CN117126095A - Releasable functional compound containing polyethylene glycol chain segment with single molecular weight, nanoparticle containing same and preparation method - Google Patents

Releasable functional compound containing polyethylene glycol chain segment with single molecular weight, nanoparticle containing same and preparation method Download PDF

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CN117126095A
CN117126095A CN202311048994.XA CN202311048994A CN117126095A CN 117126095 A CN117126095 A CN 117126095A CN 202311048994 A CN202311048994 A CN 202311048994A CN 117126095 A CN117126095 A CN 117126095A
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polyethylene glycol
compound
functional compound
ala
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刘世勇
侯明萱
刘妙一
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University of Science and Technology of China USTC
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University of Science and Technology of China USTC
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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Abstract

The invention discloses a releasable functional compound containing a polyethylene glycol chain segment with single molecular weight, which has a structure shown in a formula (I):wherein R is 1 Representing reactive groups suitable for binding to biological macromolecules; r is R 2 A drug molecule in which S-S bonds are linked through an ester bond or an ether bond; PEG (polyethylene glycol) n Represents a polyethylene glycol segment having a single molecular weight; n is an integer of 4 to 500.

Description

Releasable functional compound containing polyethylene glycol chain segment with single molecular weight, nanoparticle containing same and preparation method
Technical Field
At least one embodiment of the invention relates to a releasable functional compound, in particular to a releasable functional compound containing a single molecular weight polyethylene glycol chain segment, a nanoparticle containing the same and a preparation method.
Background
Polyethylene glycol is a high molecular polymer, has good water solubility and good compatibility with a plurality of organic components. Polyethylene glycol refers to a general method of covalently or noncovalently attaching polyethylene glycol (PEG) to small molecule drugs, contrast agents, proteins, nucleic acids, liposomes, and the like. This strategy gives the resulting pegylated drug a degree of improvement in water dispersibility, stability, pharmacokinetics and pharmacodynamics. However, conventional polydisperse PEG is a series of mixtures that may complicate the synthesis and purification of pegylated drugs and elicit unwanted immunogenic responses that ultimately affect the therapeutic effect.
In addition, although polydisperse pegylated drugs have been widely used, absorption of non-specific proteins by the pegylated drugs has been observed, and the protein corona thus formed directly determines the fate of the pegylated drugs in vivo, affecting the targeting specificity and efficiency of the pegylated drugs.
Disclosure of Invention
In view of the above, the present invention provides a releasable functional compound having high stability and a polyethylene glycol segment with a single molecular weight, nanoparticles comprising the same and a preparation method thereof, so as to prolong the acting time of a drug molecule in blood, and have lower immunogenicity and protein adsorption, and further can efficiently deliver the drug molecule into cells so as to release the drug molecule in the cells.
As one aspect of the present invention, the present invention provides a releasable functional compound comprising a polyethylene glycol segment of a single molecular weight, having a structure as shown in formula (I):
wherein R is 1 Representing reactive groups suitable for binding to biological macromolecules; r is R 2 A drug molecule in which S-S bonds are linked through an ester bond or an ether bond; PEG (polyethylene glycol) n Represents a polyethylene glycol segment having a single molecular weight; n is an integer of 4 to 500.
As another aspect of the present invention, there is provided a method for producing the above releasable functional compound, comprising:
Providing a polyethylene glycol chain segment with single molecular weight, wherein the structure of the polyethylene glycol chain segment with single molecular weight is shown as a formula (X1);
Trt-PEG n OTs (X1)
A polyethylene glycol chain segment with single molecular weight, diethanolamine and a compound R shown in a formula (X1) 1 H, reacting to obtain a compound with a secondary amine group, wherein the compound is shown as a formula (X2);
the drug molecule R having a terminal carboxyl group or hydroxyl group 2 Carrying out condensation reaction with dithiodiethanol to obtain a compound shown as a formula (X3);
subjecting a compound represented by the formula (X2) having a secondary amine group and a compound represented by the formula (X3) to an amide synthesis reaction to obtain a releasable functional compound having a structure represented by the formula (I)
As still another aspect of the present invention, the present invention provides a nano-scale example, which is assembled by using the releasable functional compound described above.
According to the releasable functional compound provided by the embodiment of the invention, the polyethylene glycol block with single molecular weight is adopted, so that the recognition of an anti-PEG antibody can be reduced, the induction of unnecessary immune rejection reaction can be avoided, and the biocompatibility of the releasable functional compound is improved.
According to the releasable functional compound provided by the embodiment of the invention, the releasable functional compound with an amide bond has high stability due to the fact that the amide bond is relatively stable.
According to the nanoparticle assembled by the releasable functional compound provided by the embodiment of the invention, the nanoparticle is injected in an injection mode, and the polyethylene glycol block in the nanoparticle passes through the active group R 1 Can be combined with albumin in blood, can increase the acting time of the nano particles in the blood, so that the nano particles have long-time stability in the blood, and are beneficial to improving the specificity and efficiency of the nano particles for targeting tumor tissues.
According to the nanoparticle assembled by the releasable functional compound provided by the embodiment of the invention, the nanoparticle enters cells, and the disulfide bond of the releasable functional compound is broken in the cells by the reduced glutathione existing in the cells so as to release the drug molecules in the cells and exert the drug effect.
Drawings
FIG. 1 shows the 5-ALA-2 (OEG) of example 1 of the invention 4 -MI);
FIG. 2 shows the 5-ALA-2 (OEG) of example 1 of the invention 4 -MI) time-of-flight mass spectrometry data;
FIG. 3 shows the 5-ALA-2 (OEG) of example 2 of the invention 4 -SI) nuclear magnetic hydrogen spectrum;
FIG. 4 shows the 5-ALA-2 (OEG) of example 2 of the invention 4 -SI) time-of-flight mass spectrometry data;
FIG. 5 shows the 5-ALA-2 (OEG) of example 1 of the invention 4 -MI) assembled nanoparticles and 5-ALA-2 (OEG) of example 2 4 -SI) particle size distribution profile of assembled nanoparticles;
FIGS. 6A-6B show 5-ALA-2 (OEG) of example 1 of the invention 4 -MI) assembled nanoparticles and implementationExample 2 5-ALA-2 (OEG) 4 -SI) cytotoxicity of the assembled nanoparticle;
FIG. 7 shows the 5-ALA-2 (OEG) of example 1 of the invention 4 -MI) assembled nanoparticles and 5-ALA-2 (OEG) of example 2 4 -SI) relative fluorescence intensity of assembled nanoparticles in vitro within cells;
FIG. 8 shows the 5-ALA-2 (OEG) of example 1 of the invention 4 -MI) assembled nanoparticles and 5-ALA-2 (OEG) of example 2 4 -SI) a picture of organelle co-localization of the assembled nanoparticle under confocal fluorescence microscopy; and
FIG. 9 shows the 5-ALA-2 (OEG) of example 1 of the invention 4 -MI) assembled nanoparticles and 5-ALA-2 (OEG) of example 2 4 SI) in-animal imaging of assembled nanoparticles.
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. In the drawings, the size of layers and regions, as well as the relative sizes, may be exaggerated for the same elements throughout.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The terms "comprises," "comprising," and/or the like, as used herein, specify the presence of stated features, steps, operations, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, or components.
In practical application, small molecular drugs often have the problems of quick metabolism, multiple injection and easy metabolism, and the existence of the defects greatly limits the practical application range of the small molecular anticancer drugs. The most used methods for preparing derivatives of anticancer small molecules include coating with liposome, grafting small molecules to high molecules, and the like to prolong the cycle time. However, the possible immune response and cellular and living toxicity due to these approaches have less application prospects.
Albumin has a free sulfhydryl group, and maleimide can perform a Michael addition reaction with albumin with high efficiency. The invention designs different connecting motifs for connecting small molecule drugs (functional molecules R 2 ) And Maleimide (MI) group, MI can not only combine with endogenous albumin in situ in blood to prolong half life of medicine, but also avoid enriching liver and spleen, target tumor, and penetrate vascular endothelial deep into tumor. During blood circulation, maleimide groups on the nanoparticle surface can be covalently coupled with serum albumin to form albumin-rich protein crowns. Nanoparticles with albumin-rich protein crowns cannot be cleared rapidly by the Kupffer cells of the liver and the nanoparticles penetrate into the tumor stroma through Gp60 receptor-mediated endocytosis. The reducing environment (GSH) triggers the partial breakdown of the nanoparticles, turning into smaller nanoparticles, to achieve tumor depth penetration. Finally, the nanoparticles can be subjected to chemotherapy by completely releasing cisplatin small molecule drugs.
Disulfide bonds (S-S bonds) are important chemical functional groups that are found primarily in various types of natural small molecule compounds and biological protein structures. In organisms, the primary roles of disulfide bonds include helping protein structure solidify, controlling cell signaling, participating in metabolic pathways, and the like. Since disulfide bonds have low toxicity in vivo and it can be broken in the presence of reduced Glutathione (GSH) in the external environment, the present invention introduces disulfide bonds into the drug structure to achieve better therapeutic effects.
At the same time, it benefits from the recent development of monodisperse polyethylene glycol synthesis. Monodisperse PEG has the same chemical structure as polydisperse PEG, but has a precise molecular weight. The invention envisages that, on the basis of the construction of precisely pegylated derivatives, the regulation of the surface protein corona of the delivery system is further achieved by modifying the groups at the end of the PEG.
The stability of the drug nano particles can be greatly improved by using the small molecule derivative and coating the small molecule derivative with polyethylene glycol. On the other hand, the use of maleimide as a part of helping molecules to better increase circulation time in vivo can provide longer duration of action and higher therapeutic efficiency for medical treatment.
In view of the above, the present invention provides a releasable functional compound having a single molecular weight polyethylene glycol segment with high stability, wherein the releasable functional compound achieves a targeting effect by designing active groups, particularly selecting maleimide, and releases drug molecules loaded by the releasable functional compound in cells through cleavage of disulfide bonds.
According to an exemplary embodiment of the present invention, there is provided a releasable functional compound having a single molecular weight polyethylene glycol segment, having a structure as shown in formula (I):
wherein R is 1 Representing reactive groups suitable for binding to biological macromolecules; r is R 2 A drug molecule in which S-S bonds are linked through an ester bond or an ether bond; PEG (polyethylene glycol) n Represents a polyethylene glycol segment having a single molecular weight, and n is an integer of 4 to 500.
According to embodiments of the invention, PEG n The structural formula of (2) is as follows:
according to an embodiment of the present invention, the degree of polymerization n may be 4, 10, 50, 100, 500. When n is less than 4, the polymerization degree of PEG is too low, so that the formed nano particles have poor water solubility; when the polymerization degree is higher than 500, the formed polymer chain is not easy to dissolve in water due to the too large molecular weight.
According to an embodiment of the invention, R 2 Selected from a drug molecule R2' linked to an S-S bond via an ester bond or a drug molecule R linked to an S-S bond via an ether bond 2 "Zhongzhong (Chinese medicine)R, R is as follows 2 ' is selected from any one of the following structures:
according to an embodiment of the invention, R2 "is selected from any one of the following structures:
r is as follows 2 The corresponding drug molecule is selected from 5-aminolevulinic acid, acetaminophen, allopurinol, topotecan, tetrahydro-guarantor, oxazepam related substance a, dihydrocarbamazepine, morphine, 7-ethyl-10-hydroxycamptothecin, berberine isomer a, atropine sulfate, testosterone, irinotecan, chloroberberine, cefoperazone, halopinavir, berberine isomer B, paclitaxel, camptothecine, daunorubicin, berberine, gemcitabine, hydroxycholesterol, hydroxycholesterate or 2-hydroxy-hydroxycholesterol.
R is as follows 2 The main therapeutic efficacy of the corresponding drug molecules are: resisting cancer, treating inflammation, inhibiting chronic gout and hypertension, acting on nervous system, relieving pain, regulating arteriosclerosis and cholelithiasis, etc.
According to an embodiment of the invention, R 2 The corresponding drug molecule is preferably paclitaxel, the paclitaxel has a mediating function in cells, and the increase of PEG can excite the immune effect in vivo.
According to an embodiment of the invention, R 1 Selected from any one of the following structures:
r is as follows 3 Selected from-H, -CH 3 Chlorine, bromine or iodine.
R is as follows 4 Selected from any one of the following structures:
according to an embodiment of the present invention, the molecular weight of the releasable functional compound is 1000 to 100000, for example, 1000, 2000, 5000, 10000.
R is as follows 1 Preferably maleimide, the maleimide with double bond can perform Michael addition reaction with albumin, thereby the albumin can be captured efficiently through covalent bond, and albumin-rich protein crowns are formed, and the maleimide is not easy to be cleared by immunity and is helpful for targeting tumor tissues.
It should be noted that the active group with carboxyl and hydroxyl can be combined with amino in the protein, and the active group with amino can form hydrogen bond with the oxygen-containing part of the protein, so that the releasable functional compound can achieve the targeting effect.
According to an embodiment of the present invention, the releasable functional compound described above has any one of the following structures:
According to an exemplary embodiment of the present invention, there is provided a method for preparing a releasable functional compound having a single molecular weight polyethylene glycol segment having the structure of formula (I) as described above, comprising: step S01 to step S04.
In the step S01, providing a polyethylene glycol chain segment with single molecular weight, wherein the structure of the polyethylene glycol chain segment with single molecular weight is shown as a formula (X1);
Trt-PEG n OTs (X1)
According to an embodiment of the present invention, the synthesis process of the polyethylene glycol segment with single molecular weight is shown in the following formula (1), and includes steps S011 to S013:
wherein n=n1+n2=4 to 100.
In step S011, a first polyethylene glycol PEG of single molecular weight is added n1 Hydrophobic end capping is carried out on the terminal hydroxyl of the polyethylene glycol to obtain hydrophobic end capped first polyethylene glycol Trt-PEG with single molecular weight n1 -OTrt；
In step S012, a hydrophobic end-capped single molecular weight first polyethylene glycol Trt-PEG n1 Selective deprotection of one end hydroxyl group of OTrt to give a single molecular weight first polyethylene glycol Trt-PEG with one end hydrophobically capped n1 -OH；
In step S013, protecting a single-molecular-weight first polyethylene glycol, one end of which is end-capped by hydrophobicity, by using p-toluenesulfonyl to obtain a single-molecular-weight first polyethylene glycol chain segment structure Trt-PEG n1 -OTs；
In step S014, the same procedure as in steps S011 to S012 was used to obtain a single molecular weight second polyethylene glycol Trt-PEG having one end hydrophobically capped n2 -OH；
In step S015, the structure Trt-PEG of the first polyethylene glycol segment of single molecular weight is prepared n1 OTs and a single molecular weight second polyethylene glycol Trt-PEG with one end hydrophobically capped n2 Condensation reaction is carried out on the-OH to obtain the third polyethylene glycol Trt-PEG with single molecular weight and end capped by hydrophobic property n1+n2 -OTrt；
In step S016, a hydrophobic end-capped third polyethylene glycol Trt-PEG of single molecular weight n1+n2 The procedure of steps S012-S016 is repeated until the structure of the single molecular weight first polyethylene glycol segment reaches the target molecular weight.
According to an embodiment of the present invention, when n is 4, the synthetic route of the polyethylene glycol segment having a single molecular weight is shown in the following formula (2), and specifically includes: a500 mL two-necked flask was charged with tetraethylene glycol (200 g, 1)mol) and toluene (100 mL), azeotropically dehydrated, under an ice bath under N 2 DIPEA (N, N-diisopropylethylamine) was added under atmosphere followed by the slow addition of TrtCl (56 g,0.2 mol) to N 2 The reaction was carried out for 4 hours under an atmosphere. After the reaction is finished, extracting with water and Ethyl Acetate (EA), washing with saturated sodium chloride, and spin-evaporating to obtain an organic Trt-PEG 4 -OH。
The Trt-PEG product was placed in a 1L flask 4 -OH and THF (250 mL), ice-bath, naOH (32 g,0.8 mol) was added to 125mL of water, ice-bath to below 10 ℃, aqueous NaOH was added to the system, and ice-bath stirred for 10 minutes. TsCl (44 g,0.23 mol) was dissolved in 100mL THF and added to the system via a constant pressure dropping funnel under ice bath, the ice bath reaction was maintained for the first 6 hours, and the room temperature reaction was resumed for 12 hours. After the reaction, EA and water are added for extraction, saturated saline water is used for washing, the organic phase is dried, and the organic phase is purified by column chromatography (PE: EA=10:1) to obtain a colorless transparent product Trt-PEG 4 -OTs。
According to an embodiment of the present invention, when n is 8, the synthetic route of the polyethylene glycol segment with single molecular weight is shown in formula (3), specifically including: taking Trt-PEG of the above product 4 OTs (0.1 mol) are dissolved in 100mL of toluene, tetraethylene glycol (0.12 mol) is added, the toluene azeotropically dehydrated, then 200mL of anhydrous THF are added, naH (1.4 mol) is slowly added at room temperature N 2 Reacting for 12h in atmosphere, adding EA and water for extraction after the reaction is finished, washing with saturated saline water, drying an organic phase, and purifying by column chromatography (PE: EA=2:1) to obtain a colorless transparent product Trt-PEG 8 -OH。
The Trt-PEG is obtained 8 -OH (1 eq.) 200mL THF was added, ice-bath, naOH (3.0 eq.) was added to 125mL water, ice-bath to below 10 ℃, aqueous NaOH was added to the system and ice-bath stirred for 10 minutes. TsCl (1.25 eq.) was dissolved in 100mL THF and added to the system via a constant pressure dropping funnel under ice bath, the ice bath reaction was maintained for the first 6 hours, after which the room temperature reaction was resumed for 12 hours. After the reaction, EA and water are added for extraction, and saturated saline water is used for washing The organic phase was dried and purified by column chromatography (PE: ea=2:1) to give the colorless transparent product Trt-PEG 8 -OTs。
According to an embodiment of the present invention, when n is 16, the synthetic route of the polyethylene glycol segment of a single molecular weight is shown as formula (4). At 7.5g (10 mmol) Trt-PEG 8 The OTs raw material is exemplified, and the preparation process specifically comprises:
7.5g (10 mmol) of Trt-PEG 8 OTs are dissolved in 100mL of toluene and 6.2g (11 mmol) of Trt-PEG are added 8 Azeotropic removal of water from-OH, toluene, followed by 200mL dry THF, slow addition of NaH (10 eq.) at room temperature N 2 Reacting for 12h in atmosphere, adding EA and water for extraction after the reaction is finished, washing with saturated saline water, drying an organic phase, and purifying by column chromatography (PE: EA=1:1) to obtain a colorless transparent product Trt-PEG 16 -OTrt。
The Trt-PEG is obtained 16 OTrt (1 eq.) was added with 100mL MeOH, tsOH (0.1 eq.) was dissolved in 10mL MeOH, added dropwise to the system at room temperature, and reacted for 12 hours at room temperature. After the reaction, DCM and water were added for extraction, the saturated brine was washed, the organic phase was dried, and purified by column chromatography (EA: meOH=10:1) to give the colorless and transparent product Trt-PEG 16 -OH。
The Trt-PEG is obtained 16 -OH (1 eq.) 200mL THF was added, ice-bath, naOH (3.0 eq.) was added to 125mL water, ice-bath to below 10 ℃, aqueous NaOH was added to the system and ice-bath stirred for 10 minutes. TsCl (1.25 eq.) was dissolved in 100mL of THF and added to the system via a constant pressure dropping funnel under ice bath, the ice bath reaction was maintained for the first 6 hours, and the reaction was resumed at room temperature for 12 hours. After the reaction, EA and water are added for extraction, saturated saline water is used for washing, an organic phase is dried, and the organic phase is purified by column chromatography (EA: PE=5:1) to obtain a colorless transparent product Trt-PEG 16 -OTs。
According to an embodiment of the present invention, when n is 24, the synthetic route of the polyethylene glycol chain segment with single molecular weight is shown in formula (5), and specifically includes:
taking the synthesized Trt-PEG 16 OTs (1.0 eq) were dissolved in 100mL toluene and Trt-PEG was added 8 -OH (1.1 eq.) and toluene were azeotropically dehydrated, followed by 200mL of anhydrous THF, slow addition of NaH (10 eq.) and N at room temperature 2 After the reaction is finished, EA and water are added for extraction, saturated saline water is used for washing, an organic phase is dried, and column chromatography purification (EA: meOH=10:1) is carried out to obtain a colorless transparent product Trt-PEG 24 -OTs。
According to the embodiment of the invention, trt-PEG is obtained according to the synthetic route shown in the formula (1) by maintaining the feeding ratio n1+n2 -OH and Trt-PEG n1+n2 OTs, where n1+n2=4 to 100.
In step S02, a single molecular weight polyethylene glycol segment represented by formula (X1), diethanolamine and a compound R 1 H, reacting to obtain a compound with a secondary amine group, wherein the compound is shown as a formula (X2);
according to an embodiment of the present invention, the synthesis process of the compound represented by formula (X2) specifically includes: step S021 to step S025.
In step S021, diethanolamine is reacted with Boc anhydride to obtain N-Boc-2CH 3 CH 2 OH。
According to an embodiment of the present invention, the synthetic route of step S021 is shown in formula (6):
Specifically, diethanolamine was added to 500mL of monoInto the flask, 100mL of THF was added for dissolution, followed by 100mL of saturated aqueous sodium bicarbonate solution. The boc anhydride was slowly added with stirring and reacted at room temperature for 12h. After the reaction is finished, spin-drying THF, adding EA and water for extraction, reserving an organic phase, adding saturated saline water for washing, and drying with anhydrous sodium sulfate to obtain colorless viscous liquid, namely the compound N-Boc-2CH 3 CH 2 OH。
In step S022, OTs- (PEG) n -Trt) 2 、N-Boc-2CH 3 CH 2 OH and NaH to obtain N-Boc- (PEG) n -Trt) 2 。
According to an embodiment of the present invention, the synthetic route of step S022 is as shown in formula (7):
specifically, OTs- (PEG) n -Trt) 2 (2.5eq.)、N-Boc-2CH 3 CH 2 OH (1 eq.) and NaH (10 eq.) were added to a 500mL single-necked flask, dissolved in 100mL THF, reacted at room temperature for 36h, cooled to 23℃and then the toluene was dried, and the organic phase was extracted with EA, washed with saturated brine and dried over anhydrous sodium sulfate. Purifying the product by column chromatography using EA and THF as mobile phase to obtain colorless oily substance, namely the compound N-Boc- (PEG) n -Trt) 2 。
In step S023, N-Boc- (PEG n -Trt) 2 Sequentially reacting with HAc, tsOH and TsCl to obtain N-Boc- (PEG) n -OTs) 2 。
According to an embodiment of the present invention, the synthetic route of step S023 is shown in formula (8):
specifically, the starting material N-Boc- (PEG) n -Trt) 2 (1 eq) was placed in a 100mL single-necked flask, a mixed solution of 40mL of acetic acid and 10mL of water was added, the mixture was heated to 40℃and reacted for 4 hours, and the completion of the reaction was confirmed by spotting. After the completion of the reaction, the reaction mixture,spin-drying acetic acid and extracting with PE:EA mixed solvent, dissolving the impurity point in organic phase, retaining water phase, extracting with dichloromethane, retaining organic phase, washing with saturated saline water, drying with anhydrous sodium sulfate to obtain pale yellow oily liquid, namely the compound N-Boc- (PEG) n -OH) 2 。
N-Boc- (PEG) n -OH) 2 (1 eq.) was added to a 250mL one-neck flask and dissolved by adding 100mL THF. NaOH (3 eq.) was dissolved in 10mL of water, cooled to room temperature in an ice bath, and slowly added to the flask in a low temperature circulation zone. TsCl (1.25 eq.) was dissolved in 10mL THF, and added dropwise to a single-necked flask with a constant pressure dropping funnel over 30min, taking care of the system temperature to be lower than 0 ℃ at any time, and the reaction was carried out in an ice bath for 12h. Adding NaOH to react for 4 hours at 40 ℃, hydrolyzing TsCl, spin-drying THF, adding dichloromethane to extract, retaining an organic phase, and spin-evaporating to obtain a light yellow transparent oily liquid, namely the compound N-Boc- (PEG) n -OTs) 2 。
In step S024, N-Boc- (PEG) n -OTs) 2 And compound R 1 H, removing Boc to obtain the compound shown as the formula (X2).
According to an embodiment of the present invention, the synthetic route of step S024 is shown in formula (9):
Specifically, N-Boc- (PEG) n -OTs) 2 (1 eq.) and Compound R i H (2.25 eq.) was added to a 50mL one-necked flask, 20mL DMF was added to dissolve, and the reaction was stirred until the reaction was dissolved. Adding K 2 CO 3 The reaction was stirred for 24h, during which time the plate confirms the progress of the reaction. After the reaction, DMF was removed by spin-evaporation, EA and water were added to extract, saturated brine was added to wash, the organic phase was kept, dried over anhydrous sodium sulfate, and after spin-drying, oily liquid was precipitated by immersing in a solution of diethyl ether: petroleum ether=10:1, to obtain a compound represented by formula (X2).
In step S03, a drug molecule R having a terminal carboxyl or hydroxyl group is prepared 2 Condensation reaction with dithio-diethanol to obtainA compound represented by the formula (X3).
R is as follows 2 Selected from drug molecules R linked to S-S bond by ester bond 2 ' or drug molecule R linked to S-S bond through ether bond 2 "one of the following.
According to an embodiment of the present invention, the synthetic route of the compound represented by formula (X3) is represented by formula (10) or formula (11):
according to an embodiment of the present invention, the synthesis process of the compound represented by formula (X3) specifically includes: the drug molecule R having a terminal carboxyl group or hydroxyl group 2 (R 2 Is R 2 ' or R 2 ",1.0 eq.) and 2,2' -dithiodiethanol (1.25 eq.) were added to a 100mL single neck flask and dissolved by adding 50mL THF solution. DMAP (1.0 eq.) and DCC (1.0 eq.) were added and the reaction was stirred at ambient temperature for 24h, during which time the plates were spotted and the progress of the reaction was confirmed with ninhydrin solution. After completion of the reaction, THF was dried by spin-drying, extracted with DCM and saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography (PE: EA) to give a white or pale yellow crystalline solid, i.e., a compound represented by the formula (X3).
In step S04, a compound represented by formula (X2) having a secondary amine group and a compound represented by formula (X3) are subjected to an amide synthesis reaction to obtain a releasable functional compound having a structure represented by formula (I).
According to an embodiment of the present invention, the synthetic route of the releasable functional compound having the structure shown in formula (I) is shown in formula (12) or formula (13):
according to an embodiment of the present invention, the synthesis process of the releasable functional compound having the structure shown in formula (I) specifically includes: the compound (1.0 eq.) and CDI (1.25 eq.) represented by formula (X3) were added to a 50mL one-neck flask, and dissolved by adding 10mL of toluene solution. At N 2 The reaction was stirred for 24h at 100℃under protection, during which time the plate was spotted and the progress of the reaction was confirmed with ninhydrin solution. After the reaction, CDI was removed by extraction with an aqueous solution of sodium dihydrogen phosphate having ph=5.0, and an organic phase was retained. The organic phase was charged into a 50mL one-necked flask, and 5mL toluene was added to dissolve the organic phase. Adding NH- (PEG) compound represented by formula (X2) dissolved in 2mL chloroform n1+n2 -R 1 ) 2 (1.1 eq.) at room temperature, stirring under nitrogen for 12h. The chloroform in the system is dried by spinning, the organic phase is remained by using DCM and water extraction, and the light yellow oily product is obtained after drying by using anhydrous sodium sulfate, namely the releasable functional compound with the structure shown as the formula (I).
The small molecule drug fragment (drug molecule R) containing the releasable functional compound having the structure of formula (I) 2 ) The PEG-disulfide bond molecule of (2) is taken as an inner core, and the polyethylene glycol block with hydrophilic property forms an outer group.
According to the releasable functional compound provided by the embodiment of the invention, the polyethylene glycol block with single molecular weight is adopted, so that the recognition of an anti-PEG antibody can be reduced, the induction of unnecessary immune rejection reaction can be avoided, and the biocompatibility of the releasable functional compound is improved.
According to the releasable functional compound provided by the embodiment of the invention, the releasable functional compound with an amide bond has high stability due to the fact that the amide bond is relatively stable.
The invention also provides a nanoparticle which is formed by assembling the releasable functional compound with the structure shown as the formula (I).
The invention also provides a preparation method of the nanoparticle, which comprises the following steps: dissolving a releasable functional compound with a structure shown as a formula (I) in dimethyl sulfoxide to obtain an organic solution; and (3) flash-sinking the organic solution into the aqueous solution of the phospholipid-polyethylene glycol to obtain the nano particles.
According to an embodiment of the invention, the concentration of the releasable functional compound in dimethyl sulfoxide is 0.5mg/mL; the mass ratio of releasable functional compound to phospholipid-poly L glycol was 1:1 to obtain stable nanoparticles.
According to the embodiment of the invention, the prepared nanoparticle containing the drug molecules can exist stably, has a better effect in cell and animal experiments, and can be applied to treatment of tumors.
According to the nanoparticle assembled by the releasable functional compound provided by the embodiment of the invention, the nanoparticle is injected in an injection mode, and the polyethylene glycol block in the nanoparticle passes through the active group R 1 Can be combined with albumin in blood, can increase the acting time of the nano particles in the blood, so that the nano particles have long-time stability in the blood, and are beneficial to improving the specificity and efficiency of the nano particles for targeting tumor tissues.
According to the nanoparticle of the releasable functional compound provided by the embodiment of the invention, the nanoparticle enters the cell, and the disulfide bond of the releasable functional compound is broken in the cell by the reduced glutathione existing in the cell, so that the drug molecule is released in the cell and the drug effect is exerted.
The following schematically illustrates a contemplated releasable functional compound and a method of preparing nanoparticles including the releasable functional compound. It should be noted that the examples are only specific embodiments of the present invention and are not intended to limit the scope of the present invention.
In addition, R is 1 Is maleimide, R 2 5-aminolevulinic acid (5-ALA) is exemplified.
Example 1
(1) Synthesizing a first intermediate compound represented by formula C1:
reference toThe drug molecule R is represented by the formula (1-1) 2 The (5-ALA) and Boc anhydride undergo an amide reaction to synthesize a first intermediate compound shown as a formula C1, namely, a tert-butoxycarbonyl (Boc) protected 5-ALA. Specifically, 5-ALA (3.0 g,22.9 mmol) was added to a 500mL single-neck flask, and 100mL aqueous sodium bicarbonate solution and 100mL Tetrahydrofuran (THF) were added and stirred until the reactants dissolved. Slowly adding Boc anhydride (di-tert-butyl dicarbonate, boc) 2 O,7.49g,34.4 mmol) was stirred at ambient temperature for 24h, during which time the plates were spotted and the progress of the reaction was confirmed with ninhydrin solution. After completion of the reaction, THF was dried by spin-drying, and the remaining Boc was removed by extraction with Petroleum Ether (PE) 2 O, the aqueous phase was retained and adjusted to ph=2 with 0.1M hydrochloric acid solution, EA was added to extract the aqueous solution, the organic phase was retained, and after spin-drying, a colorless crystalline solid was obtained, to obtain a first intermediate compound (Boc-5-ALA) as shown in formula C1.
(2) Synthesizing a second intermediate compound of formula C2:
referring to formula (1-2), the first intermediate compound (Boc-5-ALA) shown in formula C1 is subjected to esterification reaction with dithiodiethanol to synthesize a second intermediate compound shown in formula C2. Specifically, boc-5-ALA (3.0 g,22.9 mmol) and 2,2' -dithiodiethanol (3.0 g,22.9 mmol) as shown in C1 were added to a 100mL one-neck flask, and dissolved in 20mL THF solution. 4-Dimethylaminopyridine (DMAP) (7.49 g,34.4 mmol) and Dicyclohexylcarbodiimide (DCC) (7.49 g,34.4 mmol) were added thereto and reacted at room temperature with stirring for 24 hours while spotting the plate and confirming the progress of the reaction with ninhydrin solution. After completion of the reaction, THF was dried by spin-drying, extracted with Dichloromethane (DCM) and saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography (PE: ea=4:1) to give a white crystalline solid, i.e. the second intermediate compound represented by formula C2.
(3) Synthesizing furan-protected maleimide as shown in formula A3:
the synthesis of furan-protected maleimide is shown in the formula (1-3). Referring to formulas (1-3), maleimide (5 g,51.5 mmol) of formula A1 was taken in a 250mL round bottom flask, and after a further dehydration treatment in a solvent treatment system, 100mL of anhydrous toluene was taken and added with furan (C) of formula A2 4 H 4 O,14g,206 mmol), heated to 50℃and condensed at reflux for 12 hours. The reaction was cooled to room temperature, suction filtered to give a white solid, which was then washed with 20mL of toluene to give a furan-protected maleimide (8.2 g, 92.4%) as shown in formula A3.
(4) Synthesizing the tert-butoxycarbonyl-protected diethanolamine shown in the formula B2:
the process for preparing t-butoxycarbonyl-protected diethanolamine shown in formula B2 by reacting diethanolamine shown in formula B1 with Boc anhydride is shown in the following formula (1-4). Specifically, diethanolamine as shown in formula B1 was added to a 500mL single-necked flask, and 100mL of Tetrahydrofuran (THF) was added for dissolution, followed by 100mL of saturated aqueous sodium bicarbonate solution. The boc anhydride was slowly added with stirring and reacted at room temperature for 12 hours. After the reaction, spin-drying THF, adding EA and water for extraction, reserving an organic phase, adding saturated saline water for washing, and drying with anhydrous sodium sulfate to obtain colorless viscous liquid, namely the tert-butoxycarbonyl-protected diethanolamine shown in a formula B2.
(5) Synthesis of Boc-N- (OEG) of formula B3 4 -OTrt) 2 ：
Synthesis of Boc-N- (OEG) of formula B3 4 -OTrt) 2 The process of (2) is represented by the following formula (1-5). Specifically, OTs-PEG as raw material 4 Trt (5.0 g,6.5 mmol), t-butoxycarbonyl-protected diethanolamine of formula B2 (0.4 g,2.1 mmol) and NaH (1.5 g,4.3 mmol) were added to a 500mL one-neck flask, dissolved in 100mL THF, reacted at room temperature for 36h, cooled to 23℃and then dried with toluene, and the organic phase was extracted with EA, washed with saturated brine and dried over anhydrous sodium sulfate. The product was purified by column chromatography using EA and THF as mobile phases to give a colorless oil (2.4 g, 82%) which was Boc-N- (OEG) as shown in formula B3 4 -OTrt) 2 。
(6) Synthesis of Boc-N- (OEG) as shown in formula B5 4 -OTs) 2 ：
Boc-N- (OEG) as shown in formula B3 4 -OTrt) 2 Reacting with acetic acid to form ether, synthesizing Boc-N- (OEG) shown in formula B4 4 -OH) 2 The method comprises the steps of carrying out a first treatment on the surface of the Boc-N- (OEG) as shown in formula B4 4 -OH) 2 Reacting with TsCl to synthesize Boc-N- (OEG) shown in formula B5 4 -OTs) 2 . Synthesis of Boc-N- (OEG) as shown in formula B5 4 -OTs) 2 The processes of (1-6) and (1-7) are shown below.
Specifically, boc-N- (OEG) of formula B3 4 -OTrt) 2 (3.0 g,2.8 mmol) was put into a 100mL single-necked flask, a mixed solution of 40mL of acetic acid and 10mL of water was added, the reaction was heated to 40℃for 4 hours, and the completion of the reaction was confirmed by spotting. After the reaction, the acetic acid was dried by spinning and extracted with a mixed solvent of PE: ea=10:1, wherein the impurity was dissolved in the organic phase, the aqueous phase was kept and extracted with methylene chloride, the organic phase was kept, washed with saturated saline solution, and dried over anhydrous sodium sulfate to give a pale yellow oily liquid (1.12 g, 89%) which was Boc-N- (OEG) represented by formula B4 4 -OH) 2 。
Boc-N- (OEG) as shown in formula B4 4 -OH) 2 (1.5 g,2.7 mmol) was added to a 250mL single neck flask100mL of THF was added for dissolution. NaOH (1.08 g,27 mmol) was dissolved in 10mL of water, and after cooling to room temperature in an ice bath, the flask was slowly added in a low temperature circulation. TsCl (1.28 g,6.7 mmol) was dissolved in 10mL THF, and the mixture was added dropwise to a single-necked flask with a constant pressure dropping funnel over 30min, taking care that the system temperature was lower than 0℃at any time, and the reaction was carried out in an ice bath for 12h. Adding NaOH, reacting at 40deg.C for 4 hr, hydrolyzing TsCl, spin drying THF, extracting with dichloromethane, and steaming to obtain light yellow transparent oily liquid, namely Boc-N- (OEG) shown in formula B5 4 -OTs) 2 (1.6g，81％)。
(7) Synthesizing a compound shown as a formula B6:
Boc-N- (OEG) as shown in formula B5 4 -OTs) 2 Reacting with furan-protected maleimide shown as formula A3 to synthesize a compound shown as formula B6, wherein the reaction is shown as formula (2-8). Specifically, boc-N- (OEG) of formula B5 4 -OTs) 2 (1.0 g,1.1 mmol) and furan-protected maleimide (0.5 g,2.8 mmol) of formula A3 were added to a 50mL one-neck flask, dissolved in 20mL Dimethylformamide (DMF), and stirred until the reactants dissolved. Adding K 2 CO 3 The reaction was stirred for 24h, during which time the plate confirms the progress of the reaction. After the reaction, DMF is removed by spin evaporation, EA and water are added for extraction, saturated saline water is added for washing, an organic phase is reserved, anhydrous sodium sulfate is used for drying, and after spin drying, the mixture is immersed into a solution of diethyl ether and petroleum ether=10:1 to separate out oily liquid, namely the compound shown as a formula B6.
(8) Synthesizing polyethylene glycol chain segment NH--(OEG 4 -FuMI) 2 ：
And (3) reacting the compound shown as the formula B6 with dioxane hydrochloric acid solution to synthesize the polyethylene glycol chain segment shown as the formula B7. The process for synthesizing the polyethylene glycol segment represented by formula B7 is represented by the following formulas (1-9).
A50 mL one-necked flask was charged with the compound (1.2 g,1.4 mmol) represented by the formula B6, and 20mL of dioxane hydrochloric acid solution was added for dissolution. The reaction was stirred at room temperature for 12 hours. Spin-drying hydrochloric acid solution of dioxane in the system, then immersing the product into solution of petroleum ether and anhydrous ether=1:10, centrifuging, discarding upper liquid to obtain pale yellow oily product, namely synthesizing polyethylene glycol chain segment NH- (OEG) shown in formula B7 4 -FuMI) 2 。
(9) Synthesizing a third intermediate compound represented by formula C3:
the process of synthesizing the third intermediate compound shown in formula C3 by reacting the second intermediate compound shown in formula C2 with carbonyl imidazole is shown in the following formulas (1-10).
Specifically, a second intermediate compound (1.0 g,2.72 mmol) represented by formula C2 and carbonyl imidazole (CDI, 0.48g,2.99 mmol) were added to a 50mL one-neck flask, and dissolved by adding 5mL toluene solution. The reaction was stirred for 24h at 100℃under N2 protection, during which time the plates were spotted and the progress of the reaction was confirmed with ninhydrin solution. After the reaction, the CDI is removed by extraction with an aqueous solution of sodium dihydrogen phosphate having ph=5.0, and the organic phase is retained, thereby obtaining a third intermediate compound represented by formula C3.
(10) Synthesizing a fourth intermediate compound represented by formula C4:
the process of synthesizing the fourth intermediate compound shown in formula C4 by reacting the third intermediate compound shown in formula C3 with the polyethylene glycol segment shown in formula B7 is shown in the following formulas (1-11).
Specifically, a third intermediate compound (1.1 g,2.38 mmol) represented by formula C3 was charged into a 50mL one-neck flask, and 5mL toluene was added for dissolution. Polyethylene glycol segment (1.96 g,2.62 mmol) represented by formula B7 dissolved in 2mL of chloroform was added thereto, and the reaction was stirred under nitrogen for 12 hours at room temperature. The chloroform was dried over DCM and the organic phase was retained by water extraction and dried over anhydrous sodium sulfate to give the product as a pale yellow oil to give the fourth intermediate compound as shown in formula C4.
(11) Synthesis of a fifth intermediate compound Boc-5-ALA-2 (OEG) of formula C5 4 -MI)：
The process of dissolving the fourth intermediate compound represented by formula C4 in toluene to obtain the fifth intermediate compound represented by formula C5 is represented by the following formulas (1 to 12).
Specifically, a fourth intermediate compound (0.8 g,0.6 mmol) represented by formula C4 was charged into a 50mL one-neck flask, and 10mL toluene was added for dissolution. Continuously introducing nitrogen, reacting at 90 ℃ for 12 hours, and cooling to room temperature after the reaction is finished. The toluene in the system was dried by spin-drying, the organic phase was retained by extraction with DCM and water, and the product was obtained as a pale yellow oil after drying with anhydrous sodium sulfate to give a fifth intermediate compound, boc-5-ALA-2 (OEG), as shown in formula C5 4 -MI)。
(12) Synthesis of the releasable functional Compound 5-ALA-2 (OEG) having the Structure of formula (I) 4 -MI)：
The process for synthesizing the releasable functional compound having the structure of formula (I) by reacting the fifth intermediate compound represented by formula C5 with a dioxane hydrochloric acid solution is represented by the following formulas (1-13).
Specifically, the formula will beA fifth intermediate compound (0.3 g,0.4 mmol) shown in C5 was charged into a 50mL one-neck flask, and 10mL dioxane hydrochloride was added for dissolution. Stirring at room temperature under nitrogen for 12 hr, spin-drying the solvent after the reaction, extracting with DCM and water to obtain organic phase, and drying with anhydrous sodium sulfate to obtain pale yellow oily product, i.e. 5-ALA-2 (OEG) as final product 4 -MI)。
FIG. 1 shows the 5-ALA-2 (OEG) of example 1 of the invention 4 -MI).
FIG. 2 shows the 5-ALA-2 (OEG) of example 1 of the invention 4 -MI) time-of-flight mass spectrometry data.
Referring to fig. 1 and 2, the nuclear magnetic hydrogen spectrum result and the time-of-flight mass spectrum data represent successful synthesis of 5-ALA-2 (OEG) 4 -MI)。
With 5-ALA-2 (OEG) 4 -MI) is the assembly of exemplified Fan Nami particles:
weighing 5mg of 5-ALA-2 (OEG) 4 MI) (7 mmol) was dissolved in 100. Mu.L of dimethyl sulfoxide, sonicated to give a first solution, and 5mg of DSPE-PEG was dissolved in another 100. Mu.L of dimethyl sulfoxide 2000 Ultrasonic dissolution is performed to avoid a large number of bubbles from being generated by vibration and oscillation, and a second solution is obtained. And mixing the first solution and the second solution to obtain a mixed solution.
200. Mu.L of 5-ALA-2 (OEG) 4 -MI) and DSPE-PEG 2000 Is rapidly dropped into 1800 mu L of ultrapure water by flash precipitation, and is stirred for 5 minutes at the rotating speed of 500r/min to obtain clear solution which is nanoparticle solution.
Subsequently, 1mL of the nanoparticle solution was taken and placed in a dialysis membrane, and dialyzed in 5L of ultrapure water for 12 hours to obtain water-soluble nanoparticles. After lyophilization, redissolved and subjected to the next step of experiment.
Example 2
In addition, R is 1 Is succinimide, R 2 Is 5-aminolevulinic acid (5)ALA) is illustrated as an example.
5-ALA-2(OEG 4 -SI) and 5-ALA-2 (OEG) 4 -MI) differs in the synthesis process:
as shown in reference formula (2-1), boc-N- (OEG) as shown in formula B5 is used as the raw material 4 -OTs) 2 (1.0 g,1.1 mmol) and succinimide (0.3 g,2.4 mmol) were added to a 50mL single neck flask, dissolved in 20mL DMF and stirred until the reactants dissolved. Adding K 2 CO 3 The reaction was stirred for 24h, during which time the plate confirms the progress of the reaction. After the reaction, DMF is removed by spin evaporation, EA and water are added for extraction, saturated saline water is added for washing, an organic phase is reserved, anhydrous sodium sulfate is used for drying, and after spin drying, the mixture is immersed into a solution of diethyl ether and petroleum ether=10:1 to separate out oily liquid, namely the compound shown as a formula B6'.
As shown by reference to formula (2-2), the compound represented by formula B6' (0.8 g,1.0 mmol) was put into a 50mL one-neck flask, and 20mL of dioxane was added thereto for dissolution. The reaction was stirred at room temperature for 12 hours. Spin-drying hydrochloric acid solution of dioxane in the system, then immersing the product into solution of petroleum ether and anhydrous ether=1:10, centrifuging, discarding upper liquid to obtain pale yellow oily product, namely synthesizing polyethylene glycol segment NH- (OEG) shown as formula B7 4 -SI) 2 。
Referring to formula (2-3), a third intermediate compound (0.8 g,0.9 mmol) represented by formula C3 was added to a 50mL one-neck flask, and dissolved in 5mL toluene. Polyethylene glycol segment (1.0 g,1.5 mmol) as shown in formula B7' dissolved in 2mL of chloroform was added. The reaction was stirred at room temperature under nitrogen for 12 hours. The chloroform was dried over DCM and water to leave an organic phase which was dried over anhydrous sodium sulfate to give the product as a pale yellow oilTo NH-Boc- (OEG) as shown in formula C4' below 4 -SI) 2 。
Referring to formula (2-4), the compound represented by formula C4' (0.3 g,0.4 mmol) was charged into a 50mL one-neck flask, and 10mL dioxane hydrochloride was added to dissolve. Stirring at room temperature under nitrogen for 12 hr, spin-drying the solvent after the reaction, extracting with DCM and water to obtain organic phase, and drying with anhydrous sodium sulfate to obtain pale yellow oily product as final product 5-ALA-2 (OEG) 4 -SI)。
FIG. 3 shows the 5-ALA-2 (OEG) of example 2 of the invention 4 -SI).
FIG. 4 shows the 5-ALA-2 (OEG) of example 2 of the invention 4 -SI) time-of-flight mass spectrometry data.
Referring to fig. 1 and 2, the nuclear magnetic hydrogen spectrum result and the time-of-flight mass spectrum data represent successful synthesis of 5-ALA-2 (OEG) 4 -SI)。
FIG. 5 shows the 5-ALA-2 (OEG) of example 1 of the invention 4 -MI) assembled nanoparticles and 5-ALA-2 (OEG) of example 2 4 SI) particle size distribution profile of assembled nanoparticles.
Referring to FIG. 5, 5-ALA-2 (OEG 4 -MI) assembled nanoparticles with an average diameter of 182nm, a dispersion coefficient of 0.206, a particle size distribution map characterizing the 5-ALA-2 (OEG) obtained synthetically 4 -MI) the particle size distribution of the assembled nanoparticles is uniform. 5-ALA-2 (OEG) 4 -SI) the assembled nanoparticles have an average diameter of 179nm, a dispersion coefficient of 0.222, and a particle size distribution map characterizing the 5-ALA-2 (OEG) obtained synthetically 4 SI) the particle size distribution of the assembled nanoparticles is uniform.
FIGS. 6A-6B show 5-ALA-2 (OEG) of example 1 of the invention 4 -MI) assembled nanoparticlesSub-and 5-ALA-2 (OEG) of example 2 4 SI) cytotoxicity of assembled nanoparticles.
Referring to FIG. 6A, 5-ALA-2 (OEG 4 MI) the assembled nanoparticles enter HepG2 cells (one of hepatoma cells), the HepG2 cells remain highly active. In the concentration range of 3 mu M-12 mu M of drug molecules and nanoparticles, the cells are in the range of 5-ALA, 5-ALA-2 (OEG) 4 -MI) and 5-ALA-2 (OEG 4 -SI) shows good cell activity when incubated together, indicating that the drug molecule 5-ALA, 5-ALA-2 (OEG) synthesized in example 1 4 -MI), 5-ALA-2 (OEG) synthesized in example 2 4 SI) has little influence on cell activity, and has good physiological activity and safety.
Referring to FIG. 6B, the activity of HepG2 cells was reduced by photodynamic therapy (PDT), and a part of HepG2 cells died during the treatment, indicating that photodynamic therapy (PDT) can combat cancer cells. In the range of 10. Mu.M-12. Mu.M, the concentration of the nanoparticles was 5-ALA-2 (OEG 4 -MI) reaches IC 50 (half-inhibitory concentration), where half-inhibitory concentration means half of the cells are killed. In contrast to fig. 6A, it is demonstrated that cancer cells can only be killed by drugs under photodynamic therapy (PDT). That is, the nanoparticles enter the cells, but do not undergo photodynamic therapy, and do not damage the cells; after photodynamic therapy, the phenomenon of killing cells only occurs. Therefore, even if the drug-loaded nanoparticle remains in normal cells, it will not cause damage to normal cells, and the safety and practical applicability are excellent.
FIG. 7 shows the 5-ALA-2 (OEG) of example 1 of the invention 4 -MI) assembled nanoparticles and 5-ALA-2 (OEG) of example 2 4 -SI) relative fluorescence intensity of the nanoparticle in cells in vitro.
Referring to FIG. 7, 5-ALA-2 (OEG 4 -MI) assembled nanoparticle reached the peak of relative fluorescence intensity at 10h, 5-ALA drug molecule reached the peak of relative fluorescence intensity at 8h, indicating 5-ALA-2 (OEG) 4 MI) the peak value of the relative fluorescence intensity of the assembled nanoparticle with respect to the 5-ALA drug molecule is shifted backward, achieving the purpose of prolonging the action time.
FIG. 8 shows the 5-ALA-2 (OEG) of example 1 of the invention 4 -MI) assembled nanoparticles and 5-ALA-2 (OEG) of example 2 4 SI) organelle co-localization picture of assembled nanoparticles under confocal fluorescence microscopy.
Referring to FIG. 8, the region of action of 5-ALA drug molecules in cells is in-line with the granulocytes, 5-ALA-2 (OEG 4 -MI) assembled nanoparticles, 5-ALA-2 (OEG 4 -SI) the region of action of the assembled nanoparticle in the cell is also in the line with the particle, indicating 5-ALA-2 (OEG) 4 -MI) assembled nanoparticles, 5-ALA-2 (OEG 4 SI) assembled nanoparticles the same process as 5-ALA drug molecules.
FIG. 9 shows the 5-ALA-2 (OEG) of example 1 of the invention 4 -MI) assembled nanoparticles and 5-ALA-2 (OEG) of example 2 4 SI) in-animal imaging of assembled nanoparticles.
Bab/c females (6 weeks) were chosen as the main biological model for the experiment. Subcutaneous injections (50 mg/kg per injection), intramuscular injections (50 mg/kg per injection) and intravenous injections (25 mg/kg per injection) of nanoparticle solutions were performed subcutaneously at the back hind legs, intramuscularly at the front hind legs and intravenously at the tail veins of the mice, respectively. And fluorescence of intramuscular and subcutaneous injection sites (dashed circles in fig. 9) was observed in the small animal imager at different times, i.e., at 0h, 1h, 2h, 3h, 5h, 7h, 12h, respectively. Since intravenous drug molecules and nanoparticle molecules can be transmitted to the whole body from blood, fluorescence of the back of the mice can be observed in a small animal imager.
Since 5-ALA itself has fluorescence at an excitation wavelength of 445nm, 5-ALA-2 (OEG) n -MI) nanoparticles and 5-ALA-2 (OEG 4 SI) nanoparticles, the nanoparticles are metabolized into cells and then can generate fluorescence, and the observed fluorescence indicates that the nanoparticles exert efficacy in mice.
Referring to FIG. 9, a 5-ALA drug molecule, 5-ALA-2 (OEG) 4 -MI) nanoparticles and 5-ALA-2 (OEG 4 -SI) nanoparticles, intravenous injection of mice, respectively Injection, intramuscular injection and subcutaneous injection.
In the intravenous group, 5-ALA-2 (OEG 4 -MI) the assembled nanoparticle reached the strongest fluorescence intensity at 7h, indicating that the drug at this time reached the peak intracellular concentration, which was the optimal time of action for the drug; the fluorescence intensity of the 5-ALA drug molecules reaches the peak value in 3 hours, and then gradually decreases; 5-ALA-2 (OEG) 4 SI) fluorescence of the assembled nanoparticle peaks at 5 h. It follows that, in contrast to the 5-ALA drug molecule, 5-ALA-2 (OEG 4 -MI) assembled nanoparticles and 5-ALA-2 (OEG 4 SI) assembled nanoparticles all achieve the purpose of prolonged action. This is due to 5-ALA-2 (OEG) 4 -MI) assembled nanoparticles and 5-ALA-2 (OEG 4 SI) assembled nanoparticles are slowly released from the package, prolonging the duration of action.
In the intramuscular injection group, 5-ALA appeared to fluoresce significantly at the injection site (in the virtual circle) for 3-5h, but gradually disappeared after 7h, indicating that 3-5h is the optimal time of action of the 5-ALA drug molecule; 5-ALA-2 (OEG) 4 MI) assembled nanoparticles were fluorescent at 3h and were still present up to 12h, indicating that 5-ALA-2 (OEG) 4 -MI) the assembled nanoparticle has an effect of a long-term presence compared to 5-ALA; 5-ALA-2 (OEG) 4 SI) the assembled nanoparticle shows fluorescence after 2h and gradually disappears after 7 h. It follows that, in contrast to the 5-ALA drug molecule, 5-ALA-2 (OEG n -MI) assembled nanoparticles and 5-ALA-2 (OEG 4 SI) assembled nanoparticles all achieve the purpose of prolonged action. 5-ALA-2 (OEG) 4 -SI) assembled nanoparticles compared to 5-ALA-2 (OEG) 4 SI) the effect of the assembled nanoparticles on prolonged action is better.
In the subcutaneous injection group, 5-ALA fluoresced at 2h, with fluorescence substantially disappearing at 5 h; 5-ALA-2 (OEG) 4 MI) the assembled nanoparticle fluoresced at 2h, with the strongest fluorescence at 5h, and gradually decreased after 7 h; 5-ALA-2 (OEG) 4 SI) assembled nanoparticles fluoresce at 2h, with the strongest fluorescence at 7h and substantially vanish after 12 h.
It follows that both intravenous and intramuscular injections, as wellIs subcutaneously injected, compared with 5-ALA drug molecule, 5-ALA-2 (OEG) 4 -MI) assembled nanoparticles and 5-ALA-2 (OEG 4 SI) assembled nanoparticles have the effect of extending the duration of action.
The use of ordinal numbers such as "first," "second," "third," etc., in the description and the claims to modify a corresponding element does not by itself connote any ordinal number of elements or the order of manufacturing or use of the ordinal numbers in a particular claim, merely for enabling an element having a particular name to be clearly distinguished from another element having the same name.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.

Claims (10)

1. A releasable functional compound containing a polyethylene glycol segment of single molecular weight, having a structure as shown in formula (I):
wherein R is 1 Representing reactive groups suitable for binding to biological macromolecules;
R 2 a drug molecule in which S-S bonds are linked through an ester bond or an ether bond;
PEG n represents a polyethylene glycol segment having a single molecular weight;
n is an integer of 4 to 500.
2. The releasable functional compound of claim 1, wherein PEG n The structural formula of (2) is as follows:
3. the releasable functional compound of claim 1, wherein R 2 Selected from any one of the following structures:
4. the releasable functional compound of claim 1, wherein R 1 Selected from any one of the following structures:
Wherein R is 3 Selected from H, methyl, chlorine, bromine or iodine;
R 4 selected from any one of the following structures:
5. the releasable functional compound of claim 1, wherein the molecular weight of the releasable functional compound is 1000 to 100000.
6. The releasable functional compound of claim 1, wherein the releasable functional compound has any one of the following structures:
7. a method for producing the releasable functional compound according to any one of claims 1 to 6, comprising:
providing a polyethylene glycol chain segment with single molecular weight, wherein the structure of the polyethylene glycol chain segment with single molecular weight is shown as a formula (X1);
Trt-PEG n OTs (X1)
A polyethylene glycol chain segment with single molecular weight, diethanolamine and a compound R shown in a formula (X1) 1 H, reacting to obtain a compound with a secondary amine group, wherein the compound is shown as a formula (X2);
the drug molecule R having a terminal carboxyl group or hydroxyl group 2 Carrying out condensation reaction with dithiodiethanol to obtain a compound shown as a formula (X3);
subjecting a compound represented by the formula (X2) having a secondary amine group and a compound represented by the formula (X3) to an amide synthesis reaction to obtain a releasable functional compound having a structure represented by the formula (I)
8. The process according to claim 7, wherein the drug molecule R having a terminal carboxyl or hydroxyl group 2 The condensation reaction with dithiodiethanol comprises:
the drug molecule R having a terminal carboxyl group or hydroxyl group 2 Dissolving the extract and dithiodiethanol in tetrahydrofuran solution;
adding 4-dimethylaminopyridine and dicyclohexylcarbodiimide into the tetrahydrofuran solution for condensation reaction;
preferably, the amide synthesis reaction of the compound represented by the formula (X2) and the compound represented by the formula (X3) having a secondary amine group includes:
dissolving a compound represented by the formula (X3) and carbonyl imidazole in toluene, at N 2 Stirring for reaction under the protection and heating conditions;
after the reaction is completed, carbonyl imidazole in the reaction liquid is removed, an organic phase is reserved, and the organic phase is dissolved in toluene;
adding a compound represented by the formula (X2) to toluene in which an organic phase is dissolved, and adding a compound represented by the formula (X2) to toluene in an amount of N 2 Stirring for reaction under protection.
9. A nanoparticle formed by assembling the releasable functional compound according to any one of claims 1 to 6.
10. A method of preparing the nanoparticle of claim 9, comprising:
Dissolving the releasable functional compound in dimethyl sulfoxide to obtain an organic solution;
and flash-sinking the organic solution into an aqueous solution of phospholipid-polyethylene glycol to obtain the nano particles.
CN202311048994.XA 2023-08-18 2023-08-18 Releasable functional compound containing polyethylene glycol chain segment with single molecular weight, nanoparticle containing same and preparation method Pending CN117126095A (en)

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