CN109364256A - A kind of agarose micella pharmaceutical carrier - Google Patents

Abstract

The present invention relates to agarose technical field, in particular to a kind of agarose micella pharmaceutical carrier.Agarose carrier energy payload drug of the invention, drug includes antibiotic and/or anti-tumor drug, so as to effectively improve biocompatibility, the histocompatbility of drug；Slow releasing function is played when acting on body, drug toxicity can be reduced, while drug of the invention also has good antibacterial, antitumor activity energy, there are greater advantages than conventional medicament.

Description

A kind of agarose micella pharmaceutical carrier

[technical field]

The present invention relates to agarose technical field, in particular to a kind of agarose micella pharmaceutical carrier.

[background technique]

Antibiotic be by microorganism or high animals and plants in life process caused by have antipathogen or other work A kind of secondary metabolite of property, can interfere the chemical substance of other living cells development functions.It is currently known natural antibiotics Not lower ten thousand kinds.The antibacterial or bactericidal effect of the antibacterial agents such as antibiotic is applied to medically extensively, still, since antibiotic does not have There is discrimination, can also kill beneficial bacterium while killing disinfect pathogen, therefore, antibiotic dosage is improper will to will cause antibiotic Antibiotic is used for a long time in abuse, patient, it will generates drug resistance, will finally will lead to " no medicine can cure ".In addition, anti-tumor drug It is big to human body toxic side effect.Therefore, in order to reduce the toxicity of antibiotic and anti-tumor drug, in addition to the control on dosage, number System is outer, there are also many medical personal's Study of Support medicines, accomplishes single to be administered for cause of disease lesion, improves drug and cell phase Capacitive reduces drug toxicity, and still, there is presently no about the research of agarose-polyethylene glycol carrier medicine.This is because natural Agarose by D- galactolipin and 3, the chain neutral polysaccharide that 6- inner ether-L- galactolipin is constituted is (31-36 in gelling temp DEG C) have reversible at gelation, and in solution temperature (65-85 DEG C) not at gel, this feature makes agarose become base The good carrier of cause, polypeptide or protein drug and living cells, because biologically active drug and living cells can be suitable for Temperature is embedded in Ago-Gel, in addition the hydrophily of agarose, biocompatibility, will not be such that polypeptide or protein drug occurs Denaturation, is a kind of pharmaceutical carrier raw material with good prospect.But due to containing hydroxy functional group in its structural unit, easily In in structural unit hydrogen atom and segment surrounding water molecules form hydrogen bond and be dispersed in water with random coil, solution clarification is saturating It is bright, it is a kind of polymer with distinct temperature responsiveness.In the agarose that nature is isolated, its degradation is slow, it is also desirable to have Higher phase transition temperature, and it does not have adhesiveness to albumen, polypeptide, cell, thus agarose is limited in biomedicine With the application in other field.And polyethylene glycol (PEG) has low exempt to human body with good hydrophilic and biocompatibility Epidemic disease reaction, is ratified by U.S. Food and Drug Administration (FDA), can be applied to clinical biomaterial.If can use it to Agarose is modified, and prepares agarose gel beam loading drug, it will immune response of the human body to drug is reduced, it can be preferably by fine jade Lipolysaccharide is applied to field of biomedicine.But it at present and has no any and modifies agarose about using polyethylene glycol (PEG) Relevant report；Also the report of relevant agarose polyethylene glycol medicine carrier is had no.

[summary of the invention]

In view of above content, it is necessary to provide a kind of agarose micella pharmaceutical carrier, the carrier is by agarose and poly- second two Hydramine reaction is made, and by introducing hydrophilic macromolecule segment PEG (polyethylene glycol) in agarose structure, it is double to destroy agarose Helical structure is formed, and agarose phase transition temperature is reduced, which can be effectively reduced human body to antibiotic Agarose preferably can be applied to field of biomedicine by immune response.

In order to achieve the above objectives, the technical scheme adopted by the invention is that:

A kind of agarose micella pharmaceutical carrier, the carrier are the graft polymers of agarose and polyethylene glycol.

Further, the carrier is 1:16 anti-by alkalization according to mass ratio by agarose and methoxy poly (ethylene glycol) amine It should be made with nucleophilic substitution.

Further, the compatibility of the carrier and cervical cancer cell is greater than 90%.

The present invention also provides a kind of carrier medicines prepared by the pharmaceutical carrier, which is characterized in that the drug is anti- Raw element and/or anti-tumor drug.

Further, the antibiotic is vancomycin, and anti-tumor drug is adriamycin.

The present invention also provides a kind of preparation methods of carrier medicine, which is characterized in that the method includes walking as follows It is rapid:

(1) medical preconditioning: dissolving antibiotic and/or anti-tumor drug with dimethyl sulfoxide solution, triethylamine be then added, The drug solution for obtaining having pre-processed for 24 hours is uniformly rocked at room temperature；

(2) carrier loaded: to dissolve agarose micella pharmaceutical carrier with dimethyl sulfoxide solution, step (1) is then added and locates in advance The drug solution managed, after mixing in cell crushing instrument ultrasonication 20min.

(3) reaction solution after step (2) ultrasonication is fitted into bag filter and is dialysed, is lyophilized after dialysis, complete antibiotic And/or anti-tumor drug load.

Further, the dialysis bag retention molecular weight of the dialysis is 3500D.

The present invention also provides the carrier medicine antibacterial and/or it is antitumor in application.

The invention has the following beneficial effects:

Agarose carrier energy payload drug of the invention, drug include antibiotic and/or anti-tumor drug, so as to Effectively improve biocompatibility, the histocompatbility of drug；Slow releasing function is played when acting on body, drug toxicity can be reduced, Drug of the invention simultaneously also has good antibacterial, antitumor activity energy, has greater advantages than conventional medicament.Agar of the invention Sugar carrier, which is reacted by agarose with polyoxamide, to be made, by quaternization and nucleophilic substitution, by polyethylene glycol segment It is grafted on agarose, agarose-grafting-poly glycol monomethyl ether polymer (Agarose-g-mPEG) is prepared.The party Method can destroy agarose double-spiral structure and be formed, and reduce agarose phase transition temperature, reduce immune response of the human body to drug, side Method is simple, and reaction condition is mild, and processing is convenient.

[Detailed description of the invention]

Fig. 1 is the MTT evaluation figure of carrier of the embodiment of the present invention；

Fig. 2 is the haemolysis evaluation figure of carrier of the embodiment of the present invention；

Fig. 3 is the BSA circular dichroism spectrogram of carrier of the embodiment of the present invention；

Fig. 4 is the histotomy figure after H&E dyeing of the present invention；

The antibacterial micella and growth curve chart after Bacteria Culture 6h that Fig. 5 is the load vancomycin of various concentration of the present invention；

Fig. 6 is the Critical Solution micellar concentration figure of micellar solution of the present invention；

Fig. 7 is the transmission electron microscope figure of present invention load vancomycin micella；

Fig. 8 is the hydrated diameter figure of present invention load vancomycin micella；

Fig. 9 is the transmission electron microscope figure of present invention load adriamycin micella；

Figure 10 is the hydrated diameter figure of present invention load adriamycin micella；

Figure 11 is the nucleus magnetic hydrogen spectrum figure of carrier of the present invention at different temperatures.

[specific embodiment]

All features disclosed in this specification or disclosed all methods or in the process the step of, in addition to mutually exclusive Feature and/or step other than, can combine in any way.

Any feature disclosed in this specification (including any accessory claim, abstract), unless specifically stated, each Feature is an example in a series of equivalent or similar characteristics.

Embodiment 1 (preparation of load vancomycin carrier):

With the vancomycin of the dimethyl sulfoxide solution dissolution 8mg of 500 μ L, then it is added 35 μ L's (25.5mg, 44 μm of ol) Triethylamine uniformly rocks for 24 hours at room temperature, removes hydrochloride；Then with the dimethyl sulfoxide solution dissolution 20mg's of 500 μ L Agarose-g-PEG_20k-7；The Agarose-g-PEG that dimethyl sulfoxide solution is dissolved_20k- 7 and dimethyl sulfoxide solution dissolution ten thousand Ancient mycin mixing, then adds 5mL deionized water, cell crushing instrument ultrasound, and ultrasonic 5s stops 3s, duration 20min. It after sufficiently ultrasound, is transferred in the dialysis band that molecular cut off is 3500, dialysis band is placed in 1000mL distilled water and is dialysed 48h, every 12h change a water, remove free vancomycin and solvent dimethyl sulfoxide.Three parts of samples of preparation in parallel, every part of sample 300 μ L polypeptide drug-loaded micelle solutions are taken in solution, freeze-drying for 24 hours, respectively takes 2mL dimethyl sulfoxide solution to dissolve, in ultraviolet-visible spectrophotometer Under, it measures in the absorbance that wavelength is 281nm, calculates vancomycin content, be averaged.

Embodiment 2 (preparation of load adriamycin carrier):

2mg doxorubicin hydrochloride is dissolved with the dimethyl sulfoxide solution of 500 μ L, three second of 35 μ L (25.5mg, 44 μm of ol) are added Amine, uniformly concussion overnight, removes hydrochloride at room temperature.Then with the dimethyl sulfoxide solution dissolution 20mg's of 500 μ L Agarose-g-PEG_20k- 7, the Agarose-g-PEG that dimethyl sulfoxide solution is dissolved_20k- 7 and dimethyl sulfoxide containing adriamycin it is molten Liquid mixing, then adds 5mL deionized water, cell crushing instrument ultrasound, and ultrasonic 5s stops 3s, duration 20min.Wait fill It after dividing ultrasound, is transferred in the dialysis band that molecular cut off is 3500, dialysis band is placed in 1000mL distilled water the 72h that dialyses, Every 12h changes a water, removes free adriamycin and solvent dimethyl sulfoxide.Three parts of samples of preparation in parallel, in every part of sample solution 300 μ L polypeptide drug-loaded micelle solutions are taken, freeze-drying for 24 hours, respectively takes 2mL dimethyl sulfoxide solution to dissolve, under ultraviolet-visible spectrophotometer, wave A length of 480nm measures doxorubicin content, is averaged.

The embodiment of the present application 1-2 agarose micella pharmaceutical carrier (Agarose-g-PEG_20k- 7) the preparation method is as follows:

Agarose is subjected to quaternization and nucleophilic substitution, hydrophilic macromolecule segment is introduced in agarose structure Polyethylene glycol；The method of the quaternization are as follows: be heated to fine jade after mixing 0.1g low melting-point agarose and 100mL deionized water Lipolysaccharide sufficiently dissolves；Sodium hydroxide and the 1.6mL epoxychloropropane isothermal reaction of 0.5g are added after being cooled to 50-65 DEG C under room temperature 2h；The method of the nucleophilic substitution are as follows: methoxy poly (ethylene glycol) of the 5mL containing different quality is added into quaternization product Deionized water reacts 72h under the conditions of 30 DEG C；Then reaction solution is transferred to bag filter, dialysed 3 days, every 12h changes a water, freezes It is dry；The temperature of constant temperature is 30 DEG C in the quaternization；The molecular weight of the methoxy poly (ethylene glycol) amine is 20k；The agar Sugar is 1:16 with methoxy poly (ethylene glycol) amine mass ratio；The molecular weight of the bag filter retention is 50k.

The micelle medicine carrying amount and encapsulation rate of the embodiment of the present application 1-2 is calculated by following formula:

Carrier property test:

(1) MTT is evaluated

Cervical cancer cell (Hela) is inoculated in 96 orifice plates with the density in every hole 5000, in 37 DEG C, CO₂The perseverance that concentration is 5% After cultivating for 24 hours in warm incubator, the Agarose-g-PEG that concentration is 20-200 μ g/mL is added_20k- 7 carriers be incubated for altogether for 24 hours, with Then 3- (4,5- dimethylthiazole -2) -2,5- bis- that 20 μ L concentration are 5mg/mL is added as control in culture medium RPMI-1640 Phenyl tetrazole bromide (MTT) solution after being incubated for 4h, sucks culture medium, the dimethyl sulfoxide solution of 150 μ L is added, in microplate reader On rock 2min, wavelength is that 490nm surveys light absorption value.Cell survival rate is according to the following formula:

As shown in Figure 1, working as Agarose-g-PEG_20kWhen -7 concentration are 20 μ g/mL, the survival rate of cervical cancer cell (Hela) 99.89%.Improve Agarose-g-PEG_20k- 7 concentration, the survival rate of Hela cell 90% or more, show Agarose-g- PEG_20kThe toxicity of -7 couples of Hela is lower, has good biocompatibility.

(2) haemolysis is evaluated

PBS is added in the red blood cell (HRBC) of people, centrifugation (1500rpm × 10min) is washed six times, until supernatant is nothing Color, PBS solution dilute stand-by (concentration are as follows: 2.0 × 10⁸A/mL).Red blood cell (HRBC) solution of 200 μ L people is taken, is added certain The concentration of volume is 200 μ g/mLAgarose-g-PEG_20k- 7 solution and PBS.Agarose-g-PEG_20k- 7 solution ultimate densities For 0~100 μ g/mL.Negative control group is PBS, and positive controls are deionized water.It is placed in 37 DEG C of insulating boxs and is incubated for 2h.Sample containing red cell suspension is centrifuged 10min under the conditions of 1500rpm/min, supernatant is taken, passes through multifunctional enzyme mark Instrument measures its each group sample wavelength light absorption value at 541nm, calculates degree of hemolysis.Hemolysis rate is calculated as follows:

Agarose-g-PEG is evaluated in vitro by hemolytic experiment_20kWhether -7 have an impact to erythrocyte.Deionized water With PBS solution respectively as positive and negative control.As shown in Fig. 2, erythrocyte is destroyed, and is released when deionized water is added Hemoglobin is put, solution takes on a red color.In PBS solution, erythrocyte is not affected by destruction, and after centrifugation, supernatant is also colourless It is bright.Agarose-g-PEG is added_20kAfter -7, micellar concentration is the corresponding hemolysis rate of 20-100 μ g/mL are as follows: 0.32%, 0.43%, 0.05%, 0.21%, 0.19%, it is below the standard of haemolysis degree 5% as defined in National Pharmacopeia.

(3) circular dichroism spectra (CD) is tested:

It weighs 68mg bovine serum albumin(BSA) (BSA), is added 100mL phosphate buffer solution (PBS), ultrasonic disperse.Prepare 200 The Agarose-g-PEG of μ g/mL_20k- 7PBS solution takes the Agarose-g- of 400 μ L BSA solution and certain volume respectively PEG_20k- 7 solution and PBS, are sufficiently mixed, at 25 DEG C, circular dichroism spectra of the measurement wavelength in 190-250nm.The experimental results showed that As shown in figure 3, with Agarose-g-PEG_20k- 7 concentration increase, and significant change does not occur, shows Agarose- for the conformation of BSA g-PEG_20k- 7 on the conformation of BSA without influence.

(4) H&E is dyed

Compound concentration are as follows: the Agarose-g-PEG of 0.1mg/mL, 1mg/mL, 10mg/mL_20k- 7 solution (solvent 0.9% Physiological saline).12 BALB/C mices (about 20g/ every) are taken, four groups (control group and experimental groups), control group are randomly divided into With experimental group every group 3.By mouse anesthesia, every experimental mice injects the sample of 100 μ L various doses by tail vein respectively Product solution (0.5,5,50mg/kg), negative control group inject the physiological saline of 100 μ L 0.9%, and normal raising is for 24 hours.By every group In mouse select one at random, execution takes its heart, liver, spleen, lung, kidney, is fixed for 24 hours with 4% paraformaldehyde, makes tissue paraffin Gu block, slice, H&E dyeing are taken pictures.

In order to further investigate the biocompatibility of micella and tissue, pass through tail vein injection difference in BALB/C mice body The Agarose-g-PEG of concentration_20k- 7 micellar solution, for 24 hours, solution treats others for earnest, liver, spleen, lung, kidney for culture, and H&E is dyed and done tissue Slice analysis, as a result as shown in Figure 4: the experimental results showed that, the heart, liver, spleen, lung, kidney histotomy in there is no pathological tissue Damage or abnormal phenomenon, show Agarose-g-PEG under experimental study dosage_20k- 7 micella has good histocompatbility.

(5) antibacterial experiment

Antibacterial micella is added in Gram-negative bacteria Escherichia coli (E.coli) solution, is surveyed by ultraviolet specrophotometer Determine optical density at 600nm (O.D) value, evaluates the anti-microbial property of antibacterial micella.Specific step is as follows:

1. the preparation of nutrient broth and agar medium:

Solid medium: tryptone 2.5g, yeast extract 1.25g, sodium chloride 0.5g, agar 7.5g；

Fluid nutrient medium: tryptone 5g, beef extract 2.5g, sodium chloride 2.5g

2. deionized water dissolving is settled in 500mL, adjusting culture medium solution pH by sodium hydroxide is 7.4；

3. after culture solution sealing, 121 DEG C, 20min carries out high pressure sterilization；

4. taking out culture solution, appropriate to save: solid medium is placed in the environment not less than 55 DEG C, prevents from cooling and solidifying； Fluid nutrient medium is cooled to room temperature, and is placed in 4 DEG C of refrigerators and is saved backup；

5. in super-clean bench after sterilization, measuring 10mL solid medium with graduated cylinder, culture dish being placed into, in clean bench Air-drying solidifies culture medium, as saving backup in 4 DEG C of refrigerators after being wrapped culture medium with preservative film；

6. fluid nutrient medium is added by various concentration microbionation in culture bottle, cultivated at 37 DEG C for 24 hours, measurement is thin Bacteria concentration OD value；In addition, the bacterium solution of above-mentioned various concentration is smeared on a lbmc agar plate simultaneously, is cultivated at 37 DEG C for 24 hours, calculate bacterium Number is fallen, determines OD value-clump count standard curve；

7. respectively taking 200 μ L vancomycin micellar solutions, micella is respectively as follows: 0,6,20,30 and 40 μ g/ in bacterium solution emphasis concentration The empty vectors micellar solution of mL and corresponding same vehicle concentration, instill into culture bottle, cultivate 6h at 37 DEG C.

The Agarose-g-PEG of load vancomycin is investigated at Gram-negative bacteria Escherichia coli (E.coil)_20k- 7 micellas Anti-microbial property, Agarose-g-PEG_20k- 7 micellas as a control group, as shown in figure 5, research discovery 6h after, load it is mould through the ages The Agarose-g-PEG of element_20k- 7 micellas can inhibit E.coil to increase, and as the concentration of carrier micelle improves, E.coil bacterium colony Number is reduced.When carrier micelle concentration is 40 μ g/mL, it is suppressed that E.coil increases.However, there is identical load in control group In the bacterium solution of weight, E.coil sustainable growth shows blank micella without anti-microbial property.

(6) Critical Solution micellar concentration (CMC)

Critical Solution micellar concentration is the minimum concentration that polymer forms micella in the solution.Agarose is made by hydrogen bond With, formation hydrophobic cores, hydrophilic layer of the polyethylene glycol as micella.With Agarose-g-PEG_20k- 7 concentration increase, polymerization Object is acted on by hydrophobe, is self-assembled into micella, the pyrene for being insoluble in water is wrapped and is born into the hydrophobic core of micella, the glimmering of pyrene is caused Luminous intensity increases.CMC the result shows that, as shown in fig. 6, Agarose-g-PEG_20k- 7 minimum concentrations for forming micella are 31.55 μ g/mL.Vancomycin is a kind of glycopeptide antibiotic, by inhibiting the synthesis of cell wall, kills bacterium, is commonly used for research drug resistance The drug model of bacterium.Adriamycin is a kind of most popular anthracene series antineoplastic medicament, by separating adriamycin in conjunction with DNA Double helix chain, prevention form DNA purine and DNA dependent form RNA polymerase, hinder DNA and RNA synthesis, inhibit tumour cell raw It is long.By dialysis, vancomycin and adriamycin are supported on Agarose-g-PEG_20kIn -7, the drugloading rate of the micella is studied And encapsulation rate.

The calculation formula of drugloading rate is as follows:

The calculation formula of encapsulation rate is as follows:

The drugloading rate of vancomycin is calculated by above formula and encapsulation rate is respectively as follows: 1.12% and 2.58%；Adriamycin Drugloading rate and encapsulation rate are respectively as follows: 3.23% and 8.06%.Agarose-g-PEG_20kThe drugloading rate of -7 load adriamycins is higher than ten thousand The drugloading rate of ancient mycin, shows Agarose-g-PEG_20k- 7 are easier load adriamycin, this may be due to adriamycin and agar Sugar forms more hydrogen bonds.

(7) transmission electron microscope (TEM)

Pass through the exterior appearance of transmission electron microscope (TEM) observable nanoparticle.Vancomycin and adriamycin conduct Drug model observes Agarose-g-PEG_20kExterior appearance and size after -7 carrying medicaments.Load vancomycin Agarose-g-PEG_20kFor -7 micellas in the spherical shape (as shown in Figure 7) of rule, diameter is about 77.5nm, is surveyed by dynamic light scattering The hydrated diameter of micella is 85.7nm (as shown in Figure 8) out, and Zeta potential is -18.94mV.The micella for loading adriamycin is also in ball Shape (as shown in Figure 9), diameter 63.8nm, measuring the hydrated diameter of micella by dynamic light scattering is 88.2nm (such as Figure 10 institute Show), Zeta potential is -10.89mV.

(8) temperature-responsive

Agarose has special temperature-responsive.In low temperature, agarose relies on sugar chain hydrogen bond action, with double helix side Formula is mutually wound, and forms double-spiral structure, and close-packed arrays form gel, white muddiness；At high temperature, in structural unit Hydrogen bond is destroyed, and is dispersed in water with random coil, solution clear.PEG is grafted in agarose structure unit_20kSegment Afterwards, Agarose-g-PEG_20kWhether -7 there is temperature-responsive to still need to further investigate.By testing at different temperatures in this chapter Agarose-g-PEG_20k- 7¹HNMR studies Agarose-g-PEG_20kWhether -7 have temperature-responsive.

As shown in figure 11, as temperature increases, D₂The chemical shift of the solvent peak of O changes, and 25 DEG C are 4.80ppm, 4.36ppm at 65 DEG C, but Agarose-g-PEG_20kSignificant change does not occur for -7 proton intensity.Temperature is increased, is failed obvious Weaken agarose hydrogen bond action in hydrophobic segment, micella is still evenly dispersed in solution, and modified agarose micella does not have There is temperature-responsive.

In conclusion using the agarose carrier energy payload drug of the application, especially antibiotic, so as to effective The biocompatibility for improving antibiotic, plays slow releasing function, can reduce antibiotics toxic, have greater advantages than conventional medicament.

The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously Limitation of the scope of the invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art, Without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection model of the invention It encloses.Therefore, protection scope of the present invention should be determined by the appended claims.

Claims (8)

1. a kind of agarose micella pharmaceutical carrier, which is characterized in that the carrier is the graft polymerization of agarose and polyethylene glycol Object.

2. a kind of agarose micella pharmaceutical carrier according to claim 1, which is characterized in that the carrier is by agarose and first Oxygroup polyoxamide is 1:16 obtained by quaternization and nucleophilic substitution according to mass ratio.

3. a kind of agarose micella pharmaceutical carrier according to claim 1, which is characterized in that the carrier and cervical cancer cell Compatibility be greater than 90%.

4. a kind of carrier medicine of the pharmaceutical carrier as described in claim 1-3 any one preparation, which is characterized in that the drug is Antibiotic and/or anti-tumor drug.

5. carrier medicine according to claim 4, which is characterized in that the antibiotic is vancomycin, and anti-tumor drug is Adriamycin.

6. a kind of preparation method for preparing carrier medicine as claimed in claim 4, which is characterized in that the method includes walking as follows It is rapid:

(1) medical preconditioning: antibiotic and/or anti-tumor drug are dissolved with dimethyl sulfoxide solution, triethylamine, room temperature is then added The drug solution for obtaining having pre-processed for 24 hours is uniformly rocked down；

(2) carrier loaded: to dissolve agarose micella pharmaceutical carrier with dimethyl sulfoxide solution, step (1) is then added and pre-processes Drug solution, after mixing in cell crushing instrument ultrasonication 20min.

(3) reaction solution after step (2) ultrasonication is fitted into bag filter and is dialysed, is lyophilized after dialysis, complete antibiotic and/or Anti-tumor drug load.

7. preparation method according to claim 6, which is characterized in that the dialysis bag retention molecular weight of the dialysis is 3500D。

8. carrier medicine as claimed in claim 3 antibacterial and/or it is antitumor in application.