CN103285400B - 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 and a kind of there is the application as pharmaceutical carrier of the polymeric prodrugs of sensitivity to acid, nanoparticle prepared therefrom and this nanoparticle, belong to 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.The treatment of cancer be 21st century facing mankind maximum test and a difficult problem.
Along with development and the progress of science and technology, the mankind are to the continuous exploration of the related sciences such as oncology, molecular biology, pharmacy and chemistry, make there has been significant progress to the research of antitumor drug, many effective cancer therapy drugs are designed to develop.But current most cancer therapy drug is micromolecule, there is the shortcoming of the large and poorly water-soluble of toxic and side effects, thus significantly limit the application of cancer therapy drug in clinical.
In order to solve small molecule anticancer drug water solublity low (hydrophobicity is strong), bioavailability low, to be easily excreted and to problems such as human body toxic and side effects are large, people study various polymeric prodrugs or pharmaceutical carrier etc. and change mode and the distribution in vivo that cancer therapy drug enters human body.
Although polymeric prodrugs can improve the dissolubility of dewatering medicament to a great extent, but still there is as lower in drug loading, the inefficient problem of cell endocytic in several water-soluble prodrugs entering clinical trial that comprise of research at present, and medicine is the slow releasing by the degraded of polymer only, makes cancerous cell easily produce drug resistance like this; The medicament contg that polymer nanocomposite pharmaceutical carrier can load usually is high, and by the enrichment in tumor cell of EPR effect, but the problems such as body internal stability is poor, the easy premature disconnection of medicine.
People study the prodrug adopting and replace only end group bonding at the multiple drug molecule of hydrophilic polymer side chain graft, it can the self assembly prodrug micelle nanoparticle that to be formed be core with hydrophobic anticancer drug, hydrophilic polymer is shell, and cancer therapy drug can be linked with the chemical bond of stimulating responsive (cystine linkage etc. as responsive in the acetal bonds of peptide bond, phosphoric acid ester bond, acid-sensitive, hydrazone key, ester bond, amido link and reduction), make Nano medication can discharge medicine fast after arrival tumor locus, reach the object of oncotherapy.
In general, the pH value of the pathological tissues such as tumor and inflammation inside is all low than the pH value of normal body fluid, presents faintly acid.Therefore, this feature can be utilized well to carry out the design of prodrug micelle nanoparticle, by some in acid condition labile component introducing nanoparticle.The micelle that they are assembled in aqueous in neutral conditions can drug loading stable existence, but in acid condition, fracture can occur for acid sensitive group or segment makes micelle destroy, thus the medicine of its internal package can be discharged rapidly, these acid sensitive group comprise acetal radical, hydrazone key, orthoformate etc.
In the prior art, more existing reports about the prodrug micelle nanoparticle of sensitivity to acid.But, as drug material, good biocompatibility and biodegradability should be had, and, as the carrier of antitumor drug, also should have following features: can form stable polymer micelle in aqueous, its hydrophobic cores is hydrophobic anticancer drug, and hydrophilic shell plays the effect of stable micelle, raising micelle blood circulation time; When carrier micelle arrives tumor or pathological tissues, the feature that the pH value of local organization is lower can be utilized, destroy micelle, discharge cancer therapy drug rapidly.
Therefore, the polymeric prodrugs micelle nano grain seeking more acid-sensitive is needed.
Summary of the invention
The object of the invention is, there is provided a kind of and there is the polymeric prodrugs of sensitivity to acid and nanoparticle prepared therefrom, to utilize the endosome in cancerous cell/lysosomal sour environment to control the release of medicine, reach the effect of Therapeutic 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 polymeric prodrugs; In this acid-sensitive polymeric prodrugs, polymer precursor is the water solublity A-B type bi-block copolymer of vinyl ether functional, wherein the Polyethylene Glycol of A to be molecular weight be 3000 ~ 30000 Da, and B block is carboxylic polymer; Medicine is paclitaxel, mitoxantrone, curcumin or astaxanthin;
Described drug molecule is linked by acetal bonds and A-B type bi-block copolymer;
In described A-B type hydrophilic bi-block copolymer, in B block, the number of repeat unit of monomer is 10 ~ 100;
In described acid-sensitive polymeric prodrugs, the percent grafting of medicine is 0.1 ~ 0.6; A block forms hydrophilic segment, and form hydrophobic part after B block grafting medicine, hydrophilic segment molecular weight is 0.5 ~ 8 times of hydrophobic part molecular weight.
The present invention's medicine used is paclitaxel (PTX), mitoxantrone (MTO), curcumin (Cur) or astaxanthin (ASTA), and its molecular structural formula is as follows:
。
In preferred technical scheme, medicine is paclitaxel; B block is polyacrylic acid, polymethylacrylic acid, poly-ethylacrylic acid, poly-aspartate or polyglutamic acid.
In technique scheme, in water solublity A-B type bi-block copolymer precursor B block side chain carboxyl group can with glycol divinyl ether generation esterification, obtain the polymer that pendant vinyl base ether is functionalized, the double bond of vinyl ethers can with hydroxyl generation nucleophilic addition, form acetal bonds.Paclitaxel, mitoxantrone, curcumin, astaxanthin etc. can be bonded on polymer precursor by acetal bonds with the Hydrophobic small molecules cancer therapy drug of active hydroxyl groups.The structural formula of glycol divinyl ether is as follows:
。
In 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, structurally both containing hydrophobic small-molecule drug, can be used for loading another kind of hydrophobic anticancer drug; Again containing the acetal radical that can occur in acid condition to be hydrolyzed, under certain pH conditions, acetal radical is hydrolyzed, and micelle is destroyed, thus discharges the hydrophobic anticancer drug being wrapped in micelle inside rapidly.
The preparation method of above-mentioned acid-sensitive polymeric prodrugs, comprises the following steps:
First A-B type bi-block copolymer is prepared; Obtained the polymer of vinyl ether functional again by DCC/DMAP esterification reaction method, then catalytic amount p-methyl benzenesulfonic acid (
p-TSA) effect under, the prodrug polymer of a series of different pharmaceutical content can be prepared.
The prodrug nanoparticle prepared by above-mentioned acid-sensitive polymeric prodrugs, the shell of this nanoparticle is made up of block A, and core is made up of medicine; The particle diameter of nanoparticle is 50 ~ 250 nm, and distribution of sizes is 0.07 ~ 0.30.
In technique scheme, the preparation method of prodrug nanoparticle can be direct hydration method: first mixed with low molecular poly (PEG) by polymeric prodrugs, agitating heating 10 ~ 30 min at 80 ~ 110 DEG C; Then 1-5 mL PB buffer solution is added and ultrasonic; Finally dialysis removing PEG can obtain nanoparticle.
In above-mentioned preparation method, the molecular weight of Polyethylene Glycol is 350 ~ 1500Da; By the polymeric prodrugs of acetal bonds bonding Hydrophobic small molecules anticarcinogen can in aqueous self assembly formed with block A(PEG block) for hydrophilic shell, with the polymeric prodrugs nanoparticle of the pH sensitivity that is hydrophobic core by the responsive B block of the pH of acetal bonds bonding hydrophobic anticancer drug.Through this process, the prodrug micelle nanoparticle that polymer self assembles is formed, hydrophobic anticancer drug forms the core of micelle nano grain, have good biodegradability properties, the compatibility hydrophilic PEG block arrangement in outside, form the shell of micelle nano grain.
Acid-sensitive polymeric prodrugs nanoparticle prepared by the present invention has very high stability 7.4 times at pH, place it in 4 DEG C and store one month, its particle diameter does not have significant change, and under the condition of simulation human physiological environment, nanoparticle still kept stable after one week; But under mildly acidic conditions, connecting the acetal bond hydrolysis of medicine and polymer in prodrug nanoparticle, making medicine from discharging under prodrug nanoparticle comes off, such that the hydrophobicity of whole polymer declines, hydrophilic strengthens, therefore nanoparticle also can swell, and particle size distribution broadens.
Another object of the present invention is to provide the application of aforementioned prodrugs nanoparticle as anti-cancer medicament carrier.The prodrug nanoparticle of technique scheme gained can also the identical or different hydrophobic anticancer drug of physical loading as nano-carrier, is conducive to the therapeutic alliance of tumor.
Nanoparticle surface prepared by the present invention can be introduced and be improved prodrug nanoparticle by tumor cell endocytosis to targeting part such as antibody, polypeptide, folic acid and the galactose etc. of tumor cell specific, thus improves the concentration of medicine in tumor cell.Such as, the folate molecule of targeting entity tumor is introduced on prodrug nanoparticle surface, KB(population rhinocarcinoma cell with surperficial high expressed folacin receptor) test the cytotoxicity (MTT assay) of the prodrugs of paclitaxel nanoparticle of this surperficial coupling folic acid in cell, hatch the IC of 48 hours
50be 1.42 μ g/mL, and under the same conditions with the IC of the nanoparticle without targeting
50value, up to 17.16 μ g/mL, embodies targeting significantly.
Due to the enforcement of such scheme, the present invention compared with prior art, has the following advantages:
1. the acid-sensitive polymeric prodrugs that obtains of the present invention by the mode of chemical bonding in conjunction with hydrophobic anticancer drug, can effectively prevent medicine in vivo circulation time discharge too early;
2. the acid-sensitive polymeric prodrugs nanoparticle that the present invention obtains combines the advantage of prodrug and micelle simultaneously, cell is entered and by the rapid delivery of pharmaceuticals of the acid environment trigger drug in cancerous cell by the endocytosis of cell, and medicine maintains its prototype structure, there is potent ability of killing and wounding cancerous cell and drug resistant cancer cells;
3. the acid-sensitive polymeric prodrugs nanoparticle that the present invention obtains as the another kind of micromolecule hydrophobic anticancer drug of pharmaceutical carrier physically trapping, can be conducive to the therapeutic effect of the therapeutic alliance for tumor, raising tumor;
4. the acid-sensitive polymeric prodrugs nanoparticle that obtains of the present invention can further in its finishing specific tumour targeted molecular improve the endocytosis efficiency of cancerous cell to it, thus improve drug level and the kill capability to cancerous cell in cancerous cell.Therefore, nanoparticle disclosed by the invention can load and release anti-cancer medicine effectively, in the treatment of cancer, has good using value.
Accompanying drawing explanation
Fig. 1 is the synthesis schematic diagram of prodrugs of paclitaxel in embodiment one;
Fig. 2 is the synthesis schematic diagram of curcumin prodrug in embodiment five;
Fig. 3 is the result of variations figure of prodrugs of paclitaxel nanoparticle particle diameter and light scatter intensity under mildly acidic conditions in embodiment 13;
Fig. 4 is the release in vitro result of prodrugs of paclitaxel nanoparticle paclitaxel under different pH in embodiment 14;
Fig. 5 is the cytotoxicity experiment result figure of polymer precursor in embodiment 15;
Fig. 6 be in embodiment 16 prodrugs of paclitaxel nanoparticle to the toxicity data figure of HeLa cell;
Fig. 7 be in embodiment 16 prodrugs of paclitaxel nanoparticle to the toxicity data figure of KB cell;
Fig. 8 is the toxicity data figure of prodrugs of paclitaxel nanoparticle antagonism property of medicine cancerous cell in embodiment 16;
Fig. 9 is release in vitro result while two kinds of medicines in the prodrugs of paclitaxel nanoparticle being loaded with amycin in embodiment 18;
Figure 10 is that in embodiment 20, surperficial coupling has the prodrugs of paclitaxel nanoparticle of folic acid to KB(-FA) the toxicity data figure of cell.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the invention will be further described:
embodiment one
Prodrugs of paclitaxel PEG
113-P (AA
20-
g-PTX) preparation, accompanying drawing 1 be its synthesis schematic diagram:
At N
2under protection; by initiator A IBN(3.12 mg; 0.019 mmol); Macro RAFT agent PEG-DMP(0.48 g; 0.095 mmol), the solvent 1 of acrylic acid (163 μ L, 2.375 mmol) and 6 mL; 4-dioxane joins in the sealed reactor of 25mL respectively, continues logical 30 minutes N
2, then reactor to be placed in the oil bath of 70 DEG C stirring reaction 48 hours.Precipitate in ice ether after reaction terminates, 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 4-dioxane, join in 50 ml two neck round-bottomed flasks, then add DMAP (56.3 mg, 0.47 mmol), DCC (575.1 mg, 2.8 mmol), first at room temperature reacts, ethylene glycol mono vinyl ether (EGVE will be added again after activated carboxylic 12h, 625 μ L, 6.98 mmol), mixed solution continues lucifuge reaction 24h at 25 DEG C.After reaction terminates, solution is white casse shape, after filtering, filtrate is deposited in cold ether with core, filters and is placed in vacuum drying oven drying.Finally obtain the PEG that vinyl ether functionalized degree is 12
113-PAA
20white solid powder, productive rate about 71%.
1h NMR (400 MHz, CDCl
3): PEG (-CH
2-CH
2-O-: δ 3.63; CH
3, PAA (-CH-O-: δ 3.38)
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
2under protection, be the PEG of 12 by vinyl ether functionalized degree
113-PAA
20(100.0 mg, equv. 164.8 μm of ol EGVE unit) are dissolved in 10 mL dry DMF, then add paclitaxel PTX (47.0 mg, 55.0 μm of ol) successively,
p-TSA (0.32 mg, 1.65 μm of ol), 4 molecular sieves of just having dried.React after 4 days, remove unreacted paclitaxel by after reacting liquid filtering with DMF dialysis (molecular cut off 3500).Dialyse after 24 hours in DMF, then be transferred in a water and dialyse 48 hours, dialysis terminates postlyophilization, finally obtains the prodrugs of paclitaxel PEG by acetal bonds bonding
113-P (AA
20-
g-PTX) white solid of (PTX content: 21.6 wt.%), productive rate about 79%.
1h NMR (400 MHz, CDCl
3): PEG bl DEG C of k (-CH
2-CH
2-O-: δ 3.63; CH
3, PAA (-CH-O-: δ 3.38)
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, in this prodrug, the content of PTX is about 21.6 wt.%.
embodiment two
Prodrugs of paclitaxel PEG
113-P (AA
20-
g-PTX) preparation
At N
2under environment, vinyl ether functionalized degree embodiment one prepared is the PEG of 12
113-PAA
20(100.0 mg, equv. 164.8 μm of ol EGVE unit) are dissolved in 10 mL dry DMF, then add PTX (70.4 mg, 82.4 μm of ol) successively,
p-TSA (0.32 mg, 1.65 μm of ol), 4 molecular sieves of just having dried.React after 4 days, remove unreacted paclitaxel by after reacting liquid filtering with DMF dialysis (molecular cut off 3500).Dialyse after 24 hours in DMF, then be transferred in a water and dialyse 48 hours, dialysis terminates postlyophilization, finally obtains white solid, productive rate about 73.5%.Nuclear-magnetism and HPLC test result show, in this prodrug, the content of PTX is about 27.6 wt.%.
embodiment three
Prodrugs of paclitaxel PEG
113-P (AA
20-
g-PTX) preparation
At N
2under environment, vinyl ether functionalized degree embodiment one prepared is the PEG of 12
113-PAA
20(100.0 mg, equv. 164.8 μm of ol EGVE unit) are dissolved in 10 mL dry DMF, then add PTX (140.8 mg, 164.8 μm of ol) successively,
p-TSA (0.32 mg, 1.65 μm of ol), 4 molecular sieves of just having dried.React after 4 days, remove unreacted paclitaxel by after reacting liquid filtering with DMF dialysis (molecular cut off 3500).Dialyse after 24 hours in DMF, then be transferred in a water and dialyse 48 hours, dialysis terminates postlyophilization, finally obtains white solid, productive rate about 78.2%.Nuclear-magnetism and HPLC test result show, in this prodrug, the content of PTX is about 42.8 wt.%.
embodiment four
Mitoxantrone (MTO) prodrug PEG
113-P (AA
35-
g-MTO) preparation
At N
2under protection, by initiator A IBN(3.12 mg, 0.019 mmol), Macro RAFT agent PEG
113-DMP(0.48 g, 0.095 mmol), the solvent Isosorbide-5-Nitrae-dioxane of acrylic acid (326 μ L, 4.75 mmol) and 8 mL joins in the sealed reactor of 25mL respectively, continues logical 30 minutes N
2, then reactor to be placed in the oil bath of 70 DEG C stirring reaction 48 hours.Precipitate in ice ether after reaction terminates, 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 4-dioxane, join in 50 ml two neck round-bottomed flasks, then add DMAP (85.3 mg, 0.7 mmol), DCC (863.4 mg, 4.2 mmol), first at room temperature reacts, ethylene glycol mono vinyl ether (EGVE will be added again after activated carboxylic 12h, 630 μ L, 7 mmol), mixed solution continues lucifuge reaction 24h at 25 DEG C.After reaction terminates, solution is white casse shape, after filtering, filtrate is deposited in cold ether with core, filters and is placed in vacuum drying oven drying.Finally obtain vinyl ether functionalized be 12 PEG
113-PAA
35white solid powder, productive rate about 68%.
At N
2under protection, in 50 ml two neck round-bottomed flasks, by MTO (148.6 mg, 287.2 μm of ol),
p-TSA (0.273 mg, 1.435 μm of ol), is dissolved in 6 mL dry DMF, adds 4 molecular sieves just activated, by vinyl ether functionalized be 12 PEG
113-PAA
35(100.0 mg, equv. 143.5 μm of ol EGVE unit) are dissolved in 4 mL dry DMF, dropwise join in reactant liquor.React after 4 days, dialyse after reacting liquid filtering 72 hours with a water, remove unreacted mitoxantrone hydrochloride and DMF solution.Dialysis terminates postlyophilization, finally obtains the mitoxantrone prodrug PEG by acetal bonds bonding
113-P (AA
35-
g-MTO) basket color solid, productive rate about 68%.Nuclear-magnetism and ultraviolet test result show, in this prodrug, the content of MTO is about 7.5 wt.%.
1h NMR (400 MHz, DMSO): PEG bl DEG C k (-CH
2-CH
2-O-: δ 3.63; CH
3, PAA (-CH-O-: δ 3.38)
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 MTO (δ 2.74
,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) synthesis, accompanying drawing 2 be its synthesis schematic diagram:
At N
2under protection, by initiator A IBN(3.12 mg, 0.019 mmol), Macro RAFT agent PEG
680-DMP(2.85 g, 0.095 mmol), methacrylic acid (MA, 800 μ L, 9.5 mmol) and the solvent Isosorbide-5-Nitrae-dioxane of 10 mL join in the sealed reactor of 25mL respectively, continue logical 30 minutes N
2, then reactor to be placed in the oil bath of 70 DEG C stirring reaction 48 hours.Precipitate in ice ether after reaction terminates, 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), is dissolved in the anhydrous Isosorbide-5-Nitrae-dioxane of 10 mL, join in 50 ml two neck round-bottomed flasks, then DMAP (17.1 mg, 0.14 mmol) is added, DCC (173.2 mg, 0.84 mmol), first at room temperature react, ethylene glycol mono vinyl ether (EGVE, 130 μ L will be added after activated carboxylic 12h again, 1.45 mmol), mixed solution continues lucifuge reaction 24h at 25 DEG C.After reaction terminates, solution is white casse shape, after filtering, filtrate is deposited in cold ether with core, filters and is placed in vacuum drying oven drying.Finally obtain vinyl ether functionalized be 50 PEG
680-PMA
80white solid powder, productive rate about 74%.
At N
2under protection of the environment, by vinyl ether functionalized be 50 PEG
680-PMA
80(equv. 57.6 μm of ol EGVE unit) are dissolved in 10 mL dry DMF, then add curcumin (12 mg, 32.6 μm of ol) successively,
p-TSA (0.11 mg, 0.58 μm of ol), 4 molecular sieves of just having dried.React after 4 days, remove unreacted curcumin by after reacting liquid filtering with DMF dialysis (molecular cut off 3500).Dialyse after 24 hours in DMF, then be transferred in a water and dialyse 48 hours, dialysis terminates postlyophilization, finally obtains light yellow solid, productive rate about 70.9%.Nuclear-magnetism and fluorescence spectrum test result show, in this prodrug, the content of curcumin is about 39.5 wt.%.
embodiment six
Prodrugs of paclitaxel PEG
273-P (Asp
20-
g-PTX) preparation:
At N
2under protective condition, by Asp (OBzl)-NCA(0.3 g, 1.2 mmol) be dissolved in DMF(6 mL) in, by PEG
273-NH
2(0.6 g, 0.05 mmol) is dissolved in DMF(6 mL) in, then by PEG-NH
2/ DMF solution is added in Asp (OBzl)-NCA/DMF solution fast, reacts 72 h under airtight condition at 40 DEG C.Reacted rear excessive absolute ether to precipitate, core filters, and precipitation is dissolved with chloroform again, and again by excessive absolute ether precipitation, core filters, and dry 24 h of ambient temperature in vacuum, obtain target di-block copolymer PEG
273-PAsp (OBzl)
20.Productive rate is 90.3%.
At N
2under protective condition, by PEG
273-PAsp (OBzl)
20(0.5g, 0.031 mmol) is dissolved in tetrahydrofuran solution (5 mL), and then add 1 M NaOH solution (3.1 mL), stirred at ambient temperature reacts 3 days.Then proceed in bag filter (MWCO=3500) and dialyse 48 hours to a water, with 0.5 M HCl, the pH of solution is adjusted to acidity (pH 3 ~ 5), lyophilization obtains 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 10 mL dry DMF, join in 50 ml two neck round-bottomed flasks, then DMAP (34.2 mg, 0.28 mmol) is added, DCC (346.3 mg, 1.68 mmol), first at room temperature react, ethylene glycol mono vinyl ether (EGVE, 252 μ L will be added after activated carboxylic 12h again, 2.8 mmol), mixed solution continues lucifuge reaction 24h at 25 DEG C.After reaction terminates, solution is white casse shape, after filtering, filtrate is deposited in cold ether with core, filters and is placed in vacuum drying oven drying.Finally obtain the PEG that vinyl ether functionalized degree is 10
273-PAsp
20white solid powder, productive rate about 76.5%.
At N
2under environment, be the PEG of 10 by vinyl ether functionalized degree
273-PAsp
20(100.0 mg, equv. 70 μm of ol EGVE unit) are dissolved in 10 mL dry DMF, then add PTX (34.2 mg, 40 μm of ol) successively,
p-TSA (0.13 mg, 0.7 μm of ol), 4 molecular sieves of just having dried.React after 4 days, remove unreacted paclitaxel by after reacting liquid filtering with DMF dialysis (molecular cut off 3500).Dialyse after 24 hours in DMF, then be transferred in a water and dialyse 48 hours, dialysis terminates postlyophilization, finally obtains white solid, productive rate about 73.6%.Nuclear-magnetism and HPLC test result show, in this prodrug, the content of PTX is about 18.6 wt.%.
embodiment seven
Prodrugs of paclitaxel PEG
454-P (Glu
45-
g-PTX) preparation (PTX content: 13.3 wt.%)
At N
2under protective condition, by Glu (OBzl)-NCA(0.14 g, 0.53 mmol) be dissolved in DMF(5 mL) in, by PEG
454-NH
2(0.2 g, 0.01 mmol) is dissolved in DMF(3.5 mL) in, then by PEG
454-NH
2/ DMF solution is added in Asp (OBzl)-NCA/DMF solution fast, reacts 72 h under airtight condition at 40 DEG C.Reacted rear excessive absolute ether to precipitate, core filters, and precipitation is dissolved with chloroform again, and again by excessive absolute ether precipitation, core filters, and dry 24 h of ambient temperature in vacuum, obtain target di-block copolymer PEG
454-PGlu (OBzl)
45.Productive rate is 87.6%.
At N
2under protective condition, by PEG
454-PGlu (OBzl)
45(6.67 μm of ol) is dissolved in THF solution (5 mL), then adds 1 M NaOH solution (1.5 mL), room temperature for overnight.Then proceed in bag filter (MWCO=3500) and dialyse 48 hours to a water, with 0.5 M HCl, the pH of solution is adjusted to acidity (pH 3 ~ 5), lyophilization obtains 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 10 mL dry DMF, join in 50 ml two neck round-bottomed flasks, then add DMAP (15.3 mg, 0.125 mmol), DCC (154.6 mg, 0.75 mmol), first at room temperature reacts, ethylene glycol mono vinyl ether (EGVE will be added again after activated carboxylic 12h, 113 μ L, 1.25 mmol), mixed solution continues lucifuge reaction 24h at 25 DEG C.After reaction terminates, solution is white casse shape, after filtering, filtrate is deposited in cold ether with core, filters and is placed in vacuum drying oven drying.Finally obtain the PEG that vinyl ether functionalized degree is 30
454-PGlu
45white solid powder, productive rate about 75.2%.
At N
2under environment, be the PEG of 30 by vinyl ether functionalized degree
454-PGlu
45(equv. 53.8 μm of ol EGVE unit) are dissolved in 10 mL dry DMF, then add PTX (23 mg, 26.9 μm of ol) successively,
p-TSA (0.12 mg, 0.6 μm of ol), 4 molecular sieves of just having dried.React after 4 days, remove unreacted paclitaxel by after reacting liquid filtering with DMF dialysis (molecular cut off 3500).Dialyse after 24 hours in DMF, then be transferred in a water and dialyse 48 hours, dialysis terminates postlyophilization, finally obtains white solid, productive rate about 80.9%.Nuclear-magnetism and HPLC test result show, in this prodrug, the content of PTX is about 13.3 wt.%.
embodiment eight
Coupling has the prodrugs of paclitaxel FA-PEG of folic acid
136-P (AA
20-
g-PTX) synthesis of (content of taxol: 20.6 wt.%)
At N
2under protection, by initiator A IBN(3.12 mg, 0.019 mmol), Macro RAFT agent BOCH
2n-PEG
136-DMP(0.5 g, 0.083 mmol), the solvent Isosorbide-5-Nitrae-dioxane of acrylic acid (143 μ L, 2.075 mmol) and 6 mL joins in the sealed reactor of 25mL respectively, continues logical 30 minutes N
2, then reactor to be placed in the oil bath of 70 DEG C stirring reaction 48 hours.Precipitate in ice ether after reaction terminates, sample vacuum drying 24 hours, obtains target di-block copolymer BOCH
2n-PEG
136-PAA
20, productive rate is 87%.
At N
2under protection, by polymer B OCH
2n-PEG
136-PAA
20(0.4 g, 1.07 mmol COOH), are dissolved in 10 mL anhydrous 1, in 4-dioxane, join in 50 ml two neck round-bottomed flasks, then add DMAP (65.7 mg, 0.54 mmol), DCC (661.7 mg, 3.21 mmol), first at room temperature reacts, ethylene glycol mono vinyl ether (EGVE will be added again after activated carboxylic 12h, 481 μ L, 5.35 mmol), mixed solution continues lucifuge reaction 24h at 25 DEG C.After reaction terminates, solution is white casse shape, after filtering, filtrate is deposited in cold ether, filters and be placed on dry 24h in vacuum drying oven with core.Finally obtain the BOCH that vinyl ether functionalized degree is 12
2n-PEG
136-PAA
20white solid powder, productive rate about 86.4%.
Be the BOCH of 12 by vinyl ether functionalized degree
2n-PEG
136-PAA
20in (0.3 g) is dissolved in 2 mL trifluoroacetic acids and 2 mL HCl(1M), after stirred at ambient temperature 5 h, after its pH being adjusted to 5-6 by NaOH solution (1M), load bag filter (MWCO=3500) and dialyse 48 hours with a water, then the vinyl ether functionalized degree of cryodesiccated white solid is the H of 12
2n-PEG
136-PAA
20, productive rate is 92.6%.
First, folic acid (0.18 g, 0.4 mmol) in the dimethyl sulfoxine of 5 mL, DCC(0.25 g, 3.3 mmol) and NHS(0.07 g, 0.6 mmol) exist under, lucifuge activates 12 hours, is then the H of 12 by the vinyl ether functionalized degree that upper step obtains
2n-PEG
136-PAA
20(0.3 g, 0.04 mmol) is dissolved in 5 mL dimethyl sulfoxines, adds triethylamine (56 μ L, 0.4 mmol), reacts 24 hours at normal temperatures.Reaction terminates rear first mistake and filters by-product DCU, and then dialysing to dimethyl sulfoxine removes unreacted folic acid in 48 hours, and then dialyses 48 hours with water, 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 by the absworption peak of ultraviolet spectra at 363 nm places.HPLC test result shows, in this prodrug, the content of PTX is about 20.6 wt.%.
embodiment nine
Direct hydration method prepares PEG
113-P (AA
20-
g-PTX) (PTX content: 42.8 wt.%) prodrugs of paclitaxel nanoparticle
1 mg PTX prodrug is added and 15 μ L molecular weight are the Polyethylene Glycol of 1000 in 4 mL centrifuge tubes.By mixture agitating heating 10 minutes at 105 DEG C, then mixture is cooled to room temperature, adds the phosphate buffer solution (PB, 10 mM, pH 7.4) of 2 mL, ultrasonic 20min, the 12h that finally dialyses in PB buffer solution removes PEG1000.It is 248.3 nm that dynamic light scattering records nanoparticle mean diameter, and particle size distribution index is 0.30.
embodiment ten
Direct hydration method prepares PEG
113-P (AA
20-
g-PTX) (PTX content: 21.6 wt.%) prodrugs of paclitaxel nanoparticle
1 mg PTX prodrug is added and 15 μ L molecular weight are the Polyethylene Glycol of 350 in 4 mL centrifuge tubes.By mixture agitating heating 15 minutes at 95 DEG C, then mixture is cooled to room temperature, progressively adds 10,20, phosphate buffer solution (PB, 10 mM of 70,1900 μ L, pH 7.4), and after adding, ultrasonic 20min, the 12h that finally dialyses in PB buffer solution remove PEG550 at every turn.It is 158.3 nm that dynamic light scattering records nanoparticle mean diameter, and 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: 39.5 wt.%) curcumin prodrug nanoparticle
1 mg Cur prodrug is added and 15 μ L molecular weight are the Polyethylene Glycol of 550 in 4 mL centrifuge tubes.By mixture agitating heating 25 minutes at 85 DEG C, then mixture is cooled to room temperature, progressively adds 10,20, phosphate buffer solution (PB, 10 mM of 70,1900 μ L, pH 7.4), and after adding, ultrasonic 20min, the 12h that finally dialyses in PB buffer solution remove PEG550 at every turn.It is 230.3 nm that dynamic light scattering records nanoparticle mean diameter, and particle size distribution index is 0.24.
embodiment 12
Direct hydration method prepares PEG
113-P (AA
35-
g-MTO) (MTO content: 7.5 wt.%) mitoxantrone prodrug nanoparticle
1 mg MTO prodrug is added and 15 μ L molecular weight are the Polyethylene Glycol of 1500 in 4 mL centrifuge tubes.By mixture agitating heating 25 minutes at 95 DEG C, then mixture is cooled to room temperature, progressively adds 10,20, phosphate buffer solution (PB, 10 mM of 70,1900 μ L, pH 7.4), and after adding, ultrasonic 20min, the 12h that finally dialyses in PB buffer solution remove PEG550 at every turn.It is 62 nm that dynamic light scattering records nanoparticle mean diameter, and particle size distribution index is 0.11.
embodiment 13
PEG
113-P (AA
20-g-PTX) (content of taxol: 21.6 wt.%) prodrugs of paclitaxel nanoparticle under mildly acidic conditions particle diameter and light scatter intensity change mensuration
The concentration that we get that 1 mL prepares is the nanoparticle solution example of 0.5 mg/mL, its pH is adjusted to 5.0, is placed in 37 DEG C of shaking tables and rocks, and at the appointed time some dynamic light scattering (DLS) surveys the change of its particle diameter and light scatter intensity.
Accompanying drawing 3 is the particle diameter of above-mentioned nanoparticle and the change of light scatter intensity, can find out: under pH 5.0 condition, nanoparticle along with the prolongation of time can be very fast by 160 nm swells or assemble the particle (1700 nm) forming submicron and micron, particle size distribution has 0.15 to be increased to 0.45, its light scatter intensity is also 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
Extracorporeal releasing experiment HAc-NaAc(pH 5.0,10 mM at 37 DEG C of paclitaxel), 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 in the above-mentioned three kinds of buffer solution media of 25 mL respectively, puts into 37 DEG C of shaking tables, at each sampling time point, takes 8 mL release medium away and tests for HPLC, add the fresh dialysis medium that 8 mL are corresponding in addition.After the 8 mL samples taken out are concentrated into 0.5 mL by lyophilization, high performance liquid chromatography (HPLC, Agilent 1260, using the acetonitrile of volume ratio 1/1 and water as mobile phase) is utilized to measure the content of paclitaxel in the absorption intensity at 227 nm places.Each release experiment two groups is parallel, results averaged.
Accompanying drawing 4 is the cumulative release amount of PTX, can find out: for the release under physiological pH (7.4), and the rate of release from prodrugs of paclitaxel nanoparticle of PTX is in acid condition faster, and this is consistent with the result of acetal hydro; For PEG
113-P (AA
20-
g-PTX) (42.8 wt.%), in 48 hours, discharge PTX under pH 5.0 and pH 6.0 condition and be about 86.9% and 66.4% respectively, and discharge (<29%) with only having a small amount of drug slow under pH 7.4 condition.These results also show simultaneously, and PTX can avoid violent release phenomenon effectively by being chemically bonded to the prodrug nanoparticle that polymer chain is formed, and drug release also has good controllability.
embodiment 15
Polymer precursor (vinyl ether functionalized PEG
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) 37 DEG C, under 5% carbon dioxide conditions, cultivate in culture medium (DMEM), before culture medium uses, add the calf serum of 10%, 1% glutamate, Glu, antibiotics penicillin (100 IU/mL) and streptomycin (100 μ g/mL).Be layered on respectively by three kinds of cancerous cell on 96 porocyte culture plates, 5000 cells are approximately planted in each hole.24 hours later cell grow to the coverage rate of 70-80%, in the celliferous culture medium of 90 μ L, 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 bond hydrolysis the acetaldehyde (32.5 mM) of issuable maximum) continue cultivation adds 10 μ L 3-(4 after 48 hours, 5-dimethylthiazole-2) PBS solution (5 mg/mL) of-2,5-diphenyltetrazolium bromide bromine salt (MTT).Continued by culture plate to put into incubator culture fluid sucking-off after 4 hours, the purple crystal that living cells produces dissolves with 150 μ L DMSO.The optical density (OD) of sample measures at 492 nm places by microplate reader (Thermo Multiskan FC), and last the data obtained is the meansigma methods of four groups.Add in blank well with the PBS of sample same volume in contrast, be designated as 100% survival.
Accompanying drawing 5 is the relation of cell survival rate and polymer precursor concentration, can find out: polymer precursor (vinyl ether functionalized PEG-PAA) is even if when concentration reaches 1 mg/mL, also show substantially nontoxic when respective amount acetaldehyde exists, cell survival rate is all greater than 90%, illustrate that vinyl ether functionalized polymer precursor is nontoxic, and there is good biocompatibility.
embodiment 16
The active anticancer of prodrugs of paclitaxel nanoparticle measures
Cytotoxicity assay 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) 37 DEG C, under 5% carbon dioxide conditions, cultivate in culture medium (DMEM), be layered on respectively by three kinds of cancerous cell on 96 porocyte culture plates, 5000 cells are approximately planted in each hole.24 hours later cell grow to the coverage rate of 70-80%, and in the celliferous culture medium of 90 μ L, adding 10 μ L prodrugs of paclitaxel solution, (final PTX concentration is respectively 5 × 10
-5, 5 × 10
-4, 0.005,0.01,0.1,1,5,10 and 20 μ g/mL), PTX Cremophor EL and ethanol 1: 1(v/v freely) make Taxol
form as a control group, each concentration has four parallel multiple holes.Cell continues cultivation 48 hours, and processing method is same as described above.Add in blank well with the PBS of sample same volume in contrast, be designated as 100% survival.IC
50the value of (drug level that apoptosis one half is corresponding) is obtained by nonlinear regression (sigmoidal) analytical calculation.
Accompanying drawing 6,7 is the relation of cell survival rate and PTX concentration, can find out: prodrugs of paclitaxel nanoparticle shows the similar high drug effect with free PTX; It is the prodrugs of paclitaxel nanoparticle of 42.8 wt.% to PTX content, when in nanoparticle, PTX content is 1 μ g/mL, after itself and HeLa and KB cell cultivate 48 hours respectively, the survival rate of HeLa cell have decreased to 41.9%(Fig. 6), survival rate only remaining 22.7%(Fig. 7 of KB cell).The high Anti-tumor angiogenesis of PTX prodrug nanoparticle describes PTX and can be discharged in the nucleus of HeLa cell and KB cell by conveying effectively.
In addition, as accompanying drawing 8, MTT experiment shows the cancerous cell A549/PTX of this prodrugs of paclitaxel nanoparticle antagonism property of medicine
ralso stronger kill capability is had.Such as, acid-sensitive prodrugs of paclitaxel nanoparticle and A549/PTX
rafter hatching 48 hours, be 10 μ g/mL in PTX equivalent concentration, cell survival rate is only 50.3%, and its IC50 value is about 8.2 μ g/mL; And free paclitaxel concentration 20 μ g/mL, hatch 48 hours after, A549/PTX
rcell shows obvious Drug resistance, and cell survival rate still reaches more than 80%, and its IC50 value is 175.8 μ g/mL.And it should be noted that the free paclitaxel of same concentrations and normal A549 cell incubation are after 48 hours, its IC
50value is only 0.2 μ g/mL, and both differ by more than 800 times, and this can illustrate the A549/PTX that we use well
rcell maintains good drug resistance.Experiment shows that this prodrugs of paclitaxel nanoparticle connected by acetal bonds can overcome the drug resistance of tumor cell generation effectively, has good application prospect in field of cancer.
embodiment 17
PTX prodrug nanoparticle parcel amycin (DOX)
PTX prodrug (1 mg) is put into 4 mL centrifuge tubes, adds 15 μ L PEG550, then stir 25 minutes at 95 DEG C.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, stir 20 minutes at 60 DEG C.Add 20,70 and 1900 μ L PB buffer solution subsequently successively, and after adding ultrasonic 20 minutes at every turn.Finally by DOX and PEG550 that removing in 12 hours of dialysing in PB buffer solution does not have to wrap, the concentration of drug-carrying nanometer particle is 0.5 mg/mL.
The drug loading of DOX and envelop rate are by fluorescent spectrophotometer assay (FLS 920, excitation wavelength: 480 nm, emission wavelength: 560 nm).Concrete steps to add ultrasound destruction after 3 mL DMSO after 100 μ L medicine-carried nano particles solution lyophilizations, then to be calculated by the standard curve of DOX/DMSO.Drug loading (DLC) and envelop rate (DLE) are calculated by formula below:
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, in theoretical drug loading (i.e. DOXHCl/(polymer+DOXHCl) ratio) when being 5,10 and 20 wt. %, three kinds of PTX prodrug nanoparticles have higher envelop rate (DLE) to DOX, and we find that the envelop rate of DOX can reduce along with the increase of PTX content, it may be the cause because PTX content makes the core of nanoparticle comparatively loosen 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 25 DEG C, pH measured by DLS for 7.4 times.
embodiment 18
Be loaded with the extracorporeal releasing experiment of the PTX prodrug nanoparticle of DOX
While DOX and PTX extracorporeal releasing experiment at 37 DEG C, three kinds of different media: (i) hac buffer, pH 5.0; (ii) hac buffer, pH 6.0; (iii) phosphate buffer solution, measures in pH 7.4.The concentration of these three kinds of media is all 10 mM.The nanoparticle sample being loaded with DOX prepared is divided into three parts (every part of 0.5 mL), then transfers in bag filter (MWCO=12000 ~ 14000), bag filter is placed in the corresponding buffer medium of 25mL, then put into 37 DEG C of constant-temperature tables.The time point of specifying, takes out the release medium of 8 mL from delivery systme, then supplement the fresh medium of equal volume, wherein 4mL is by the content of fluoremetry DOX, other 4 mL measure the content of PTX by HPLC, and parallel two groups of this release experiment, result gets its meansigma methods.
Accompanying drawing 9 is the relation of release amount of medicine and time, can find out: for the release under physiological pH (7.4), the rate of release of DOX and PTX is in acid condition faster, and this is mainly due to the hydrolysis of acetal bonds on main polymer chain; For PEG-P (AA-
g-PTX) (21.6 wt.%), in 48 hours, pH 5.0 times, the DOX of nearly 82.8% and the PTX of 85.3% discharge from nanoparticle, and under the same conditions, pH only has discharging (<30%) of a small amount of drug slow for 7.4 times.
embodiment 19
Direct hydration method prepares the prodrugs of paclitaxel nanometer of surperficial coupling folic acid
0.8 mg prodrugs of paclitaxel PEG is added in 4 mL centrifuge tubes
113-P(AA
20-
g-PTX) (content of taxol: 42.8 wt.%), 0.2 mg coupling folic acid prodrugs of paclitaxel FA-PEG
136-P (AA
20-
g-PTX) and 15 μ L molecular weight be 550 Polyethylene Glycol (PEG550).By mixture agitating heating 25 minutes at 95 DEG C, then mixture is cooled to room temperature, adds 10 respectively, 20, phosphate buffer solution (PB, 10 mM of 70,1900 μ L, pH 7.4), and after adding, ultrasonic 20min, the 12h that finally dialyses in PB buffer solution remove PEG550 at every turn.It is 182.5 nm that dynamic light scattering records nanoparticle mean diameter, and particle size distribution index is 0.12.
0.8 mg prodrugs of paclitaxel PEG-P(AA-is added in 4 mL centrifuge tubes
g-PTX) and 0.2 mg coupling folic acid FA-PEG
136-PLA with 15 μ L molecular weight be 550 Polyethylene Glycol (PEG550).By mixture agitating heating 25 minutes at 95 DEG C, then mixture is cooled to room temperature, adds 10 respectively, 20, phosphate buffer solution (PB, 10 mM of 70,1900 μ L, pH 7.4), and after adding, ultrasonic 20min, the 12h that finally dialyses in PB buffer solution remove PEG550 at every turn.It is 188.4 nm that dynamic light scattering records nanoparticle mean diameter, and particle size distribution index is 0.19.
embodiment 20
The active anticancer of the prodrugs of paclitaxel nanoparticle of surface coupling folic acid measures
The cytotoxicity assay of the prodrugs of paclitaxel nanoparticle of surface coupling folic acid is with similar above.The people nasopharynx epidermal carcinoma cell (KB cell) of surface containing folacin receptor 37 DEG C, under 5% carbon dioxide conditions, cultivate in culture medium (DMEM), be layered on by cell on 96 porocyte culture plates, 5000 cells are approximately planted in each hole.24 hours later cell grow to the coverage rate of 70-80%, and in the celliferous culture medium of 90 μ L, adding the prodrugs of paclitaxel nanoparticle solution of the prodrugs of paclitaxel nanoparticle solution of 10 μ L surface coupling folic acid and surperficial unmodified folic acid, (final PTX concentration is respectively 5 × 10
-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 first cultivates 3 hours, then changes fresh culture medium, and continue to hatch 45 hours, processing method is same as described above.Add in blank well with the PBS of sample same volume in contrast, be designated as 100% survival.IC
50the value of (drug level that apoptosis one half is corresponding) is obtained by nonlinear regression (sigmoidal) analytical calculation.
Accompanying drawing 10 is the relation of cell survival rate and PTX concentration, can find out: surface has coupling to have the prodrugs of paclitaxel nanoparticle of folic acid to show higher cytotoxicity; Effects on surface has coupling to have the prodrugs of paclitaxel nanoparticle of folic acid, and when in nanoparticle, PTX content is 10 μ g/mL, the survival rate of cell is 34.9%, its IC
50value is 1.42 μ g/mL; And the prodrugs of paclitaxel nanoparticle of effects on surface unmodified folic acid, its cell survival rate is 52.6%, IC
50value is 17.16 μ g/mL.Surface has coupling to have the high Anti-tumor angiogenesis of the PTX prodrug nanoparticle of folic acid to describe nanoparticle can more effectively to enter cell and discharge antiradiation drug after targeted molecular in modification, improve the kill capability of nanoparticles on tumor cells.
embodiment 21
Surface coupling has release experiment in the endocytosis being loaded with the PTX nanoparticle of DOX of folic acid and cell
Surface coupling has the PTX nanoparticle being loaded with DOX of folic acid at KB(-FA) endocytosis in cell and release behavior observe sign by confocal laser microscope (CLSM).KB(-FA) cell is at 37 DEG C, and under 5% carbon dioxide conditions, containing 10% serum, do not containing in the RPMI-1640 of folic acid and cultivate, cell density is 5 × 10
4individual/hole.After 24 hours, adding 50 μ L surface couplings has the PTX nanoparticle solution being loaded with DOX of folic acid, the PTX nanoparticle solution being loaded with DOX of the non-coupling folic acid in surface or DOXHCl solution freely, and the concentration of final DOX all remains on 5 μ g/mL.Respectively with cell incubation 3,6 and 12 hours, wherein, the sample of 6 hours and 12 hours needs after 3 hours to change fresh culture adding sample solution, then continues to cultivate 3 hours and 9 hours respectively.After cultivation terminates, sop up the culture medium of cultivating in plate hole, the cell phosphate buffer solution in culture plate washs three times, then fixes 20min with the paraformaldehyde of 4%, and nucleus DAPI dyes.The CLSM photo of cell measures (TCS SP2) by confocal laser scanning microscope, CLSM.
Surface coupling has the PTX nanoparticle being loaded with DOX and the KB(-FA of folic acid) cell is after 6 hours hatch, we just can see the red fluorescence having obvious DOX around nucleus, illustrate that nanoparticle can be ingested very soon and effectively discharge fast in cell.After hatching 12 hours, in nucleus, occurred stronger DOX red fluorescence, and similar with the fluorescence of free DOX, and after MCF-7 cell incubation 12 hours, still only there is cell peripheral in the fluorescence of DOX.This has absolutely proved, surface coupling have the PTX nanoparticle being loaded with DOX of folic acid due to can with the folacin receptor specific binding of KB (-FA) cell surface, by this receptor-mediated effect, nanoparticle can enter cell more quickly and effectively, and discharges medicine rapidly.
Claims (3)
1. an acid-sensitive polymeric prodrugs, it is characterized in that: in described acid-sensitive polymeric prodrugs, polymer precursor is the water solublity A-B type bi-block copolymer of vinyl ether functional, the wherein Polyethylene Glycol of A block to be molecular weight be 3000 ~ 30000 Da, B block is polyacrylic acid, polymethylacrylic acid, poly-ethylacrylic acid, poly-aspartate or polyglutamic acid; Medicine is paclitaxel, mitoxantrone, curcumin or astaxanthin;
Described drug molecule is linked by acetal bonds and A-B type bi-block copolymer;
In described water solublity A-B type bi-block copolymer, in B block, the number of repeat unit of monomer is 10 ~ 100;
In described acid-sensitive polymeric prodrugs, the percent grafting of medicine is 0.1 ~ 0.6; A block forms hydrophilic segment, and form hydrophobic part after B block grafting medicine, hydrophilic segment molecular weight is 0.5 ~ 8 times of hydrophobic part molecular weight.
2. acid-sensitive polymeric prodrugs according to claim 1, is characterized in that: medicine is paclitaxel.
3. the prodrug nanoparticle prepared by any one acid-sensitive polymeric prodrugs in claim 1 ~ 2, is characterized in that: the shell of described prodrug nanoparticle is made up of block A, and core is made up of medicine; The particle diameter of described nanoparticle is 50 ~ 250 nm.
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