CN111973756B - Dimethyl curcumin prodrug nanoparticles, nano preparation, preparation method and application thereof - Google Patents

Dimethyl curcumin prodrug nanoparticles, nano preparation, preparation method and application thereof Download PDF

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CN111973756B
CN111973756B CN202010853775.9A CN202010853775A CN111973756B CN 111973756 B CN111973756 B CN 111973756B CN 202010853775 A CN202010853775 A CN 202010853775A CN 111973756 B CN111973756 B CN 111973756B
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dimethyl curcumin
dmc
dimethyl
prodrug
curcumin
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CN111973756A (en
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徐德锋
赖超
胡航
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Changzhou University
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
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Abstract

The invention relates to the technical field of drug delivery systems, in particular to water-soluble dimethyl curcumin prodrug nanoparticles, a nano preparation, and a preparation method and application thereof. Methoxy polyethylene glycol aldehyde group and dimethyl curcumin are covalently coupled through hydrazine hydrate to form a compound, and the dimethyl curcumin prodrug nano-particle is provided. The dimethyl curcumin prodrug nanoparticles provided by the invention can further load free dimethyl curcumin with high encapsulation rate and drug-loading capacity. In vitro drug release research shows that the prodrug nanoparticles loaded with the free dimethyl curcumin have the property of pH-sensitive drug release, and in vitro tumor inhibition research shows that the killing effect of the prodrug nanoparticles loaded with the free dimethyl curcumin on tumor cells is stronger than that of the free dimethyl curcumin.

Description

Dimethyl curcumin prodrug nanoparticles, nano preparation, preparation method and application thereof
Technical Field
The invention relates to the technical field of drug delivery systems, and in particular relates to water-soluble dimethyl curcumin prodrug nanoparticles, a nano preparation, and a preparation method and application thereof.
Background
Cancer is an important disease that threatens human health in today's society. The current tumor clinical treatment medicines still have a plurality of defects, such as high toxicity to normal cells when the anti-tumor effect is exerted, easily-generated multi-drug resistance phenomenon, incapability of effectively preventing the metastasis of cancer cells, high clinical recurrence rate, high chemical toxicity and the like. Compared with the traditional medicine, the nano-medicine can enhance the dissolution rate of the medicine, stabilize the medicine effect, enhance the action targeting property of the medicine, reduce the toxic and side effects of the medicine on normal tissues, and the research, development and utilization of the nano-medicine in pharmaceutics become hot spots.
Curcumin has long medicinal history and wide pharmacological activity, but has low solubility, unstable metabolism and poor bioavailability. Dimethyl curcumin (DMC) is a lipophilic structural analogue of curcumin, hydroxyl groups of benzene rings on two sides are simultaneously substituted by methoxy groups on the molecular structural formula of the curcumin, phenolic hydroxyl groups at potential oxidation positions on the benzene rings of the curcumin are eliminated, and a large number of research results show that: DMC can exert drug effect through multiple ways, has stronger activity for inhibiting proliferation of various tumor cells than curcumin, and shows obviously stronger therapeutic potential than curcumin on various disease models. However, dimethyl curcumin has the problems of extremely poor water solubility, low bioavailability and the like, and the application of dimethyl curcumin is severely limited.
The hydrophilic polymer polyethylene glycol (PEG) is a particle surface modification material which is most widely applied and is approved by FDA to be used for human bodies, the PEG and the medicament are combined by a chemical bond with pH responsiveness, the bioavailability and targeted enrichment of the medicament can be improved, the medicament is released in a targeted manner in a special pH environment of a focus part, and the toxic and side effects on other normal tissues are reduced. However, how to effectively combine the dimethyl curcumin with the PEG carrier through a pH-responsive chemical bond so as to improve the water solubility and bioavailability of the dimethyl curcumin and further improve the antitumor activity is not reported yet.
Disclosure of Invention
The invention provides a water-soluble dimethyl curcumin prodrug nanoparticle. The dimethyl curcumin prodrug nanoparticle is a compound formed by covalently coupling methoxy polyethylene glycol aldehyde group and dimethyl curcumin with hydrazine hydrate.
The second object of the invention provides a preparation method of dimethyl curcumin prodrug nanoparticles, which comprises the following steps:
(1) preparation of dimethyl curcumin derivative: dimethyl curcumin (DMC) and hydrazine hydrate are directly heated and reacted in a solvent to obtain the dimethyl curcumin derivative. The dimethyl curcumin derivative can be prepared by DMC ═ N-NH2And (4) showing.
In a preferred embodiment of the invention, the preparation method specifically comprises the following steps: dissolving dimethyl curcumin in a solvent, adding hydrazine hydrate, heating and reacting under the protection of nitrogen, removing the solvent by rotary evaporation after the reaction is finished, freezing at-80 ℃ for 2 hours, and transferring to a freeze dryer for photophobic freeze drying to obtain a dimethyl curcumin derivative;
wherein the molar ratio of the dimethyl curcumin to the hydrazine hydrate is 1: 1-1: 10.
The heating reaction temperature is 25-80 ℃.
The heating reaction time is 12-48 hours.
The solvent is absolute methanol or absolute ethanol.
(2) Preparing dimethyl curcumin prodrug nanoparticles: and (2) reacting the dimethyl curcumin derivative prepared in the step (1) with methoxy polyethylene glycol aldehyde group (mPEG-CHO) to obtain the dimethyl curcumin prodrug nanoparticles. The resulting dimethylcurcumin prodrug nanoparticles can be represented by mPEG-CH ═ N-N ═ DMC.
In a preferred embodiment of the invention, the specific preparation method comprises the following steps: dissolving the dimethyl curcumin derivative prepared in the step (1) and methoxy polyethylene glycol aldehyde group in a solvent, heating for reaction, after the reaction is finished, removing the solvent by rotary evaporation, adding distilled water for dissolving, transferring into a dialysis bag with the molecular weight cutoff of 3500Da, dialyzing in water, after the dialysis is finished, centrifuging dialysate, taking supernatant, freezing at minus 80 ℃ for 2h, transferring into a freeze dryer for freeze drying, and obtaining the dimethyl curcumin prodrug nanoparticles.
Wherein the average molecular weight of the methoxy polyethylene glycol aldehyde group is 1 kDa-20 kDa;
the molar ratio of the methoxy polyethylene glycol aldehyde group to the dimethyl curcumin derivative is 1: 1-1: 10.
The heating reaction temperature is 25-80 ℃.
The heating reaction time is 12-48 hours.
The solvent is absolute methanol or absolute ethanol.
The dialysis treatment time is 48-96 hours.
The specific synthetic route for preparing the dimethyl curcumin prodrug nano-particles is shown in the attached figure 1.
The dimethyl curcumin and hydrazine hydrate are directly heated to react to generate dimethyl curcumin derivative (DMC ═ N-NH) shown in the formula (I)2)；
Figure BDA0002645694140000031
The dimethyl curcumin derivative and methoxy polyethylene glycol aldehyde group are directly heated to react to generate a dimethyl curcumin prodrug (mPEG-CH-N-DMC) with a structure shown in a formula (II).
Figure BDA0002645694140000041
The dimethyl curcumin prodrug nanoparticles provided by the invention can load free dimethyl curcumin with high encapsulation rate and drug-loading rate, and the nano preparation loaded with the free dimethyl curcumin has the pH-sensitive drug release property and has a strong tumor cell killing effect.
The third purpose of the invention is to provide a preparation method of the dimethyl curcumin prodrug nanoparticles for preparing water-soluble dimethyl curcumin nano preparation, namely, the nano preparation loaded with free dimethyl curcumin is prepared by a film hydration method.
In a preferred embodiment of the invention, the specific preparation method comprises the following steps:
adding the dimethyl curcumin prodrug nanoparticles and the free dimethyl curcumin into a solvent, stirring and dissolving at room temperature, removing the solvent by rotary evaporation and forming a film, adding ultrapure water, performing ultrasonic dispersion, centrifuging, taking supernatant, and freeze-drying to obtain the nano preparation loaded with the free dimethyl curcumin.
The mass ratio of the dimethyl curcumin prodrug nanoparticles to the dimethyl curcumin is 1: 0-1: 0.3;
stirring for 2-5 hours;
the solvent is absolute methanol or absolute ethanol;
freezing the supernatant at-80 deg.C for 2 hr, and lyophilizing in a lyophilizer in dark to obtain the nanometer preparation loaded with free dimethyl curcumin.
The prodrug nanoparticles or the nano preparation prepared by the method are applied to resisting prostate cancer and liver cancer.
In general, compared with the prior art, the technical scheme of the invention can achieve the following beneficial effects:
(1) the invention couples dimethyl curcumin and methoxy polyethylene glycol aldehyde group through hydrazone bond. The compound can be self-assembled into nanoparticles in ultrapure water, and can load free dimethyl curcumin with high encapsulation rate and drug-loading rate.
(2) The nano preparation loaded with the free dimethyl curcumin has the property of pH-sensitive drug release, and can promote the release of the loaded drug in a meta-acid microenvironment in tumor cells.
(3) The nano preparation loaded with the free dimethyl curcumin has good biocompatibility, and has stronger killing effect on tumor cells than the free dimethyl curcumin.
Drawings
FIG. 1 is a scheme showing the synthesis of a compound of formula (II) according to the present invention;
fig. 2 is a nuclear magnetic resonance hydrogen spectrum of the dimethyl curcumin prodrug prepared in example 1 of the present invention;
fig. 3 is an infrared spectrum of a dimethyl curcumin prodrug prepared in example 1 of the present invention;
fig. 4 is a distribution diagram of the particle size of nanoparticles self-assembled from a dimethyl curcumin prodrug prepared in example 1 of the present invention in ultrapure water;
fig. 5 is an electron microscope image of self-assembly of a dimethyl curcumin prodrug prepared in example 1 of the present invention into nanoparticles in ultrapure water;
fig. 6 is a drug release profile of prodrug nanoparticles loaded with free dimethylcurcumin prepared in experimental example 3 of the present invention;
fig. 7 is a graph showing the tumor cell killing effect of the prodrug nanoparticles loaded with free dimethylcurcumin prepared in example 3 of the present invention.
Detailed Description
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
This example provides a dimethyl curcumin prodrug, comprising the following steps:
(1) dissolving 48mg (0.12mmol) of dimethyl curcumin in 3mL of methanol, adding 16.5mg (0.33mmol) of hydrazine hydrate, and stirring and reacting for 24 hours at 50 ℃ in the dark under the protection of nitrogen to obtain reaction liquid A;
(2) carrying out rotary evaporation on the reaction liquid A obtained in the step (1), removing the solvent, transferring to a freeze dryer, and freeze-drying in a dark place to obtain a dark yellow solid which is a dimethyl curcumin derivative;
(3) dissolving the dimethyl curcumin derivative obtained in the step (2) in 3mL of methanol, adding 200mg (0.10mmol) of methoxy polyethylene glycol aldehyde group, and stirring and reacting for 24 hours at 50 ℃ in the dark to obtain a reaction solution B;
(4) and (3) carrying out rotary evaporation on the reaction liquid B obtained in the step (3), removing the solvent, adding 6mL of ultrapure water for dissolving, dialyzing for 3 days by using the ultrapure water, wherein the molecular weight cut-off of a dialysis bag is 3500Da, freezing for 2 hours in a refrigerator at the temperature of-80 ℃ after the dialysis is finished, moving to a freeze dryer for dark freeze drying, and obtaining a dark yellow solid which is the dimethyl curcumin prodrug nano-particle (164.5mg, yield: 80.8%).
Example 2
This example provides a dimethyl curcumin prodrug, comprising the following steps:
(1) dissolving 48mg (0.12mmol) of dimethyl curcumin in 3mL of methanol, adding 7.2mg (0.12mmol) of hydrazine hydrate, and stirring and reacting for 24 hours at the temperature of 50 ℃ in the dark under the protection of nitrogen to obtain reaction liquid A;
(2) carrying out rotary evaporation on the reaction liquid A obtained in the step (1), removing the solvent, transferring to a freeze dryer, and freeze-drying in a dark place to obtain a dark yellow solid which is a dimethyl curcumin derivative;
(3) dissolving the dimethyl curcumin derivative obtained in the step (2) in 3mL of methanol, adding 200mg (0.10mmol) of methoxy polyethylene glycol aldehyde group, and stirring and reacting for 24 hours at 50 ℃ in the dark to obtain a reaction solution B;
(4) and (3) carrying out rotary evaporation on the reaction liquid B obtained in the step (3), removing the solvent, adding 6mL of ultrapure water for dissolving, dialyzing for 3 days by using the ultrapure water, wherein the molecular weight cut-off of a dialysis bag is 3500Da, freezing for 2 hours in a refrigerator at the temperature of-80 ℃ after the dialysis is finished, moving to a freeze dryer for dark freeze drying, and obtaining a dark yellow solid which is the dimethyl curcumin prodrug (145.3mg, yield: 71.4%).
Example 3
This example provides a dimethyl curcumin prodrug, comprising the following steps:
(1) dissolving 48mg (0.12mmol) of dimethyl curcumin in 3mL of methanol, adding 7.2mg (0.60mmol) of hydrazine hydrate, and stirring and reacting for 24 hours at the temperature of 50 ℃ in the dark under the protection of nitrogen to obtain reaction liquid A;
(2) carrying out rotary evaporation on the reaction liquid A obtained in the step (1), removing the solvent, transferring to a freeze dryer, and freeze-drying in a dark place to obtain a dark yellow solid which is a dimethyl curcumin derivative;
(3) dissolving the dimethyl curcumin derivative obtained in the step (2) in 3mL of methanol, adding 200mg (0.10mmol) of methoxy polyethylene glycol aldehyde group, and stirring and reacting for 24 hours at 50 ℃ in the dark to obtain a reaction solution B;
(4) and (3) carrying out rotary evaporation on the reaction liquid B obtained in the step (3), removing the solvent, adding 6mL of ultrapure water for dissolving, dialyzing for 3 days by using the ultrapure water, wherein the molecular weight cut-off of a dialysis bag is 3500Da, freezing for 2 hours in a refrigerator at the temperature of-80 ℃ after the dialysis is finished, moving to a freeze dryer for dark freeze drying, and obtaining a dark yellow solid which is the dimethyl curcumin prodrug (153.3mg, yield: 75.3%).
Example 4
This example provides a dimethyl curcumin prodrug, comprising the following steps:
(1) dissolving 48mg (0.12mmol) of dimethyl curcumin in 3mL of methanol, adding 7.2mg (1.20mmol) of hydrazine hydrate, and stirring and reacting for 24 hours at 50 ℃ in the dark under the protection of nitrogen to obtain reaction liquid A;
(2) performing rotary evaporation on the reaction liquid A obtained in the step (1), removing the solvent, transferring to a freeze dryer, and freeze-drying in a dark place to obtain a dark yellow solid which is a dimethyl curcumin derivative;
(3) dissolving the dimethyl curcumin derivative obtained in the step (2) in 3mL of methanol, adding 200mg (0.10mmol) of methoxy polyethylene glycol aldehyde group, and stirring for reaction for 24 hours at 50 ℃ in a dark place to obtain a reaction solution B;
(4) and (3) carrying out rotary evaporation on the reaction liquid B obtained in the step (3), removing the solvent, adding 6mL of ultrapure water for dissolving, dialyzing for 3 days by using the ultrapure water, wherein the molecular weight cut-off of a dialysis bag is 3500Da, freezing for 2 hours in a refrigerator at the temperature of-80 ℃ after the dialysis is finished, moving to a freeze dryer for dark freeze drying, and obtaining a dark yellow solid which is the dimethyl curcumin prodrug (156.3mg, yield: 76.8%).
Example 5
This example provides a dimethyl curcumin prodrug, comprising the following steps:
the steps (1) to (2) are the same as in example 1.
(3) Dissolving the dimethyl curcumin derivative obtained in the step (2) in 3mL of methanol, adding 240mg (0.12mmol) of methoxy polyethylene glycol aldehyde group, and stirring and reacting for 24 hours at 50 ℃ in the dark to obtain a reaction solution B;
(4) and (3) carrying out rotary evaporation on the reaction liquid B obtained in the step (3), removing the solvent, adding 6mL of ultrapure water for dissolving, dialyzing for 3 days by using the ultrapure water, wherein the molecular weight cut-off of a dialysis bag is 3500Da, freezing for 2 hours in a refrigerator at the temperature of-80 ℃ after the dialysis is finished, moving to a freeze dryer for dark freeze drying, and obtaining a dark yellow solid which is the dimethyl curcumin prodrug (188.0mg, the yield: 66.2%).
Example 6
This example provides a dimethyl curcumin prodrug, comprising the steps of:
the steps (1) to (2) are the same as in example 1.
(3) Dissolving the dimethyl curcumin derivative obtained in the step (2) in 3mL of methanol, adding 48mg (0.024mmol) of methoxy polyethylene glycol aldehyde group, and stirring and reacting for 24 hours at 50 ℃ in the dark to obtain a reaction solution B;
(4) and (3) carrying out rotary evaporation on the reaction liquid B obtained in the step (3), removing the solvent, adding 6mL of ultrapure water for dissolving, dialyzing for 3 days by using the ultrapure water, wherein the molecular weight cut-off of a dialysis bag is 3500Da, freezing for 2 hours in a refrigerator at the temperature of-80 ℃ after the dialysis is finished, moving to a freeze dryer for dark freeze drying, and obtaining a dark yellow solid which is the dimethyl curcumin prodrug (36.2mg, yield: 74.0%).
Example 7
This example provides a dimethyl curcumin prodrug, comprising the steps of:
the steps (1) to (2) are the same as in example 1.
(3) Dissolving the dimethyl curcumin derivative obtained in the step (2) in 3mL of methanol, adding 24mg (0.012mmol) of methoxy polyethylene glycol aldehyde group, and stirring and reacting for 24 hours at 50 ℃ in the dark to obtain a reaction solution B;
(4) and (4) carrying out rotary evaporation on the reaction liquid B obtained in the step (3), removing the solvent, adding 6mL of ultrapure water for dissolving, dialyzing for 3 days by using the ultrapure water, wherein the molecular weight cut-off of a dialysis bag is 3500Da, freezing for 2 hours at the temperature of-80 ℃ after the dialysis is finished, moving to a freeze dryer for photophobic freeze-drying, and obtaining a dark yellow solid which is the dimethyl curcumin prodrug (10.6mg, yield: 43.5%).
Example 8
This example provides a dimethyl curcumin prodrug, comprising the following steps:
(1) dissolving 48mg (0.12mmol) of dimethyl curcumin in 3mL of methanol, adding 16.5mg (0.33mmol) of hydrazine hydrate, and stirring and reacting for 24 hours at 25 ℃ in the dark under the protection of nitrogen to obtain reaction liquid A;
(2) carrying out rotary evaporation on the reaction liquid A obtained in the step (1), removing the solvent, transferring to a freeze dryer, and freeze-drying in a dark place to obtain a dark yellow solid which is a dimethyl curcumin derivative;
(3) dissolving the dimethyl curcumin derivative obtained in the step (2) in 3mL of methanol, adding 200mg (0.10mmol) of methoxy polyethylene glycol aldehyde group, and stirring and reacting for 24 hours at 25 ℃ in the dark to obtain a reaction solution B;
(4) and (3) carrying out rotary evaporation on the reaction liquid B obtained in the step (3), removing the solvent, adding 6mL of ultrapure water for dissolving, dialyzing for 3 days by using the ultrapure water, wherein the molecular weight cut-off of a dialysis bag is 3500Da, freezing for 2 hours in a refrigerator at the temperature of-80 ℃ after the dialysis is finished, moving to a freeze dryer for dark freeze drying, and obtaining a dark yellow solid which is the dimethyl curcumin prodrug (124.4mg, yield: 61.1%).
Example 9
This example provides a dimethyl curcumin prodrug, comprising the following steps:
(1) dissolving 48mg (0.12mmol) of dimethyl curcumin in 3mL of ethanol, adding 16.5mg (0.33mmol) of hydrazine hydrate, and stirring and reacting for 24 hours at 80 ℃ in the dark under the protection of nitrogen to obtain reaction liquid A;
(2) carrying out rotary evaporation on the reaction liquid A obtained in the step (1), removing the solvent, transferring to a freeze dryer, and freeze-drying in a dark place to obtain a dark yellow solid which is a dimethyl curcumin derivative;
(3) dissolving the dimethyl curcumin derivative obtained in the step (2) in 3mL of ethanol, adding 200mg (0.10mmol) of methoxy polyethylene glycol aldehyde group, and stirring for reaction for 24 hours at 80 ℃ in a dark place to obtain a reaction solution B;
(4) and (3) carrying out rotary evaporation on the reaction liquid B obtained in the step (3), removing the solvent, adding 6mL of ultrapure water for dissolving, dialyzing for 3 days by using the ultrapure water, wherein the molecular weight cut-off of a dialysis bag is 3500Da, freezing for 2 hours in a refrigerator at the temperature of-80 ℃ after the dialysis is finished, moving to a freeze dryer for dark freeze drying, and obtaining a dark yellow solid which is the dimethyl curcumin prodrug (160.2mg, yield: 78.7%).
Example 10
This example provides a dimethyl curcumin prodrug, comprising the steps of:
(1) dissolving 48mg (0.12mmol) of dimethyl curcumin in 3mL of methanol, adding 16.5mg (0.33mmol) of hydrazine hydrate, and stirring for reaction for 12 hours at 50 ℃ in the dark under the protection of nitrogen to obtain reaction liquid A;
(2) carrying out rotary evaporation on the reaction liquid A obtained in the step (1), removing the solvent, transferring to a freeze dryer, and freeze-drying in a dark place to obtain a dark yellow solid which is a dimethyl curcumin derivative;
(3) dissolving the dimethyl curcumin derivative obtained in the step (2) in 3mL of methanol, adding 200mg (0.10mmol) of methoxy polyethylene glycol aldehyde group, and stirring and reacting for 12 hours at 50 ℃ in a dark place to obtain a reaction solution B;
(4) and (3) carrying out rotary evaporation on the reaction liquid B obtained in the step (3), removing the solvent, adding 6mL of ultrapure water for dissolving, dialyzing for 3 days by using the ultrapure water, wherein the molecular weight cut-off of a dialysis bag is 3500Da, freezing for 2 hours in a refrigerator at the temperature of-80 ℃ after the dialysis is finished, moving to a freeze dryer for dark freeze drying, and obtaining a dark yellow solid which is the dimethyl curcumin prodrug (147.4mg, yield: 72.4%).
Example 11
This example provides a dimethyl curcumin prodrug, comprising the following steps:
(1) dissolving 48mg (0.12mmol) of dimethyl curcumin in 3mL of methanol, adding 16.5mg (0.33mmol) of hydrazine hydrate, and stirring and reacting for 36 hours at 50 ℃ in the dark under the protection of nitrogen to obtain reaction liquid A;
(2) carrying out rotary evaporation on the reaction liquid A obtained in the step (1), removing the solvent, transferring to a freeze dryer, and freeze-drying in a dark place to obtain a dark yellow solid which is a dimethyl curcumin derivative;
(3) dissolving the dimethyl curcumin derivative obtained in the step (2) in 3mL of methanol, adding 200mg (0.10mmol) of methoxy polyethylene glycol aldehyde group, and stirring and reacting for 36 hours at 50 ℃ in a dark place to obtain a reaction solution B;
(4) and (3) carrying out rotary evaporation on the reaction liquid B obtained in the step (3), removing the solvent, adding 6mL of ultrapure water for dissolving, dialyzing for 3 days by using the ultrapure water, wherein the molecular weight cut-off of a dialysis bag is 3500Da, freezing for 2 hours in a refrigerator at the temperature of-80 ℃ after the dialysis is finished, moving to a freeze dryer for dark freeze drying, and obtaining a dark yellow solid which is the dimethyl curcumin prodrug (165.3mg, yield: 81.2%).
Example 12
This example provides a dimethyl curcumin prodrug, comprising the following steps:
(1) dissolving 48mg (0.12mmol) of dimethyl curcumin in 3mL of methanol, adding 16.5mg (0.33mmol) of hydrazine hydrate, and stirring for reaction for 48 hours at 50 ℃ in the dark under the protection of nitrogen to obtain a reaction solution A;
(2) carrying out rotary evaporation on the reaction liquid A obtained in the step (1), removing the solvent, transferring to a freeze dryer, and freeze-drying in a dark place to obtain a dark yellow solid which is a dimethyl curcumin derivative;
(3) dissolving the dimethyl curcumin derivative obtained in the step (2) in 3mL of methanol, adding 200mg (0.10mmol) of methoxy polyethylene glycol aldehyde group, and stirring for reaction for 48 hours at 50 ℃ in a dark place to obtain a reaction solution B;
(4) and (3) carrying out rotary evaporation on the reaction liquid B obtained in the step (3), removing the solvent, adding 6mL of ultrapure water for dissolving, dialyzing for 3 days by using the ultrapure water, wherein the molecular weight cut-off of a dialysis bag is 3500Da, freezing for 2 hours in a refrigerator at the temperature of-80 ℃ after the dialysis is finished, moving to a freeze dryer for freeze-drying in the dark place, and obtaining a dark yellow solid which is the dimethyl curcumin prodrug (163.9mg, yield: 80.5%).
Example 13
This example provides a dimethyl curcumin prodrug, comprising the following steps:
the steps (1) to (3) are the same as in example 1.
(4) And (4) carrying out rotary evaporation on the reaction liquid B obtained in the step (3), removing the solvent, adding 6mL of ultrapure water for dissolving, dialyzing with ultrapure water for 2 days, wherein the molecular weight cut-off of a dialysis bag is 3500Da, freezing for 2 hours at the temperature of-80 ℃ after dialysis is finished, transferring to a freeze dryer for freeze-drying in the dark place, and obtaining a dark yellow solid which is the dimethyl curcumin prodrug (141.3mg, yield: 69.4%).
Example 14
This example provides a dimethyl curcumin prodrug, comprising the following steps:
the steps (1) to (3) are the same as in example 1.
(4) And (4) carrying out rotary evaporation on the reaction liquid B obtained in the step (3), removing the solvent, adding 6mL of ultrapure water for dissolving, dialyzing for 4 days by using the ultrapure water, wherein the molecular weight cut-off of a dialysis bag is 3500Da, freezing for 2 hours at the temperature of-80 ℃ after the dialysis is finished, moving to a freeze dryer for light-shielding freeze drying, and obtaining a dark yellow solid which is the dimethyl curcumin prodrug (123.2mg, yield: 60.5%).
Test example 1
Chemical structure confirmation of the target compound dimethyl curcumin prodrug in example 1:
the chemical structure of the target compound prepared in example 1 was confirmed by hydrogen nuclear magnetic resonance spectroscopy and infrared spectroscopy. Fig. 2 and 3 are a nuclear magnetic resonance hydrogen spectrum and an infrared spectrum of the target compound prepared in example 1 of the present invention.
As can be seen from FIG. 2, the nuclear magnetic spectra of the dimethyl curcumin correspond to methyl hydrogen in methoxy group, aromatic hydrogen in benzene ring and double bond hydrogen in double bond respectively at 3.78-3.81 and 6.08-6.09, 6.79-6.84, 6.98-7.00, 7.23-7.26, 7.33 and 7.55-7.58. Peaks of nuclear magnetic spectrograms of methoxy polyethylene glycol aldehyde groups at 3.24-3.26 ppm, 3.51-3.56 ppm, 3.85-3.86 ppm and 9.64ppm respectively correspond to methyl hydrogen and methylene hydrogen in polyethylene glycol and aldehyde hydrogen in aldehyde groups. The nuclear magnetic spectrum of the target compound of example 1 showed the disappearance of nuclear magnetic signals of aldehyde hydrogens and the appearance of nuclear magnetic signals of aromatic hydrogens, double bond hydrogens, methyl hydrogens, indicating that dimethyl curcumin successfully bound to methoxy polyethylene glycol aldehyde groups.
As can be seen from FIG. 3, compared with the infrared spectrum of methoxypolyethylene glycol aldehyde group, the infrared spectrum of dimethyl curcumin isMedicine at 1518cm-1A new absorption peak appears at 1719cm due to the skeleton vibration of aromatic ring in the dimethyl curcumin-1The absorption peak at (a) disappeared due to the reaction between the aldehyde group and the dimethyl curcumin derivative.
Test example 2
Self-assembly behavior of the dimethyl curcumin prodrug obtained in example 1:
preparation of experimental solution: weighing 2mg of the dimethyl curcumin prodrug prepared in the embodiment 1 of the invention, adding 2mL of ultrapure water, and ultrasonically dispersing for 5min to prepare 1mg/mL of aqueous solution; 20mg of phosphotungstic acid is weighed and dissolved in 1mL of ultrapure water to prepare 2% phosphotungstic acid aqueous solution.
The particle size distribution of the dimethylcurcumin prodrug assembly prepared in this example was measured by dynamic light scattering using 1mL of the aqueous solution of the dimethylcurcumin prodrug prepared in this example.
Fig. 4 is a particle size distribution diagram of the dimethylcurcumin prodrug assembly prepared in this example. The experimental result shows that the particle size of the dimethyl curcumin prodrug assembly prepared by the embodiment is in normal distribution, the particle size distribution is narrow, and the average diameter is about 100 nm.
50 mu L of the aqueous solution of the dimethyl curcumin prodrug prepared in the embodiment is dripped on a copper mesh covered with a carbon support film, is naturally dried in the air, is dyed with 2% phosphotungstic acid for 2min, and the morphology of the dimethyl curcumin prodrug assembly prepared in the embodiment is observed by a transmission electron microscope.
Fig. 5 is an electron microscope image of the dimethylcurcumin prodrug assembly prepared in this example. The experimental result shows that the dimethyl curcumin prodrug assembly prepared by the embodiment is approximately spherical, has the morphological characteristics of nanoparticles, is uniform in particle size distribution and has the diameter of about 100 nm.
Test example 3
Drug loading behavior of the self-assembled nanoparticles of the dimethyl curcumin prodrug:
the drug loading method comprises the following steps: weighing 10mg of the dimethyl curcumin prodrug prepared in the embodiment 1 of the invention and 1.0mg of dimethyl curcumin, dissolving in 3mL of methanol, stirring for 4 hours at room temperature, removing the solvent by rotary evaporation, adding 2mL of ultrapure water, carrying out ultrasonic treatment for 5 minutes, and centrifuging to obtain a supernatant to obtain a DMC @ mPEG-DMC (15.5%) nanoparticle solution.
Weighing 10mg of the dimethyl curcumin prodrug prepared in the embodiment 1 of the invention and 2.0mg of dimethyl curcumin, dissolving in 3mL of methanol, stirring for 4 hours at room temperature, removing the solvent by rotary evaporation, adding 2mL of ultrapure water, carrying out ultrasonic treatment for 5 minutes, and centrifuging to obtain a supernatant to obtain a DMC @ mPEG-DMC (18.6%) nanoparticle solution.
Weighing 10mg of the dimethyl curcumin prodrug prepared in the embodiment 1 of the invention and 3.0mg of dimethyl curcumin, dissolving in 3mL of methanol, stirring for 4 hours at room temperature, removing the solvent by rotary evaporation, adding 2mL of ultrapure water, carrying out ultrasonic treatment for 5 minutes, and centrifuging to obtain a supernatant to obtain a DMC @ mPEG-DMC (22.0%) nanoparticle solution.
The drug loading of the drug-loaded nanoparticles prepared in this example was detected by high performance liquid chromatography. The drug-loaded nanoparticles prepared in this example were weighed to determine the mass W1The mass W of the dimethyl curcumin in the drug-loaded nanoparticles prepared in the example was measured by high performance liquid chromatography2The Drug Loading (DLC) is represented by the formula DLC (%) ═ W2/W1×100％。
The dosage of the dimethyl curcumin is set as W3The dosage of the dimethyl curcumin prodrug prepared in the embodiment 1 of the invention is set as W4Theoretical Drug Loading (DLC)t) Using the formula DLCt(％)＝W3/(W3+W4)×100％。
The Encapsulation Efficiency (EE) adopts the formula EE (%) (DLC/DLC)t×100％。
The drug loading and encapsulation efficiency of dimethylcurcumin in the DMC @ mPEG-DMC (15.5%) nanoparticles prepared in this example were 15.5% and 96.0%, respectively, the drug loading and encapsulation efficiency of dimethylcurcumin in the DMC @ mPEG-DMC (18.6%) nanoparticles prepared in this example were 18.6% and 68.7%, respectively, and the drug loading and encapsulation efficiency of dimethylcurcumin in the DMC @ mPEG-DMC (22.0%) nanoparticles prepared in this example were 22.0% and 62.5%, respectively.
Test example 4
Particle size and charge characterization of mPEG-DMC prepared in example 1 (7.4%), DMC @ mPEG-DMC prepared in test example 3 (15.5%), DMC @ mPEG-DMC (18.6%), DMC @ mPEG-DMC (22.0%):
sample preparation: 2mg of each of mPEG-DMC (7.4%) prepared in inventive example 1, DMC @ mPEG-DMC (15.5%), DMC @ mPEG-DMC (18.6%), and DMC @ mPEG-DMC (22.0%) prepared in Experimental example 3 was weighed, dissolved in 2mL of ultrapure water, and dispersed by sonication for 5min to prepare a 1mg/mL aqueous solution.
The particle size and charge of the aqueous mPEG-DMC (7.4%), DMC @ mPEG-DMC (15.5%), DMC @ mPEG-DMC (18.6%), DMC @ mPEG-DMC (22.0%) solutions prepared in this example were measured by dynamic light scattering using 1mL of the aqueous mPEG-DMC (7.4%), DMC @ mPEG-DMC (15.5%), DMC @ mPEG-DMC (18.6%), DMC @ mPEG-DMC (22.0%) solutions prepared in this example.
As can be seen from Table 1, with the increase of the drug loading, the particle size of the sample is increased, the absolute value of the potential is increased, and the polydispersity indexes of the samples are all less than 0.3, which shows that the dispersibility of the samples is good.
TABLE 1 particle size and charge characterization results for mPEG-DMC prepared in example 1 (7.4%), DMC @ mPEG-DMC prepared in test example 3 (15.5%), DMC @ mPEG-DMC (18.6%), DMC @ mPEG-DMC (22.0%).
Figure BDA0002645694140000151
Figure BDA0002645694140000161
Test example 5
Drug release behavior of mPEG-DMC prepared in example 1 (7.4%), DMC @ mPEG-DMC prepared in test example 3 (15.5%), DMC @ mPEG-DMC (18.6%) nanoparticles:
preparing a medicine solution: the mPEG-DMC (7.4%) prepared in inventive example 1, the DMC @ mPEG-DMC (15.5%) prepared in test example 3, and the DMC @ mPEG-DMC (18.6%) solution were diluted with ultrapure water to prepare an aqueous solution having a dimethylcurcumin concentration of 180. mu.g/mL.
Preparing release liquid:
(1) release solution I (PBS pH7.4, Tween 80 content 1.0%): 2.16g of disodium hydrogen phosphate dodecahydrate, 0.26g of potassium dihydrogen phosphate and 10g of tween-80 were weighed, dissolved in ultrapure water, and the volume was adjusted to 1000mL, and then the pH was adjusted to 7.4 with dilute hydrochloric acid.
(2) Release solution II (PBS pH5.0, Tween 80 content of 1.0%): 2.16g of disodium hydrogen phosphate dodecahydrate, 0.26g of potassium dihydrogen phosphate and 10g of tween-80 were weighed, dissolved in ultrapure water, and the volume was adjusted to 1000mL, and then the pH was adjusted to 5.0 with dilute hydrochloric acid.
1mL of the drug solution was put into a dialysis bag (cut-off: 3500Da), sealed, and then the dialysis bag containing the drug solution was immersed in 30mL of the release solution, and then put into a shaker at 37 ℃ and 180 rpm. 1mL of release solution was removed at 4, 8, 24, 36, 48, 60 and 72h time points, respectively, and 1mL of the corresponding release solution was replenished. Drug release experiments in each release solution were performed in parallel in three groups. The whole process of the drug release experiment is carried out under the condition of keeping out of the light. The concentration of the drug in the taken-out release solution is measured by a microplate reader, and the cumulative release amount is calculated. Cumulative release results for the mPEG-DMC prepared in example 1 (7.4%), DMC @ mPEG-DMC prepared in test example 3 (15.5%), DMC @ mPEG-DMC (18.6%) solutions are shown in Table 2.
TABLE 2 cumulative release results for mPEG-DMC prepared in example 1 (7.4%), DMC @ mPEG-DMC prepared in test example 3 (15.5%), DMC @ mPEG-DMC (18.6%).
Figure BDA0002645694140000171
As can be seen from Table 2, the release rate of mPEG-DMC (7.4%) prepared in example 1 in release liquid I and release liquid II was slow and the total release was only 4.70% and 4.99%. Test example 3 solutions of DMC @ mPEG-DMC (15.5%), DMC @ mPEG-DMC (18.6%) released significantly faster in release liquid II at pH5.0 than release liquid I at pH7.4 and released more completely in release liquid II. The total drug release of the DMC @ mPEG-DMC (15.5%), DMC @ mPEG-DMC (18.6%) solutions prepared in test example 3 was only 14.05% and 15.56% at 72h under release solution I condition at pH7.4, while the total drug release of the DMC @ mPEG-DMC (15.5%), DMC @ mPEG-DMC (18.6%) solutions prepared in test example 3 at 72h could reach 29.74%, 32.50% under release solution II condition at pH 5.0. This indicates that the DMC @ mPEG-DMC (15.5%) and DMC @ mPEG-DMC (18.6%) solutions prepared in inventive test example 3 have pH responsive drug release characteristics. FIG. 6 is a drug release profile of mPEG-DMC (7.4%) prepared in example 1, DMC @ mPEG-DMC (15.5%) prepared in test example 3, DMC @ mPEG-DMC (18.6%) solution.
Test example 6
In vitro antitumor activity of the dimethylcurcumin prodrug mPEG-DMC (7.4%) prepared in experimental example 1, DMC @ mPEG-DMC (15.5%) nanoparticles prepared in experimental example 3 and DMC @ mPEG-DMC (18.6%) nanoparticles:
preparation of experimental drugs: dissolving the dimethyl curcumin prodrug prepared in the experimental example 1 in ultrapure water, and performing ultrasonic treatment for 5min to prepare a 13.5mg/mL aqueous solution (wherein the content of the dimethyl curcumin is 1 mg/mL); dissolving free dimethyl curcumin in dimethyl sulfoxide to obtain 4mg/mL dimethyl curcumin solution.
The HepG-2 cell killing experiment was divided into four groups, free dimethyl curcumin experimental group (DMC), dimethyl curcumin prodrug experimental group mPEG-DMC (7.4%) prepared in experimental example 1, DMC @ mPEG-DMC (15.5%) nanoparticle experimental group prepared in experimental example 3, and DMC @ mPEG-DMC (18.6%) nanoparticle experimental group. Each experimental group was diluted from the prepared high concentration solution to a concentration of 0.1, 1, 5, 10 and 20 μ g/mL by DMEM medium and added to a 96-well plate to neutralize the cell incubation, the number of cells per well was about 5000, and each concentration in each experimental group was repeated for 4 wells, followed by culturing for 48h and 72 h.
Cell viability was measured by the MTT method, with cells incubated with blank medium as 100% viability. Free dimethyl curcumin experimental group (DMC), dimethyl curcumin prodrug experimental group mPEG-DMC (7.4%) prepared in experimental example 1, DMC @ mPEG-DMC (15.5%) nanoparticle experimental group prepared in experimental example 3, and DMC @ mPEG-DMC (18.6%) nanoparticle experimental group to the killing effect of HepG-2 cells are shown in FIG. 7.
As can be seen from FIG. 7, the cell survival rates of the experiment group incubated for 48h, the free dimethyl curcumin experiment group, the DMC @ mPEG-DMC (15.5%) nanoparticle experiment group and the DMC @ mPEG-DMC (18.6%) nanoparticle experiment group are close to each other, the HepG-2 cell growth inhibition effect is obvious, and the dimethyl curcumin prodrug has no obvious killing effect on HepG-2 cells. Compared with a free dimethyl curcumin experimental group, the DMC @ mPEG-DMC (15.5%) nanoparticle experimental group and the DMC @ mPEG-DMC (18.6%) nanoparticle experimental group prepared in the experimental example 3 have approximate or even lower cell survival rates and obvious HepG-2 cell growth inhibition effect. Experimental example 3 the DMC @ mPEG-DMC (15.5%) nanoparticle experimental group and the DMC @ mPEG-DMC (18.6%) nanoparticle experimental group prepared in Experimental example have similar HepG-2 cell killing effect than free dimethyl curcumin. But the dimethyl curcumin prodrug has no obvious killing effect on HepG-2 cells. Overall, the DMC @ mPEG-DMC (15.5%) nanoparticle experimental group and the DMC @ mPEG-DMC (18.6%) nanoparticle experimental group killed HepG-2 cells less than 22Rv1 cells.
The 22Rv1 cell killing experiment was divided into four groups, free dimethyl curcumin experimental group (DMC), dimethyl curcumin prodrug experimental group mPEG-DMC (7.4%) prepared in experimental example 1, DMC @ mPEG-DMC (15.5%) nanoparticle experimental group prepared in experimental example 3, and DMC @ mPEG-DMC (18.6%) nanoparticle experimental group. Each experimental group was diluted from the prepared high concentration solution to a concentration of 0.1, 1, 5, 10 and 20 μ g/mL by 1640 medium and added to a 96-well plate to neutralize cell incubation, the number of cells per well was about 5000, and each concentration in each experimental group was repeated for 4 wells, followed by culture for 48h and 72 h.
Cell viability was measured by the MTT method, with cells incubated with blank medium as 100% viability. The killing effect of free dimethyl curcumin experimental group (DMC), dimethyl curcumin prodrug experimental group mPEG-DMC (7.4%) prepared in experimental example 1, DMC @ mPEG-DMC (15.5%) nanoparticle experimental group prepared in experimental example 3 and DMC @ mPEG-DMC (18.6%) nanoparticle experimental group on 22Rv1 cells is shown in FIG. 7.
As can be seen from FIG. 7, after incubation for 48h, the cell viability of the DMC @ mPEG-DMC (15.5%) nanoparticle experimental group and the cell viability of the DMC @ mPEG-DMC (18.6%) nanoparticle experimental group are close to each other, and the cell viability of the DMC @ mPEG-DMC (18.6%) nanoparticle experimental group is higher than that of the free dimethylcurcumin experimental groupThe rate is lower, and the effect of inhibiting the growth of 22Rv1 cells is more obvious. Comparative free dimethyl curcumin experimental group (IC)5010.7 μ g/mL), the half inhibitory concentrations of the DMC @ mPEG-DMC (15.5%) and DMC @ mPEG-DMC (18.6%) nanoparticle test groups were significantly reduced (IC)50Only 3.6 μ g/mL and 4.4 μ g/mL, respectively), indicating that the DMC @ mPEG-DMC (15.5%) nanoparticle test group and the DMC @ mPEG-DMC (18.6%) nanoparticle test group prepared in test example 3 have stronger cell killing effect at 22Rv1 than free dimethyl curcumin. The free dimethyl curcumin prodrug also has obvious killing effect on 22Rv1 cells. After the experiment group is incubated for 72 hours, the cell survival rates of the free dimethyl curcumin experiment group, the DMC @ mPEG-DMC (15.5%) nanoparticle experiment group and the DMC @ mPEG-DMC (18.6%) nanoparticle experiment group are close to each other, and the effect of inhibiting the growth of 22Rv1 cells is obvious. Comparative free dimethyl curcumin experimental group (IC)508.0. mu.g/mL), slightly lower half Inhibitory Concentrations (IC) for the DMC @ mPEG-DMC (15.5%) nanoparticle test group and the DMC @ mPEG-DMC (18.6%) nanoparticle test group504.5 μ g/mL and 7.0 μ g/mL, respectively), indicating that the DMC @ mPEG-DMC (15.5%) nanoparticle experimental group and the DMC @ mPEG-DMC (18.6%) nanoparticle experimental group prepared in test example 3 have better cell killing effect of 22Rv1 than free dimethyl curcumin. The dimethyl curcumin prodrug also has obvious killing effect on 22Rv1 cells.
Finally, the method of the present invention is only a preferred embodiment, and is not intended to limit the scope of the present invention. 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 (9)

1. The dimethyl curcumin prodrug nanoparticles are characterized in that the dimethyl curcumin prodrug nanoparticles are prepared from methoxy polyethylene glycol aldehyde groups and dimethyl curcumin through covalent coupling with hydrazine hydrate;
the structural formula of the compound is
Figure FDA0003653365910000011
2. The preparation method of the dimethyl curcumin prodrug is characterized by comprising the following steps:
(1) adding dimethyl curcumin DMC and hydrazine hydrate into a solvent, and heating and stirring the mixture to react in a dark place under the atmosphere of nitrogen; after the reaction is finished, removing the solvent by rotary evaporation, transferring to a freeze dryer for freeze drying in a dark place to obtain the dimethyl curcumin derivative;
the structural formula of the dimethyl curcumin derivative is shown as
Figure FDA0003653365910000012
(2) Adding the dimethyl curcumin derivative prepared in the step (1) and methoxy polyethylene glycol aldehyde group mPEG-CHO into a solvent, heating in a dark place, and stirring for reaction; after the reaction is finished, removing the solvent by rotary evaporation, adding distilled water to dissolve the solvent, moving the solvent into a dialysis bag with the molecular weight cutoff of 3500Da, dialyzing the solvent in water, centrifuging the dialysate after the dialysis is finished, taking the supernatant, and freeze-drying to obtain the dimethyl curcumin prodrug; the structural formula is as follows:
Figure FDA0003653365910000021
3. the method for preparing the dimethyl curcumin prodrug as claimed in claim 2, wherein the molar ratio of the dimethyl curcumin to the hydrazine hydrate in the step (1) is 1:1 to 1: 10; the solvent is absolute methanol or absolute ethanol.
4. The method for preparing a dimethylcurcumin prodrug as claimed in claim 2, wherein the temperature of the heating reaction in the step (1) is 25 to 80 ℃; the heating reaction time is 12-48 hours.
5. The method for preparing a dimethylcurcumin prodrug as claimed in claim 2, wherein the average molecular weight of said methoxypolyethyleneglycol aldehyde group of step (2) is 1kDa to 20 kDa; the molar ratio of the methoxy polyethylene glycol aldehyde group to the dimethyl curcumin derivative is 1: 1-1: 10; the solvent is absolute methanol or absolute ethanol.
6. The method for preparing a dimethylcurcumin prodrug as claimed in claim 2, wherein the temperature of the heating reaction in the step (2) is 25 to 80 ℃, and the time of the heating reaction is 12 to 48 hours; the dialysis treatment time is 48 to 96 hours.
7. A preparation method of a nano preparation loaded with free dimethyl curcumin is characterized by comprising the following steps: adding the dimethyl curcumin prodrug nanoparticles and the free dimethyl curcumin into a solvent, stirring and dissolving at room temperature, removing the solvent by rotary evaporation and forming a film, adding ultrapure water, carrying out ultrasonic dispersion, centrifuging to obtain a supernatant, and carrying out freeze drying to obtain the nano preparation loaded with the free dimethyl curcumin, wherein the structural formula of the dimethyl curcumin prodrug is shown in the specification
Figure FDA0003653365910000031
8. The method for preparing free dimethyl curcumin prodrug nanoparticle-loaded nanoparticles as claimed in claim 7, wherein the mass ratio of the dimethyl curcumin prodrug nanoparticles to the free dimethyl curcumin is 1: 0-1: 0.3; the solvent is absolute methanol or absolute ethanol; the stirring time is 2-5 hours.
9. The prodrug prepared by the method of claims 2 to 6 and the nano preparation prepared by the method of claim 7 or claim 8 are applied to the preparation of drugs for resisting prostate cancer and liver cancer.
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透明质酸-姜黄素pH响应型纳米粒响应型纳米粒的制备与研究;叶俊贤;《万方数据库》;20200416;第1-54页 *

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