathological tissues inside such as tumor and inflammation, presents faintly acid.Therefore, can utilize these characteristics to carry out the design of prodrug micelle nano grain well, some labile components under acid condition are introduced in the nanoparticle.The micelle that they are assembled in aqueous solution can drug loading and stable existence under neutrallty condition, but under acid condition, fracture can take place for sensitivity to acid group or segment destroys micelle, thereby the medicine of its internal package can be discharged rapidly, these sensitivity to acid groups comprise acetal radical, hydrazone key, orthoformate etc.
In the prior art, more existing reports about the prodrug micelle nano grain of sensitivity to acid.But, as drug material, should have excellent biological compatibility and biodegradability, and, carrier as antitumor drug, also should have following feature: can form the stable polymer micelle in aqueous solution, its hydrophobic cores is hydrophobic anticancer drug, and the hydrophilic shell plays the effect of stable micelle, raising micelle blood circulation time; When carrier micelle arrives tumor or pathological tissues, can utilize the lower characteristics of pH value of local organization, destroy micelle, discharge cancer therapy drug rapidly.
Therefore, need seek the polymeric prodrugs micelle nano grain of more acid-sensitive sense.
Summary of the invention
The objective of the invention is, a kind of polymeric prodrugs and nanoparticle prepared therefrom with sensitivity to acid is provided, to utilize the endosome/lysosomal sour environment in the cancerous cell to control the release of medicine, reach the effect for the treatment of cancer, avoid cancerous cell to produce drug resistance.
For achieving the above object, the concrete technical scheme of the present invention is: a kind of acid-sensitive sensitive polymeric prodrug; In this acid-sensitive sensitive polymeric prodrug, polymer precursor is the water solublity A-B type bi-block copolymer of vinyl ether functionalization, and wherein A is that molecular weight is the Polyethylene Glycol of 3000～30000 Da, and the B block is carboxylic polymer; Medicine is paclitaxel, mitoxantrone, curcumin or astaxanthin;
Described drug molecule links by acetal bonds and A-B type bi-block copolymer;
In the described A-B type hydrophilic bi-block copolymer, the number of repeat unit of monomer is 10～100 in the B block;
In the described acid-sensitive sensitive polymeric prodrug, the percent grafting of medicine is 0.1～0.6; The A block constitutes hydrophilic segment, constitutes hydrophobic part behind the B block graft medicine, and the hydrophilic segment molecular weight is 0.5～8 times of hydrophobic part molecular weight.
The used medicine of the present invention is paclitaxel (PTX), mitoxantrone (MTO), curcumin (Cur) or astaxanthin (ASTA), and its molecular structural formula is as follows:
In the optimized technical scheme, medicine is paclitaxel; The B block is polyacrylic acid, polymethylacrylic acid, poly-ethylacrylic acid, poly-aspartate or polyglutamic acid.
In the technique scheme, in the water solublity A-B type bi-block copolymer precursor side chain carboxyl group of B block can with glycol divinyl ether generation esterification, obtain the polymer of side chain vinyl ether functionalization, the enough and hydroxyl generation nucleophilic addition of two bond energys of vinyl ethers forms acetal bonds.The hydrophobic micromolecule cancer therapy drug that paclitaxel, mitoxantrone, curcumin, astaxanthin etc. have active hydroxyl can be bonded on the polymer precursor by acetal bonds.The structural formula of glycol divinyl ether is as follows:
In the technique scheme, the group with sensitivity to acid is acetal radical, and its chemical structural formula is:
This biodegradable prodrug nanoparticle with sensitivity to acid had structurally both contained hydrophobic small-molecule drug, can be used for loading another kind of hydrophobic anticancer drug; Contain the acetal radical that hydrolysis can take place again under acid condition, acetal radical generation hydrolysis under certain pH conditions, micelle is destroyed, thereby discharges the hydrophobic anticancer drug that is wrapped in micelle inside rapidly.
The preparation method of above-mentioned acid-sensitive sensitive polymeric prodrug may further comprise the steps:
At first prepare A-B type bi-block copolymer; Obtain the polymer of vinyl ether functionalization again by the DCC/DMAP esterification reaction method, then the p-methyl benzenesulfonic acid of catalytic amount (
p-TSA) effect can prepare the prodrug polymer of a series of different pharmaceutical content down.
A kind of prodrug nanoparticle that is prepared by above-mentioned acid-sensitive sensitive polymeric prodrug, the shell of this nanoparticle is made of block A, and nuclear is made of medicine; The particle diameter of nanoparticle is 50～250 nm, and distribution of sizes is 0.07～0.30.
In the technique scheme, the preparation method of prodrug nanoparticle can be direct hydration method: at first polymeric prodrugs is mixed with low molecular poly (PEG), at 80～110 ℃ of following agitating heating 10～30 min; Add 1-5 mL PB buffer solution and ultrasonic then; Dialysis is at last removed PEG and can be obtained nanoparticle.
In the above-mentioned preparation method, the molecular weight of Polyethylene Glycol is 350～1500Da; Polymeric prodrugs by the hydrophobic micromolecule anticarcinogen of acetal bonds bonding can be in aqueous solution self assembly form with block A(PEG block) be hydrophilic shell, be the pH sensitive polymers prodrug nanoparticle of hydrophobic core with the pH sensitivity B block by the hydrophobic anticarcinogen of acetal bonds bonding.Through this process, the prodrug micellar nanoparticles that the polymer self assembly forms, hydrophobic cancer therapy drug constitutes the nuclear of micelle nano grain, has the hydrophilic PEG block arrangement of good biodegradability properties, the compatibility in the outside, forms the shell of micelle nano grain.
The acid-sensitive sensitive polymeric prodrug nanoparticle of the present invention's preparation has very high stability 7.4 times at pH, place it in 4 ℃ and store one month, its particle diameter does not have significant change, and under the condition of simulation human body physiological environment, and nanoparticle still keeps stable after through a week; But under solutions of weak acidity, connect the acetal bonds hydrolysis of medicine and polymer in the prodrug nanoparticle, medicine is come off down on the medicine nanoparticle in the past and discharge, make that the hydrophobicity of whole polymer descends, hydrophilic strengthens, therefore nanoparticle also can swell, and particle size distribution broadens.
Another object of the present invention provides the aforementioned prodrugs nanoparticle as the application of anti-cancer medicament carrier.The prodrug nanoparticle of technique scheme gained can also the identical or different hydrophobic cancer therapy drug of physical loading as nano-carrier, is conducive to the therapeutic alliance of tumor.
Targeting part such as antibody, polypeptide, folic acid and galactose that the nanoparticle surface of the present invention's preparation can be introduced tumor cell specific wait to improve the prodrug nanoparticle by the tumor cell endocytosis, thereby improve the concentration of medicine in tumor cell.For example, introduce the folate molecule of targeting entity tumor on prodrug nanoparticle surface, KB(population rhinocarcinoma cell with surperficial high expressed folacin receptor) tests the cytotoxicity (MTT assay) of prodrugs of paclitaxel nanoparticle that should surface coupling folic acid in the cell, hatched 48 hours IC
50Be 1.42 μ g/mL, and under the same conditions with the IC of the nanoparticle of no targeting
50Value has embodied targeting significantly up to 17.16 μ g/mL.
Because the enforcement of such scheme, the present invention compared with prior art has the following advantages:
1. the acid-sensitive sensitive polymeric prodrug of the present invention's acquisition in conjunction with hydrophobic cancer therapy drug, can effectively prevent that medicine from discharging in vivo circulation time too early by the mode of chemical bonding;
2. the acid-sensitive sensitive polymeric prodrug nanoparticle of the present invention's acquisition combines the advantage of prodrug and micelle simultaneously, endocytosis by cell enters cell also triggers medicine by the acid environment in the cancerous cell rapid delivery of pharmaceuticals, and medicine has kept its prototype structure, has potent ability of killing and wounding cancerous cell and drug resistance cancerous cell;
3. the acid-sensitive sensitive polymeric prodrug nanoparticle of the present invention's acquisition can be conducive to therapeutic alliance, raising tumor treatment effect for tumor as the hydrophobic cancer therapy drug of the another kind of micromolecule of pharmaceutical carrier physically trapping;
The acid-sensitive sensitive polymeric prodrug nanoparticle that obtains of the present invention can be further in its finishing the specific tumour targeted molecular improve cancerous cell to its endocytosis efficient, thereby improve drug level in the cancerous cell and to the kill capability of cancerous cell.Therefore, nanoparticle disclosed by the invention can load and release anti-cancer medicine effectively, aspect treatment for cancer, has excellent application value.
Description of drawings
Fig. 1 is the synthetic sketch map of prodrugs of paclitaxel among the embodiment one;
Fig. 2 is the synthetic sketch map of curcumin prodrug among the embodiment five;
Fig. 3 is the result of variations figure of prodrugs of paclitaxel nanoparticle particle diameter and light scattering intensity under solutions of weak acidity among the embodiment 13;
Fig. 4 is the release in vitro result of prodrugs of paclitaxel nanoparticle paclitaxel under different pH among the embodiment 14;
Fig. 5 is the cytotoxicity experiment figure as a result of polymer precursor among the embodiment 15;
Fig. 6 be among the embodiment 16 the prodrugs of paclitaxel nanoparticle to the toxicity of HeLa cell figure as a result;
Fig. 7 be among the embodiment 16 the prodrugs of paclitaxel nanoparticle to the toxicity of KB cell figure as a result;
Fig. 8 is the toxicity figure as a result of prodrugs of paclitaxel nanoparticle antagonism property of medicine cancerous cell among the embodiment 16;
Fig. 9 is release in vitro result two kinds of medicines time the in the prodrugs of paclitaxel nanoparticle that is loaded with amycin among the embodiment 18;
Figure 10 is that surperficial coupling has the prodrugs of paclitaxel nanoparticle of folic acid to KB(-FA among the embodiment 20) toxicity of cell figure as a result.
The specific embodiment
Below in conjunction with embodiment and accompanying drawing the present invention is further described:
Embodiment one
Prodrugs of paclitaxel PEG
113-P (AA
20-
g-PTX) preparation, accompanying drawing 1 is its synthetic sketch map:
At N
2Protection down; with initiator A IBN(3.12 mg; 0.019 mmol); macromole RAFT reagent PEG-DMP(0.48 g; 0.095 mmol), the solvent 1 of acrylic acid (163 μ L, 2.375 mmol) and 6 mL; the 4-dioxane joins respectively in the sealed reactor of 25mL, continues logical 30 minutes N
2, then reactor was placed in 70 ℃ the oil bath stirring reaction 48 hours.Reaction finishes the back and precipitates in the ice ether, and sample vacuum drying 24 hours obtains target di-block copolymer PEG
113-PAA
20, productive rate is 85%.
1H NMR (400 MHz, D
2O): PEG (-CH
2-CH
2-O-: δ 3.63; CH
3-O-: δ 3.38), PAA (-CH
2-CH-COO-): δ 2.37; -CH
2-CH-COO-: δ 1.6-1.9).
M n (
1H NMR) = 6.4 kg/mol。
Polymer P EG
113-PAA
20(0.3 g, 0.93 mmol COOH) is dissolved in 10 mL anhydrous 1, in the 4-dioxane, join in 50 ml, the two neck round-bottomed flasks, add DMAP (56.3 mg then, 0.47 mmol), DCC (575.1 mg, 2.8 mmol), reaction at room temperature earlier, glycol monomethyl vinyl ethers (EGVE will be added behind the activated carboxylic 12h again, 625 μ L, 6.98 mmol), mixed solution continues lucifuge reaction 24h down at 25 ℃.After reaction finished, the solution muddy shape that is white in color after filtering with core, was deposited in filtrate in the cold ether, filtered to place vacuum drying oven dry.Finally obtain vinyl ethers functionalization degree and be 12 PEG
113-PAA
20The white solid powder, productive rate about 71%.
1H NMR (400 MHz, CDCl
3): PEG (CH
2-CH
2-O-: δ 3.63; CH
3-O-: δ 3.38), PAA (CH
2-CH-COO-): δ 2.37;-CH
2-CH-COO-: δ 1.6-1.9), EGVE unit (COO-CH
2-CH
2-O-: δ 4.27;-COO-CH
2-CH
2-O-3.87;-O-CH=CH
2: δ 4.05,4.18 and 6.50).
M n(
1H NMR)=7.3 kg/mol,
M n(GPC)=9.0 kg/mol, PDI (GPC)=1.03.
At N
2Protecting down, is 12 PEG with vinyl ethers functionalization degree
113-PAA
20(100.0 mg, equv. 164.8 μ mol EGVE unit) are dissolved in the 10 mL dry DMF, add paclitaxel PTX (47.0 mg, 55.0 μ mol) then successively,
p-TSA (0.32 mg, 1.65 μ mol), just 4 molecular sieves of oven dry.React after 4 days, will remove unreacted paclitaxel with DMF dialysis (molecular cut off 3500) behind the reacting liquid filtering., be transferred in a water again and dialysed 48 hours after 24 hours in dialysis in the DMF, dialysis finishes postlyophilization, obtains the prodrugs of paclitaxel PEG by the acetal bonds bonding at last
113-P (AA
20-
g-PTX) (PTX content: white solid 21.6 wt.%), productive rate about 79%.
1H NMR (400 MHz, CDCl
3): PEG bl ℃ of k (CH
2-CH
2-O-: δ 3.63; CH
3-O-: δ 3.38), PAA (CH
2-CH-COO-): δ 2.37;-CH
2-CH-COO-: δ 1.6-1.9), EGVE unit (COO-CH
2-CH
2-O-: δ 4.27;-COO-CH
2-CH
2-O-3.87;-O-CH=CH
2: δ 4.05,4.18 and 6.50), and PTX (δ 1.1-1.2
,δ 2.2-2.4, δ 3.5-3.8, δ 7.3-8.4), acetal bond (O-CH (CH
3)-O-: δ 4.37;-O-CH (CH
3)-O-: δ 1.36).
M n(
1H NMR)=9.3 kg/mol,
M n(GPC)=17.0 kg/mol, PDI (GPC)=1.42. nuclear-magnetism and HPLC test result show that the content of PTX is about 21.6 wt.% in this prodrug.
Embodiment two
Prodrugs of paclitaxel PEG
113-P (AA
20-
g-PTX) preparation
At N
2Under the environment, be 12 PEG with the vinyl ethers functionalization degree of embodiment one preparation
113-PAA
20(100.0 mg, equv. 164.8 μ mol EGVE unit) are dissolved in the 10 mL dry DMF, add PTX (70.4 mg, 82.4 μ mol) then successively,
p-TSA (0.32 mg, 1.65 μ mol), just 4 molecular sieves of oven dry.React after 4 days, will remove unreacted paclitaxel with DMF dialysis (molecular cut off 3500) behind the reacting liquid filtering., be transferred in a water again and dialysed 48 hours after 24 hours in dialysis in the DMF, dialysis finishes postlyophilization, obtains white solid at last, productive rate about 73.5%.Nuclear-magnetism and HPLC test result show that the content of PTX is about 27.6 wt.% in this prodrug.
Embodiment three
Prodrugs of paclitaxel PEG
113-P (AA
20-
g-PTX) preparation
At N
2Under the environment, be 12 PEG with the vinyl ethers functionalization degree of embodiment one preparation
113-PAA
20(100.0 mg, equv. 164.8 μ mol EGVE unit) are dissolved in the 10 mL dry DMF, add PTX (140.8 mg, 164.8 μ mol) then successively,
p-TSA (0.32 mg, 1.65 μ mol), just 4 molecular sieves of oven dry.React after 4 days, will remove unreacted paclitaxel with DMF dialysis (molecular cut off 3500) behind the reacting liquid filtering., be transferred in a water again and dialysed 48 hours after 24 hours in dialysis in the DMF, dialysis finishes postlyophilization, obtains white solid at last, productive rate about 78.2%.Nuclear-magnetism and HPLC test result show that the content of PTX is about 42.8 wt.% in this prodrug.
Embodiment four
Mitoxantrone (MTO) prodrug PEG
113-P (AA
35-
g-MTO) preparation
At N
2Under the protection, with initiator A IBN(3.12 mg, 0.019 mmol), macromole RAFT reagent PEG
113-DMP(0.48 g, 0.095 mmol), the solvent 1 of acrylic acid (326 μ L, 4.75 mmol) and 8 mL, the 4-dioxane joins respectively in the sealed reactor of 25mL, continues logical 30 minutes N
2, then reactor was placed in 70 ℃ the oil bath stirring reaction 48 hours.Reaction finishes the back and precipitates in the ice ether, and sample vacuum drying 24 hours obtains target di-block copolymer PEG
113-PAA
35, productive rate is 80%.
1H NMR (400 MHz, D
2O): PEG (-CH
2-CH
2-O-: δ 3.63; CH
3-O-: δ 3.38), PAA (-CH
2-CH-COO-): δ 2.37; -CH
2-CH-COO-: δ 1.6-1.9).
M n (
1H NMR) = 6.8 kg/mol。
Polymer P EG
113-PAA
35(0.3 g, 1.4 mmol COOH) are dissolved in 10 mL anhydrous 1, in the 4-dioxane, join in 50 ml, the two neck round-bottomed flasks, add DMAP (85.3 mg then, 0.7 mmol), DCC (863.4 mg, 4.2 mmol), reaction at room temperature earlier, glycol monomethyl vinyl ethers (EGVE will be added behind the activated carboxylic 12h again, 630 μ L, 7 mmol), mixed solution continues lucifuge reaction 24h down at 25 ℃.After reaction finished, the solution muddy shape that is white in color after filtering with core, was deposited in filtrate in the cold ether, filtered to place vacuum drying oven dry.Finally obtain the vinyl ethers function and turn to 12 PEG
113-PAA
35The white solid powder, productive rate about 68%.
At N
2Under the protection, in 50 ml, the two neck round-bottomed flasks, with MTO (148.6 mg, 287.2 μ mol),
p-TSA (0.273 mg, 1.435 μ mol) is dissolved in the 6 mL dry DMF, adds 4 good molecular sieves of activation just, the vinyl ethers function is turned to 12 PEG
113-PAA
35(100.0 mg, equv. 143.5 μ mol EGVE unit) are dissolved in the 4 mL dry DMF, dropwise join in the reactant liquor.React after 4 days, with dialysing 72 hours with a water behind the reacting liquid filtering, remove unreacted mitoxantrone hydrochloride and DMF solution.Dialysis finishes postlyophilization, obtains the mitoxantrone prodrug PEG by the acetal bonds bonding at last
113-P (AA
35-
g-MTO) basket color solid, productive rate about 68%.Nuclear-magnetism and ultraviolet test result show that the content of MTO is about 7.5 wt.% in this prodrug.
1H NMR (400 MHz, DMSO): PEG bl ℃ of k (CH
2-CH
2-O-: δ 3.63; CH
3-O-: δ 3.38), PAA (CH
2-CH-COO-): δ 2.37;-CH
2-CH-COO-: δ 1.6-1.9), EGVE unit (COO-CH
2-CH
2-O-: δ 4.27;-COO-CH
2-CH
2-O-3.87;-O-CH=CH
2: δ 4.05,4.18 and 6.50), (δ 2.74 for and MTO
,δ 3.18, δ 6.5-6.85), acetal bond (O-CH (CH
3)-O-: δ 4.37;-O-CH (CH
3)-O-: δ 1.36).
M n(
1H NMR)=9.2 kg/mol.
Embodiment five
Curcumin prodrug PEG
680-P (MA
80-
g-Cur) synthetic, accompanying drawing 2 is its synthetic sketch map:
At N
2Under the protection, with initiator A IBN(3.12 mg, 0.019 mmol), macromole RAFT reagent PEG
680-DMP(2.85 g, 0.095 mmol), the solvent 1 of methacrylic acid (MA, 800 μ L, 9.5 mmol) and 10 mL, the 4-dioxane joins respectively in the sealed reactor of 25mL, continues logical 30 minutes N
2, then reactor was placed in 70 ℃ the oil bath stirring reaction 48 hours.Reaction finishes the back and precipitates in the ice ether, and sample vacuum drying 24 hours obtains target di-block copolymer PEG
680-PMA
80, productive rate is 78%.
Polymer P EG
680-PMA
80(equ.0.28 mmol COOH), it is anhydrous 1 to be dissolved in 10 mL, in the 4-dioxane, join in 50 ml, the two neck round-bottomed flasks, add DMAP (17.1 mg, 0.14 mmol) then, DCC (173.2 mg, 0.84 mmol), reaction at room temperature will add glycol monomethyl vinyl ethers (EGVE, 130 μ L again behind the activated carboxylic 12h earlier, 1.45 mmol), mixed solution continues lucifuge reaction 24h down at 25 ℃.After reaction finished, the solution muddy shape that is white in color after filtering with core, was deposited in filtrate in the cold ether, filtered to place vacuum drying oven dry.Finally obtain the vinyl ethers function and turn to 50 PEG
680-PMA
80The white solid powder, productive rate about 74%.
At N
2Protect under the environment, the vinyl ethers function is turned to 50 PEG
680-PMA
80(equv. 57.6 μ mol EGVE unit) are dissolved in the 10 mL dry DMF, add curcumin (12 mg, 32.6 μ mol) then successively,
p-TSA (0.11 mg, 0.58 μ mol), just 4 molecular sieves of oven dry.React after 4 days, will remove unreacted curcumin with DMF dialysis (molecular cut off 3500) behind the reacting liquid filtering., be transferred in a water again and dialysed 48 hours after 24 hours in dialysis in the DMF, dialysis finishes postlyophilization, obtains light yellow solid at last, productive rate about 70.9%.Nuclear-magnetism and fluorescence spectrum test result show that the content of curcumin is about 39.5 wt.% in this prodrug.
Embodiment six
Prodrugs of paclitaxel PEG
273-P (Asp
20-
g-PTX) preparation:
At N
2Under the protective condition, with Asp (OBzl)-NCA(0.3 g, 1.2 mmol) be dissolved in DMF(6 mL) in, with PEG
273-NH
2(0.6 g, 0.05 mmol) is dissolved in DMF(6 mL) in, then with PEG-NH
2/ DMF solution is added in Asp (OBzl)-NCA/DMF solution fast, reacts 72 h down in 40 ℃ under the airtight condition.Reacted the back and precipitated with excessive absolute ether, core filters, the dissolving of precipitation reuse chloroform, and again with excessive absolute ether precipitation, the core filtration, vacuum drying 24 h under the room temperature obtain target di-block copolymer PEG
273-PAsp (OBzl)
20Productive rate is 90.3%.
At N
2Under the protective condition, with PEG
273-PAsp (OBzl)
20(0.5g, 0.031 mmol) is dissolved in the tetrahydrofuran solution (5 mL), adds 1 M NaOH solution (3.1 mL) then, and stirring reaction is 3 days under the room temperature.Change over to then in the bag filter (MWCO=3500) a water dialysis 48 hours, with 0.5 M HCl the pH of solution is transferred to acidity (pH 3 ~ 5), lyophilization gets white solid PEG
273-PAsp
20, productive rate is 86.8%.
Get polymer P EG
273-PAsp
20(0.4 g, 0.56 mmol COOH) is dissolved in the 10 mL dry DMF, join in 50 ml, the two neck round-bottomed flasks, add DMAP (34.2 mg, 0.28 mmol) then, DCC (346.3 mg, 1.68 mmol), reaction at room temperature will add glycol monomethyl vinyl ethers (EGVE, 252 μ L again behind the activated carboxylic 12h earlier, 2.8 mmol), mixed solution continues lucifuge reaction 24h down at 25 ℃.After reaction finished, the solution muddy shape that is white in color after filtering with core, was deposited in filtrate in the cold ether, filtered to place vacuum drying oven dry.Finally obtain vinyl ethers functionalization degree and be 10 PEG
273-PAsp
20The white solid powder, productive rate about 76.5%.
At N
2Under the environment, be 10 PEG with vinyl ethers functionalization degree
273-PAsp
20(100.0 mg, equv. 70 μ mol EGVE unit) are dissolved in the 10 mL dry DMF, add PTX (34.2 mg, 40 μ mol) then successively,
p-TSA (0.13 mg, 0.7 μ mol), just 4 molecular sieves of oven dry.React after 4 days, will remove unreacted paclitaxel with DMF dialysis (molecular cut off 3500) behind the reacting liquid filtering., be transferred in a water again and dialysed 48 hours after 24 hours in dialysis in the DMF, dialysis finishes postlyophilization, obtains white solid at last, productive rate about 73.6%.Nuclear-magnetism and HPLC test result show that the content of PTX is about 18.6 wt.% in this prodrug.
Embodiment seven
Prodrugs of paclitaxel PEG
454-P (Glu
45-
g-PTX) preparation (PTX content: 13.3 wt.%)
At N
2Under the protective condition, with Glu (OBzl)-NCA(0.14 g, 0.53 mmol) be dissolved in DMF(5 mL) in, with PEG
454-NH
2(0.2 g, 0.01 mmol) is dissolved in DMF(3.5 mL) in, then with PEG
454-NH
2/ DMF solution is added in Asp (OBzl)-NCA/DMF solution fast, reacts 72 h down in 40 ℃ under the airtight condition.Reacted the back and precipitated with excessive absolute ether, core filters, the dissolving of precipitation reuse chloroform, and again with excessive absolute ether precipitation, the core filtration, vacuum drying 24 h under the room temperature obtain target di-block copolymer PEG
454-PGlu (OBzl)
45Productive rate is 87.6%.
At N
2Under the protective condition, with PEG
454-PGlu (OBzl)
45(6.67 μ mol) is dissolved in the THF solution (5 mL), adds 1 M NaOH solution (1.5 mL) then, stirs under the room temperature and spends the night.Change over to then in the bag filter (MWCO=3500) a water dialysis 48 hours, with 0.5 M HCl the pH of solution is transferred to acidity (pH 3 ~ 5), lyophilization gets white solid PEG
454-PGlu
45, productive rate is 90.1%.
Get polymer P EG
454-PGlu
45(eqv. 0.25 mmol COOH), be dissolved in the 10 mL dry DMF, join in 50 ml, the two neck round-bottomed flasks, add DMAP (15.3 mg then, 0.125 mmol), DCC (154.6 mg, 0.75 mmol), reaction at room temperature earlier, glycol monomethyl vinyl ethers (EGVE will be added behind the activated carboxylic 12h again, 113 μ L, 1.25 mmol), mixed solution continues lucifuge reaction 24h down at 25 ℃.After reaction finished, the solution muddy shape that is white in color after filtering with core, was deposited in filtrate in the cold ether, filtered to place vacuum drying oven dry.Finally obtain vinyl ethers functionalization degree and be 30 PEG
454-PGlu
45The white solid powder, productive rate about 75.2%.
At N
2Under the environment, be 30 PEG with vinyl ethers functionalization degree
454-PGlu
45(equv. 53.8 μ mol EGVE unit) are dissolved in the 10 mL dry DMF, add PTX (23 mg, 26.9 μ mol) then successively,
p-TSA (0.12 mg, 0.6 μ mol), just 4 molecular sieves of oven dry.React after 4 days, will remove unreacted paclitaxel with DMF dialysis (molecular cut off 3500) behind the reacting liquid filtering., be transferred in a water again and dialysed 48 hours after 24 hours in dialysis in the DMF, dialysis finishes postlyophilization, obtains white solid at last, productive rate about 80.9%.Nuclear-magnetism and HPLC test result show that the content of PTX is about 13.3 wt.% in this prodrug.
Embodiment eight
Coupling has the prodrugs of paclitaxel FA-PEG of folic acid
136-P (AA
20-
g-PTX) (content of taxol: 20.6 wt.%) synthetic
At N
2Under the protection, with initiator A IBN(3.12 mg, 0.019 mmol), macromole RAFT reagent BOCH
2N-PEG
136-DMP(0.5 g, 0.083 mmol), the solvent 1 of acrylic acid (143 μ L, 2.075 mmol) and 6 mL, the 4-dioxane joins respectively in the sealed reactor of 25mL, continues logical 30 minutes N
2, then reactor was placed in 70 ℃ the oil bath stirring reaction 48 hours.Reaction finishes the back and precipitates in the ice ether, and sample vacuum drying 24 hours obtains target di-block copolymer BOCH
2N-PEG
136-PAA
20, productive rate is 87%.
At N
2Under the protection, with polymer B OCH
2N-PEG
136-PAA
20(0.4 g, 1.07 mmol COOH) are dissolved in 10 mL anhydrous 1, in the 4-dioxane, join in 50 ml, the two neck round-bottomed flasks, add DMAP (65.7 mg then, 0.54 mmol), DCC (661.7 mg, 3.21 mmol), reaction at room temperature earlier, glycol monomethyl vinyl ethers (EGVE will be added behind the activated carboxylic 12h again, 481 μ L, 5.35 mmol), mixed solution continues lucifuge reaction 24h down at 25 ℃.After reaction finished, the solution muddy shape that is white in color after filtering with core, was deposited in filtrate in the cold ether, filters and is placed on dry 24h in the vacuum drying oven.Finally obtain vinyl ethers functionalization degree and be 12 BOCH
2N-PEG
136-PAA
20The white solid powder, productive rate about 86.4%.
Be 12 BOCH with vinyl ethers functionalization degree
2N-PEG
136-PAA
20(0.3 g) is dissolved in 2 mL trifluoroacetic acids and 2 mL HCl(1M) in, after stirring 5 h under the room temperature, after with NaOH solution (1M) its pH being transferred to 5-6, the bag filter of packing into (MWCO=3500) was with a water dialysis 48 hours, and cryodesiccated white solid vinyl ethers functionalization degree is 12 H then
2N-PEG
136-PAA
20, productive rate is 92.6%.
At first, folic acid (0.18 g, 0.4 mmol) in the dimethyl sulfoxine of 5 mL, DCC(0.25 g, 3.3 mmol) with NHS(0.07 g, 0.6 mmol) exist down, lucifuge activation 12 hours will be gone up vinyl ethers functionalization degree that the step obtains and be 12 H then
2N-PEG
136-PAA
20(0.3 g, 0.04 mmol) is dissolved in the 5 mL dimethyl sulfoxines, adds triethylamine (56 μ L, 0.4 mmol), reacts at normal temperatures 24 hours.Remove by filter by-product DCU earlier after reaction finishes, dialysis was removed unreacted folic acid in 48 hours to dimethyl sulfoxine then, and then water dialysis 48 hours, and then lyophilization obtains yellow target product FA-PEG
136-P(AA
20-PTX), productive rate: 80.6 %.The amount of folic acid is determined at the absworption peak at 363 nm places by ultraviolet spectra.The HPLC test result shows that the content of PTX is about 20.6 wt.% in this prodrug.
Embodiment nine
Direct hydration method prepares PEG
113-P (AA
20-
g-PTX) (PTX content: prodrugs of paclitaxel nanoparticle 42.8 wt.%)
Adding 1 mg PTX prodrug and 15 μ L molecular weight are 1000 Polyethylene Glycol in 4 mL centrifuge tubes.Mixture 105 ℃ of following agitating heating 10 minutes, is cooled to room temperature with mixture then, adds the phosphate buffer solution (pH 7.4 for PB, 10 mM) of 2 mL, ultrasonic 20min, dialysis 12h removes PEG1000 in PB buffer solution at last.It is 248.3 nm that dynamic light scattering records the nanoparticle mean diameter, and the particle size distribution index is 0.30.
Embodiment ten
Direct hydration method prepares PEG
113-P (AA
20-
g-PTX) (PTX content: prodrugs of paclitaxel nanoparticle 21.6 wt.%)
Adding 1 mg PTX prodrug and 15 μ L molecular weight are 350 Polyethylene Glycol in 4 mL centrifuge tubes.Mixture 95 ℃ of following agitating heating 15 minutes, is cooled to room temperature with mixture then, progressively adds 10,20, the phosphate buffer solution of 70,1900 μ L (PB, 10 mM, pH 7.4), and at each ultrasonic 20min in back that adds, dialysis 12h removes PEG550 in PB buffer solution at last.It is 158.3 nm that dynamic light scattering records the nanoparticle mean diameter, and the particle size distribution index is 0.17.The critical micelle concentration that fluorescence spectrum records this prodrug nanoparticle is 3.16 mg/L.
Embodiment 11
Direct hydration method prepares PEG
680-P (MA
80-
g-Cur) (curcumin content: curcumin prodrug nanoparticle 39.5 wt.%)
Adding 1 mg Cur prodrug and 15 μ L molecular weight are 550 Polyethylene Glycol in 4 mL centrifuge tubes.Mixture 85 ℃ of following agitating heating 25 minutes, is cooled to room temperature with mixture then, progressively adds 10,20, the phosphate buffer solution of 70,1900 μ L (PB, 10 mM, pH 7.4), and at each ultrasonic 20min in back that adds, dialysis 12h removes PEG550 in PB buffer solution at last.It is 230.3 nm that dynamic light scattering records the nanoparticle mean diameter, and the particle size distribution index is 0.24.
Embodiment 12
Direct hydration method prepares PEG
113-P (AA
35-
g-MTO) (MTO content: mitoxantrone prodrug nanoparticle 7.5 wt.%)
Adding 1 mg MTO prodrug and 15 μ L molecular weight are 1500 Polyethylene Glycol in 4 mL centrifuge tubes.Mixture 95 ℃ of following agitating heating 25 minutes, is cooled to room temperature with mixture then, progressively adds 10,20, the phosphate buffer solution of 70,1900 μ L (PB, 10 mM, pH 7.4), and at each ultrasonic 20min in back that adds, dialysis 12h removes PEG550 in PB buffer solution at last.It is 62 nm that dynamic light scattering records the nanoparticle mean diameter, and the particle size distribution index is 0.11.
Embodiment 13
PEG
113-P (AA
20-g-PTX) (content of taxol: the mensuration of prodrugs of paclitaxel nanoparticle particle diameter and light scattering Strength Changes under solutions of weak acidity 21.6 wt.%)
The concentration that we get that 1 mL prepares is the nanoparticle solution example of 0.5 mg/mL, and its pH is transferred to 5.0, be placed in 37 ℃ of shaking tables and rock, and the variation of at the appointed time surveying its particle diameter and light scattering intensity with dynamic light scattering (DLS).
Accompanying drawing 3 is the particle diameter of above-mentioned nanoparticle and the variation of light scattering intensity, as can be seen: under pH 5.0 conditions, nanoparticle along with the prolongation meeting of time very fast by 160 nm rise big or assemble forms submicron and micron particle (1700 nm), particle size distribution has 0.15 to be increased to 0.45, its light scattering intensity also is reduced to about 80 kcps by 450 original kcps gradually, can observe flocculent substance simultaneously and separate out.
Embodiment 14
The extracorporeal releasing experiment of prodrugs of paclitaxel nanoparticle
The extracorporeal releasing experiment of paclitaxel is at 37 ℃ of following HAc-NaAc(pH 5.0,10 mM), HAc-NaAc(pH 6.0,10 mM) and PB(pH 7.4,10 mM) carry out under three kinds of conditions.The concentration of nanoparticle is 0.5 mg/mL, gets 0.5 mL nanoparticle solution and puts into release bag filter (Spectra/Pore
, MWCO 12000) in, bag filter immerses respectively in the above-mentioned three kinds of buffer solution media of 25 mL, puts into 37 ℃ of shaking tables, at each sampling time point, takes 8 mL release medium away and is used for the HPLC test, adds the fresh dialysis medium of 8 mL correspondences in addition.After the 8 mL samples that take out are concentrated into 0.5 mL by lyophilization, utilize high performance liquid chromatography (HPLC, Agilent 1260, with the acetonitrile of volume ratio 1/1 and water as mobile phase) measure the content of paclitaxel in the absorption intensity at 227 nm places.Two groups of each release experiment are parallel, results averaged.
Accompanying drawing 4 is the cumulative release amount of PTX, and as can be seen: for the release under the physiological pH (7.4), the rate of release from the prodrugs of paclitaxel nanoparticle of the PTX under acid condition is faster, and the result of this and acetal hydrolysis is consistent; For PEG
113-P (AA
20-
g-PTX) (42.8 wt.%) in 48 hours, discharges PTX and is about 86.9% and 66.4% respectively under pH 5.0 and pH 6.0 conditions, and discharges (＜29%) under pH 7.4 conditions with having only a spot of drug slow.These results show that also PTX can avoid the violent release phenomenon effectively by being chemically bonded to the prodrug nanoparticle that forms on the polymer chain, and drug release also has excellent controllability simultaneously.
Embodiment 15
Polymer precursor (the PEG of vinyl ethers functionalization
113-PAA
20, PEG
1818-PMA
80, PEG
273-PAsp
20, PEG
1136-PGlu
45) cytotoxicity experiment (MTT)
The cytotoxicity experiment of polymer precursor adopts mtt assay.People's nasopharynx epidermal carcinoma cell (KB cell), cervical cancer cell (HeLa cell) and breast cancer cell (MCF-7) are under 37 ℃, 5% carbon dioxide conditions, in culture medium (DMEM), cultivate, culture medium will be added 10% calf serum, 1% glutamate, Glu, antibiotic penicillin (100 IU/mL) and streptomycin (100 μ g/mL) before using.Three kinds of cancerous cell are layered on respectively on the 96 porocyte culture plates, and 5000 cells are approximately planted in each hole.The long coverage rate to 70-80% of cell after 24 hours, add polymer precursor solution (concentration is 0.05 ~ 1 mg/mL) cell that 10 μ L contain the variable concentrations of respective amount acetaldehyde (be equivalent to the maximum prodrug of content of dispersion when maximum concentration 1 mg/mL during all acetal bonds hydrolysis the acetaldehyde (32.5 mM) of issuable maximum) in the celliferous culture medium of 90 μ L and continue to cultivate the 3-(4 that adds 10 μ L after 48 hours, 5-dimethylthiazole-2)-2, the PBS solution of 5-diphenyl tetrazole bromine salt (MTT) (5 mg/mL).With culture plate continue to put into incubator after 4 hours the culture fluid sucking-off, the purple crystal that living cells produces dissolves with 150 μ L DMSO.The optical density (OD) of sample is measured at 492 nm places with microplate reader (Thermo Multiskan FC), and last gained data are four groups meansigma methods.The PBS of adding and sample equal volume is designated as 100% survival in contrast in the blank well.
Accompanying drawing 5 is the relation of cell survival rate and polymer precursor concentration, as can be seen: polymer precursor (PEG-PAA of vinyl ethers functionalization) is even when concentration reaches 1 mg/mL, when respective amount acetaldehyde exists, also show nontoxicly substantially, cell survival rate is all greater than 90%, the polymer precursor that the vinyl ethers functionalization is described is nontoxic, and has good biocompatibility.
Embodiment 16
The active anticancer of prodrugs of paclitaxel nanoparticle is measured
Cytotoxicity method of testing and the embodiment 15 of prodrugs of paclitaxel nanoparticle are similar.Human lung carcinoma cell (the A549/PTX of people's nasopharynx epidermal carcinoma cell (KB cell), cervical cancer cell (HeLa cell) and the anti-paclitaxel of reverse
R) under 37 ℃, 5% carbon dioxide conditions, in culture medium (DMEM), cultivate, three kinds of cancerous cell are layered on respectively on the 96 porocyte culture plates, 5000 cells are approximately planted in each hole.The long coverage rate to 70-80% of cell after 24 hours, (final PTX concentration is respectively 5 * 10 to add 10 μ L prodrugs of paclitaxel solution in the celliferous culture medium of 90 μ L
-5, 5 * 10
-4, 0.005,0.01,0.1,1,5,10 and 20 μ g/mL), PTX is with Cremophor EL and ethanol 1 freely: 1(v/v) make Taxol
Form organize in contrast, each concentration has four parallel multiple holes.Cell continues to cultivate 48 hours, and processing method is same as described above.The PBS of adding and sample equal volume is designated as 100% survival in contrast in the blank well.IC
50The value of (drug level of apoptosis one half correspondence) obtains by nonlinear regression (sigmoidal) analytical calculation.
Accompanying drawing 6,7 is the relation of cell survival rate and PTX concentration, and as can be seen: the prodrugs of paclitaxel nanoparticle shows the similar high drug effect with free PTX; Be the prodrugs of paclitaxel nanoparticle of 42.8 wt.% to PTX content, when PTX content in the nanoparticle is 1 μ g/mL, after itself and HeLa and KB cell were cultivated 48 hours respectively, the survival rate of HeLa cell had dropped to 41.9%(Fig. 6), the survival rate of KB cell is remaining 22.7%(Fig. 7 only).The high antitumor cell activity of PTX prodrug nanoparticle has illustrated that PTX can be carried effectively and has been discharged in the nucleus of HeLa cell and KB cell.
In addition, as accompanying drawing 8, the MTT experiment shows the cancerous cell A549/PTX of this prodrugs of paclitaxel nanoparticle antagonism property of medicine
RStronger kill capability is also arranged.Such as, acid-sensitive sense prodrugs of paclitaxel nanoparticle and A549/PTX
RAfter hatching 48 hours, be 10 μ g/mL in PTX equivalent concentration, cell survival rate only is 50.3%, and its IC50 value is about 8.2 μ g/mL; And free paclitaxel concentration at 20 μ g/mL, hatch 48 hours after, A549/PTX
RCell has shown tangible Drug resistance, and cell survival rate still reaches more than 80%, and its IC50 value is 175.8 μ g/mL.And after free paclitaxel that it should be noted that same concentrations is hatched 48 hours with normal A549 cell, its IC
50Value only is 0.2 μ g/mL, and both differ and surpass 800 times, and this can illustrate the A549/PTX that we use well
RCell has kept drug resistance preferably.Experiment shows that this prodrugs of paclitaxel nanoparticle that is connected by acetal bonds can overcome the drug resistance that tumor cell produces effectively, has good application prospects in field of cancer.
Embodiment 17
PTX prodrug nanoparticle parcel amycin (DOX)
PTX prodrug (1 mg) is put into 4 mL centrifuge tubes, add 15 μ L PEG550, then stirred 25 minutes down at 95 ℃.Then mixture is cooled to room temperature, adds DMSO solution (5.0 mg/mL) and the 10 μ L PB buffer solution (pH 7.4,10 mM) of 20 μ L DOX, stirred 20 minutes down at 60 ℃.Add 20,70 and 1900 μ L PB buffer solution subsequently successively, and added the back ultrasonic 20 minutes each.Removed DOX and the PEG550 that does not have bag to enter in 12 hours by dialysis in PB buffer solution at last, the concentration of drug-carrying nanometer particle is 0.5 mg/mL.
(FLS 920, excitation wavelength: 480 nm, emission wavelength: 560 nm) by fluorescent spectrophotometer assay for the drug loading of DOX and envelop rate.Concrete steps are that the standard curve by DOX/DMSO calculates then with ultrasound destruction behind the adding 3 mL DMSO after the 100 μ L medicine-carried nano particles solution lyophilizations.Drug loading (DLC) and envelop rate (DLE) calculate by following formula:
Drug loading (wt%)=(drug loading quality/(polymer quality+drug loading quality) * 100%
Envelop rate (%)=(drug loading quality/medicine always drops into quality) * 100%
As table 1, at theoretical drug loading (be DOXHCl/(polymer+DOXHCl) ratio) when being 5,10 and 20 wt. %, three kinds of PTX prodrug nanoparticles have higher entrapment (DLE) to DOX, and the envelop rate that we find DOX be along with the increase of PTX content can reduce, and may be because PTX content makes the loose cause of nuclear of nanoparticle more.
Table 1: the sign of the PTX prodrug nanoparticle of parcel DOX
a
aThe ultimate density of nanoparticle is 0.5 mg/mL;
bMean diameter (nm) and particle size distribution are measured by DLS 25 ℃, pH 7.4 times.
Embodiment 18
Be loaded with the extracorporeal releasing experiment of the PTX prodrug nanoparticle of DOX
DOX with PTX the time extracorporeal releasing experiment under 37 ℃, three kinds of different media: (i) hac buffer, pH 5.0; (ii) hac buffer, pH 6.0; (iii) phosphate buffer solution is measured among the pH 7.4.The concentration of these three kinds of media all is 10 mM.The nanoparticle sample that is loaded with DOX for preparing is divided into three parts (every part of 0.5 mL), transfers to then in the bag filter (MWCO=12000 ~ 14000), and bag filter is placed the corresponding buffer medium of 25mL, puts into 37 ℃ of constant temperature shaking tables then.At the time point of appointment, from the release medium that delivery systme takes out 8 mL, replenish the fresh medium of equal volume then, wherein 4mL is by the content of fluoremetry DOX, other 4 mL measure the content of PTX by HPLC, parallel two groups of this release experiment, and the result gets its meansigma methods.
Accompanying drawing 9 is release amount of medicine and time relation, and as can be seen: for the release under the physiological pH (7.4), the DOX under acid condition and the rate of release of PTX are faster, and this mainly is because the hydrolysis of acetal bonds on the main polymer chain; For PEG-P (AA-
g-PTX) (21.6 wt.%), in 48 hours, pH 5.0 times, nearly 82.8% DOX and 85.3% PTX discharge from nanoparticle, and under the same conditions, pH has only discharge (＜30%) of a spot of drug slow for 7.4 times.
Embodiment 19
Direct hydration method prepares the prodrugs of paclitaxel nanometer of surperficial coupling folic acid
In 4 mL centrifuge tubes, add 0.8 mg prodrugs of paclitaxel PEG
113-P(AA
20-
g42.8 wt.%), the prodrugs of paclitaxel FA-PEG of 0.2 mg coupling folic acid-PTX) (content of taxol:
136-P (AA
20-
g-PTX) and 15 μ L molecular weight be 550 Polyethylene Glycol (PEG550).Mixture 95 ℃ of following agitating heating 25 minutes, is cooled to room temperature with mixture then, adds 10,20 respectively, the phosphate buffer solution of 70,1900 μ L (PB, 10 mM, pH 7.4), and at each ultrasonic 20min in back that adds, dialysis 12h removes PEG550 in PB buffer solution at last.It is 182.5 nm that dynamic light scattering records the nanoparticle mean diameter, and the particle size distribution index is 0.12.
In 4 mL centrifuge tubes, add 0.8 mg prodrugs of paclitaxel PEG-P(AA-
g-PTX) and 0.2 mg coupling folic acid FA-PEG
136-PLA with 15 μ L molecular weight be 550 Polyethylene Glycol (PEG550).Mixture 95 ℃ of following agitating heating 25 minutes, is cooled to room temperature with mixture then, adds 10,20 respectively, the phosphate buffer solution of 70,1900 μ L (PB, 10 mM, pH 7.4), and at each ultrasonic 20min in back that adds, dialysis 12h removes PEG550 in PB buffer solution at last.It is 188.4 nm that dynamic light scattering records the nanoparticle mean diameter, and the particle size distribution index is 0.19.
The active anticancer of the prodrugs of paclitaxel nanoparticle of surface coupling folic acid is measured
The cytotoxicity method of testing of the prodrugs of paclitaxel nanoparticle of surface coupling folic acid and top similar.People's nasopharynx epidermal carcinoma cell (KB cell) that folacin receptor is contained on the surface is cultivated in culture medium (DMEM) under 37 ℃, 5% carbon dioxide conditions, and cell is layered on the 96 porocyte culture plates, and 5000 cells are approximately planted in each hole.The long coverage rate to 70-80% of cell after 24 hours, (final PTX concentration is respectively 5 * 10 to add the prodrugs of paclitaxel nanoparticle solution of 10 μ L surface coupling folic acid and the prodrugs of paclitaxel nanoparticle solution of surperficial unmodified folic acid in the celliferous culture medium of 90 μ L
-5, 5 * 10
-4, 0.005,0.01,0.1,1,5,10 and 20 μ g/mL), each concentration has four parallel multiple holes.Cell was cultivated 3 hours earlier, changed fresh culture medium then, continued to hatch 45 hours, and processing method is same as described above.The PBS of adding and sample equal volume is designated as 100% survival in contrast in the blank well.IC
50The value of (drug level of apoptosis one half correspondence) obtains by nonlinear regression (sigmoidal) analytical calculation.
Accompanying drawing 10 is the relation of cell survival rate and PTX concentration, and as can be seen: the surface has coupling to have the prodrugs of paclitaxel nanoparticle of folic acid to show higher cytotoxicity; Have coupling that the prodrugs of paclitaxel nanoparticle of folic acid is arranged to the surface, when PTX content in the nanoparticle was 10 μ g/mL, the survival rate of cell was 34.9%, its IC
50Value is 1.42 μ g/mL; And to the prodrugs of paclitaxel nanoparticle of surperficial unmodified folic acid, its cell survival rate is 52.6%, IC
50Value is 17.16 μ g/mL.The surface has coupling to have the high antitumor cell activity of the PTX prodrug nanoparticle of folic acid to illustrate that nanoparticle can more effectively enter cell and discharge antiradiation drug after the targeted molecular, has improved the kill capability of nanoparticle to tumor cell on modifying.
Embodiment 21
The surface coupling has endocytosis and the interior release experiment of cell of the PTX nanoparticle that is loaded with DOX of folic acid
The surface coupling has the PTX nanoparticle that is loaded with DOX of folic acid at KB(-FA) endocytosis and release behavior in the cell observe sign by confocal laser microscope (CLSM).KB(-FA) cell is at 37 ℃, under 5% carbon dioxide conditions, containing 10% serum, do not containing among the RPMI-1640 of folic acid and cultivate, and cell density is 5 * 10
4Individual/hole.After 24 hours, add the PTX nanoparticle solution that is loaded with DOX, surface that the coupling of 50 μ L surface has folic acid not the PTX nanoparticle solution that is loaded with DOX or the DOXHCl solution freely of coupling folic acid, the concentration of final DOX all remains on 5 μ g/mL.Hatched 3,6 and 12 hours with cell respectively, wherein, the sample of 6 hours and 12 hours needs to change fresh culture adding sample solution after 3 hours, continue then to cultivate respectively 3 hours and 9 hours.After cultivating end, sop up the culture medium of cultivating in the plate hole, the cell in the culture plate washs three times with phosphate buffer solution, and the paraformaldehyde of reuse 4% is 20min fixedly, and nucleus dyes with DAPI.The CLSM photo of cell is measured (TCS SP2) by confocal laser scanning microscope, CLSM.
The surface coupling has the PTX nanoparticle and the KB(-FA that are loaded with DOX of folic acid) after cell hatched through 6 hours, we just can see the red fluorescence that apparent in view DOX is arranged around nucleus, illustrate that nanoparticle can be ingested and discharge fast effectively in cell very soon.After hatching 12 hours, occurred stronger DOX red fluorescence in the nucleus, and with the fluorescence of free DOX after similar and MCF-7 cell was hatched 12 hours, still only there is cell peripheral in the fluorescence of DOX.This has proved absolutely, the surface coupling has the PTX nanoparticle that is loaded with DOX of folic acid because can (FA) the folacin receptor specificity of cell surface is combined with KB, by this receptor-mediated effect, nanoparticle can enter cell more quickly and effectively, and discharges medicine rapidly.
Claims (5)
1. acid-sensitive sensitive polymeric prodrug, it is characterized in that: in the described acid-sensitive sensitive polymeric prodrug, polymer precursor is the water solublity A-B type bi-block copolymer of vinyl ether functionalization, and wherein the A block is that molecular weight is the Polyethylene Glycol of 3000～30000 Da, and the B block is carboxylic polymer; Medicine is paclitaxel, mitoxantrone, curcumin or astaxanthin;
Described drug molecule links by acetal bonds and A-B type bi-block copolymer;
In the described water solublity A-B type bi-block copolymer, the number of repeat unit of monomer is 10～100 in the B block;
In the described acid-sensitive sensitive polymeric prodrug, the percent grafting of medicine is 0.1～0.6; The A block constitutes hydrophilic segment, constitutes hydrophobic part behind the B block graft medicine, and the hydrophilic segment molecular weight is 0.5～8 times of hydrophobic part molecular weight.
2. acid-sensitive sensitive polymeric prodrug according to claim 1, it is characterized in that: medicine is paclitaxel.
3. acid-sensitive sensitive polymeric prodrug according to claim 1 is characterized in that: the B block is polyacrylic acid, polymethylacrylic acid, poly-ethylacrylic acid, poly-aspartate or polyglutamic acid.
4. prodrug nanoparticle that is prepared by any one acid-sensitive sensitive polymeric prodrug in the claim 1～3, it is characterized in that: the shell of described prodrug nanoparticle is made of block A, and nuclear is made of medicine; The particle diameter of described prodrug nanoparticle is 50～250 nm.
5. the described prodrug nanoparticle of claim 4 is as the application of anti-cancer medicament carrier.
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