CN109224082B - Macromolecule prodrug nano-drug, preparation method and application thereof - Google Patents

Macromolecule prodrug nano-drug, preparation method and application thereof Download PDF

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CN109224082B
CN109224082B CN201811079938.1A CN201811079938A CN109224082B CN 109224082 B CN109224082 B CN 109224082B CN 201811079938 A CN201811079938 A CN 201811079938A CN 109224082 B CN109224082 B CN 109224082B
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polyethylene glycol
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李新松
凌龙兵
何伟
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Southeast University
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Abstract

The invention discloses a macromolecular prodrug nano-drug, a preparation method and application thereof. The macromolecule prodrug nano-drug is a reduced lipoic acid-polyethylene glycol amphiphilic molecule bonded with two hydrophobic drug molecules, the shell of the formed intermolecular cross-linked disulfide bond is a polyethylene glycol hydrophilic macromolecule layer, the inner core of the formed intermolecular cross-linked disulfide bond is nano-particles of the hydrophobic drug, and the size of the nano-drug is 10-1000 nanometers. The pharmaceutical composition nanoparticles can be used as liquid preparation, solid preparation and semisolid preparation, have low toxicity, and can be used for treating tumor.

Description

Macromolecule prodrug nano-drug, preparation method and application thereof
Technical Field
The invention relates to a macromolecular prodrug nano-drug with tumor treatment effect, a preparation method and application thereof, relating to the technical field of medicines.
Background
Many small molecule chemical drugs are too hydrophobic or too hydrophilic and poorly lipophilic to penetrate cell membranes, resulting in poor efficacy in treating diseases.
Some plant-derived drugs with strong physiological activity have strong hydrophobicity, difficult administration and limited application. Camptothecin (CPT) and 10-Hydroxycamptothecin (HCPT) have strong cytotoxic activity. The greatest drawback of camptothecin compounds for the treatment of tumors is their poor water solubility, making it difficult to obtain solutions of high effective concentration. In order to improve the hydrophobicity defects of CPT and HCPT, research institutions and enterprises modify the chemical structure of camptothecin to synthesize hundreds of camptothecin derivatives. To date, 2 camptothecin derivatives with certain hydrophilicity have been approved for clinical tumor therapy, irinotecan and topotecan, respectively, but their water solubility is still low.
Paclitaxel is also an alkaloid and is currently the only drug that promotes microtubule polymerization and stabilizes polymerized microtubules. Studies have shown that paclitaxel binds to polymerized microtubules and does not react with unpolymerized tubulin dimers. Paclitaxel is mainly suitable for ovarian cancer and breast cancer, and has therapeutic effect on lung cancer, carcinoma of large intestine, melanoma, head and neck cancer, lymphoma, and cerebroma. However, paclitaxel has poor water solubility, and needs to be solubilized by polyoxyethylene castor oil and ethanol, and the polyoxyethylene castor oil may cause allergic reaction, and patients need to take antiallergic drugs before taking the drugs. Research shows that the application of the polyoxyethylene castor oil reduces the antitumor activity of the paclitaxel. Meanwhile, paclitaxel has the defects of high toxicity, easy generation of drug resistance, incapability of penetrating through a blood brain barrier and the like due to poor selectivity.
Controlled release by encapsulation in a carrier is an important mode of administration. The nano-drug is prepared by embedding drug molecules in a polymer nano-carrier, and has the following advantages: 1) the solubility of the hydrophobic drug is obviously improved, the circulation time in the drug body is prolonged, and the bioavailability is improved; 2) the drug is specifically targeted to tumor tissues through passive targeting (EPR effect) or active targeting, so that the distribution of the drug in normal tissues is reduced, and the toxic and side effects of the drug are reduced. Doxil, an anticancer drug for the treatment of various cancers, is a liposome drug entrapping adriamycin, with a polyethylene glycol (PEG) hydration layer on the surface. Clinical application shows that Doxil can prolong the circulation time of medicine in body, enrich medicine in tumor and reduce cardiac toxicity of medicine. In addition, a number of PEG-based polymeric nanomedicines have entered clinical or different clinical trials. For example, a polyethylene glycol-polylactic acid copolymer (PEG-PLA) Paclitaxel (PTX) -loaded nanomicelle drug (Genexol-PM) has been used in korea for clinical treatment of breast cancer, lung cancer, ovarian cancer, and the like since 2007.
However, existing nano-drugs also face numerous challenges: 1) the nano medicine has low entrapment efficiency (less than 10 percent), and after being injected into a body, the nano medicine is easily decomposed or accumulated due to the action of being diluted and different components in blood, so that the entrapped medicine is leaked or prematurely released; 2) the PEG nano-drug has too slow release speed or insufficient release after entering the tumor tissue, and the drug utilization rate is low.
Therefore, it is necessary to develop a new nano-drug release system, improve the solubility, stability and targeting property of the drug, and endow the drug with the function of reducing sensitivity and releasing drug quickly, thereby achieving the purposes of reducing toxic and side effects and improving bioavailability.
Disclosure of Invention
The technical problem is as follows: the invention provides a macromolecule prodrug nano-drug and a preparation method and application thereof.A hydrophobic drug molecule is connected with a low molecular weight polyethylene glycol hydrophilic macromolecule through a disulfide bond to form the macromolecule prodrug, the prodrug is assembled into the nano-drug, and is crosslinked through the disulfide bond to form an inner core, a shell is a low molecular weight polyethylene glycol hydrophilic macromolecule brush, and the nano-drug has the drug release function of reducing sensitive targeted tumor cells, has better stability and does not leak the drug.
The technical scheme is as follows: the macromolecular prodrug nano-drug comprises an amphiphilic macromolecular prodrug of a general formula (1) formed by connecting two hydrophobic drug molecules through lipoic acid and low molecular weight polyethylene glycol by disulfide bonds, wherein the amphiphilic macromolecular prodrug is self-assembled and cross-linked through intermolecular disulfide bonds to form nanoparticles with drug-containing cores and low molecular weight polyethylene glycol hydrophilic macromolecular brushes as shells, and the size of the nanoparticles is 10-1000 nm:
Figure GDA0003321021240000021
wherein X is serinol or 3-amino glycerol, D is a hydrophobic drug, and Y is CH2CH2OCOO or CH2CH2OCONH, n is 6-80.
In a preferred embodiment of the nano-drug of the present invention, the hydrophobic drug is paclitaxel, docetaxel, cabazitaxel, doxorubicin, epirubicin, daunorubicin, demethoxydaunorubicin, aclarubicin, pirarubicin, zorubicin, camptothecin, 7-ethyl-10-hydroxycamptothecin, topotecan, irinotecan, rubitecan, belotecan, cephalotaxine, isotrichodermin, rapamycin, maytansine, everolimus or dasatinib.
In a preferred embodiment of the nano-drug of the present invention, the nano-drug further comprises a pharmaceutically acceptable carrier.
In the preferable scheme of the nano-medicament, the nano-medicament also comprises an auxiliary agent. In a further preferred embodiment, the auxiliary agent is a fat or a phospholipid.
In the preferable scheme of the nano-medicament, the nano-medicament is a liquid preparation, a solid preparation, a semi-solid preparation, a capsule, a granule, a gel, an injection, a sustained release preparation or a controlled release preparation.
From the screening of in vitro pharmacological activity and the like, the macromolecular prodrug nano-drug has good biological activity such as anti-tumor and the like. Experiments show that the toxicity in vivo of the macromolecular prodrug nano-drug is less than that of the original drug. Therefore, the compound can be used as an anti-tumor drug for patients.
The preparation method of the macromolecular prodrug nano-drug comprises the steps of assembling the macromolecular prodrug of the general formula (1) into a nano-micelle with the diameter of 10-1000 nanometers in an aqueous solution, adding hydrogen peroxide or oxidizing sulfydryl in the air to cause intermolecular crosslinking to form crosslinked nano-particles with a hydrophobic drug in an inner core and a hydrophilic polyethylene glycol brush in an outer layer, and freeze-drying to obtain the crosslinked nano-particle freeze-dried powder wrapping the drug.
The invention also provides application of the macromolecular prodrug nano-drug in preparation of antitumor drugs.
The preparation method of the macromolecular prodrug nano-drug is to prepare the macromolecular prodrug or the mixture of the macromolecular prodrug and an auxiliary agent by a film dispersion method, a reverse phase evaporation method, a freeze drying method, an ultrasonic dispersion method, a spray drying method, a film extrusion method, a high-pressure homogenization method and the like.
The invention self-assembles nano particles by utilizing the amphipathy before the macromolecule, forms an inner core by utilizing the sulfydryl crosslinking in the macromolecule, forms a shell by utilizing the low molecular weight polyethylene glycol brush, prepares the nano medicament with the inner core crosslinking and the hydrophilic shell, and improves the medicament loading rate and the stability of the nano medicament; the nano-drug of the invention has long half-life period and the anti-tumor activity is obviously superior to that of the original drug.
The macromolecular prodrug nano-drug is not only a prodrug, but also a drug carrier, has the functions of long circulation, tumor targeting and sensitive release, and has low toxic and side effects.
Has the advantages that: compared with the prior art, the invention has the following advantages:
the molecular of the prodrug of the macromolecular prodrug nano-drug only has two drug molecular groups, the hydrophilic part is polyethylene glycol with low molecular weight, and the prodrug has low molecular weight and clear structure.
The macromolecular prodrug nano-drug is self-assembled into nano-particles by utilizing the amphipathy of the macromolecular prodrug, the inner core is formed by utilizing the cross-linking of sulfydryl in the macromolecular prodrug molecules, the outer shell is formed by the low molecular weight polyethylene glycol brush, the nano-drug with the cross-linked inner core and the hydrophilic outer shell is prepared, and the drug loading rate is high.
The shell of the macromolecular prodrug nano-drug is a low molecular weight polyethylene glycol brush, so that the stability of nano-particles is good, and the half life period of in vivo circulation is long.
According to the macromolecular prodrug nano-drug, hydrophobic drug molecules are bonded to a macromolecular chain through disulfide bonds, the drug molecules cannot leak, the drug molecules have glutathione reduction sensitivity, the drug is quickly released mainly in tumor tissues or tumor cells, the drug molecules in normal tissues are difficult to release, and the tumor targeting property is realized.
The macromolecular prodrug of the macromolecular prodrug nano-drug is crosslinked by disulfide bonds to form an inner core, is not easy to disintegrate in vivo, has reduction sensitivity, and can disintegrate quickly in tumor tissues or tumor cells to realize quick drug release.
The macromolecular prodrug of the macromolecular prodrug nano-drug adopts low-molecular-weight polyethylene glycol, and the low-molecular-weight polyethylene glycol released after in vivo degradation has short in vivo retention time, is easy to be quickly discharged out of a body, is not retained in the body, and does not generate polyethylene glycol antibodies in the body.
The macromolecule prodrug nano-particle overcomes the defect that the medicine in the physical packaging type nano-medicine is easy to leak.
The part of the macromolecular prodrug nano-drug containing the drug comprises disulfide bonds, can be degraded by glutathione and the like to be rapidly broken, and mercapto groups and the like in fragments containing drug molecules after being broken attack carbonyl groups of the molecules through 'back biting', so that the original drug is rapidly released, the defect that the ordinary spacer arm is difficult to rapidly break and cannot rapidly release the original drug is overcome, and the drug effect is stronger.
The macromolecular prodrug nano-drug has a passive targeting effect and realizes targeted release.
The macromolecular prodrug nano-drug can be used as a liquid preparation, a solid preparation and a semi-solid preparation.
The macromolecular prodrug nano-drug is combined with a targeting group, and has an active targeting effect.
The macromolecule prodrug of the invention is assembled and then is crosslinked into nano particles through oxygen oxidation, and the preparation process is simple.
Drawings
FIG. 1. lipoic acid-serinol-polyethylene glycol synthetic route
FIG. 2. synthetic route of Bicamptothecine-lipoic acid-serinol-polyethylene glycol
FIG. 3. Synthesis route of Taxol-lipoic acid-serinol-polyethylene glycol
FIG. 4. Synthesis route of bisacodyl-lipoic acid-serinol-polyethylene glycol
FIG. 5. Synthesis route of bisacodyl-N-lipoic acid-serinol-polyethylene glycol
FIG. 6. synthetic route of cabazitaxel-lipoic acid-serinol-polyethylene glycol
FIG. 7. Synthesis route of ditertiline-lipoic acid-serinol-polyethylene glycol
FIG. 8 is a synthetic route of bimetacin-lipoic acid-serinol-polyethylene glycol
FIG. 9. Synthesis route of everolimus-lipoic acid-serinol-polyethylene glycol
FIG. 10. Synthesis route of didaxatinib-lipoic acid-serinol-polyethylene glycol
FIG. 11. the synthetic route of the didaxatinib-lipoic acid-amino glycerol-polyethylene glycol
FIG. 12 is a schematic representation of thiol oxidative crosslinking in macromolecular prodrugs
FIG. 13 dynamic light scattering measurement of nano-particle size of Bicamptothecin-lipoic acid-serinol-polyethyleneglycol macromolecule prodrug (A) and transmission electron microscope picture (B) thereof
FIG. 14 in vitro degradation of Bicamptothecine-lipoic acid-serinol-polyethylene glycol macromolecular prodrug nanoparticles
FIG. 15 shows the in vitro anti-tumor activity of the nano-particles of the macromolecule prodrug of Bicamptothecine-lipoic acid-serinol-polyethylene glycol, (A) MCF-7 of breast cancer cells, and (B) HepG-2 of liver cancer cells
FIG. 16 blood concentration of Bicamptothecine-lipoic acid-serinol-polyethyleneglycol macromolecule prodrug nanoparticles (A) and distribution of drug in tumor and tissue (B)
Figure 17. the antitumor effect (a) and the change in body weight (B) of the animals of the dicamptothecin-lipoic acid-serinol-polyethylene glycol macromolecular prodrug nanoparticles in animals.
Detailed Description
The technical scheme of the invention is further explained in detail by combining the drawings and the embodiments in the specification.
The invention relates to a macromolecular prodrug nano-drug which is characterized in that two hydrophobic drug molecules are connected with a low molecular weight polyethylene glycol hydrophilic macromolecule through lipoic acid by disulfide bonds to form the macromolecular prodrug of a general formula (1), the prodrug is self-assembled and crosslinked through the disulfide bonds to form nanoparticles, the inner core of the nanoparticles is a crosslinked structure containing drugs, the outer shell of the nanoparticles is a low molecular weight polyethylene glycol hydrophilic macromolecule brush, and the size of the nanoparticles is 10-1000 nanometers.
Figure GDA0003321021240000041
Wherein X is serinol or 3-amino glycerol, D is a hydrophobic drug, and Y is CH2CH2OCOO or CH2CH2OCONH, n is 6-80.
The hydrophobic medicine B is taxane medicine comprising paclitaxel, docetaxel and cabazitaxel, the anthraquinone medicine comprising adriamycin, epirubicin, daunorubicin, demethoxydaunorubicin, aclarubicin, pirarubicin and zorubicin, the camptothecin medicine comprising camptothecin, 7-ethyl-10-hydroxycamptothecin, topotecan, irinotecan, rubitecan and belotecan, and the cephalotaxoid medicine comprising cephalotaxine, isotetracycline, maytansine, everolimus and dasatinib.
The macromolecular prodrug nano-drug comprises a carrier or an auxiliary agent which is acceptable in pharmacodynamics, wherein the auxiliary agent is one of fat and phospholipid.
The preparation method of the macromolecular prodrug nano-drug comprises the steps of assembling the macromolecular prodrug of the general formula (1) or (2) into a nano-micelle with the diameter of 10-1000 nanometers in an aqueous solution, adding hydrogen peroxide or oxidizing sulfydryl in the air to cause intermolecular crosslinking to form crosslinked nano-particles with a hydrophobic drug in an inner core and a hydrophilic polyethylene glycol brush in an outer layer, and freeze-drying to obtain the crosslinked nano-particle freeze-dried powder wrapping the drug.
The invention relates to application of a macromolecular prodrug nano-drug in preparation of an anti-tumor drug.
In a preferred embodiment of the pharmaceutical composition of the present invention, the pharmaceutical composition is a liquid preparation, a solid preparation, a semisolid preparation, a capsule, a granule, a gel, an injection, a sustained release preparation or a controlled release preparation.
From the screening of in vitro pharmacological activity and the like, the nano-drug of the invention has good biological activity such as anti-tumor and the like. The test shows that the in vivo toxicity of the compound of the invention is less than that of the original drug. Therefore, the compound can be used as an anti-tumor drug for patients.
The nano-drug prepared by the invention has strong transmembrane capability, has the characteristic of forming liquid preparation, solid preparation and semisolid preparation, is used for treating tumors and the like, and has a targeting function; the nano-drug of the invention can be degraded in vivo to release the drug rapidly, exerts the drug effect and has low toxic and side effects.
The application of the nano-drug in the preparation of anti-tumor drugs and other drugs, the macromolecule prodrug nano-drug and a carrier acceptable in pharmacodynamics are prepared into a medicament. The nano-drugs of the present invention may be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants to make a suitable administration form or dosage form for human use. Generally, the pharmaceutical composition of the present invention contains 0.1 to 100% by weight of the nano-drug of the present invention.
The administration route of the nano-drug can be intestinal tract or parenteral tract, such as oral administration, muscle, subcutaneous administration, nasal cavity, oral mucosa, skin, peritoneum or rectum, and the like. Can be administered by injection, including intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, and acupoint injection. The administration dosage form can be liquid dosage form or solid dosage form. For example, the liquid dosage form can be true solution, colloid, microparticle, emulsion, or suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, etc.
The compound can be prepared into common preparations, sustained release preparations, controlled release preparations, targeting preparations and various microparticle drug delivery systems.
The nano-drug of the invention has disulfide bond spacer arms or cross-linking bonds. The spacer arm or the cross-linking bond is acted by glutathione and the like in tumor tissues or cells to be rapidly broken, so that the proto-type drug is rapidly released to play the drug effect.
The nano-drug of the invention improves the drug wrapping efficiency and the drug stability, and simultaneously, the nano-drug is easy to be taken into cells to exert the drug effect.
The synthetic reagents in the examples are abbreviated as follows:
tetrabutylammonium fluoride trihydrate: TBAF
2- (pyridyl-disulfanyl) ethanol:
1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride: EDC & HCl
Lipoic acid: LA
Diisopropylethylamine: DIPEA
1-hydroxybenzotriazole: HOBt (China general microbiological culture Bt)
4- (dimethylamino) pyridine: DMAP
Triphosgene: BTC
T-butoxycarbonyl group: BOC
Comparative example 1:
synthesis of reduced lipoic acid-serinol-polyethylene glycol (LA-SPEG) (see FIG. 1 for synthetic route)
Dithiooctanoic acid-serine ester (diLA-Ser) synthesis: 2.97g Lipoic Acid (LA), 3.31g 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl) were added to a 100mL round bottom flask, and 40mL CH was added2Cl2Dissolving and activating for 30 min. Will dissolve in 20mL CH2Cl21.25g of Boc-serinol and 2.21g of 4- (dimethylamino) pyridine (DMAP) were added to the above mixture and reacted for 12 hours. After the reaction is finished, filtering, fully washing the filtrate by using excessive 0.1mol/L hydrochloric acid, standing and discarding the water phase. Drying the oil phase with sodium sulfate, and purifying by column chromatography (eluent: CHCl)3/CH3OH 20/1, v/v) to give di-Boc-LA-Ser as a pale yellow oil (3.19g, 86% yield). 1g di-Boc-LA-Ser in 10mL CH2Cl2To the reaction solution, the reaction solution was cooled to 0 ℃ and 0.41g of trifluoroacetic acid (TFA) was slowly added dropwise thereto, followed by reaction for 2 hours. The mixture was concentrated in vacuo to give the crude diLA-Ser.
Synthesis of reduced lipoic acid-serinol-polyethylene glycol (LA-SPEG): the crude dilA-Ser product was dissolved in 20mL of anhydrous CH2Cl23g of methoxypolyethylene glycol succinic acid monoester (polyethylene glycol molecular weight 4000, 2000, 1000, 300, respectively) and 0.42g E were addedDC.HCl, 0.19g of 1-hydroxybenzotriazole (HOBt) and 0.85g of Diisopropylethylamine (DIPEA) were reacted at room temperature for 12 hours. 50mL of CH was added2Cl2Dilute and wash 3 times with 0.1M HCl. Collecting and combining organic phase, anhydrous Na2SO4Drying and vacuum concentrating. Chromatography on silica gel Column (CH)2 Cl 220/1, v/v) to give a yellow solid or liquid, approximately 3 g. The intermediate product was dissolved in 15mL of a mixture of methanol and water (1/4, v/v), cooled to 0 deg.C, and 0.10g NaBH added4The reaction was continued for 2 hours until the solution became colorless. After the reaction was completed, the pH was adjusted to 3.0, and the oligomer was purified by dialysis. Lyophilizing to obtain white solid or viscous liquid reduced thioctic acid-serinol-polyethylene glycol (LA-SPEG) each about 2.5 g. The products were designated LA-SPEG4K, LA-SPEG2K, LA-SPEG1K, LA-SPEG300, respectively, based on the polyethylene glycol molecular weight.
Comparative example 1:
synthesis of reduced lipoic acid-amino glycerol-polyethylene glycol (LA-GPEG)
Dithiooctanoic acid-aminoglyceride Synthesis: 3g Lipoic Acid (LA), 3.31g 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl) were added to a 100mL round bottom flask, and 40mL CH was added2Cl2Dissolving and activating for 30 min. Will dissolve in 20mL CH2Cl21.25g of Boc-3-aminoglycerol and 2g of 4- (dimethylamino) pyridine (DMAP) were added to the above mixture and reacted for 12 hours. After the reaction is finished, filtering, fully washing the filtrate by using excessive 0.1mol/L hydrochloric acid, standing and discarding the water phase. Drying the oil phase with sodium sulfate, and purifying by column chromatography (eluent: CHCl)3/CH3OH 20/1, v/v) to give a pale yellow oily liquid. The intermediate product was dissolved in 10mL CH2Cl2To the reaction solution, the reaction solution was cooled to 0 ℃ and 0.41g of trifluoroacetic acid (TFA) was slowly added dropwise thereto, followed by reaction for 2 hours. The mixture was concentrated in vacuo to give crude dithiooctanoic acid-aminoglyceride.
Synthesizing reduced lipoic acid-amino glycerol-polyethylene glycol: the crude dithiooctanoic acid-aminoglyceride product was dissolved in 20mL of anhydrous CH2Cl23g of methoxypolyethylene glycol succinic acid monoester (polyethylene glycol molecular weights 4000, 2000, 1000, 300, respectively), 0.42g of EDC. HCl, 0.19g of 1-hydroxybenzotriazole (HOBt) and 0.85g Diisopropylethylamine (DIPEA) at room temperature for 12 h. 50mL of CH was added2Cl2Dilute and wash 3 times with 0.1M HCl. Collecting and combining organic phase, anhydrous Na2SO4Drying and vacuum concentrating. Chromatography on silica gel Column (CH)2 Cl 220/1, v/v) to yield a yellow solid or liquid methoxypolyethylene glycol succinoylamino glyceryl lipoate, approximately 3 g. The intermediate product was dissolved in 15mL of a mixture of methanol and water (1/4, v/v), cooled to 0 deg.C, and 0.10g NaBH added4The reaction was continued for 2 hours until the solution became colorless. After the reaction was completed, the pH was adjusted to 3.0, and the oligomer was purified by dialysis. The reduced lipoic acid-amino glycerol-polyethylene glycol LA-GPEG is lyophilized to obtain white solid or viscous liquid, each about 2.5 g. According to the molecular weight of polyethylene glycol, products are respectively marked as LA-GPEG4K, LA-GPEG2K, LA-GPEG1K and LA-GPEG 300.
Example 1:
synthesis of Dicamptothecin-lipoic acid-serinol-polyethylene glycol (DicPteria-SPEG) (synthetic route shown in FIG. 2)
A mixture of 0.8g Camptothecin (CPT) and 0.3g triphosgene (BTC) was dissolved or suspended in anhydrous 40mL CH2Cl2In, N20.6g of DMAP CH is slowly added dropwise under protection2Cl2And (3) solution. After 2 hours at room temperature, 0.4g of 2- (pyridyl-disulfanyl) ethanol was added in one portion and stirred overnight. The reaction mixture was washed with deionized water and concentrated in vacuo. The crude product was purified by silica gel column chromatography (eluent: EtOAc/DCM. RTM. 1/1, v/v) to give 0.6g of camptothecin dithiopyridine (CPT-SS-Pyr) as a pale yellow solid in 50% yield.
Respectively taking 0.06g of LA-SPEG4K, LA-SPEG2K, LA-SPEG1K and LA-SPEG300 of the control list 1, respectively adding 0.1g of CPT-SS-Pyr into 2mL of methanol/dimethyl sulfoxide (2/3, v/v) mixed solution, stirring and reacting for 48h at 37 ℃, introducing nitrogen and removing oxygen into deionized water for dialysis for 12h, and freeze-drying to respectively obtain light yellow powder of the camptothecin-lipoic acid-serinol-polyethylene glycol, wherein the light yellow powder of the camptothecin-SPEG-lipoic acid-serinol-polyethylene glycol is respectively marked as dicTLA-SPEG 4K, dicTLA-SPEG 2K, dicTLA-SPEG 1K and dicTLA-SPEG 300 according to the molecular weight of the polyethylene glycol and is respectively about 0.08 g.
Example 2:
synthesis of Ditaxusol-lipoic acid-serinol-polyethylene glycol (DiptXLA-SPEG) (see FIG. 3 for synthetic route)
A mixture of 0.3g of paclitaxel-2 '-tert-butyldimethylsilane (PTX-2' -TBDMS) and 0.12g of triphosgene (BTC) was dissolved or suspended in 40mL of anhydrous CH2Cl2In, N2Under the protection, 0.29g of CH of DMAP is slowly added dropwise2Cl2The solution was reacted at room temperature for 2 hours. Subsequently, 0.20g of 2- (pyridyl-disulfanyl) ethanol was added in one portion, and stirred overnight. Washing the mixed solution system by deionized water, and concentrating in vacuum. The crude product was purified by silica gel column chromatography (EtOAc/DCM ═ 1/1, v/v). The crude product was dissolved in 20mL of dichloromethane and 0.5 g of tetrabutylammonium fluoride was added for deprotection. The reaction mixture was concentrated and purified by silica gel column chromatography (eluent: EtOAc/DCM-1/1, v/v). To obtain light yellow solid paclitaxel dithiopyridine (PTX-SS-Pyr)0.35 g.
0.06g of each of LA-SPEG4K, LA-SPEG2K, LA-SPEG1K and LA-SPEG300 in the control 1 was added to 0.1g of PTX-SS-Pyr dissolved in 2mL of methanol/dimethylsulfoxide (2/3, v/v) mixture, and after stirring at 37 ℃ for 48 hours, the mixture was dialyzed for 12 hours in deionized water containing nitrogen to remove oxygen. Lyophilizing to obtain white powders of paclitaxel-thioctic acid-serinol-polyethylene glycol, which are respectively marked as DIPTXLA-SPEG4K, DIPTXLA-SPEG2K, DIPTXLA-SPEG1K, and DIPTXLA-SPEG300 according to the molecular weight of polyethylene glycol, and each is about 0.07 g.
Example 3:
synthesis of Doxorubicin-lipoic acid-serinol-polyethylene glycol (didoXLA-SPEG) (see FIG. 4 for synthetic route)
0.20g 2- (pyridyl-disulfanyl) ethanol was dissolved in anhydrous 40mL CH2Cl2To a mixture of 0.12g of triphosgene (BTC), N2Under the protection, 0.3g of CH of DMAP is slowly added dropwise2Cl2The solution was reacted at room temperature for 4 hours. 40mL CH with 0.3g doxorubicin hydrochloride (DOXHCl) was added2Cl2Suspension and reaction for 4 hours. Subsequently, the crude product was purified by silica gel column chromatography (EtOAc/DCM ═ 1/1, v/v) to give doxorubicin dithiopyridine (DOX-SS-Pyr)0.35g as a red solid powder.
0.06g of LA-SPEG1K and 0.1g of DOX-SS-Pyr are weighed and dissolved in 2mL of methanol/dimethyl sulfoxide (2/3, v/v) mixed solution, stirred for 48h at 37 ℃, and dialyzed for 12h in deionized water with nitrogen for removing oxygen. Freeze-drying to obtain diadoxine-thioctic acid-serinol-polyethylene glycol (diDOXLA-SPEG1K) as red solid powder.
Example 4:
synthesis of Doxorubicin-N-lipoic acid-serinol-polyethylene glycol (didoXLA-SPEG) (see FIG. 5 for synthetic route)
0.20g 2- (pyridyl-disulfanyl) ethanol was dissolved in anhydrous 40mL CH2Cl2To a mixture of 0.12g of triphosgene (BTC), N2Under the protection, 0.3g of CH of DMAP is slowly added dropwise2Cl2The solution was reacted at room temperature for 4 hours. 40mL CH with 0.3g Adriamycin (DOX) added2Cl2Suspension and reaction for 4 hours. Subsequently, the crude product was purified by silica gel column chromatography (EtOAc/DCM ═ 1/1, v/v) to give doxorubicin dithiopyridine (DOX-SS-Pyr)0.35g as a red solid powder.
0.06g of LA-SPEG1K and 0.1g of DOX-SS-Pyr are weighed and dissolved in 2mL of methanol/dimethyl sulfoxide (2/3, v/v) mixed solution, stirred for 48h at 37 ℃, and dialyzed for 12h in deionized water with nitrogen for removing oxygen. Freeze-drying to obtain red solid powder of bisacodyl-N-lipoic acid-serinol-polyethylene glycol (diDOXNLA-SPEG 1K).
Example 5:
synthesis of cabazitaxel-lipoic acid-serinol-polyethylene glycol (the synthetic route is shown in figure 6)
A mixture of 0.3g Cabazitaxel (CAB) and 0.12g triphosgene was dissolved or suspended in anhydrous 40mL CH2Cl2In N2Under the protective atmosphere, 0.29g of CH of DMAP is slowly added dropwise2Cl2Solution, hydroxyl group of cabazitaxel reacts with triphosgene at room temperature. Subsequently, 0.20g of 2- (pyridyl-disulfanyl) ethanol was added in one portion, and stirred overnight. Washing the mixed solution system by deionized water, and concentrating in vacuum. The crude product was purified by silica gel column chromatography (EtOAc/DCM ═ 1/1, v/v). Light yellow solid adriamycin dithiopyridine (CAB-SS-Pyr)0.25g is obtained.
0.06g of LA-SPEG1K and 0.1g of CAB-SS-Pyr are weighed and dissolved in 2mL of methanol/dimethyl sulfoxide (2/3, v/v) mixed solution, stirred for 24h at 37 ℃, and dialyzed for 12h in deionized water with nitrogen for removing oxygen. Freeze drying to obtain white powder of dicabazitaxel-thioctic acid-serinol-polyethylene glycol 1K (diballa-SPEG 1K).
Example 6:
synthesis of Biharringtonine-lipoic acid-serinol-polyethylene glycol (see FIG. 7 for synthetic route)
A mixture of 0.3g Harringtonine (HAR) and 0.12g triphosgene was dissolved or suspended in 40mL anhydrous CH2Cl2In N2Under the protective atmosphere, 0.29g of CH of DMAP is slowly added dropwise2Cl2The solution, hydroxyl at room temperature, was reacted with triphosgene. Subsequently, 0.20g of 2- (pyridyl-disulfanyl) ethanol was added in one portion, and stirred overnight. Washing the mixed solution system by deionized water, and concentrating in vacuum. The crude product was purified by silica gel column chromatography (EtOAc/DCM ═ 1/1, v/v). Light yellow solid harringtonine dithiopyridine (HAR-SS-Pyr)0.25g is obtained.
0.06g of LA-SPEG1K and 0.1g of HAR-SS-Pyr are weighed and dissolved in 2mL of methanol/dimethyl sulfoxide (2/3, v/v) mixed solution, stirred for 24h at 37 ℃, and dialyzed for 12h in deionized water with nitrogen for removing oxygen. Freeze drying to obtain white powder product ditertiline-thioctic acid-serinol-polyethylene glycol 1K (diHARLA-SPEG 1K).
Example 7:
synthesis of bimetasone-lipoic acid-serinol-polyethylene glycol 1K (see FIG. 8 for synthetic route)
Dissolving 0.3g Maytansine (MAY) in anhydrous 40mL CH2Cl2In N2Under the protective atmosphere, 0.29g of CH of DMAP is slowly added dropwise2Cl2To the solution, 0.20g of 2, 2' -dithiodipyridine was added and stirred overnight. Washing the mixed solution system by deionized water, and concentrating in vacuum. The crude product was purified by silica gel column chromatography (EtOAc/DCM ═ 1/1, v/v). Thus, 0.25g of maytansinoid dithiopyridine (MAY-SS-Pyr) was obtained as a pale yellow solid.
0.06gLA-SPEG 1K-and 0.1g MAY-SS-Pyr are weighed and dissolved in 2mL methanol/dimethyl sulfoxide (2/3, v/v) mixed solution, stirred for 24h at 37 ℃, and dialyzed for 12h in deionized water with nitrogen for removing oxygen. Freeze drying to obtain white powder product bimatoin-lipoic acid-serinol-polyethylene glycol 1K (diMAYLA-SPEG 1K).
Example 8:
synthesis of Dieverolimus-lipoic acid-serinol-polyethylene glycol (DiEVELA-SPEG) (see FIG. 9 for synthetic route)
A mixture of 0.3g of tert-butyldimethylsiloxane-protected everolimus (EVE) and 0.12g of triphosgene was dissolved or suspended in 40mL of anhydrous CH2C12In N2Under the protective atmosphere, 0.29g of CH of DMAP is slowly added dropwise2Cl2Solution, hydroxyl group of everolimus reacts with triphosgene at room temperature. Subsequently, 0.20g of 2- (pyridyl-disulfanyl) ethanol was added in one portion, and stirred overnight. Washing the mixed solution system by deionized water, and concentrating in vacuum. The crude product was dissolved in 20mL of dichloromethane and 0.5 g of tetrabutylammonium fluoride was added for deprotection. The reaction mixture was concentrated and purified by silica gel column chromatography (eluent: EtOAc/DCM-1/1, v/v). Thus obtaining light yellow solid everolimus dithiopyridine (EVE-SS-Pyr)0.25 g.
0.06g of LA-SPEG 1K-and 0.1g of EVE-SS-Pyr were weighed and dissolved in 2mL of methanol/dimethyl sulfoxide (2/3, v/v) mixture, stirred at 37 ℃ for 24 hours, and dialyzed in deionized water containing nitrogen to remove oxygen for 12 hours. Freeze-drying to obtain white powder product dileverolimus-thioctic acid-serinol-polyethylene glycol 1K (diEVELA-SPEG 1K).
Example 9:
synthesis of bis-dasatinib-lipoic acid-polyethylene glycol (diDASLA-SPEG) (see the synthetic route in figure 10)
A mixture of 0.3g Dasatinib (DAS) and 0.12g triphosgene was dissolved or suspended in anhydrous 40mL CH2Cl2In N2Under the protective atmosphere, 0.29g of CH of DMAP is slowly added dropwise2Cl2And (3) reacting hydroxyl of the dasatinib with triphosgene at room temperature. Subsequently, 0.20g of 2- (pyridyl-disulfanyl) ethanol was added in one portion, and stirred overnight. Washing the mixed solution system by deionized water, and concentrating in vacuum. The crude product was purified by silica gel column chromatography (EtOAc/DCM ═ 1/1, v/v). Obtaining light yellow solid dasatinib dithiopyridine (DAS-SS-Pyr)0.25 g.
0.06g of LA-SPEG 1K-and 0.1g of DAS-SS-Pyr are weighed and dissolved in 2mL of methanol/dimethyl sulfoxide (2/3, v/v) mixed solution, stirred for 24h at 37 ℃, and dialyzed for 12h in deionized water with nitrogen for removing oxygen. Freeze drying to obtain white powder product of didaxatinib-thioctic acid-serinol-polyethylene glycol 1K (didala-SPEG 1K).
Example 10:
synthesis of bis-dasatinib-lipoic acid-amino glycerol-polyethylene glycol (diDASLA-GPEG) (the synthetic route is shown in figure 11)
A mixture of 0.3g Dasatinib (DAS) and 0.12g triphosgene was dissolved or suspended in anhydrous 40mL CH2Cl2In N2Under the protective atmosphere, 0.29g of CH of DMAP is slowly added dropwise2Cl2And (3) reacting hydroxyl of the dasatinib with triphosgene at room temperature. Subsequently, 0.20g of 2- (pyridyl-disulfanyl) ethanol was added in one portion, and stirred overnight. Washing the mixed solution system by deionized water, and concentrating in vacuum. The crude product was purified by silica gel column chromatography (EtOAc/DCM ═ 1/1, v/v). Obtaining light yellow solid dasatinib dithiopyridine (DAS-SS-Pyr)0.25 g.
Respectively taking 0.06g of LA-GPEG4K, LA-GPEG2K, LA-GPEG1K and LA-GPEG300 of the control list 2, respectively adding 0.1g of DAS-SS-Pyr into 2mL of methanol/dimethyl sulfoxide (2/3, v/v) mixed solution, respectively stirring and reacting for 12h at 37 ℃, dialyzing for 12h in deionized water with nitrogen for removing oxygen, and respectively freezing and drying to obtain white powder didaxatinib-lipoic acid-amino glycerol-polyethylene glycol, wherein the white powder didaxatinib-GPEG 4K, didaLA-GPEG 2K, didaLA-GPEG 1K and didaLA-GPEG 300 are respectively marked as 0.08g according to the molecular weight of the polyethylene glycol.
Example 12:
preparation of macromolecular prodrug nanoparticles
Examples 1-11 macromolecular prodrugs of different polyethylene glycol molecular weights Bicamptothecine-serinol-polyethylene glycol (DiCPTLA-SPEG), Bitaxel-lipoic acid-serinol-polyethylene glycol (DiPTXLA-SPEG), Biadriamycin-lipoic acid-serinol-polyethylene glycol (DiDOXLA-SPEG), Biadriamycin-N-lipoic acid-serinol-polyethylene glycol (DiDOXNLA-SPEG), Bicarbamazepine-lipoic acid-serinol-polyethylene glycol (DiCBALA-SPEG), Bitricuspid ester-lipoic acid-serinol-polyethylene glycol (DiHARLA-SPEG), Bimetrytin-lipoic acid-serinol-polyethylene glycol (DiMAYLA-SPEG), Bieverol-lipoic acid-serinol-polyethylene glycol (DiELA-SPEG), 0.05g of each of the didaxatinib-lipoic acid-polyethylene glycol (diDASLA-SPEG) and didaxatinib-lipoic acid-amino glycerol-polyethylene glycol (diDASLA-GPEG) is dispersed in 10mL of water which is filled with nitrogen and is used for removing oxygen, and the mixture is slowly stirred for 20 minutes. The thiol group in the macromolecule prodrug is oxidized and crosslinked by a small amount of oxygen (the crosslinking scheme is shown in figure 12), and the macromolecule prodrug nanoparticle solution is obtained. Freeze drying to obtain the nanometer powder of the macromolecule prodrug.
The particle size of the nanoparticles is detected by a Dynamic Light Scattering (DLS) method, and the result shows that the average particle size of all the various macromolecular prodrug nanoparticles is 50-100 nanometers. The transmission electron microscope measures the shape of the nano-particles, and the result shows that all the macromolecular prodrug nano-particles show a spherical structure. Fig. 13(a) is a graph of dynamic light scattering method for measuring average particle size of the dicamptothecin-lipoic acid-serinol-polyethylene glycol 1K macromolecular prodrug nanoparticle, which is 75nm, and fig. 13(B) is the nanoparticle morphology, which is in a regular sphere shape.
Example 13:
in vitro degradation of dicamptothecin-lipoic acid-serinol-polyethylene glycol (dicplta-SPEG) macromolecular prodrug nanoparticles
Sample preparation: the bicrine-lipoic acid-serinol-polyethylene glycol 1K (dipoPtRA-SPEG 1K) macromolecular prodrug nanoparticles prepared in example 12 were dissolved in an appropriate amount of PBS solution to prepare a solution with a concentration of 0.1 mmol.
The samples were divided into 2 portions, one of which was 0.5mL of Glutathione (GSH) in PBS buffer (GSH concentration 20mM), and the other was added in PBS buffer, and the samples were incubated in an incubator at 37 ℃ and the camptothecin content was measured by HPLC (Agilent 1100 LC, Zorbax reverse phase C18 column, 150X 4.6mM, 5 μm, 20 μ L of the amount to be added, column temperature 25 ℃, detection wavelength. lamda. cndot.254 nm; gradient elution: 2-90% buffer B/A, flow rate 1.0mL/min, buffer A: deionized water of 0.1% TFA, buffer B: acetonitrile of 0.1% TFA).
The results show that: the results are shown in FIG. 14. The PBS solution of GSH-treated bichin-lipoic acid-serinol-polyethylene glycol (dicplla-SPEG 1K) macromolecular prodrug nano-particles releases camptothecin raw drug which reaches 70% of the total amount after 0.5 hour, and releases camptothecin raw drug which reaches 90% of the total amount after 1 hour; the camptothecin raw drug released after 1 hour from the macromolecular prodrug nanoparticle solution without GSH treatment is only 10 percent of the total amount.
It is clear that the nanoparticles of the macromolecular prodrug of dicplla-SPEG 1K have GSH sensitivity. Under the action of GSH, disulfide bonds of the dicpTLA-SPEG1K macromolecular prodrug nanoparticles are broken, and carbonyl groups of carbonate bonds are attacked by broken end-group sulfydryl, so that camptothecin raw medicines are released rapidly.
Example 14:
in vitro degradation of doxorubicin macromolecular prodrug nanoparticles
Sample preparation: the bisacodyl-lipoic acid-serinol-polyethylene glycol 1K (diDOXLA-SPEG1K), and bisacodyl-N-lipoic acid-serinol-polyethylene glycol 1K (diDOXLA-SPEG1K) macromolecular prodrug nanoparticles prepared in example 12 were dissolved in an appropriate amount of PBS solution to prepare a solution with a concentration of 0.1 mmol.
The two samples were divided into 2 portions, one of which was added with 0.5mL of Glutathione (GSH) in PBS buffer (GSH concentration 20mM), the other was added with PBS buffer, and the samples were incubated in an incubator at 37 ℃ and subjected to high performance liquid chromatography to detect the camptothecin content (Agilent 1100 LC, Zorbax reverse phase C18 column, 150X 4.6mM, 5 μm, 20 μ L of the sample, 25 ℃ of the column temperature, detection wavelength. lamda. cndot.254 nm; gradient elution: 2-90% buffer B/A, flow rate 1.0mL/min, buffer A: deionized water with 0.1% TFA, buffer B: acetonitrile with 0.1% TFA).
The results show that: PBS solution of GSH-treated didOXLA-SPEG1K and didOXNLA-SPEG1K macromolecule prodrug nano-particles releases camptothecin raw drug which reaches 70 percent of the total amount after 0.1 hour, and releases doxorubicin raw drug which reaches 85 percent of the total amount after 1 hour; the release of doxorubicin prodrug from the GSH-untreated macromolecular prodrug nanoparticle solution after 1 hour was less than 10% of the total.
It is evident that the diDOXLA-SPEG1K, diDOXLA-SPEG1K macromolecular prodrug nanoparticles have GSH sensitivity. Under the action of GSH, disulfide bonds of the didOXLA-SPEG1K and didOXNLA-SPEG1K macromolecular prodrug nanoparticles are broken, and the broken end-group sulfydryl attacks carbonyl groups of carbonate bonds or carbamate bonds, so that doxorubicin prodrug is rapidly released.
Experimental example 15:
pharmacological experiments: MTT method cancer cell viability assay
Antitumor Activity of Bicamptothecine-lipoic acid-serinol-polyethylene glycol 1K (DicPtra-SPEG 1K) Macro-prodrug nanoparticles
MCF-7 human breast cancer cell and liver cancer cell HepG2 at 8 x 103The inoculum size of each well was inoculated in 96-well culture plates with 5% CO2After 24h of incubation in an incubator at 37 ℃, 100 μ L of each of the dicplla-SPEG 1K macromolecular prodrug nanoparticle solution of example 12 and a control camptothecin drug solution (camptothecin dissolved in physiological saline and solubilized with DMSO) with different concentrations were added to each well so that the final concentrations of the finally screened drugs were 1, 2, 5, 10 and 20 μ g/mL, and the incubation was continued for 24 h; respectively adding 50 mu LMTT for incubation for 4h, discarding the culture medium, adding 150 mu LDMSO, shaking up on a plate shaker, reading the plate at 495nm by a microplate reader, and calculating the cell inhibition rate according to the measured absorbance value. Data are presented as mean ± SD (n ═ 6).
The result is shown in fig. 15, the dicplla-SPEG 1K macromolecule prodrug nanoparticle has good growth inhibition and killing effects on breast cancer cells MCF-7 and liver cancer cells HepG-2, retains strong antitumor activity of camptothecin, has IC50 values of 2.06 μ g/mL and 1.89 μ g/mL corresponding to two tumor cells respectively, and has enhanced cytotoxicity with the increase of drug concentration and concentration dependence. Under the same drug dose, the nano-particles of the dichiptla-SPEG 1K macromolecular prodrug have weaker cytotoxicity than the free camptothecin prodrug. This is because the dicamptothecin-lipoic acid-polyethylene glycol 1K macromolecular prodrug nanoparticles self-assemble into stable nanoparticles in PBS (pH 7.4), and may have a certain sustained release effect compared to the release of free camptothecin drugs. Therefore, the nano-particles of the macromolecular prodrug of the dicplla-SPEG 1K show lower antitumor activity in 24h compared with the free drug.
Experimental example 16:
pharmacological experiments: MTT method cancer cell viability assay
Antitumor activity of doxorubicin macromolecular prodrug nanoparticles
MCF-7 human breast cancer cell and liver cancer cell HepG2 at 8 x 103The inoculum size of each well was inoculated in 96-well culture plates with 5% CO2After culturing in an incubator at 37 ℃ for 24 hours, adding 100 μ L of each of the bisacodyl-lipoic acid-serinol-polyethylene glycol 1K (didoXLA-SPEG1K), bisacodyl-N-lipoic acid-serinol-polyethylene glycol 1K (didoXNLA-SPEG1K) macromolecular prodrug nanoparticle solution and a control adriamycin solution (adriamycin is dissolved in physiological saline and solubilized by DMSO) of example 12 with different concentrations into each well, so that the final concentration of the finally screened drug is 1, 2, 5, 10 and 20 μ g/mL respectively, and continuing culturing for 24 hours; respectively adding 50 mu LMTT for incubation for 4h, discarding the culture medium, adding 150 mu LDMSO, shaking up on a plate shaker, reading the plate at 495nm by a microplate reader, and calculating the cell inhibition rate according to the measured absorbance value. Data are presented as mean + SD (n-6).
The results show that the Doxorubicin-lipoic acid-serinol-polyethylene glycol 1K (DiDOXLA-SPEG1K) and Doxorubicin-N-lipoic acid-serinol-polyethylene glycol 1K (DiDOXNLA-SPEG1K) macromolecular prodrug nanoparticles have good growth inhibition and killing effects on breast cancer cells MCF-7 and liver cancer cells HepG-2, retain the strong antitumor activity of the Doxorubicin, have IC50 values similar to those of the Doxorubicin corresponding to two kinds of tumor cells, have enhanced cytotoxicity along with the increase of the drug concentration and have concentration dependence. Under the same drug dosage, the macromolecular prodrug nano-particles show weaker cytotoxicity than the doxorubicin original drug in a free state. This is because the macromolecular prodrug nanoparticles self-assemble into stable nanoparticles in PBS (pH 7.4), which may have some sustained release effect compared to the release of the free form of the doxorubicin drug. Therefore, the didoXLA-SPEG1K and didoXNLA-SPEG1K macromolecular prodrug nanoparticles show lower antitumor activity in 48h compared with free drugs.
Example 17:
pharmacokinetic study of Bicamptothecine-lipoic acid-serinol-polyethylene glycol 1K (DicPtra-SPEG 1K) macromolecular prodrug nanoparticles
The size of the implanted tumor is 100-150mm3Female BALB/c nude mice, MCF-7, were randomly divided into irinotecan and a panel of dicplla-SPEG 1K macromolecular prodrug nanoparticles (n ═ 3). Blood samples were collected by tail vein injection of 5mg CPT/kg irinotecan and dicPLLA-SPEG 1K macromolecule prodrug nanoparticle solutions at 0.5h, 1h, 2h, 4h, 6h, 8h and 24h, respectively, and centrifuged at 3,000rpm for 10min to obtain plasma. The plasma was then extracted with acetonitrile for the amount of irinotecan and unhydrolyzed diCPTLA-SPEG1K macromolecular prodrug. The method comprises the following specific steps: 100 μ L of plasma samples were placed in a 2mL centrifuge tube, 1mL acetonitrile containing 0.5% acetic acid was added and mixed well. After the mixture was centrifuged at 11,000rpm for 10 minutes, the obtained supernatant was subjected to HPLC analysis.
To evaluate the in vivo tissue distribution of the pharmaceutical preparations, BALB/c nude mice loaded with MCF-7 tumor were sacrificed by cervical dislocation method after injection of 5mg CPT/kg irinotecan and a solution of the large prodrug nanoparticles of dicTLA-SPEG 1K for 0.5h, 2h and 6h, major organs including heart, liver, spleen, lung, kidney and tumor were excised, rinsed with 0.9% physiological saline and weighed, and homogenized. CPT was extracted from the homogenate using the same procedure as for plasma extraction, drug concentrations were determined by HPLC, and the corresponding CPT content in the tissue was calculated accordingly.
The results are shown in FIG. 13 (A). Within 4-8 hours after administration, free irinotecan is clearly cleared from the blood circulation. This result is in marked contrast to the nanoparticles of the macromolecular prodrug of dicplla-SPEG 1K, which prolongs the blood circulation of the drug. This is because the nanoparticles of the macromolecular prodrug of dicplla-SPEG 1K are advantageous in avoiding the rapid uptake by the reticuloendothelial system (RES). Second, the large prodrug of dicplla-SPEG 1K retains its good stability without decomposition in physiological environments. Third, the presence of PEG will further prolong blood circulation. Moreover, short-chain PEG is not easy to cause the body to generate PEG antibodies, thereby reducing the plasma elimination rate of the drug.
The distribution of nanoparticles in BALB/c nude mouse tumors and other organs transplanted with MCF-7 tumor is shown in FIG. 16 (B). It can be seen that the nanoparticles of the macromolecular prodrug of dicplla-SPEG 1K distribute primarily after 2 hours to the reticuloendothelial system (RES) organs, such as the lung, spleen, and particularly the liver, while the corresponding irinotecan levels are lower. The greater accumulation of the nanocplla-SPEG 1K macromolecular prodrug nanoparticles in these organs is likely due to the nanoscale nature of the formulation, which may provide long-term therapeutic effects. More importantly, the experimental group treated with the nanoparticles of the macromolecular prodrug of dicplla-SPEG 1K 1K showed higher concentrations of CPT in the tumor (p < 0.01) than the irinotecan-treated group. It can be seen that the nanocplla-SPEG 1K macromolecular prodrug nanoparticles can reduce the rapid capture of the RES system and accumulate passively in tumors due to the EPR effect. Thus, compared to irinotecan alone, the nanoparticle of macromolecular prodrug from dicytla-SPEG 1K can prolong plasma half-life and significantly enhance distribution to tumor tissue in vivo.
Experimental example 18:
in vivo efficacy and toxicity experiments of dicamptothecin-lipoic acid-serinol-polyethylene glycol 1K (dicplla-SPEG 1K) macromolecular prodrug nanoparticles
Preparation of a nude mouse model: the cultured HepG2 cell suspension was collected at a concentration of 1X 107Each 0.1ml of each of the cells was inoculated subcutaneously into the right axilla of a nude mouse. Grouping and administration: measuring the diameter of the transplanted tumor of the nude mouse by using a vernier caliper, and enabling the tumor to grow to 75mm3Animals were randomly grouped. Meanwhile, each group of nude mice starts to be dosed, the dosing scheme is shown in the group and the dosing scheme, and the antitumor effect of the tested sample is dynamically observed by using a method for measuring the tumor size. The formula for Tumor Volume (TV) is: TV 1/2 × a × b2(formula 3-2), wherein a and b represent the length and width, respectively.
Groups and dosing regimens: blank group: saline, i.v. once every three days, 0.2ml in volume for 3 consecutive weeks.
Control group: irinotecan was dissolved in physiological saline (trace amount of DMSO as a solubilizer, dose 10mg/kg) and injected intravenously every three days in a volume of 0.2ml for 3 consecutive weeks.
Drug group: the solution of the macromolecular prodrug nanoparticles of dicplla-SPEG 1K of example 12 (administered in an amount equivalent to 10mg/kg of camptothecin) was injected intravenously every three days in a volume of 0.2ml for 3 consecutive weeks. During this period, the body weight change was measured.
The results of antitumor activity and body weight change are shown in FIG. 17. From the view point of antitumor activity (fig. 17A), the nano-particles of the macromolecular prodrug of dicplla-SPEG 1K of the present invention have good tumor growth inhibition effect, and the animal body weight is not reduced (fig. 17B), showing no toxicity.
Experimental example 19:
doxorubicin-lipoic acid-serinol-polyethylene glycol macromolecule prodrug nanoparticle in-vivo efficacy and toxicity test
Preparation of a nude mouse model: the cultured HepG2 cell suspension was collected at a concentration of 1X 107Each 0.1ml of each of the cells was inoculated subcutaneously into the right axilla of a nude mouse. Grouping and administration: measuring the diameter of the transplanted tumor of the nude mouse by using a vernier caliper, and enabling the tumor to grow to 75mm3Animals were randomly grouped. Meanwhile, each group of nude mice starts to be dosed, the dosing scheme is shown in the group and the dosing scheme, and the antitumor effect of the tested sample is dynamically observed by using a method for measuring the tumor size. The formula for Tumor Volume (TV) is: TV 1/2 × a × b2(formula 3-2), wherein a and b represent the length and width, respectively.
Groups and dosing regimens: blank group: saline, i.v. once every three days, 0.2ml in volume for 3 consecutive weeks.
Control group: doxorubicin was dissolved in physiological saline (trace DMSO as adjuvant, dose 10mg/kg), injected intravenously every three days with a volume of 0.2ml for 3 consecutive weeks.
Drug group: the doxorubicin-lipoic acid-serinol-polyethylene glycol 1K (diDOXLA-SPEG1K), doxorubicin-N-lipoic acid-serinol-polyethylene glycol 1K (diDOXLA-SPEG1K) macromolecular prodrug nanoparticle solution of example 12 (administered at an amount equivalent to 10mg/kg of camptothecin) was injected intravenously once every three days in a volume of 0.2ml for 3 consecutive weeks. During this period, the body weight change was measured.
The results show that the tumor volume of animals administered with the bisacodyl-lipoic acid-serinol-polyethylene glycol 1K (didOXLA-SPEG1K) and the bisacodyl-N-lipoic acid-serinol-polyethylene glycol 1K (didOXNLA-SPEG1K) macromolecular prodrug nanoparticles is far smaller than that of an adriamycin control group, and the macromolecular prodrug nanoparticles have a good tumor growth inhibition effect; animals in the nanoparticle-administered group did not lose weight (fig. 17B), showing lower toxicity than doxorubicin; animal myocardial immunohistochemistry shows that the animal myocardial tissues of the macromolecular prodrug nanoparticle group have no obvious change, and the animal myocardial of the adriamycin group has obvious histological change.
Example 20:
preparation of macromolecular prodrug nanoparticles
Macromolecular prodrugs of the polyethylene glycol of examples 1, 2, 3, respectively, dicamptothecin-lipoic acid-serinol-polyethylene glycol (dichTLA-SPEG), dicataxol-lipoic acid-serinol-polyethylene glycol (dichPTXLA-SPEG), and bisacodyl-lipoic acid-serinol-polyethylene glycol (didoXLA-SPEG) were added in an amount of 0.05g each, and the mixture was dispersed in 10mL of water containing nitrogen and oxygen, and stirred slowly for 20 minutes. The thiol group in the macromolecule prodrug is oxidized and crosslinked by a small amount of oxygen (the crosslinking scheme is shown in figure 12), and a type 3 macromolecule prodrug nanoparticle solution is obtained. Freeze drying to obtain 3 kinds of nanometer prodrug powder. The particle size of the nanoparticles is detected by a Dynamic Light Scattering (DLS) method, and the result shows that the average particle size of all the various macromolecular prodrug nanoparticles is 50-300 nanometers.

Claims (5)

1. The nano-drug of the macromolecular prodrug is characterized by comprising two amphipathic macromolecular prodrugs of a general formula (1) formed by connecting two hydrophobic drug molecules through lipoic acid and low molecular weight polyethylene glycol through disulfide bonds, wherein the amphipathic macromolecular prodrugs are self-assembled and cross-linked through intermolecular disulfide bonds to form nanoparticles with drug-containing cores and low molecular weight polyethylene glycol hydrophilic macromolecular brushes as shells, and the size of the nanoparticles is 10-1000 nm:
Figure FDA0003274355480000011
wherein X is serinol or 3-amino glycerol, D is a hydrophobic drug, and Y is CH2CH2OCOO or CH2CH2OCONH, n is 6-80, the hydrophobic drug is paclitaxel, docetaxel, cabazitaxel, camptothecin, 7-ethyl-10-hydroxycamptothecin, topotecan, irinotecan, rubitecan, belotecan, adriamycin, epirubicin, daunorubicin, demethoxydaunorubicin, aclarubicin, pirarubicin, zorubicin, cephalotaxine, isotrichacharidine, maytansine, everolimus or dasatinib.
2. The macromolecular prodrug nanomedicine of claim 1, further comprising a pharmaceutically acceptable carrier or adjuvant, wherein the adjuvant is a fat or phospholipid.
3. The macromolecular prodrug nanomedicine according to claim 1 or 2, wherein the nanomedicine is a liquid, solid or semi-solid formulation.
4. A preparation method of a macromolecular prodrug nano-drug is characterized in that the preparation method comprises the steps of assembling the macromolecular prodrug of a general formula (1) into a nano-micelle with the diameter of 10-1000 nanometers in an aqueous solution, adding hydrogen peroxide or oxidizing sulfydryl in the air to cause intermolecular crosslinking to form crosslinked nano-particles with a hydrophobic drug in an inner core and a hydrophilic polyethylene glycol brush in an outer layer, and freeze-drying to obtain the crosslinked nano-particle freeze-dried powder wrapping the drug:
Figure FDA0003274355480000012
wherein X is serinol or 3-amino glycerol, D is a hydrophobic drug, and Y is CH2CH2OCOO or CH2CH2OCONH, n is 6-80.
5. Use of the macromolecular prodrug nanomedicine of claim 1, 2 or 3 in the preparation of an anti-tumor drug.
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