Releasable functional compounds containing single molecular weight polyethylene glycol segments, nanoparticles comprising the same, and methods of preparation

Abstract

This invention discloses a releasable functional compound containing a single molecular weight polyethylene glycol (PEG) segment, having the structure shown in formula (I): Formula (I) wherein R1 represents an active group suitable for binding to biomolecules; R2 represents a drug molecule connected by an S-S bond via an ester bond or an ether bond; PEG n This represents a polyethylene glycol segment with a single molecular weight; n is an integer from 4 to 500.

Description

Technical Field

[0001] At least one embodiment of the present invention relates to a releasable functional compound, and more particularly to a releasable functional compound containing a single molecular weight polyethylene glycol segment, nanoparticles containing the same, and a method for its preparation. Background Technology

[0002] Polyethylene glycol (PEG) is a high molecular weight polymer with good water solubility and compatibility with many organic components. PEGylation refers to a common method of covalently or non-covalently linking polyethylene glycol (PEG) to small molecule drugs, contrast agents, proteins, nucleic acids, liposomes, and other pharmaceuticals. This strategy imparts certain improvements to the water dispersibility, stability, pharmacokinetics, and pharmacodynamics of the resulting PEGylated drugs. However, traditional polydisperse PEGs are a series of mixtures, which can complicate the synthesis and purification of PEGylated drugs and trigger unwanted immunogenic reactions, ultimately affecting therapeutic efficacy.

[0003] Furthermore, although polydisperse PEGylated drugs have been widely used, the absorption of non-specific proteins by PEGylated drugs has still been observed. The resulting protein crown directly determines the fate of PEGylated drugs in vivo, affecting their targeting specificity and efficiency. Summary of the Invention

[0004] In view of this, the present invention provides a highly stable releaseable functional compound containing a single molecular weight polyethylene glycol segment, nanoparticles containing the same, and a preparation method thereof, to prolong the action time of drug molecules in the blood and have lower immunogenicity and protein adsorption, thereby enabling efficient delivery of drug molecules into cells to release drug molecules into the cells.

[0005] As one aspect of the present invention, the present invention provides a releasable functional compound containing a single molecular weight polyethylene glycol segment, having a structure as shown in formula (I):

[0006] Formula (I)

[0007] Wherein, R1 represents the active group, suitable for binding with biological macromolecules; R2 represents a drug molecule linked by an ester or ether bond; PEG n This represents a polyethylene glycol segment with a single molecular weight; n is an integer from 4 to 500.

[0008] As another aspect of the present invention, the present invention provides a method for preparing the above-mentioned releasable functional compound, comprising:

[0009] Provide polyethylene glycol segments of a single molecular weight, the structure of which is shown in formula (X1);

[0010] Equation (X1)

[0011] A single molecular weight polyethylene glycol segment as shown in formula (X1), diethanolamine, and compound R1H are reacted to obtain a compound as shown in formula (X2) having a secondary amine group.

[0012] Equation (X2)

[0013] Drug molecule R2 with terminal carboxyl or hydroxyl groups is condensed with dithiodiethanol to obtain a compound as shown in formula (X3).

[0014] Equation (X3)

[0015] The compounds of formula (X2) and (X3) having secondary amine groups were subjected to an amide synthesis reaction to obtain a releasable functional compound having the structure shown in formula (I).

[0016] Formula (I).

[0017] As another aspect of the present invention, the present invention provides a nano-example which is assembled using the above-described releasable functional compound.

[0018] According to the above embodiments of the present invention, the releaseable functional compound uses a single molecular weight polyethylene glycol block, which can reduce the recognition of anti-PEG antibodies, thereby avoiding the induction of unnecessary immune rejection reactions and improving the biocompatibility of the releaseable functional compound.

[0019] According to the above embodiments of the present invention, the releasable functional compound having amide bonds has high stability due to the relatively stable amide bonds.

[0020] The nanoparticles assembled from the releasable functional compounds provided in the above embodiments of the present invention are injected by injection. The polyethylene glycol block in the nanoparticles can bind to albumin in the blood through the active group R1, which increases the duration of action of the nanoparticles in the blood and makes the nanoparticles stable in the blood for a long time, which helps to improve the specificity and efficiency of the nanoparticles in targeting tumor tissues.

[0021] The nanoparticles formed by assembling the releaseable functional compounds according to the above embodiments of the present invention enter the cells, and the reduced glutathione present in the cells causes the disulfide bonds of the releaseable functional compounds to break in the cells, thereby releasing drug molecules into the cells and exerting a drug effect. Attached Figure Description

[0022] Figure 1 The 1H NMR spectrum of 5-ALA-2(PEG4-MI) of Example 1 of the present invention is shown;

[0023] Figure 2 The time-of-flight mass spectrometry data of 5-ALA-2 (PEG4-MI) in Embodiment 1 of the present invention are shown;

[0024] Figure 3 The 1H NMR spectrum of 5-ALA-2(PEG4-SI) of Example 2 of the present invention is shown;

[0025] Figure 4 The time-of-flight mass spectrometry data of 5-ALA-2 (PEG4-SI) in Example 2 of the present invention are shown;

[0026] Figure 5 The particle size distribution diagrams of the 5-ALA-2 (PEG4-MI) assembled nanoparticles of Example 1 and the 5-ALA-2 (PEG4-SI) assembled nanoparticles of Example 2 are shown.

[0027] Figures 6A-6B The cytotoxicity of the 5-ALA-2 (PEG4-MI) assembled nanoparticles of Example 1 and the 5-ALA-2 (PEG4-SI) assembled nanoparticles of Example 2 of the present invention is shown.

[0028] Figure 7 The relative fluorescence intensities of the 5-ALA-2 (PEG4-MI) assembled nanoparticles of Example 1 and the 5-ALA-2 (PEG4-SI) assembled nanoparticles of Example 2 are shown in vitro and in cells.

[0029] Figure 8 Images of organelle colocalization of 5-ALA-2 (PEG4-MI) assembled nanoparticles of Example 1 and 5-ALA-2 (PEG4-SI) assembled nanoparticles of Example 2 are shown under a confocal fluorescence microscope; and

[0030] Figure 9 Animal intracellular imaging is shown of the 5-ALA-2 (PEG4-MI) assembled nanoparticles of Example 1 and the 5-ALA-2 (PEG4-SI) assembled nanoparticles of Example 2 of the present invention. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and the accompanying drawings. However, this invention can be implemented in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to make the invention thorough and complete, and to fully convey the scope of the invention to those skilled in the art. In the accompanying drawings, for clarity, the dimensions and relative dimensions of layers and regions may be exaggerated, and the same reference numerals denote the same elements throughout.

[0032] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The terms “comprising,” “including,” etc., as used herein indicate the presence of the stated features, steps, operations, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, or components.

[0033] Small molecule drugs often suffer from problems in practical applications, such as rapid metabolism, the need for multiple injections, and easy metabolism. These drawbacks significantly limit the practical application of small molecule anticancer drugs. The most common methods for preparing derivatives of anticancer small molecules include liposome encapsulation and grafting small molecules onto polymers to extend circulation time. However, due to the potential for immune responses, as well as cellular and in vivo toxicity, these methods have limited application prospects.

[0034] Albumin possesses a free sulfhydryl group, and maleimide can undergo a highly efficient Michael addition reaction with albumin. This invention designs different linker units to connect a small molecule drug (functional molecule R2) to a maleimide (MI) group. MI not only binds to endogenous albumin in situ in the blood, prolonging the drug's half-life, but also avoids liver and spleen accumulation, targeting tumors and facilitating penetration of vascular endothelium into the tumor interior. During blood circulation, the maleimide groups on the nanoparticle surface covalently couple with serum albumin, forming an albumin-rich protein crown. Nanoparticles with this albumin-rich protein crown cannot be rapidly cleared by Kupffer cells in the liver; instead, they penetrate into the tumor stroma via Gp60 receptor-mediated endocytosis. A reducing environment (GSH) triggers partial decomposition of the nanoparticles, transforming them into smaller nanoparticles for deeper tumor penetration. Ultimately, the nanoparticles can deliver chemotherapy by completely releasing the cisplatin small molecule drug.

[0035] Disulfide bonds (SS bonds) are important chemical functional groups, mainly found in various natural small molecule compounds and the structures of proteins in organisms. In vivo, the main functions of disulfide bonds include helping to solidify protein structures, controlling cell signal transduction, and participating in metabolic pathways. Because disulfide bonds have low toxicity in vivo and can be broken in the presence of reduced glutathione (GSH) in the external environment, this invention introduces disulfide bonds into the drug structure to achieve better therapeutic effects.

[0036] Meanwhile, thanks to recent advancements in the synthesis of monodisperse polyethylene glycol (PEG), monodisperse PEG, which shares the same chemical structure as polydisperse PEG but with a more precise molecular weight, is now available. This invention envisions further regulating the protein crown on the surface of the delivery system by modifying the terminal groups of PEG, based on the construction of precisely PEGylated derivatives.

[0037] Using small molecule derivatives and coating them with polyethylene glycol can significantly improve the stability of drug nanoparticles. On the other hand, using maleimide as a component that helps the molecule better increase its circulation time in vivo can provide longer duration of action and higher therapeutic efficiency for medical treatments.

[0038] In view of this, the present invention provides a highly stable releaseable functional compound containing a single molecular weight polyethylene glycol segment. Through the design of the active group, especially the selection of maleimide, the releaseable functional compound achieves targeted action and releases the drug molecule loaded with the releaseable functional compound into the cell through the breaking of disulfide bonds.

[0039] According to an exemplary embodiment of the present invention, the present invention provides a releasable functional compound containing a single molecular weight polyethylene glycol segment, having a structure as shown in formula (I):

[0040] Formula (I)

[0041] Wherein, R1 represents the active group, suitable for binding with biological macromolecules; R2 represents a drug molecule linked by an ester or ether bond; PEG n This represents a polyethylene glycol segment with a single molecular weight, where n is an integer from 4 to 500.

[0042] According to an embodiment of the present invention, PEG n The structural formula is shown below:

[0043] .

[0044] According to embodiments of the present invention, the degree of polymerization n can be 4, 10, 50, 100, or 500. It should be noted that when n is less than 4, the degree of polymerization of PEG is too low, resulting in poor water solubility of the formed nanoparticles; while when the degree of polymerization is greater than 500, the formed polymer chains are too large in molecular weight and are not easily soluble in water.

[0045] According to embodiments of the present invention, R2 is selected from either drug molecule R2' connected by an ester bond or drug molecule R2'" connected by an ether bond, and R2' is selected from any of the following structures:

[0046] .

[0047] According to an embodiment of the present invention, R2” is selected from any of the following structures:

[0048] .

[0049] It should be noted that the drug molecule corresponding to R2 is selected from 5-aminolevulinic acid, acetaminophen, allopurinol, topotecan, tetrahydro-silverinol, oxazepam-related substance A, dihydrocarbamazepine, morphine, 7-ethyl-10-hydroxycamptothecin, berberine isomer A, atropine sulfate, testosterone, irinotecan, chloroberberine, cefoperazone, chrysanthemumone, lopinavir, berberine isomer B, paclitaxel, camptothecin, daunorubicin, berberine, gemcitabine, hydroxycholesterol, hydroxycholesterol ester, or 2-hydroxy-hydroxycholesterol.

[0050] It should be noted that the main therapeutic effects of the drug molecule corresponding to R2 are: fighting cancer, treating inflammation, inhibiting chronic gout and hypertension, acting on the nervous system and relieving pain, regulating arteriosclerosis and gallstones, etc.

[0051] According to an embodiment of the present invention, the drug molecule corresponding to R2 is preferably paclitaxel, which has a mediating function in cells, and the increase of PEG can stimulate the immune effect in vivo.

[0052] According to an embodiment of the present invention, R1 is selected from any of the following structures:

[0053]

[0054] It should be noted that R3 is selected from -H, -CH3, chlorine, bromine or iodine.

[0055] It should be noted that R4 is selected from any of the following structures:

[0056] .

[0057] According to embodiments of the present invention, the molecular weight of the above-mentioned releasable functional compound is 1,000 to 100,000, for example, it can be 1,000, 2,000, 5,000, or 10,000.

[0058] It should be noted that R1 is preferably maleimide. Maleimide with double bonds can undergo a highly efficient Michael addition reaction with albumin, thereby efficiently capturing albumin through covalent bonds to form an albumin-rich protein crown that is not easily cleared by the immune system, which helps to target tumor tissue.

[0059] It should be noted that active groups with carboxyl and hydroxyl groups can bind to amino groups in proteins, and active groups with amino groups can form hydrogen bonds with oxygen-containing parts of proteins, enabling releaseable functional compounds to achieve targeted effects.

[0060] According to embodiments of the present invention, the above-described releasable functional compound has any one of the following structures:

[0061] .

[0062] According to an exemplary embodiment of the present invention, the present invention provides a method for preparing a releasable functional compound having the structure of formula (I) as described above, comprising: steps S01 to S04.

[0063] In step S01, a polyethylene glycol segment of a single molecular weight is provided, the structure of which is shown in formula (X1).

[0064] Equation (X1)

[0065] According to an embodiment of the present invention, the synthesis process of a single molecular weight polyethylene glycol segment is shown in the following formula (1), including steps S011 to S013:

[0066]

[0067] Equation (1)

[0068] Where n = n1 + n2 = 4 ~ 100.

[0069] In step S011, the first polyethylene glycol (PEG) of a single molecular weight is... n1 Hydrophobic capping of the terminal hydroxyl groups yields hydrophobically capped, single-molecular-weight first polyethylene glycol Trt-PEG. n1 -OTrt;

[0070] In step S012, hydrophobically end-capped single molecular weight polyethylene glycol Trt-PEG is... n1-OTrt selectively deprotects one end of its hydroxyl group to obtain a single molecular weight polyethylene glycol Trt-PEG with one end hydrophobically capped. n1 -OH;

[0071] In step S013, a single molecular weight polyethylene glycol with one end hydrophobically capped is protected with a p-toluenesulfonyl group to obtain the structure Trt-PEG of a single molecular weight polyethylene glycol segment. n1 -OTs;

[0072] In step S014, the same operation as in steps S011-S012 is used to obtain a single molecular weight second polyethylene glycol Trt-PEG with one end hydrophobically capped. n2 -OH;

[0073] In step S015, the structure of the first polyethylene glycol segment with a single molecular weight, Trt-PEG, is... n1 -OTs and a single-molecular-weight second polyethylene glycol Trt-PEG with one end hydrophobically capped. n2 The -OH groups undergo a condensation reaction to yield hydrophobically capped, single-molecular-weight third polyethylene glycol Trt-PEG. n1+n2 -OTrt;

[0074] In step S016, hydrophobically capped single molecular weight third polyethylene glycol Trt-PEG is... n1+n2 -OTrt iterates to a new hydrophobically capped single molecular weight first polyethylene glycol, and repeats steps S012 to S016 until the structure of the single molecular weight first polyethylene glycol segment reaches the target molecular weight.

[0075] According to an embodiment of the present invention, when n is 4, the synthetic route of the polyethylene glycol segment with a single molecular weight is shown in formula (2) below, specifically including: adding tetraethylene glycol (200g, 1mol) and toluene (100mL) to a 500mL two-necked flask, azeotropically removing water, adding DIPEA (N,N-diisopropylethylamine) under N2 atmosphere in an ice bath, and then slowly adding TrtCl (56g, 0.2mol), reacting for 4 hours under N2 atmosphere. After the reaction is completed, the mixture is extracted with water and ethyl acetate (EA), washed with saturated sodium chloride water, and rotary evaporated to obtain the organic compound Trt-PEG4-OH.

[0076] Add the above product Trt-PEG4-OH and THF (250 mL) to a 1 L flask, and place in an ice bath. Add NaOH (32 g, 0.8 mol) to 125 mL of water, and place in an ice bath below 10°C. Add the NaOH aqueous solution to the system and stir in an ice bath for 10 minutes. Dissolve TsCl (44 g, 0.23 mol) in 100 mL of THF and add it to the system through a constant pressure dropping funnel while continuously in an ice bath. Maintain the reaction in an ice bath for the first 6 hours, and then allow the reaction to proceed to room temperature for 12 hours. After the reaction is complete, extract with EA and water, wash with saturated brine, dry the organic phase, and purify by column chromatography (PE:EA = 10:1) to obtain the colorless and transparent product Trt-PEG4-OTs.

[0077]

[0078] Equation (2)

[0079] According to an embodiment of the present invention, when n is 8, the synthetic route of the single molecular weight polyethylene glycol segment is shown in formula (3), specifically including: dissolving the above product Trt-PEG4-OTs (0.1 mol) in 100 mL of toluene, adding tetraethylene glycol (0.12 mol), azeotropically removing water from the toluene, then adding 200 mL of anhydrous THF, slowly adding NaH (1.4 mol), reacting at room temperature under N2 atmosphere for 12 h, after the reaction is completed, adding EA and water for extraction, washing with saturated brine, drying the organic phase, and purifying by column chromatography (PE:EA=2:1) ​​to obtain the colorless and transparent product Trt-PEG8-OH.

[0080] Take 1 eq. of the obtained Trt-PEG8-OH and add it to 200 mL of THF. Place the mixture in an ice bath. Add 3.0 eq. of NaOH to 125 mL of water and place the mixture in an ice bath below 10°C. Add the NaOH aqueous solution to the system and stir in an ice bath for 10 minutes. Dissolve 1.25 eq. of TsCl in 100 mL of THF and add it to the system through a constant-pressure dropping funnel while continuously in an ice bath. Maintain the reaction in an ice bath for the first 6 hours, then allow the reaction to proceed to room temperature for 12 hours. After the reaction is complete, extract with EA and water, wash with saturated brine, dry the organic phase, and purify by column chromatography (PE:EA = 2:1) to obtain the colorless and transparent product Trt-PEG8-OTs.

[0081]

[0082] Equation (3)

[0083] According to an embodiment of the present invention, when n is 16, the synthetic route for a single molecular weight polyethylene glycol segment is shown in formula (4). Taking 7.5 g (10 mmol) Trt-PEG8-OTs raw material as an example, the preparation process specifically includes:

[0084] 7.5 g (10 mmol) of Trt-PEG8-OTs was dissolved in 100 mL of toluene, and 6.2 g (11 mmol) of Trt-PEG8-OH was added. The mixture was azeotropically dehydrated in toluene, followed by the addition of 200 mL of anhydrous THF and the slow addition of NaH (10 eq.). The reaction was carried out at room temperature under a N2 atmosphere for 12 h. After the reaction was completed, EA and water were added for extraction, followed by washing with saturated brine. The organic phase was dried and purified by column chromatography (PE:EA = 1:1) to obtain the colorless and transparent product Trt-PEG. 16 -OTrt.

[0085] The obtained Trt-PEG 16 -OTrt (1 eq.) was added to 100 mL of MeOH, and TsOH (0.1 eq.) was dissolved in 10 mL of MeOH. The solutions were added dropwise to the system at room temperature, and the reaction was allowed to proceed for 12 hours at room temperature. After the reaction was complete, DCM and water were added for extraction, followed by washing with saturated brine. The organic phase was dried and purified by column chromatography (EA:MeOH = 10:1) to obtain the colorless and transparent product Trt-PEG. 16 -OH.

[0086] The obtained Trt-PEG 16 Add 1 eq. of -OH to 200 mL of THF and heat in an ice bath. Add 3.0 eq. of NaOH to 125 mL of water and heat in an ice bath below 10°C. Add the NaOH aqueous solution to the system and stir in an ice bath for 10 minutes. Dissolve 1.25 eq. of TsCl in 100 mL of THF and add it to the system through a constant pressure dropping funnel while continuously heating in an ice bath. Maintain the reaction in an ice bath for the first 6 hours, then allow the reaction to proceed to room temperature for 12 hours. After the reaction is complete, extract with EA and water, wash with saturated brine, dry the organic phase, and purify by column chromatography (EA:PE = 5:1) to obtain the colorless and transparent product Trt-PEG. 16 -OTs.

[0087]

[0088] Equation (4)

[0089] According to an embodiment of the present invention, when n is 24, the synthetic route of a single molecular weight polyethylene glycol segment is shown in formula (5), specifically including:

[0090] Take the synthesized Trt-PEG 16-OTs (1.0 eq) were dissolved in 100 mL of toluene, and Trt-PEG8-OH (1.1 eq.) was added. The mixture was azeotropically treated with toluene to remove water, followed by the addition of 200 mL of anhydrous THF and the slow addition of NaH (10 eq.). The reaction was carried out at room temperature under a nitrogen atmosphere for 12 h. After the reaction was completed, EA and water were added for extraction, followed by washing with saturated brine. The organic phase was dried and purified by column chromatography (EA:MeOH = 10:1) to obtain the colorless and transparent product Trt-PEG. 24 -OTs.

[0091] Equation (5)

[0092] According to an embodiment of the present invention, maintaining the feed ratio, Trt-PEG is obtained according to the synthesis route shown in formula (1). n1+n2 -OH and Tlt-PEG n1+n2 -OTs, where n1+n2=4~100.

[0093] In step S02, a single molecular weight polyethylene glycol segment as shown in formula (X1), diethanolamine, and compound R1H are reacted to obtain a compound as shown in formula (X2) having a secondary amine group.

[0094] Equation (X2).

[0095] According to an embodiment of the present invention, the synthesis process of the compound as shown in formula (X2) specifically includes steps S021 to S025.

[0096] In step S021, diethanolamine is reacted with boc anhydride to obtain N-Boc-2CH3CH2OH.

[0097] According to an embodiment of the present invention, the synthetic route of step S021 is shown in formula (6):

[0098]

[0099] Equation (6)

[0100] Specifically, diethanolamine was added to a 500 mL single-necked flask, dissolved in 100 mL of THF, followed by 100 mL of saturated sodium bicarbonate aqueous solution. Under stirring, Boc anhydride was slowly added, and the reaction was carried out at room temperature for 12 h. After the reaction was complete, the THF was evaporated to dryness, and the mixture was extracted with EA and water, retaining the organic phase. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate to obtain a colorless, viscous liquid, which is the compound N-Boc-2CH3CH2OH.

[0101] In step S022, OTs-(PEG) n-Trt)2、N-Boc-2CH3CH2OH reacts with NaH to give N-Boc-(PEG) n -Trt)2.

[0102] According to an embodiment of the present invention, the synthetic route of step S022 is shown in formula (7):

[0103]

[0104] Equation (7)

[0105] Specifically, OTs-(PEG) n 2.5 eq. of N-Boc-2CH3CH2OH (1 eq.) and 10 eq. of NaH were added to a 500 mL single-necked flask, dissolved in 100 mL of THF, and reacted at room temperature for 36 h. After cooling to 23 °C, toluene was evaporated to dryness, 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 the mobile phase to give a colorless oily substance, which is the compound N-Boc-(PEG). n -Trt)2.

[0106] In step S023, N-Boc-(PEG) n -Trt)2 reacts sequentially with HAc, TsOH, and TsCl to give N-Boc-(PEG) n -OTs)2.

[0107] According to an embodiment of the present invention, the synthetic route of step S023 is shown in formula (8):

[0108]

[0109] Equation (8)

[0110] Specifically, the raw material N-Boc-(PEG) n -Trt)2 (1 eq) was added to a 100 mL single-necked flask, followed by a mixture of 40 mL acetic acid and 10 mL water. The mixture was heated to 40 °C and reacted for 4 h. TLC was then performed to confirm the completeness of the reaction. After the reaction was complete, the acetic acid was evaporated and extracted with a PE:EA mixed solvent. Impurities dissolved in the organic phase. The aqueous phase was retained and extracted with dichloromethane. The organic phase was retained, washed with saturated brine, and dried over anhydrous sodium sulfate to obtain a pale yellow oily liquid, which is the compound N-Boc-(PEG). n -OH)2.

[0111] N-Boc-(PEG) nAdd 1 eq. of N-Boc-(PEG)2 to a 250 mL single-necked flask and dissolve it in 100 mL of THF. Dissolve 3 eq. of NaOH in 10 mL of water, cool to room temperature in an ice bath, and slowly add the solution to the flask in a low-temperature circulating zone. Dissolve 1.25 eq. of TsCl in 10 mL of THF and add the solution dropwise to the single-necked flask over 30 min using a constant-pressure dropping funnel, ensuring the system temperature remains below 0 °C. React in an ice bath for 12 h. Add NaOH and react at 40 °C for 4 h to hydrolyze off the TsCl. Dry the THF, extract with dichloromethane, retain the organic phase, and rotary evaporate to obtain a pale yellow transparent oily liquid, which is the compound N-Boc-(PEG). n -OTs)2.

[0112] In step S024, N-Boc-(PEG) n -OTs)2 reacts with compound R1H to remove Boc, yielding the compound shown in formula (X2).

[0113] According to an embodiment of the present invention, the synthetic route of step S024 is shown in formula (9):

[0114]

[0115] Equation (9)

[0116] Specifically, N-Boc-(PEG) n -OTs)2 (1 eq.) and compound R1H (2.25 eq.) were added to a 50 mL single-necked flask, and 20 mL of DMF was added to dissolve them. The mixture was stirred until the reactants were dissolved. K2CO3 was added and the mixture was stirred for 24 h, during which the reaction progress was monitored by TLC. After the reaction was completed, DMF was removed by rotary evaporation, and the mixture was extracted with EA and water. The mixture was washed with saturated brine, and the organic phase was retained. The organic phase was dried with anhydrous sodium sulfate, and after rotary evaporation, it was precipitated in a solution of diethyl ether: petroleum ether = 10:1 to precipitate an oily liquid, yielding the compound shown in formula (X2).

[0117] In step S03, the drug molecule R2, which has a terminal carboxyl group or hydroxyl group, is condensed with dithiodiethanol to obtain a compound as shown in formula (X3).

[0118] It should be noted that R2 is selected from either drug molecule R2' which is connected to the SS bond via an ester bond or drug molecule R2” which is connected to the SS bond via an ether bond.

[0119] According to embodiments of the present invention, the synthetic route of the compound represented by formula (X3) is shown in formula (10) or formula (11):

[0120] Equation (10)

[0121] Equation (11).

[0122] According to an embodiment of the present invention, the synthesis process of the compound shown in formula (X3) specifically includes: adding a drug molecule R2 (R2 is R2' or R2”, 1.0 eq.) having a terminal carboxyl or hydroxyl group and 2,2'-dithiodiethanol (1.25 eq.) to a 100 mL single-necked flask, and dissolving it in 50 mL of THF solution. Adding DMAP (1.0 eq.) and DCC (1.0 eq.), the mixture is stirred at room temperature for 24 h, during which time TLC is performed and the reaction progress is confirmed using ninhydrin solution. After the reaction is complete, the THF is evaporated, and the mixture is extracted with DCM and saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography (PE:EA) to obtain a white or pale yellow crystalline solid, which is the compound shown in formula (X3).

[0123] In step S04, the compound with a secondary amine group as shown in formula (X2) and the compound with a secondary amine group as shown in formula (X3) are subjected to an amide synthesis reaction to obtain a releasable functional compound with the structure shown in formula (I).

[0124] According to embodiments of the present invention, the synthetic route for a releaseable functional compound with the structure shown in formula (I) is shown in formula (12) or formula (13):

[0125]

[0126] Equation (12)

[0127]

[0128] Equation (13).

[0129] According to an embodiment of the present invention, the synthesis process of the releasable functional compound with the structure shown in formula (I) specifically includes: adding the compound (1.0 eq.) shown in formula (X3) and CDI (1.25 eq.) to a 50 mL single-necked flask, and dissolving them in 10 mL of toluene solution. The reaction is carried out under N2 protection at 100 °C with stirring for 24 h, during which TLC is performed and the reaction progress is confirmed with ninhydrin solution. After the reaction is complete, the mixture is extracted with an aqueous solution of sodium dihydrogen phosphate at pH 5.0 to remove CDI, retaining the organic phase. The organic phase is added to a 50 mL single-necked flask, and dissolving it in 5 mL of toluene. The compound NH-(PEG) shown in formula (X2) dissolved in 2 mL of chloroform is added. n1+n2 -R1)2 (1.1 eq.) was stirred at room temperature under nitrogen protection for 12 h. The chloroform in the system was evaporated to dryness, and the organic phase was retained by extraction with DCM and water. After drying with anhydrous sodium sulfate, a pale yellow oily product was obtained, which is the releasable functional compound with the structure of formula (I).

[0130] It should be noted that the releasable functional compound with the structure of formula (I) has a PEG-disulfide bond molecule containing a small molecule drug fragment (drug molecule R2) as the core, and a hydrophilic polyethylene glycol block as the outer group.

[0131] According to the above embodiments of the present invention, the releaseable functional compound uses a single molecular weight polyethylene glycol block, which can reduce the recognition of anti-PEG antibodies, thereby avoiding the induction of unnecessary immune rejection reactions and improving the biocompatibility of the releaseable functional compound.

[0132] According to the above embodiments of the present invention, the releasable functional compound having amide bonds has high stability due to the relatively stable amide bonds.

[0133] The present invention also provides nanoparticles, which are assembled using the above-described releasable functional compounds having a structure as shown in formula (I).

[0134] The present invention also provides a method for preparing the above-mentioned nanoparticles, comprising: dissolving a releasable functional compound having a structure of formula (I) in dimethyl sulfoxide to obtain an organic solution; and flash-precipitating the organic solution into an aqueous solution of phospholipid-polyethylene glycol to obtain nanoparticles.

[0135] According to an embodiment of the present invention, the concentration of the releaseable functional compound in dimethyl sulfoxide is 0.5 mg / mL; the molar ratio of the releaseable functional compound to phospholipid-polyethylene glycol is 1:1 to obtain stable nanoparticles.

[0136] According to embodiments of the present invention, the prepared nanoparticles containing drug molecules are stable and exhibit good effects in cell and animal experiments, and can be applied to the treatment of tumors.

[0137] The nanoparticles assembled from the releasable functional compounds provided in the above embodiments of the present invention are injected by injection. The polyethylene glycol block in the nanoparticles can bind to albumin in the blood through the active group R1, which increases the duration of action of the nanoparticles in the blood and makes the nanoparticles stable in the blood for a long time, which helps to improve the specificity and efficiency of the nanoparticles in targeting tumor tissues.

[0138] According to the above embodiments of the present invention, the nanoparticles of the releaseable functional compound enter the cell, and the reduced glutathione present in the cell causes the disulfide bonds of the releaseable functional compound to break in the cell, so as to release the drug molecule into the cell and exert its drug effect.

[0139] The following illustrative description illustrates the designed releaseable functional compound and the preparation method of nanoparticles comprising the releaseable functional compound. It should be noted that this illustration is merely a specific embodiment of the present invention and does not limit the scope of protection of the present invention.

[0140] It should be noted that the explanation is based on the example of R1 being maleimide and R2 being 5-aminolevulinic acid (5-ALA).

[0141] Example 1

[0142] (1) Synthesis of the first intermediate compound as shown in formula C1:

[0143] Referring to formula (1-1), drug molecule R2 (5-ALA) undergoes an amide reaction with Boc anhydride to synthesize the first intermediate compound shown in formula C1, namely tert-butyloxycarbonyl (Boc) protected 5-ALA. Specifically, 5-ALA (3.0 g, 22.9 mmol) was added to a 500 mL single-necked flask, along with 100 mL of sodium bicarbonate aqueous solution and 100 mL of tetrahydrofuran (THF), and stirred until the reactants dissolved. Boc anhydride (di-tert-butyl dicarbonate, Boc2O, 7.49 g, 34.4 mmol) was slowly added, and the reaction was stirred at room temperature for 24 h. The reaction progress was monitored by TLC and ninhydrin solution during this period. After the reaction was completed, the THF was evaporated to dryness, and the remaining Boc2O was removed by extracting the aqueous solution with petroleum ether (PE). The aqueous phase was retained and the pH was adjusted to 2 with 0.1M hydrochloric acid solution. EA was added to extract the aqueous solution, and the organic phase was retained. After evaporation to dryness, a colorless crystalline solid was obtained, which is the first intermediate compound (Boc-5-ALA) as shown in formula C1.

[0144]

[0145] Equation (1-1)

[0146] R2 C1

[0147] (2) Synthesize the second intermediate compound as shown in formula C2:

[0148] Referring to formulas (1-2), the first intermediate compound (Boc-5-ALA) shown in formula C1 was esterified with dithiodiethanol to synthesize the 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 100 mL single-necked flask and dissolved in 20 mL of THF solution. 4-Dimethylaminopyridine (DMAP) (7.49 g, 34.4 mmol) and dicyclohexylcarbodiimide (DCC) (7.49 g, 34.4 mmol) were added, and the mixture was stirred at room temperature for 24 h. The reaction progress was monitored by TLC and ninhydrin solution during the reaction. After the reaction was completed, the THF was evaporated to dryness, and the mixture was extracted with dichloromethane (DCM) and saturated brine. After drying with anhydrous sodium sulfate, the mixture was purified by column chromatography (PE:EA=4:1) to obtain a white crystalline solid, which is the second intermediate compound shown in formula C2.

[0149]

[0150] C1 C2 Equation (1-2)

[0151] (3) Synthesis of furan-protected maleimide as shown in Formula A3:

[0152] The synthesis of furan-protected maleimide is shown in formula (1-3). Referring to formula (1-3), maleimide (5 g, 51.5 mmol) as shown in formula A1 was placed in a 250 mL round-bottom flask. After further dehydration in a solvent treatment system, 100 mL of anhydrous toluene was added, followed by the addition of furan (C4H4O, 14 g, 206 mmol) as shown in formula A2. The mixture was heated to 50 °C and refluxed for 12 hours. The reaction system was cooled to room temperature, filtered, and a white solid was obtained. This solid was then washed with 20 mL of toluene to obtain a white solid, which was furan-protected maleimide (8.2 g, 92.4%) as shown in formula A3.

[0153]

[0154] A1 A2 A3

[0155] Equation (1-3)

[0156] (4) Synthesize diethanolamine protected by tert-butyloxycarbonyl as shown in formula B2:

[0157] The process for preparing tert-butyloxycarbonyl-protected diethanolamine as shown in Formula B2 by reacting diethanolamine as shown in Formula B1 with Boc anhydride is shown in equations (1-4). Specifically, diethanolamine as shown in Formula B1 is added to a 500 mL single-necked flask, dissolved in 100 mL of tetrahydrofuran (THF), followed by 100 mL of saturated sodium bicarbonate aqueous solution. Under stirring, Boc anhydride is slowly added, and the reaction is carried out at room temperature for 12 hours. After the reaction is complete, the THF is evaporated to dryness, and the mixture is extracted with EA and water, retaining the organic phase. The organic phase is washed with saturated brine and dried over anhydrous sodium sulfate to obtain a colorless viscous liquid, which is the tert-butyloxycarbonyl-protected diethanolamine as shown in Formula B2.

[0158]

[0159] B1 B2 Equation (1-4)

[0160] (5) Synthesis of Boc-N-(PEG4-OTrt)2 as shown in Formula B3:

[0161] The synthesis of Boc-N-(PEG4-OTrt)2 as shown in Formula B3 is illustrated in equations (1-5). Specifically, the starting material OTs-PEG4-Trt (5.0 g, 6.5 mmol), diethanolamine protected by tert-butyloxycarbonyl as shown in Formula B2 (0.4 g, 2.1 mmol), and NaH (1.5 g, 4.3 mmol) were added to a 500 mL single-necked flask, dissolved in 100 mL of THF, and reacted at room temperature for 36 h. After cooling to 23 °C, toluene was evaporated to dryness, 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 the mobile phase to obtain a colorless oil (2.4 g, 82%), which is Boc-N-(PEG4-OTrt)2 as shown in Formula B3.

[0162]

[0163] B2 B3 Equation (1-5)

[0164] (6) Synthesis of Boc-N-(PEG4-OTs)2 as shown in Formula B5:

[0165] Boc-N-(PEG4-OTrt)2 as shown in Formula B3 was etherified with acetic acid to synthesize Boc-N-(PEG4-OH)2 as shown in Formula B4; Boc-N-(PEG4-OH)2 as shown in Formula B4 was reacted with TsCl to synthesize Boc-N-(PEG4-OTs)2 as shown in Formula B5. The process for synthesizing Boc-N-(PEG4-OTs)2 as shown in Formula B5 is shown in Formulas (1-6) and (1-7) below.

[0166] Specifically, Boc-N-(PEG4-OTrt)2 (3.0 g, 2.8 mmol) as shown in Formula B3 was added to a 100 mL single-necked flask, along with a mixed solution of 40 mL acetic acid and 10 mL water. The mixture was heated to 40 °C and reacted for 4 h, with TLC confirming complete reaction. After the reaction was complete, the acetic acid was evaporated and extracted with a mixed solvent of PE:EA = 10:1, where impurities dissolved in the organic phase. The aqueous phase was retained and extracted with dichloromethane. The organic phase was retained, washed with saturated brine, and dried with anhydrous sodium sulfate to obtain a pale yellow oily liquid (1.12 g, 89%), which is Boc-N-(PEG4-OH)2 as shown in Formula B4.

[0167] Add 1.5 g (2.7 mmol) of Boc-N-(PEG4-OH)2 as shown in Formula B4 to a 250 mL single-necked flask and dissolve it in 100 mL of THF. Dissolve 1.08 g (27 mmol) of NaOH in 10 mL of water, cool to room temperature in an ice bath, and slowly add it to the flask in a low-temperature circulating zone. Dissolve 1.28 g (6.7 mmol) of TsCl in 10 mL of THF and add it dropwise to the single-necked flask over 30 min using a constant-pressure dropping funnel, keeping the system temperature below 0 °C at all times. React in an ice bath for 12 h. Add NaOH and react at 40 °C for 4 h to hydrolyze off TsCl. Dry the THF, extract with dichloromethane, retain the organic phase, and rotary evaporate to obtain a pale yellow transparent oily liquid, which is Boc-N-(PEG4-OTs)2 (1.6 g, 81%) as shown in Formula B5.

[0168]

[0169] B3 B4 Equation (1-6)

[0170]

[0171] B4 B5 Equation (1-7)

[0172] (7) Synthesize the compound shown in formula B6:

[0173] The compound shown in Formula B6 was synthesized by reacting Boc-N-(PEG4-OTs)2 (as shown in Formula B5) with a furan-protected maleimide (as shown in Formula A3), as shown in Formulas (2-8). Specifically, Boc-N-(PEG4-OTs)2 (1.0 g, 1.1 mmol) and furan-protected maleimide (0.5 g, 2.8 mmol) (as shown in Formula A3) were added to a 50 mL single-necked flask, and 20 mL of dimethylformamide (DMF) was added to dissolve them. The mixture was stirred until dissolved. K2CO3 was added and the mixture was stirred for 24 h, during which the reaction progress was monitored by TLC. After the reaction was completed, DMF was removed by rotary evaporation, and the mixture was extracted with EA and water. After washing with saturated brine, the organic phase was retained, dried with anhydrous sodium sulfate, and then precipitated in a solution of diethyl ether: petroleum ether = 10:1 to form an oily liquid, which is the compound shown in Formula B6.

[0174]

[0175] B5 B6 Equation (1-8)

[0176] (8) Synthesis of polyethylene glycol segment NH-(PEG4-FuMI)2 as shown in Formula B7:

[0177] The compound shown in Formula B6 was reacted with dioxane hydrochloric acid solution to synthesize the polyethylene glycol segment shown in Formula B7. The process for synthesizing the polyethylene glycol segment shown in Formula B7 is shown in formulas (1-9).

[0178] The compound shown in Formula B6 (1.2 g, 1.4 mmol) was added to a 50 mL single-necked flask, and 20 mL of dioxane hydrochloric acid solution was added to dissolve it. The reaction was stirred at room temperature for 12 hours. The dioxane hydrochloric acid solution in the system was evaporated to dryness, and the product was then precipitated into a solution of petroleum ether: anhydrous diethyl ether = 1:10. After centrifugation, the supernatant was discarded to obtain a pale yellow oily product, which is the synthesis of the polyethylene glycol segment NH-(PEG4-FuMI)2 shown in Formula B7.

[0179]

[0180] B6 B7 Equation (1-9)

[0181] (9) Synthesize the third intermediate compound as shown in formula C3:

[0182] The process of reacting the second intermediate compound as shown in formula C2 with carbonyl imidazole to synthesize the third intermediate compound as shown in formula C3 is shown in formula (1-10).

[0183] Specifically, the second intermediate compound (1.0 g, 2.72 mmol) as shown in formula C2 and carbonyl imidazole (CDI, 0.48 g, 2.99 mmol) were added to a 50 mL single-necked flask, and 5 mL of toluene solution was added to dissolve them. The reaction was carried out under N2 protection at 100 °C with stirring for 24 h, during which the reaction was monitored by TLC and ninhydrin solution. After the reaction was completed, the mixture was extracted with an aqueous solution of sodium dihydrogen phosphate at pH 5.0 to remove CDI, and the organic phase was retained to obtain the third intermediate compound as shown in formula C3.

[0184]

[0185] C2 C3 Equation (1-10)

[0186] (10) Synthesize the fourth intermediate compound as shown in formula C4:

[0187] The process of reacting the third intermediate compound as shown in Formula C3 with the polyethylene glycol segment as shown in Formula B7 to synthesize the fourth intermediate compound as shown in Formula C4 is shown in Formula (1-11).

[0188] Specifically, the third intermediate compound (1.1 g, 2.38 mmol) as shown in Formula C3 was added to a 50 mL single-necked flask and dissolved in 5 mL of toluene. A polyethylene glycol segment (1.96 g, 2.62 mmol) as shown in Formula B7, dissolved in 2 mL of chloroform, was added, and the mixture was stirred at room temperature under nitrogen protection for 12 hours. The chloroform in the system was evaporated to dryness, and the organic phase was extracted with DCM and water to retain the organic phase. After drying with anhydrous sodium sulfate, a pale yellow oily product was obtained, yielding the fourth intermediate compound as shown in Formula C4.

[0189]

[0190] C3 C4 Equation (1-11)

[0191] (11) Synthesis of the fifth intermediate compound Boc-5-ALA-2(PEG4-MI) as shown in formula C5:

[0192] The process of dissolving the fourth intermediate compound as shown in formula C4 in toluene to obtain the fifth intermediate compound as shown in formula C5 is shown in formula (1-12).

[0193] Specifically, the fourth intermediate compound (0.8 g, 0.6 mmol) as shown in Formula C4 was added to a 50 mL single-necked flask, and 10 mL of toluene was added to dissolve it. The reaction was carried out at 90 °C for 12 h under continuous nitrogen purging, and then cooled to room temperature after the reaction was completed. The toluene in the system was evaporated to dryness, and the organic phase was extracted with DCM and water to retain the organic phase. After drying with anhydrous sodium sulfate, a pale yellow oily product was obtained, which is the fifth intermediate compound Boc-5-ALA-2(PEG4-MI) as shown in Formula C5.

[0194]

[0195] C4 C5 Equation (1-12)

[0196] (12) Synthesis of a releaseable functional compound 5-ALA-2(PEG4-MI) having the structure of formula (I):

[0197] The process of reacting the fifth intermediate compound, as shown in formula C5, with dioxane hydrochloric acid solution to synthesize a releasable functional compound with the structure of formula (I) is shown in formula (1-13).

[0198] Specifically, the fifth intermediate compound (0.3 g, 0.4 mmol) as shown in Formula C5 was added to a 50 mL single-necked flask, and 10 mL of dioxane hydrochloride was added to dissolve it. The mixture was stirred under nitrogen protection at room temperature for 12 hours. After the reaction was completed, the solvent was evaporated, and the organic phase was extracted with DCM and water to retain the organic phase. After drying with anhydrous sodium sulfate, a pale yellow oily product was obtained, which is the final product 5-ALA-2 (PEG4-MI) as shown in Formula C5.

[0199]

[0200] C5 I Equation (1-13)

[0201] Figure 1 The 1H NMR spectrum of 5-ALA-2 (PEG4-MI) of Example 1 of the present invention is shown.

[0202] Figure 2 The time-of-flight mass spectrometry data of 5-ALA-2 (PEG4-MI) in Example 1 of the present invention are shown.

[0203] refer to Figure 1 , Figure 2 As shown, the results of 1H NMR spectroscopy and time-of-flight mass spectrometry indicate that 5-ALA-2 (PEG4-MI) was successfully synthesized.

[0204] Using 5-ALA-2(PEG4-MI) as an example, the assembly of nanoparticles is demonstrated:

[0205] Weigh 5 mg of 5-ALA-2 (PEG4-MI) (7 mmol), dissolve it in 100 μL of dimethyl sulfoxide, and sonicate to obtain the first solution. Dissolve 5 mg of DSPE-PEG in another 100 μL of dimethyl sulfoxide. 2000 The solution is dissolved by sonication, avoiding the generation of large amounts of bubbles from shaking, to obtain a second solution. The first and second solutions are then mixed to obtain a mixed solution.

[0206] Take 200 μL of 5-ALA-2 (PEG4-MI) and DSPE-PEG 2000 The 1:1 mixed solution was rapidly added dropwise to 1800 μL of ultrapure water by flash precipitation, and stirred at 500 r / min for 5 minutes to obtain a clear solution, which is a nanoparticle solution.

[0207] Then, 1 mL of the nanoparticle solution was placed in a dialysis membrane and dialyzed in 5 L of ultrapure water for 12 h to obtain water-soluble nanoparticles. After lyophilization, the nanoparticles were redissolved and used for the next experiment.

[0208] Example 2

[0209] It should be noted that the explanation is based on R1 being succinimide and R2 being 5-aminolevulinic acid (5-ALA).

[0210] The synthesis process of 5-ALA-2(PEG4-SI) differs from that of 5-ALA-2(PEG4-MI) in that:

[0211] Referring to formula (2-1), the starting materials Boc-N-(PEG4-OTs)2 (1.0 g, 1.1 mmol) and succinimide (0.3 g, 2.4 mmol) as shown in formula B5 were added to a 50 mL single-necked flask, and 20 mL of DMF was added to dissolve them. The mixture was stirred until the reactants dissolved. K2CO3 was added and the mixture was stirred for 24 h, during which the reaction progress was monitored by TLC. After the reaction was completed, DMF was removed by rotary evaporation, and the mixture was extracted with EA and water. The mixture was washed with saturated brine, and the organic phase was retained. The organic phase was dried with anhydrous sodium sulfate, and after rotary evaporation, it was precipitated in a solution of diethyl ether: petroleum ether = 10:1 to form an oily liquid, which is the compound shown in formula B6'.

[0212]

[0213] B5 B6' Equation (2-1)

[0214] Referring to formula (2-2), 0.8 g (1.0 mmol) of the compound shown in formula B6' was added to a 50 mL single-necked flask, and 20 mL of dioxane hydrochloric acid solution was added to dissolve it. The reaction was stirred at room temperature for 12 hours. The dioxane hydrochloric acid solution in the system was evaporated to dryness, and then the product was precipitated into a solution of petroleum ether: anhydrous diethyl ether = 1:10. After centrifugation, the supernatant was discarded to obtain a pale yellow oily product, which is the synthesis of polyethylene glycol segment NH-(PEG4-SI)2 as shown in formula B7'.

[0215]

[0216] B6' B7' Equation (2-2)

[0217] Referring to formula (2-3), the third intermediate compound (0.8 g, 0.9 mmol) as shown in formula C3 was added to a 50 mL single-necked flask and dissolved in 5 mL of toluene. A polyethylene glycol segment (1.0 g, 1.5 mmol) as shown in formula B7' was added dissolved in 2 mL of chloroform. The reaction was stirred for 12 hours at room temperature under nitrogen protection. The chloroform in the system was evaporated to dryness, and the organic phase was extracted with DCM and water to retain the organic phase. After drying with anhydrous sodium sulfate, a pale yellow oily product was obtained, yielding NH-Boc-(PEG4-SI)2 as shown in formula C4'.

[0218]

[0219] C3 C4' Equation (2-3)

[0220] Referring to formula (2-4), 0.3 g (0.4 mmol) of the compound shown in formula C4' was added to a 50 mL single-necked flask, and 10 mL of dioxane hydrochloride was added to dissolve it. The mixture was stirred under nitrogen protection at room temperature for 12 hours. After the reaction was completed, the solvent was evaporated, and the organic phase was extracted with DCM and water. After drying with anhydrous sodium sulfate, a pale yellow oily product was obtained, which is the final product 5-ALA-2 (PEG4-SI) as shown in formula (I).

[0221]

[0222] C4' I (2-4)

[0223] Figure 3 The 1H NMR spectrum of 5-ALA-2 (PEG4-SI) of Example 2 of the present invention is shown.

[0224] Figure 4 The time-of-flight mass spectrometry data of 5-ALA-2 (PEG4-SI) in Example 2 of the present invention are shown.

[0225] refer to Figure 1 , Figure 2 As shown, the results of 1H NMR spectroscopy and time-of-flight mass spectrometry indicate that 5-ALA-2 (PEG4-SI) was successfully synthesized.

[0226] Figure 5 The particle size distribution diagrams of the 5-ALA-2 (PEG4-MI) assembled nanoparticles of Example 1 and the 5-ALA-2 (PEG4-SI) assembled nanoparticles of Example 2 are shown.

[0227] refer to Figure 5 As shown, the nanoparticles assembled with 5-ALA-2(PEG4-MI) have an average diameter of 182 nm and a dispersion index of 0.206. The particle size distribution diagram indicates that the synthesized 5-ALA-2(PEG4-MI) assembled nanoparticles have a uniform particle size distribution. The nanoparticles assembled with 5-ALA-2(PEG4-SI) have an average diameter of 179 nm and a dispersion index of 0.222. The particle size distribution diagram also indicates that the synthesized 5-ALA-2(PEG4-SI) assembled nanoparticles have a uniform particle size distribution.

[0228] Figures 6A-6B The cytotoxicity of the 5-ALA-2 (PEG4-MI) assembled nanoparticles of Example 1 and the 5-ALA-2 (PEG4-SI) assembled nanoparticles of Example 2 of the present invention is shown.

[0229] refer to Figure 6A As shown, after the nanoparticles assembled with 5-ALA-2 (PEG4-MI) entered HepG2 cells (a type of liver cancer cell), the HepG2 cells maintained high activity. Within the concentration range of 3 μM to 12 μM of the drug molecule and nanoparticles, the cells exhibited good cell activity when co-incubated with 5-ALA, 5-ALA-2 (PEG4-MI), and 5-ALA-2 (PEG4-SI), indicating that the drug molecule 5-ALA, the 5-ALA-2 (PEG4-MI) synthesized in Example 1, and the 5-ALA-2 (PEG4-SI) synthesized in Example 2 had minimal impact on cell activity and possessed good physiological activity and safety.

[0230] refer to Figure 6B As shown, after photodynamic therapy (PDT), HepG2 cell activity decreased, and some HepG2 cells died during treatment, indicating that PDT can fight cancer cells. At nanoparticle concentrations in the range of 10 μM-12 μM, 5-ALA-2 (PEG4-MI) achieved an IC50 threshold. 50 (Half-inhibitory concentration), where half-inhibitory concentration represents the concentration at which half of the cells are killed. Figure 6A This contrast demonstrates that cancer cells can only be killed by drugs under photodynamic therapy (PDT). In other words, nanoparticles enter cells but do not cause damage without photodynamic therapy; cell-killing only occurs after PDT. Therefore, even if drug-loaded nanoparticles remain within normal cells, they will not harm them, demonstrating excellent safety and practical applicability.

[0231] Figure 7 The relative fluorescence intensities of the 5-ALA-2 (PEG4-MI) assembled nanoparticles of Example 1 and the 5-ALA-2 (PEG4-SI) nanoparticles of Example 2 in vitro and in cells are shown.

[0232] refer to Figure 7 As shown, the nanoparticles assembled with 5-ALA-2(PEG4-MI) reached the peak of relative fluorescence intensity at 10h, while the 5-ALA drug molecule reached the peak of relative fluorescence intensity at 8h. This indicates that the peak of relative fluorescence intensity of the nanoparticles assembled with 5-ALA-2(PEG4-MI) relative to the 5-ALA drug molecule was shifted later, thus achieving the purpose of prolonging the action time.

[0233] Figure 8 Images of organelle colocalization of 5-ALA-2 (PEG4-MI) assembled nanoparticles of Example 1 and 5-ALA-2 (PEG4-SI) assembled nanoparticles of Example 2 are shown under a confocal fluorescence microscope.

[0234] refer to Figure 8 As shown, the intracellular action region of the 5-ALA drug molecule is in the mitochondria. The intracellular action regions of the 5-ALA-2 (PEG4-MI) assembled nanoparticles and the 5-ALA-2 (PEG4-SI) assembled nanoparticles are also in the mitochondria, indicating that the action process of the 5-ALA-2 (PEG4-MI) assembled nanoparticles and the 5-ALA-2 (PEG4-SI) assembled nanoparticles is the same as that of the 5-ALA drug molecule.

[0235] Figure 9 Animal intracellular imaging is shown of the 5-ALA-2 (PEG4-MI) assembled nanoparticles of Example 1 and the 5-ALA-2 (PEG4-SI) assembled nanoparticles of Example 2 of the present invention.

[0236] Bab / c female mice (6 weeks old) were selected as the main biological model for the experiment. Nanoparticle solution was administered subcutaneously (50 mg / kg per mouse), intramuscularly (50 mg / kg per mouse), and intravenously (25 mg / kg per mouse) to the hind legs on the back, muscles on the hind legs on the front, and vein in the tail, respectively. The injection sites of the intramuscular and subcutaneous injections were observed using a small animal imaging system at different time points: 0h, 1h, 2h, 3h, 5h, 7h, and 12h. Figure 9 The fluorescence of the dotted circle in the image is observed. Since intravenously injected drug molecules and nanoparticle molecules are transported throughout the body via the bloodstream, the fluorescence on the back of a mouse can be observed using a small animal imaging system.

[0237] Since 5-ALA itself is fluorescent at an excitation wavelength of 445 nm, after injecting 5-ALA-2 (PEG4-MI) nanoparticles and 5-ALA-2 (PEG4-SI) nanoparticles into mice, the nanoparticles can produce fluorescence after being metabolized in the mitochondria of the cells. The observation of fluorescence indicates that the nanoparticles have exerted their effects in mice.

[0238] refer to Figure 9 As shown, 5-ALA drug molecules, 5-ALA-2 (PEG4-MI) nanoparticles, and 5-ALA-2 (PEG4-SI) nanoparticles were administered to mice via intravenous, intramuscular, and subcutaneous injections, respectively.

[0239] In the intravenous injection group, the 5-ALA-2(PEG4-MI) assembled nanoparticles reached their peak fluorescence intensity at 7 hours, indicating that the drug concentration in cells reached its peak at this time, representing the optimal time for drug action. The 5-ALA drug molecule reached its peak fluorescence intensity at 3 hours, subsequently decreasing gradually. The fluorescence of the 5-ALA-2(PEG4-SI) assembled nanoparticles peaked at 5 hours. Therefore, compared to the 5-ALA drug molecule, both the 5-ALA-2(PEG4-MI) and 5-ALA-2(PEG4-SI) assembled nanoparticles achieved a prolonged duration of action. This is because the 5-ALA-2(PEG4-MI) and 5-ALA-2(PEG4-SI) assembled nanoparticles are slowly released from their encapsulation, thus extending the duration of action.

[0240] In the intramuscular injection group, 5-ALA showed significant fluorescence at the injection site (within the dashed circle) for 3-5 hours, which gradually disappeared after 7 hours, indicating that 3-5 hours was the optimal action time for the 5-ALA drug molecule. The 5-ALA-2(PEG4-MI) assembled nanoparticles showed fluorescence after 3 hours and the fluorescence persisted until 12 hours, indicating that the 5-ALA-2(PEG4-MI) assembled nanoparticles had a longer duration of action compared to 5-ALA. The 5-ALA-2(PEG4-SI) assembled nanoparticles showed fluorescence after 2 hours, which gradually disappeared after 7 hours. Therefore, compared to the 5-ALA drug molecule, both the 5-ALA-2(PEG4-MI) and 5-ALA-2(PEG4-SI) assembled nanoparticles achieved the goal of prolonging the action time. The 5-ALA-2(PEG4-SI) assembled nanoparticles showed a better effect in prolonging the action time.

[0241] In the subcutaneous injection group, 5-ALA showed fluorescence at 2 hours and the fluorescence basically disappeared at 5 hours; the nanoparticles assembled with 5-ALA-2(PEG4-MI) showed fluorescence at 2 hours, the fluorescence was strongest at 5 hours, and it gradually weakened after 7 hours; the nanoparticles assembled with 5-ALA-2(PEG4-SI) showed fluorescence at 2 hours, the fluorescence was strongest at 7 hours, and the fluorescence basically disappeared after 12 hours.

[0242] Therefore, it can be seen that, whether injected intravenously, intramuscularly, or subcutaneously, nanoparticles assembled with 5-ALA-2 (PEG4-MI) and nanoparticles assembled with 5-ALA-2 (PEG4-SI) have the effect of prolonging the duration of action compared to 5-ALA drug molecules.

[0243] The use of ordinal numbers such as "first," "second," "third," etc., in the specification and claims to modify the corresponding elements does not imply that the element has any ordinal number, nor does it represent the order of one element with another element, or the order of manufacturing methods. The use of these ordinal numbers is only to enable a named element to be clearly distinguished from another element with the same name.

[0244] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A releasable functional compound containing a single molecular weight polyethylene glycol segment, having the structure shown in formula (I): Equation (I) in, PEG n This indicates a polyethylene glycol segment with a single molecular weight; n is an integer from 4 to 500; R1 is selected from maleimide group and succinimide group; R2 is 5-aminoacetylpropionyl.

2. The releasable functional compound according to claim 1, characterized in that, PEG n The structural formula is shown below: 。 3. The releasable functional compound according to claim 1, characterized in that, The molecular weight of the releasable functional compound is 1,000 to 100,000.

4. A method for preparing a releasable functional compound as described in any one of claims 1 to 3, characterized in that, include: Provide a polyethylene glycol segment of a single molecular weight, the structure of which is shown in formula (X1); Equation (X1) A single molecular weight polyethylene glycol segment as shown in formula (X1), diethanolamine, and compound R1H are reacted to obtain a compound as shown in formula (X2) having a secondary amine group. Equation (X2) The drug molecule R2 with a terminal carboxyl group was condensed with dithiodiethanol to obtain the compound shown in formula (X3). Equation (X3) The compounds of formula (X2) and (X3) having secondary amine groups were subjected to an amide synthesis reaction to obtain a releasable functional compound having the structure shown in formula (I). Equation (I).

5. The preparation method according to claim 4, characterized in that, The condensation reaction of drug molecule R2 with terminal carboxyl groups and dithiodiethanol includes: The drug molecule R2, which has a terminal carboxyl group, is dissolved in a tetrahydrofuran solution along with dithiodiethanol. 4-Dimethylaminopyridine and dicyclohexylcarbodiimide were added to the tetrahydrofuran solution to carry out a condensation reaction.

6. The preparation method according to claim 4, characterized in that, The amide synthesis reaction of compounds having a secondary amine group as shown in formula (X2) and compounds shown in formula (X3) includes: The compound shown in formula (X3) and carbonyl imidazole were dissolved in toluene and reacted under N2 protection and heating conditions with stirring. After the reaction is complete, remove the carbonyl imidazole from the reaction solution, retain the organic phase, and dissolve the organic phase in toluene; The compound shown in formula (X2) is added to toluene containing the organic phase, and the reaction is stirred under N2 protection.

7. A nanoparticle formed by assembling a releasable functional compound as described in any one of claims 1 to 3.

8. A method for preparing nanoparticles as described in claim 7, characterized in that, include: The releasable functional compound was dissolved in dimethyl sulfoxide to obtain an organic solution; The organic solution was flash-precipitated into an aqueous solution of phospholipid-polyethylene glycol to obtain the nanoparticles.

Images