CN103083682B - Folic acid modified chitosan quaternary ammonium salt-taxol polymer medicine, as well as preparation method and application thereof - Google Patents
Folic acid modified chitosan quaternary ammonium salt-taxol polymer medicine, as well as preparation method and application thereof Download PDFInfo
- Publication number
- CN103083682B CN103083682B CN201310029010.3A CN201310029010A CN103083682B CN 103083682 B CN103083682 B CN 103083682B CN 201310029010 A CN201310029010 A CN 201310029010A CN 103083682 B CN103083682 B CN 103083682B
- Authority
- CN
- China
- Prior art keywords
- quaternary ammonium
- ammonium salt
- paclitaxel
- chitosan quaternary
- tmc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention belongs to the technical field of medicines and specifically provides a folic acid modified chitosan quaternary ammonium salt-taxol polymer medicine, as well as a preparation method and an application of the polymer medicine. The polymer medicine provided by the invention is formed by connecting taxol to chitosan quaternary ammonium salt in the form of covalent linkage by succinyl ester and connecting tumor target ligand folic acid. The preparation method comprises the following steps of: connecting succinic anhydride open loop with taxol to synthesize succinyl taxol, modifying chitosan by quaternization to obtain chitosan quaternary ammonium salt; and under the action of a catalyst, connecting succinyl taxol to chitosan quaternary ammonium salt in the form of covalent linkage, and introducing target ligand folic acid. The polymer medicine can be assembled automatically in water to form nanoparticles, wherein hydrophobic medicine is used as the inner core and chitosan quaternary ammonium salt is used as a hydrophilic shell. The polymer medicine is simple in preparation method, excellent in repeatability and security, and can enhance the targeting and tumor inhibiting effect of taxol by two administration routes consisting of oral taking and intravenous injection.
Description
Technical field
The invention belongs to medical technical field, be specifically related to chitosan quaternary ammonium salt-paclitaxel polymeric medicine of a kind of modified with folic acid and its preparation method and application.
Background technology
Anti-tumor medicine thing because of non-specific distribution in poorly water-soluble, body, toxic and side effects larger etc., cannot give full play to its therapeutic efficiency.Paclitaxel, as First-line chemotherapy medicine, can promote microtubule to assemble and stop tubulin depolymerization, thus quick division and the propagation of effective anticancer.But paclitaxel is insoluble in water, the paclitaxel injection of clinical use is using polyoxyethylene castor oil/ethanol (1:1, v/v) as solvent, can cause allergic reaction, the toxic and side effects such as Cardiovascular Toxicity after intravenous injection.Adopt drug delivery system can overcome above-mentioned defect, the polymeric medicine that wherein water-soluble polymer carrier and paclitaxel chemistry are connected to form can improve taxol solubility, controls drug release, improves tumor-targeting, alleviates toxic and side effects.Paclitaxel can directly be connected with polymer support, but may be because of excessively slow (the Shahin M et al. of factor drug release rate such as sterically hindered, Int J Pharm 2010,389:213-222), after paclitaxel first can being connected with the micromolecule such as dicarboxylic acids or aminoacid, be indirectly connected to polymer support, connection can improve the reactivity of medicine indirectly, increases drug hydrolysis site.
The polymeric medicine based on paclitaxel of research is many through intravenous administration at present, and after intravenous injection, medicine can directly enter blood circulation; In the long therapeutic procedure of cancer, oral convenient with respect to intravenous injection, can improve patient compliance.The small intestinal poor permeability of paclitaxel, is vulnerable to the drug efflux of enterocyte surface P-glycoprotein mediation and the drug metabolism that Cytochrome P450 relies on, and oral administration biaavailability is very low.Can improve the oral absorption of medicine by delivery vector, wherein chitosan is with good physicochemical property and be easy to carry out structure and functionalized modification and be widely used in the preparation of oral delivery vehicle, but chitosan poorly water-soluble under physiological environment, application is restricted.Chitosan can be low-molecular weight chitoglycan (Mw<10 kDa) through hydrolysis or enzymolysis, water solublity strengthens, by paclitaxel through succinyl ester bond and the covalently bound formation low-molecular weight chitoglycan-paclitaxel of low-molecular weight chitoglycan polymeric medicine, can improve paclitaxel dissolubility, improve paclitaxel oral administration biaavailability (Lee E et al., J Med Chem 2008,51:6442-6449), in the body of oral low-molecular weight chitoglycan-paclitaxel, tumour inhibiting rate is suitable with intravenous injection paclitaxel solution.But it is generally acknowledged that molecular weight is greater than the molecule of 50 kDa or the particle suitable with it (for example nanoparticle) has obvious enhancing infiltration to be detained (EPR) effect, can passive target tumor tissues.The molecular weight of low-molecular weight chitoglycan-paclitaxel polymeric medicine and can not self assembly form nanoparticle, cannot utilize EPR effect to improve antitumor efficacy.
Utilize receptor-specific recognition reaction on part and cell membrane, drug delivery vehicle is carried out to the ligand modified picked-up of cell to drug delivery vehicle, the enhancing active targeting effect of increasing of targeting.Intestinal epithelial cell surface folacin receptor high expressed, the nanoparticle of modified with folic acid can significantly improve the small intestinal turn-over capacity (Roger E et al., Mol Pharmaceutics 2012,9:2103-2110) of medicine; Folacin receptor is significantly higher than normal cell at kinds of tumor cells (as ovarian cancer, hepatocarcinoma, breast carcinoma etc.) surperficial high expressed and activity, the nanoparticle of modified with folic acid can be accumulated at tumor tissues camber, there is good tumor cell targeting (Wang J et al., Biomacromolecules 2011,12:228-234).
Summary of the invention
The object of the invention is to overcome the defect of prior art, provide a kind of and there is self assembly performance, can be used for chitosan quaternary ammonium salt-paclitaxel polymeric medicine of the modified with folic acid of multiple route of administration and its preparation method and application.
Basic scheme of the present invention is as follows:
Succinic anhydrides, through pyridine catalysis open loop, is connected ambroin acyl paclitaxel with paclitaxel; Chitosan and iodomethane reaction synthesize chitosan quaternary ammonium salt; Chitosan quaternary ammonium salt and succinyl paclitaxel be through catalyzing and condensing, and targeting part folic acid is connected to chitosan quaternary ammonium salt, makes chitosan quaternary ammonium salt-paclitaxel polymeric medicine of modified with folic acid.This polymeric medicine, because containing hydrophilic polymer chain and hydrophobic drug, can self assembly form nanoparticle in water.The present invention is according to effects such as hydrophilic, mucosa adhesion and the cell adhesions of chitosan quaternary ammonium salt, and paclitaxel is covalently bound to chitosan quaternary ammonium salt by succinyl ester bond, improves medicine water solublity, controls drug release, strengthens medicine tumor-targeting; The folic acid of active targeting according to to(for) kinds of tumor cells, to chitosan quaternary ammonium salt, realizes the initiatively targeting of tumor tissues of polymeric medicine by covalently bound folic acid, reduces the toxic and side effects of medicine normal tissue.Modified with folic acid chitosan quaternary ammonium salt-paclitaxel polymeric medicine prepared by the present invention can improve by the multiple route of administration such as oral and intravenous injection the antitumor efficacy of medicine.
Particularly, chitosan quaternary ammonium salt-paclitaxel polymeric medicine of modified with folic acid provided by the invention, that antitumor drug paclitaxel is covalently bound to chitosan quaternary ammonium salt through succinyl ester bond, targeting part folic acid is connected to the polymeric medicine of chitosan quaternary ammonium salt formation, this polymeric medicine can self assembly form nanoparticle in water again.Wherein chitosan molecule weight range is 10-500 kDa, and paclitaxel carried medicine amount is 3-25%.
Chitosan quaternary ammonium salt-paclitaxel polymeric medicine of described modified with folic acid, the nanoparticle mean diameter that self assembly forms is 100-500 nm.
The preparation method of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of modified with folic acid of the present invention is as follows:
(1) succinic anhydrides is suspended in dichloromethane, adds paclitaxel, stirs, and adds anhydrous pyridine, stirring reaction 24-72 h; Distilling under reduced pressure, adds water washing, centrifugal collecting precipitation, and vacuum drying, obtains succinyl paclitaxel;
(2) according to the synthetic chitosan quaternary ammonium salt of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83);
(3) succinyl paclitaxel is dissolved in dimethyl sulfoxide, adds catalyst, stirs; Chitosan quaternary ammonium salt is water-soluble, add in the dimethyl sulfoxide that contains succinyl paclitaxel reaction 6-24 h;
(4) folic acid is dissolved in to dimethyl sulfoxide, adds catalyst, stir, add in the reaction system that step (3) obtains, lucifuge reaction 6-24 h; Purification, lyophilization, obtains chitosan quaternary ammonium salt-paclitaxel polymeric medicine of modified with folic acid.
In step (1), the mass ratio of succinic anhydrides and paclitaxel is 1:1-1:10, the 0.25-2.0%(v/v that pyridine consumption is reaction system).
In step (2), the synthetic chitosan quaternary ammonium salt response time is 45-150 min; Gained chitosan quaternary ammonium salt is with Cl
-type anion exchange resin purification, lyophilization; The quaternized degree that wherein reacts the chitosan quaternary ammonium salt of 45,120 and 150 min gained is respectively 15%, 30% and 45%.
In step (3), reaction used catalyst is EDC hydrochlorate and N-hydroxy-succinamide, and the mol ratio of succinyl paclitaxel and EDC hydrochlorate and N-hydroxy-succinamide is 1:1-1:10, the mass ratio of succinyl paclitaxel and chitosan quaternary ammonium salt is 0.05:1-0.4:1, and chitosan quaternary ammonium salt concentration of aqueous solution is 10-30 mg/ml.
In step (4), the mass ratio of folic acid and chitosan quaternary ammonium salt is 0.05:1-0.3:1, reaction used catalyst is EDC hydrochlorate and N-hydroxy-succinamide, and the mol ratio of folic acid and EDC hydrochlorate and N-hydroxy-succinamide is 1:1-1:10.
Chitosan quaternary ammonium salt-paclitaxel polymeric medicine of modified with folic acid of the present invention, can be used for by oral and two kinds of route of administration of vein the treatment of the kinds cancers such as hepatocarcinoma.
The invention has the advantages that:
(1) according to advantages such as the good water solublity of chitosan quaternary ammonium salt, adhesion and promotion infiltrations, by covalently bound to chitosan quaternary ammonium salt paclitaxel connecting key of cleavable under physiological condition, and targeting part folic acid is connected to chitosan quaternary ammonium salt, prepare a kind of novel folic acid beautify chitosan quaternary ammonium salt-paclitaxel polymeric medicine.Modified with folic acid chitosan quaternary ammonium salt-paclitaxel polymeric medicine of the present invention can strengthen the little Intestinal permeability of medicine, not only can be enriched in tumor tissues by EPR effect, also can be by identification, the combination of targeting part folic acid and tumor cell surface specific receptor, increasing tumor tissues Chinese medicine distributes, improve the antitumor efficacy of medicine, reduce the toxicity to other internal organs.
(2) in chitosan quaternary ammonium salt-paclitaxel polymeric medicine water of modified with folic acid of the present invention, self assembly forms nanoparticle, and particle size distribution is even, and dispersibility is better, and particle diameter is stable under different pH environment.
(3) chitosan quaternary ammonium salt-paclitaxel polymeric medicine of modified with folic acid of the present invention slowly release in vivo, can prevent that medicine from arriving the toxicity of the front normal tissue of target spot; Under acid condition, polymeric medicine is more stable, and drug release rate increases with the increase of release medium pH, can prevent that medicine from losing at stomach, is conducive to the intestinal absorption of polymeric medicine.
(4) the small intestinal mucosa adherence rate of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of modified with folic acid of the present invention, higher than chitosan quaternary ammonium salt, can significantly improve by opening the tight connection of small intestine epithelium and epithelial cell picked-up the small intestinal transport efficacy of paclitaxel.
(5) chitosan quaternary ammonium salt-paclitaxel polymeric medicine of modified with folic acid of the present invention can be used for by oral and two kinds of route of administration of intravenous injection the treatment of the kinds cancers such as hepatocarcinoma; In the research of H22 liver cancer model mice tumor killing effect, oral and tumour inhibiting rate intravenous injection modified with folic acid chitosan quaternary ammonium salt-paclitaxel polymeric medicine group is all significantly higher than intravenous injection paclitaxel solution group.
(6) connection paclitaxel and folic acid can reduce the positive charge of chitosan quaternary ammonium salt, strengthen blood compatibility.
(7) the various raw materials of the present invention are cheap and easy to get, and the method for introducing targeting group is simple and convenient, and connecting key is firm, and synthesis technique is simple, and condition is controlled, reproducible.
Term used herein:
As used herein, term " PTX " refers to paclitaxel.
As used herein, term " TMC " refers to N, N, N-N-trimethyl chitosan TMC quaternary ammonium salt.
As used herein, term " EDC " refers to EDC hydrochlorate.
As used herein, term " NHS " refers to N-hydroxy-succinamide.
As used herein, term " FA " refers to folic acid.
As used herein, term " TMC-PTX " refers to chitosan quaternary ammonium salt-paclitaxel polymeric medicine.
As used herein, term " FA-TMC-PTX " refers to modified with folic acid chitosan quaternary ammonium salt-paclitaxel polymeric medicine.
Brief description of the drawings
Fig. 1 is the scanning electron microscope (SEM) photograph (embodiment 13) of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of modified with folic acid.Scale 2 μ m in figure.
Fig. 2 is chitosan quaternary ammonium salt-paclitaxel polymeric medicine (embodiment 12,13,14) of modified with folic acid particle diameter under different pH environment.
Fig. 3 is chitosan quaternary ammonium salt-paclitaxel polymeric medicine (embodiment 13) release conditions (A) in 10 days in pH 7.4 PBS of modified with folic acid; Release conditions (B) in different pH release medium, wherein, Ph value corresponding to curve is from the bottom up followed successively by: 1.2,4.5,5.0,5.3,5.7,6.0,6.5,7.0,7.4.
Detailed description of the invention
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only, for the present invention is described, limit the scope of the invention and be not used in.The experimental technique of unreceipted actual conditions in lower routine embodiment, conventionally according to normal condition, or the condition of advising according to manufacturer.Unless otherwise indicated, concentration is quality percentage by volume (w/v).
The preparation of embodiment 1 succinyl paclitaxel
Take 3 mg succinic anhydrides, add 4 mL dichloromethane, stir; Add 30 mg PTX, stir; Add 10 μ L anhydrous pyridines, reaction system becomes clarification, stirs 24 h; 50 ° of C distilling under reduced pressure, add 10 mL water, stir 15 min, centrifugal collecting precipitation; Add 30 mL water washing precipitations, centrifugal collecting precipitation, vacuum drying.
The preparation of embodiment 2 succinyl paclitaxels
Take 6 mg succinic anhydrides, add 4 mL dichloromethane, stir; Add 30 mg PTX, stir; Add 20 μ L anhydrous pyridines, reaction system becomes clarification, stirs 40 h; 50 ° of C distilling under reduced pressure, add 10 mL water, stir 15 min, centrifugal collecting precipitation; Add 30 mL water washing precipitations, centrifugal collecting precipitation, vacuum drying.
The preparation of embodiment 3 succinyl paclitaxels
Take 10 mg succinic anhydrides, add 4 mL dichloromethane, stir; Add 30 mg PTX, stir; Add 30 μ L anhydrous pyridines, reaction system becomes clarification, stirs 55 h; 50 ° of C distilling under reduced pressure, add 10 mL water, stir 15 min, centrifugal collecting precipitation; Add 30 mL water washing precipitations, centrifugal collecting precipitation, vacuum drying.
The preparation of embodiment 4 succinyl paclitaxels
Take 20 mg succinic anhydrides, add 4 mL dichloromethane, stir; Add 30 mg PTX, stir; Add 50 μ L anhydrous pyridines, reaction system becomes clarification, stirs 65 h; 50 ° of C distilling under reduced pressure, add 10 mL water, stir 15 min, centrifugal collecting precipitation; Add 30 mL water washing precipitations, centrifugal collecting precipitation, vacuum drying.
The preparation of embodiment 5 succinyl paclitaxels
Take 30 mg succinic anhydrides, add 4 mL dichloromethane, stir; Add 30 mg PTX, stir; Add 80 μ L anhydrous pyridines, reaction system becomes clarification, stirs 72 h; 50 ° of C distilling under reduced pressure, add 10 mL water, stir 15 min, centrifugal collecting precipitation; Add 30 mL water washing precipitations, centrifugal collecting precipitation, vacuum drying.
The preparation of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 6 modified with folic acid
According to the synthetic TMC of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83), chitosan weight average molecular weight is 10 kDa, and the response time is 45 min, and the quaternized degree of gained TMC is 15%; Cl
-type anion exchange resin purification, lyophilization.Take 200 mg TMC and be dissolved in 20 mL water; Take 40 mg succinyl paclitaxels (embodiment 3) and be dissolved in 40 mL dimethyl sulfoxide, add NHS and EDC, the mol ratio of succinyl paclitaxel, NHS and EDC is 1:1:1, stirs; Add TMC solution, reaction 6 h prepare TMC-PTX; Take 22 mg FA and be dissolved in dimethyl sulfoxide, add NHS and EDC, FA, NHS and EDC mol ratio are 1:4:4, stir, and add in TMC-PTX reaction system, and lucifuge is reacted 12 h; With 3 days (MWCO 3,500 Da) of water dialysis, lyophilization.
The preparation of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 7 modified with folic acid
According to the synthetic TMC of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83), chitosan weight average molecular weight is 30 kDa, and the response time is 120 min, and the quaternized degree of gained TMC is 30%; Cl
-type anion exchange resin purification, lyophilization.Take 200 mg TMC and be dissolved in 20 mL water; Take 40 mg succinyl paclitaxels (embodiment 3) and be dissolved in 40 mL dimethyl sulfoxide, add NHS and EDC, the mol ratio of succinyl paclitaxel, NHS and EDC is 1:3:3, stirs; Add TMC solution, reaction 12 h prepare TMC-PTX; Take 22 mg FA and be dissolved in dimethyl sulfoxide, add NHS and EDC, the mol ratio of FA, NHS and EDC is 1:4:4, stirs, and adds in TMC-PTX reaction system, and lucifuge is reacted 12 h; With 3 days (MWCO 3,500 Da) of water dialysis, lyophilization.
The preparation of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 8 modified with folic acid
According to the synthetic TMC of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83), chitosan weight average molecular weight is 100 kDa, and the response time is 150 min, and the quaternized degree of gained TMC is 45%; Cl
-type anion exchange resin purification, lyophilization.Take 200 mg TMC and be dissolved in 20 mL water; Take 40 mg succinyl paclitaxels (embodiment 3) and be dissolved in 40 mL dimethyl sulfoxide, add NHS and EDC, the mol ratio of succinyl paclitaxel, NHS and EDC is 1:5:5, stirs; Add TMC solution, reaction 24 h prepare TMC-PTX; Take 22 mg FA and be dissolved in dimethyl sulfoxide, add NHS and EDC, the mol ratio of FA, NHS and EDC is 1:4:4, stirs, and adds in TMC-PTX reaction system lucifuge stirring reaction 12 h; With 3 days (MWCO 3,500 Da) of water dialysis, lyophilization.
The preparation of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 9 modified with folic acid
According to the synthetic TMC of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83), chitosan weight average molecular weight is 200 kDa, and the response time is 120 min, and the quaternized degree of gained TMC is 30%; Cl
-type anion exchange resin purification, lyophilization.Take 200 mg TMC and be dissolved in 20 mL water; Take 40 mg succinyl paclitaxels (embodiment 3) and be dissolved in 40 mL dimethyl sulfoxide, add NHS and EDC, the mol ratio of succinyl paclitaxel, NHS and EDC is 1:7:7, stirs; Add TMC solution, reaction 12 h prepare TMC-PTX; Take 22 mg FA and be dissolved in dimethyl sulfoxide, add NHS and EDC, the mol ratio of FA, NHS and EDC is 1:4:4, stirs, and adds in TMC-PTX reaction system, and lucifuge is reacted 12 h; With 3 days (MWCO 3,500 Da) of water dialysis, lyophilization.
The preparation of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 10 modified with folic acid
According to the synthetic TMC of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83), chitosan weight average molecular weight is 500 kDa, and the response time is 120 min, and the quaternized degree of gained TMC is 30%; Cl
-type anion exchange resin purification, lyophilization.Take 200 mg TMC and be dissolved in 20 mL water; Take 40 mg succinyl paclitaxels (embodiment 3) and be dissolved in 40 mL dimethyl sulfoxide, add NHS and EDC, the mol ratio of succinyl paclitaxel, NHS and EDC is 1:10:10, stirs; Add TMC solution, reaction 12 h prepare TMC-PTX; Take 22 mg FA and be dissolved in dimethyl sulfoxide, add NHS and EDC, the mol ratio of FA, NHS and EDC is 1:4:4, stirs, and adds in TMC-PTX reaction system, and lucifuge is reacted 12 h; With 3 days (MWCO 3,500 Da) of water dialysis, lyophilization.
The preparation of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 11 modified with folic acid
According to the synthetic TMC of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83), chitosan weight average molecular weight is 100 kDa, and the response time is 120 min, and the quaternized degree of gained TMC is 30%; Cl
-type anion exchange resin purification, lyophilization.Take 200 mg TMC and be dissolved in 20 mL water; Take 10 mg succinyl paclitaxels (embodiment 3) and be dissolved in 40 mL dimethyl sulfoxide, add NHS and EDC, the mol ratio of succinyl paclitaxel, NHS and EDC is 1:5:5, stirs; Add TMC solution, reaction 12 h prepare TMC-PTX; Take 22 mg FA and be dissolved in dimethyl sulfoxide, add NHS and EDC, the mol ratio of FA, NHS and EDC is 1:4:4, stirs, and adds in TMC-PTX reaction system, and lucifuge is reacted 12 h; With 3 days (MWCO 3,500 Da) of water dialysis, lyophilization.
The preparation of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 12 modified with folic acid
According to the synthetic TMC of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83), chitosan weight average molecular weight is 100 kDa, and the response time is 120 min, and the quaternized degree of gained TMC is 30%; Cl
-type anion exchange resin purification, lyophilization.Take 200 mg TMC and be dissolved in 20 mL water; Take 20 mg succinyl paclitaxels (embodiment 3) and be dissolved in 40 mL dimethyl sulfoxide, add NHS and EDC, the mol ratio of succinyl paclitaxel, NHS and EDC is 1:5:5, stirs; Add TMC solution, reaction 12 h prepare TMC-PTX; Take 22 mg FA and be dissolved in dimethyl sulfoxide, add NHS and EDC, the mol ratio of FA, NHS and EDC is 1:4:4, stirs, and adds in TMC-PTX reaction system, and lucifuge is reacted 12 h; With 3 days (MWCO 3,500 Da) of water dialysis, lyophilization.
The preparation of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 13 modified with folic acid
According to the synthetic TMC of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83), chitosan weight average molecular weight is 100 kDa, and the response time is 120 min, and the quaternized degree of gained TMC is 30%; Cl
-type anion exchange resin purification, lyophilization.Take 200 mg TMC and be dissolved in 20 mL water; Take 40 mg succinyl paclitaxels (embodiment 3) and be dissolved in 40 mL dimethyl sulfoxide, add NHS and EDC, the mol ratio of succinyl paclitaxel, NHS and EDC is 1:5:5, stirs; Add TMC solution, reaction 12 h prepare TMC-PTX; Take 22 mg FA and be dissolved in dimethyl sulfoxide, add NHS and EDC, the mol ratio of FA, NHS and EDC is 1:4:4, stirs, and adds in TMC-PTX reaction system, and lucifuge is reacted 12 h; With 3 days (MWCO 3,500 Da) of water dialysis, lyophilization.
The preparation of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 14 modified with folic acid
According to the synthetic TMC of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83), chitosan weight average molecular weight is 100 kDa, and the response time is 120 min, and the quaternized degree of gained TMC is 30%; Cl
-type anion exchange resin purification, lyophilization.Take 200 mg TMC and be dissolved in 20 mL water; Take 60 mg succinyl paclitaxels (embodiment 3) and be dissolved in 40 mL dimethyl sulfoxide, add NHS and EDC, the mol ratio of succinyl paclitaxel, NHS and EDC is 1:5:5, stirs; Add TMC solution, reaction 12 h prepare TMC-PTX; Take 22 mg FA and be dissolved in dimethyl sulfoxide, add NHS and EDC, the mol ratio of FA, NHS and EDC is 1:4:4, stirs, and adds in TMC-PTX reaction system, and lucifuge is reacted 12 h; With 3 days (MWCO 3,500 Da) of water dialysis, lyophilization.
The preparation of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 15 modified with folic acid
According to the synthetic TMC of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83), chitosan weight average molecular weight is 100 kDa, and the response time is 120 min, and the quaternized degree of gained TMC is 30%; Cl
-type anion exchange resin purification, lyophilization.Take 200 mg TMC and be dissolved in 20 mL water; Take 80 mg succinyl paclitaxels (embodiment 3) and be dissolved in 40 mL dimethyl sulfoxide, add NHS and EDC, the mol ratio of succinyl paclitaxel, NHS and EDC is 1:5:5, stirs; Add TMC solution, reaction 12 h prepare TMC-PTX; Take 22 mg FA and be dissolved in dimethyl sulfoxide, add NHS and EDC, the mol ratio of FA, NHS and EDC is 1:4:4, stirs, and adds in TMC-PTX reaction system, and lucifuge is reacted 12 h; With 3 days (MWCO 3,500 Da) of water dialysis, lyophilization.
The preparation of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 16 modified with folic acid
According to the synthetic TMC of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83), chitosan weight average molecular weight is 10 kDa, and the response time is 120 min, and the quaternized degree of gained TMC is 30%; Cl
-type anion exchange resin purification, lyophilization.Take 200 mg TMC and be dissolved in 20 mL water; Take 40 mg succinyl paclitaxels (embodiment 3) and be dissolved in 10 mL dimethyl sulfoxide, add NHS and EDC, the mol ratio of succinyl paclitaxel, NHS and EDC is 1:5:5, stirs; Add TMC solution, reaction 12 h prepare TMC-PTX; Take 22 mg FA and be dissolved in dimethyl sulfoxide, add NHS and EDC, the mol ratio of FA, NHS and EDC is 1:4:4, stirs, and adds in TMC-PTX reaction system, and lucifuge is reacted 12 h; With 3 days (MWCO 3,500 Da) of water dialysis, lyophilization.
The preparation of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 17 modified with folic acid
According to the synthetic TMC of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83), chitosan weight average molecular weight is 30 kDa, and the response time is 120 min, and the quaternized degree of gained TMC is 30%; Cl
-type anion exchange resin purification, lyophilization.Take 200 mg TMC and be dissolved in 10 mL water; Take 40 mg succinyl paclitaxels (embodiment 3) and be dissolved in 10 mL dimethyl sulfoxide, add NHS and EDC, the mol ratio of succinyl paclitaxel, NHS and EDC is 1:5:5, stirs; Add TMC solution, reaction 12 h prepare TMC-PTX; Take 22 mg FA and be dissolved in dimethyl sulfoxide, add NHS and EDC, the mol ratio of FA, NHS and EDC is 1:4:4, stirs, and adds in TMC-PTX reaction system, and lucifuge is reacted 12 h; With 3 days (MWCO 3,500 Da) of water dialysis, lyophilization.
The preparation of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 18 modified with folic acid
According to the synthetic TMC of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83), chitosan weight average molecular weight is 100 kDa, and the response time is 120 min, and the quaternized degree of gained TMC is 30%; Cl
-type anion exchange resin purification, lyophilization.Take 200 mg TMC and be dissolved in 6.7 mL water; Take 40 mg succinyl paclitaxels (embodiment 3) and be dissolved in 13.3 mL dimethyl sulfoxide, add NHS and EDC, the mol ratio of succinyl paclitaxel, NHS and EDC is 1:5:5, stirs; Add TMC solution, reaction 12 h prepare TMC-PTX; Take 22 mg FA and be dissolved in dimethyl sulfoxide, add NHS and EDC, the mol ratio of FA, NHS and EDC is 1:4:4, stirs, and adds in TMC-PTX reaction system, and lucifuge is reacted 12 h; With 3 days (MWCO 3,500 Da) of water dialysis, lyophilization.
The preparation of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 19 modified with folic acid
According to the synthetic TMC of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83), chitosan weight average molecular weight is 200 kDa, and the response time is 120 min, and the quaternized degree of gained TMC is 30%; Cl
-type anion exchange resin purification, lyophilization.Take 200 mg TMC and be dissolved in 20 mL water; Take 40 mg succinyl paclitaxels (embodiment 3) and be dissolved in 60 mL dimethyl sulfoxide, add NHS and EDC, the mol ratio of succinyl paclitaxel, NHS and EDC is 1:5:5, stirs; Add TMC solution, reaction 12 h prepare TMC-PTX; Take 22 mg FA and be dissolved in dimethyl sulfoxide, add NHS and EDC, the mol ratio of FA, NHS and EDC is 1:4:4, stirs, and adds in TMC-PTX reaction system, and lucifuge is reacted 12 h; With 3 days (MWCO 3,500 Da) of water dialysis, lyophilization.
The preparation of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 20 modified with folic acid
According to the synthetic TMC of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83), chitosan weight average molecular weight is 500 kDa, and the response time is 120 min, and the quaternized degree of gained TMC is 30%; Cl
-type anion exchange resin purification, lyophilization.Take 200 mg TMC and be dissolved in 20 mL water; Take 40 mg succinyl paclitaxels (embodiment 3) and be dissolved in 80 mL dimethyl sulfoxide, add NHS and EDC, the mol ratio of succinyl paclitaxel, NHS and EDC is 1:5:5, stirs; Add TMC solution, reaction 12 h prepare TMC-PTX; Take 22 mg FA and be dissolved in dimethyl sulfoxide, add NHS and EDC, the mol ratio of FA, NHS and EDC is 1:4:4, stirs, and adds in TMC-PTX reaction system, and lucifuge is reacted 12 h; With 3 days (MWCO 3,500 Da) of water dialysis, lyophilization.
The preparation of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 21 modified with folic acid
According to the synthetic TMC of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83), chitosan weight average molecular weight is 100 kDa, and the response time is 120 min, and the quaternized degree of gained TMC is 30%; Cl
-type anion exchange resin purification, lyophilization.Take 200 mg TMC and be dissolved in 20 mL water; Take 40 mg succinyl paclitaxels (embodiment 3) and be dissolved in 40 mL dimethyl sulfoxide, add NHS and EDC, the mol ratio of succinyl paclitaxel, NHS and EDC is 1:5:5, stirs; Add TMC solution, reaction 12 h prepare TMC-PTX; Take 12 mg FA and be dissolved in dimethyl sulfoxide, add NHS and EDC, the mol ratio of FA, NHS and EDC is 1:1:1, stirs, and adds in TMC-PTX reaction system, and lucifuge is reacted 6 h; With 3 days (MWCO 3,500 Da) of water dialysis, lyophilization.
The preparation of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 22 modified with folic acid
According to the synthetic TMC of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83), chitosan weight average molecular weight is 100 kDa, and the response time is 120 min, and the quaternized degree of gained TMC is 30%; Cl
-type anion exchange resin purification, lyophilization.Take 200 mg TMC and be dissolved in 20 mL water; Take 40 mg succinyl paclitaxels (embodiment 3) and be dissolved in 40 mL dimethyl sulfoxide, add NHS and EDC, the mol ratio of succinyl paclitaxel, NHS and EDC is 1:5:5, stirs; Add TMC solution, reaction 12 h prepare TMC-PTX; Take 22 mg FA and be dissolved in dimethyl sulfoxide, add NHS and EDC, the mol ratio of FA, NHS and EDC is 1:2:2, stirs, and adds in TMC-PTX reaction system, and lucifuge is reacted 12 h; With 3 days (MWCO 3,500 Da) of water dialysis, lyophilization.
The preparation of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 23 modified with folic acid
According to the synthetic TMC of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83), chitosan weight average molecular weight is 100 kDa, and the response time is 120 min, and the quaternized degree of gained TMC is 30%; Cl
-type anion exchange resin purification, lyophilization.Take 200 mg TMC and be dissolved in 20 mL water; Take 40 mg succinyl paclitaxels (embodiment 3) and be dissolved in 40 mL dimethyl sulfoxide, add NHS and EDC, the mol ratio of succinyl paclitaxel, NHS and EDC is 1:5:5, stirs; Add TMC solution, reaction 12 h prepare TMC-PTX; Take 40 mg FA and be dissolved in dimethyl sulfoxide, add NHS and EDC, the mol ratio of FA, NHS and EDC is 1:4:4, stirs, and adds in TMC-PTX reaction system, and lucifuge is reacted 24 h; With 3 days (MWCO 3,500 Da) of water dialysis, lyophilization.
The preparation of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 24 modified with folic acid
According to the synthetic TMC of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83), chitosan weight average molecular weight is 100 kDa, and the response time is 120 min, and the quaternized degree of gained TMC is 30%; Cl
-type anion exchange resin purification, lyophilization.Take 200 mg TMC and be dissolved in 20 mL water; Take 40 mg succinyl paclitaxels (embodiment 3) and be dissolved in 40 mL dimethyl sulfoxide, add NHS and EDC, the mol ratio of succinyl paclitaxel, NHS and EDC is 1:5:5, stirs; Add TMC solution, reaction 12 h prepare TMC-PTX; Take 50 mg FA and be dissolved in dimethyl sulfoxide, add NHS and EDC, the mol ratio of FA, NHS and EDC is 1:6:6, stirs, and adds in TMC-PTX reaction system, and lucifuge is reacted 12 h; With 3 days (MWCO 3,500 Da) of water dialysis, lyophilization.
The preparation of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 25 modified with folic acid
According to the synthetic TMC of bibliographical information method (Eur J Pharm Biopharm 2004,57:77-83), chitosan weight average molecular weight is 100 kDa, and the response time is 120 min, and the quaternized degree of gained TMC is 30%; Cl
-type anion exchange resin purification, lyophilization.Take 200 mg TMC and be dissolved in 20 mL water; Take 40 mg succinyl paclitaxels (embodiment 3) and be dissolved in 40 mL dimethyl sulfoxide, add NHS and EDC, the mol ratio of succinyl paclitaxel, NHS and EDC is 1:5:5, stirs; Add TMC solution, reaction 12 h prepare TMC-PTX; Take 60 mg FA and be dissolved in dimethyl sulfoxide, add NHS and EDC, the mol ratio of FA, NHS and EDC is 1:10:10, stirs, and adds in TMC-PTX reaction system, and lucifuge is reacted 12 h; With 3 days (MWCO 3,500 Da) of water dialysis, lyophilization.
Embodiment 26 scanning electron microscopies characterize the form of modified with folic acid chitosan quaternary ammonium salt-paclitaxel polymeric medicine
After the FA-TMC-PTX of embodiment 13 is disperseed by suitable quantity of water, drop on mica sheet, natural drying at room temperature, sticks in Tong Tai, metal spraying, and scanning electron microscope (SEM) is observed its pattern, sees accompanying drawing 1.FA-TMC-PTX can self assembly form nanoparticle as seen from the figure, nanoparticle almost spherical, and smooth surface, particle diameter is 100-200 nm, particle size distribution is even and dispersibility is better.
Particle diameter, the electromotive force of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 27 modified with folic acid
The FA-TMC-PTX of embodiment 12,13 and 14 is disperseed by suitable quantity of water, and Zetasizer Nano electromotive force and particle size analyzer are measured particle diameter and Zeta electric potential.Particle size determination parameter: He-Ne laser (wavelength 635 nm), index of refraction and viscosity are respectively n=1.333 and η=0.933 cp, measure 25 ° of C of temperature.Potential determination parameter: He-Ne laser (wavelength 635 nm), scatteringangleθ=14 °, measure 25 ° of C of temperature.Every duplicate samples parallel assay 3 times.Measurement result is in table 1.As shown in table 1, the nanoparticle particle diameter that in water, FA-TMC-PTX forms first reduces rear increase with the increase of succinyl paclitaxel inventory, and nanoparticle electromotive force is all greater than 30 mV, shows that nanoparticle is more stable.
The particle diameter of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of modified with folic acid under the different pH environment of embodiment 28
The FA-TMC-PTX of embodiment 12,13 and 14 is scattered in respectively in pH 1.2 HCl, pH 5.0 PBS, pH 6.8 PBS and pH 7.4 PBS, and particle size analyzer is measured particle diameter.Particle size determination parameter: He-Ne laser (wavelength 635 nm), index of refraction and viscosity are respectively n=1.333 and η=0.933 cp, measure 25 ° of C of temperature.Every duplicate samples parallel assay 3 times.Measurement result is shown in accompanying drawing 2.By nanoparticle particle diameter that under the visible pH 1.2-7.4 environment of accompanying drawing 2, FA-TMC-PTX forms without significant change; After FA-TMC-PTX is oral, enter blood the final tumor tissues that arrives through gastrointestinal tract, experienced different pH environment, and FA-TMC-PTX can keep stable nano shape under different pH environment.
The drug loading of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 29 modified with folic acid
Accurate preparation 1 mg/mL FA-TMC-PTX(embodiment 12,13,14), precision measures 200 μ L, is diluted with water to 2 mL, measures 227 nm place light absorption values, calculates the drug loading (%) of polymeric medicine.As shown in table 1, the drug loading of FA-TMC-PTX increases with the increase of succinyl paclitaxel inventory.
Particle diameter, electromotive force and the drug loading (n=3) of table 1 modified with folic acid chitosan quaternary ammonium salt-paclitaxel polymeric medicine
| Sample | Particle diameter (nm) | Polydispersity | Electromotive force (mV) | Drug loading (%) |
| FA-TMC-PTX (embodiment 12) | 311.1 ± 19.6 | 0.360 ± 0.066 | 33.2 ± 0.7 | 5.0 ± 0.3 |
| FA-TMC-PTX (embodiment 13) | 186.9 ± 21.5 | 0.290 ± 0.022 | 30.7 ± 1.3 | 10.5 ± 0.3 |
| FA-TMC-PTX (embodiment 14) | 344.7 ± 9.1 | 0.187 ± 0.031 | 32.3 ± 1.3 | 17.2 ± 0.3 |
The release in vitro of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 30 modified with folic acid
Take appropriate FA-TMC-PTX(embodiment 13) be dispersed in pH 7.4 PBS, measure 4 mL, put in MWCO 3,500 Da bag filters, bag filter is put 100 mL containing 0.1%(w/v) and in the pH 7.4 PBS solution of Tween 80,37 ° of C, 100 rpm vibration incubations; Sample 0.5 mL respectively at 1,2,3,4,6,8,12,24 h and 2,3,4,5,6,7,8,9,10 d, add 0.5 mL release medium; Centrifugal 10 min of sample 12,000 rpm, supernatant is with 0.22 μ m membrane filtration, and HPLC measures PTX concentration.
Prepare 1 mg/mL FA-TMC-PTX(embodiment 13), measure 100 μ L, add respectively containing 0.1%(w/v) pH 4.5,5.0,5.3,5.7,6.0,6.5,7.0,7.4 PBS and pH 1.2 HCl solution dilution to 1 mL of Tween 80,37 ° of C, 100 rpm vibration incubations, respectively at 1,2,4,6,8,24,48 h 12, centrifugal 10 min of 000 rpm, supernatant is with 0.22 μ m membrane filtration, and HPLC measures PTX concentration.
HPLC chromatographic condition: chromatographic column: (5 μ m) for the Dalian Chemistry and Physics Institute of the Chinese Academy of Sciences, 200 × 4.6 mm for Hypersil ODS2 post; Mobile phase: acetonitrile: water (52:48, v/v); Flow velocity: 1 mL/min; Detect wavelength: 227 nm; Sample size: 20 μ L.
Calculate PTX amount in polymeric medicine by embodiment 29, be calculated as follows cumulative release percentage rate:
Cumulative release percentage rate (%)=(C
i× V/PTX amount) × 100
Wherein C
ifor PTX concentration in sample point release medium (μ g/mL), V is release medium volume.
As shown in accompanying drawing 3A, FA-TMC-PTX(embodiment 13 in pH 7.4 PBS) without obviously dashing forward and release, sustainable slow Slow release reaches 10 d, can prevent that medicine from arriving the toxicity of the front normal tissue of target spot, slows down the clearance rate of medicine.As shown in accompanying drawing 3B, FA-TMC-PTX(embodiment 13) more stable under acid condition, release amount of medicine increases with release medium pH, can prevent that medicine from losing at stomach, is conducive to the intestinal absorption of polymeric medicine; In addition, tumor cell can absorb nano grade polymer medicine through endocytosis, after entering born of the same parents, enter in endosome or the interior cell of lysosome born of the same parents (pH 4-6), under weak acid environment, slowly discharge PTX, polymeric medicine is escaped endosome or lysosome enters Cytoplasm (pH 7.2), rate of releasing drug increases, and is conducive to microtubule effect performance effect in PTX and its target spot kytoplasm.
Sticking at body small intestinal of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 31 modified with folic acid
Take 3 mg RhB, add 1 mL pH 4.5 HCl solution and dissolve, add 1.5 mg NHS and 3 mg EDC, lucifuge stirs 1 h; Take 0.3 g TMC and be dissolved in and add 9 mL water, add in RhB system, lucifuge stirs 24 h; Reactant liquor is with 3 days (MWCO 3,500 Da) of water dialysis, and lyophilization obtains the TMC of RhB labelling.TMC with RhB labelling substitutes TMC, prepares the FA-TMC-PTX of RhB labelling by embodiment 13.
SD rat, fasting 12 hours, can freely drink water, weigh, by 350 mg/kg lumbar injection chloral hydrate solution (3.5%, w/v) anesthetized rat, open abdominal cavity along ventrimeson, get approximately 10 cm ileum intestinal segments, normal saline flushing is to clean, two ends become sealing intestinal ring with surgical thread ligation, inject 1 TMC of mL RhB labelling or the FA-TMC-PTX(1 mg/mL of RhB labelling in intestinal ring); Intestinal segment is put back to abdominal cavity, after 2 h, put to death rat, cut off intestinal ring and collect intestinal juice, add normal saline flushing intestinal segment, merge intestinal juice and flushing liquor, the TMC of microplate reader mensuration RhB labelling and the FA-TMC-PTX content of RhB labelling (
λ ex=560 nm,
λ em=590 nm).Calculate small intestinal adhesive force (%) by following formula:
Small intestinal adhesive force (%)=(A-B) × 100/A
Wherein A is the theoretical content of the FA-TMC-PTX of RhB labelling in injection liquid; B is the content of collecting the FA-TMC-PTX of RhB labelling in intestinal juice.
Visible, after 2 h, have 89.0 ± 2.9% FA-TMC-PTX(embodiment 13) be adhered to small intestinal, be significantly higher than TMC(48.8 ± 1.0%).Compared with TMC, FA-TMC-PTX positive charge weakens, and Interchain interaction reduces, and pliability increases, and hydrophobicity strengthens, and can be combined through hydrophobic interaction power with the mucoprotein and intestinal epithelial cell of mucosa, and small intestinal adhesive force improves.
The small intestinal of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 32 modified with folic acid is transported in vitro
PTX solution preparation: with polyoxyethylene castor oil/dehydrated alcohol (1:1, v/v) preparation 1 mg/mL PTX stock solution, be diluted to 100 μ g/mL with Kreb ' s-Ringer buffer when use.The preparation of PTX/TMC mixed liquor: preparation 10 mg/mL TMC solution, when use, PTX stock solution, TMC solution and Kreb ' s-Ringer buffer are pressed to 1:1:8(v/v/v) mix.
SD rat, fasting 12 hours, can freely drink water.Cervical vertebra dislocation is put to death, open abdominal cavity along ventrimeson and take out ileum, clean with Kreb ' s-Ringer buffer solution for cleaning, be cut into approximately 7 cm long, approximately 1 cm place, two ends forms sealing intestinal ring with surgical thread ligation, injects respectively 0.5 mL PTX solution, PTX/TMC mixed liquor, FA-TMC-PTX(embodiment 13 in intestinal ring) (concentration is counted 100 μ g/mL by PTX).Intestinal ring is put in tall regular weighing bottle, adds Kreb ' the s-Ringer buffer that 37 ° of C oxygen of 5 mL are saturated, makes intestinal environmental protection hold extended state, 37 ° of C, 100 rpm vibration incubations.Sample 200 μ L respectively at 30,60,90,120,180 min, add 37 ° of C Kreb ' s-Ringer buffer 200 μ L.Centrifugal 15 min of sample 12,000 rpm, supernatant is with 0.22 μ m membrane filtration, PTX content in HPLC working sample, be calculated as follows PTX apparent infiltration coefficient (
p app).
Wherein A is rat small intestine in vitro intestinal segment surface area, C
0for medicine initial concentration in intestinal ring.
PTX solution, PTX/TMC mixed liquor and FA-TMC-PTX(embodiment 13 in rat small intestine)
p appbe respectively (1.76 ± 0.41) × 10
-7cm/s, (4.74 ± 0.55) × 10
-7cm/s and (3.30 ± 0.25) × 10
-6cm/s, shows that FA-TMC-PTX can significantly promote the small intestinal transhipment of PTX.PTX/TMC mixed liquor
p appfor 2.7 times of PTX solution, but significantly lower than FA-TMC-PTX, show that FA-TMC-PTX absorbs the small intestinal transhipment amount that increases PTX by epithelial cell.
Chitosan quaternary ammonium salt-paclitaxel polymeric medicine tumor killing effect in the body of H-22 liver cancer model mice of embodiment 33 modified with folic acid
PTX solution preparation: with polyoxyethylene castor oil/dehydrated alcohol (1:1, v/v) preparation 10 mg/mL PTX solution, add 10 times of normal saline dilutions when use.
Female kunming mice (18-22g) forelimb oxter inoculation H-22 hepatic ascites, treats that tumor grows to 100 mm
3, random packet, 6 every group.Every group of tumor-bearing mice be oral (p.o.) or the following sample of intravenous injection (i.v.) respectively: normal saline (p.o.), normal saline (i.v.), PTX solution (i.v.), FA-TMC-PTX(embodiment 13) (p.o.) with FA-TMC-PTX(embodiment 13) (i.v.).Dosage is 10 mg/kg by paclitaxel.Be administered once every other day, successive administration five times.So that administration time is as the 0th d for the first time, to the 16th day, execution mice, took out tumor and also weighs, and calculates tumor killing effect, in table 2.
As shown in table 2, oral and intravenous injection FA-TMC-PTX(embodiment 13) remarkable to the anti-tumor in vivo effect of H-22 liver cancer model mice, tumour inhibiting rate is respectively 69.4% and 87.5%, is significantly higher than intravenous injection PTX solution (tumour inhibiting rate 34.52%); Intravenous injection FA-TMC-PTX tumour inhibiting rate is significantly higher than oral FA-TMC-PTX.
Table 2 polymeric medicine and paclitaxel solution are in the anti-tumor in vivo effect of H22 liver cancer model mice
*:
p<0.05 vs PTX solution (i.v.) group
#:
p<0.05 vs FA-TMC-PTX(p.o.) group.
The hemolysis rate of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 34 modified with folic acid
Get healthy kunming mice whole blood 3 mL, put in the centrifuge tube of heparin sodium anticoagulant, centrifugal 10 min of 3,000 rpm, abandon supernatant, and normal saline Washed Red Blood Cells are precipitated to supernatant redfree, and normal saline is mixed with 2%(v/v) red cell suspension.Measure 0.5 mL red cell suspension, add respectively TMC and the FA-TMC-PTX(embodiment 13 of 0.5 mL normal saline preparation) (0.5,1,2 mg/mL), respectively with 0.5 mL water and the positive contrast of normal saline and negative control, 37 ° of C, 100 rpm vibration incubation 1 h, centrifugal 10 min of 3,000 rpm, get supernatant, measure the light absorption value at 540 nm places, be calculated as follows hemolysis rate (%):
The hemolysis rate of TMC shows concentration dependent, and the hemolysis rate of 0.5,1,2 mg/mL TMC is respectively 1.21 ± 0.23%, 4.98 ± 0.36% and 11.22 ± 0.73%, and 2 mg/mL TMC hemolysis rates are greater than 5%; And the hemolysis rate of 0.5,1,2 mg/mL FA-TMC-PTX is respectively 1.48 ± 0.19%, 3.61 ± 0.39% and 4.00 ± 0.26%, all lower than 5%, blood compatibility property improvement.
The tissue toxicity of chitosan quaternary ammonium salt-paclitaxel polymeric medicine of embodiment 35 LDH test determination modified with folic acid
SD rat, fasting 12 h, can freely drink water, weigh, by 350 mg/kg lumbar injection chloral hydrate solution (3.5%, w/v) anesthetized rats, open abdominal cavity along ventrimeson, take out approximately 10 cm ileum intestinal segments, two ends become sealing intestinal ring with surgical thread ligation, inject the FA-TMC-PTX(embodiment 13 of 1 mL normal saline preparation in intestinal ring) (1 mg/mL); The negative contrast of normal saline, the positive contrast of 10 mg/mL NaTDC.Intestinal segment is put back to abdominal cavity, after 2 h, put to death rat, cut off intestinal ring along ligature, collect intestinal juice, centrifugal 10 min of 12,000 rpm, measure supernatant 20 μ L, LDH kit measurement LDH content (U/mL).
Negative control, positive control and FA-TMC-PTX(embodiment 13) group LDH burst size be respectively 2.55 ± 0.62 U/mL, 7.14 ± 0.05 U/mL, 3.69 ± 0.35 U/mL, FA-TMC-PTX group LDH burst size there was no significant difference compared with normal saline group, but significantly lower than positive controls, show that polymeric medicine tissue toxicity is low, safety is good.
Claims (9)
1. chitosan quaternary ammonium salt-paclitaxel polymeric medicine of a modified with folic acid, it is characterized in that being prepared by following process: chitosan makes hydrophilic chitosan quaternary ammonium salt through quaternized modification, succinic anhydrides open loop is connected and makes hydrophobicity succinyl paclitaxel with paclitaxel chemistry, chitosan quaternary ammonium salt and succinyl paclitaxel are covalently bound, the covalently bound folic acid of chitosan quaternary ammonium salt; Wherein the mass ratio of succinyl paclitaxel and chitosan quaternary ammonium salt is 0.05:1-0.4:1, and the mass ratio of folic acid and chitosan quaternary ammonium salt is 0.05:1-0.3:1.
2. chitosan quaternary ammonium salt-paclitaxel polymeric medicine of modified with folic acid according to claim 1, is characterized in that chitosan molecule weight range is 10-500 kDa.
3. chitosan quaternary ammonium salt-paclitaxel polymeric medicine of modified with folic acid according to claim 1, is characterized in that paclitaxel carried medicine amount is 3-25%.
4. according to chitosan quaternary ammonium salt-paclitaxel polymeric medicine of the modified with folic acid one of claim 1-3 Suo Shu, it is characterized in that the nanoparticle that formed by this medicine self assembly, particle diameter is 100-500 nm.
5. a preparation method for the modified with folic acid chitosan quaternary ammonium salt-paclitaxel polymeric medicine as described in one of claim 1-4, is characterized in that concrete steps are as follows:
(1) succinic anhydrides is suspended in dichloromethane, adds paclitaxel, stirs, and adds anhydrous pyridine, stirring reaction 24-72 hour; Distilling under reduced pressure, adds water washing, centrifugal collecting precipitation, and vacuum drying, obtains succinyl paclitaxel;
(2) synthetic chitosan quaternary ammonium salt;
(3) succinyl paclitaxel is dissolved in dimethyl sulfoxide, adds catalyst, stirs; Chitosan quaternary ammonium salt is water-soluble, adds in the dimethyl sulfoxide that contains succinyl paclitaxel reaction 6-24 hour;
(4) folic acid is dissolved in dimethyl sulfoxide, adds catalyst, stirs, and adds in the reaction system of (3) lucifuge reaction 6-24 hour; Purification, lyophilization, obtains modified with folic acid chitosan quaternary ammonium salt-paclitaxel polymeric medicine.
6. preparation method according to claim 5, is characterized in that the mass ratio of the middle succinic anhydrides of step (1) and paclitaxel is 1:1-1:10, the 0.25-2.0% that pyridine consumption is reaction system, v/v.
7. preparation method according to claim 5, is characterized in that in step (2) that the response time is 45-150 min; Gained chitosan quaternary ammonium salt is with Cl
-type anion exchange resin purification, lyophilization; The quaternized degree that wherein reacts the chitosan quaternary ammonium salt of 45,120 and 150 min gained is respectively 15%, 30% and 45%.
8. preparation method according to claim 5, the mass ratio that it is characterized in that the middle succinyl paclitaxel of step (3) and chitosan quaternary ammonium salt is 0.05:1-0.4:1, chitosan quaternary ammonium salt concentration of aqueous solution is 10-30 mg/mL, reaction used catalyst is EDC hydrochlorate and N-hydroxy-succinamide, and the mol ratio of succinyl paclitaxel and EDC hydrochlorate and N-hydroxy-succinamide is 1:1-1:10.
9. preparation method according to claim 5, the mass ratio that it is characterized in that step (4) Folic Acid and chitosan quaternary ammonium salt is 0.05:1-0.3:1, reaction used catalyst is EDC hydrochlorate and N-hydroxy-succinamide, and the mol ratio of folic acid and EDC hydrochlorate and N-hydroxy-succinamide is 1:1-1:10.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310029010.3A CN103083682B (en) | 2013-01-25 | 2013-01-25 | Folic acid modified chitosan quaternary ammonium salt-taxol polymer medicine, as well as preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310029010.3A CN103083682B (en) | 2013-01-25 | 2013-01-25 | Folic acid modified chitosan quaternary ammonium salt-taxol polymer medicine, as well as preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103083682A CN103083682A (en) | 2013-05-08 |
| CN103083682B true CN103083682B (en) | 2014-10-29 |
Family
ID=48197153
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310029010.3A Expired - Fee Related CN103083682B (en) | 2013-01-25 | 2013-01-25 | Folic acid modified chitosan quaternary ammonium salt-taxol polymer medicine, as well as preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103083682B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104274830B (en) * | 2013-07-04 | 2016-08-17 | 复旦大学 | A kind of nasal cavity immunity carrier based on antigen covalent bond chitosan nano |
| CN104784153B (en) * | 2015-04-29 | 2019-08-13 | 苏州大学 | A kind of sulphonic acid betaine-chitosan nano particle of modified with folic acid and its preparation method and application |
| CN107158404A (en) * | 2017-05-26 | 2017-09-15 | 中国医学科学院生物医学工程研究所 | It is a kind of applied to Liver targeting pH sensitivity nanoparticles delivery systems of chemotherapy of hepatocellular carcinoma administering drug combinations and preparation method thereof |
| CN111603445B (en) * | 2020-04-17 | 2021-02-19 | 华南农业大学 | Nano emulsion drug carrier and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101780042A (en) * | 2010-03-26 | 2010-07-21 | 天津大学 | Taxol nano targeting slow-release long-circulating liposome and preparation method thereof |
-
2013
- 2013-01-25 CN CN201310029010.3A patent/CN103083682B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101780042A (en) * | 2010-03-26 | 2010-07-21 | 天津大学 | Taxol nano targeting slow-release long-circulating liposome and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103083682A (en) | 2013-05-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhong et al. | Hyaluronic acid-shelled acid-activatable paclitaxel prodrug micelles effectively target and treat CD44-overexpressing human breast tumor xenografts in vivo | |
| Chiang et al. | Reactive oxygen species and glutathione dual redox-responsive micelles for selective cytotoxicity of cancer | |
| Li et al. | Redox-sensitive micelles self-assembled from amphiphilic hyaluronic acid-deoxycholic acid conjugates for targeted intracellular delivery of paclitaxel | |
| Li et al. | Supramolecular PEGylated dendritic systems as pH/redox dual-responsive theranostic nanoplatforms for platinum drug delivery and NIR imaging | |
| Guo et al. | pH-triggered intracellular release from actively targeting polymer micelles | |
| Zhang et al. | Tumor-targeting micelles based on folic acid and α-tocopherol succinate conjugated hyaluronic acid for paclitaxel delivery | |
| KR101444274B1 (en) | Block copolymer for drug complex and pharmaceutical composition | |
| CN102060991B (en) | Amphiphilic prodrug of 7- ethyl-10-hydroxycamptothecin and preparation method thereof | |
| CN101791411A (en) | Preparation and application of amphiphilic polysaccharide conjugate and medicinal compositions thereof | |
| WO2009152691A1 (en) | A polyglycol modified chitosan oligosaccharide fatty acid graft, preparation method thereof and use of the same | |
| CN110229323B (en) | Reduction-sensitive reversible-crosslinked polymersome with asymmetric membrane structure and application thereof in preparation of liver cancer treatment drugs | |
| CN101254309A (en) | Folacin receptor mediated targeted acetyl pullulan polysaccharide nano granule and preparation thereof | |
| Yin et al. | Redox-sensitive hyaluronic acid–paclitaxel conjugate micelles with high physical drug loading for efficient tumor therapy | |
| Liu et al. | Bio-responsive Bletilla striata polysaccharide-based micelles for enhancing intracellular docetaxel delivery | |
| Zhou et al. | Engineered borate ester conjugated protein-polymer nanoconjugates for pH-responsive drug delivery | |
| CN103656650B (en) | A kind of brain tumor twin-stage targeted nano delivery system of pH sensitivity and preparation method and application | |
| Jaiswal et al. | Chitosan modified by organo-functionalities as an efficient nanoplatform for anti-cancer drug delivery process | |
| CN104971353B (en) | The preparation and application of the amphiphilic polysaccharide derivative carrier and its pharmaceutical compositions of target tumor new vessels | |
| CN103242517A (en) | Preparation of multifunctional linear-dendritic segmented copolymer and application in pharmaceutics thereof | |
| CN109010846A (en) | Polyethylene glycol-chitosan-curcumin polymer and its medicine-carried nano particles and preparation method | |
| KR20110074583A (en) | Method of preparing polyglutamate conjugates | |
| CN109966507A (en) | PH and the macromolecular nano-prodrug of redox double-response of a kind of cancer target and the preparation method and application thereof | |
| ES2547326T3 (en) | Cellulose-based nanoparticles for drug delivery | |
| Han et al. | Free paclitaxel-loaded E-selectin binding peptide modified micelle self-assembled from hyaluronic acid-paclitaxel conjugate inhibit breast cancer metastasis in a murine model | |
| CN103083682B (en) | Folic acid modified chitosan quaternary ammonium salt-taxol polymer medicine, as well as preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20141029 Termination date: 20180125 |