CN103656653A - Polyelectrolyte compound based on hyaluronic acid drug-loading nano particles, preparation method and application thereof - Google Patents
Polyelectrolyte compound based on hyaluronic acid drug-loading nano particles, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a polyelectrolyte compound based on hyaluronic acid (HA) drug-loading nano particles, a preparation method and applications thereof. Hyaluronic acid base polymers coat and carry drugs so as to form electronegative nano particles, and then the nano particles compound with polycation static so as to form the polyelectrolyte compound, wherein the hyaluronic acid base polymers are prepared by subjecting hyaluronic acid to a hydrophobic modification treatment, namely a hydrophobic side chain is grafted to a carboxyl bond in the hyaluronic acid (HA) so as to form a grafted hyaluronic acid, wherein the mass content of the hydrophobic side chain is 10 to 60%, the number average molecular weight of the hydrophobic side chain is 200 to 20000, the ratio of the positive charge of the polycation to the negative charge of the hyaluronic acid base polymer is in a range of 1.5 to 6.0, the number average molecular weight of hyaluronic acid (HA) is 2000 to 200000, and the particle size of the compound is less than 500 nm. The compound is developed to promote the oral delivery effect of drugs and especially to improve the accumulation of anti-tumor drugs in the tumor sites, so as to improve the curative effect and reduce the toxic and side effect.
Description
Technical field
The present invention relates to a kind of compound polyelectrolyte and preparation method and application based on hyaluronic acid drug-carrying nanometer particle, belong to the application technology of macromolecular material in pharmaceutical preparation.
Background technology
Improve the oral administration biaavailability of insoluble drug and promote that medicine is still one of difficult problem of oral drug preparation through the absorption of intestinal mucosa.The main barrier that the oral digestive tract of dewatering medicament absorbs, mainly comes from the obstruction of the low aqueous solubility of medicine and the cell membrane of gastrointestinal epithelial cells.The nanoparticle of diameter <500nm, can, by the mode of simulation Chylomicron, realize the gastrointestinal epithelial cells that nanoparticle is complete and absorb.By the drug encapsulation technology of nanoparticulate carriers, hydrophobic drug is loaded in nanoparticle hydrophobic inner core, can strengthen the water solublity of medicine, but mucoadhesive is poor, cause its oral administration biaavailability always very low, as oral for dewatering medicament of (PLGA) or the amphipathic copolymer of PEG-polyester.Also have the nano-carrier design of pH sensitivity, carrying medicament carries out oral, and the lower protection medicine of stomach acidity environment (pH=1.0～3.0) does not discharge, and at the lower medicine that discharges of intestinal weakly alkaline environment (pH=7.4～8.0), promotes the intestinal absorption of medicine.But still there is the problem that is difficult to enter by intestinal mucosa body circulation in the drug molecule being discharged in intestinal environment.Chitosan, as a kind of natural cationic polysaccharide, has good intestinal mucosa adhesion, thereby is conducive to promote medicine through the absorption of intestinal mucosa.Utilize pH sensitive nanoparticles that chitosan and polyglutamic acid be compounded to form by static to be widely studied (Y.H.Lin for the oral research of the hydrophilic medicaments such as insulin, K.Sonaje, K.M.Lin, J.H.Juang, F.L.Mi, H.W.Yang, H.W.Sung, Journal of Controlled Release, 2008, 132:141-149), under pH3.0-6.0, two kinds of polyelectrolyte form nanoparticle, pharmaceutical pack is rolled in nanoparticle, nanoparticle disintegrate under pH7.4, thereby release medicine, but utilize the targeted orally-administered dewatering medicament of sending of polyelectrolyte composite nano-granule, so far rarely has report.Applicant has reported a kind of compound polyelectrolyte that contains hydrophobic drug (201210192591.8) in earlier stage, has the characteristic that pH sensitivity discharges drug-carrying nanometer particle and medicine, for the oral administration of dewatering medicament.The present invention is in this technical foundation, in order further to promote the oral delivery effect of medicine, especially improve antitumor drug in the gathering of tumor locus, prepare and evaluated based on hyaluronic drug-carrying nanometer particle, its structure is optimized, and prepares oral compound polyelectrolyte with the nanoparticle of the hyaluronic acid carrying medicament of structure optimization.The advantage that not only possesses compound polyelectrolyte described in document (CN201210192591.8), but also increased the selectively targeted effect of tumor cell CD44 that hyaluronic acid (HA) has, and utilize the degradability of hyaluronidase to HA in tumor tissues and cell, and then can promote complex to drug release function in the targeted delivery of antitumor drug and tumor cell, therefore the oral administration that, is particularly useful for antitumor drug.In addition, research is found, the characteristic that the complex in the present invention has colon position to assemble is suitable for colon targeting drug administration application.
Summary of the invention
The object of the present invention is to provide a kind of compound polyelectrolyte based on hyaluronic acid (HA) drug-carrying nanometer particle, this compound polyelectrolyte is applicable to the exploitation of multi-medicament oral formulations, the oral administration that is particularly useful for antitumor hydrophobic drug, has good application prospect.
The present invention is realized by the following technical programs:
A compound polyelectrolyte based on hyaluronic acid drug-carrying nanometer particle, so hyaluronic acid based polyalcohol bag medicine carrying thing forms electronegative nanoparticle, then the compound polyelectrolyte being compounded to form with polycation static; Hyaluronic acid based polyalcohol is the hyaluronic acid of hydrophobically modified, is the hyaluronic acid of the graft type that formed by the carboxyl keyed jointing hydrophobic side chain on hyaluronic acid HA, and wherein the mass content of hydrophobic side chain accounts for 10-60%; The number-average molecular weight of hydrophobic side chain is at 200-20000; In complex, the positive and negative charge of polycation and hyaluronic acid based polyalcohol is than between 1.5-6.0; The number-average molecular weight of HA is 2000-200000; The particle diameter of complex is less than 500nm.
The upper hydrophobic side chain of described HA is selected from degradable polyester, deoxycholic acid, cholesterol or enoxolone; During hydrophobic side chain number-average molecular weight 200-600, the upper hydrophobic side chain percent grafting of HA is 20-60%, and during hydrophobic side chain number-average molecular weight 600-20000, the upper hydrophobic side chain percent grafting of HA is 1-30%.
Described hydrophobic side chain is homopolymer or the copolymer degradable polyester of lactone, lactide; Lactone is the lactone that carries out ring-opening polymerization, and lactide is lactide or Acetic acid, hydroxy-, bimol. cyclic ester;
Described hydrophobic side chain lactone is caprolactone, Isosorbide-5-Nitrae, the homopolymer of 8-trioxa spiral shell [4.6]-9-hendecanone, P-Dioxane ketone or their copolymer; The copolymer that described own hydrophobic side chain lactone is lactone and TOSUO, the mass percent that TOSUO unit accounts for hydrophobic side chain is 5-30%.
Polycation in described complex is selected from chitosan and derivant, polylysine, cationic starch derivative, gelatin, cationic protein or polypeptide, cationic polyester, cation poly phosphate, polyvinyl pyrroles salt, N, homopolymer and the copolymer of (methyl) acrylate that N-replaces.
Described polycation is selected from chitosan, and the number-average molecular weight of chitosan is 2000-200000.
Compound polyelectrolyte of the present invention, wherein the hydrophobic drug of institute's load is to be suitable for oral water-insoluble drug; Preferably from antitumor drug, antibacterial, antiviral, anti-inflammatory type hydrophobic drug.
The medicine of institute's load is antitumor drug, is preferably paclitaxel, Docetaxel, amycin, hydroxy camptothecin or curcumin.
The preparation method of compound polyelectrolyte of the present invention, step is as follows:
1) the hydrophobically modified hyaluronic acid of formula ratio and dewatering medicament are dissolved into wiring solution-forming in cosolvent, then install in bag filter, with the PBS of pH7.4, dialyse to solvent and be removed completely, centrifugal, remove precipitate, obtain the hyaluronic nanoparticle aqueous dispersions of hydrophobically modified of load dewatering medicament;
2), by the further lyophilizing of nanoparticle aqueous dispersions, obtain the lyophilized powder of nanoparticle;
3) under stirring, 1) in the hyaluronic nanoparticle aqueous dispersions of hydrophobically modified or 2 of the carrying medicament that obtains) in the nano-granule freeze-dried powder redispersion that obtains to the nanoparticle aqueous dispersions forming in water, be added drop-wise in the aqueous solution that contains formula ratio polycation, carry out static compound, make the aqueous dispersions of the compound polyelectrolyte nanoparticle of load hydrophobic drug;
4) by 3) the aqueous dispersions lyophilizing of the compound polyelectrolyte nanoparticle prepared or mix lyophilizing again with lyophilizing stabilizing agent, obtain the lyophilized powder of compound polyelectrolyte; Lyophilizing stabilizing agent is selected from a kind of of mannitol, Polyethylene Glycol, glucose, lactose, dextran, sucrose, sodium chloride, Glycine sodium, sodium deoxycholate, sodium dihydrogen phosphate and sodium hydrogen phosphate, sorbitol or dextran or their combination in any.
The application of the compound polyelectrolyte that contains hydrophobic drug of the present invention, described complex is distributed to the aqueous humour forming in aqueous medium with nanoparticle state, or its lyophilized powder is loaded in capsule, or is prepared into tablet, as oral drug preparation; Or composite with other medicines or material, for the preparation of oral drug preparation.
Advantage of the present invention is the complex containing medicine being compounded to form by static; outside positive charge shell can be protected the stability of complex under stomach acidity environment; and complex dissociation discharges the hydrophobically modified HA nanoparticle of the medicine carrying including under the slight alkali environment of intestinal; by the saturating intestinal mucosa effect of nanoparticle; promote medicine through the absorption of intestinal mucosa; and the polycation shell dissociating has mucoadhesive, can prolong drug in time of staying of gastrointestinal mucosa.Therefore, be applicable to dewatering medicament oral administration, increase the bioavailability of hydrophobic drug, reduce and be administered to the misery that will bring to patient.
In addition, the tumor cell CD44 receptor-specific targeting that hyaluronic acid (HA) has, can promote the targeted delivery of complex to antitumor drug, and the Degradation of the hyaluronidase of tumor tissues and the interior higher concentration of cell to HA, can promote the release of medicine in tumor cell.Therefore, the technology of the present invention is particularly useful for the oral administration application of hydrophobic anticancer drug, is conducive to overcome hydrophobic anticancer drug oral administration biaavailability low, the problem that antitumous effect is poor.
Accompanying drawing explanation
Fig. 1. (A) PCL, the nuclear magnetic spectrogram of the HA-g-PCL of (B, C) embodiment 1 preparation in deuterated dimethyl sulfoxide and deuterated water.From spectrogram B, can calculate grafting segment molecule amount and percent grafting, in C, can't see the proton peak of hydrophobic section, prove that HA-g-PCL has formed the nanoparticle of hydrophobic section in core in water.
Fig. 2 A is that HAg-1 nanoparticle, Fig. 2 B are that HAg-1 compares particle size distribution and the TEM photo of the nanoparticle (II-1) for being compounded to form with chitosan (CS) under 4:1, pH=6 at positive and negative charge.HAg-1 is solid spherical, and II-1 has nucleocapsid structure, is respectively kernel and CS shell.
Fig. 3. under condition of different pH, the transmission electron microscope photo of composite nano-granule (II-1): (A) pH=1.2, (B) pH=5.0, (C) pH=5.8, (D) pH=6.8, (E) pH=7.4, the nanoparticle in the supernatant of (F) separating under pH=7.4.Show under acid condition, II-1 can keep stable spherical-like morphology and composite construction layer by layer.When under pH7.4 condition, outer field chitosan dissociates, and discharges the HAg-1 nanoparticle including.
The blank nanoparticle nanoparticle of HAg-13 of Fig. 4 (A) embodiment 13; (B) be paclitaxel loaded HAg-13 nanoparticle (II-16); (C) particle size distribution of chitosan (CS) and HAg-13 composite nano-granule (II-16) aqueous dispersions.
Fig. 5. the release of Complex II-1 nanoparticle under different pH, does not have hyaluronidase in medium.This is the drug release under simulation gastrointestinal HAg-1 road environment, visible, and Complex II-1 all discharges the seldom medicine of amount in gastrointestinal tract, keeps the stability of medicine in gastrointestinal tract.
The drug release curve of Fig. 6 .HAg-1 nanoparticle (HA-g-PCL/PTX) under different hyaluronic acid enzyme concentrations.Result shows, HAg-1 nanoparticle (HA-g-PCL/PTX) is under without hyaluronidase (Hyal-1), and drug release seldom.Under Hyal-1 exists, drug release rate increases rapidly, and along with Hyal-1 concentration increases and improves.
Fig. 7. after being the different preparation administrations of paclitaxel, mouse tumor growth curve.The negative matched group of normal saline (Saline), with paclitaxel clinical preparation (Taxol, taxol) the positive matched group of lumbar injection, hyaluronic acid g-polycaprolactone and chitosan composite nano-granule (CS/HA-g-PCL) are blank, and HAg-1 nanoparticle (HA-g-PCL/PTX) and II-1 complex nanometer granule (CS/HA-g-PCL/PTX) are test set.Negative control group does not have any antitumous effect, and tumor growth is obvious.In four groups of administration groups, the direct oral group of antitumous effect of formulation for paclitaxel is the poorest, illustrate that the direct oral administration biaavailability of paclitaxel solution is very low, formulation for paclitaxel lumbar injection can to a certain degree suppress the growth of tumor, do not there is any antitumous effect with the compound effect of nano-paclitaxel HAg-1 of chitosan, illustrate that not the HAg-1 nanoparticle solution direct oral administration biaavailability compound with chitosan is very low.Oral group of II-1 complex nanometer granule and the comparison of formulation for paclitaxel lumbar injection group, II-1 complex nanometer granule can significantly suppress the growth of gross tumor volume, presents better anti-tumor activity.
Mice survival rate after the different preparation lumbar injections of Fig. 8 paclitaxel and oral (gavage) administration.Because the toxic and side effects of formulation for paclitaxel is very strong, although formulation for paclitaxel is oral, than injection toxicity, reduce, but still have toxicity.II-1 complex nanometer granule group experimental session is without animal dead, and this proof can effectively reduce its toxic and side effects by the paclitaxel of hydrophobic interaction load, extends the time-to-live of animal.
The specific embodiment
A kind ofly based on hyaluronic compound polyelectrolyte, (be designated hereinafter simply as: compound polyelectrolyte), it is characterized in that HA based polyalcohol and derivant bag medicine carrying thing thereof form electronegative nanoparticle, then the compound polyelectrolyte being compounded to form by static with polycation.
So long as the electronegative nanoparticle in surface that HA based polyalcohol bag medicine carrying thing forms is all suitable for the preparation of compound polyelectrolyte in the present invention.Preferably, the nanoparticle forming with the HA self assembly of hydrophobically modified is more suitable for load hydrophobic drug and is prepared into compound polyelectrolyte.
The hyaluronic acid of hydrophobically modified is the hyaluronic acid of the graft type that formed by the carboxyl keyed jointing hydrophobic side chain on hyaluronic acid (HA), and wherein the mass content of hydrophobic side chain accounts for 10-50%, is preferably 20-40%; The number-average molecular weight of hydrophobic side chain is at 200-20000, preferably 200-5000; In described complex, the positive and negative charge of polycation and hyaluronic acid based polyalcohol compares between 1.5-6.0, preferably 2.0-4.0; The number-average molecular weight of HA is 2000-200000, is preferably 2000-100000, further preferred 2000-50000; The particle diameter of described complex is less than 500nm.
In the present invention, the mass content of hydrophobic side chain accounts for the hyaluronic 10-50% of hydrophobically modified, is preferably 20-40%; Hydrophobic side chain is preferably from degradable polyester, deoxycholic acid, cholesterol, enoxolone.During hydrophobic side chain number-average molecular weight 200-600, the upper hydrophobic side chain percent grafting of HA is preferably 20-60%, and during hydrophobic side chain number-average molecular weight 600-20000, the upper hydrophobic side chain percent grafting of HA is preferably 1-30%.Hydrophobic section percent grafting refers to that the surveyor's chain molal quantity of the upper grafting of HA accounts for the percentage ratio of HA construction unit molal quantity.
Described hydrophobic side chain is degradable polyester, and described degradable polyester is homopolymer or the copolymer of lactone, lactide; Described lactone is the lactone that can carry out ring-opening polymerization, and described lactide is preferably lactide, Acetic acid, hydroxy-, bimol. cyclic ester.
In the present invention, hydrophobic side chain is caprolactone, Isosorbide-5-Nitrae, the homopolymer of 8-trioxa spiral shell [4.6]-9-hendecanone (TOSUO), P-Dioxane ketone or their copolymer; The copolymer of caprolactone and TOSUO more preferably, the mass percent that TOSUO unit accounts for hydrophobic side chain is 5-30%, is preferably 5-20%.
Form compound polyelectrolyte of the present invention, outer polycation used can be any in aqueous medium the material with positive charge, preferably from chitosan and derivant thereof, polylysine, cationic starch derivative, gelatin, cationic protein or polypeptide, cationic polyester, cation poly phosphate, polyvinyl pyrroles salt, N, homopolymer and the copolymer of (methyl) acrylate that N-replaces.
Described poly-electrolysis polycation is chitosan more preferably, the chitosan of preferred number average molecular weight 5000-100000 and derivant thereof, and the number-average molecular weight of chitosan is 10000-100000 more preferably.The deacetylation degree of chitosan is preferably greater than 90%.
Matter complex, wherein contained hydrophobic drug is to be suitable for oral water-insoluble drug; Be selected from antitumor drug, preferably paclitaxel, Docetaxel, amycin, hydroxy camptothecin, curcumin; Or be selected from antibacterial, antiviral, anti-inflammatory type hydrophobic drug, be selected from arteannuin, indomethacin, dexamethasone, prednisolone acetate.
Drug quality content in electronegative nanoparticle can reach 20%, preferably 0.5-15%.
The preparation of described compound polyelectrolyte is the nanoparticle of first preparing the hydrophobically modified HA of medicine carrying, the preparation method of this nanoparticle can adopt the preparation method of conventional amphiphilic nanoparticle, as solvent evaporated method, nanometer sedimentation, dialysis etc.At this, only provide the hydrophobically modified HA nanoparticle that adopts dialysis to prepare medicine carrying, then static compound action is prepared complex.Key step is: 1) the hydrophobically modified hyaluronic acid of formula ratio and dewatering medicament are dissolved into wiring solution-forming in cosolvent, then install in bag filter, with PBS(pH7.4) dialysing to solvent is removed completely, centrifugal, remove precipitate, obtain the hyaluronic nanoparticle aqueous dispersions of hydrophobically modified of load dewatering medicament.2) further lyophilizing of nanoparticle aqueous dispersions, obtains the lyophilized powder of nanoparticle; 3) under stirring, 1) in the hyaluronic nanoparticle aqueous dispersions of hydrophobically modified or 2 of the carrying medicament that obtains) in the nano-granule freeze-dried powder redispersion that obtains to the nanoparticle aqueous dispersions forming in water, be added drop-wise in the aqueous solution that contains formula ratio polycation, carry out static compound, make the aqueous dispersions of the compound polyelectrolyte nanoparticle of load hydrophobic drug; 4) by 3) the aqueous dispersions lyophilizing of the compound polyelectrolyte nanoparticle prepared or with lyophilizing stabilizing agent as Polyethylene Glycol mixes lyophilizing again, obtain the lyophilized powder of described compound polyelectrolyte.
Complex of the present invention is distributed to the aqueous humour forming in aqueous medium with nanoparticle state, or its lyophilized powder is loaded in capsule, or is prepared into tablet, as oral drug preparation; Described complex also can be composite with other medicines or material, for the preparation of oral drug preparation; Be preferred for antitumor oral administration and colon targeting drug administration.
With embodiment, the present invention is further illustrated again below.
The hyaluronic acid of higher molecular weight (10000-200000) is commercially available prod, and lower molecular weight hyaluronic acid is prepared by Co irradiation and hyaluronidase biodegrading process.
Embodiment 1:
Polyanion is connected by ester bond with hydrophobic section and prepared by drug-carrying nanometer particle:
In dry reaction bottle, add 0.1g hyaluronic acid (HA) (number-average molecular weight 2.9KDa), 33mg polycaprolactone (PCL, 3.3KDa) with 82.52mg dicyclohexylcarbodiimide (DCC), dimethyl formamide (DMF) dissolves, and finally adds 48.87mg4-dimethylamino naphthyridine (DMAP).Evacuation, inflated with nitrogen.At room temperature react 24h.The reactant liquor 24h that dialyses in DMF, then at distill water dialysis 24h, afterwards by product lyophilization 24h, obtains the hyaluronic acid of ester bond bonding hydrophobic section.
25mg HAg-1 and 20mg paclitaxel are dissolved into wiring solution-forming in 10ml DMF, then install in bag filter (molecular cut off 3500), spend PBS(pH7.4) dialysis 48h, centrifugal HA-g-PCL copolymer nano particle (HAg-1) aqueous dispersions that obtains bag year paclitaxel.
The hydrophobically modified hyaluronic acid drug-carrying nanometer particle that table 1 connects by ester bond
The relative molecular mass of MHA:HA; Mg: the relative molecular mass of graft side chain; r
g: the percent grafting of side chain; PCL: polycaprolactone; PLA: polylactic acid; PLGA: Poly(D,L-lactide-co-glycolide; PGA: polyglycolic acid; PCTm: polycaprolactone-Isosorbide-5-Nitrae, 8-trioxa spiral shell [4.6]-9-hendecanone copolymer, m is the quality percentage composition of TOSUO in PET.PDK: poly-para-dioxanone.PTX: paclitaxel; DOX: amycin; DM: indomethacin; HCPT: hydroxy camptothecin; TXT: Docetaxel
The drug-carrying nanometer particle that HA is connected by amido link with hydrophobic section:
In dry reaction bottle, add 0.1g deoxycholic acid (DA), after dissolving with DMF, add 0.197g1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC.HCL) and 0.23g N-hydroxy-succinamide, activated carboxyl 2h at room temperature, add 0.5ml ethylenediamine, continue reaction 24h, by the deionized water precipitating of appropriate amount, sucking filtration, vacuum drying.
In dry reaction bottle, add 0.1g hyaluronic acid (HA) (number-average molecular weight 2.9KDa), the amidized deoxycholic acid of 33mg (DA) and 0.197g1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC.HCL) 0.23g N-hydroxy-succinamide, dimethyl formamide (DMF) dissolves, evacuation, inflated with nitrogen.At room temperature react 24h.The reactant liquor 24h that dialyses in DMF, then at distill water dialysis 24h, afterwards by product lyophilization 24h, obtains the hyaluronic acid target product HA-g-DA of ester bond bonding hydrophobic section.
Appropriate HA-g-DA and 20mg paclitaxel are dissolved into wiring solution-forming in 10ml DMF, then install in bag filter (molecular cut off 3500), spend PBS(pH7.4) dialysis 48h, centrifugal HA-g-PCL copolymer nano particle (HAg-1) aqueous dispersions that obtains bag year paclitaxel.
Change HA molecular weight, hydrophobic section content and kind, adjust the consumption of solvent and catalyst, and change DA into enoxolone (CH), cholesterol, can prepare the hydrophobically modified hyaluronic acid of the amido link keyed jointing of different structure.
Adopt the hydrophobically modified hyaluronic acid of prepared amido link keyed jointing to carry out drug loading, prepare drug-carrying nanometer particle, method is with embodiment 1, the polymer and the drug taxol that are amount of calculation are dissolved in DMF equal solvent, wiring solution-forming, then install in bag filter (molecular cut off 3500), spend PBS(pH7.4) dialysis 48h, the centrifugal nanoparticle aqueous dispersions that obtains wrapping medicine carrying thing, specifically forms structure in Table 2.
Table 2 connects hydrophobically modified hyaluronic acid drug-carrying nanometer particle by amido link
DA: deoxycholic acid; GA: enoxolone; CH: cholesterol.
Embodiment 27:
The preparation of the compound polyelectrolyte that contains hydrophobic drug:
5mg chitosan (CS) (100KDa, degree of deacetylation 95%) is dissolved in to 5mL pH5.0(0.01M) acetum in, by 2mL
HAg-1 nanoparticle aqueous dispersions (pH7.4,0.01M), dropwise joins in CS solution, dropwises and stirs 1h, obtains paclitaxel loaded compound polyelectrolyte (II-1) aqueous dispersions.
Embodiment 28-56: press embodiment 55 methods, by changing polycation, nanoparticle and proportioning, can obtain the multiple compound polyelectrolyte that contains hydrophobic drug, as shown in table 3.
The compound polyelectrolyte that table 3 contains hydrophobic drug
CS200KDa, 50KDa, 10KDa, the deacetylation degree of 5KDa is respectively 96%, 98%, 97%, 98; PL: polylysine; PDMAEMA: polymethylacrylic acid N, N-dimethylaminoethyl; PEI: polymine; PDMAM: poly-N, N-dimethylmethacryl amide; PVPs: polyvinyl pyrroles hydrochlorate.
Characterizing method:
Particle diameter and form: the pH value of nanoparticle aqueous dispersions is adjusted to respectively pH3.0,5.0,7.4 with dilute hydrochloric acid or NaOH.Zetasizer3000HS (Malvern Instrument, Inc.Worcestershire, UK) particle size analyzer determination nanoparticle particle diameter, transmission electron microscope (TEM, Hitachi, H-7650) is observed particle shape.Result as shown in Figure 1-2.
Drug release:
After adjusting pH value, the complex nanometer granule aqueous dispersions (1mg/mL) (release medium) that the drug-carrying nanometer particle of hydrophobically modified HA or itself and polycation are formed is encapsulated in bag filter; be put in the buffer (receiving liquid) that contains 0.1% (w/v) Tween 80 of the identical pH of 40ml; then be placed in 37 ℃ of agitators; interval different time takes out 20mL buffer detection of drugs concentration, the fresh buffer of simultaneously supplementing same amount.
Drug release under Hyal-1 enzyme catalysis is in discharging liquid and release medium, all to add the Hyal-1 of same concentrations, carries out drug release experiment.As Fig. 3,4.
Accumulative total release amount of medicine (Er) calculates (take paclitaxel as example) by following formula:
Where m
pTXrepresent PTX content in nanoparticle, V
0the volume (V of release medium
0=40mL), Ve is sample volume (Ve=20mL), and and Ci is the concentration that detects PTX in sample, and each experimental group is established parallel 3 groups, averages.
The experiment of cell endocytosis:
To contain l0% hyclone (FBS; Biochrom Ag, Germany) DMEM(Sigma-Aldrich, USA) be basic culture solution, by cell (EC109 and NIH3T3, China Medical College radiological study institute) with 1 * 10
5individual/mL cell concentration is inoculated in 96 orifice plates, is placed in 37 ℃, 5%CO
2, cultivate under saturated humidity condition.Normal saline (control), DOX, I-2 and II-3 solution dilute with DMEM, obtain concentration at the solution of 10～100ng/ml.The above-mentioned solution of getting 100 each concentration of μ L is added to respectively alternative original fluid in the orifice plate of inoculating cell.The cell of cultivation in DMEM as a control group.Cultivate respectively after a period of time, take out culture plate, with laser confocal microscope, detect the endocytosis ability of cell to material.
Result shows: EC109 cell and NIH3T3 cell be to endocytosis ability (concentration 0.5ug/mL) incubation time of free amycin 2 hours, as seen EC109 cell to the endocytosis ability of HAg-1 nanoparticle far away higher than normal cell.
EC109 cell is competed endocytosis (doxorubicin concentration 0.5ug/mL, hyaluronic acid concentration 10mg/ml) incubation time 2 hours to the receptor-mediated endocytosis of HAg-2 nanoparticle and free hyaluronic acid.Visible EC109 cell weakens the endocytosis of HAg-2 nanoparticle, illustrates with hyaluronic acid receptor closely related.
EC109 cell to HAg-2 nanoparticle endocytosis after, amycin is in intracellular release and distribution.Along with time lengthening, at endonuclear amycin, obviously increase.
Experiment distributes in body:
The nude mice of subcutaneous vaccination anthropogenic pulmonary carcinoma tumor, fasting 12 hours, use respectively doxorubicin hydrochloride aqueous dispersions, HAg-1 nanoparticle and Complex II-1 nanoparticle solution carry out gavage (doxorubicin concentration: 74ug/mL), after 4 hours, mice is anaesthetized lethal with heavy dose of pentobarbital sodium solution, then mice is dissected, mouse heart after dissection, liver, spleen, lung, kidney, stomach, small intestinal, colon, tumor are separated, then according to order from left to right, be placed in sample panel, carry out fluorescence imaging observation, take pictures.Mice is bought from Beijing dimension tonneau China laboratory animal company limited, and is all male, is 4-6 age in week.In zoopery process, mice is all raised the environment at SPF, and manages and operate in strict accordance with < < zoopery management rules > >.Result shows: after oral 5 hours, the distribution of each organ is all high than doxorubicin hydrochloride and HAg-1 nanoparticle in vivo for II-1 nanoparticle group, especially in the gathering at tumor and colon position higher than other organ, and along with the drug level in time lengthening (2-24h) tumor significantly improves.Illustrate that compound polyelectrolyte in the present invention can promote medicine sending in the absorption of intestinal and body, especially has cancer target delivery functions.
Anti-tumor in vivo effect:
Get healthy Bab/c mice, female, body weight 19 ± 2g, adopt the tumor strain animal of going down to posterity, take out EC109 cell, ascites cells and normal saline are diluted in 1:3 ratio, under aseptic condition, inoculate fast, in mice right rear leg subcutaneous injection glucagonoma liquid 0.2mL/ only, cell concentration is 5.0 * 10
6/ only.After tumor liquid is inoculated one week, gross tumor volume reaches 100mm
3(V=1/2 (a * b
2)) time, mice is divided into 6 groups at random, be normal saline matched group (per injection 300 μ L), PTX preparation lumbar injection group (is equivalent to 15mg PTX/Kg, per injection 300 μ L), blank nanoparticle matched group (II-1) (each 300 μ L), PTX preparation oral group (is equivalent to 15mg PTX/Kg, each 300 μ L), oral group of II-1 nanoparticle solution (is equivalent to 15mg PTX/Kg, each 300 μ L), after oral group of (being equivalent to 15mg PTX/Kg, each 300 μ L) administration of HAg-1 nanoparticle solution, animal tracking is observed.Investigate the variation of 24 days gross tumor volumes of administration, gross tumor volume is by vernier caliper measurement, according to V=1/2 (a * b
2) calculate, a represents the length of tumor here, b represents the wide of tumor, stops observing when having tumor to occur diabrosis.Result is as Fig. 5, and 6.
Claims (10)
1. the compound polyelectrolyte based on hyaluronic acid drug-carrying nanometer particle, it is characterized in that hyaluronic acid based polyalcohol bag medicine carrying thing forms electronegative nanoparticle, then the compound polyelectrolyte being compounded to form with polycation static; Hyaluronic acid based polyalcohol is the hyaluronic acid of hydrophobically modified, is the hyaluronic acid of the graft type that formed by the carboxyl keyed jointing hydrophobic side chain on hyaluronic acid HA, and wherein the mass content of hydrophobic side chain accounts for 10-60%; The number-average molecular weight of hydrophobic side chain is 200-20000; In complex, the positive and negative charge of polycation and hyaluronic acid based polyalcohol is than between 1.5-6.0; The number-average molecular weight of HA is 2000-200000; The particle diameter of complex is less than 500nm.
2. compound polyelectrolyte claimed in claim 1, is characterized in that, the upper hydrophobic side chain of described HA is selected from degradable polyester, deoxycholic acid, cholesterol or enoxolone; When hydrophobic side chain number-average molecular weight is 200-600, the upper hydrophobic side chain percent grafting of HA is 20-60%, and during hydrophobic side chain number-average molecular weight 600-20000, the upper hydrophobic side chain percent grafting of HA is 1-30%.
3. compound polyelectrolyte claimed in claim 2, is characterized in that, described hydrophobic side chain is homopolymer or the copolymer degradable polyester of lactone, lactide; Lactone is the lactone that carries out ring-opening polymerization, and lactide is lactide or Acetic acid, hydroxy-, bimol. cyclic ester.
4. compound polyelectrolyte claimed in claim 3, is characterized in that, described hydrophobic side chain lactone is caprolactone, Isosorbide-5-Nitrae, the homopolymer of 8-trioxa spiral shell [4.6]-9-hendecanone, P-Dioxane ketone or their copolymer; The copolymer that described own hydrophobic side chain lactone is lactone and TOSUO, the mass percent that TOSUO unit accounts for hydrophobic side chain is 5-30%.
5. compound polyelectrolyte claimed in claim 1, it is characterized in that, polycation in described complex is selected from chitosan and derivant, polylysine, cationic starch derivative, gelatin, cationic protein or polypeptide, cationic polyester, cation poly phosphate, polyvinyl pyrroles salt, N, homopolymer and the copolymer of (methyl) acrylate that N-replaces.
6. compound polyelectrolyte claimed in claim 5, is characterized in that described polycation is selected from chitosan, and the number-average molecular weight of chitosan is 2000-200000.
7. any one compound polyelectrolyte described in claim 1～6, wherein the hydrophobic drug of institute's load is to be suitable for oral water-insoluble drug; Preferably from antitumor drug, antibacterial, antiviral, anti-inflammatory type hydrophobic drug.
8. compound polyelectrolyte claimed in claim 7, feature is that the medicine of institute's load is antitumor drug, is preferably paclitaxel, Docetaxel, amycin, hydroxy camptothecin or curcumin.
9. the preparation method of compound polyelectrolyte claimed in claim 1, is characterized in that step is as follows:
1) the hydrophobically modified hyaluronic acid of formula ratio and dewatering medicament are dissolved into wiring solution-forming in cosolvent, then install in bag filter, with the PBS of pH7.4, dialyse to solvent and be removed completely, centrifugal, remove precipitate, obtain the hyaluronic nanoparticle aqueous dispersions of hydrophobically modified of load dewatering medicament;
2), by the further lyophilizing of nanoparticle aqueous dispersions, obtain the lyophilized powder of nanoparticle;
3) under stirring, 1) in the hyaluronic nanoparticle aqueous dispersions of hydrophobically modified or 2 of the carrying medicament that obtains) in the nano-granule freeze-dried powder redispersion that obtains to the nanoparticle aqueous dispersions forming in water, be added drop-wise in the aqueous solution that contains formula ratio polycation, carry out static compound, make the aqueous dispersions of the compound polyelectrolyte nanoparticle of load hydrophobic drug;
4) by 3) the aqueous dispersions lyophilizing of the compound polyelectrolyte nanoparticle prepared or mix lyophilizing again with lyophilizing stabilizing agent, obtain the lyophilized powder of compound polyelectrolyte; Lyophilizing stabilizing agent is selected from a kind of of mannitol, Polyethylene Glycol, glucose, lactose, dextran, sucrose, sodium chloride, Glycine sodium, sodium deoxycholate, sodium dihydrogen phosphate and sodium hydrogen phosphate, sorbitol or dextran or their combination in any.
10. the application of the compound polyelectrolyte that contains hydrophobic drug described in claim 1～8 any one, be characterised in that described complex is distributed to the aqueous humour forming in aqueous medium with nanoparticle state, or its lyophilized powder is loaded in capsule, or be prepared into tablet, as oral drug preparation; Or composite with other medicines or material, for the preparation of oral drug preparation.
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Cited By (8)
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| CN105694120A (en) * | 2014-11-28 | 2016-06-22 | 中国海洋大学 | Preparation method of chitosan oligosaccharide-hyaluronic acid composite nanometer particle |
| CN106432746A (en) * | 2016-09-30 | 2017-02-22 | 华中科技大学 | Amphipathy hydroxyethyl-starch-coupled-polylactic-acid copolymer and preparing method and application thereof |
| CN107308457A (en) * | 2017-05-19 | 2017-11-03 | 四川大学 | A kind of deep layer degraded with tumor microenvironment response penetrates nanoscale medicine delivery system |
| CN108030928A (en) * | 2017-12-18 | 2018-05-15 | 青岛大学附属医院 | Gene target delivery system and application for CD44 high expression type tumours |
| CN110124054A (en) * | 2019-06-21 | 2019-08-16 | 天津科技大学 | A kind of preparation method and application of the targeted nano-particle of LBL self-assembly |
| CN111423591A (en) * | 2020-04-10 | 2020-07-17 | 黑龙江大学 | Amphiphilic graft copolymer based on hyaluronic acid and preparation method and application thereof |
| CN111518356A (en) * | 2020-05-20 | 2020-08-11 | 中国科学院长春应用化学研究所 | Modified polypropylene and preparation method thereof |
| CN113769174A (en) * | 2021-09-16 | 2021-12-10 | 上海玮沐医疗科技有限公司 | Hyaluronic acid composite microsphere containing levorotatory polylactic acid and preparation method thereof |
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| CN105694120A (en) * | 2014-11-28 | 2016-06-22 | 中国海洋大学 | Preparation method of chitosan oligosaccharide-hyaluronic acid composite nanometer particle |
| CN105694120B (en) * | 2014-11-28 | 2019-07-05 | 中国海洋大学 | A kind of preparation method of chitosan oligosaccharide hyaluronic acid composite nano particle |
| CN106432746A (en) * | 2016-09-30 | 2017-02-22 | 华中科技大学 | Amphipathy hydroxyethyl-starch-coupled-polylactic-acid copolymer and preparing method and application thereof |
| CN106432746B (en) * | 2016-09-30 | 2019-05-10 | 华中科技大学 | A kind of amphipathic hydroxyethyl starch coupling copolymer of poly lactic acid and its preparation method and application |
| CN107308457A (en) * | 2017-05-19 | 2017-11-03 | 四川大学 | A kind of deep layer degraded with tumor microenvironment response penetrates nanoscale medicine delivery system |
| CN108030928A (en) * | 2017-12-18 | 2018-05-15 | 青岛大学附属医院 | Gene target delivery system and application for CD44 high expression type tumours |
| CN110124054A (en) * | 2019-06-21 | 2019-08-16 | 天津科技大学 | A kind of preparation method and application of the targeted nano-particle of LBL self-assembly |
| CN110124054B (en) * | 2019-06-21 | 2022-02-18 | 天津科技大学 | Preparation method and application of targeted nano particles self-assembled layer by layer |
| CN111423591A (en) * | 2020-04-10 | 2020-07-17 | 黑龙江大学 | Amphiphilic graft copolymer based on hyaluronic acid and preparation method and application thereof |
| CN111518356A (en) * | 2020-05-20 | 2020-08-11 | 中国科学院长春应用化学研究所 | Modified polypropylene and preparation method thereof |
| CN113769174A (en) * | 2021-09-16 | 2021-12-10 | 上海玮沐医疗科技有限公司 | Hyaluronic acid composite microsphere containing levorotatory polylactic acid and preparation method thereof |
| CN113769174B (en) * | 2021-09-16 | 2022-09-02 | 上海玮沐医疗科技有限公司 | Hyaluronic acid composite microsphere containing levorotatory polylactic acid and preparation method thereof |
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