CN107595805A - PEG6000-PHDCA loads the preparation method of adriamycin nano-particles - Google Patents

Abstract

The invention discloses a kind of preparation method of PEG6000-PHDCA load adriamycin nano-particles：Adriamycin is dissolved into 2.5mgmL with deionized water^‑1Interior aqueous phase storing solution；PEG6000-PHDCA is made into 25mgmL^‑1Organic phase；Interior aqueous phase storing solution is added in organic phase, ultrasonic emulsification is disperseed, and forms colostrum；The aqueous solution for the PLURONICS F87 that mass concentration is 0.5% is prepared, regulation pH to 8.5~9.5 forms outer aqueous phase, under magnetic stirring, outer aqueous phase is added dropwise in colostrum, and interior aqueous phase storing solution, organic phase, the volume ratio of outer aqueous phase are 0.5:2:10, after dripping off, continue stirring and form emulsion, emulsion vacuum rotary steam is removed into organic solvent and part water, PEG6000-PHDCA load adriamycin nano-particles suspension is made.The nanoparticle for carrying adriamycin is prepared in the present invention, and drugloading rate is 2.17 ± 0.67%, and envelop rate is 79.54 ± 4.66%.With obvious slow release characteristic, there is good security and biocompatibility.

Description

PEG6000-PHDCA loads the system of adriamycin nano-particles Preparation Method

Technical field

The present invention relates to a kind of preparation of PEG6000-PHDCA load adriamycin nano-particles Method.

Background technology

With the continuous development of nanometer technology, exploitation novel nano preparation is opened up to diagnose and treating the diseases such as malignant tumour Direction.Wherein, nanoparticle (Nanoparticles, NPs) is used as a kind of new drug carrier, can be by drug encapsulation in load Internal portion, there is enhancing medicine stability, improve insoluble drug solubility, increase drug bioavailability, control medicine to release Put and the advantages that targeting drug delivery.Material difference according to nanoparticle is prepared can be divided into inorganic material (mesoporous silicon, CNT, four Fe 3 O, Jenner's grain of rice etc.) and organic polymer (PLA, PLGA, PCL, PAMAM etc.), inorganic material is often limited to biology Degraded in vivo is difficult and is difficult to clinical practice the problems such as bio-toxicity, on the contrary, organic polymer can degrade in vivo with Catabolite does not have the advantages of toxic side effect, and nanometer formulation prepared by existing numerous organic polymers enters clinical test.Therefore, New organic polymer and its application in nanometer formulation also increasingly become the study hotspot of medical personal.

PEG6000-PHDCA

(monomethoxy-polyethyleneglycolcyanoacrylate-co-poly- Hexadecylcyanoacrylate, mPEG-PHDCA) it is a kind of new organic polymer, with BCA, cyanogen Base isobutyl acrylate etc. compares, and it possesses the biocompatibility that the degree of polymerization is controllable, toxicity is smaller, good.By mPEG-PHDCA Carry medicine and be prepared into NPs, the advantages that it delays controlled release characteristics, and the hydrophily of polyglycol chain and flexible imparting can be assigned The long circulating characteristics of NPs in blood, can change pharmacokinetics behavior inside medicine, prevent from being removed by reticuloendothelial system, Extend the blood circulation time of medicine, improve indirect targeting, mitigate toxic side effect of the medicine to human normal tissue.

The content of the invention

The present invention has synthesized organic polymer mPEG-PHDCA, using clinical antineoplastic medicine adriamycin as model drug, prepares and carries The mPEG-PHDCA nanoparticles (mPEG-PHDCA-NPs) of adriamycin, its pharmacy characteristic of preliminary examinations and its external biological of evaluation Characteristic is learned, internal pharmacodynamics is evaluated for next step and pharmacokinetics lays the foundation.

The technical solution adopted by the present invention is：

A kind of preparation method of PEG6000-PHDCA load adriamycin nano-particles, the side Method comprises the following steps：

(1) adriamycin is dissolved into 2.5mgmL with deionized water^-1Interior aqueous phase storing solution；The poly- hexadecane of Pegylation Base cyanoacrylate is dissolved with organic solvent, is made into 25mgmL^-1Organic phase；The organic solvent is dichloromethane, second Acetoacetic ester volume ratio 1:1 mixing；Interior aqueous phase storing solution is added in organic phase, interior aqueous phase storing solution, the volume of organic phase Than for 0.5:2, Probe Ultrasonic Searching emulsion dispersion, form colostrum (W/O)；The PEG6000-PHDCA Mean molecule quantity be 5800~6000；

(2) aqueous solution for the PLURONICS F87 that mass concentration is 0.5%, regulation pH to 8.5~9.5 (preferably 9.0) are prepared Outer aqueous phase is formed, under magnetic stirring, outer aqueous phase is added dropwise in colostrum, interior aqueous phase storing solution, organic phase, the body of outer aqueous phase Product is than being 0.5:2:10, after dripping off, continue to stir 30min, form emulsion (W/O/W), emulsion vacuum rotary steam is removed organic molten Agent and part water, PEG6000-PHDCA load adriamycin nano-particles (mPEG-PHDCA- is made NPs) suspension.

In the present invention, the PEG6000-PHDCA of use is preferably made by the following method：

(a) synthesis of cetyl cyan-acetic ester (HDCA)

Weigh hexadecanol 200mmol and cyanoacetic acid 240mmol is placed in 500mL three-neck flasks, add 120mL CH₂Cl₂ With 20mL ethyl acetate, magnetic agitation dissolving, it is slowly added dropwise dissolved with 300mmol condensing agent DCC and 20mmol catalyst DMAP's 60mL CH₂Cl₂Solution, nitrogen protection stirring room temperature reaction 24h, adds distillation water destruct DCC, filters, collect filtrate, filter residue is used Ethyl acetate is washed to white, and merging filtrate puts separatory funnel extraction, is washed with supersaturated NaCl solution, adds anhydrous slufuric acid Sodium, the moisture in organic phase is removed, rotate organic solvent, separate out HDCA crude products, gained crude product is polished, is beaten with absolute ethyl alcohol Slurry recrystallization, purifying obtain sterling HDCA faint yellow solids；

(b) synthesis of polyethylene glycol cyan-acetic ester (mPEG-CA)

Weigh polyethylene glycol 10mmol, DCC15mmol and DMAP10mmol to be placed in 250mL three-neck flask, add 150mL CH₂Cl₂With 25mL ethyl acetate, magnetic agitation dissolving；Weigh the CH that cyanoacetic acid 12mmol is dissolved in 30mL₂Cl₂With In 10mL ethyl acetate, it is slowly added dropwise into flask, at room temperature nitrogen protection stirring reaction 24h；Distilled water is added, is destroyed DCC, through suction filtration, CH₂Cl₂Washing, separatory funnel extraction, collected organic layer, the washing of supersaturated NaCl solution, anhydrous sodium sulfate remove Water, revolving organic solvent, obtain mPEG-CA crude products；Crude product is polished, and sterling is obtained after being beaten recrystallization purifying with ethyl acetate MPEG-CA white waxy solids；

(c) synthesis of poly- cetyl cyanoacrylate (PHDCA)

PHDCA is that monomer HDCA is polymerized：Take HDCA 10g to be placed in 100mL three-neck flasks, add 30mLCH₂Cl₂With 10mL absolute ethyl alcohols, magnetic agitation dissolving, 3.6mL formaldehyde (concentration 37%) solution and 0.2mL pyrroles are added dropwise into reaction solution Alkane, at room temperature nitrogen protection stirring reaction 24h；Reaction solution is transferred in separatory funnel, successively with 10% salt acid elution, distillation Water washing, finally washed with supersaturated NaCl solution, collected organic layer, the moisture added in anhydrous sodium sulfate removing organic phase, Organic solvent is rotated, separates out poly- PHDCA crude products；Crude product is polished, and sterling is obtained after being beaten crystallization purifying with absolute ethyl alcohol PHDCA yellow solids；

(d) synthesis of the poly- cetyl cyanoacrylate (mPEG-PHDCA) of Pegylation

MPEG-PHDCA is the experimental implementation by being formed through Knoevenagel reactions and anionic polymerisation：Weigh PHDCA 22g and mPEG-CA 10g are placed in three-neck flask, add 100mLCH₂Cl₂It is molten with 50mL ethyl acetate, magnetic agitation 1.8mL formaldehyde (concentration 37%) solution and 0.1mL pyrrolidines are added dropwise after solution, adds DCC and DMAP catalysis, nitrogen is protected at room temperature Protect stirring reaction 48h；Reaction solution is transferred in separatory funnel, successively with 10% salt acid elution, distills water washing, it is last used Saturation NaCl solution is washed, collected organic layer, the moisture added in anhydrous sodium sulfate removing organic phase, rotates organic solvent, analysis Go out mPEG-PHDCA crude products；By crude product with a small amount of CH₂Cl₂After dissolving, ice ether precipitates 2 times, and solid vacuumizes drying through oil pump, Obtain faint yellow sterling mPEG-PHDCA.Obtained mPEG-PHDCA mean molecule quantities are 5800~6000.

The beneficial effects of the present invention are synthesized mPEG- using Knoevenagel reactions and anionic polymerisation PHDCA, it is model drug to select adriamycin, is prepared for carrying the mPEG-PHDCA nanoparticles of adriamycin using double emulsion-solvent evaporation technique, Efficient liquid phase determines its drugloading rate and envelop rate, and particle instrument determines its particle diameter and current potential, its microscopic appearance of transmission electron microscope observing, thoroughly Analysis bag method investigates its release behaviour in vitro, and hemolytic experiment outside polymeric acceptor is investigated from rabbit erythrocyte, from Hela, MCF-7 and MCF-7/ADR cell lines investigate the cytotoxicity of polymer and nanoparticle.As a result pass through¹H-NMR and GPC is characterized, and is successfully synthesized MPEG-PHDCA, molecular weight are about 6K, and molecular weight distribution curve is uniform, PDI 1.13.The nanometer for carrying adriamycin is prepared Grain, particle diameter is 94.61 ± 3.91nm, is distributed homogeneous, soilless sticking phenomenon, and rounding spherical structure is presented through transmission electron microscope observing, carries Dose is 2.17 ± 0.67%, and envelop rate is 79.54 ± 4.66%.Preparation reaches 85.38% in tablets in vitro 48h, With obvious slow release characteristic, drug release profiles are distributed in Weibull equations；Polymer shows in the range of 0~1.0mM without haemolysis As there is good security and biocompatibility to Hela, MCF-7 and MCF-7/ADR cell line in the range of 0~0.5mM； Drug-carrying nanometer particle has certain cytotoxicity to Hela and MCF-7 cells, but effect is less than adriamycin active compound；In mdr cell It is in MCF-7/ADR, medicament-carried nano granulocyte toxicity is significantly higher than adriamycin active compound.Conclusion successfully synthesizes polymer mPEG- PHDCA, there are the gentle controlled release characteristics of good drug carrying ability, biocompatibility, be expected to pass drug carrier as novel nano.

Brief description of the drawings

The synthetic reaction formula of Fig. 1 polymer.

Fig. 2 polymer¹H-NMR collection of illustrative plates, wherein A figures are HDCA, and B figures are mPEG-CA, and C figures are PHDCA, and D figures are mPEG-PHDCA。

Fig. 3 polymer molecular weights scatter chart and polymer molecular weight and profile exponent figure, wherein A figures are polymer point Son amount scatter chart, B figures are polymer molecular weight and profile exponent.

Fig. 4 mPEG-PHDCA-NPs grain size distributions and transmission electron microscope picture, wherein A figures are grain size distribution, and B figures are transmission Electron microscope.

Fig. 5 vitro cumulatives drug release percentage-time changing curve figure, n=3.

Fig. 6 polymer concentrations-erythrocyte hemolysis rate curve map.

Fig. 7 mPEG-PHDCA-NPs cytotoxicity compares figure, and in Fig. 7, A figures are blank mPEG-PHDCA-NPs, concentration Measured by mPEG-PHDCA；B, C, D figure are load medicine mPEG-PHDCA-NPs tri- kinds thin to Hela, MCF-7 and MCF-7/ADR respectively The cytotoxicity of born of the same parents system, concentration are measured by adriamycin.

Embodiment

Technical scheme is described further with specific embodiment below, but protection scope of the present invention is not It is limited to this.

Embodiment 1

1 material

1.1 instrument

Bruker Avance-400 types NMR (German Bruker companies)；JEM-1200EX transmission electron microscopes (Japan Electronics Co., Ltd)；The laser particle size analyzers of Nano-ZS 90 (Malvern companies of Britain)；Waters2695 type efficient liquid phases Chromatograph, Waters1515 types gel permeation chrommatograph (Waters, US)；ST16R desk centrifuges (U.S. Thermo Fisher Scientific companies)；Mill-Q ultra-pure waters instrument (Millpore companies of the U.S.)；SpectraMaxM₅Multifunctional enzyme Mark instrument (Molecular Devices companies of the U.S.)；Thermo Scientific Forma II CO₂Incubator (the U.S. Thermo companies)；MD200 nitrogen evaporators (Hangzhou Ao Sheng Instrument Ltd.)；DZF-6020 vacuum drying chambers (Guangzhou Kang Heng instruments Co., Ltd)；CP225D electronic balances (Beijing Sai Duolisi Instrument Ltd.)；Deng.

1.2 medicines and reagent

Hexadecanol, cyanoacetic acid, formaldehyde, pyrrolidines, mono methoxy polyethylene glycol (mPEG, molecular weight 2K), dicyclohexyl Carbodiimide (DCC), dimethylamino naphthyridine (DMAP), tetrazolium bromide (MTT) (Shanghai Aladdin reagent Co., Ltd)；RPMI1640 Culture medium (10% hyclone of addition, 100umL^-1Penicillin and 100umL^-1Streptomysin), 0.25% trypsase it is (beautiful Gibco companies of state)；Adriamycin (Zhejiang Hai Zheng pharmaceutical Co. Ltds present, purity ＞ 98%)；Adriamycin reference substance (China's food Product drug assay research institute, lot number 130509-200306)；Methanol, acetonitrile (Honeywell Corp. USA, chromatographically pure)；Other Reagent is that domestic analysis is pure.

1.3 experimental cell

Rabbit erythrocyte (Zhejiang University of Traditional Chinese Medicine's Experimental Animal Center provides)；Human cervical carcinoma Hela cell system, human breast carcinoma MCF-7 cell lines and human breast carcinoma resistance MCF-7/ADR cell lines (come from Shanghai Inst. of Life Science, CAS cell Resource center), MCF-7/ADR cell lines add the μ gmL of adriamycin 1 in subculture^-1To maintain its drug resistance.

2 methods

The synthesis of 2.1 carrier materials and sign

2.1.1 the synthesis of cetyl cyan-acetic ester (HDCA)

HDCA synthesis：Weigh hexadecanol 200mmol and cyanoacetic acid 240mmol is placed in 500mL three-neck flasks, add 120mL CH₂Cl₂With 20mL ethyl acetate, magnetic agitation dissolving.It is slowly added dropwise and is urged dissolved with 300mmol condensing agents DCC and 20mmol Agent DMAP 60mL CH₂Cl₂Solution, nitrogen protection stirring room temperature reaction 24h.100mL distillation water destruct DCC are added, are filtered, Filtrate is collected, filter residue is washed to white with ethyl acetate, and merging filtrate puts separatory funnel extraction, with appropriate supersaturated NaCl solution Washing 2 times, anhydrous sodium sulfate is added, remove the moisture in organic phase, rotate organic solvent, separate out HDCA crude products.Gained crude product It is polished, it is beaten and is recrystallized with absolute ethyl alcohol, is purified 3 times and obtain sterling HDCA faint yellow solids.

2.1.2 the synthesis of polyethylene glycol cyan-acetic ester (mPEG-CA)

MPEG-CA synthesis：Weigh three necks that polyethylene glycol 10mmol, DCC15mmol and DMAP10mmol are placed in 250mL In flask, 150mL CH are added₂Cl₂With 25mL ethyl acetate, magnetic agitation dissolving.Weigh cyanoacetic acid 12mmol and be dissolved in 30mL CH₂Cl₂In 10mL ethyl acetate, it is slowly added dropwise into flask, at room temperature nitrogen protection stirring reaction 24h.Add 100mL distilled water, DCC is destroyed, through suction filtration, CH₂Cl₂Washing, separatory funnel extraction, collected organic layer, supersaturated NaCl solution are washed Wash, anhydrous sodium sulfate water removal, revolving organic solvent, obtain mPEG-CA crude products.Crude product is polished, and weight is beaten with ethyl acetate After crystallization purifying 3 times sterling mPEG-CA white waxy solids.

2.1.3 the synthesis of poly- cetyl cyanoacrylate (PHDCA)

PHDCA is that monomer HDCA is polymerized：Take HDCA 10g to be placed in 100mL three-neck flasks, add 30mLCH₂Cl₂With 10mL absolute ethyl alcohols, magnetic agitation dissolving, 3.6mL formaldehyde (concentration 37%) solution and 0.2mL pyrroles are added dropwise into reaction solution Alkane, at room temperature nitrogen protection stirring reaction 24h.Reaction solution is transferred in separatory funnel, with 10% salt acid elution, every time 30mL, wash 3 times, distill water washing, each 20mL, wash 2 times, finally washed 2 times, collected with appropriate supersaturated NaCl solution Organic layer, the moisture added in anhydrous sodium sulfate removing organic phase, rotates organic solvent, separates out PHDCA crude products.Crude product is ground Afterwards, sterling PHDCA yellow solids are obtained after being beaten crystallization purifying 3 times with appropriate absolute ethyl alcohol.

2.1.4 the synthesis of the poly- cetyl cyanoacrylate (mPEG-PHDCA) of Pegylation

MPEG-PHDCA is by being formed through Knoevenagel reactions and anionic polymerisation^[4], experimental implementation：Weigh PHDCA22g and mPEG-CA 10g are placed in three-neck flask, add 100mLCH₂Cl₂With 50mL ethyl acetate, magnetic agitation dissolving 1.8mL formaldehyde (concentration 37%) solution and 0.1mL pyrrolidines are added dropwise afterwards, adds appropriate DCC and DMAP catalysis, at room temperature nitrogen Protect stirring reaction 48h.Reaction solution is transferred in separatory funnel, with 10% salt acid elution, each 30mL, washed 3 times, distillation Water washing, each 20mL, wash 2 times, finally washed 2 times with appropriate supersaturated NaCl solution, collected organic layer, add appropriate nothing Aqueous sodium persulfate removes the moisture in organic phase, rotates organic solvent, separates out mPEG-PHDCA crude products.By crude product with a small amount of CH₂Cl₂ After dissolving, ice ether precipitates 2 times, and solid vacuumizes drying through oil pump, obtains faint yellow sterling mPEG-PHDCA.It is total to by nuclear-magnetism Vibration Meter measure products therefrom proton nmr spectra (¹H-NMR), the molecule of detection resulting polymers is determined using gel permeation chrommatograph Amount and molecular weight distribution curve.

The preparation of 2.2 nanoparticles and sign

On the basis of technique is investigated in early stage, mPEG-PHDCA-NPs is prepared using double emulsion-solvent evaporation technique：Weigh certain DOX is measured, 2.5mgmL is dissolved into deionized water^-1Interior aqueous phase storing solution；50mg mPEG-PHDCA are weighed, being dissolved in 2mL has Solvent (dichloromethane/ethyl acetate v/v=1:1) organic phase is formed in；Aqueous phase storing solution in 0.5mL is added to organic phase In, Probe Ultrasonic Searching emulsion dispersion, form colostrum (W/O)；It is another to take 10mL mass concentrations as the water-soluble of 0.5% PLURONICS F87 Liquid, regulation pH to 9.0 or so form outer aqueous phase；Under magnetic stirring, outer aqueous phase is added dropwise in colostrum, drop finishes, and continues to stir 30min is mixed, forms emulsion (W/O/W)；Emulsion is placed in 40 DEG C of vacuum rotary steams and removes organic solvent and part water, is finally settled to 10mL, obtain the mPEG-PHDCA-NPs suspensions of orange red opalescence.Same procedure, DOX is not added with, prepares blank mPEG- PHDCA-NPs suspensions.

Taking a small amount of mPEG-PHDCA-NPs suspensions, distilled water diluting, particle instrument determines its average grain diameter in sample cell And Zeta potential.A drop mPEG-PHDCA-NPs suspensions drop is taken, with 2.0% Salkowski's solution negative staining, to transmit electricity on copper mesh Micro- its exterior appearance of sem observation of son.Precision takes 1mL mPEG-PHDCA-NPs suspensions to be placed in centrifuge tube, in 20000rpm 30min is centrifuged, takes supernatant to determine free doxorubicin concentration, computational envelope rate and drugloading rate through HPLC.

2.3 tablets in vitro are investigated

Select pH7.4PBS buffer solutions that it is special to investigate mPEG-PHDCA-NPs tablets in vitro using dialysis as dissolution medium Property：Doxorubicin solution (DOX-sol) and mPEG-PHDCA-NPs suspensions appropriate (equivalent DOX amounts are 1mg) is taken to pour into respectively Analyse in bag, with being placed in after dialysis clamp opening in 200mL dissolution mediums, be put in constant temperature in 37 DEG C of water bath with thermostatic control vibrations (60rpm) and shake Swing, dissolution medium sample 1mL is taken respectively at 0.25,0.5,1,2,3,4,6,8,12,24,48h, add equivalent blank release and be situated between Matter, DOX contents are determined through HPLC after sample pretreating, calculate cumulative release percentage (Q), draw and be fitted tablets in vitro curve.

2.4 hemolysis in vitro are tested

The fresh rabbit blood after anti-freezing is taken, adds centrifuge tube, upper serum and albumen are removed in 2000rpm centrifugations 5min Afterwards, it is resuspended with PBS and rinses and centrifuge 3 times, until the not aobvious red of supernatant, last PBS dilution is prepared red thin Born of the same parents' concentration is about every milliliter 10⁸Individual storing solution.The mPEG-CA or PHDCA or mPEG- of certain mass concentration are added in centrifuge tube PHDCA, PBS are diluted to graded series concentration so that cumulative volume is 800 μ L, is eventually adding 200 μ L red blood cell deposit Liquid.Centrifuge tube is put into 37 DEG C of constant-temperature tables and vibrated, after 2h, in 2000rpm centrifugations 5min isolate do not rupture it is red thin Born of the same parents.The μ L of upper liquid 100 are collected, its absorbance (A) at 540nm is determined using ELIASA.Feminine gender is used as using corresponding PBS solution Control, pure water solution calculate hemolysis rate (Hemolysis Ratio), Hemolysis Ratio=(A as positive control_Sample- A_{It is cloudy})/(A_Sun-A_{It is cloudy}) * 100%.

2.5 cytotoxicity experiment

Take the logarithm growth period Hela, MCF-7 and MCF-7/ADR cell, after 0.25% Trypsin Induced, collection, centrifugation, It is to contain 2 × 10 per 1mL to be diluted to density with RPMI1640 culture mediums⁴It is individual, 96 orifice plates are taken, after the 190 μ L of addition per hole, stable 12h, Experimental group is separately added into the blank of various concentrations or carries the μ L of medicine mPEG-PHDCA-NPs suspensions 10, makes ultimate density for series ladder Degree, control group add the PBS of equivalent, parallel 6 hole.10 μ L 5mgmL is added after 48h per hole^-1MTT solution, jog for several times after Continuous to be incubated 4h, centrifugation adds 150 μ L DMSO after abandoning supernatant, is placed on oscillator and vibrates 3min, and 570nm ripples are determined with ELIASA Long optical density (OD) value.The mean OD value in 6 holes is taken to calculate the survival rate (Cell Viability) of cell, Cell Viability=OD_{Experimental group}/OD_{Control group}× 100%.

3 results

The sign of 3.1 carrier materials

Polymer HDCA, mPEG-CA, PHDCA and mPEG-PHDCA's¹H-NMR is as shown in Fig. 2 deuterated reagent is CDCl₃, It is marked in structure according to the quantity of diverse location hydrogen and displacement in polymer.In Fig. 2, A figures are HDCA, and B figures are mPEG- CA, C figure are PHDCA, and D figures are mPEG-PHDCA.In figure displacement 0.9 or so be hexadecanol on alkyl chain methyl characteristic peak, position 3.4 or so the characteristic peaks for being polyethylene glycol upper end methoxyl group are moved, using 3 hydrogen on the two characteristic peaks as standard value, are used for Integrating peak areas and the purity or molecular weight for calculating each polymer of assessment.It is computed, HDCA and mPEG-CA synthesis purity are all higher than 95%, and PHDCA and mPEG-PHDCA purity is all higher than 90%.In polymer mPEG-PHDCA, mPEG molecular weight is 2k, repeat unit-C in its structure₂H₄Corresponding about 180 hydrogen of-O-, the corresponding displacement peak integrations about 15 of Fig. 2 D, show mPEG- Repeat unit HDCA in PHDCA is about 12 (180/15=12), therefore the molecular weight for extrapolating mPEG-PHDCA is about 6K (i.e. 2000+85-18+12*309+12*30-12*18=5919).Using gel permeation chrommatograph determine each polymer molecular weight and Molecular weight distribution curve, as shown in figure 3, HDCA and mPEG-CA molecular weight distributions are homogeneous, and mPEG-PHDCA molecular weight distributions are bent Acromion is opened up before having one in line, it may be possible to due to there is a small amount of PHDCA straight chain bilateral even-couplings mPEG-CA in polymerisation. MPEG-PHDCA weight average molecular weight is 5.85K, close to the result of nuclear-magnetism estimation, PDI 1.13.Fig. 2A is HDCA's¹H-NMR (CDCl₃, 400Hz, δ ppm)：0.88(CH₃-, 3H), 1.26 (- C₁₃H₂₆-, 26H), 1.68 (- CH₂-CH₂- OCO-, 2H), 3.45 (-OCO-CH₂- CN, 2H), 4.20 (- CH₂-CH₂- OCO-, 2H).Fig. 2 B are mPEG-CA's¹H-NMR(CDCl₃, 400Hz, δ ppm)：3.42(CH₃- O-, 3H), 3.50~3.75 (- C₂H₄- O-, 182H), 3.81 (- OCO-CH₂- CN, 2H), 4.36 (- CH₂- OCO-, 2H).Fig. 2 C are PHDCA's¹H-NMR(CDCl₃, 400Hz, δ ppm)：0.88(CH₃-, 3H), 1.28 (- C₁₃H₂₆-, 26H), 1.75 (- CH₂-CH₂- OCO-, 2H), 2.20~2.55 (- C-CH₂- C- ,-C-CH₂-CH₂- C- ,-OCO-CH (CN)- CH₂-, 3.46H), 4.27 (- CH₂-CH₂- OCO-, 2H).Fig. 2 D are mPEG-PHDCA's¹H-NMR(CDCl₃, 400Hz, δ ppm)： 0.88(CH₃-, 3H), 1.26 (- C₁₃H₂₆-, 26H), 1.71 (- CH₂-CH₂- OCO-, 2H), 2.10~2.55 (- C-CH₂- C- ,-C- CH₂-CH₂- C- ,-OCO-CH (CN)-CH₂-, 3.05H), 3.42 (CH₃- O-, 0.29H), 3.50~3.75 (- C₂H₄- O-, 14.52H), 4.26 (- CH₂- OCO-, 2.36H).

The sign of 3.2 nanoparticles

The mPEG-PHDCA-NPs being prepared is presented orange red and is had obvious opalescence, average after particle instrument determines Particle diameter is (94.61 ± 3.91) nm, and Zeta potential is (- 11.68 ± 0.83) mV.With transmission electron microscope observing (Fig. 4 B), mPEG- PHDCA-NPs is in ball particle, and surface is round and smooth, uniform in size, and distribution is good, and adhesion and agglomeration are had no between particle.Through HPLC determines envelop rate and drugloading rate, as a result show drugloading rate that mPEG-PHDCA-NPs contains adriamycin be 2.17 ± 0.67%, envelop rate is 79.54 ± 4.66%, has preferable Drug loading capacity.

The release behaviour in vitro of 3.3 nanoparticles

Tablets in vitro curve maps of the DOX-sol and mPEG-PHDCA-NPs in PBS is shown in Fig. 5.As seen from the figure, DOX-sol releases the drug quickly in dissolution medium, and medicine substantially all release during 4h, cumulative release percentage reaches 95.32%. MPEG-PHDCA-NPs drug release behaviors can be divided into it is prominent releases and be sustained two-phase, during beginning release comparatively fast, in 3h the accumulation of medicine release It is respectively 42.25% to put percentage, may be adsorbed due to some drugs in the release of nanoparticle surface comparatively fast, and subsequent drug release profiles Gradually steady, slowly drug release, reaches 85.38% in preparation in 48h.Respectively with zero level, First order dynamic model, Higuchi models, Weibull models are fitted to its tablets in vitro behavior, and mPEG-PHDCA-NPs is in PBS Drug release behavior meets Weibull equations, is lnln (1/1-Q)=0.6582lnt-1.3154 (R²=0.9794).

The tablets in vitro curvilinear equation of table 1 is fitted

3.4 hemolysis in vitro are investigated

Hemolysis in vitro experiment is one of important indicator for evaluating preparation security, commonly uses external test tube method and AAS Detected, due to the slightly higher rear presentation white " milky " of polymer solution concentration, external test tube method can not be used to carry out accurate judgement, Therefore using absorbance of the release hemoglobin at 540nm after spectrophotometry red blood cell rupture.Polymer concentration-red For cell hemolysis rate curve map as shown in fig. 6, when concentration is less than 0.1mM, each polymer will not significantly cause erythrocyte hemolysis； When concentration increases to 1mM, PHDCA starts to cause more serious haemolysis, mainly may be due to Polyacrylate materials There is stronger bioadhesion to act on for itself, causes cell membrane disorderly when being largely adhered to cell membrane surface and ruptures, and by It is disorderly in the hydrophily of polyglycol chain and the flexible adhesion that can be protected between gentle depolymerization compound and cell membrane and cell membrane, MPEG-CA and mPEG-PHDCA will not cause cell haemolysis.

3.5 vitro cytotoxicities are investigated

Blank is investigated using mtt assay and carries medicine mPEG-PHDCA-NPs to tri- kinds of cells of Hela, MCF-7 and MCF-7/ADR The cytotoxicity of system, as shown in Figure 7 A, for blank mPEG-PHDCA-NPs when concentration reaches 0.5mM, the survival rate of cell is still More than 90% is maintained at, without obvious cytotoxicity, shows that mPEG-PHDCA has higher biocompatibility.Carry medicine MPEG-PHDCA-NPs has certain cyto-inhibition to Hela, MCF-7 and MCF-7/ADR, using SPSS softwares, probability Per unit system is fitted its IC₅₀Respectively 7.14 μM, 7.65 μM, 8.53 μM, with DOX-sol (IC₅₀Respectively 3.49 μM, 2.86 μM, 14.92 μM) compare, medicine mPEG-PHDCA-NPs is carried to Hela and MCF-7 cytotoxicity significantly less than DOX-sol, and it is right MCF-7/ADR cytotoxicity is noticeably greater than DOX-sol.Because mPEG-PHDCA-NPs drug releases are slow, make really to play drug effect Medicine be far below actual concentrations, therefore be less than DOX-sol to Hela and MCF-7 cytotoxicity, and MCF-7/ADR belong to Ah Mycin drug-resistant type cell line, the cell line overexpression P- glycoprotein, adriamycin can be arranged outside, and nanoparticle has reverse P- sugar The function of albumen drug resistance, so carry medicine mPEG-PHDCA-NPs is more than DOX-sol to MCF-7/ADR cytotoxicity.

4 discuss

Although nanometer formulation brings dawn to diagnose and treating the diseases such as malignant tumour, clinical examination can be eventually entered into The product tested or even push application to is but very rare, the EPR effects that one side of tracing it to its cause nanometer formulation plays a role relied on In clinical pathology and unobvious, animal experiment is set to be differed greatly with clinical test results；Further aspect is that the nanometer system of preparation The carrier material of agent can not meet clinical requirement, and inorganic material possesses higher stability and Drug loading capacity, while is dropped by hardly possible The restriction of solution property and toxicity, organic polymer possess excellent biocompatibility and degradability, but often stability difference and load Dose is low and is difficult to apply.Therefore, the research of nanometer formulation had both been needed using more accurate clinical rationale as guidance, while was also needed Perfect carrier material is wanted to rely on, the mPEG-PHDCA carrier materials of synthesis and investigation are also by for nanometer formulation in this research Development provides reference.

In nanoparticle technique is prepared, compare and other method (self-emulsifying solvent diffusion method, nanoprecipitation method, emulsion Polymerization etc.), this studies the double emulsion-solvent evaporation technique of selection advantageously in preparing water soluble medicament-entrapping nanoparticle, and it is former Reason emulsifies to form W/O/W type emulsion droplets, then by vacuum rotary steam removing have using water soluble drug solution as interior aqueous phase by two steps Solvent, polymer support separates out the process of assembling balling-up while contains the medicine of interior aqueous phase, wherein stable colostrum is the method Successfully crucial, integrated artistic is more complicated, it is desirable to higher.Because the meeting that water soluble drug is difficult to avoid that in preparation process is outside Aqueous phase leaks, or is adsorbed during revolving removes organic solvent on nanoparticle surface, causes drug carrying ability to reduce, but compare In report document, carrier material mPEG-PHDCA still has preferable envelop rate and drugloading rate.

To sum up, this research successfully synthesizes organic polymer using Knoevenagel reactions and anionic polymerisation MPEG-PHDCA, double emulsion-solvent evaporation technique are prepared for mPEG-PHDCA nanoparticles, have preferable envelop rate and drugloading rate, Uniform particle diameter, good dispersion, there are necessarily slow controlled release characteristics, and tentatively show good biocompatibility, be expected to turn into Nanometer passs drug carrier material, but its vivo biodistribution security needs further to be studied.

Claims (3)

1. a kind of preparation method of PEG6000-PHDCA load adriamycin nano-particles, its feature exist In the described method comprises the following steps：

(1) adriamycin is dissolved into 2.5mgmL with deionized water^-1Interior aqueous phase storing solution；The poly- cetyl cyanogen of Pegylation Base acrylate is dissolved with organic solvent, is made into 25mgmL^-1Organic phase；The organic solvent is dichloromethane, acetic acid second Ester volume ratio 1:1 mixing；Interior aqueous phase storing solution is added in organic phase, interior aqueous phase storing solution, the volume ratio of organic phase are 0.5:2, Probe Ultrasonic Searching emulsion dispersion, form colostrum；The average mark of the PEG6000-PHDCA Son amount is 5800~6000；

(2) aqueous solution for the PLURONICS F87 that mass concentration is 0.5% is prepared, regulation pH to 8.5~9.5 forms outer aqueous phase, Under magnetic agitation, outer aqueous phase is added dropwise in colostrum, interior aqueous phase storing solution, organic phase, the volume ratio of outer aqueous phase are 0.5:2: 10, after dripping off, continue to stir 30min, form emulsion, emulsion vacuum rotary steam is removed into organic solvent and part water, poly- second is made Diolation poly- cetyl cyanoacrylate load adriamycin nano-particles suspension.

2. the method as described in claim 1, it is characterised in that the pH value of the outer aqueous phase is 9.0.

3. the method as described in claim 1, it is characterised in that the PEG6000-PHDCA is pressed Following methods are made：

(a) cetyl cyan-acetic ester HDCA synthesis

Weigh hexadecanol 200mmol and cyanoacetic acid 240mmol is placed in 500mL three-neck flasks, add 120mL CH₂Cl₂With 20mL ethyl acetate, magnetic agitation dissolving, is slowly added dropwise dissolved with 300mmol condensing agent DCC and 20mmol catalyst DMAP's 60mL CH₂Cl₂Solution, nitrogen protection stirring room temperature reaction 24h, adds distillation water destruct DCC, filters, collect filtrate, filter residue is used Ethyl acetate is washed to white, and merging filtrate puts separatory funnel extraction, is washed with supersaturated NaCl solution, adds anhydrous slufuric acid Sodium, the moisture in organic phase is removed, rotate organic solvent, separate out HDCA crude products, gained crude product is polished, is beaten with absolute ethyl alcohol Slurry recrystallization, purifying obtain sterling HDCA faint yellow solids；

(b) polyethylene glycol cyan-acetic ester mPEG-CA synthesis

Weigh polyethylene glycol 10mmol, DCC15mmol and DMAP10mmol to be placed in 250mL three-neck flask, add 150mL CH₂Cl₂With 25mL ethyl acetate, magnetic agitation dissolving；Weigh the CH that cyanoacetic acid 12mmol is dissolved in 30mL₂Cl₂With 10mL second In acetoacetic ester, it is slowly added dropwise into flask, at room temperature nitrogen protection stirring reaction 24h；Distilled water is added, DCC is destroyed, through taking out Filter, CH₂Cl₂Washing, separatory funnel extraction, collected organic layer, the washing of supersaturated NaCl solution, anhydrous sodium sulfate water removal, revolving have Solvent, obtain mPEG-CA crude products；Crude product is polished, white with sterling mPEG-CA is obtained after ethyl acetate mashing recrystallization purifying Color waxy solid；

(c) poly- cetyl cyanoacrylate PHDCA synthesis

PHDCA is that monomer HDCA is polymerized：Take HDCA 10g to be placed in 100mL three-neck flasks, add 30mLCH₂Cl₂And 10mL Absolute ethyl alcohol, magnetic agitation dissolving, it is 37% formalin and 0.2mL pyrrolidines that 3.6mL mass concentrations, which are added dropwise, into reaction solution, Nitrogen protection stirring reaction 24h at room temperature；Reaction solution is transferred in separatory funnel, successively with 10% salt acid elution, distilled water Washing, is finally washed, collected organic layer with supersaturated NaCl solution, the moisture added in anhydrous sodium sulfate removing organic phase, rotation Organic solvent is steamed, separates out poly- PHDCA crude products；Crude product is polished, and sterling PHDCA is obtained after being beaten crystallization purifying with absolute ethyl alcohol Yellow solid；

(d) the poly- cetyl cyanoacrylate mPEG-PHDCA of Pegylation synthesis

Weigh PHDCA 22g and mPEG-CA 10g to be placed in three-neck flask, add 100mLCH₂Cl₂With 50mL ethyl acetate, magnetic It is 37% formalin and 0.1mL pyrrolidines that 1.8mL mass concentrations are added dropwise after power stirring and dissolving, adds DCC and DMAP catalysis, room The lower nitrogen protection stirring reaction 48h of temperature；Reaction solution is transferred in separatory funnel, successively with 10% salt acid elution, distillation washing Wash, finally washed with supersaturated NaCl solution, collected organic layer, the moisture added in anhydrous sodium sulfate removing organic phase, revolving Organic solvent, separate out mPEG-PHDCA crude products；By crude product with a small amount of CH₂Cl₂After dissolving, ice ether precipitates 2 times, and solid is through oil pump Drying is vacuumized, obtains faint yellow sterling mPEG-PHDCA；Obtained mPEG-PHDCA mean molecule quantities are 5800~6000.