CN113651965A - High molecular compound, preparation method and application thereof - Google Patents

High molecular compound, preparation method and application thereof Download PDF

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CN113651965A
CN113651965A CN202110830340.7A CN202110830340A CN113651965A CN 113651965 A CN113651965 A CN 113651965A CN 202110830340 A CN202110830340 A CN 202110830340A CN 113651965 A CN113651965 A CN 113651965A
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polycaprolactone
interpolymer
drug
hydroxyethyl starch
icg
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CN113651965B (en
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胡豫
梅恒
唐宇翔
陈钊钊
姚惟琦
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Tongji Medical College of Huazhong University of Science and Technology
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/804—Blood cells [leukemia, lymphoma]

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a high molecular compound, a preparation method and application thereof. The interpolymer has the general formula shown in FIG. 4. A drug delivery system comprising said interpolymer and a drug effect enhancing formulation loaded on the interpolymer. A process for preparing an interpolymer comprising the steps of: activating the carboxyl of polycaprolactone, carrying out esterification reaction on the carboxyl-activated polycaprolactone and a hydroxyethyl starch solution, and purifying to obtain the hydroxyethyl starch-polycaprolactone copolymer. The copolymer is used in preparing medicine for preventing and treating tumor, and the medicine carrying system is used in preparing antitumor medicine. The copolymer can be better used for drug loading, is beneficial to CAR-T treatment of tumors, and LY/ICG @ HES-PCL has a good tumor accumulation effect on a raji cell lymphoma model, realizes the photothermal effect of ICG, can obviously improve the density of tumor extracellular matrix, improves the infiltration of CAR-T on lymphoma tissues, and improves the proliferation and activity of CAR-T.

Description

High molecular compound, preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a macromolecular compound, a preparation method and application thereof.
Background
Lymphoma is a malignant tumor which is originally generated in lymph nodes and extranodal tissues, is one of ten malignant tumors in China, and seriously harms human health. Clinical first-line lymphoma therapy mainly comprises chemotherapy (rituximab, cyclophosphamide, adriamycin, vincristine, prednisone and the like), but has poor treatment effect, the effective remission rate is 18% -45%, the median survival time of patients is 3-9 months, and part of patients have drug resistance. CAR-T is collectively known as chimeric antigen receptor T cell immunotherapy and holds promise for lymphoma treatment. Clinical studies have shown that CAR-T therapy is aimed at increasing median survival to 12.9 months in recurrent non-hodgkin's lymphoma (NHL) in children and adults. Although the efficacy of CAR-T for treating lymphoma is improved to a certain extent, an effective scheme is still urgently needed to further improve the efficacy of CAR-T treatment. Lymphoma tissues have tumor immunosuppressive microenvironment, abnormal vascular microenvironment and cross-linked compact matrix barriers, so that homing, infiltration, proliferation and activation of CAR-T lymphoma tissues can be hindered, and the tumor immunosuppressive microenvironment, abnormal vascular microenvironment and cross-linked compact matrix barriers are important reasons for poor drug effect of CAR-T lymphoma treatment.
The nano-drug is a drug body which is prepared by 0-1000 nm of drug and carrier through a nano-drug loading technology, and can increase the accumulation of the drug in a tumor part by enhancing retention and penetration effects (EPR effects) so as to enhance the efficacy and reduce the toxicity of the drug; meanwhile, the blood stability of the medicine is improved, the medicine is prevented from being excreted by the kidney too fast, the half-life period of the loaded medicine is prolonged, and the bioavailability of the medicine is improved.
Disclosure of Invention
Aiming at the problems, the invention provides a high molecular compound, a preparation method and application thereof, and mainly solves the problems that the existing medicine for enhancing the curative effect of CAR-T on lymphoma cannot better improve the tumor immunosuppression microenvironment, abnormal blood vessel microenvironment, infiltration of CAR-T on lymphoma tissues, tumor extracellular matrix density and the like.
In order to solve the problems, the invention adopts the following technical scheme:
an interpolymer having the formula:
Figure BDA0003175200830000021
wherein the content of the first and second substances,
n is 100-5000, m is 50-500, i is 2-9, further, n is 150-250, m is 75-150, i is 4-7, and m, n and i are positive integers in any of the above corresponding ranges; preferably, n is 200, m is 100, i is 6;
R1-R4 are any one of halogeno, -OH and-OR ', and R' is halogeno; R1-R4 are respectively any one of halogeno, -OH and-OR', and R1-R4 can be the same OR different and are in the protection scope of the invention;
r5 is any one of halogeno, -OH and-OR, -R' halogeno OR- (CH2) kOH, and k is a positive integer.
When i is 6, the formula is as follows:
Figure BDA0003175200830000022
in some modes, the grafting rate of the polycaprolactone on the hydroxyethyl starch is 0.61-1;
preferably, the grafting ratio of the polycaprolactone on the hydroxyethyl starch is 0.75,
preferably, R1-R4 are all-OH.
A process for preparing an interpolymer comprising the steps of:
the carboxyl group of the polycaprolactone is activated,
the carboxyl activated polycaprolactone and the hydroxyethyl starch solution are subjected to esterification reaction,
purifying to obtain the hydroxyethyl starch-polycaprolactone copolymer.
In some embodiments, the step of activating the carboxyl group of the polycaprolactone is: dissolving polycaprolactone in anhydrous dimethyl sulfoxide, adding 1-hydroxybenzotriazole and N-N' -dicyclohexylcarbodiimide, and performing carboxyl activation reaction; and/or
The preparation method of the hydroxyethyl starch solution comprises the following steps: dissolving hydroxyethyl starch into anhydrous dimethyl sulfoxide, preferably, adopting inert gas for protection in the dissolving process; and/or
The esterification reaction temperature is 40-80 ℃, and preferably, inert gas is adopted for protection in the reaction process.
In some forms, the inert gas is helium; the temperature for dissolving polycaprolactone by using anhydrous dimethyl sulfoxide is 50-70 ℃.
In some embodiments, the purification step is: dialyzing the copolymer mixture obtained by the esterification reaction through a PBS solution, freezing the liquid obtained by the dialysis, and further freeze-drying to obtain the hydroxyethyl starch-polycaprolactone copolymer.
In some embodiments, the purification step is specifically:
dialyzing the copolymer mixture obtained by the esterification reaction through a PBS solution, removing unreacted 1-hydroxybenzotriazole and/or N-N' -dicyclohexylcarbodiimide and/or polycaprolactone and/or DMSO solvent,
freezing the liquid obtained by dialysis at the temperature of-20 to-25 ℃ for 3 to 5 hours,
and (3) putting the mixture into a freezing and drying device at the temperature of-40 to-60 ℃ to obtain the hydroxyethyl starch-polycaprolactone copolymer freeze-dried powder.
The copolymer is applied to preparing a medicine for preventing and treating tumors.
Some embodiments, the use is as a vehicle for a pharmacodynamic-enhancing formulation in CAR-T therapy; preferably, the pharmacodynamic-enhancing agent is a TGF- β inhibitor and/or ICG (indocyanine green), more preferably, the TGF- β inhibitor is LY.
A drug delivery system comprising the interpolymer and a drug effect enhancing formulation carried by the interpolymer; preferably, the pharmacodynamic-enhancing agent is a TGF- β inhibitor and/or ICG, more preferably, the TGF- β inhibitor is LY.
In some embodiments, the drug delivery system has a drug particle size of 100-200nm, preferably 150 nm;
the drug loading rate of the drug loading system is 6 wt% -20 wt%, preferably, the drug loading rate of ICG is 3 wt% -10 wt%, and the drug loading rate of LY is 3 wt% -10 wt%, more preferably, the drug loading rates of ICG and LY are both 5 wt%.
Some methods, comprising the steps of:
dissolving the copolymer in water, adding a trichloromethane emulsion solution blended by LY and ICG, and carrying out ultrasonic crushing to obtain an emulsion; preferably, the crushing process is ice-bath (ice-bath is not limited to be used, and other similar means are also equivalent and are all within the scope of the invention);
homogenizing the obtained emulsion for a plurality of times under 400-1000bar, and purifying to obtain the amphiphilic hydroxyethyl starch-polycaprolactone high molecular compound drug-loading system co-encapsulating LY and ICG.
The drug-carrying system is applied to preparing anti-tumor drugs.
In some embodiments, the use is for the manufacture of an adjunct medicament for CAR-T therapy.
In some embodiments, the adjunct agent is a CAR-T adjunct agent for treating a tumor;
preferably, the auxiliary agent is a tumor accumulation aid; and/or the presence of a gas in the gas,
the auxiliary agent is CAR-T proliferation promoter and/or activity promoter;
preferably, the tumor is a lymphoma.
The invention has the beneficial effects that:
the copolymer can be better used for drug loading, and is beneficial to CAR-T treatment of tumors, LY/ICG @ HES-PCL has good tumor accumulation effect on a raji (lymphoma cell) cell lymphoma model, and the photo-thermal effect of ICG is realized, so that the tumor extracellular matrix density can be obviously improved, the infiltration of CAR-T on lymphoma tissues is improved, and the proliferation and activity of CAR-T are improved.
Drawings
FIG. 1 is a graph of the HES-PCL structure characterized by H-NMR and FTIR;
FIG. 2 is an FTIR hydrogen spectrum;
FIG. 3 is a DLS and TEM image of the prepared ICG/LY @ HES-PCL;
FIG. 4 is a general structural formula of the interpolymer;
FIG. 5 is a graph showing the effect of ICG/LY @ HES-PCL on proliferation and activity of CD-19+ CAR-T;
FIG. 6 is a graph of the results of CAR-T tumor accumulation experiments.
Detailed Description
The invention is further illustrated below:
aiming at the defects of CAR-T for treating lymphoma, the invention provides a hydroxyethyl starch-polycaprolactone nano-carrier and a preparation method and application thereof. The nano drug-carrying system which carries ICG and LY together is prepared by using the amphiphilic high molecular compound through ultrasonic emulsification and high-pressure homogenization technology, so that the problem of lymphoma tissue co-delivery of ICG and LY is solved.
In one aspect, the interpolymer has the formula:
Figure BDA0003175200830000051
wherein the content of the first and second substances,
n is 100-5000, m is 50-500, i is 2-9, preferably, i is 4-7, n is 200, and m is 100;
R1-R4 are any one of halogeno, -OH and-OR ', and R' is halogeno;
r5 is any one of halogeno, -OH and-OR, -R' is halogeno OR- (CH)2) k is a positive integer.
Wherein the grafting rate of the polycaprolactone on the hydroxyethyl starch is 0.61-1;
preferably, the grafting ratio of the polycaprolactone on the hydroxyethyl starch is 0.75,
preferably, R1-R4 are all-OH.
The degree of PCL substitution in HES-PCL (MSPCL) was calculated by NMR spectroscopy (deuterated DMSO as solvent) as
Figure BDA0003175200830000061
Wherein IbIs the integral of the peaks of hydrogen on the two methylene groups in the caprolactone repeat unit identified as b in FIG. 1, NCLTo average the number of caprolactone recurring units (CL) per PCL chain, IAGUIs the integration of the peaks (4.4-6.0ppm) for the three hydroxyl hydrogens in the hydroxyethyl starch anhydroglucose unit and the hydrogen on carbon No. 1. By rearranging the formula, the grafting ratio of PCL in HES-PCL is calculated to be 0.75.
Where i is preferably 6, other carbon chain lengths, as long as they have similar properties to the present invention, are within the scope of the present invention. For compounds with different carbon chain lengths represented by i, different raw materials are correspondingly adopted in the preparation process, the reaction conditions are adjusted according to conditions, and repeated description is omitted, and the compounds with the same and similar drug loading capacity are equivalent.
Meanwhile, the invention also has other parts which need to be explained, as follows:
first, a method for preparing the interpolymer comprises the following steps:
the carboxyl group of the polycaprolactone is activated,
the carboxyl activated polycaprolactone and the hydroxyethyl starch solution are subjected to esterification reaction,
purifying to obtain the hydroxyethyl starch-polycaprolactone copolymer.
Specifically, the step of activating the carboxyl group of polycaprolactone comprises the following steps: dissolving polycaprolactone in anhydrous dimethyl sulfoxide, adding 1-hydroxybenzotriazole and N-N' -dicyclohexylcarbodiimide, and performing carboxyl activation reaction; and/or
The preparation method of the hydroxyethyl starch solution comprises the following steps: dissolving hydroxyethyl starch into anhydrous dimethyl sulfoxide, preferably, adopting inert gas for protection in the dissolving process; and/or
The esterification reaction temperature is 40-80 ℃, and preferably, inert gas is adopted for protection in the reaction process.
In the optimization scheme, the inert gas is helium; dissolving polycaprolactone in anhydrous dimethyl sulfoxide at 50-70 deg.C; the purification steps are as follows: dialyzing the copolymer mixture obtained by the esterification reaction through a PBS solution, freezing the liquid obtained by the dialysis, and further freeze-drying to obtain the hydroxyethyl starch-polycaprolactone copolymer.
One specific scheme of the purification step is as follows:
dialyzing the copolymer mixture obtained by the esterification reaction through a PBS solution, removing unreacted 1-hydroxybenzotriazole and/or N-N' -dicyclohexylcarbodiimide and/or polycaprolactone and/or DMSO solvent,
freezing the liquid obtained by dialysis at the temperature of-20 to-25 ℃ for 3 to 5 hours,
and (3) putting the mixture into a freezing and drying device at the temperature of-40 to-60 ℃ to obtain the hydroxyethyl starch-polycaprolactone copolymer freeze-dried powder.
The copolymer is applied to preparing a medicine for preventing and treating tumors.
The application is the application as a drug effect enhancing preparation carrier in CAR-T treatment; preferably, the pharmacodynamic-enhancing agent is a TGF- β inhibitor and/or ICG, more preferably, the TGF- β inhibitor is LY (LY 2157299).
A drug delivery system comprising a drug effect enhancing formulation of said interpolymer in interpolymer; preferably, the pharmacodynamic-enhancing agent is a TGF- β inhibitor and/or ICG, more preferably, the TGF- β inhibitor is LY.
Solves the problems that LY is difficult to dissolve in water and administer in vivo, and ICG can not be delivered to the tumor part in a targeted way.
Preferably, the particle size of the drug delivery system is 100-200nm, and more preferably 150 nm;
the drug loading rate of the drug loading system is 6 wt% -20 wt%, preferably, the drug loading rate of ICG is 3 wt% -10 wt%, and the drug loading rate of LY is 3 wt% -10 wt%, more preferably, the drug loading rates of ICG and LY are both 5 wt%.
The preparation method of the drug-carrying system comprises the following steps:
dissolving the copolymer in water, adding a trichloromethane emulsion solution blended by LY and ICG, and carrying out ultrasonic crushing to obtain an emulsion; preferably, the crushing process is ice-bath;
homogenizing the obtained emulsion for a plurality of times under 400-1000bar, and purifying to obtain the amphiphilic hydroxyethyl starch-polycaprolactone high molecular compound drug-loading system co-encapsulating LY and ICG.
The drug-carrying system is applied to preparing anti-tumor drugs.
The application is the application in the preparation of an auxiliary medicament for CAR-T treatment.
The auxiliary agent is an auxiliary agent for treating tumors by CAR-T;
preferably, the auxiliary agent is a tumor accumulation aid; and/or the presence of a gas in the gas,
the auxiliary agent is CAR-T proliferation promoter and/or activity promoter;
preferably, the tumor is a lymphoma.
In some cases, R5 appears primarily as-O (CH)2)2OH, the remaining R1-R4 being replaceable substituents, the interpolymer having the formula:
Figure BDA0003175200830000081
wherein n is 100 to 5000, m is 50 to 500,
r1 is any one of halo OR-OR ', R2 is any one of halo OR-OR', R3 is any one of halo OR-OR ', R4 is any one of halo OR-OR', R '(-R') is halo,
preferably, n is 200 and m is 100.
Wherein R5 is-O (CH)2)2On an OH basis, the interpolymers are sometimes two as shown below
Figure BDA0003175200830000091
The copolymer of the formula I and II can be prepared from hydroxyethyl starch and polycaprolactone, wherein before OR during the reaction, one of the polycaprolactone is partially substituted by hydroxyl group such as R1, R2, R3, R4(-R1, -R2, -R3, -R4), OR R5(-R5) is any one of-OH and-OR', and one of the hydroxyl groups is-OCH2CH2OH; the copolymer of the invention is not limited to the structures in the formulas I and II;
more particularly, a particular form of the interpolymer is an amphiphilic hydroxyethyl starch-polycaprolactone (or polymer such as a homologue thereof) interpolymer (when R1, R2, R3, R4 are all-OH) having the general formula:
Figure BDA0003175200830000092
wherein n is 100 to 5000, m is 50 to 500,
preferably, n is 200 and m is 100.
Preferably, the grafting ratio of the polycaprolactone on the hydroxyethyl starch is 0.61-1, more preferably 0.75, and the better use effect is achieved when the grafting ratio is 0.75.
On the basis of the description of another aspect of the invention, the invention provides a preparation method of an amphiphilic hydroxyethyl starch-polycaprolactone macromolecular compound, and the amphiphilic hydroxyethyl starch-polycaprolactone copolymer is obtained by carrying out esterification reaction on carboxylated polycaprolactone and hydroxyethyl starch.
Preferably, the method comprises the following steps, and the invention also provides a preparation method of the hydroxyethyl starch-polycaprolactone copolymer, which comprises the following steps:
1) dissolving polycaprolactone and activating its carboxyl group: dissolving polycaprolactone by using anhydrous dimethyl sulfoxide, then adding 1-hydroxybenzotriazole and N-N' -dicyclohexylcarbodiimide to perform carboxyl activation reaction, and stirring at room temperature for 1-4 hours to obtain a carboxyl-terminated activated polycaprolactone solution;
2) dissolving hydroxyethyl starch: under the condition of helium protection, completely dissolving hydroxyethyl starch in anhydrous dimethyl sulfoxide at 50-70 ℃ to obtain a dimethyl sulfoxide solution of the hydroxyethyl starch:
3) esterification reaction: mixing the carboxyl activated polycaprolactone solution obtained in the step 1) with the dimethyl sulfoxide solution of hydroxyethyl starch obtained in the step 2), performing esterification reaction for 24-72 hours under the protection of nitrogen at the temperature of 40-80 ℃, and purifying to obtain a copolymer mixture;
4) and (3) purification: co-dialyzing the copolymer mixture for 1-5 days by using a PBS (phosphate buffer solution), removing unreacted 1-hydroxybenzotriazole, N-N' -dicyclohexylcarbodiimide, polycaprolactone and DMSO (dimethyl sulfoxide) solvents, freezing the liquid in a dialysis bag for 3-5 hours at the temperature of-20 to-25 ℃ after dialysis is finished, then freeze-drying at the temperature of-40 to-60 ℃, and obtaining the hydroxyethyl starch grafted polycaprolactone copolymer freeze-dried powder after freeze-drying; namely the hydroxyethyl starch grafted polycaprolactone copolymer.
According to another aspect of the invention, the invention is described in more detail based on the hydroxyethyl starch-polycaprolactone copolymer, and relates to a nano drug delivery system of the hydroxyethyl starch-polycaprolactone copolymer, wherein the nano drug delivery system comprises hydroxyethyl starch-polycaprolactone high molecular compound and LY and ICG. The particle size of the nano-drug is 100-200nm, preferably 150nm, preferably, the drug loading rate of the ICG of the nano-drug delivery system is 3-10 wt%, the drug loading rate of LY is 3-10 wt%, and further preferably 5 wt%.
When n or m is other numerical values, the preparation can be realized by adopting a similar preparation method and then controlling the raw material proportion and the like by the technicians in the field; hydroxyethyl starch polycondensates and polycaprolactone polycondensates with different condensation degrees are adopted for reaction, and further description is omitted. For example, n is 150, 175, 225, 250, m is 75, 125, or 150, different raw materials are selected according to different group requirements, and the specific preparation steps are locally adjusted adaptively on the basis of the current embodiment of the invention; it is to be understood that the terms "m" and "n" are not intended to limit the scope of the present invention, and that the terms "m" and "n" are equivalent to the present invention.
The preparation method of the nanometer drug-loading system of the hydroxyethyl starch-polycaprolactone copolymer comprises the following steps:
(1) dissolving hydroxyethyl starch-polycaprolactone high molecular compound in water, carrying out ultrasonic crushing in an ice bath, and simultaneously adding a trichloromethane emulsion solution blended by LY and ICG to obtain an ultrasonic emulsion;
(2) and (3) placing the emulsion obtained in the step (1) into a high-pressure homogenizer, homogenizing for 1-6 times under 400-1000bar, and purifying to obtain the amphiphilic hydroxyethyl starch-polycaprolactone macromolecular compound nano drug-loading system co-encapsulating LY and ICG.
The application of the hydroxyethyl starch-polycaprolactone copolymer nano drug delivery system co-encapsulating LY/ICG is applied to the synergistic CAR-T treatment of lymphoma.
Example 1:
the amphiphilic hydroxyethyl starch-polycaprolactone macromolecular compound is synthesized by the following steps:
(1) dissolving polycaprolactone and activating its carboxyl group: dissolving 0.5g folic acid in 5ml anhydrous dimethyl sulfoxide, adding 1-hydroxy benzotriazole and N-N' -dicyclohexyl carbodiimide, performing carboxyl activation reaction at 60 ℃ for 30min, and stirring at room temperature for 2-4 h to obtain a terminal carboxyl activated folic acid solution; wherein the feeding molar ratio of polycaprolactone, 1-hydroxybenzotriazole and N-N' -dicyclohexylcarbodiimide is 1:1: 1;
2) dissolving polysaccharide: under the protection of helium, 0.5g of hydroxyethyl starch with molecular weight of 5kDa is fully dissolved in anhydrous dimethyl sulfoxide at 50 ℃ to obtain a dimethyl sulfoxide solution of the hydroxyethyl starch:
3) esterification reaction: mixing the carboxyl activated polycaprolactone solution obtained in the step 1) with the dimethyl sulfoxide solution of hydroxyethyl starch obtained in the step 2), carrying out esterification reaction for 48 hours under the protection of nitrogen and at the temperature of 48 ℃, and purifying to obtain a copolymer mixture;
4) and (3) purification: dialyzing the copolymer mixture by using a PBS solution, dialyzing for 35 days by using a dialysis bag with the molecular weight of 3500Da, removing unreacted 1-hydroxybenzotriazole, N-N' -dicyclohexylcarbodiimide, hydroxyethyl starch and a DMSO solvent, transferring the liquid in the dialysis bag into a culture dish after dialysis is finished, freezing for 4 hours at the temperature of-20 ℃, then freeze-drying at the temperature of-50 ℃, and obtaining the HES-PCL copolymer freeze-dried powder after freeze-drying. The structure of the HES-PCL was characterized by H-NMR and FTIR, and the results are shown in FIG. 1.
The NMR spectrum of HES-PCL showed new peaks at chemical shifts 1.30, 1.55, 2.28 and 3.99ppm, assigned to the methylene hydrogen on PCL, indicating successful synthesis of HES-PCL (FIG. 1). FTIR results indicated 1730cm-1The PCL ester bond peak (FIG. 2) appears, further illustrating the structure of HES-PCL.
Preparing a nano drug delivery system of amphiphilic hydroxyethyl starch coupled polycaprolactone: dissolving purified HES-PCL into water, adding ICG and LY under the condition of ice bath by using a ultrasonicator and carrying out ultrasonic treatment at the same time, and adding the mixture of 1:1, carrying out ultrasonic treatment for 5min in total, adding the emulsion into a high-pressure homogenizer after the ultrasonic treatment, homogenizing for 2 times at 500bar to finally obtain a suspension of ICG/LY @ HES-PCL, ICG and LY, centrifuging for 30min at 5000rpm to remove the unencapsulated LY, placing the suspension of ICG and ICG/LY @ HES-PCL into a dialysis bag of 3500Da, dialyzing for 2 days by using deionized water, freeze-drying the nanoparticle aqueous solution obtained by dialysis to obtain the ICG/LY @ HES-CH freeze-dried powder, and carrying out the whole step under the condition of keeping out of the sun.
Detecting the drug loading amount of LY and ICG in ICG/LY @ HES-PCL by an ultraviolet spectrophotometry, weighing the ICG/LY @ HES-PCL by a weight W1, measuring the LY mass W2 and the ICG mass W3 by the ultraviolet spectrophotometry, calculating the drug loading amount of LY by adopting a formula W2/W1 by 100% to obtain 5.5 wt%, and calculating the drug loading amount of ICG by adopting a formula W3/W1 by 100% to obtain 4.3 wt%.
Preparing 1mg/ml ICG/LY @ HES-PCL, dissolving in ultrapure water, and performing ultrasonic treatment for 10min for later use.
The copper mesh was placed in a petri dish covered with filter paper, 10. mu.l of nanoparticle dispersion at 1mg/ml was dropped on the copper mesh, naturally dried at room temperature, and the morphology was observed by a transmission electron microscope (H-7000FA, HITACHI). The particle size distribution of ICG/LY @ HES-PCL was measured using a laser particle sizer (Nano-ZS90, Malvern). FIG. 3 shows the DLS and TEM images of ICG/LY @ HES-PCL prepared by the present invention, and the nanoparticles have a particle size of 130nm, are uniform and are spherical. The DLS results were consistent with the TEM results.
Example 2:
the invention utilizes transwell experiment to research the CD-19 pair ICG/LY @ HES-PCL+The effects of CAR-T proliferation and activity were as follows: raji cells and LY/ICG @ HES-CH nanoparticles prepared in example 1 were mixed in a 24-well plate in advance, irradiated with near infrared light for 5min, and then transferred to the upper chamber (pore size ≈ 1 μm) of a transwell chamber, in which CAR-T cells were seeded, the transwell chamber was incubated at 37 ℃ under 5% CO2 for 3 days, and then CD-19+ CAR-T proliferation was analyzed by flow-assay, and CD-19 was detected by enzyme-linked immunosorbent assay+CAR-T cultured 3 days endocrine IL-2 and TNF-gamma to CD-19+CAR-T activity was assayed. The ICG, ICG @ HES-PCL nano-drug and near infrared light irradiation group is set as a control group in the experiment.
After 3 days, the number of CAR T in LY/ICG @ HES-PCL group was 2.7 times that in the control group. Both photothermal and release of LY promoted proliferation of CAR T, LY being a more potent stimulus (4); the experimental group obviously improves the CAR T activity and promotes the IL-2 and IFN-lambda release of CAR T, and after 3 days, the IL-2 and IFN-lambda release amount of the CAR T in the LY/ICG @ HES-PCL group is 2.5 and 2.1 times that of the CAR T in the control group (figure 5). LY is a major factor in CAR T activation.
Example 3:
the invention evaluates the relation of LY/ICG @ HES-PCL to CD-19 through an in-animal experiment+Effect of the accumulating Effect of CAR-T lymphoma, in particularThe operation is as follows: constructing an NSG mouse lymphoma subcutaneous tumor model, injecting a LY/ICG @ HES-PCL nanoparticle tail vein into Raji tumor-bearing NSG mouse, and irradiating the tumor with near infrared light according to the time point of the highest accumulation amount of the LY/ICG @ HES-PCL nanoparticles obtained by imaging the small animal. After irradiation, the Raji tumor-bearing NSG mouse model was injected with CD-19 for 24h+CAR-T treatment of NSG mice. 48 hours later, the mice were sacrificed, the separated tumor tissues were harvested, minced, the tumors were dispersed into cell monomers using collagenase, and CD-19 was detected by flow cytometry in the different tumor tissues after the above treatments+Number of CAR-T. The ICG, ICG @ HES-PCL nano-drug and near infrared light irradiation group is set as a control group in the experiment.
The results show that: (1) LY/ICG @ HES-PCL is 4.45 times of the number of the control group CAR-T, so that the number of CART at tumor parts is obviously increased; (2) the photothermal group increased CAR-T numbers by 2.96-fold, higher than the non-photothermal group (2.22-fold), suggesting that photothermal may improve tumor stroma, further increasing CAR-T tumor accumulation (fig. 6).
It will be apparent to those skilled in the art that various modifications may be made to the above embodiments without departing from the general spirit and concept of the invention. All falling within the scope of protection of the present invention. The protection scheme of the invention is subject to the appended claims.

Claims (10)

1. An interpolymer characterized by the formula:
Figure FDA0003175200820000011
wherein the content of the first and second substances,
n is 100 to 5000, m is 50 to 500, i is 2 to 9, preferably n is 150 to 250, m is 75 to 150, i is 4 to 7, more preferably n is 200, m is 100, i is 6,
R1-R4 are all any one of halogeno, -OH and-OR ', R' is halogeno,
r5 is any one of halogeno, -OH and-OR ', R' halogeno OR- (CH)2)kOH and k are positive integers.
2. The interpolymer of claim 1, wherein each of R1-R4 is-OH and R5 is-O (CH)2)2OH。
3. The interpolymer of claim 1, wherein the grafting ratio of the polycaprolactone on the hydroxyethyl starch is 0.61-1;
preferably, the grafting ratio of the polycaprolactone on the hydroxyethyl starch is 0.75.
4. Use of the interpolymer of any one of claims 1-3 in the preparation of a medicament for the control of tumors.
5. Use of the interpolymer of any of claims 1-3 in the preparation of a CAR-T therapeutic efficacy enhancing formulation; preferably, the interpolymer is a pharmaceutical carrier and the pharmacodynamic-enhancing formulation comprises a TGF- β inhibitor and/or ICG, more preferably, the TGF- β inhibitor is LY.
6. Use of the interpolymer of any one of claims 1-3 in the preparation of a formulation vehicle for improving the immunosuppressive microenvironment and/or the angiopathic microenvironment of a lymphoma tissue.
7. A drug delivery system comprising the interpolymer of any one of claims 1-3.
8. The drug delivery system of claim 7, further comprising a drug effect enhancing formulation incorporated in the interpolymer; preferably, the drug effect enhancing preparation is a preparation for improving the immunosuppressive microenvironment and/or the vascular abnormal microenvironment of the lymphoma tissue; preferably, the pharmacodynamic-enhancing agent is a TGF- β inhibitor and/or ICG, more preferably, the TGF- β inhibitor is LY;
the particle size of the drug delivery system is 100-200nm, preferably 150 nm;
the drug loading rate of the drug loading system is 6 wt% -20 wt%, preferably, the drug loading rate of ICG is 3 wt% -10 wt%, and the drug loading rate of LY is 3 wt% -10 wt%, more preferably, the drug loading rates of ICG and LY are both 5 wt%.
9. The method of preparing the drug delivery system of claim 7 or 8, comprising the steps of:
dissolving the interpolymer of any one of claims 1-3 in water, adding a chloroform emulsion solution blended with LY and ICG, and crushing to obtain an emulsion; preferably, the crushing process is ice-bath;
homogenizing the obtained emulsion for a plurality of times under 400-1000bar, and purifying to obtain the amphiphilic hydroxyethyl starch-polycaprolactone high molecular compound drug-loading system co-encapsulating LY and ICG.
10. The use of the drug delivery system of claim 7 in the preparation of a carrier for a drug for the prevention and treatment of tumors or the use of the drug delivery system of claim 8 in the preparation of a drug for the prevention and treatment of tumors.
Use of the drug delivery system of claim 7 or 8 for the manufacture of an adjuvant for CAR-T therapy.
The use according to claim 10, wherein the adjuvant is a CAR-T adjuvant for the treatment of tumors;
preferably, the auxiliary agent is a tumor accumulation aid; and/or the presence of a gas in the gas,
the auxiliary agent is CAR-T proliferation promoter and/or activity promoter;
preferably, the tumor is a lymphoma.
The process for preparing the interpolymer of claim 2, comprising the steps of:
the carboxyl group of the polycaprolactone is activated,
the carboxyl activated polycaprolactone and the hydroxyethyl starch solution are subjected to esterification reaction,
purifying to obtain the hydroxyethyl starch-polycaprolactone copolymer.
The method of producing the interpolymer according to claim 13, wherein,
the step of activating the carboxyl of polycaprolactone comprises the following steps: dissolving polycaprolactone in anhydrous dimethyl sulfoxide, adding 1-hydroxybenzotriazole and N-N' -dicyclohexylcarbodiimide, and performing carboxyl activation reaction; and/or
The preparation method of the hydroxyethyl starch solution comprises the following steps: dissolving hydroxyethyl starch into anhydrous dimethyl sulfoxide, preferably, adopting inert gas for protection in the dissolving process; and/or
The esterification reaction temperature is 40-80 ℃, and preferably, inert gas is adopted for protection in the reaction process.
The process for preparing the interpolymer of claim 14, wherein the inert gas is helium; the temperature for dissolving polycaprolactone by using anhydrous dimethyl sulfoxide is 50-70 ℃.
The method of making the interpolymer of claim 13, wherein the purification step comprises:
dialyzing the copolymer mixture obtained by the esterification reaction through a PBS solution, freezing the liquid obtained by the dialysis, and further freeze-drying to obtain the hydroxyethyl starch-polycaprolactone copolymer.
The method of preparing the interpolymer of claim 16, wherein the purification step comprises:
dialyzing the copolymer mixture obtained by the esterification reaction through a PBS solution, removing unreacted 1-hydroxybenzotriazole and/or N-N' -dicyclohexylcarbodiimide and/or polycaprolactone and/or DMSO solvent,
freezing the liquid obtained by dialysis at the temperature of-20 to-25 ℃ for 3 to 5 hours,
and (3) putting the mixture into a freezing and drying device at the temperature of-40 to-60 ℃ to obtain the hydroxyethyl starch-polycaprolactone copolymer freeze-dried powder.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113321812A (en) * 2021-05-31 2021-08-31 华中科技大学 Polylactic acid-hydroxyethyl starch-folic acid macromolecular compound, drug delivery system, preparation method and application thereof
CN114939171A (en) * 2022-04-29 2022-08-26 华中科技大学同济医学院附属协和医院 Nano drug-loading system and preparation method and application thereof
CN115025064A (en) * 2022-05-17 2022-09-09 华中科技大学同济医学院附属协和医院 Application of nano drug delivery system in inhibiting lymphoma recurrence

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996020220A1 (en) * 1994-12-23 1996-07-04 Basf Aktiengesellschaft Biodegradable thermoplastic starches
CN101230139A (en) * 2008-02-03 2008-07-30 中山大学 Polycaprolactone/amylose amphiphilic block polymer as well as preparation method and use thereof
CN102397236A (en) * 2010-09-16 2012-04-04 同济大学 Method for preparing shell-sheddable polymer micelle drug carrier
CN106432746A (en) * 2016-09-30 2017-02-22 华中科技大学 Amphipathy hydroxyethyl-starch-coupled-polylactic-acid copolymer and preparing method and application thereof
CN106674615A (en) * 2016-11-22 2017-05-17 汪逸凡 Preparation method of enhanced type polycaprolactone biodegradable plastics
CN107308500A (en) * 2017-05-12 2017-11-03 广州迈普再生医学科技有限公司 A kind of tissue repair film and preparation method thereof and the load medicine tissue repair film being made
MX2017005889A (en) * 2017-05-04 2018-11-09 Univ Guanajuato Synthesis of a biodegradable graft copolymer capable of forming low adsorption film.

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996020220A1 (en) * 1994-12-23 1996-07-04 Basf Aktiengesellschaft Biodegradable thermoplastic starches
CN101230139A (en) * 2008-02-03 2008-07-30 中山大学 Polycaprolactone/amylose amphiphilic block polymer as well as preparation method and use thereof
CN102397236A (en) * 2010-09-16 2012-04-04 同济大学 Method for preparing shell-sheddable polymer micelle drug carrier
CN106432746A (en) * 2016-09-30 2017-02-22 华中科技大学 Amphipathy hydroxyethyl-starch-coupled-polylactic-acid copolymer and preparing method and application thereof
CN106674615A (en) * 2016-11-22 2017-05-17 汪逸凡 Preparation method of enhanced type polycaprolactone biodegradable plastics
MX2017005889A (en) * 2017-05-04 2018-11-09 Univ Guanajuato Synthesis of a biodegradable graft copolymer capable of forming low adsorption film.
CN107308500A (en) * 2017-05-12 2017-11-03 广州迈普再生医学科技有限公司 A kind of tissue repair film and preparation method thereof and the load medicine tissue repair film being made

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CAI J , FANG L , HUANG Y , ET AL.: "Simultaneous overactivation of Wnt/β-catenin and TGFβ signalling by miR-128-3p confers chemoresistance-associated metastasis in NSCLC", 《NATURE COMMUNICATIONS》 *
HU H , XIAO C , WU H , ET AL: "Nanocolloidosomes with Selective Drug Release for Active Tumor-Targeted Imaging-Guided Photothermal/Chemo Combination Therapy", 《ACS APPLIED MATERIALS & INTERFACES》 *
RONGHUA TAN: "Hydroxyethyl starch and its derivatives as nanocarriers for delivery of diagnostic and therapeutic agents towards cancers", 《BIOMATERIRALS TRANSLATIONAL》 *
徐玲等: "脂肪酶催化合成淀粉/聚己内酯接枝共聚物", 《高分子材料科学与工程》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113321812A (en) * 2021-05-31 2021-08-31 华中科技大学 Polylactic acid-hydroxyethyl starch-folic acid macromolecular compound, drug delivery system, preparation method and application thereof
CN113321812B (en) * 2021-05-31 2022-03-11 华中科技大学 Polylactic acid-hydroxyethyl starch-folic acid macromolecular compound, drug delivery system, preparation method and application thereof
CN114939171A (en) * 2022-04-29 2022-08-26 华中科技大学同济医学院附属协和医院 Nano drug-loading system and preparation method and application thereof
CN114939171B (en) * 2022-04-29 2023-05-30 华中科技大学同济医学院附属协和医院 Nanometer medicine carrying system and preparation method and application thereof
CN115025064A (en) * 2022-05-17 2022-09-09 华中科技大学同济医学院附属协和医院 Application of nano drug delivery system in inhibiting lymphoma recurrence

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