CN102357079A - Carboxymethyl chitosan nanoparticles modified with glycyrrhizic acid, preparation method and application thereof - Google Patents
Carboxymethyl chitosan nanoparticles modified with glycyrrhizic acid, preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of nano materials, and in particular relates to carboxymethyl chitosan nanoparticles modified with glycyrrhizic acid, as well as a preparation method and application thereof. The nanoparticles disclosed by the invention are in a core-shell type; a hydrophobic polymer serves as a kernel packaged medicament; and carboxymethyl chitosan serves as a hydrophilic shell and is covalently linked with hepatic targeting ligand glycyrrhizic acid. The preparation method comprises the following steps of: firstly grating and copolymerizing carboxymethyl chitosan and hydrophobic monomers and self-assembling to form nanoparticles in the action of an initiator; secondly, oxidizing glycyrrhizic acid to obtain a glycyrrhizic acid aldehyde solution, mixing the glycyrrhizic acid aldehyde solution with the nanoparticles at a certain proportion and introducing targeting groups, and carrying out ultrasonic loading on the medicament. Compared with the prior art, the preparation method is simple and easy to control and has good repeatability and better stability and safety; and the targeting property and the tumor-inhabiting effect of the anti-tumor medicament can be strengthened remarkably. The nanoparticles serve as a targeting delivery vector for hydrophobic medicaments or molecular probes and can be used for treatment and diagnosis of liver diseases.
Description
Technical field
The invention belongs to targeted nanometer material technology field, be specifically related to a kind of liver tumor targeting property glycyrrhizic acid and modify nanometer carboxymethyl chitosan particle.
Background technology
Hepatic disease such as hepatocarcinoma, hepatitis is the commonly encountered diseases and the frequently-occurring disease of serious threat human health, and the medicine of regular dosage form is behind vein, oral or local injection, and drug distribution is in whole body, and toxic and side effects is bigger.Targeting drug delivery system can be delivered to drug selectivity the diseased region of liver through carrier material, reduces the toxic and side effects to normal structure, reduces dosage and administration number of times, improves drug effect.Nanoparticle is by particle diameter 10～1000 nm solid colloid particles natural or that synthesized polymer material is processed; Its dissolubility that can improve medicine as novel drug administration carrier with stability, change in the medicine body distribute, prolong drug action time, and have good slow controlled-release effect.The active targeting of medicine can be realized through the modification of ligands specific in the nanoparticle surface.
Parenchyma is the key component and the metabolism place of liver, and most pathological changes of liver all betide parenchyma.Glycyrrhizic acid is the active component that is present in licorice, stem.Mammal hepatic parenchymal cells film surface has been proved and has had glycyrrhizic acid specific receptor (Negishi M et al. Biochim Biophys Acta 1991; 1066 (1): 77-82); And (the Ilicheva TN et al.Vopr Onko 1998,44 (4): 390-394) of overexpression in tumor tissues.Existing both at home and abroad research shows, can accumulate at the liver camber with liposome or nanoparticle that glycyrrhizic acid is modified, and have good hepatic parenchymal cells targeting property (Osaka S et al. Biol Pharm Bull 1994,17 (7): 940-943; Lin AH et al. Int J Pharmaceut 2008,359 (1-2): 247-253).
Chitosan is the alkaline polysaccharide of the unique existence of nature, have excellent biological compatibility and non-toxic degradable, but chitosan is only soluble in acid solution, and physiological condition is unstable down, is prone to aggregate and precipitate, and the soluble derivative of chitosan can effectively improve character in its body.Chitosan and derivant thereof have been widely used in fields such as biological medicine, food, cosmetics, can be used as the composition material of nano-medicament carrier.Chinese patent CN1760223 prepares mucoadhesive property nanoparticle with water soluble derivatives of chitosan, surperficial adsorbable hydrophilic medicament.Glycyrrhizic acid is less as the research of the ligand modified chitosan nano carrier of targeting; Chinese patent CN101006983A discloses the method for preparing of a kind of glycyrrhetate beautify chitosan/using carboxyl chitosan composite drug-loaded nanoparticles; Form nanoparticle through electrostatic interaction, only be suitable for water soluble medicament-entrapping; But only prove this nanoparticle targeting to hepatic parenchymal cells, do not show that it has initiatively targeting property to HCC; And compare with chitosan, the cellular uptake of glycyrrhetate beautify chitosan/using carboxyl chitosan composite nano-granule only improves about 2 times, and the active targeting of performance glycyrrhizic acid not yet in effect.
Summary of the invention
The objective of the invention is to overcome the defective of prior art, provide a kind of stability and good reproducibility, safe and reliable hud typed liver tumor targeting property glycyrrhizic acid to modify nanometer carboxymethyl chitosan particle.
Basic scheme of the present invention is following:
Through the initiator effect, glycerol polymerization obtains amphipathic copolymer in aqueous solution for hydrophilic carboxymethyl chitosan and hydrophobic alkyl methacrylate, alkyl acrylate or alkyl cyanoacrylate; This amphipathic copolymer self assembly forms the polymer nanometer carboxymethyl chitosan particle with hydrophobic inner core, hydrophilic outer shell; Hydrophobic drug is written into the nanoparticle kernel through hydrophobic interaction.The present invention with the covalently bound water-wetted surface to this nanometer carboxymethyl chitosan particle of glycyrrhizic acid molecule, realizes the active targeting of nanoparticle to HCC according to the height targeting property of glycyrrhizic acid to hepatic parenchymal cells, reduces the toxic and side effects of medicine to normal structure.Preparation of the present invention avoids the use of organic solvent, and in the scope of pH 3 ~ 12, all can not produce the deposition separate out.
Particularly; Liver tumor targeting property glycyrrhizic acid provided by the invention is modified nanometer carboxymethyl chitosan particle; Be the nuclear shell structure nano grain with hydrophobic inner core, hydrophilic outer shell that is formed by hydrophilic carboxymethyl chitosan and hydrophobic polyalkyl methacrylate, polyalkyl acrylate or Polyalkylcyanoacrylanano graft copolymerization, hydrophilic outer shell surperficial covalently bound has glycyrrhizic acid;
Wherein, the mass volume ratio of carboxymethyl chitosan and hydrophobic monomer alkyl methacrylate, alkyl acrylate or alkyl cyanoacrylate is 1:0.25 ~ 1:4 (g/ml), and the content of glycyrrhizic acid group is 2 ~ 40% of nanoparticle quality.
Described carboxymethyl chitosan is an oxygen substituted carboxymethyl chitosan, and molecular weight ranges is 10,000 ~ 500,000 Da, deacetylation>85%.
Described alkyl methacrylate is one or more in methyl methacrylate, EMA, propyl methacrylate, butyl methacrylate, isobutyl methacrylate and the N-Hexyl methacrylate.
Described alkyl acrylate is one or more in acrylic acid methyl ester., ethyl acrylate, propyl acrylate, butylacrylate, Isobutyl 2-propenoate, the Hexyl 2-propenoate.
Described alkyl cyanoacrylate is one or more in methyl 2-cyanoacrylate, cyanacrylate, alpha-cyanoacrylate propyl ester, Tisuacryl, isobutylcyanoacrylate, the own ester of alpha-cyanoacrylate.
It is 100 ~ 300 nm that described liver tumor targeting property glycyrrhizic acid is modified the nanometer carboxymethyl chitosan particle particle diameter.
The method for preparing that liver tumor targeting property glycyrrhizic acid of the present invention is modified nanometer carboxymethyl chitosan particle is following:
(1) carboxymethyl chitosan is dissolved in the water, is heated to 30 ~ 50 ℃, add hydrophobic alkyl methacrylate, alkyl acrylate or alkyl cyanoacrylate; Stir, add initiator, be warming up to 70 ~ 85 ℃; Reaction 2 ~ 24 h; Purification, lyophilization gets nanometer carboxymethyl chitosan particle;
(2) glycyrrhizic acid is dissolved in the carbonate buffer solution of pH 8.0 ~ 10.0, adds sodium periodate solution, lucifuge stirs 6 ~ 12 h under the ice bath, gets glycyrrhizic acid oxidation product glycyrrhizic acid aldehyde;
(3) the nanoparticle product with step (1) is scattered in the water, adds in the glycyrrhizic acid aldehyde solution of step (2), and lucifuge stirs 12 ~ 36 h under the room temperature; Add sodium borohydride aqueous solution, lucifuge stirs 12 ~ 36 h under the room temperature; Purification, lyophilization gets glycyrrhizic acid and modifies nanometer carboxymethyl chitosan particle.
Described carboxymethyl chitosan is an oxygen substituted carboxymethyl chitosan, and molecular weight ranges is 10,000 ~ 500,000 Da, deacetylation>85%.
Described alkyl methacrylate is one or more in methyl methacrylate, EMA, propyl methacrylate, butyl methacrylate, isobutyl methacrylate and the N-Hexyl methacrylate.
Described alkyl acrylate is one or more in acrylic acid methyl ester., ethyl acrylate, propyl acrylate, butylacrylate, Isobutyl 2-propenoate, the Hexyl 2-propenoate.
Described alkyl cyanoacrylate is one or more in methyl 2-cyanoacrylate, cyanacrylate, alpha-cyanoacrylate propyl ester, Tisuacryl, isobutylcyanoacrylate, the own ester of alpha-cyanoacrylate.
In the step (1), the mass volume ratio of carboxymethyl chitosan and hydrophobicity alkyl methacrylate, alkyl acrylate or alkyl cyanoacrylate is 1:0.25 ~ 1:4 (g/ml).
Initiator in the step (1) is a kind of of Ammonium persulfate. or potassium peroxydisulfate, and consumption is 0.01 ~ 0.2% of a reaction system, g/mL.Initiator is crossed reaction at least not exclusively, and polyreaction is too fast at most excessively, and preferred 0.05%.
In the step (2), the mol ratio of sodium metaperiodate and glycyrrhizic acid is 1:1 ~ 4:1.
In the step (3), glycyrrhizic acid aldehyde and nanoparticle mass ratio are 0.25:1 ~ 4:1, and when mass ratio was higher than 4:1, nanoparticle was prone to produce deposition, and preferred glycyrrhizic acid aldehyde and nanoparticle mass ratio are 1:1 ~ 4:1.
In the step (3), the mol ratio of sodium borohydride and glycyrrhizic acid aldehyde is 1:1 ~ 4:1.
As the application that above-mentioned liver tumor targeting property glycyrrhizic acid is modified nanometer carboxymethyl chitosan particle, the present invention also provides a kind of liver tumor targeting property glycyrrhizic acid to modify the carboxymethyl chitosan drug-carrying nanometer particle.This drug-carrying nanometer particle is a carrier with above-mentioned nanoparticle, and bag carries hydrophobic drug to be processed; Wherein, the mass ratio of hydrophobic drug and liver tumor targeting property glycyrrhizic acid modification nanometer carboxymethyl chitosan particle is 1:5 ~ 1:20.Wherein, said hydrophobic drug is one or more the mixture in cancer therapy drug and the molecular probe.Said cancer therapy drug is one or more the mixture in paclitaxel, amycin, epirubicin, 5-fluorouracil, all-trans-retinoic acid, the camptothecine; Said hydrophobic molecule probe is fluorescent marker, Geigers, magnetic molecule, responsive to temperature type or pH responsive type molecule etc.
The method for preparing that above-mentioned liver tumor targeting property glycyrrhizic acid is modified the carboxymethyl chitosan drug-carrying nanometer particle is following:
Liver tumor targeting property glycyrrhizic acid is modified nanometer carboxymethyl chitosan particle to be scattered in the phosphate buffer of pH 7.4; Add hydrophobic drug, stirring at room 0.5 ~ 4 h, pulsating ultrasound under the ice bath with organic solvent dissolution; Residual organic solvent is removed in dialysis, gets the drug-carrying nanometer particle suspension.
Wherein, the mass ratio of hydrophobic drug and liver tumor targeting property glycyrrhizic acid modification nanometer carboxymethyl chitosan particle is 1:5 ~ 1:20.
Described organic solvent is ethanol, acetonitrile, acetone, oxolane, N, a kind of in the dinethylformamide.
The condition of described pulsating ultrasound is output 100 W, 2 s that work, intermittently 2 s.
Liver tumor targeting property glycyrrhizic acid of the present invention is modified nanometer carboxymethyl chitosan particle and can be used as the intravenous administration carrier that bag carries hydrophobic anticancer drug and molecular probe, is used for the treatment of hepatic disease.
The invention has the advantages that:
(1) the liver property that becomes with glycyrrhizic acid combines with the good biology performance of chitosan derivatives nanoparticle, has prepared a kind of novel liver tumor targeting property drug administration carrier, has passive target and active targeted double effect concurrently.Nanoparticle surface hydrophilic of the present invention, and glycyrrhizic acid modifies and can increase its hydrophilic, avoids the removing of reticuloendothelial system, realizes long circulation in the body, and drug-carrying nanometer particle can accumulate in tumor tissues through the EPR effect in the tumor neogenetic blood vessels leakages.Targeting group (glycyrrhizic acid) on the nanoparticle can be by the identification of tumor cell of liver surface specific receptor, combination; Significantly improve cellular uptake efficient, compare with the unmodified nanometer carboxymethyl chitosan particle, the cellular uptake that glycyrrhizic acid is modified nanometer carboxymethyl chitosan particle improves 10 times; More effectively bring into play the active targeting of glycyrrhizic acid; Thereby increase the intracellular concentration of medicine, improve drug effect, reach the purpose of targeted therapy.
(2) nanoparticle glycyrrhizic acid degree of modification of the present invention is higher, is prone to combine with the HCC surface receptor, and the targeting ability is stronger.
(3) nanoparticle of the present invention is uniformly dispersed, and polydispersity coefficient is all less than 0.2, and distribution is narrower.Simultaneously, nanoparticle of the present invention all has stronger surface charge, and is all stable under room temperature preservation and the physiological condition, is difficult for producing cohesion and deposition.
(4) nanoparticle compact structure of the present invention, can through ultrasonic directly, Fast Packet carries hydrophobic drug, the protection pharmaceutically active.Contained medicine discharges slowly in vivo, can prevent that medicine from arriving the preceding toxicity to normal structure of target spot.
(5) nanoparticle of the present invention is framework material with the carboxymethyl chitosan, nontoxic and non-immunogenicity, and biocompatibility is good, degradable in vivo, catabolite is harmless to organism.
(6) contain carboxyl in the carboxymethyl chitosan, have the enzyme effect of pressing down.
(7) the various low in raw material cost of the present invention are easy to get, and the nanoparticle synthesis technique is simple, and condition is controlled, good reproducibility.Nanoparticle can be in aqueous solution forms through the copolymer self assembly, and the method for introducing the targeting group is simple and convenient, and connecting key is firm, and need not to use organic solvent such as surfactant in the preparation process, and is safe and reliable.
Term used herein:
" CMCNP " refers to nanometer carboxymethyl chitosan particle, and it consists of the amphipathic copolymer of carboxymethyl chitosan-methyl methacrylate.
" CMCNP-GL " refers to glycyrrhizic acid modification nanometer carboxymethyl chitosan particle.
" PTX/CMCNP " refers to carry the paclitaxel nanometer carboxymethyl chitosan particle.
" PTX/CMCNP-GL " refers to carry the paclitaxel glycyrrhizic acid and modifies nanometer carboxymethyl chitosan particle.
Description of drawings
Fig. 1 is the transmission electron microscope picture of nanoparticle, (A) nanometer carboxymethyl chitosan particle (embodiment 1), and (B) glycyrrhizic acid is modified nanometer carboxymethyl chitosan particle (embodiment 18).Scale is 100 nm among the figure.
Fig. 2 is the serum stability that nanometer carboxymethyl chitosan particle (embodiment 1) and glycyrrhizic acid are modified nanometer carboxymethyl chitosan particle (embodiment 17,18,19).
Fig. 3 is that nanometer carboxymethyl chitosan particle (embodiment 1) is modified the cellular uptake of nanometer carboxymethyl chitosan particle (embodiment 17,18,19) at HCC 2 h with glycyrrhizic acid.
Fig. 4 cultivates the vitro cytotoxicity of 24 h altogether for paclitaxel solution and year paclitaxel nano grain (embodiment 24,25,26,27) and HCC.
The specific embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used to explain the present invention, and are not used in restriction scope of the present invention.The following experimental technique of unreceipted actual conditions among the routine embodiment, usually according to normal condition, or the condition of advising according to manufacturer.Unless otherwise indicated, concentration is mass and size percent (w/v).
The preparation of embodiment 1 nanometer carboxymethyl chitosan particle
Get the carboxymethyl chitosan 1g of molecular weight 10,000 Da, add in the 100 mL water, stirring at room is to dissolving fully.Be heated to 40 ℃, add 0.5 mL methyl methacrylate, stir 20 min after, add 0.05 g Ammonium persulfate., slowly be warming up to 70 ℃, continue reaction 24 h.The milky nanoparticle suspension that reaction obtains is put the purification of dialysing in 14, the 000 Da semipermeable membranes, lyophilization.
The preparation of embodiment 2 nanometer carboxymethyl chitosan particles
Get the carboxymethyl chitosan 1g of molecular weight 10,000 Da, add in the 100 mL water, stirring at room is to dissolving fully.Be heated to 30 ℃, add 0.25 mL methyl methacrylate, stir 10 min after, add 0.01 g Ammonium persulfate., slowly be warming up to 75 ℃, continue reaction 12 h.The milky nanoparticle suspension that reaction obtains is put the purification of dialysing in 14, the 000 Da semipermeable membranes, lyophilization.
The preparation of embodiment 3 nanometer carboxymethyl chitosan particles
Get the carboxymethyl chitosan 1g of molecular weight 10,000 Da, add in the 100 mL water, stirring at room is to dissolving fully.Be heated to 50 ℃, add 4 mL methyl methacrylates, stir 30 min after, add 0.2 g Ammonium persulfate., slowly be warming up to 85 ℃, continue reaction 2 h.The milky nanoparticle suspension that reaction obtains is put the purification of dialysing in 14, the 000 Da semipermeable membranes, lyophilization.
The preparation of embodiment 4 nanometer carboxymethyl chitosan particles
Get the carboxymethyl chitosan 1g of molecular weight 10,000 Da, add in the 100 mL water, stirring at room is to dissolving fully.Be heated to 50 ℃, add 1 mL acrylic acid methyl ester., stir 30 min after, add 0.1 g potassium peroxydisulfate, slowly be warming up to 80 ℃, continue reaction 8 h.The milky nanoparticle suspension that reaction obtains is put the purification of dialysing in 14, the 000 Da semipermeable membranes, lyophilization.
The preparation of embodiment 5 nanometer carboxymethyl chitosan particles
Get the carboxymethyl chitosan 1g of molecular weight 100,000 Da, add in the 100 mL water, stirring at room is to dissolving fully.Be heated to 45 ℃, add 2 mL EMAs, stir 30 min after, add 0.05 g Ammonium persulfate., slowly be warming up to 70 ℃, continue reaction 6 h.The milky nanoparticle suspension that reaction obtains is put the purification of dialysing in 14, the 000 Da semipermeable membranes, lyophilization.
The preparation of embodiment 6 nanometer carboxymethyl chitosan particles
Get the carboxymethyl chitosan 1g of molecular weight 100,000 Da, add in the 100 mL water, stirring at room is to dissolving fully.Be heated to 35 ℃, add 3 mL ethyl acrylate, stir 20 min after, add 0.02 g Ammonium persulfate., slowly be warming up to 75 ℃, continue reaction 4 h.The milky nanoparticle suspension that reaction obtains is put the purification of dialysing in 14, the 000 Da semipermeable membranes, lyophilization.
The preparation of embodiment 7 nanometer carboxymethyl chitosan particles
Get the carboxymethyl chitosan 1g of molecular weight 100,000 Da, add in the 100 mL water, stirring at room is to dissolving fully.Be heated to 40 ℃, add 1 mL methyl 2-cyanoacrylate, stir 10 min after, add 0.05 g Ammonium persulfate., slowly be warming up to 80 ℃, continue reaction 10 h.The milky nanoparticle suspension that reaction obtains is put the purification of dialysing in 14, the 000 Da semipermeable membranes, lyophilization.
The preparation of embodiment 8 nanometer carboxymethyl chitosan particles
Get the carboxymethyl chitosan 1g of molecular weight 100,000 Da, add in the 100 mL water, stirring at room is to dissolving fully.Be heated to 50 ℃, add 0.5 mL propyl methacrylate, stir 20 min after, add 0.15 g potassium peroxydisulfate, slowly be warming up to 85 ℃, continue reaction 14 h.The milky nanoparticle suspension that reaction obtains is put the purification of dialysing in 14, the 000 Da semipermeable membranes, lyophilization.
The preparation of embodiment 9 nanometer carboxymethyl chitosan particles
Get the carboxymethyl chitosan 1g of molecular weight 300,000 Da, add in the 100 mL water, stirring at room is to dissolving fully.Be heated to 30 ℃, add 0.25 mL butyl methacrylate, stir 20 min after, add 0.1 g Ammonium persulfate., slowly be warming up to 70 ℃, continue reaction 16 h.The milky nanoparticle suspension that reaction obtains is put the purification of dialysing in 14, the 000 Da semipermeable membranes, lyophilization.
The preparation of embodiment 10 nanometer carboxymethyl chitosan particles
Get the carboxymethyl chitosan 1g of molecular weight 300,000 Da, add in the 100 mL water, stirring at room is to dissolving fully.Be heated to 45 ℃, add 4 mL isobutylcyanoacrylates, stir 30 min after, add 0.2 g potassium peroxydisulfate, slowly be warming up to 80 ℃, continue reaction 18 h.The milky nanoparticle suspension that reaction obtains is put the purification of dialysing in 14, the 000 Da semipermeable membranes, lyophilization.
The preparation of embodiment 11 nanometer carboxymethyl chitosan particles
Get the carboxymethyl chitosan 1g of molecular weight 500,000 Da, add in the 100 mL water, stirring at room is to dissolving fully.Be heated to 40 ℃, add 2 mL Hexyl 2-propenoates, stir 10 min after, add 0.2 g Ammonium persulfate., slowly be warming up to 75 ℃, continue reaction 20 h.The milky nanoparticle suspension that reaction obtains is put the purification of dialysing in 14, the 000 Da semipermeable membranes, lyophilization.
The preparation of embodiment 12 nanometer carboxymethyl chitosan particles
Get the carboxymethyl chitosan 1g of molecular weight 500,000 Da, add in the 100 mL water, stirring at room is to dissolving fully.Be heated to 50 ℃, add 0.5 mL cyanacrylate, stir 20 min after, add 0.05 g potassium peroxydisulfate, slowly be warming up to 85 ℃, continue reaction 22 h.The milky nanoparticle suspension that reaction obtains is put the purification of dialysing in 14, the 000 Da semipermeable membranes, lyophilization.
The preparation of embodiment 13 glycyrrhizic acid aldehyde
Glycyrrhizic acid (GL) is dissolved in the carbonic acid buffer of pH 9.0, gets NaIO
4Water-soluble in right amount, glycyrrhizic acid and NaIO
4Molar concentration is 0.05 mol/L.Stir down NaIO
4Solution slowly drops in the above-mentioned glycyrrhizic acid solution, NaIO
4With the mol ratio of glycyrrhizic acid be 1:1.The ice bath lucifuge stirs 6 h, gets the glycyrrhizic acid oxidation product, is designated as glycyrrhizic acid aldehyde.
The preparation of embodiment 14 glycyrrhizic acid aldehyde
Glycyrrhizic acid (GL) is dissolved in the carbonic acid buffer of pH 8.0, gets NaIO
4Water-soluble in right amount, glycyrrhizic acid and NaIO
4Molar concentration is 0.05 mol/L.Stir down NaIO
4Solution slowly drops in the above-mentioned glycyrrhizic acid solution, NaIO
4With the mol ratio of glycyrrhizic acid be 2:1.The ice bath lucifuge stirs 9 h, gets the glycyrrhizic acid oxidation product, is designated as glycyrrhizic acid aldehyde.
The preparation of embodiment 15 glycyrrhizic acid aldehyde
Glycyrrhizic acid (GL) is dissolved in the carbonic acid buffer of pH 10.0, gets NaIO
4Water-soluble in right amount, glycyrrhizic acid and NaIO
4Molar concentration is 0.05 mol/L.Stir down NaIO
4Solution slowly drops in the above-mentioned glycyrrhizic acid solution, NaIO
4With the mol ratio of glycyrrhizic acid be 4:1.The ice bath lucifuge stirs 12 h, gets the glycyrrhizic acid oxidation product, is designated as glycyrrhizic acid aldehyde.
The detection of embodiment 16 aldehyde radicals
Cu (OH) with new system
2Solution and GL aldehyde product solution Hybrid Heating have red precipitate (Cu
2O) prove that aldehyde radical exists.
Embodiment 17 glycyrrhizic acids are modified the preparation of nanometer carboxymethyl chitosan particle
The nanometer carboxymethyl chitosan particle of embodiment 1 is dispersed in the water, under stirring the nanoparticle suspension is slowly added in the glycyrrhizic acid aldehyde solution of embodiment 13, the mass ratio of glycyrrhizic acid aldehyde and nanometer carboxymethyl chitosan particle is 1:1, and the room temperature lucifuge stirs 24 h.With NaBH
4Water-soluble, dropwise add above-mentioned reaction system, NaBH
4With the mol ratio of glycyrrhizic acid be 1:1, stirring at room 24 h.After ultrasonic product is put the purification of dialysing in 3, the 500 Da semipermeable membranes, lyophilization.
Embodiment 18 glycyrrhizic acids are modified the preparation of nanometer carboxymethyl chitosan particle
The nanometer carboxymethyl chitosan particle of embodiment 1 is dispersed in the water, under stirring the nanoparticle suspension is slowly added in the glycyrrhizic acid aldehyde solution of embodiment 13, the mass ratio of glycyrrhizic acid aldehyde and nanometer carboxymethyl chitosan particle is 2:1, and the room temperature lucifuge stirs 24 h.With NaBH
4Water-soluble, dropwise add above-mentioned reaction system, NaBH
4With the mol ratio of glycyrrhizic acid be 1:1, stirring at room 24 h.After ultrasonic product is put the purification of dialysing in 3, the 500 Da semipermeable membranes, lyophilization.
Embodiment 19 glycyrrhizic acids are modified the preparation of nanometer carboxymethyl chitosan particle
The nanometer carboxymethyl chitosan particle of embodiment 1 is dispersed in the water, under stirring the nanoparticle suspension is slowly added in the glycyrrhizic acid aldehyde solution of embodiment 13, the mass ratio of glycyrrhizic acid aldehyde and nanometer carboxymethyl chitosan particle is 4:1, and the room temperature lucifuge stirs 24 h.With NaBH
4Water-soluble, dropwise add above-mentioned reaction system, NaBH
4With the mol ratio of glycyrrhizic acid be 1:1, stirring at room 24 h.After ultrasonic product is put the purification of dialysing in 3, the 500 Da semipermeable membranes, lyophilization.
The nanometer carboxymethyl chitosan particle of embodiment 3 is dispersed in the water, under stirring the nanoparticle suspension is slowly added in the glycyrrhizic acid aldehyde solution of embodiment 14, the mass ratio of glycyrrhizic acid aldehyde and nanometer carboxymethyl chitosan particle is 0.25:1, and the room temperature lucifuge stirs 12 h.With NaBH
4Water-soluble, dropwise add above-mentioned reaction system, NaBH
4With the mol ratio of glycyrrhizic acid be 2:1, stirring at room 36 h.After ultrasonic product is put the purification of dialysing in 3, the 500 Da semipermeable membranes, lyophilization.
Embodiment 21 glycyrrhizic acids are modified the preparation of nanometer carboxymethyl chitosan particle
The nanometer carboxymethyl chitosan particle of embodiment 6 is dispersed in the water, under stirring the nanoparticle suspension is slowly added in the glycyrrhizic acid aldehyde solution of embodiment 15, the mass ratio of glycyrrhizic acid aldehyde and nanometer carboxymethyl chitosan particle is 0.25:1, and the room temperature lucifuge stirs 12 h.With NaBH
4Water-soluble, dropwise add above-mentioned reaction system, NaBH
4With the mol ratio of glycyrrhizic acid be 4:1, stirring at room 12 h.After ultrasonic product is put the purification of dialysing in 3, the 500 Da semipermeable membranes, lyophilization.
Embodiment 22 glycyrrhizic acids are modified the preparation of nanometer carboxymethyl chitosan particle
The nanometer carboxymethyl chitosan particle of embodiment 9 is dispersed in the water, under stirring the nanoparticle suspension is slowly added in the glycyrrhizic acid aldehyde solution of embodiment 14, the mass ratio of glycyrrhizic acid aldehyde and nanometer carboxymethyl chitosan particle is 0.5:1, and the room temperature lucifuge stirs 36 h.With NaBH
4Water-soluble, dropwise add above-mentioned reaction system, NaBH
4With the mol ratio of glycyrrhizic acid be 1:1, stirring at room 12 h.After ultrasonic product is put the purification of dialysing in 3, the 500 Da semipermeable membranes, lyophilization.
Embodiment 23 glycyrrhizic acids are modified the preparation of nanometer carboxymethyl chitosan particle
The nanometer carboxymethyl chitosan particle of embodiment 12 is dispersed in the water, under stirring the nanoparticle suspension is slowly added in the glycyrrhizic acid aldehyde solution of embodiment 15, the mass ratio of glycyrrhizic acid aldehyde and nanometer carboxymethyl chitosan particle is 0.5:1, and the room temperature lucifuge stirs 36 h.With NaBH
4Water-soluble, dropwise add above-mentioned reaction system, NaBH
4With the mol ratio of glycyrrhizic acid be 1:1, stirring at room 36 h.After ultrasonic product is put the purification of dialysing in 3, the 500 Da semipermeable membranes, lyophilization.
The preparation of 24 years paclitaxel nanometer carboxymethyl chitosan particles of embodiment
The nanometer carboxymethyl chitosan particle of embodiment 1 is dispersed in phosphate buffer (0.2 M; PH 7.4); Paclitaxel is dissolved in dehydrated alcohol; Under stirring paclitaxel solution is dropped to glycyrrhizic acid and modify in the nanometer carboxymethyl chitosan particle suspension, the mass ratio of paclitaxel and nanometer carboxymethyl chitosan particle is 1:5.Stirring at room 1 h, pulsating ultrasound appearance ultrasonic (output 100 W, 2 s that work, intermittently 2 s) under the ice bath, product is put in 3, the 500 Da semipermeable membranes, the dialysis purification.
The preparation of embodiment paclitaxel glycyrrhizic acid modification in 25 years nanometer carboxymethyl chitosan particle
The glycyrrhizic acid of embodiment 17 is modified nanometer carboxymethyl chitosan particle be dispersed in phosphate buffer (0.2 M; PH 7.4); Paclitaxel is dissolved in dehydrated alcohol; Under stirring paclitaxel solution is dropped to glycyrrhizic acid and modify in the nanometer carboxymethyl chitosan particle suspension, the mass ratio that paclitaxel and glycyrrhizic acid are modified nanometer carboxymethyl chitosan particle is 1:5.Stirring at room 1 h, pulsating ultrasound appearance ultrasonic (output 100 W, 2 s that work, intermittently 2 s) under the ice bath, product is put in 3, the 500 Da semipermeable membranes, the dialysis purification.
The preparation of embodiment paclitaxel glycyrrhizic acid modification in 26 years nanometer carboxymethyl chitosan particle
The glycyrrhizic acid of embodiment 18 is modified nanometer carboxymethyl chitosan particle be dispersed in phosphate buffer (0.2 M; PH 7.4); Paclitaxel is dissolved in dehydrated alcohol; Under stirring paclitaxel solution is dropped to glycyrrhizic acid and modify in the nanometer carboxymethyl chitosan particle suspension, the mass ratio that paclitaxel and glycyrrhizic acid are modified nanometer carboxymethyl chitosan particle is 1:5.Stirring at room 1 h, pulsating ultrasound appearance ultrasonic (output 100 W, 2 s that work, intermittently 2 s) under the ice bath, product is put in 3, the 500 Da semipermeable membranes, the dialysis purification.
The preparation of embodiment paclitaxel glycyrrhizic acid modification in 27 years nanometer carboxymethyl chitosan particle
The glycyrrhizic acid of embodiment 19 is modified nanometer carboxymethyl chitosan particle be dispersed in phosphate buffer (0.2 M; PH 7.4); Paclitaxel is dissolved in dehydrated alcohol; Under stirring paclitaxel solution is dropped to glycyrrhizic acid and modify in the nanometer carboxymethyl chitosan particle suspension, the mass ratio that paclitaxel and glycyrrhizic acid are modified nanometer carboxymethyl chitosan particle is 1:5.Stirring at room 1 h, pulsating ultrasound appearance ultrasonic (output 100 W, 2 s that work, intermittently 2 s) under the ice bath, product is put in 3, the 500 Da semipermeable membranes, the dialysis purification.
The preparation of embodiment paclitaxel glycyrrhizic acid modification in 28 years nanometer carboxymethyl chitosan particle
The glycyrrhizic acid of embodiment 20 is modified nanometer carboxymethyl chitosan particle be dispersed in phosphate buffer (0.2 M; PH 7.4); Paclitaxel is dissolved in anhydrous acetonitrile; Under stirring paclitaxel solution is dropped to glycyrrhizic acid and modify in the nanometer carboxymethyl chitosan particle suspension, the mass ratio that paclitaxel and glycyrrhizic acid are modified nanometer carboxymethyl chitosan particle is 1:10.Stirring at room 0.5 h, pulsating ultrasound appearance ultrasonic (output 100 W, 2 s that work, intermittently 2 s) under the ice bath, product is put in 3, the 500 Da semipermeable membranes, the dialysis purification.
The preparation of embodiment paclitaxel glycyrrhizic acid modification in 29 years nanometer carboxymethyl chitosan particle
The glycyrrhizic acid of embodiment 21 is modified nanometer carboxymethyl chitosan particle be dispersed in phosphate buffer (0.2 M; PH 7.4); Paclitaxel is dissolved in anhydrous propanone; Under stirring paclitaxel solution is dropped to glycyrrhizic acid and modify in the nanometer carboxymethyl chitosan particle suspension, the mass ratio that paclitaxel and glycyrrhizic acid are modified nanometer carboxymethyl chitosan particle is 1:10.Stirring at room 2 h, pulsating ultrasound appearance ultrasonic (output 100 W, 2 s that work, intermittently 2 s) under the ice bath, product is put in 3, the 500 Da semipermeable membranes, the dialysis purification.
The preparation of embodiment paclitaxel glycyrrhizic acid modification in 30 years nanometer carboxymethyl chitosan particle
The glycyrrhizic acid of embodiment 22 is modified nanometer carboxymethyl chitosan particle be dispersed in phosphate buffer (0.2 M; PH 7.4); Paclitaxel is dissolved in oxolane; Under stirring paclitaxel solution is dropped to glycyrrhizic acid and modify in the nanometer carboxymethyl chitosan particle suspension, the mass ratio that paclitaxel and glycyrrhizic acid are modified nanometer carboxymethyl chitosan particle is 1:20.Stirring at room 0.5 h, pulsating ultrasound appearance ultrasonic (output 100 W, 2 s that work, intermittently 2 s) under the ice bath, product is put in 3, the 500 Da semipermeable membranes, the dialysis purification.
The preparation of embodiment paclitaxel glycyrrhizic acid modification in 31 years nanometer carboxymethyl chitosan particle
The glycyrrhizic acid of embodiment 23 is modified nanometer carboxymethyl chitosan particle be dispersed in phosphate buffer (0.2 M; PH 7.4); Paclitaxel is dissolved in N; Dinethylformamide drops to glycyrrhizic acid with paclitaxel solution under stirring and modifies in the nanometer carboxymethyl chitosan particle suspension, and the mass ratio that paclitaxel and glycyrrhizic acid are modified nanometer carboxymethyl chitosan particle is 1:20.Stirring at room 4 h, pulsating ultrasound appearance ultrasonic (output 100 W, 2 s that work, intermittently 2 s) under the ice bath, product is put in 3, the 500 Da semipermeable membranes, the dialysis purification.
The preparation of embodiment amycin glycyrrhizic acid modification in 32 years nanometer carboxymethyl chitosan particle
The glycyrrhizic acid of embodiment 18 is modified nanometer carboxymethyl chitosan particle be dispersed in phosphate buffer (0.2 M; PH 7.4); Amycin is dissolved in dehydrated alcohol; Under stirring the amycin drips of solution is added to glycyrrhizic acid and modify in the nanometer carboxymethyl chitosan particle suspension, the mass ratio that amycin and glycyrrhizic acid are modified nanometer carboxymethyl chitosan particle is 1:5.Stirring at room 1 h, pulsating ultrasound appearance ultrasonic (output 100 W, 2 s that work, intermittently 2 s) under the ice bath, product is put in 3, the 500 Da semipermeable membranes, the dialysis purification.
The preparation of embodiment camptothecine glycyrrhizic acid modification in 33 years nanometer carboxymethyl chitosan particle
The glycyrrhizic acid of embodiment 18 is modified nanometer carboxymethyl chitosan particle be dispersed in phosphate buffer (0.2 M; PH 7.4); Camptothecine is dissolved in dehydrated alcohol; Under stirring the camptothecine drips of solution is added to glycyrrhizic acid and modify in the nanometer carboxymethyl chitosan particle suspension, the mass ratio that camptothecine and glycyrrhizic acid are modified nanometer carboxymethyl chitosan particle is 1:5.Stirring at room 1 h, pulsating ultrasound appearance ultrasonic (output 100 W, 2 s that work, intermittently 2 s) under the ice bath, product is put in 3, the 500 Da semipermeable membranes, the dialysis purification.
The preparation of embodiment 5-fluorouracil glycyrrhizic acid modification in 34 years nanometer carboxymethyl chitosan particle
The glycyrrhizic acid of embodiment 18 is modified nanometer carboxymethyl chitosan particle be dispersed in phosphate buffer (0.2 M; PH 7.4); 5-fluorouracil is dissolved in dehydrated alcohol; Under stirring the 5-fluorouracil drips of solution is added to glycyrrhizic acid and modify in the nanometer carboxymethyl chitosan particle suspension, the mass ratio that camptothecine and glycyrrhizic acid are modified nanometer carboxymethyl chitosan particle is 1:5.Stirring at room 1 h, pulsating ultrasound appearance ultrasonic (output 100 W, 2 s that work, intermittently 2 s) under the ice bath, product is put in 3, the 500 Da semipermeable membranes, the dialysis purification.
Embodiment 35 transmission electron microscopes characterize the nanoparticle form
Drop on the copper mesh that is loaded with carbon film after the nanometer carboxymethyl chitosan particle that the glycyrrhizic acid of the nanometer carboxymethyl chitosan particle of embodiment 1 and embodiment 18 is modified disperses with suitable quantity of water, dry under the room temperature, transmission electron microscope observing is also taken pictures, and sees accompanying drawing 1.It is spherical all to be the nucleocapsid shape by the visible nanoparticle of figure, is uniformly dispersed big or small homogeneous.Compare with nanometer carboxymethyl chitosan particle, glycyrrhizic acid is modified back nanoparticle particle diameter and is reduced, the thickening of surface hydrophilic layer.
Embodiment 36 glycyrrhizic acids are modified particle diameter, the electromotive force of nanometer carboxymethyl chitosan particle
Zetasizer Nano electromotive force and particle size analyzer are measured the particle diameter and the Zeta electric potential of nanometer carboxymethyl chitosan particle (embodiment 1) and glycyrrhizic acid modification nanometer carboxymethyl chitosan particle (embodiment 17,18,19).The particle size determination parameter: He-Ne laser (wavelength 633nm), 23 ° of C of temperature are measured in scatteringangle=90 °.The Zeta electric potential location parameter: He-Ne laser (wavelength 653nm), 23 ° of C of temperature are measured in scatteringangle=14 °.Every duplicate samples parallel assay 3 times.Measure the result and see table 1.
As shown in table 1, glycyrrhizic acid is modified nanometer carboxymethyl chitosan particle electromotive force absolute value all greater than 25mV, shows that nanoparticle is more stable.The particle size range of nanoparticle is 102-202 nm, can be engulfed by liver earlier in the distributed process in the body, is easy to act on the tumor locus gathering through the permeability and the delay of diseased region.The nanoparticle size receives the influence of glycyrrhizic acid percent grafting.A spot of glycyrrhizic acid is modified the nanometer carboxymethyl chitosan particle particle diameter is increased, and with the raising of GL modification amount, particle diameter reduces.
Particle diameter, electromotive force, envelop rate and the drug loading of embodiment paclitaxel glycyrrhizic acid modification in 37 years nanometer carboxymethyl chitosan particle
Particle diameter, Zeta electric potential:
Zetasizer Nano electromotive force and particle size analyzer are measured and are carried paclitaxel nanometer carboxymethyl chitosan particle (embodiment 24) and the particle diameter and the Zeta electric potential that carry paclitaxel glycyrrhizic acid modification nanometer carboxymethyl chitosan particle (embodiment 25,26,27).The particle size determination parameter: He-Ne laser (wavelength 633nm), 23 ° of C of temperature are measured in scatteringangle=90 °.The Zeta electric potential location parameter: He-Ne laser (wavelength 653nm), 23 ° of C of temperature are measured in scatteringangle=14 °.Every duplicate samples parallel assay 3 times.Measure the result and see table 1.
Paclitaxel envelop rate and drug loading:
Silica gel for chromatography dress post (60 ~ 100 order) drains bubble.To carry paclitaxel nanometer carboxymethyl chitosan particle (embodiment 24) and carry the paclitaxel glycyrrhizic acid and modify and nanometer carboxymethyl chitosan particle (embodiment 25,26,27) is scattered in respectively make the nanoparticle suspension in the water; Respectively get 0.5 mL and go up appearance; With water elution; Peak value according to WXJ-9388 type Ultraviolet Detector 280 nm places show is collected the nanoparticle eluent, accurately measures its volume.Get proper amount of nano grain eluent, with 10 times of volumes methanol dissolvings, vortex 2 min, centrifugal 20 min of 12,000 rpm get supernatant 20 μ L and cross 0.45 μ m filter membrane, and HPLC (HPLC) is measured bag and is stated from the paclitaxel concentration in the nanoparticle.Reuse ethanol and water mixed solvent gradient elution are collected the free paclitaxel eluent with method, accurately measure its volume.HPLC measures free paclitaxel concentration.Be calculated as follows the paclitaxel envelop rate:
Envelop rate (%)=A * 100/ (A+B), drug loading (%)=A * 100/ (A+C)
Wherein A is stated from the content of taxol (mg) in the nanoparticle for bag; B is for being present in free content of taxol (mg) in the nanoparticle suspension; C is the quality (mg) of nanoparticle.
As shown in table 1, to compare with blank nanoparticle, each nanoparticle particle diameter all slightly increases behind the medicine carrying, the equal no change of Zeta electric potential.After the GL finishing, CMCNP significantly improves the envelop rate and the drug loading of paclitaxel, and increases with the increase of GL degree of modification.
Table 1 glycyrrhizic acid is modified nanometer carboxymethyl chitosan particle and the particle diameter, electromotive force, the envelop rate (n=3) that carry paclitaxel glycyrrhizic acid modification nanometer carboxymethyl chitosan particle
Embodiment 38 glycyrrhizic acids are modified the plasma stability of nanometer carboxymethyl chitosan particle
Nanometer carboxymethyl chitosan particle (embodiment 1) and glycyrrhizic acid are modified nanometer carboxymethyl chitosan particle (embodiment 17,18,19) be scattered among the PBS that contains 10% blood plasma, measure the particle diameter and the Zeta electric potential of nanoparticle respectively at 12 h, 24 h, 48 h, 72 h, 96 h, 120 h.The result sees accompanying drawing 2.
As shown in Figure 2; Change modified the particle diameter of nanometer carboxymethyl chitosan particle (embodiment 17,18,19) in 120 h and electromotive force there are no significant by nanometer carboxymethyl chitosan particle (embodiment 1) and glycyrrhizic acid; There are not deposition and flocculation phenomenon, more stable under physiological condition.
Embodiment 39 glycyrrhizic acids are modified the picked-up of nanometer carboxymethyl chitosan particle in the SMMC-7721 HCC
(Biomaterials 2010,31:3657-3666) preparation rhodamine fluorescent labeling nanoparticle according to the bibliographical information method.The take the logarithm SMMC-7721 cell of trophophase is with 1 * 10
5The density of cells/well is inoculated on 24 orifice plates, 37 ℃, 5% CO
2Change fresh DMEM culture medium (containing 10% hyclone) after cultivating 24 h; Every hole adds fluorescently-labeled nanometer carboxymethyl chitosan particle (embodiment 1) and fluorescently-labeled glycyrrhizic acid is modified nanometer carboxymethyl chitosan particle (embodiment 17,18,19), and the concentration of every hole nanoparticle is 200 μ g/mL.Liquid in the hole is abandoned in suction after cultivating 2 h; With phosphate buffer (0.2 M, pH 7.4) washing three times, with the pH 8.0 NaOH solution cell lysis that contain 0.5% SDS; ELIASA is measured the content (Ex 530 nm, Em 590 nm) of fluorescent labeling nanoparticle in the lysate.The Lowry method is measured the protein content in the lysate, and the cellular uptake amount contains μ g fluorescent labeling nanoparticle with every mg albumen and representes that accompanying drawing 3 is seen in parallel 3 holes of every duplicate samples.
As shown in Figure 3; Glycyrrhizic acid is modified nanometer carboxymethyl chitosan particle (embodiment 17,18,19) intake in the SMMC-7721 cell and is respectively 75.88 ± 3.62 μ g/mg, 131.67 ± 11.13 μ g/mg and 27.84 ± 2.57 μ g/mg; And corresponding nanometer carboxymethyl chitosan particle (embodiment 1) intake in the SMMC-7721 cell is merely 13.12 ± 1.77 μ g/mg; Show that the glycyrrhizic acid modification has significantly improved the cellular uptake ability of nanometer carboxymethyl chitosan particle; Glycyrrhizic acid content is that 28% nanoparticle intake is the highest, is about 10 times of nanometer carboxymethyl chitosan particle.
40 years paclitaxel glycyrrhizic acids of embodiment are modified the toxicity of nanometer carboxymethyl chitosan particle to the SMMC-7721 HCC
The take the logarithm SMMC-7721 cell of trophophase is with 1 * 10
4The density of cells/well is inoculated on 96 orifice plates, 37 ℃, 5% CO
2Change fresh DMEM culture medium (containing 10% hyclone) after cultivating 24 h, add the paclitaxel solution Taxol of variable concentrations
, carry paclitaxel nanometer carboxymethyl chitosan particle (embodiment 24) and carry the paclitaxel glycyrrhizic acid and modify nanometer carboxymethyl chitosan particle (embodiment 25,26,27), the addition of every hole nanoparticle is definite according to paclitaxel concentration.Negative control group adds 200 μ L fresh cultures; Positive controls adds 20 μ L, 220 mmol/L H
2O
2After cultivating 24 h, inhale and to abandon the liquid hole in, with phosphate buffer (0.2 M, pH 7.4) washing three times, every hole adds the 5 mg/mL MTT solution that 200 μ L fresh cultures and 20 μ L prepare with phosphate buffer (0.2 M, pH 7.4).After cultivating 4 h, inhale and abandon liquid in the hole, every hole adds 200 mL DMSO, and room temperature jolting 20 min, ELIASA measure 570 nm place light absorption values.Parallel 3 holes of every duplicate samples are 100% cell survival rate with the negative control group light absorption value, calculate the cell survival rate (%) after each sample treatment, see accompanying drawing 4.
As shown in Figure 4; When paclitaxel concentration is low (2.5 mg/mL), the tumor cell survival rate of handling through the drug-carrying nanometer particle of glycyrrhizic acid modification is about 50%, when paclitaxel concentration is 25 mg/mL; The tumor cell survival rate is merely 18%, is significantly higher than paclitaxel solution Taxol
Under the same medicine dosage, the carboxymethyl chitosan steamed bun stuffed with sugar carries the cytotoxicity that paclitaxel can strengthen medicine, and glycyrrhizic acid is modified carboxymethyl chitosan and can further improve the lethality of medicine to tumor cell as the paclitaxel drug administration carrier.
41 years paclitaxel glycyrrhizic acids of embodiment are modified nanometer carboxymethyl chitosan particle tumor killing effect in the body of H-22 liver cancer model mice
Female Kunming mouse (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 distinguished tail vein injection saline, paclitaxel solution Taxol
, carry paclitaxel nanometer carboxymethyl chitosan particle (embodiment 24) and carry paclitaxel glycyrrhizic acid modification nanometer carboxymethyl chitosan particle (embodiment 25,26,27).Dosage is 10 mg/mL by paclitaxel.Be administered once successive administration three times in three days.With the first time administration time be the 0th d, measured a gross tumor volume at a distance from one day, to the 16th day, put to death mice, take out tumor and also weigh, observe and also calculate tumor killing effect.See table 2.
As shown in table 2, it is remarkable to the anti-tumor in vivo effect of H-22 liver cancer model mice to carry paclitaxel glycyrrhizic acid modification nanometer carboxymethyl chitosan particle, and tumour inhibiting rate is about 90%, is significantly higher than nanometer carboxymethyl chitosan particle (tumour inhibiting rate 64.01%) and paclitaxel solution Taxol
(tumour inhibiting rate 34.52%).
Table 2 year paclitaxel nano grain and paclitaxel solution are in the anti-tumor in vivo effect of H-22 liver cancer model mice
a:
p<0.001 vs Taxol
Group
b:
p<0.001 vs PTXCMCNP group.
Claims (13)
1. a liver tumor targeting property glycyrrhizic acid is modified nanometer carboxymethyl chitosan particle; It is characterized in that it being the nuclear shell structure nano grain with hydrophobic inner core, hydrophilic outer shell that is formed by hydrophilic carboxymethyl chitosan and hydrophobic polyalkyl methacrylate, polyalkyl acrylate or Polyalkylcyanoacrylanano graft copolymerization, hydrophilic outer shell surperficial covalently bound has glycyrrhizic acid;
Wherein, the mass volume ratio of carboxymethyl chitosan and hydrophobic monomer alkyl methacrylate, alkyl acrylate or alkyl cyanoacrylate is 1:0.25 ~ 1:4 g/ml, and the content of glycyrrhizic acid group is 2 ~ 40% of nanoparticle quality.
2. nanoparticle according to claim 1 is characterized in that described carboxymethyl chitosan is an oxygen substituted carboxymethyl chitosan, and molecular weight ranges is 10,000 ~ 500,000 Da.
3. nanoparticle according to claim 1 is characterized in that described alkyl methacrylate is one or more in methyl methacrylate, EMA, propyl methacrylate, butyl methacrylate, isobutyl methacrylate and the N-Hexyl methacrylate; Described alkyl acrylate is one or more in acrylic acid methyl ester., ethyl acrylate, propyl acrylate, butylacrylate, Isobutyl 2-propenoate, the Hexyl 2-propenoate; Described alkyl cyanoacrylate is one or more in methyl 2-cyanoacrylate, cyanacrylate, alpha-cyanoacrylate propyl ester, Tisuacryl, isobutylcyanoacrylate, the own ester of alpha-cyanoacrylate.
4. according to the described nanoparticle of one of claim 1-3, it is characterized in that nanoparticle is spherical, particle diameter is 100 ~ 300 nm.
5. method for preparing of modifying nanometer carboxymethyl chitosan particle like the described liver tumor targeting property of one of claim 1-4 glycyrrhizic acid is characterized in that concrete steps are following:
(1) carboxymethyl chitosan is dissolved in the water, is heated to 30 ~ 50 ℃, add hydrophobic alkyl methacrylate, alkyl acrylate or alkyl cyanoacrylate; Stir, add initiator, be warming up to 70 ~ 85 ℃; Reaction 2 ~ 24 h; Purification, lyophilization gets nanometer carboxymethyl chitosan particle;
(2) glycyrrhizic acid is dissolved in the carbonate buffer solution of pH 8.0 ~ 10.0, adds sodium periodate solution, lucifuge stirs 6 ~ 12 h under the ice bath, gets glycyrrhizic acid oxidation product glycyrrhizic acid aldehyde solution;
(3) the nanoparticle product with step (1) is scattered in the water, adds in the glycyrrhizic acid aldehyde solution of step (2), and lucifuge stirs 12 ~ 36 h under the room temperature; Add sodium borohydride aqueous solution, lucifuge stirs 12 ~ 36 h under the room temperature; Purification, lyophilization gets glycyrrhizic acid and modifies nanometer carboxymethyl chitosan particle; Wherein, the mass ratio of glycyrrhizic acid aldehyde and nanometer carboxymethyl chitosan particle is 0.25:1 ~ 4:1 g/ml.
6. method for preparing according to claim 5 is characterized in that described initiator is a kind of of Ammonium persulfate. or potassium peroxydisulfate, and consumption is 0.01 ~ 0.2% g/mL of reaction system.
7. the method for preparing of nanoparticle according to claim 5, the mol ratio that it is characterized in that sodium metaperiodate and glycyrrhizic acid is 1:1 ~ 4:1, the mol ratio of sodium borohydride and glycyrrhizic acid aldehyde is 1:1 ~ 4:1.
8. a liver tumor targeting property glycyrrhizic acid is modified the carboxymethyl chitosan drug-carrying nanometer particle, it is characterized in that with the described nanoparticle of one of claim 1 ~ 4 be carrier, and bag carries hydrophobic drug to be processed; Wherein, the mass ratio of hydrophobic drug and liver tumor targeting property glycyrrhizic acid modification nanometer carboxymethyl chitosan particle is 1:5 ~ 1:20.
9. drug-carrying nanometer particle according to claim 8 is characterized in that said hydrophobic drug is one or more the mixture in cancer therapy drug and the molecular probe.
10. drug-carrying nanometer particle according to claim 9 is characterized in that said cancer therapy drug is one or more the mixture in paclitaxel, amycin, epirubicin, 5-fluorouracil, all-trans-retinoic acid, the camptothecine; Said hydrophobic molecule probe is fluorescent marker, Geigers, magnetic molecule, responsive to temperature type or pH responsive type molecule.
11. method for preparing like one of claim 8-10 described drug-carrying nanometer particle; It is characterized in that concrete steps are following: the described liver tumor targeting property of one of claim 1-4 glycyrrhizic acid is modified nanometer carboxymethyl chitosan particle be scattered in the phosphate buffer of pH 7.4; Add hydrophobic drug, stirring at room 0.5 ~ 4 h, pulsating ultrasound under the ice bath with organic solvent dissolution; Residual organic solvent is removed in dialysis, gets the drug-carrying nanometer particle suspension.
12. the method for preparing of drug-carrying nanometer particle according to claim 11 is characterized in that described organic solvent is ethanol, acetonitrile, acetone, oxolane, N, a kind of in the dinethylformamide.
13. the method for preparing of drug-carrying nanometer particle according to claim 11, the condition that it is characterized in that said pulsating ultrasound is output 100 W, 2 s that work, intermittently 2 s.
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