CN103751795A - Preparation and application of hyaluronic acid-antitumor drug conjugate and composite nanoparticle composition - Google Patents
Preparation and application of hyaluronic acid-antitumor drug conjugate and composite nanoparticle composition Download PDFInfo
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- CN103751795A CN103751795A CN201310186800.2A CN201310186800A CN103751795A CN 103751795 A CN103751795 A CN 103751795A CN 201310186800 A CN201310186800 A CN 201310186800A CN 103751795 A CN103751795 A CN 103751795A
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- Prior art keywords
- hyaluronic acid
- antineoplastic agent
- linking arm
- conjugate
- antitumor drug
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Abstract
The present invention relates to a preparation method and an application of a hyaluronic acid-antitumor drug conjugate and composite nanoparticle composition with characteristics of active targeting antitumor effect and biodegradability. The preparation method is characterized by comprising: (1) a synthesis method for conjugating an antitumor drug and a spacer and conjugating a targeting ligand hyaluronic acid or an ammonium salt thereof and the antitumor drug-spacer; and (2) a new technology for assembling the hyaluronic acid-antitumor drug conjugate and amphiphilic polyester block copolymer composite nanoparticles. The composition has effects of substantially increased drug loading, efficacy improving, in vivo long-circulating effect achievement, active tumor targeting property and drug toxic-side effect reduction. The macromolecular conjugate and complex nanoparticle composition can be used for injection administration, oral administration or mucosal administration. In addition, the preparation method has characteristics of mature process and high yield, and is suitable for industrial production.
Description
Technical field
The present invention relates to chemical field, pharmaceutic adjuvant or field of pharmaceutical preparations.Be specifically related to the preparation method of hyaluronic acid-antitumor drug conjugate and complex nanometer granule.Comprise antitumor drug and linking arm coupling, the preparation method of target ligand hyaluronic acid or its ammonium salt and antitumor drug-linking arm; The invention still further relates to the assembling new technique with active target head hyaluronic acid-antitumor drug conjugate and amphipathic ester block copolymer composite nano-granule.
Background technology
Cancer has become one of principal disease of serious threat human life health.Due to cancer early discovery and comparatively difficulty of diagnosis, chemotherapy plays an important role clinically, but still has variety of issue and facing the challenge, as poorly soluble in antitumor drug, toxic and side effects large and the problem such as drug resistance.Along with developing rapidly of oncology, molecular biology and high molecular polymer, the targeting of antitumor drug carries administration to become study hotspot.Polymer-drug conjugates (Polymer-drug delivery system, PDDS) has many advantages, has developed into the novel field of drug delivery system.
Polymer-antineoplastic agent conjugate is that small-molecule drug is connected with water-soluble polymer by covalent bond, drug conjugates can significantly improve the dissolubility of insoluble drug, polymer is hydrolyzed or enzymatic degradation by simple in body circulation, half-life in extension body in blood plasma, thereby arrive target site performance drug effect, the particularly important is antitumor conjugate and there is EPR effect and can accumulate at tumor locus.Main feature has: (1) improves the water solublity of insoluble drug; (2) improve medicine tumor-targeting prodrug composite nano-granule and there is nano-scale, by the EPR effect of tumor, concentrate in tumor tissues, there is passive targeting; The nucleocapsid structure of composite nano-granule is versatile and flexible, can connect at macromolecule surface-part, as glycosyl (HA), peptidyl, folic acid, antibody etc., by ligand molecular and the effect of target cell surface receptor molecular specific, strengthen its specific recognition to tissue and organ, realize initiatively targeting; (3) the physically trapping nano-micelle system initial stage release And Spread of Solute of dashing forward is avoided in the stable transhipment that realizes medicine, medicine is taken to target site and realizes intelligent release, thereby reach the effect that strengthens drug effect, lowers toxic and side effects.
Adopt biocompatible polymer material and PTX coupling, building targeted drug transmission system (DDS) has become current study hotspot.Conjugate Chinese medicine molecule combines with main polymer chain by covalent bond, in aqueous medium, form hud typed micelle, can increase drug solubility, improve bioavailability, stability in blood circulation, avoid medicine to arrive target site front by enzymes metabolism, inactivation, medicine is transported to target site to greatest extent, reduces the toxic and side effects of medicine.
HA is water-soluble natural macromolecule polysaccharide, has multi-functional physical chemistry and biological property, and as biocompatibility, biodegradability and immunogenicity, can be absorbed by specific cancer cell selectivity extensive use diversification carrier system.In strand, contain a large amount of active groups, as carboxyl, hydroxyl, N-acetyl group and reduction end all can be to its chemical modifications.Main method of modifying has the reactions such as esterification, crosslinked, grafting, and wherein hydroxyl and carboxyl are common covalent modification sites.Polymer-drug conjugates has enough functional groups, thereby can carry out chemical coupling.
Most of malignant tumor and around matrix group are woven with the hyaluronic acid (HA) of high concentration, and overexpression HA receptor CD44 or RAHMM, as distortion galactophore epithelial cell, ovarian tumor cell, colon cancer, pulmonary carcinoma, acute leukemia cells etc.The conjugate building take HA as part can, by collaborative to passive target and receptor-mediated active targeting, make medicine orientation be gathered in tumor locus, changes ingestion of medicines mechanism, reaches initiatively targeting.
Chytil etc. form macromole conjugate (HPMA-DOX) by hydrazone key by amycin (DOX) and large hydrophilic molecular HPMA covalent coupling, vitro cytotoxicity experiment shows, HPMA-DOX is to mouse T cell lymphoma EL4 cell, mouse B cell lymphoma 38C13 cell, the toxicity of human large intestine cancer SW620 cell etc. all free amycin is strong, pharmacodynamic study shows its antitumor curative effect [Chytil that increases in body, P., Etrych, T., Konak, C., Sirova, M., Mrkvan, T., Rihova, B., Ulbrich, K., Properties of HPMA copolymer-doxorubicin conjugates with pH-controlled activation:effect of polymer chain modification.J.Controlled Release.2006, 115 (1), 26-36.].A kind of hyaluronic acid-hydroxy camptothecin (HA-CPT) conjugate has been synthesized in the designs such as Norbedo Stefano, its toxicity to HT-29 cell, H460 cell and H460-M2 cell is obviously strong compared with irinotecan, adopt hyaluronic acid decorated hydroxy camptothecin, can increase the combination of medicine and cell, thereby improve cellular uptake [WO2009074678A2].
In recent years, amphipathic polyester has been a great concern as pharmaceutical carrier, and the hydrophobic core of internal layer is as non-covalent vesicle, and outer field hydrophilic shell prevents protein adsorption, it can self assembly nucleation-shell nanoparticle, thereby avoids being absorbed by non-specific reticuloendothelial system (RES).By by hydrophilic PEG and hydrophobic group synthetic copolymer, as with polylactic acid (PLA), polycaprolactone (PCL), polylactic-co-glycolic acid (PLGA) chemical coupling block copolymer.PEGization strategy can reduce liver uptake ratio, improves tumor-targeting.PEGization HA nanoparticle can shield HA surface in cyclic process, by absorbing front exposure HA target spot, is more conducive to improve the therapeutical effect of tumour medicine.Based on the drug delivery system of HA, for the treatment of some tumors, there is great potential using value, Novel Drug Delivery Systems provides theoretical foundation to the clinical practice of anticarcinogen.
Prodrug layout strategy take paclitaxel as parent, for the wide clinical application of paclitaxel provides new opportunity.The present invention is take targeting part hyaluronic acid as water solublity framework material, by different linking arms, antitumor drug is become to amphipathic polymer-anticarcinogen conjugate as paclitaxel, Docetaxel, amycin, camptothecine with hyaluronic acid coupling, adopt self-assembling technique to be prepared by macromole conjugate and amphipathic nature block polymer to the targeting drug delivery system of Novel composite nano grain, optionally target tumor position, improve the interior characteristics of pharmacokinetics of body of chemotherapeutics, reduce toxic and side effects, holdup time in extension body, improve antitumor curative effect.
Summary of the invention
Another object of the present invention is to provide the composite nano-granule Novel Drug Delivery Systems that a kind of above-mentioned macromole conjugate is combined with block copolymer, avoids engulfing and renal excretion of reticuloendothelial system, the holdup time in extension body.
For achieving the above object, the present invention is realized by the following technical programs.Utilize amphipathic PEG block copolymer and tumor targeting macromolecule conjugate to carry out self assembly and form novel composite Nano kernel structure.Hydrophilic segment PEG, by decorated nanometer grain system, has long circulating action and avoids the function of reticuloendothelial system phagocytic, and hydrophobic chain segment can insert nanoparticle core core, forms more stable nanoparticle drug-supplying system.This Multifunction composite nano-granule drug-supplying system, can improve blood stability, improves tumor-targeting, reduces toxic and side effects, the holdup time in extension body, strengthen antitumor curative effect, and be more suitable for clinical quoting, for anticancer strategy provides new Research Thinking.
The invention provides a kind of or serial macromole hyaluronic acid-antineoplastic agent conjugate and the composite nano-granule compositions with tumor-targeting and multi-function action, it is characterized in that: this conjugate is active group hydroxyl, amino and hydroxyl, amino or carboxyl (the OH ,-NH that utilizes antitumor drug
2with-OH ,-NH
2,-COOH), by linking arm, form antineoplastic agent-linking arm intermediate product, the hydroxyl of hyaluronic acid or its ammonium salt or carboxyl react with intermediate product, by covalent bonds, form hyaluronic acid-antineoplastic agent conjugate.
Hyaluronic acid-antineoplastic agent conjugate schematic arrangement is as Fig. 1;
The object of the invention is hyaluronic acid-antineoplastic agent conjugate to be assembled into nanoparticle, prior object is, by self-assembling technique, hyaluronic acid-antineoplastic agent conjugate and amphipathic nature block polymer are assembled into multi-functional composite nano-granule by electrostatic interaction, and described multi-functional composite nano-granule structural representation is as Fig. 2;
Wherein: Spacer is linking arm, AB, ABA, ABC are amphipathic-polyester block copolymer; Amphipathic group is Polyethylene Glycol, single methoxy ether Polyethylene Glycol, polyamino acid;
The combination weight of hyaluronic acid-antineoplastic agent conjugate and amphipathic ester copolymer is: 1: 0.2～5, and the weight ratio of hyaluronic acid and antineoplastic agent coupling is 1: 0.05～1;
The mol ratio of hyaluronic acid and antitumor drug is 1: 20～1: 1, and hyaluronic acid substitution value is 3%～48%; Preferred molar ratio 1: 5～1: 2, preferably clear matter acid substitution value is 12%～35%.
The linking arm (Spacer) of described hyaluronic acid and antineoplastic agent is the carboxylic acid that dicarboxylic anhydride, dicarboxylic acids, end have amino or hydroxyl; For the antitumor drug that contains amino or carboxyl, select end to have the linking arm of carboxyl to connect to form ester bond or amido link, for the antitumor drug that contains carboxyl, select to have the linking arm of hydroxyl or primary amino radical to form ester bond or amido link; Described link arm is selected from aminoacid, succinic anhydrides, cis equisetic acid, the aconitic anhydride of taking advantage of a situation, hemisuccinic acid ester;
It is Pidolidone, ALANINE, METHIONINE, L-Phe, L-Leu, ILE, L-PROLINE, 1B or glycine that described aminoacid is selected aminoacid.
There is hyaluronic acid-antineoplastic agent conjugate of tumor-targeting, it is characterized in that, described antitumor drug is mainly paclitaxel, Docetaxel, amycin, daunorubicin, epirubicin, camptothecin, methotrexate, mitoxantrone or derivatives thereof, preferably paclitaxel, Docetaxel, amycin, camptothecin.
There is hyaluronic acid-antineoplastic agent conjugate of tumor-targeting, it is characterized in that, described hyaluronan molecule amount is 4500～580000, hyaluronic acid or hyaluronic acid are treated that ammonium salt is selected from hyaluronic acid-TBAH salt (HA-TBA), hyaluronic acid-cetyl trimethyl ammonium bromide salt (HA-CTA), hyaluronic acid-adipic dihydrazide (HA-ADH), and the preparation method of its ammonium salt is as follows:
A. taking hyaluronate sodium, to be dissolved in pure water appropriate, and the hydrochloric acid that adds 0.1mol/L carries out acidify, and through cation exchange resin processing, lyophilization, obtains hyaluronic acid (HA).
B.HA-TBA: take hyaluronate sodium appropriate, be dissolved in the hydrochloric acid solution of 0.1mol/L, acidification 15h under 4 ℃ of conditions.Add TBAH (TBA) and regulate pH to 8.0, the dialysis treatment removal of impurity in 3 days, lyophilization, obtains HA-TBA.
C.HA-CTA: take cetyl trimethyl ammonium bromide (CTAB), 40 ℃ are dissolved in (a phase) in pure water; Separately take hyaluronate sodium and be dissolved in right amount pure water (b phase); A is dropped in b, and 40 ℃ of reaction 2h, separate out white precipitate, filter to obtain solid, and vacuum drying, obtains HA-CTA.
D.HA-ADH: hyaluronate sodium is dissolved in pure water, add adipic dihydrazide (ADH), with 0.05mol/L hydrochloric acid, solution being adjusted to pH is 4.75, add carbodiimide hydrochloride (EDCHCl), reaction 2～4h, adds 0.01mol/LNaOH and regulates pH to neutral, deionized water dialysis (MWCO=6000), lyophilization, obtains HA-ADH.
The preparation method with tumor-targeting and multi-functional hyaluronic acid-antineoplastic agent conjugate provided by the invention comprises that step is as follows:
A. contain antitumor drug-HA conjugate preparation of hydroxyl
(1) medicine-linking arm intermediate product: the antitumor drug that contains hydroxyl is dissolved in organic solvent, take Fmoc-aminoacid, dicarboxylic anhydride or half ester as linking arm or take Boc-aminoacid, dicarboxylic anhydride as linking arm, add catalyst and dehydrant to carry out Steglich reaction, temperature is controlled at 0～30 ℃, magnetic agitation reaction 0.5～50h, add inert organic solvents precipitation medicine-linking arm product, adopt column chromatography separation and purification intermediate product, obtain pharmacological activation-linking arm intermediate;
Hyaluronic acid or hyaluronic acid ammonium salt activator: hyaluronate sodium is dissolved in to the hydrochloric acid solution of 0.1mol/L, acidify obtains hyaluronic acid; Hyaluronic acid is added to TBAH or cetyl trimethyl ammonium bromide, reaction 0.25～4h, vacuum drying 2～4h, obtains hyaluronic acid ammonium salt activator;
(2) hyaluronic acid or hyaluronic acid ammonium salt activator and medicine-linking arm intermediate product are dissolved in to organic solvent, add catalyst and dehydrant, temperature is controlled at 10～50 ℃, magnetic agitation reaction 2～60h, reactant is put to bag filter, the removal of impurity in 3 days of deionized water dialysis treatment, lyophilization, obtains hyaluronic acid-antineoplastic agent conjugate; Or
B. contain antitumor drug-HA conjugate preparation of amino or hydroxyl
(1) antitumor drug that contains amino or hydroxyl is dissolved in organic solvent, adds 9-fluorenylmethyloxycarbonyl chlorine, react with aminoacid, dicarboxylic anhydride or half ester, add catalyst and dehydrant, obtain N-Fmoc-antitumor drug intermediate;
Hyaluronic acid or hyaluronic acid ammonium salt activator: hyaluronate sodium is dissolved in to solvent and acidify obtains hyaluronic acid; Hyaluronic acid is added to TBAH or cetyl trimethyl ammonium bromide, reaction 0.25～6h, lyophilization, obtains hyaluronic acid ammonium salt activator;
(2) derivatives of hyaluronic acids activator and medicine-linking arm intermediate product are dissolved in to organic solvent, add catalyst and dehydrant, add piperidines, temperature is controlled at 10～50 ℃, magnetic agitation reaction 2～60h.Adopt cross-linking dextran G-25 purified product, lyophilization, obtains hyaluronic acid-antineoplastic agent conjugate; Or
C. contain carboxyl antitumor drug-HA conjugate preparation
(1) antitumor drug that contains carboxyl is dissolved in solvent, the carboxylic acid that adopts Boc-aminoacid or end to have sulfydryl is linking arm, carries out Steglich reaction under the effect of catalyst and dehydrant, obtains antitumor drug-linking arm intermediate;
Hyaluronate sodium is dissolved in pure water, adds 0.05mol/L hydrochloric acid, obtain HA; Add adipic dihydrazide, add dehydrant reaction 0.25～6h, with 0.01mol/LNaOH adjusting pH, to neutral, deionized water is dialysed, and lyophilization, obtains hyaluronic acid-adipic dihydrazide activator;
(2) hyaluronic acid-adipic dihydrazide activator and medicine-linking arm intermediate product are dissolved in to organic solvent, effect at dehydrant and catalyst is reacted, temperature is 10～50 ℃, magnetic agitation reaction 2～60h, reactant is put to bag filter, the deionized water dialysis removal of impurity in 3 days, lyophilization, obtains hyaluronic acid-antitumor drug conjugate.
The preparation process of hyaluronic acid-antineoplastic agent conjugate, described dehydrant is selected from DIC (DIC), N, N-dicyclohexylcarbodiimide (DCC), 1-ethyl (3-dimethylamino-propyl) carbodiimide or carbodiimide analog derivative; Catalyst is selected from DMAP (DMAP), DMA, 1-hydroxyl phenylpropyl alcohol triazole, 4-pyrrolidinyl pyridine (PPY), piperidines, triethylamine;
Wherein, organic solvent is Methanamide, DMF, dimethyl sulfoxine, oxolane; Described inertia organic solvent is methanol, ethanol, ether, acetone.
There is hyaluronic acid-antineoplastic agent conjugate and the composite nano-granule compositions of tumor-targeting and multi-function action, it is characterized in that, described amphipathic-polyester is AB, ABA or ABC type di-block copolymer, triblock copolymer or graft copolymer, wherein di-block copolymer is selected from mPEG-PLA, mPEG-PCL, mPEG-PLGA, mPEG-PAA, and triblock copolymer is selected from mPEG-PLLA-PMMD, PLA-PEG-PLA, PCL-PEG-PCL, PLGA-PEG-PLGA or PAA-PEG-PAA.
Hyaluronic acid-antineoplastic agent conjugate and the composite nano-granule compositions with tumor-targeting and multi-function action of the present invention, is characterized in that, the preparation method of described composite nano-granule compositions is as follows:
(1) adopt solvent diffuse-film dispersion method-dialysis: hyaluronic acid-antineoplastic agent conjugate is dissolved or is dispersed in pure water, amphipathic polyester is dissolved in organic solvent, by above-mentioned biphase mixing magnetic agitation 0.5～4h, 30～50 ℃ of rotary evaporations are except organic solvent, Probe Ultrasonic Searching 5～60min, deionized water dialysis (MWCO is 6000), obtains composite nano-granule suspension;
(2) Freeze Drying Technique: composite nano-granule suspension is added to freeze drying protectant, and lyophilization, obtains composite nano-granule lyophilized powder;
Wherein said organic solvent is one or more mixed solvents of dichloromethane, chloroform, methanol, ethanol, acetonitrile, acetone etc.; Wherein described in dialysis, solvent is deionized water, purified water, and dialysis time is 12～120h, preferably 24～72h.
Described composite nano-granule freeze drying protectant is selected from one or more in mannitol, glucose, lactose, sucrose or trehalose.
Hyaluronic acid-antineoplastic agent conjugate provided by the present invention and composite nano-granule compositions; it is characterized in that; described hyaluronic acid-antineoplastic agent conjugate or assemble single nanoparticle or be assembled into composite nano-granule with amphipathic polyester; there is the anticarcinogen of improvement dissolubility; drug loading is greater than 12%; maximum drug loading is 35%, and mean diameter is less than 200nm, the multiple advantage such as good stability.Can biodegradation in body, this composite nano-granule, can avoid engulfing and the excretion of kidney of reticuloendothelial system (RES), has long circulating action, and in body, drug effect is apparently higher than the agent of antineoplastic agent normal injection.Its clinically acceptable route of administration have drug administration by injection, oral administration or mucosal drug delivery; Dosage form is injection, infusion solution, frozen powder for injection injection, tablet, capsule or powder spray.
Hyaluronic acid-antineoplastic agent conjugate provided by the present invention and composite nano-granule compositions, is characterized in that this macromole conjugate nanoparticle or composite nano-granule can be used for treating the single or drug combination of the malignant tumor such as nonsmall-cell lung cancer, breast carcinoma, ovarian cancer, cervical cancer, gastric cancer, hepatocarcinoma, leukemia.Novel Drug Delivery Systems provides experiment basis and theoretical foundation to the clinical practice of anticarcinogen, has great clinical meaning.
Accompanying drawing explanation
Fig. 1 is hyaluronic acid-antineoplastic agent conjugate schematic arrangement
Fig. 2 is the multi-functional composite nano-granule structural representation of paclitaxel
Fig. 3 is paclitaxel injection (PTX-injection), hyaluronic acid-paclitaxel conjugate (HA-PTX) and composite nano-granule (mPP-HA-PTX-NPs) cytotoxicity experiment figure (n=3)
Fig. 4 is the external enzymolysis lab diagram of hyaluronic acid-paclitaxel composite nano-granule (n=3)
The specific embodiment
Embodiment 1
Hyaluronic acid-paclitaxel conjugate (HA-PTX) preparation (linking arm is valine)
(1) 100mg fluorenylmethyloxycarbonyl-valine (Fmoc-Val) and 170mg paclitaxel (PTX) are dissolved in dichloromethane, add 24mg dimethylamino naphthyridine (DMAP) and 46mg carbodiimide hydrochloride (EDCHCl), ice bath reaction 1h, stirs 8h.With hydrochloric acid solution and the deionized water wash reactant liquor of 0.05mol/L, use anhydrous magnesium sulfate drying organic layer, filter, concentrate, add ketopyrrolidine dichloromethane solution, stir 3h.Vacuum drying obtains crude product.Adopt column chromatography purification product, obtain 2 '-valyl paclitaxel (2 '-Val-PTX).
(2) hyaluronate sodium is dissolved in purified water, cation exchange resin processing, lyophilization obtains hyaluronic acid (HA).600mg hyaluronic acid (HA) is dissolved in Methanamide; add 38.4mg carbodiimide hydrochloride (EDCHCl) and 23mgN-N-Hydroxysuccinimide (NHS); room temperature activation 30min; 2 '-Val-PTX is dissolved in appropriate DMF; under nitrogen current protection, added in HA solution; stir 24h; add acetone except intermediate; 4 ℃ of standing 1h; sucking filtration, redissolves and gets precipitation, dialysis treatment 72h (MWCO=6000) with deionized water; lyophilization, obtains hyaluronic acid-paclitaxel conjugate (HA-PTX).
Through proton nmr spectra
1h NMR and infrared FT-IR identify its structure:
1H?NMR(400Hz，D
20)：8.12(dd，J=7.08，1.44，2H)，7.75(dd，J=7.08，1.44，2H)，7.61(s，1H)，7.58-7.49(m，4H)，7.47-7.32(m，6H)，7.13(d，9.21H
2，1H)，6.30(s，1H)，6.24(t，J=9.0，1H)，6.03(dd，J=92H
2，3.5H
2，1H)，5.67(d，J=7.11，1H)，5.54(d，J=3.6H2，1H，C2)，4.98(dd，J=9.57，1.44，1H)，4.45(m，1H)，4.30(d，J=8.46，1H)，4.21(d，J=8.46，1H)，3.81(d，J=6.9H2，1H)，3.73(m，1H，α-Met)，2.59(m，2H，γ-Met)，2.38(s，3H)，2.23(s，3H)，2.12(m，2H，β-Met)，2.01(s，3H，δ-Met)，1.95(s，3H)，1.68(s，3H)，1.24(s，3H)，1.15(s，3H)。
FT-IR：3423cm
-1、2961cm
-1、2927cm
-1、1655cm
-1、1490cm
-1、1155cm
-1、1082cm
-1、1048cm
-1、949cm
-1、711cm
-1。
Embodiment 2
Hyaluronic acid-paclitaxel conjugate (HA-PTX) preparation (linking arm is methionine)
(1) 110mg fluorenylmethyloxycarbonyl-methionine (Fmoc-Met) and 170mg paclitaxel (PTX) are dissolved in dichloromethane, add 24mg dimethylamino naphthyridine (DMAP) and 46mg carbodiimide hydrochloride (EDCHCl), ice bath reaction 1h, stirring at room 8h.By hydrochloric acid solution and the deionized water wash reactant liquor of 0.05mol/L, anhydrous magnesium sulfate drying organic layer use, filter, concentrate, add ethanolamine dichloromethane solution, stir 4h.Vacuum drying obtains crude product, adopts column chromatography purification product, obtains 2 '-methionyl paclitaxel (2 '-Met-PTX).
(2) take cetyl trimethyl ammonium bromide (CTAB), under 40 ℃ of conditions, be dissolved in (A phase) in purified water; Separately take hyaluronate sodium and be dissolved in purified water (B phase); 40 ℃ add to A in B, and reaction 2h, filters, and obtains white solid, with hot water injection, precipitates 2～3 times, and vacuum drying, obtains HA-CTA.1.2g HA-CTA is dissolved in DMF; add 38.4mg carbodiimide hydrochloride (EDCHCl) and 23mg N-hydroxy-succinamide (NHS); activation 30min; 2 '-Met-PTX is dissolved in DMF; under nitrogen current protection, added in HA-CTA; stir 24h; add acetone to remove intermediate; place 1h for 4 ℃; sucking filtration, redissolves and gets precipitation, dialysis treatment 72h (MWCO=6000) with deionized water; lyophilization, obtains hyaluronic acid-paclitaxel conjugate (HA-PTX).Through proton nmr spectra
1h NMR and infrared FT-IR identify its structure.
Embodiment 3
Hyaluronic acid-Docetaxel conjugate (HA-DTX) preparation (linking arm is leucine)
(1) 105mg fluorenylmethyloxycarbonyl-leucine (Fmoc-Leu) and 160mg Docetaxel (DTX) are dissolved in dichloromethane, add 24mg dimethylamino naphthyridine (DMAP) and 46mg carbodiimide hydrochloride (EDCHCl), condition of ice bath reaction 1h, stirring at room 8h.Use respectively hydrochloric acid solution and the deionized water wash of 0.05mol/L, anhydrous magnesium sulfate drying, filters, concentrates, and adds strong aqua ammonia dioxane/4M NaOH (30: 9: 1), stirs 2h.Vacuum drying, obtains crude product, adopts column chromatography purification product, obtains 2 '-leucyl paclitaxel (2 '-Leu-DTX).Through proton nmr spectra
1h NMR and infrared FTIR identify its structure.
(2) hyaluronate sodium is dissolved in 0.1mol/L hydrochloric acid solution to 4 ℃ of acidify 15h.Add TBA (TBAH) and regulate pH to 8.0, dialysis treatment 3 days, the removal of impurity, lyophilization, obtains product HA-TBA.1.0g HA-TBA is dissolved in DMF, adds 38.4mg carbodiimide hydrochloride (EDCHCl) and 23mgN-N-Hydroxysuccinimide (NHS), ice bath activation 30min.The DMF solution of 2 '-Leu-PTX is added in HA-TBA solution, and 35 ℃ are stirred 24h, deionized water dialysis treatment 72h (MWCO=6000), and lyophilization, obtains hyaluronic acid-paclitaxel conjugate (HA-DTX).Through proton nmr spectra
1h NMR and infrared FT-IR identify its structure.
Embodiment 4
Hyaluronic acid-amycin conjugate (HA-DOX) preparation (linking arm is cis-aconitic acid anhydride)
(1) by 50mg doxorubicin hydrochloride (DOX) and the molten (NaHCO of 8mg sodium bicarbonate
3) in purified water, stir; 29mg9-fluorenylmethyloxycarbonyl chlorine (9-Fmoc) is dissolved in ethyl acetate, adds to (pH is 7～8) in above-mentioned solution, add saturated NaHCO
3solution, reaction 6h, extracts ethyl acetate layer, recrystallization in 0.1% trifluoroacetic acid, ice ester rinses crystallization, and vacuum drying, obtains N-Fmoc-DOX.Added and contained in 36mg cis-aconitic acid anhydride (CAA), 12mg dimethylamino naphthyridine (DMAP) DMF, stir 12h, vacuum drying, adopt silicagel column to carry out purification, ethyl acetate: normal hexane (90: 10) eluting, obtains N-fluorenylmethyloxycarbonyl-13-Immuno toxin amycin (N-Fmoc-CA-DOX).Through proton nmr spectra
1hNMR and infrared FT-IR identify its structure.
(2) hyaluronic acid is prepared with embodiment 1.600mg hyaluronic acid (HA) is dissolved in to distilled water, add 16mg DIC (DIC) and 23mg N-hydroxy-succinamide (NHS), ice bath activation 30min, under 50 ℃ of conditions, add N-Fmoc-DOX-CA product, 50 ℃ are stirred 12h, add piperidines appropriate, stir 2h.Adopt cross-linking dextran G-25 purified product, obtain hyaluronic acid-A can mycin conjugate (HA-DOX).Through proton nmr spectra
1h NMR and infrared FT-IR identify its structure.
Embodiment 5
Synthetic (linking arm is succinic anhydrides) of hyaluronic acid-amycin conjugate (HA-DOX)
(1) by 50mg doxorubicin hydrochloride (DOX) and 8mg sodium bicarbonate (NaHCO
3) be dissolved in purified water, stir; 29mg9-fluorenylmethyloxycarbonyl chlorine (9-Fmoc) is dissolved in ethyl acetate, drops to above-mentioned aqueous solution (pH is 7～8), add saturated NaHCO
3solution, reaction 6h, extracts ethyl acetate layer, recrystallization in 0.1% trifluoroacetic acid, ice ester rinses the residual 9-Fmoc of clean surface, and vacuum drying, obtains N-Fmoc-DOX.Be added to containing in 8mg succinic anhydrides (SA), 12mg dimethylamino naphthyridine (DMAP) DMF stirring at room 12h, vacuum drying.Adopt silicagel column to carry out purification, ethyl acetate: normal hexane (95: 5) eluting, obtains N-fluorenylmethyloxycarbonyl-13-succinyl amycin (N-Fmoc-SA-DOX).
(2) 600mg hyaluronic acid (HA) is dissolved in to purified water, add 38.4mg carbodiimide hydrochloride (EDCHCl) and 23mg N-hydroxy-succinamide (NHS), ice bath activation 30min, add N-Fmoc-SA-DOX, 50 ℃ are stirred 24h, add piperidines 3mL, stirring at room 2h.Adopt cross-linking dextran G-25 purified product, obtain hyaluronic acid-amycin conjugate (HA-DOX).Through proton nmr spectra
1h NMR and infrared FT-IR identify its structure.
Embodiment 6
Hyaluronic acid-camptothecine conjugate (HA-CPT) preparation (linking arm is hemisuccinic acid ester)
(1) 37mg hemisuccinic acid list tertiary butyl ester and 50mg camptothecine (CPT) are dissolved in dichloromethane, add 24mg dimethylamino naphthyridine (DMAP) and DIC, ice bath reaction 1h, room temperature magnetic agitation 8h.With hydrochloric acid solution and the deionized water wash reactant liquor of 0.05mol/L, anhydrous magnesium sulfate drying filters, and rotary evaporation is removed dichloromethane, residue adds acetone recrystallization, filters vacuum drying, obtain solids, add in the dichloromethane solution of the trifluoroacetic acid that contains 40% standing 1h, evaporation drying, add recrystallizing methanol, filter, vacuum drying, obtains 20-hemisuccinic acid ester-CPT.Through proton nmr spectra
1h NMR and infrared FT-IR identify its structure.
(2) HA-CTA is prepared with embodiment 2.1.2g hyaluronic acid-cetyl trimethyl ammonium bromide (HA-CTA) is dissolved in DMF, add 26.6mg N, N-dicyclohexylcarbodiimide (DCC) and 23mg N-hydroxy-succinamide (NHS), ice bath activation 30min.Add the DMF solution of 20-hemisuccinic acid ester-CPT to above-mentioned HA-CTA solution, 35 ℃ are stirred 24h, add saturated nacl aqueous solution, stir 30min, add 50mL dehydrated alcohol, filter, with after DMF and washing with alcohol, residue is dissolved in deionized water, dialysis purification (MWCO=6000), lyophilization, obtains hyaluronic acid-camptothecine conjugate (HA-CPT).Through proton nmr spectra
1h NMR and infrared FT-IR identify its structure.
Embodiment 7
Hyaluronic acid-camptothecine conjugate (HA-CPT) preparation (linking arm is the aconitic anhydride of taking advantage of a situation)
(1) 50mg camptothecine (CPT) and 55mg are taken advantage of a situation aconitic anhydride (CAA) is dissolved in dichloromethane; add 12mg dimethylamino naphthyridine (DMAP) and 23mg carbodiimide hydrochloride (EDCHCl); ice bath reaction 1h; be transferred to room temperature; under nitrogen protection, stir 24h; with 0.05mol/L hcl as extraction agent reactant liquor; rotation is steamed except dichloromethane; obtain solid product; silica gel column chromatography; dichloromethane: methanol (100: 1) purification, obtains cis-aconitic anhydride-camptothecine (CA-CPT).Through proton nmr spectra
1h NMR and infrared FT-IR identify its structure.
(2) hyaluronic acid is prepared with embodiment 1.Getting 600mg hyaluronic acid (HA) is dissolved in distilled water, add 260mg adipic dihydrazide (ADH), with 0.05mol/L hydrochloric acid, adjusting pH is 4.75, add 50mg carbodiimide hydrochloride (EDCHCl), room temperature reaction 2.5h, adds 0.01mol/LNaOH and regulates pH to neutral, deionized water dialysis, lyophilization, obtains HA-ADH.HA-ADH is dissolved in to DMF, add CA-CPT, 38.4mg carbodiimide hydrochloride (EDCHCl) and 23mg N-hydroxy-succinamide (NHS), the lower 35 ℃ of activation 30min of N2, magnetic agitation 24h, deionized water dialysis treatment 72h (MWCO=6000), lyophilization, obtains hyaluronic acid-paclitaxel conjugate (HA-CPT).Through proton nmr spectra
1hNMR and infrared FT-IR identify its structure.
Embodiment 8
The preparation of hyaluronic acid-paclitaxel and amphipathic polyester composite nano-granule
40mg HA-Met-PTX conjugate is scattered in purified water, add in the acetonitrile solution of 40mg mPEG-PLA, stirring at room 24h, Probe Ultrasonic Searching 20min, 37 ℃ of rotary evaporations are removed acetonitrile, Probe Ultrasonic Searching 10min, obtains mPEG-PLA-HA-PTX composite nano-granule (mPP-HA-PTX-NPs).Adopt ZetaSizer laser particle size analyzer to measure particle size distribution and Zeta potential.Its particle diameter is 146.2 ± 7.8nm, and PI is 0.12, and Zeta potential is-26.7mV.
Embodiment 9
The preparation of hyaluronic acid-Docetaxel and amphipathic polyester composite nano-granule
40mg HA-DTX is dissolved in purified water water, getting 20mg mPEG-PLGA is dissolved in dichloromethane, both mix, stir 3h, Probe Ultrasonic Searching 30min, 37 ℃ of hydration 2h, rotary evaporation is removed dichloromethane, Probe Ultrasonic Searching 10min, obtains mPEG-PLGA-HA-DTX composite nano-granule (mPP-HA-DTX-NPs).Adopt ZetaSizer laser particle size analyzer to measure particle size distribution and Zeta potential.Result shows, its mean diameter is 238.9 ± 12.3nm, and PI is 0.25, and Zeta potential is-21.7mV.
Embodiment 10
The preparation of hyaluronic acid-amycin and amphipathic polyester composite nano-granule
40mg HA-DOX is dissolved in distilled water, separately getting 40mg mPEG-PCL is dissolved in chloroform, both mix, stir 3h, Probe Ultrasonic Searching 15min, 37 ℃ of hydration 1h, rotary evaporation is removed chloroform, Probe Ultrasonic Searching 20min, obtains mPEG-PCL-HA-DOX composite nano-granule (mPP-HA-DOX-NPs).Adopt ZetaSizer laser particle size analyzer to measure particle diameter and Zeta potential, its mean diameter is 184.2 ± 9.3nm, and PI is 0.18, and Zeta potential is-22.7mV.
Embodiment 11
The external enzymolysis experiment of hyaluronic acid-paclitaxel composite nano-granule
HA-PTX conjugate is dissolved in to normal saline in right amount, crosses 0.8 μ m filter membrane, add hyaluronidase (50IU/mL) and esterase (20IU/mL), be placed in bag filter and dialyse.With 0.2% Tween 80 phosphate buffer (PBS, pH5.0, pH6.8 and pH7.4), it is release medium.Bag filter is placed in to release medium, 37 ℃ of water-bath vibration dialysis treatment (MWCO=3000), respectively at 0.5,1,2,4,8,12,24,48,72h samples 5mL, add equivalent medium simultaneously, adopt ultraviolet spectrophotometer 230nm place working sample absorbance, investigate the enzymolysis behavior of HA-PTX conjugate under different pH condition.
Result as shown in Figure 4.Under the effect of hyaluronidase and esterase, HA-PTX can reach and discharge fast paclitaxel, and the burst size of dissociating respectively in 3 days is 82.1% (pH7.4), 90.3% (pH6.8) and 96.7% (pH5.0).The fastest in acid condition (pH5.0) hydrolysis, 24h is 90% enzymolysis; With pH value, raise, enzymolysis speed slows down, and illustrates that hyaluronidase is active best under pH5.0 condition.Result shows that HA-PTX is easy to enzymolysis at the tumor tissues position of slant acidity, is conducive to the performance of antitumor action.
Embodiment 12
Vitro cytotoxicity research
The take the logarithm non-small cell lung cancer cell strain A549 cell of trophophase, 1 × 10
4individual/hole is inoculated in 96 orifice plates, cultivates after 24h, and every hole adds the medicine (PTX injection, HA-PTX and mPP-HA-PTX-NPs) of respective concentration, respectively organizes sample paclitaxel concentration and is respectively 0.1,1,5,10,20 μ g/mL.Continue to hatch 72h, add MTT solution (1mg/mL) 0.1mL, hatch after 4h, absorb supernatant in hole, each hole adds DMSO100 μ L, in microplate reader working sample absorbance (OD for 570nm
test).And measure in the same way the absorbance of blank group (acellular strain) and matched group (n.s), be designated as respectively OD
blankand OD
control.Calculate according to the following equation cell strain survival rate, the cytotoxicity of assess sample.
As shown in Figure 3, along with the increase of concentration, the cell survival rate of PTX injection, HA-PTX and mPP-HA-PTX-NPs all declines result, shows to have the cytotoxicity of concentration dependent, is concentration-effect relation, its IC
50value is respectively 13.71 ± 1.09 μ g/mL, 3.02 ± 0.25 μ g/mL and 6.52 ± 0.51 μ g/mL.In 0.1～20 μ g/mL concentration range, the ability of three kinds of preparation inhibition A549 Growth of Cells has significant difference (P<0.01), HA-PTX and mPP-HA-PTX-NPs have stronger cytotoxicity to A549, the modification of HA can make nanoparticle enter in a large number cell by the receptor-mediated endocytosis of CD44, make HA-PTX-NPs under the effect of desmoenzyme, to degrade and to dissociate active pharmaceutical ingredient, performance antitumor curative effect.The anti tumor activity in vitro of mPP-HA-PTX-NPs is lower than HA-PTX-NPs, active in impact on the anti-tumor in vivo of mPP-HA-TPX-NPs.
Embodiment 13
Pharmacodynamic study in body
Experimental program: S180 mice with tumor is divided into 4 groups at random, every group 5, tail vein injection saline (0.15mL), PTX injection (7.5mg/kg), HA-PTX (dosage 7.5mg/kg) and mPP-HA-PTX-NPs (dosage 7.5mg/kg) respectively, respectively at tail vein injection administration on the 0th, 2,4,6,8.In the 9th day, mice is put to death, strip separation tumor, weigh, calculate tumour inhibiting rate, carry out pharmacodynamic evaluation.
The pharmacodynamic evaluation (n=5) of the different formulation for paclitaxel of table 1.
By table 1, illustrated, HA-PTX and mPP-HA-PTX-NPs have obvious inhibition to S180 mice with tumor, and Mice Body weight average has phenomenal growth, show that toxicity significantly declines.MPP-HA-PTX-NPs has better anti-tumor in vivo activity and lower toxicity (mPP-HA-PTX-NPs weight of mice is organized high by 11.28% than HA-PTX) compared with HA-PTX.Pharmacodynamics in vitro experiment deducibility PEG-polyester can increase the stability of composite nano-granule.MPP-HA-PTX-NPs can strengthen tumor-targeting, reduces toxic and side effects, improves antitumor drug effect.
Claims (10)
1. there is hyaluronic acid-antineoplastic agent conjugate and the composite nano-granule compositions of tumor-targeting and multi-function action, it is characterized in that: this conjugate is active group hydroxyl, amino and hydroxyl, amino or the carboxyl that utilizes antitumor drug, by linking arm, form antineoplastic agent-linking arm intermediate product, the hydroxyl of hyaluronic acid or its ammonium salt or carboxyl react with intermediate product, by covalent bonds, form hyaluronic acid-antineoplastic agent conjugate;
The structural representation that described hyaluronic acid-antineoplastic agent conjugate and amphipathic nature block polymer self assembly are multi-functional composite nano-granule is as follows:
Wherein: Spacer is linking arm, AB, ABA, ABC are amphipathic-polyester block copolymer, and amphipathic group is Polyethylene Glycol, single methoxy ether Polyethylene Glycol, polyamino acid;
The combination weight of hyaluronic acid-antineoplastic agent conjugate and amphipathic ester copolymer is: 1: 0.2～5, and the weight ratio of hyaluronic acid and antineoplastic agent coupling is 1: 0.05～1;
The rate of charge of hyaluronic acid and antitumor drug is 1: 20～1: 1, and hyaluronic acid substitution value is 3%～48%; Preferred molar ratio 1: 5～1: 2, preferably clear matter acid substitution value is 12%～35%.
2. hyaluronic acid-antineoplastic agent conjugate according to claim 1, is characterized in that, described linking arm is the carboxylic acid that dicarboxylic anhydride, dicarboxylic acids, end have amino or hydroxyl; For the antitumor drug that contains amino or carboxyl, select end to have the linking arm of carboxyl to connect to form ester bond or amido link, for the antitumor drug that contains carboxyl, select to have the linking arm of hydroxyl or primary amino radical to form ester bond or amido link;
Described linking arm is selected aminoacid, succinic anhydrides, cis equisetic acid, the aconitic anhydride of taking advantage of a situation, hemisuccinic acid ester;
It is Pidolidone, ALANINE, METHIONINE, L-Phe, L-Leu, ILE, L-PROLINE, 1B or L-glycine that described aminoacid is selected aminoacid.
3. hyaluronic acid-antineoplastic agent conjugate according to claim 1, is characterized in that described antitumor drug is paclitaxel, Docetaxel, amycin, daunorubicin, epirubicin, camptothecin, methotrexate, mitoxantrone or derivatives thereof.
4. hyaluronic acid-antineoplastic agent conjugate according to claim 1, it is characterized in that, described hyaluronan molecule amount is 4500～580000, and hyaluronic acid or hyaluronic acid are treated that ammonium salt is selected from hyaluronic acid-TBAH salt, hyaluronic acid-cetyl trimethyl ammonium bromide salt, hyaluronic acid-adipic dihydrazide.
5. described in claim 1～4, have tumor-targeting and multi-functional hyaluronic acid-antineoplastic agent conjugate, it is characterized in that, preparation process comprises the steps:
A. contain antitumor drug-HA conjugate preparation of hydroxyl
(1) medicine-linking arm intermediate product: the antitumor drug that contains hydroxyl is dissolved in organic solvent, take Fmoc-aminoacid, dicarboxylic anhydride or half ester as linking arm or take Boc-aminoacid, dicarboxylic anhydride as linking arm, add catalyst and dehydrant to carry out Steglich reaction, temperature is controlled at 0～30 ℃, magnetic agitation reaction 0.5～50h, add inert organic solvents precipitation medicine-linking arm product, adopt column chromatography separation and purification intermediate product, obtain pharmacological activation-linking arm intermediate;
Hyaluronic acid or hyaluronic acid ammonium salt activator: hyaluronate sodium is dissolved in to the hydrochloric acid solution of 0.1mol/L, acidify obtains hyaluronic acid; Hyaluronic acid is added to TBAH or cetyl trimethyl ammonium bromide, reaction 0.25～4h, vacuum drying 2～4h, obtains hyaluronic acid ammonium salt activator;
(2) hyaluronic acid or hyaluronic acid ammonium salt activator and medicine-linking arm intermediate product are dissolved in to organic solvent, add catalyst and dehydrant, temperature is controlled at 10～50 ℃, magnetic agitation reaction 2～60h, reactant is put to bag filter, the removal of impurity in 3 days of deionized water dialysis treatment, lyophilization, obtains hyaluronic acid-antineoplastic agent conjugate; Or
B. contain antitumor drug-HA conjugate preparation of amino or hydroxyl
(1) antitumor drug that contains amino or hydroxyl is dissolved in organic solvent, adds 9-fluorenylmethyloxycarbonyl chlorine, react with aminoacid, dicarboxylic anhydride or half ester, add catalyst and dehydrant, obtain N-Fmoc-antitumor drug intermediate;
Hyaluronic acid or hyaluronic acid ammonium salt activator: hyaluronate sodium is dissolved in to solvent and acidify obtains hyaluronic acid; Hyaluronic acid is added to TBAH or cetyl trimethyl ammonium bromide, reaction 0.25～6h, lyophilization, obtains hyaluronic acid ammonium salt activator;
(2) derivatives of hyaluronic acids activator and medicine-linking arm intermediate product are dissolved in to organic solvent, add catalyst and dehydrant, add piperidines, temperature is controlled at 10～50 ℃, magnetic agitation reaction 2～60h.Adopt cross-linking dextran G-25 purified product, lyophilization, obtains hyaluronic acid-antineoplastic agent conjugate; Or
C. contain carboxyl antitumor drug-HA conjugate preparation
(1) antitumor drug that contains carboxyl is dissolved in solvent, the carboxylic acid that adopts Boc-aminoacid or end to have sulfydryl is linking arm, carries out Steglich reaction under the effect of catalyst and dehydrant, obtains antitumor drug-linking arm intermediate;
Hyaluronate sodium is dissolved in pure water, adds 0.05mol/L hydrochloric acid, obtain HA; Add adipic dihydrazide, add dehydrant reaction 0.25～6h, with 0.01mol/LNaOH adjusting pH, to neutral, deionized water is dialysed, and lyophilization, obtains hyaluronic acid-adipic dihydrazide activator;
(2) hyaluronic acid-adipic dihydrazide activator and medicine-linking arm intermediate product are dissolved in to organic solvent, effect at dehydrant and catalyst is reacted, temperature is 10～50 ℃, magnetic agitation reaction 2～60h, reactant is put to bag filter, the deionized water dialysis removal of impurity in 3 days, lyophilization, obtains hyaluronic acid-antitumor drug conjugate.
6. according to hyaluronic acid-antineoplastic agent conjugate described in claim 1～5 with tumor-targeting and multi-function action, it is characterized in that, described conjugate is prepared described dehydrant and is selected from DIC, N, N-dicyclohexylcarbodiimide, 1-ethyl (3-dimethylamino-propyl) carbodiimide or carbodiimide analog derivative; Catalyst is selected from DMAP, DMA, 1-hydroxyl phenylpropyl alcohol triazole, 4-pyrrolidinyl pyridine, piperidines, triethylamine;
Wherein said organic solvent is Methanamide, DMF, dimethyl sulfoxine, oxolane; Described inertia organic solvent is one or both of methanol, ethanol, ether, acetone, acetonitrile.
7. there is according to claim 1 tumor-targeting and hyaluronic acid-antineoplastic agent conjugate and composite nano-granule compositions, it is characterized in that, described amphipathic-polyester is AB, ABA or ABC type di-block copolymer, triblock copolymer or graft copolymer, wherein di-block copolymer is selected from mPEG-PLA, mPEG-PCL, mPEG-PLGA, mPEG-PAA, and triblock copolymer is selected from mPEG-PLLA-PMMD, PLA-PEG-PLA, PCL-PEG-PCL, PLGA-PEG-PLGA or PAA-PEG-PAA; PMMD is polyalanine, polylysine, polyglycine, polyglutamic acid, and m is 500～20000.
8. according to having tumor-targeting and hyaluronic acid-antineoplastic agent conjugate and composite nano-granule compositions described in claim 1～7, it is characterized in that, the preparation method of described composite nano-granule compositions is as follows:
(1) adopt solvent diffuse-film dispersion method-dialysis: hyaluronic acid-antineoplastic agent conjugate is dissolved or is dispersed in pure water, amphipathic polyester is dissolved in organic solvent, by above-mentioned biphase mixing magnetic agitation 0.5～4h, 30～50 ℃ of rotary evaporations are except organic solvent, Probe Ultrasonic Searching 5～50min, deionized water dialysis, obtains composite nano-granule suspension;
(2) Freeze Drying Technique: composite nano-granule suspension is added to freeze drying protectant, and lyophilization, obtains composite nano-granule lyophilized powder;
Wherein said organic solvent is one or both mixed solvents of methanol, ethanol, acetonitrile, acetone, ether, dichloromethane, chloroform etc.;
Described composite nano-granule freeze drying protectant is selected from one or more in mannitol, glucose, lactose, sucrose or trehalose.
9. hyaluronic acid-antineoplastic agent conjugate and composite nano-granule compositions according to Claim 8, it is characterized in that, described hyaluronic acid-antineoplastic agent conjugate or assemble single nanoparticle or be assembled into composite nano-granule with amphipathic polyester, its clinically acceptable route of administration have drug administration by injection, oral administration or mucosa delivery; Dosage form is injection, infusion solution, frozen powder for injection injection, tablet, capsule or powder spray.
10. hyaluronic acid-antineoplastic agent conjugate and composite nano-granule compositions according to claim 8; it is characterized in that, this nanoparticle or composite nano-granule compositions can be used for treating the single or drug combination of the malignant tumor such as nonsmall-cell lung cancer, breast carcinoma, ovarian cancer, cervical cancer, gastric cancer, hepatocarcinoma, leukemia.
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| CN115487171A (en) * | 2022-11-10 | 2022-12-20 | 郑州大学 | Hyaluronic acid-isoliquiritigenin conjugate, dissolving type microneedle patch, preparation method and application |
| CN116763725B (en) * | 2023-08-16 | 2023-11-24 | 四川大学华西医院 | Intelligent response type injectable hydrogel and preparation method and application thereof |
| CN116763725A (en) * | 2023-08-16 | 2023-09-19 | 四川大学华西医院 | Intelligent response type injectable hydrogel and preparation method and application thereof |
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