CN112156191B - Lysine modified drug carrier and preparation method thereof - Google Patents

Lysine modified drug carrier and preparation method thereof Download PDF

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CN112156191B
CN112156191B CN202011188301.3A CN202011188301A CN112156191B CN 112156191 B CN112156191 B CN 112156191B CN 202011188301 A CN202011188301 A CN 202011188301A CN 112156191 B CN112156191 B CN 112156191B
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秦大伟
谢军
高峰
常书华
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SHANDONG SIX BEST EXCIPIENTS Co.,Ltd.
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Abstract

A lysine modified drug carrier and its preparation method are provided. The invention provides a preparation method of novel nano hydrogel, which comprises the steps of grafting succinic anhydride modified beta-cyclodextrin to chitosan, and then modifying with lysine to prepare the nano hydrogel. The reaction efficiency is improved by utilizing the ultrasonic reaction of the intelligent temperature control dual-frequency ultrasonic reactor. The successful synthesis of the nano hydrogel is judged through infrared and GPC, a transmission electron microscope and an atomic force electron microscope are used for observing the morphology, and the performance research of the nano hydrogel is carried out through an in-vitro degradation experiment, a drug-loading and drug-releasing experiment and a cytotoxicity MTT experiment, and the result shows that the prepared nano hydrogel is suitable for being used as a drug carrier.

Description

Lysine modified drug carrier and preparation method thereof
[ technical field ] A method for producing a semiconductor device
The invention relates to a lysine modified drug carrier and a preparation method thereof, wherein the lysine modified drug carrier is nano hydrogel prepared by grafting carboxymethyl chitosan with beta-cyclodextrin and then modifying with lysine, and the preparation method thereof is also disclosed.
[ background of the invention ]
The drug carrier can improve the utilization rate, safety and timeliness of the drug, reduce the drug administration frequency, improve the unpleasant odor of the drug, improve the accuracy of the drug administration dosage and the accurate drug release to the target tissues and organs, thereby being widely concerned by people. The development of nanotechnology promotes the research of drug carriers, and the research and application of nanoscale drug carriers make great contribution in the field of medicine.
The hydrogel is a multi-element system consisting of a high molecular three-dimensional network and water, is a hydrophilic polymer with a network cross-linking structure, only swells but does not dissolve in water, and is a substance form commonly existing in nature. Nanogel refers to a nano-sized hydrogel composed of a network of cross-linked polymers. The nanogel has the advantages of strong reactivity, simple drug loading, strong drug loading capability, high physical stability, strong design universality, good stability of encapsulated drugs, controlled release of anti-inflammatory, antibacterial and anticancer drugs and the like.
The nano hydrogel has the following characteristics: (1) the nano hydrogel has the particle size of 10-600nm, small particle size and large specific surface area; (2) the surface contains a plurality of functional groups which can be coupled with components with specific functions and can generate the responsiveness to external environment stimulation; (3) similar to human tissues, the biological compatibility is good, and the degradation is easy in vivo; (4) the inside of the micelle has cross-linked structures with different degrees, and the micelle can be dissolved in water to form a micelle with good stability.
The material for preparing the nano hydrogel mainly comprises natural high molecular materials, such as lipide, biological polysaccharide, polypeptide protein and the like. As is well known, saccharides, lipids and proteins are three essential nutrients for human body, and the application of these biological macromolecules in preparing nanometer hydrogel can ensure the safety of medicine carrier, raise the biocompatibility and degrading capacity of nanometer hydrogel and produce no cytotoxicity.
Chitosan (CS) is second to cellulose in quantity, has active hydroxyl and amino in molecules, has strong chemical reaction capacity, and is difficult to dissolve in water. Carboxymethyl chitosan (CMC) is a water-soluble chitosan derivative, which is obtained by reacting chloroacetic acid with chitosan in the presence of alkali, and has stable properties and pharmacological effects of resisting bacteria, infection, lipid and arteriosclerosis.
Cyclodextrin (CD for short) is formed by connecting 6-12D-type glucopyranoses end to end through alpha-1, 4 glycosidic bonds, and the shape of the cyclodextrin is a cone-shaped cylinder. Beta-cyclodextrin (beta-CD) is formed by 7 glucopyranoses, a large number of hydroxyl groups exist outside a conical cavity to form hydrophilicity, and oxygen atoms on glycosidic bonds on the inner surface of the conical cavity are replaced by C3And C5The structure and the characteristic of the beta-cyclodextrin show the advantage of drug loading. However, cyclodextrin itself is difficult to form a hydrogel, and must be modified to be combined with other polymers to form a hydrogel.
The team has obtained a plurality of research results in earlier research, wherein the granted technical schemes are respectively the technical schemes of chinese patent CN107383393 and chinese patent CN109044963, the effects of which have been verified, but after further load and drug release experiments, the two nanogels also have unstable pH values, so that the drug loading and drug release processes are also unstable, and after further research, the main problem is that when the hydrogel is modified, the adopted modifying group is single, and the prospect of improving stability by further modifying on the basis of the modifying group is not clear, so that the problem needs to be solved by properly improving the basic structure and selecting a better modifying group.
In the field of nano hydrogel research, drug carriers in the form of nano hydrogels with amino acids have been reported. Amino acid is a substance which can not be lost in human body synthetic protein for body operation, and the amino acid molecule has two functional groups, amino group and carboxyl group. The nano hydrogel has amino and carboxyl, and can form different ionic bonds in different pH environments to realize pH regulation.
[ summary of the invention ]
The invention aims to solve the problems in the prior art and provides a lysine modified drug carrier which has the advantages of good biocompatibility, stable system, high drug loading rate, good drug slow-control property and high degradation rate.
Aiming at the problems that in the prior art, the hydrogel prepared from beta-cyclodextrin and chitosan is single in modified group and few in functional active site, the basic structure of the hydrogel is correspondingly improved, hydroxypropyl chitosan is replaced by carboxymethyl chitosan, so that a carboxyl group is added to the whole structure, the active site of the whole structure is added on the premise of not influencing the performance of the whole structure, and a new modified group is introduced to the site; meanwhile, compared with hydroxypropyl chitosan, carboxymethyl chitosan has the advantage of pH sensitivity, and after the carboxymethyl chitosan is replaced into the overall structure of hydrogel, the prepared nano hydrogel has pH sensitivity, and the drug loading rate and the drug release rate are obviously improved compared with the existing nano hydrogel;
after a great deal of experiments and verification, the inventor finds that on the premise of replacing hydroxypropyl chitosan with carboxymethyl chitosan, from a plurality of alternative modified substances, lysine is preferably modified, and because the molecular structure of lysine has two amino groups and one carboxyl group, the unit molecular group is increased by one amino group and one carboxyl group after the basic structure is introduced during modification.
Through the improvement, the condition that the pH is unstable in the prior art is determined, and the main reason is that the amino group carried by the amino group is lost to a certain extent in the polymerization process and is positioned in the main chain of the polymer, so that the induction of the amino group to the external pH is poor, and the amino group has certain delay; a new amino group is introduced into the structure of the polymer, the amino group is positioned on a branched chain of the polymer, carboxyl and the amino group are positioned on the branched chain as well, and the two groups are distributed more uniformly, so that the steric hindrance is greatly reduced, and the sensitivity of the polymer to the environmental pH is fundamentally improved.
Based on the above inventive concept, we further provide the lysine modified carboxymethyl chitosan nano hydrogel (Lyc-CMC-g- β -CD nano hydrogel), which has the following structural formula:
Figure GDA0003149194850000031
according to the structural formula, lysine of the modified carboxymethyl chitosan is positioned on a polymer branched chain, carboxyl and amino are symmetrically distributed, and n is 25-35.
The invention also aims to provide a preparation method of the Lyc-CMC-g-beta-CD nano hydrogel, which comprises the following steps:
(1) preparation of Suc-beta-CD
The preparation method of the succinic anhydride (Suc) modified beta-CD comprises the following specific steps: a500 mL three-neck flask is placed in an intelligent temperature control dual-frequency ultrasonic reactor, and the frequency of an ultrasonic instrument is adjusted to be 30KHz and 40 KHz. 250mLN, N-Dimethylformamide (DMF) is weighed and added into a three-neck flask, the temperature is set to be 70-95 ℃, certain mass of purified beta-CD is slowly added, certain mass of succinic anhydride (weighed according to different mol ratios with the beta-CD) is added, and different reaction times are set. After the reaction, the reaction mixture was poured into a 1000mL beaker and cooled to room temperature. And adding chloroform into the beaker to obtain a large amount of white flocculent precipitate, and performing suction filtration by using a vacuum pump to obtain a white solid. Washing with a large amount of acetone for 3 times, and drying the washed solid in a vacuum drying oven. Thus obtaining the succinic anhydride modified beta-CD (Suc-beta-CD). The mechanism for the preparation of β -CD (Suc- β -CD) is as follows:
Figure GDA0003149194850000041
(2) preparation of CMC-g-Suc-beta-CD copolymer
A clean 250mL three-neck flask was placed in a constant temperature reaction tank set at 37 ℃. Adding 100ml of NaCl solution into a flask, weighing the Suc-beta-CD obtained in the step (1) into the flask, and adding a certain mass of activating agent. After activating for a period of time, adding a certain mass of CMC, and reacting at 37 ℃. After the reaction is finished, transferring the reaction solution into a dialysis bag with the molecular cut-off of 5000Da, dialyzing the reaction solution in 500mL of ultrapure water for 72h, and replacing the ultrapure water once every 8 h; after dialysis was complete, a small amount of solution was retained for characterization testing. Transferring the liquid in the dialysis bag to a glass culture dish, and freeze-drying for 24h to obtain the freeze-dried CMC-g-Suc-beta-CD. The mechanism for preparing the CMC-g-Suc-beta-CD copolymer is as follows:
Figure GDA0003149194850000042
wherein the principle of preparing the copolymer by EDC/NHS condensation reaction is as follows:
Figure GDA0003149194850000043
(3) preparation of Lyc-CMC-g-beta-CD nano hydrogel
A clean 250mL three-neck flask was placed in a constant temperature reaction tank with a temperature setting of 37 ℃. Adding 100mL of NaCl solution into a flask, weighing a certain mass of CMC-g-Suc-beta-CD obtained in the step (2) into the 250mL flask, adding a certain mass of activating agent, performing activation reaction for 25min, adding a certain mass of lysine, and reacting for a period of time at 37 ℃. After the reaction is finished, the solution is transferred into a dialysis bag with the molecular cut-off of 5000Da for dialysis for 72h, and ultrapure water is replaced once after 8 h. After the dialysis was completed, the fluid in the dialysis bag was transferred to a glass petri dish and freeze-dried for 24h using a vacuum freeze-drying oven. The freeze-dried Lyc-CMC-g-beta-CD nano hydrogel is prepared by the following mechanism:
Figure GDA0003149194850000051
after further optimization, the corresponding technical scheme parameters are as follows:
the reaction temperature in the step (1) is 80 ℃, and the ultrasonic reaction time is controlled to be 6 h;
the mol ratio of the beta-CD to the succinic anhydride in the step (1) is 1: 1;
the concentration of the NaCl solution in the step (2) is 0.1 mol.L-1；
The mass of the Suc-beta-CD in the step (2) is 2.4636 g;
the mass ratio of Suc-beta-CD to CMC in the step (2) is 1: 2;
in the step (2), the activating agent is EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) and NHS (N-hydroxysuccinimide) which are used together at intervals of 30 min; the molar ratio of EDC to NHS is 1: 1; the molar ratio of Suc-beta-CD to activator EDC is 1: 1;
the reaction time of the step (2) is 12 hours;
the concentration of the NaCl solution in the step (3) is 0.1 mol.L-1；
The mass of the CMC-g-beta-CD in the step (3) is 2.0000 g;
the mass ratio of CMC-g-beta-CD to lysine is 5: 1;
in the step (3), the activating agent is EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) and NHS (N-hydroxysuccinimide) which are used together at intervals of 30 min; the molar ratio of EDC to NHS is 1: 1; the molar ratio of lysine to activator EDC is 1: 1;
under the mass ratio, the lysine has the best modification effect, and the pH sensitivity of the prepared nano hydrogel is the best.
The reaction time of the step (3) is 10 hours;
and (3) adding a mannitol freeze-drying protective agent into the prepared nano hydrogel, and then carrying out freeze drying, wherein the concentration of mannitol is 7%, and the ratio of mannitol solution to dialysate is 2: 3.
After the optimization, the finally obtained technical scheme is as follows:
(1) preparation of Suc-beta-CD
Placing a 500mL three-neck flask in an intelligent temperature control dual-frequency ultrasonic reactor, adjusting the frequency of an ultrasonic instrument to be 40KHz, measuring 250mLN, adding N-dimethylformamide into the three-neck flask, setting the temperature to be 80 ℃, slowly adding 22.7000g of purified beta-CD, adding 2.0014g of succinic anhydride, and setting the reaction time to be 6 h; after the reaction is finished, pouring the reaction liquid into a 1000mL beaker, cooling to room temperature, adding trichloromethane into the beaker to obtain a large amount of white flocculent precipitate, performing suction filtration by using a vacuum pump to obtain white solid, washing by using 300mL of acetone for 3 times, taking the washed solid, putting the solid into a vacuum drying oven, and drying for 12 hours at the corresponding temperature set in the reaction;
(2) preparation of CMC-g-Suc-beta-CD copolymer
Taking a clean 250mL three-neck flask, and putting the flask in a constant-temperature reaction tank, wherein the temperature of the reaction tank is set to be 37 ℃; adding 100mL of NaCl solution into a flask, weighing 2.4636g of the modified beta-CD obtained in the step (1) into the 250mL flask, adding 0.3834g of EDC for activating carboxyl on the succinic anhydride modified beta-CD, adding 0.2302g of NHS after 30min, reacting for 25min, adding 4.9272g of CMC, and reacting for 12h at 37 ℃; after the reaction is finished, transferring the reaction solution into a dialysis bag with the molecular cut-off of 5000Da, dialyzing the reaction solution in 500mL of ultrapure water for 72h, and replacing the ultrapure water once every 8 h; after the dialysis is finished, transferring the liquid in the dialysis bag to a glass culture dish, and freeze-drying for 24 hours to obtain freeze-dried CMC-g-Suc-beta-CD;
(3) preparation of Lyc-CMC-g-beta-CD nano hydrogel
Placing a clean 250mL three-neck flask in a constant-temperature reaction tank, and setting the temperature to be 37 ℃; adding 100mL of NaCl solution into a flask, weighing 2.0000g of CMC-g-Suc-beta-CD obtained in the step 2 into the 250mL flask, adding 0.3834g of EDC, adding 0.2302g of NHS after 30min of reaction, adding 0.4000g of lysine after 25min of reaction, and reacting for 10h at 37 ℃; after the reaction is finished, transferring the solution into a dialysis bag with the molecular cut-off of 5000Da, dialyzing the solution in 500mL of ultrapure water for 72h, and replacing the ultrapure water once every 8 h; after the dialysis is finished, transferring the liquid in the dialysis bag to a glass culture dish, and freeze-drying for 24 hours by using a vacuum freeze-drying box; obtaining the freeze-dried Lyc-CMC-g-beta-CD nano hydrogel.
Because lysine is adopted for modification, compared with the Chinese patent CN107383393, the reaction time in the modification step is shortened to 10 hours, the overlong reaction time required by using an ultrasonic peristaltic pump in the prior art is avoided, the preparation steps are greatly simplified while the advantages of the prior art are ensured, and more functional characteristics are added on the basis of the prior art. In addition, the reaction temperature is adjusted to 37 ℃, the temperature of the drug carrier is more suitable for the temperature of a human body, and the drug carrier is prevented from being damaged and degraded due to the difference of environmental temperatures in practical application. Because lysine is essential amino acid for human body, the prepared nano hydrogel has no harm to human body after being degraded in vivo, and can supplement lysine required by human body.
The lysine modified carboxymethyl chitosan nano hydrogel obtained by the method is a drug carrier with more excellent performance, and the performance results are as follows:
the prepared nano hydrogel is spherical in shape, relatively uniform in size, and suitable for drug loading due to a specific cavity structure of the raw materials; the particle size of the nano hydrogel can change along with the change of the environment pH environment, the particle size is the smallest when the pH value is 7.0, the particle size is 140.1nm, and the nano hydrogel has pH sensitivity; the in vitro simulated degradation is easy and short; drug loading and release rates are improved over the inventor's prior applications and other prior art; in vitro cell experiments prove that the carrier has no cytotoxicity to normal cells.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the invention uses succinic acid glycoside to modify beta-cyclodextrin to increase modifiable carboxyl, then grafts chitosan through the reaction of carboxyl and amino, selects human amino acid with double-amino single carboxyl to modify on the basis, grafts different raw materials together by taking covalent bond as a bridge, and increases the uniformly distributed amino and carboxyl with strong symmetry to make the prepared hydrogel sensitive to specific pH, and the raw materials are all taken from natural substances and have high biocompatibility.
(2) The nano hydrogel prepared by the invention has good stability, uniform particle size distribution, uniform size, good dispersibility, safety and no toxic or side effect, can fully utilize the cavity to load hydrophobic drugs, effectively improves the bioavailability on the basis of reducing the drug frequency, utilizes the peculiar swelling drug release mechanism of the hydrogel to realize slow release and achieves the purpose of long-acting drug delivery.
(3) The lysine modified carboxymethyl chitosan nano hydrogel drug carrier prepared by the invention is not only suitable for berberine hydrochloride, but also suitable for other hydrophobic drugs, and has wide application prospect.
[ description of the drawings ]
FIG. 1 is a comparison graph of Fourier spectra of Suc- β -CD and β -CD;
FIG. 2 is a comparison graph of Fourier spectra of CMC-g-Suc-beta-CD and Lyc-CMC-g-beta-CD nanohydrogel;
FIG. 3 is a comparative transmission electron microscope image of CMC-g-Suc-beta-CD and Lyc-CMC-g-beta-CD nanohydrogel, (a) CMC-g-Suc-beta-CD, (b) Lyc-CMC-g-beta-CD;
FIG. 4 is an atomic force electron microscope image of Lyc-CMC-g-beta-CD nano hydrogel;
FIG. 5 is a graph showing the particle size change of Lyc-CMC-g-beta-CD nano hydrogel at different pH values (a) and the Zeta potential value (b);
FIG. 6 is a graph of the in vitro degradation curve of a Lyc-CMC-g-beta-CD nanohydrogel;
FIG. 7 is a graph of the cumulative release of the Lyc-CMC-g-beta-CD nano hydrogel to berberine hydrochloride;
FIG. 8 is a graph showing the result of cytotoxicity test of Lyc-CMC-g-beta-CD nano hydrogel.
[ detailed description ] embodiments
The present invention will be further described with reference to the following specific examples. It is to be understood that the following examples are illustrative of the present invention only and are not intended to limit the scope of the present invention.
Example 1
The preparation method of the Lyc-CMC-g-beta-CD nano hydrogel comprises the following specific steps:
(1) preparation of Suc-beta-CD
Placing a 500mL three-neck flask in an intelligent temperature control dual-frequency ultrasonic reactor, adjusting the frequency of an ultrasonic instrument to be 40KHz, measuring 250mLN, adding N-Dimethylformamide (DMF) into the three-neck flask, setting the temperature to be 80 ℃, slowly adding 22.7000g (20mmol) of purified beta-CD, weighing 2.0014g (20mmol) of succinic anhydride, and setting the reaction time to be 6 h. After the reaction, the reaction mixture was poured into a 1000mL beaker and cooled to room temperature. And adding chloroform into the beaker to obtain a large amount of white flocculent precipitate, and performing suction filtration by using a vacuum pump to obtain a white solid. The solid was washed 3 times with 300mL of acetone and the washed solid was taken out and placed in a vacuum oven and dried for 12h at the corresponding temperature set for the reaction.
(2) Preparation of CMC-g-Suc-beta-CD copolymer
A clean 250mL three-neck flask was placed in a constant temperature reaction tank set at 37 ℃. 100ml of NaCl solution was added to the flask, 2.4636g of the modified beta-CD obtained in step 1 was weighed into the flask, 0.3835g of EDC was added to activate the carboxyl group on the succinic anhydride-modified beta-CD, 0.2301g of NHS was added after 30min (the molar ratio of EDC to NHS was determined to be 1:1), 4.9272g of CMC was added after 25min of reaction, and the reaction was carried out at 37 ℃ for 12 h. After completion of the reaction, the reaction solution was transferred into a dialysis bag having a molecular weight cut-off of 5000Da and dialyzed in 500mL of ultrapure water for 72 hours, and the ultrapure water was replaced every 8 hours. After the dialysis is finished, the liquid in the dialysis bag is transferred to a glass culture dish and is frozen and dried for 24 hours to obtain the freeze-dried CMC-g-Suc-beta-CD.
(3) Preparation of Lyc-CMC-g-beta-CD nano hydrogel
A clean 250mL three-neck flask was placed in a constant temperature reaction tank with a temperature setting of 37 ℃. Adding 100mL of NaCl solution into a flask, weighing 2.0000g of CMC-g-Suc-beta-CD obtained in the step 2, adding 0.3834g of EDC into a 250mL flask, adding 0.2302g of NHS after 30min, adding 0.4000g of lysine after 25min of reaction, and reacting at 37 ℃ for 10 h. After the reaction was completed, the solution was transferred into a dialysis bag having a molecular cut-off of 5000Da and dialyzed in 500mL of ultrapure water for 72 hours, and the ultrapure water was replaced every 8 hours. After the dialysis was completed, the fluid in the dialysis bag was transferred to a glass petri dish and freeze-dried for 24h using a vacuum freeze-drying oven. Obtaining the freeze-dried Lyc-CMC-g-beta-CD nano hydrogel.
The lysine modified carboxymethyl chitosan nano hydrogel obtained by the method has the following structural formula:
Figure GDA0003149194850000091
example 2
The preparation method of the Lyc-CMC-g-beta-CD nano hydrogel comprises the following specific steps:
(1) preparation of Suc-beta-CD
Placing a 500mL three-neck flask in an intelligent temperature control dual-frequency ultrasonic reactor, adjusting the frequency of an ultrasonic instrument to be 40KHz, measuring 250mLN, adding N-Dimethylformamide (DMF) into the three-neck flask, setting the temperature to be 80 ℃, slowly adding 22.7000g (20mmol) of purified beta-CD, weighing 2.0014g (20mmol) of succinic anhydride, and setting the reaction time to be 6 h. After the reaction, the reaction mixture was poured into a 1000mL beaker and cooled to room temperature. And adding chloroform into the beaker to obtain a large amount of white flocculent precipitate, and performing suction filtration by using a vacuum pump to obtain a white solid. The solid was washed 3 times with 400mL of acetone and the washed solid was taken out and placed in a vacuum oven and dried for 12h at the corresponding temperature set for the reaction.
(2) Preparation of CMC-g-Suc-beta-CD copolymer
A clean 250mL three-neck flask was placed in a constant temperature reaction tank set at 37 ℃. A NaCl solution was added to the flask, and modified beta-CD 2.4636g obtained in step 1 was weighed in a 250mL flask, and 4.9272g of CMC was added thereto, and reacted at 37 ℃ for 12 hours. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular cut-off of 5000Da for dialysis for 72 hours, and purified water was replaced every 8 hours. After dialysis was complete, a small amount of solution was retained for characterization testing. Transferring the liquid in the dialysis bag to a glass culture dish, and freeze-drying for 24h to obtain the freeze-dried CMC-g-Suc-beta-CD.
(3) Preparation of Lyc-CMC-g-beta-CD nano hydrogel
A clean 250mL three-neck flask was placed in a constant temperature reaction tank with a temperature setting of 37 ℃. 100mL of NaCl solution was added to the flask, 2.0000g of CMC-g-Suc-. beta. -CD obtained in step 2 was weighed in a 250mL flask, and 0.4000g of lysine was added thereto to react at 37 ℃ for 10 hours. After the reaction is finished, the solution is transferred into a dialysis bag with the molecular cut-off of 5000Da for dialysis for 72h, and ultrapure water is replaced once after 8 h. After the dialysis was completed, the fluid in the dialysis bag was transferred to a glass petri dish and freeze-dried for 24h using a vacuum freeze-drying oven. Obtaining the freeze-dried Lyc-CMC-g-beta-CD nano hydrogel.
Example 3
The preparation method of the Lyc-CMC-g-beta-CD nano hydrogel comprises the following specific steps:
(1) preparation of Suc-beta-CD
Placing a 500mL three-neck flask in an intelligent temperature control dual-frequency ultrasonic reactor, adjusting the frequency of an ultrasonic instrument to be 40KHz, measuring 250mLN, adding N-Dimethylformamide (DMF) into the three-neck flask, setting the temperature to be 70 ℃, slowly adding 22.7000g (20mmol) of purified beta-CD, weighing 2.0014g (20mmol) of succinic anhydride, and setting the reaction time to be 5 h. After the reaction, the reaction mixture was poured into a 1000mL beaker and cooled to room temperature. And adding chloroform into the beaker to obtain a large amount of white flocculent precipitate, and performing suction filtration by using a vacuum pump to obtain a white solid. The solid was washed 3 times with 500mL of acetone and the washed solid was placed in a vacuum oven. Drying for 12h at the corresponding temperature set during the reaction to obtain the succinic anhydride modified beta-CD.
(2) Preparation of CMC-g-Suc-beta-CD copolymer
A clean 250mL three-neck flask was placed in a constant temperature reaction tank set at 37 ℃. A NaCl solution (100mL of 0.1 mol. L-1) was added to the flask, 2.4636g of the modified beta-CD obtained in step 1 was weighed into a 250mL flask, 0.3835g of EDC was added to activate the carboxyl group on the succinic anhydride-modified beta-CD, and after 30min, 0.2302g of NHS was added (the molar ratio of EDC to NHS was determined to be 1:1), and after 25min, 4.9272g of CMC was added and reacted at 37 ℃ for 6 h. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular cut-off of 5000Da for dialysis for 72 hours, and purified water was replaced every 8 hours. After dialysis was complete, a small amount of solution was retained for characterization testing. Transferring the liquid in the dialysis bag to a glass culture dish, and freeze-drying for 24h to obtain the freeze-dried CMC-g-Suc-beta-CD.
(3) Preparation of Lyc-CMC-g-beta-CD nano hydrogel
A clean 250mL three-neck flask was placed in a constant temperature reaction tank with a temperature setting of 37 ℃. Adding 100mL of NaCl solution into a flask, weighing 2.0000g of CMC-g-Suc-beta-CD obtained in the step 2 into a 250mL flask, adding 0.3834g of EDC, adding 0.2302g of NHS after 30min, adding 0.4000g of lysine after 25min of reaction, and reacting for 5h at 37 ℃. After the reaction is finished, the solution is transferred into a dialysis bag with the molecular cut-off of 5000Da for dialysis for 72h, and ultrapure water is replaced once after 8 h. After the dialysis was completed, the fluid in the dialysis bag was transferred to a glass petri dish and freeze-dried for 24h using a vacuum freeze-drying oven. Obtaining the freeze-dried Lyc-CMC-g-beta-CD nano hydrogel.
Examples of the experiments
Characterization and performance research of Lyc-CMC-g-beta-CD nano hydrogel
For example 1, we characterized the structural properties of the product by the following analytical means. We performed Fourier transform infrared spectroscopy tests on Suc-beta-CD and beta-CD, FIG. 1 is a comparison of the Fourier transform infrared spectra of Suc-beta-CD and beta-CD, 3380cm-1At position is an O-H stretching vibration absorption peak, 2916cm-1The position is the stretching vibration absorption peak of the C-H bond. The characteristic absorption of alpha-type glycosidic bond is 1013cm-1On the left and right, it indicates that beta-CD is connected by alpha-1, 4 glycosidic bond. Comparing the two figures, the Fourier infrared spectrogram of Suc-beta-CD is higher than that of beta-CD by 1732cm-1The characteristic peak at (a) corresponds to C ═ O, which indicates that carboxyl groups were introduced on β -CD, and it can be judged that β -CD was successfully modified with succinic anhydride.
The molecular weight of each sample is represented by weight average molecular weight (Mw), the molecular weight of CMC is 41327Da, the copolymer is 46587Da, the difference is 5260Da, the weight of grafted Suc-beta-CD is used as the weight, and the CMC-g-Suc-beta-CD can be judged to be successfully grafted, and the GPC related molecular weights of CMC and CMC-g-Suc-beta-CD are as follows:
Figure GDA0003149194850000121
the test of the Fourier infrared spectrum of CMC-g-Suc-beta-CD and Lyc-CMC-g-beta-CD nano hydrogel is carried out, figure 2 is the Fourier infrared spectrum contrast diagram of CMC-g-Suc-beta-CD and Lyc-CMC-g-beta-CD nano hydrogel, and the Fourier infrared spectrum diagram of CMC-g-Suc-beta-CD is smaller than that of Lyc-CMC-g-beta-CD by 1700cm-1Characteristic peak at 1610cm-1The characteristic peak is also enhanced, which shows that the new amido bond is grafted on the Lyc-CMC-g-beta-CD, and the functional lysine grafted CMC-g-Suc-beta-CD can be judged.
The CMC-g-Suc-beta-CD and the Lyc-CMC-g-beta-CD nano hydrogel are subjected to a transmission electron microscope test and an atomic force microscope test, some round spherical particles can be seen in fig. 3(a), and fig. 3(b) is greatly improved, which is attributed to the introduction of amino and carboxyl in lysine, so that the surface property of the nano hydrogel is improved, and in addition, a 7% mannitol freeze-drying protective agent is added in the freeze-drying process to play a role. FIG. 4 shows that the size distribution of the copolymer is in the range of 140-160nm, the size is small enough and uniformly distributed, the specific surface area is large, and higher drug loading efficiency can be realized.
Analyzing the particle size change of the Lyc-CMC-g-beta-CD at different pH values, wherein a graph 5(a) is a graph of the particle size change of the Lyc-CMC-g-beta-CD nano hydrogel at different pH values, and the particle size difference is large at different pH values, which shows that the prepared nano hydrogel has pH sensitivity, the particle size is 140.1nm at the minimum, the pH value is 7.0 at the moment, and (b) is a Zeta potential value change of the Lyc-CMC-g-beta-CD nano hydrogel at different pH values, the isoelectric point is between pH6.5 and 7.5, the nano hydrogel has no charge at the moment, no internal charge repulsion force exists, the particle size is minimum, and the conclusion is consistent with the conclusion that the minimum particle size is around pH 7.0 in particle size test analysis.
And carrying out an in-vitro simulated degradation experiment on the Lyc-CMC-g-beta-CD nano hydrogel. Fig. 6 is an in vitro degradation curve of the Lyc-CMC-g- β -CD nano hydrogel, a PBS buffer solution with a ph of 7.4 is configured to simulate a human body environment, a quantitative Lyc-CMC-g- β -CD nano hydrogel is dissolved in the buffer solution, dialyzed by a dialysis bag with a molecular cutoff of 30000Da, the solution in the dialysis bag is taken at a specific time interval for freeze drying, and the residual mass is weighed, fig. 6 shows the residual mass percentage of the nano hydrogel at different time intervals, and thus, the Lyc-CMC-g- β -CD nano hydrogel can be degraded by more than 85% in 12 days.
Selecting hydrophobic berberine hydrochloride as model drug, loading with Lyc-CMC-g-beta-CD nano hydrogel, and simulating berberine hydrochloride water solution standard curve y 0.1102x +0.0485 (R)20.99801), calculating the encapsulation rate of the Lyc-CMC-g-beta-CD nano hydrogel to berberine hydrochloride to be 51.13% and the drug loading rate to be 29.03%. The accumulative release amount of berberine hydrochloride is researched for 0-48h, fig. 7 is a accumulative release curve of the Lyc-CMC-g-beta-CD nano hydrogel to the berberine hydrochloride, the accumulative release amount of the berberine hydrochloride reaches 40.2%, and the result shows that the Lyc-CMC-g-beta-CD hydrogel has obvious slow release effect on the berberine hydrochloride and is suitable for pharmaceutical application within 24 hours.
And performing cytotoxicity detection on the Lyc-CMC-g-beta-CD nano hydrogel, selecting human endothelial cells as normal cell experimental objects, and determining the toxicity of the human endothelial cells on normal cells by an MTT method. The recovered human endothelial cells were Cultured (CO) in DMEM high-glucose medium (5% double antibody and 10% fetal bovine serum)2Incubators, 37 ℃), washing the culture medium with Phosphate Buffered Saline (PBS), adding appropriate amount of trypsin, in CO2Digest for 60s in the incubator. Adding a proper amount of culture medium and blowing the adherent cells by a rubber head dropper for suspension. Directly counting the cells under microscope, and controlling the density of the cells to 5 × 10 by calculating and supplementing DMEM medium4one/mL. Adding 200 μ L of cell sap into 96-well cell culture plate, and continuously placing CO into the 96-well plate2The incubator was incubated for 24h with the incubator temperature set at 37 ℃. Then taking out a 96-well plate, adding the Lyc-CMC-g-beta-CD nano hydrogel prepared in the example 1 into each well after PBS (phosphate buffer solution) cleaning, setting 5 concentration gradients, andparallel experiments were performed 3 times, and two control groups were set. Setting concentration gradient: 50. 100, 150, 200 and 250 mu g/mL. In CO2After 48h incubation in the incubator, 0.5% MTT reagent was added. At O2The culture was continued for 4 hours in the incubator, and after the mixed solution in the 96-well plate was poured out, 200. mu.L of dimethyl sulfoxide was added to each well. Oscillating for 10min in a constant temperature oscillator at 37 ℃, measuring the light absorption value of each hole at 490nm corresponding to the sample by using an enzyme-labeling instrument, calculating the cell survival rate, and judging whether the cell has cytotoxicity according to the cell survival rate.
Fig. 8 shows the results of the MTT experiments on cytotoxicity of the nano hydrogel, the cell survival rates were all higher than 85%, the MTT experiments showed that the prepared nano hydrogel was not toxic to normal cells, and the addition of lysine facilitated the growth of cells after the nano hydrogel was degraded. Through performance experiments, the Lyc-CMC-g-beta-CD nano hydrogel can be judged to be suitable for being used as a drug carrier, can carry out drug loading and slow release in a human body environment, and realizes the effect of treating corresponding diseases.

Claims (4)

1. A lysine modified drug carrier is lysine modified carboxymethyl chitosan nano hydrogel, which is characterized in that lysine and beta-cyclodextrin are grafted to carboxymethyl chitosan, and the structural formula of the lysine modified drug carrier is as follows:
Figure FDA0003149194840000011
2. the process for preparing a lysine-modified pharmaceutical carrier of claim 1, wherein: the method comprises the following steps:
(1) preparation of Suc-beta-CD
Placing a 500mL three-neck flask in an intelligent temperature control dual-frequency ultrasonic reactor, adjusting the frequency of an ultrasonic instrument to be 40KHz, measuring 250mLN, adding N-dimethylformamide into the three-neck flask, setting the temperature to be 80 ℃, slowly adding 22.7000g of purified beta-CD, adding 2.0014g of succinic anhydride, and setting the reaction time to be 6 h; after the reaction is finished, pouring the reaction liquid into a 1000mL beaker, cooling to room temperature, adding trichloromethane into the beaker to obtain a large amount of white flocculent precipitate, performing suction filtration by using a vacuum pump to obtain white solid, washing by using 300mL of acetone for 3 times, taking the washed solid, putting the solid into a vacuum drying oven, and drying for 12 hours at the corresponding temperature set in the reaction;
(2) preparation of CMC-g-Suc-beta-CD copolymer
Taking a clean 250mL three-neck flask, and putting the flask in a constant-temperature reaction tank, wherein the temperature of the reaction tank is set to be 37 ℃; adding 100mL of NaCl solution into a flask, weighing 2.4636g of the modified beta-CD obtained in the step (1) into the 250mL flask, adding 0.3834g of EDC for activating carboxyl on the succinic anhydride modified beta-CD, adding 0.2302g of NHS after 30min, reacting for 25min, adding 4.9272g of CMC, and reacting for 12h at 37 ℃; after the reaction is finished, transferring the reaction solution into a dialysis bag with the molecular cut-off of 5000Da, dialyzing the reaction solution in 500mL of ultrapure water for 72h, and replacing the ultrapure water once every 8 h; after the dialysis is finished, transferring the liquid in the dialysis bag to a glass culture dish, and freeze-drying for 24 hours to obtain freeze-dried CMC-g-Suc-beta-CD;
(3) preparation of Lyc-CMC-g-beta-CD nano hydrogel
Placing a clean 250mL three-neck flask in a constant-temperature reaction tank, and setting the temperature to be 37 ℃; adding 100mL of NaCl solution into a flask, weighing 2.0000g of CMC-g-Suc-beta-CD obtained in the step 2 into the 250mL flask, adding 0.3834g of EDC, adding 0.2302g of NHS after 30min of reaction, adding 0.4000g of lysine after 25min of reaction, and reacting for 10h at 37 ℃; after the reaction is finished, transferring the solution into a dialysis bag with the molecular cut-off of 5000Da, dialyzing the solution in 500mL of ultrapure water for 72h, and replacing the ultrapure water once every 8 h; after the dialysis is finished, transferring the liquid in the dialysis bag to a glass culture dish, and freeze-drying for 24 hours by using a vacuum freeze-drying box; obtaining the freeze-dried Lyc-CMC-g-beta-CD nano hydrogel.
3. The method of claim 2, wherein:
the reaction temperature in the step (1) is 80 ℃, and the ultrasonic reaction time is controlled to be 6 h;
the mol ratio of the beta-CD to the succinic anhydride in the step (1) is 1: 1;
the concentration of the NaCl solution in the steps (2) and (3) is 0.1 mol.L-1；
The mass ratio of Suc-beta-CD to CMC in the step (2) is 1: 2;
in the step (2), the activating agent is EDC (EDC), namely 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) and NHS (NHS), namely N-hydroxysuccinimide, which are used together at intervals of 30 min; the molar ratio of EDC to NHS is 1: 1; the molar ratio of Suc-beta-CD to activator EDC is 1: 1;
the mass ratio of CMC-g-beta-CD to lysine is 5: 1;
in the step (3), the activating agent is EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) and NHS (N-hydroxysuccinimide) which are used together at intervals of 30 min; the molar ratio of EDC to NHS is 1: 1; the molar ratio of lysine to activator EDC was 1: 1.
4. The method of claim 2, wherein:
and (3) adding a mannitol freeze-drying protective agent into the prepared nano hydrogel, and then carrying out freeze drying, wherein the concentration of mannitol is 7%, and the ratio of mannitol solution to dialysate is 2: 3.
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