CN115737832A - Hyaluronic acid-dolastatin conjugate and application thereof in tumor resistance - Google Patents

Hyaluronic acid-dolastatin conjugate and application thereof in tumor resistance Download PDF

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CN115737832A
CN115737832A CN202211378445.4A CN202211378445A CN115737832A CN 115737832 A CN115737832 A CN 115737832A CN 202211378445 A CN202211378445 A CN 202211378445A CN 115737832 A CN115737832 A CN 115737832A
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mmae
hyaluronic acid
oil
conjugate
dolastatin
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谭海宁
孙凤
高迪迪
李妍
侯慧文
王洁
唐雯
卢鲁
符家爱
刘增美
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Shandong University
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Abstract

The invention belongs to the field of biomedicine, and provides a hyaluronic acid-dolastatin conjugate and application thereof in tumor resistance. The preparation method comprises the following steps: the method comprises the following steps of constructing the MMAE nano liposome, carrying out encapsulation efficiency and drug loading capacity measurement on the obtained MMAE nano liposome, modifying the MMAE nano liposome by hyaluronic acid, evaluating the physicochemical properties of the product hyaluronic acid-MMAE conjugate, including morphology, particle size and potential, and evaluating the drug effect of the product hyaluronic acid-MMAE conjugate, including in vitro release condition and in vitro cytotoxicity. The result shows that the conjugate has lower toxicity than D10 and better targeting property than MMAE, in addition, the problem of indissolvability of the drug can be solved by coating the drug by lipid nanoparticles, the biodegradation of the drug is effectively avoided, the release and the biodistribution of the encapsulated drug are improved, and the conjugate has good application prospect.

Description

Hyaluronic acid-dolastatin conjugate and application thereof in tumor resistance
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a hyaluronic acid-dolastatin conjugate, a preparation method thereof, a pharmaceutical composition containing the conjugate, and application of the conjugate and the pharmaceutical composition in tumor resistance.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Malignant tumors (cancer) have become one of the major public health problems that severely threaten the health of people, and statistics have shown that in recent years, the incidence of malignant tumors has increased by about 3.9% every year and the mortality has increased by 2.5% every year. The patient base will show a steady increase over a long period of time, and finding suitable drugs to address the malignancy is currently of great importance.
Dolastatin 10 (D10) is a natural marine extract, a potent antimitotic, highly cytotoxic polypeptide. The anticancer mechanism is similar to that of vinblastine, and the anticancer mechanism is combined with amino acid residue of beta-tubulin to strongly inhibit the formation and polymerization of microtubules, and also inhibit the hydrolysis of tubulin depending on GTP to arrest mitosis. Further research also finds that D10 can induce apoptosis of various cancer cells and has strong anti-angiogenesis effect. D10 shows encouraging anti-tumor effect in preclinical research, but also has strong killing effect on normal cells in vivo due to the excessively strong activity, so that strong side effect is generated.
The derivative has strong inhibition capacity on various tumor cells, shows excellent effect in clinic as an antitumor drug, but fails clinical tests because the toxicity is too high, and then, the derivative MMAE of the dolastatin 10 is developed and is applied to the market at present. The structural formula is as follows:
Figure BDA0003927774190000021
although the toxic and side effects of MMAE compared with D10 are greatly improved, the MMAE also has a killing effect on normal cells, so that a modifier for reducing the toxicity of the MMAE and improving the targeting property of the MMAE is urgently needed.
Hyaluronic acid and its derivatives are a hot point of research as drug delivery systems, and it is based on that hyaluronic acid can bind with some specific receptors on cell surface, so that it can be used as drug carrier to raise targeting property of medicine, prolong action time of medicine in vivo, raise bioavailability and raise therapeutic effect. The hyaluronic acid has wide molecular weight range, mainly 4000-10MDa, and researches show that different molecular weights have different effects, the high molecular weight has the effect of resisting the vascular proliferation and has no immunogenicity, and the low molecular weight has opposite effects. At present, high molecular weight hyaluronic acid is used as an active targeting factor in the research, and is combined with a specific receptor on the surface of a tumor cell to mediate the medicine to enter the cell and release the medicine in the cell. However, there is currently no study of Hyaluronic Acid (HA) modified MMAE conjugates.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the MMAE conjugate modified by the Hyaluronic Acid (HA), so that the medicament is targeted to tumor cells, the concentration of the medicament at a tumor part is increased, the medicament effect can be improved, the administration dosage is reduced, and the adverse reaction of the medicament is reduced.
Drug carrier materials can be divided into two categories: polymer carrier and lipid carrier, lipid carrier has good biocompatibility and biodegradability, and is widely used in research. Lipid-based carriers include: solid lipid nanoparticles, lipid nanosuspensions, liposomes, and the like. Namely, the preparation of the nanoparticles by solid lipid drug loading is always a hotspot of drug delivery system research since the application. Although it has many advantages, such as the suitability for encapsulation of hydrophilic hydrophobic drugs, it has disadvantages, one of which is primarily low drug loading. In recent years, the emergence of nano lipid carriers has overcome this defect, and the drug-loading rate of drugs has been increased, and the nano lipid carriers have become the next-generation lipid carriers.
According to the advantages, the hyaluronic acid modified MMAE-loaded nano lipid carrier system is constructed, firstly, the nano lipid carrier is adopted to load MMAE, the solubility of the medicine is increased, the use of a solubilizer is avoided, and the toxicity of a drug delivery system is reduced; then hyaluronic acid is used as a targeting factor, and the nano lipid carrier is subjected to targeting modification through the charge adsorption effect, so that the aim of actively targeting the tumor is fulfilled.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a hyaluronic acid-dolastatin conjugate of the formula HA-MMAE, wherein hyaluronic acid is covalently linked to dolastatin 10 derivative.
In a second aspect of the present invention, there is provided a method for preparing the hyaluronic acid-dolastatin conjugate, comprising:
preparing an MMAE loaded nano lipid carrier by a melting emulsification low-temperature curing method;
the hyaluronic acid modified MMAE nano lipid carrier is prepared by adopting a charge adsorption method.
In a third aspect of the present invention, there is provided a pharmaceutical composition comprising: the hyaluronic acid-dolastatin 10 derivative conjugate.
In a fourth aspect of the present invention, the hyaluronic acid-dolastatin 10 derivative conjugates and the use of the pharmaceutical compositions in the preparation of anti-tumor products are provided.
Based on the research steps, the MMAE nano-liposome modified by hyaluronic acid can reduce toxicity on one hand, has longer blood circulation in blood vessels on the other hand, actively targets tumors, and can effectively exert the effects of inhibiting the growth of cancer cells and the like in the anti-tumor field.
The invention has the advantages of
Based on above-mentioned nanometer drug delivery system, its beneficial effect: (1) The use of organic solvents is avoided, the toxicity of a drug delivery system is reduced, and the adverse reaction of the drug is reduced; (2) Adopts nanometer technology to achieve the purpose of passive targeting, improves the distribution of the medicine and increases the medicine effect. Because MMAE has no active targeting property and lacks selectivity for tumor cells, adverse reactions occur: (3) The liposome can not be completely solidified, the precipitation of the drug is reduced, the entrapment rate is improved, the stability is improved, the entrapment rate of the drug is up to 90 percent, and compared with other nano-liposome entrapped drugs, the entrapment rate is improved by 10 percent.
Modification based on hyaluronic acid, its beneficial effects: the active targeting property of the medicine is increased, so that the medicine can be accumulated at the tumor part to the maximum, the action time of the medicine in the body is further prolonged, the killing effect of the tumor is increased, and the killing toxicity to the surrounding normal cells is reduced, which is the key for reducing the adverse reaction of the chemotherapy medicine. And the hyaluronic acid also has many advantages, good water solubility, biodegradability, good biocompatibility, no toxicity, no immunogenicity, easy chemical modification and the like.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are included to explain the exemplary embodiments of the invention and the description and are not intended to limit the invention.
FIG. 1 is a comparison of drug encapsulated in different nanoliposomes;
FIG. 2 Transmission Electron microscopy images after lyophilization;
FIG. 3 particle size plot after lyophilization;
figure 4 particle size of the lyophilized formulation.
FIG. 5 evaluation results of antitumor activity.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
A hyaluronic acid-dolastatin conjugate HAs a chemical formula of HA-MMAE, wherein hyaluronic acid and dolastatin 10 derivative are connected by covalent bond.
Based on the conjugate, the invention also researches the dosage of the hyaluronic acid, the dolastatin and other components required in the hyaluronic acid-dolastatin conjugate. Proved by verification, the HA concentration is 0.5-0.6 mg/ml, and the mass ratio of the HA to the MMAE nano-liposome is 1: 3-4, the constructed hyaluronic acid modified MMAE-loaded nano lipid carrier system has the advantages of high stability, high drug entrapment rate and active targeting property.
A method for preparing a hyaluronic acid-dolastatin conjugate, comprising:
preparing an MMAE loaded nano lipid carrier by a melting emulsification low-temperature curing method;
the hyaluronic acid modified MMAE nano lipid carrier is prepared by a charge adsorption method.
In some embodiments, the specific steps of preparing the MMAE loaded nanoliposome carrier comprise:
adding ethanol into MMAE, glyceryl monostearate, injectable soybean lecithin and soybean oil, and ultrasonic dissolving to form oil phase;
taking a CTAB aqueous solution to form a water phase, heating the water phase to 70-80 ℃ in a water bath, adding an oil phase into the water phase under the stirring condition, continuously stirring and emulsifying, and finally stirring and solidifying the mixture in an ice bath to obtain the CTAB emulsion.
In some embodiments, the specific conditions for preparing the MMAE loaded nanoliposome carrier are: the medicine-fat ratio is 1: 30-40, the lipid concentration is 1-1.5 mg/ml, the solid-liquid lipid ratio is 3-4: 1, the oil-water ratio is 1:20 to 25 percent, the concentration of the surface active agent is 0.1 to 0.15 percent, the emulsification temperature is 70 to 80 ℃, the dropping speed is 18 to 20ml/h, and the stirring speed is 600 to 650rpm.
In some embodiments, the specific steps of preparing the hyaluronic acid-modified MMAE nanoliposome carrier comprise: dropping the MMAE nano lipid carrier into the hyaluronic acid solution for reaction to obtain the product.
In some embodiments, the specific process conditions for preparing the hyaluronic acid modified MMAE nano lipid carrier are as follows: the HA molecular weight is 30 ten thousand, the HA concentration is 0.5-0.6 mg/ml, the mass ratio of the HA to the MMAE nano-liposome is 1:3 to 4, the dropping speed is 20 to 25ml/h, and the stirring speed is 600 to 650rpm.
A pharmaceutical composition comprising the hyaluronic acid-MMAE conjugate of the first aspect.
The pharmaceutical composition may be in the form of the following formulation: powder, capsule, tablet, and pellicle in solid preparation form; solutions, mixtures, syrups, tinctures, injections in the form of liquid preparations; and ointment and suppository of semisolid preparation. Preferably, the pharmaceutical composition further comprises an essential carrier. The "essential carrier" in the present application mainly means auxiliary materials necessary in the pharmaceutical product, and mainly includes an oil phase, a water phase, excipients and the like.
Further, the oil phase includes, for example, hydrocarbons, esters, animal and vegetable oils and fats, waxes, acerola oil, higher fatty acids, higher alcohols, silicones, sterols and resins, and the above-mentioned materials treated by enzymes (e.g., hydrolysis and transesterification) or chemicals (e.g., transesterification and hydrogenation); specific examples are almond oil, nut oil, avocado oil, baobab oil, camellia oil, carrot oil, castor oil, citronella oil, coconut oil, cranberry oil, grape seed oil, hemp seed oil, jojoba oil, macadamia nut oil, meadow foam seed oil, oat emollient, raspberry seed oil, rosehip oil, soybean oil, rapeseed oil, corn oil, sesame seed oil, cottonseed oil, safflower oil, sunflower seed oil, peanut oil, germ oil, macadamia nut oil, garlic oil, camellia oil, palm oil, olive oil, linseed oil, eucalyptus oil, evening primrose oil, tortoise oil, mink oil, lard, tallow, horse oil, snake oil, fish oil, egg oil; <xnotran> , , , , , , , , , 2- , 2- , -2- , , , -2- , , (3,5,5- -3', 5', 5' - ), 12- , / , 2- , , , , , , , , , , , , , , , , 12- , 10- , , , , , , , , , , , , , , , , , , , , , , , , , , , , . </xnotran>
The specific selection of the above components in the carrier depends on the preparation form of the pharmaceutical composition, and the oil phase component in solid form such as ointment and emulsion can be selected from one or more of vegetable oil, animal oil, butter and wax. The water soluble ointment preparation comprises at least one polyethylene glycol component.
Further, the excipient is a compound including, but not limited to, a thickener, a gelling agent, a neutralizing agent, an occluding agent, an antioxidant, a buffering agent, a pH adjusting agent, a filler, an emulsifier, a co-emulsifier, an emollient, a solvent, a stabilizer, a solubilizer, a hardening agent, a suspending agent, a binder, a tackifier, a penetration enhancer, a preservative, a chelating agent, a disintegrant, a plasticizer, a humectant, a perfume, and the like.
Examples of such antioxidants are ascorbic acid (vitamin C), glutathione, lipoic acid, uric acid, tocopherol (vitamin E), butylated Hydroxytoluene (BHT), butylated Hydroxyanisole (BHA), carotene or panthenol (coenzyme Q).
Examples of such gelling agents are tragacanth, sodium alginate, pectin, silicates (e.g. fumed silica), gelatin, cellulose derivatives (e.g. methylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose), carbomers, guar gum, polyvinyl alcohol clays and the like.
Examples of such penetration enhancers are oleic acid, lecithin, urea, clove oil, isopropyl myristate, menthol, carvacrol, linalool, limonene, geraniol, nerol, propylene glycol dipentaerythritol or cyclodextrins.
Examples of the antibacterial preservative are methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzoic acid, phenylmercuric nitrate, benzalkonium chloride, chlorhexidine acetate, benzyl alcohol or mercury.
Examples of such chelating agents are citric acid or maleic acid.
Examples of such lubricants are polyethylene glycol, glycerol or sorbitol.
Examples of such fragrances are lavender oil, rose oil, lemon oil, lime oil, bergamot oil, orange oil, petgrain oil, tangerine oil, chamomile oil, cinnamon oil, sage oil, cypress oil, geranium oil, ginger oil, juniper oil, marjoram oil, myrtle oil, neroli oil, pine oil, rosewood oil, ylang-ylang oil, cedar wood oil, jasmine oil, frankincense oil, myrrh oil, patchouli oil, sandalwood oil or acanthopanax oil.
Examples of such emulsifiers are alkyl sulfates, soaps, dodecylbenzene sulfonate, lactates, sulfosuccinates, monoglyceride sulfonates, phosphate esters, silicones, taurates, and the like.
In a fourth aspect of the invention, there is provided a hyaluronic acid-MMAE conjugate of the first aspect, and a pharmaceutical composition of the third aspect, for use in the treatment of tumors.
In the technical scheme, the drug has an effective mitosis inhibition effect by inhibiting tubulin polymerization, is used for various tumors including but not limited to melanoma, breast cancer, small cell lung cancer, ovarian cancer, colon cancer, prostate cancer, endometrial cancer and the like, and has a wide anti-tumor effect range.
The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.
Example 1 establishment of MMAE nanoliposomes
In the production process of the solid lipid nanoparticles, the drug is dissolved in liquid lipid melted at high temperature in the initial step, and after the temperature is reduced, the solid lipid is solidified, and at the moment, the drug is crystallized in the solid lipid to cause the drug to be separated out. The lipid carrier with the nano structure is added with liquid lipid, so that the lipid can not be completely solidified after the temperature is reduced, and the drug precipitation is reduced. Compared with solid lipid nanoparticles, the nanostructure lipid carrier has the advantages of improving drug-loading rate and realizing sustained and controlled release of drugs.
The carrier is prepared by a melting emulsification low-temperature solidification method. The nano-lipid carrier physically entraps MMAE. Accurately weighing MMAE, glyceryl monostearate, injectable soybean lecithin and soybean oil, adding a certain amount of ethanol, and ultrasonically dissolving to form an oil phase. 40ml of an aqueous solution containing 0.3% CTAB was measured out to form an aqueous phase, which was placed in a 100ml beaker and heated to 70 ℃ in a water bath. Dropwise adding the oil phase into the water phase at a stirring speed of 600rpm at a speed of 20ml/h, continuously stirring and emulsifying for 15min after dropwise adding, and finally stirring and curing for 30min in an ice bath to obtain the MMAE-loaded nano lipid carrier.
The preparation method also comprises a process of determining the encapsulation efficiency and drug loading of the product.
The method comprises the following specific steps: mu.l of MMAE nano-lipid carrier suspension was precisely weighed into a tube, and then 4.9mL of 2% Tween 80-PBS (0.05 mmol/mL, pH = 7.4) solution was added, and vortexed for 5min to completely dissolve free drug MMAE. The suspension is placed in a high speed centrifuge and centrifuged for 30min at a speed of 15000 r/min. Taking a certain amount of supernatant, diluting the supernatant by a proper amount, and measuring the amount of the free medicine in the supernatant; simultaneously, demulsifying the same amount of suspension carrying the MMAE nano lipid carrier with methanol, diluting to a proper amount, filtering with a 0.22 μm filter membrane, injecting 20 μ l of sample, and measuring the total amount of the drug.
The encapsulation efficiency of the drug is calculated according to the following formula:
EE％＝(W general assembly -W Supernatant fluid )/W General (1) ×100
DL％＝(W General (1) -W Supernatant fluid )/W NLC ×100
Wherein W General (1) Is the total amount of drug in MMAE nano lipid carrier suspension, W Supernatant fluid Amount of free drug in supernatant, W NLC Is the total mass of the glycerin monostearate vinegar, the phospholipid, the soybean oil and the medicament.
The encapsulation efficiency of the obtained nanoliposome was calculated to be 91%.
The optimal prescription and process determined by the MMAE nano-liposome are as follows: the medicine-fat ratio is 1:30, the lipid concentration is 1mg/ml, the solid-liquid lipid ratio is 3:1, oil-water ratio of 1:20, the concentration of the surfactant is 0.1 percent, the emulsification temperature is 70 ℃, the dropping speed is 18ml/h, and the stirring speed is 600rpm.
Example 2 hyaluronic acid modified MMAE nanoliposomes
In the embodiment, HA is used as a targeting factor, and the MMAE-NLC surface is subjected to targeting modification by utilizing the charge adsorption effect so as to achieve the purposes of high efficiency, low toxicity and active orientation. The HA surface contains carboxyl which interacts with positive charges on the surface of MMAE-NLC and is adsorbed on the surface, the preparation is simple, the raw materials are easy to obtain, chemical synthesis is not needed, and the time and the cost are saved.
The hyaluronic acid modified MMAE nano lipid carrier is prepared by a charge adsorption method. Measuring 10ml of 0.5% hyaluronic acid solution, placing in a beaker, dripping a certain volume of MMAE nano lipid carrier into the hyaluronic acid solution at a dripping speed of 20ml/h at a stirring speed of 600rpm, and continuously stirring for 10min after dripping to obtain the hyaluronic acid modified MMAE nano lipid carrier (HA-MMAE).
The optimal prescription and process determined by the hyaluronic acid modified MMAE nano-liposome are as follows: the HA molecular weight is 30 thousands, the HA concentration is 0.5mg/ml, and the mass ratio of the HA to the MMAE nano-liposome is 1:3, the dropping speed is 20ml/h, and the stirring speed is 600rpm.
Aiming at the entrapment rate of different nanoliposome drugs, the physicochemical properties of the conjugate of hyaluronic acid-MMAE, including morphology, particle size and potential, were evaluated, and the test results are shown in FIGS. 1 to 5.
The results show that the particle size after lyophilization is about 150 μm. The HA-MMAE after freeze-drying HAs stable properties, and the physicochemical properties of the HA-MMAE are basically unchanged compared with those before freeze-drying.
The drug effects of the hyaluronic acid-MMAE conjugate including in vitro release and in vitro cytotoxicity were evaluated:
(1) In vitro release profile study: precisely measuring 1ml of each of an HA group, an MMAE-NLC group and an HA-MMAE group, placing the HA group, the MMAE group and the HA-MMAE group into a dialysis bag (with molecular weight cut-off of 8000-10000, and then placing the HA group, the MMAE group and the HA-MMAE group into 30ml of PBS (0.05 mol/L and pH = 7.4) solution containing 1M sodium salicylate, oscillating the solution at constant temperature of 37 ℃, with the oscillation speed of 100rpm/min, taking out 1ml of medium at different time points, simultaneously discarding the rest medium, replacing 30ml of new release medium again, measuring the concentration of the drug by using an HPLC method through the taken-out medium, calculating the cumulative release rate, drawing a release curve, and fitting a release mathematical model.
(2) In vitro cytotoxicity examination: selecting three tumor cell lines, determining the growth inhibition of the three tumor cell lines by using an MTT method to determine the growth inhibition of an HA group, an MMAE-NLC group and an HA-MMAE group, and setting a control group and a blank group.
100 μ l (about 4000) of the two tumor cell suspensions in the logarithmic growth phase were taken and placed in 96-well culture plates, respectively. Then is placed in CO 2 In the incubator, the environment of the incubator is kept constant: temperature 37 ℃ CO 2 Content 5%, saturated humidity. After 24h of cell culture, 100. Mu.l of HA, MMAE-NLC and HA-MMAE at different concentrations (0.002, 0.02,0.2, 1. Mu. Mol/ml) were added, and the culture was continued for 96h.Then MTT was added to each 96-well plate at 20. Mu.l/well and incubation was continued for 4h. Finally the supernatant in the wells was discarded, 200. Mu.l DMSO was added and shaken on a shaker for 5min, each set of 6 parallel wells and repeated 3 times. Finally, the absorbance was measured by an enzyme linked immunosorbent assay, and the in vitro antitumor activity of each group of the preparations was examined, and the test results are shown in fig. 5.
Therefore, the Hyaluronic Acid (HA) -modified MMAE conjugate can target the medicine to tumor cells, increase the concentration of the medicine at tumor parts, improve the medicine effect and reduce the administration dosage.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. The hyaluronic acid-dolastatin conjugate is characterized in that the chemical formula of the conjugate is HA-MMAE, wherein hyaluronic acid and dolastatin 10 derivatives are connected through covalent bonds, the HA concentration is 0.5-0.6 mg/ml, and the mass ratio of HA to MMAE nano-liposomes is 1:3 to 4.
2. A method of preparing the hyaluronic acid-dolastatin conjugate of claim 1, comprising:
preparing an MMAE loaded nano lipid carrier by a melting emulsification low-temperature curing method;
the hyaluronic acid modified MMAE nano lipid carrier is prepared by adopting a charge adsorption method.
3. The method for preparing hyaluronic acid-dolastatin conjugate according to claim 2, wherein the specific steps for preparing MMAE nano lipid carrier comprise:
adding ethanol into MMAE, glyceryl monostearate, injectable soybean lecithin and soybean oil, and ultrasonic dissolving to form oil phase;
taking a CTAB aqueous solution to form a water phase, heating the water phase to 70-80 ℃ in a water bath, adding an oil phase into the water phase under the stirring condition, continuously stirring and emulsifying, and finally stirring and solidifying the mixture in an ice bath to obtain the CTAB emulsion.
4. The method for preparing hyaluronic acid-dolastatin conjugate according to claim 2, wherein the MMAE nano lipid carrier is prepared by the following specific conditions: the medicine-fat ratio is 1: 30-40, the lipid concentration is 1-1.5 mg/ml, the solid-liquid lipid ratio is 3-4: 1, the oil-water ratio is 1:20 to 25 percent, the concentration of the surface active agent is 0.1 to 0.15 percent, the emulsification temperature is 70 to 80 ℃, the dropping speed is 18 to 20ml/h, and the stirring speed is 600 to 650rpm.
5. The method for preparing the hyaluronic acid-dolastatin conjugate according to claim 2, wherein the specific steps for preparing the hyaluronic acid modified MMAE nano lipid carrier comprise: and (3) dripping the MMAE nano lipid carrier into a hyaluronic acid solution for reaction to obtain the nano-emulsion.
6. The method for preparing the hyaluronic acid-dolastatin conjugate according to claim 4, wherein the specific process conditions for preparing the hyaluronic acid modified MMAE nano lipid carrier are as follows: the HA molecular weight is 30 ten thousand, the HA concentration is 0.5-0.6 mg/ml, the mass ratio of the HA to the MMAE nano-liposome is 1:3 to 4, the dropping speed is 20 to 25ml/h, and the stirring speed is 600 to 650rpm.
7. A pharmaceutical composition comprising the hyaluronic acid-dolastatin 10 derivative conjugate of claim 1.
8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is in the form of a formulation comprising: powder, capsule, tablet, and pellicle in solid preparation form; solutions, mixtures, syrups, tinctures, injections in the form of liquid preparations; ointments and suppositories for semisolid preparations;
the pharmaceutical composition also comprises necessary carriers including an oil phase, a water phase or an excipient;
or, the oil phase comprises hydrocarbons, esters, animal and vegetable oils and fats, waxes, acerola oil, higher fatty acids, higher alcohols, silicones, sterols and resins, and the above by enzymatic or chemical treatment;
or, the excipient is selected from the group consisting of, but not limited to, a thickening agent, a gelling agent, a neutralizing agent, an occlusive agent, an antioxidant, a buffering agent, a pH adjusting agent, a filler, an emulsifier, a co-emulsifier, an emollient, a solvent, a stabilizer, a solubilizer, a hardening agent, a suspending agent, a binder, a viscosity increasing agent, a penetration enhancer, a preservative, a chelating agent, a disintegrating agent, a plasticizer, a humectant, or a fragrance.
9. Use of the hyaluronic acid-dolastatin 10 derivative conjugate of claim 1, and the pharmaceutical composition of claim 7 or 8 for preparing an anti-tumor product.
10. The use of claim 9, wherein the tumor includes, but is not limited to, melanoma, breast cancer, small cell lung cancer, ovarian cancer, colon cancer, prostate cancer, endometrial cancer.
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