A polyethylene glycol-ginsenoside covalent conjugate, self-assembled nanoparticles thereof and a preparation method thereof

Abstract

The present application belongs to the technical field of medicinal chemistry and novel drug delivery system, and particularly relates to a polyethylene glycol-ginsenoside covalent conjugate, self-assembled nanoparticles and a preparation method thereof. The polyethylene glycol-ginsenoside covalent conjugate is formed by bonding at least one carboxyl group in HOOC-PEG-COOH to ginsenoside through an ester bond, or by bonding at least one amino group in NH2-PEG-NH2 to ginsenoside through an amide bond. The present application connects ginsenoside with polyethylene glycol through esterification or amidation reaction to construct single-end or double-end modified PEGylated ginsenoside. The nanoparticles are prepared by self-assembly, which can significantly improve the water solubility, stability and bioavailability of ginsenoside. The method is simple and mild, and is suitable for structural modification and nano-formulation development of various ginsenosides.

Description

Technical Field

[0001] This invention belongs to the technical field of medicinal chemistry and novel drug delivery systems, specifically relating to a polyethylene glycol-ginsenoside covalent conjugate, its self-assembled nanoparticles, and a preparation method thereof. Background Technology

[0002] Ginsenosides, such as Rg3, Rb1, and Rd, are highly bioactive triterpenoid saponin compounds extracted from plants of the genus *Panax* in the Araliaceae family. These compounds exhibit significant pharmacological potential in areas such as immunomodulation, antitumor activity, anti-inflammation, antioxidation, anti-fatigue, and neuroprotection. However, their conversion into clinically effective drugs has been severely limited by their inherent physicochemical properties: ginsenoside molecules typically possess a large, rigid steroidal skeleton and multiple hydroxyl groups, resulting in extremely poor water solubility and difficulty in fully dissolving under the physiological pH environment of the gastrointestinal tract. Simultaneously, their molecular structure is easily degraded or isomerized under the action of gastric acid and intestinal enzymes, resulting in oral bioavailability generally below 5%. Furthermore, ginsenoside molecules are highly polar, making it difficult for them to passively cross the lipid bilayer of intestinal epithelial cells. Additionally, some saponins are considered substrates of the efflux transport protein P-glycoprotein (P-gp) and are actively pumped out of cells, further limiting their intestinal absorption.

[0003] To overcome these bottlenecks, researchers have explored the following strategies: The first category is physical encapsulation techniques, such as encapsulating ginsenosides in liposomes, polymer micelles, solid lipid nanoparticles, or cyclodextrin inclusions. These carrier systems can improve solubility to some extent, protect the drug from degradation, and provide a certain sustained-release effect. However, these techniques generally suffer from low drug loading (usually <10%), the need for large amounts of exogenous excipients (which may introduce potential toxicity or immunogenicity), complex preparation processes, and difficulty in controlling storage and in vivo stability. The second category is prodrug strategies and chemical modifications, which improve the lipid solubility or targeting of ginsenosides by derivatizing functional groups such as hydroxyl groups (e.g., esterification, glycosylation). However, such modifications may alter the original active conformation, and whether the modified molecule can effectively release the original drug in vivo is a key challenge.

[0004] Currently, most nanodelivery systems rely on exogenous carrier materials to construct nanostructures. Drugs are merely loaded onto these structures as "passengers." This approach has inherent drawbacks: (1) limited drug loading space, as the carrier material itself is inactive yet occupies most of the nanoparticle volume and mass; and (2) carrier-related biosafety risks. Therefore, developing a delivery system that does not require an exogenous carrier and allows active drug molecules to be designed and directly assembled into nanoparticles has become a highly attractive research direction. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a polyethylene glycol-ginsenoside covalent conjugate, its self-assembled nanoparticles, and a preparation method thereof. The polyethylene glycol-ginsenoside covalent conjugate provided by this invention achieves drug-carrier integration, requires no exogenous carrier, and can self-assemble into nanoparticles.

[0006] This invention provides a polyethylene glycol-ginsenoside covalent conjugate, which is formed by at least one carboxyl group in HOOC-PEG-COOH being bonded to ginsenoside via an ester bond; Alternatively, it can be formed by bonding at least one amino group in NH2-PEG-NH2 with ginsenosides via an amide bond.

[0007] Preferably, the ginsenosides include one or more of ginsenoside Rg3, ginsenoside Rb1, and ginsenoside Rd.

[0008] This invention provides a method for preparing the polyethylene glycol-ginsenoside covalent conjugate described above, comprising method one or method two, wherein method one includes the following steps: After the first carboxyl group is activated by mixing HOOC-PEG-COOH, the first condensing agent, the first catalyst and the organic solvent, the mixture is mixed with ginsenosides and subjected to esterification reaction to obtain polyethylene glycol-ginsenoside covalent conjugates. The second method includes the following steps: Ginsenosides, succinic anhydride, a second catalyst and an organic solvent were mixed and subjected to a ring-opening esterification reaction to obtain carboxylated ginsenoside intermediates. The carboxylated ginsenoside intermediate, the second condensing agent, and the activator were mixed and activated with the second carboxyl group. Then, the mixture was mixed with NH2-PEG-NH2 to carry out an amidation reaction to obtain polyethylene glycol-ginsenoside covalent conjugate.

[0009] Preferably, the first condensing agent is dicyclohexylcarbodiimide; the first catalyst is 4-dimethylaminopyridine; and the molar ratio of the carboxyl group, the first condensing agent, and the first catalyst in the HOOC-PEG-COOH is 1:(2.0~4.5):(0.1~0.4).

[0010] Preferably, the molar ratio of HOOC-PEG-COOH to ginsenoside is 1:(2.2~8.8).

[0011] Preferably, the molar ratio of the carboxylated ginsenoside intermediate, the second condensing agent, and the activator is 1:(1.1~1.5):(1.1~1.5).

[0012] Preferably, the molar ratio of NH2-PEG-NH2 to the carboxylated ginsenoside intermediate is 1:(2~5).

[0013] The present invention also provides polyethylene glycol-ginsenoside self-assembled nanoparticles, which are formed by the self-assembly of the polyethylene glycol-ginsenoside covalent conjugate described in the above technical solution or the polyethylene glycol-ginsenoside covalent conjugate obtained by the above preparation method in an aqueous medium.

[0014] This invention also provides a method for preparing polyethylene glycol-ginsenoside self-assembled nanoparticles as described above, comprising the following steps: A polyethylene glycol-ginsenoside covalent conjugate was mixed with a lower alcohol to obtain an organic phase; the organic phase was then injected into an aqueous phase for self-assembly to obtain polyethylene glycol-ginsenoside self-assembled nanoparticles.

[0015] This invention also provides the application of the polyethylene glycol-ginsenoside self-assembled nanoparticles described in the above technical solution or the polyethylene glycol-ginsenoside self-assembled nanoparticles obtained by the above preparation method in the preparation of antitumor drugs or immunomodulatory drugs.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a polyethylene glycol-ginsenoside covalent conjugate, which is formed by at least one carboxyl group in HOOC-PEG-COOH being bonded to ginsenoside via an ester bond; or by at least one amino group in NH2-PEG-NH2 being bonded to ginsenoside via an amide bond.

[0017] This invention employs a chemical modification strategy, utilizing bifunctional polyethylene glycol (HOOC-PEG-COOH or NH2-PEG-NH2) as a hydrophilic linker to covalently couple hydrophobic ginsenosides via ester or amide bonds, constructing an amphiphilic molecule. This molecule can spontaneously assemble into core-shell structured nanoparticles in an aqueous medium. This invention enables "carrier-free" delivery of ginsenosides, achieving 100% drug loading without the need for exogenous carriers. This invention provides two strategies: esterification and amidation. Ester bonds exhibit pH responsiveness and esterase sensitivity, facilitating rapid intracellular drug release; amide bonds possess high biological stability, beneficial for long-term circulation. This invention solves the problems of premature drug leakage or uncontrollable release when using exogenous carriers.

[0018] This invention provides a method for preparing the polyethylene glycol-ginsenoside covalent conjugate described in the above technical solution. By controlling the feed ratio of ginsenoside to polyethylene glycol, products with single-end or double-end modification can be prepared, thereby adjusting the hydrophilicity-hydrophobicity balance (HLB) of the molecules and optimizing the assembly behavior of nanoparticles. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Rg3-PEG 1000 MALDI-TOF MS spectrum of -COOH (ester bond); Figure 2 HOOC-PEG 1000 -COOH hydrogen spectrum; Figure 3 HOOC-PEG 1000 Carbon spectrum of -COOH; Figure 4 The hydrogen spectrum of Rg3; Figure 5 The carbon spectrum of Rg3; Figure 6 Rg3-PEG 1000 -COOH (ester bond) 1H NMR spectrum; Figure 7 Rg3-PEG 1000 Carbon spectrum of -COOH (ester bond); Figure 8 Rg3-PEG 1000 -COOH (ester bond), Rg3, HOOC-PEG 1000 FT-IR comparison of COOH and its three components; Figure 9 Rg3-PEG 1000 -Rg3 (ester bond), Rg3, HOOC-PEG 1000 FT-IR comparison of COOH and its three components; Figure 10 Rg3-PEG 1000 -NH2 (amide bond), Rg3, NH2-PEG 1000 FT-IR comparison chart of the three NH2 components; Figure 11 Rg3-PEG 1000 -Rg3 (amide bond), Rg3, H2N-PEG 1000 FT-IR comparison chart of the three NH2 components; Figure 12 Statistical graph of zeta potential of LP-1, LP-2, LP-3, and LP-4 nanoparticles after 7 days of storage in PBS; Figure 13Particle size distribution of LP-1, LP-2, LP-3, and LP-4 nanoparticles after 7 days of storage in PBS; Figure 14 Statistical graph of zeta potential of LP-1, LP-2, LP-3, and LP-4 nanoparticles after 7 days of incubation in serum; Figure 15 The particle size distribution of LP-1, LP-2, LP-3, and LP-4 nanoparticles after incubation in serum for 7 days is shown in the figure. Detailed Implementation

[0021] This invention provides a polyethylene glycol-ginsenoside covalent conjugate, which is formed by at least one carboxyl group in HOOC-PEG-COOH being bonded to ginsenoside via an ester bond; Alternatively, it can be formed by bonding at least one amino group in NH2-PEG-NH2 with ginsenosides via an amide bond.

[0022] In this invention, the ginsenosides preferably include one or more of ginsenoside Rg3, ginsenoside Rb1, and ginsenoside Rd.

[0023] In this invention, when the ginsenoside is ginsenoside Rg3, the number average molecular weight of HOOC-PEG-COOH (dicarboxyl-terminated polyethylene glycol) and NH2-PEG-NH2 (diamino-terminated polyethylene glycol) is preferably 1000 Da. When the ginsenoside is ginsenoside Rb1, the number average molecular weight of HOOC-PEG-COOH and NH2-PEG-NH2 is preferably 1500-2000 Da. When the ginsenoside is ginsenoside Rd, the number average molecular weight of HOOC-PEG-COOH and NH2-PEG-NH2 is preferably 1000-1500 Da. The polyethylene glycol molecular weight described in this invention does not produce a shielding effect. The polyethylene glycol (PEG) chain, through its large hydration volume, can effectively shield the hydrophobic regions of drug molecules, significantly improving water solubility, and can prolong blood circulation time through a "steric stabilization effect."

[0024] In this invention, the polyethylene glycol-ginsenoside covalent conjugate preferably includes a single-terminated esterified polyethylene glycol-ginsenoside conjugate (taking ginsenoside Rg3 as an example, denoted as Rg3-OOC-PEG-COOH), a double-terminated esterified polyethylene glycol-ginsenoside conjugate (taking ginsenoside Rg3 as an example, denoted as Rg3-OOC-PEG-COO-Rg3), a single-terminated amidated polyethylene glycol-ginsenoside conjugate (taking ginsenoside Rg3 as an example, denoted as Rg3-CONH-PEG-NH2), or a double-terminated amidated polyethylene glycol-ginsenoside conjugate (taking ginsenoside Rg3 as an example, denoted as Rg3-CONH-PEG-NHCO-Rg3).

[0025] In this invention, the amide bond is preferably a succinamide bond.

[0026] Ginsenosides contain hydroxyl groups, which can react with the carboxyl groups in HOOC-PEG-COOH to form ester bonds. The carbonyl carbon in the ester bond (-OC(=O)-) is linked to PEG, and the oxygen atom is linked to the ginsenoside. This invention first carboxylates the ginsenoside, allowing it to react with the amino group in NH2-PEG-NH2 to form an amide bond. The amino nitrogen in the amide bond (-OC(=O)-(CH2)2-C(=O)-NH-) is linked to PEG, and the oxygen atom is linked to the ginsenoside. Simply mixing or physically encapsulating ginsenosides with PEG cannot fundamentally solve the problems of unstable drug-carrier binding and easy leakage, and it cannot self-assemble into nanoparticles.

[0027] The present invention also provides a method for preparing the polyethylene glycol-ginsenoside covalent conjugate described in the above technical solution, comprising method one or method two, wherein method one comprises the following steps: After the first carboxyl group is activated by mixing HOOC-PEG-COOH, the first condensing agent, the first catalyst and the organic solvent, the mixture is mixed with ginsenosides and subjected to esterification reaction to obtain polyethylene glycol-ginsenoside covalent conjugates. The second method includes the following steps: Ginsenosides, succinic anhydride, a second catalyst and an organic solvent were mixed and subjected to a ring-opening esterification reaction to obtain carboxylated ginsenoside intermediates. The carboxylated ginsenoside intermediate, the second condensing agent, and the activator were mixed and activated with the second carboxyl group. Then, the mixture was mixed with NH2-PEG-NH2 to carry out an amidation reaction to obtain polyethylene glycol-ginsenoside covalent conjugate.

[0028] Unless otherwise specified, all materials and equipment used in this invention are commercially available products in the field.

[0029] Method 1 (for constructing ester-linked polyethylene glycol-ginsenoside covalent conjugates): In this invention, HOOC-PEG-COOH, a first condensing agent, a first catalyst, and an organic solvent are mixed and activated with a first carboxyl group, and then mixed with ginsenosides for esterification to obtain polyethylene glycol-ginsenoside covalent conjugates.

[0030] In this invention, the first condensing agent is preferably dicyclohexylcarbodiimide (DCC); the first catalyst is preferably 4-dimethylaminopyridine (DMAP); and the organic solvent is preferably anhydrous pyridine or anhydrous dimethyl sulfoxide (DMSO). The mixing of HOOC-PEG-COOH, the first condensing agent, the first catalyst, and the organic solvent is preferably carried out in an ice-water bath (0~4℃), with stirring and nitrogen protection, and the stirring time is preferably 10~20 minutes. The activation temperature of the first carboxyl group is preferably room temperature (25℃), and the activation time of the first carboxyl group is preferably 2~4 hours.

[0031] In this invention, the molar ratio of the carboxyl group, the first condensing agent, and the first catalyst in the HOOC-PEG-COOH is preferably 1:(2.0~4.5):(0.1~0.4), specifically 1:2.2:0.1, 1:2.2:0.2, 1:2.2:0.3, 1:2.2:0.4, 1:2.5:0.1, 1:4.4:0.2, or 1:4.4:0.4.

[0032] In this invention, the ginsenosides are preferably used in the form of a ginsenoside solution, and the solvent of the ginsenoside solution is preferably anhydrous DMSO, with a concentration preferably of 0.044 mmol / L. Preferably, the ginsenoside solution is added dropwise to the system obtained by the first carboxyl activation. Taking 5 mL of ginsenoside solution as an example, the addition time is preferably 10-20 minutes. The esterification reaction is preferably carried out under anhydrous, light-protected, and nitrogen-protected conditions; the temperature of the esterification reaction is preferably room temperature (25°C) or 30-40°C, and the time is preferably 12-24 hours.

[0033] In this invention, the molar ratio of HOOC-PEG-COOH to ginsenoside is preferably 1:(2.2~8.8). When preparing a single-terminated esterified polyethylene glycol-ginsenoside conjugate, the molar ratio of HOOC-PEG-COOH to ginsenoside is preferably 1:(2.2~4.4), specifically 1:4.4; when preparing a double-terminated esterified polyethylene glycol-ginsenoside conjugate, the molar ratio of COOH-PEG-COOH to ginsenoside is preferably 1:(4.4~8.8), specifically 1:8.8.

[0034] In this invention, the esterification reaction preferably includes sequential purification and freeze-drying, and the purification preferably uses a dialysis bag with a molecular weight cutoff of 1500 Da.

[0035] Method 2 (for constructing succinamide-linked polyethylene glycol-ginsenoside covalent conjugates): This invention involves mixing ginsenosides, succinic anhydride, a second catalyst, and an organic solvent to carry out a ring-opening esterification reaction to obtain carboxylated ginsenoside intermediates.

[0036] In this invention, the second catalyst is preferably 4-dimethylaminopyridine (DMAP); the organic solvent is preferably anhydrous pyridine or anhydrous dimethyl sulfoxide (DMSO).

[0037] In this invention, the molar ratio of ginsenoside to succinic anhydride is preferably 1:(1~4), specifically 1:3.

[0038] In this invention, the ring-opening esterification reaction is preferably carried out at a temperature of 40-60°C for 8-12 hours. The reaction preferably includes the removal of the reaction solvent after the ring-opening esterification reaction; the method for removing the reaction solvent is preferably rotary evaporation, and the rotary evaporation temperature is preferably 40°C.

[0039] The carboxylated ginsenoside intermediate was obtained. In this invention, the carboxylated ginsenoside intermediate, a second condensing agent and an activator were mixed and activated with a second carboxyl group, and then mixed with NH2-PEG-NH2 to carry out an amidation reaction to obtain a polyethylene glycol-ginsenoside covalent conjugate.

[0040] In this invention, the second condensing agent is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), and the activator is preferably N-hydroxysuccinimide (NHS). The second carboxyl activation is preferably carried out in a MES buffer containing a very small amount of anhydrous DMSO at pH 6.0, with the volume ratio of the MES buffer to DMSO preferably being 40:0.5; the activation temperature is preferably 25-26°C (room temperature), and the activation time is preferably 3 hours.

[0041] In this invention, the molar ratio of the carboxylated ginsenoside intermediate, the second condensing agent, and the activator is preferably 1:(1.1~1.5):(1.1~1.5), specifically 1:1.2:1.2.

[0042] In this invention, the molar ratio of NH2-PEG-NH2 to the carboxylated ginsenoside intermediate is 1:(2~5). When preparing a single-terminated amidated polyethylene glycol-ginsenoside conjugate, the molar ratio of NH2-PEG-NH2 to the carboxylated ginsenoside intermediate is preferably 1:(2~2.5), specifically 1:2.1; when preparing a double-terminated amidated polyethylene glycol-ginsenoside conjugate, the molar ratio of NH2-PEG-NH2 to the carboxylated ginsenoside intermediate is preferably 1:(2.6~4.4), specifically 1:4.2.

[0043] In this invention, the amidation reaction preferably further includes sequential purification and freeze-drying, wherein the purification is preferably performed using a dialysis bag with a molecular weight cutoff of 1500 Da.

[0044] The present invention also provides polyethylene glycol-ginsenoside self-assembled nanoparticles, which are formed by the self-assembly of the polyethylene glycol-ginsenoside covalent conjugate described in the above technical solution or the polyethylene glycol-ginsenoside covalent conjugate obtained by the above preparation method in an aqueous medium.

[0045] In this invention, the nanoparticles possess a core-shell structure. The "shell" is a dense hydration layer formed by the outward extension of covalently coupled bifunctional polyethylene glycol hydrophilic blocks, which maintains the colloidal stability of the nanoparticles and imparts stealth properties through steric hindrance. The "core" is a dense region formed by the tight aggregation and collapse of the aglycone portions of multiple hydrophobic ginsenoside molecules through hydrophobic interactions, resulting in an aggregated structure formed by the self-accumulation of the active pharmaceutical ingredient, ginsenoside. The nanoparticles of this invention differ significantly from traditional drug-loaded nanoparticles; the core is the drug itself, requiring no exogenous carrier filling, achieving 100% drug loading. The average particle size of the nanoparticles is 80-150 nm, more preferably 90-120 nm, with a polydispersity index (PDI) of less than 0.20; the zeta potential is negative, resulting in minimal disturbance to cell membranes, low toxicity, and no adsorption of plasma proteins leading to hemolysis.

[0046] In this invention, the aqueous medium preferably includes water.

[0047] This invention also provides a method for preparing polyethylene glycol-ginsenoside self-assembled nanoparticles as described above, comprising the following steps: A polyethylene glycol-ginsenoside covalent conjugate was mixed with a lower alcohol to obtain an organic phase; the organic phase was then injected into an aqueous phase for self-assembly to obtain polyethylene glycol-ginsenoside self-assembled nanoparticles.

[0048] In this invention, the lower alcohol is a good solvent for the polyethylene glycol-ginsenoside covalent conjugate; the lower alcohol preferably includes volatile methanol or ethanol; the concentration of the polyethylene glycol-ginsenoside conjugate in the organic phase is preferably 3 mg / mL.

[0049] In this invention, the aqueous phase preferably comprises water, and the water is preferably ultrapure water. The aqueous phase also preferably includes a surfactant, and the surfactant is preferably polyvinyl alcohol (PVA), with the concentration of the surfactant in the aqueous phase preferably being 0.1 mg / mL.

[0050] In this invention, the organic phase is preferably injected into the aqueous phase under stirring and an ice-water bath, with the stirring rate preferably being 1200 rpm; the volume ratio of the organic phase to the aqueous phase is preferably 1:6. The self-assembly is preferably carried out under stirring, with the stirring speed preferably being 1200 rpm and the time preferably being 2 hours. The polyethylene glycol-ginsenoside covalent conjugate is an amphiphilic covalent conjugate. After the organic phase is injected into the aqueous phase, the rapid change in the solvent environment drives the aggregation of the hydrophobic ginsenoside segment (such as Rg3) and the extension of the hydrophilic PEG segment, thereby spontaneously forming nanomicelles. In this invention, self-assembly is carried out under stirring and an ice-water bath. The stirring speed provides sufficient shear force to control the particle size distribution, and maintaining the low temperature of the system prevents the aggregation of already formed nanoparticles or potential ester bond hydrolysis, ensuring the stable completion of the self-assembly process.

[0051] In this invention, the self-assembly process preferably further includes: sequentially purifying and freeze-drying the obtained nano-suspension; the purification preferably uses a dialysis bag with a molecular weight cutoff of 3500 Da, and the dialysis can remove organic solvents and free molecules.

[0052] This invention prepares ginsenosides into nanoparticles, which can improve the solubility, antitumor activity and bioavailability of ginsenosides.

[0053] This invention also provides the application of the polyethylene glycol-ginsenoside self-assembled nanoparticles described in the above technical solution or the polyethylene glycol-ginsenoside self-assembled nanoparticles obtained by the above preparation method in the preparation of antitumor drugs or immunomodulatory drugs.

[0054] The embodiments of the present invention prepared a variety of polyethylene glycol-ginsenoside amphiphilic conjugates based on ester bonds or amide bonds, and a carrier-free nanodelivery system constructed by self-assembly, which can improve the bioavailability of ginsenosides.

[0055] To further illustrate the present invention, the polyethylene glycol-ginsenoside covalent conjugate, its self-assembled nanoparticles, and preparation method provided by the present invention are described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0056] In this embodiment of the invention, ginsenoside Rg3 (purity ≥98%), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), succinic anhydride, etc., were all analytical grade reagents, and the molecular weight cutoff (MWCO) of the dialysis bag was 1500 Da and 3500 Da. Polyethylene glycol with an average molecular weight of 1000: HOOC-PEG. 1000-COOH: Maclean's reagent, catalog number P902466-1g, CAS number 39927-08-7; H2N-PEG 1000 -NH2: Maclean's reagent, catalog number N902461-1g.

[0057] Example 1: Single-terminal ester bond conjugate (Rg3-OOC-PEG) 1000 Preparation of -COOH) This embodiment adopts the strategy of "activating PEG first, then coupling saponins" in the first approach.

[0058] 1. Activation: Add HOOC-PEG to a dry 50 mL reaction flask. 1000 -COOH (50 mg, 0.05 mmol) and 5 mL of anhydrous DMSO were dissolved by stirring. Under ice-water bath and nitrogen protection, DMSO solutions of DCC (45.4 mg, 0.22 mmol) and DMAP (1.22 mg, 0.01 mmol) were added sequentially, and the carboxyl groups were activated by stirring at room temperature for 4 hours.

[0059] 2. Coupling: Weigh ginsenoside Rg3 (172.7 mg, 0.22 mmol) and dissolve it in 5 mL of anhydrous DMSO, protecting it from light and at a temperature below 35°C. Slowly add this solution dropwise to the above activated PEG solution over 20 minutes, maintaining a light-protected, anhydrous, and nitrogen-filled environment. After the addition is complete, stir the mixture at room temperature for 24 hours.

[0060] 3. Transfer the reaction solution into an MWCO 1500 Da dialysis bag and dialyze using the following method: 5 L of ultrapure water, dialysis for 72 hours, changing the water every 6 hours for the first 24 hours, and then every 12 hours thereafter; freeze-dry to obtain a white powder product Rg3-OOC-PEG. 1000 -COOH (denoted as Rg3-PEG) 1000 -COOH (ester bond)).

[0061] The product was subjected to FT-IR, 1 H NMR, 1 The structure was characterized and confirmed by C NMR and MALDI-TOF MS.

[0062] Figure 1 Rg3-PEG 1000 The MALDI-TOF MS spectrum of -COOH (ester bond) shows a typical PEG structure (adjacent peaks differ by one PEG unit), and the horizontal axis of the highest peak corresponds to the molecular weight of the target product: 1000+785 (the molecular weight of Rg3 is 785).

[0063] Figure 2 HOOC-PEG1000 -COOH hydrogen spectrum; Figure 3 HOOC-PEG 1000 Carbon spectrum of -COOH.

[0064] Figure 4 The hydrogen spectrum of Rg3; Figure 5 This is the carbon spectrum of Rg3.

[0065] Figure 6 Rg3-PEG 1000 -COOH (ester bond) 1H NMR spectrum; Figure 7 Rg3-PEG 1000 Carbon spectrum of -COOH (ester bond).

[0066] Figure 8 Rg3-PEG 1000 -COOH (ester bond), Rg3, HOOC-PEG 1000 FT-IR comparison of the three COOH groups. Rg3-PEG 1000 -COOH at 3332 cm -1 A broad peak appears, 2931 / 2856 cm. -1 The stretching vibration of the PEG backbone is -CH2-, 1630 cm⁻¹. -1 The appearance of a new C=O absorption accompanied by a shift in carboxyl-related peaks, while retaining the characteristic absorption peaks of Rg3, indicates that Rg3 has been successfully covalently grafted to PEG at one end. The 1H NMR spectrum shows two peaks at 4.5 ppm: the high field represents the outer sugar ring of Rg3, and the low field represents the inner sugar ring. The outer sugar ring exhibits significant asymmetry after grafting; based on its position, the grafting site is presumably the hydroxyl group at position 4 of the outer sugar ring.

[0067] Example 2: Bi-terminal ester bond conjugate (Rg3-OOC-PEG) 1000 Preparation of -COO-Rg3) Referring to the method in Example 1, the feed amount was adjusted according to the bifunctionality: HOOC-PEG 1000 -COOH 50 mg, ginsenoside Rg3 345.4 mg, DCC 90.8 mg, DMAP 4.88 mg. The reaction and purification conditions were the same as in Example 1. A white powder product Rg3-OOC-PEG was obtained. 1000 -COO-Rg3 (denoted as Rg3-PEG) 1000 -Rg3 (ester bond)).

[0068] Figure 9 Rg3-PEG 1000 -Rg3 (ester bond), Rg3, HOOC-PEG 1000FT-IR comparison of the three COOH groups. Rg3-PEG 1000 -Rg3 at 3331 cm -1 Retains broad peaks, 2929 / 2854 cm -1 The characteristic peak of PEG is -CH2-, 1630~1570 cm⁻¹. -1 The enhanced absorption and disappearance of the characteristic peak of the free carboxyl group, along with the simultaneous presence of characteristic absorptions of PEG and Rg3, indicate that both ends of PEG are successfully coupled with Rg3, forming a bi-terminal substituted structure.

[0069] Example 3: Single-ended amide bond coupling (Rg3-CONH-PEG) 1000 Synthesis of -NH2) This embodiment adopts the strategy of "first activating saponins, then coupling PEG" in the second path.

[0070] 1. Preparation of the carboxylation intermediate: In a dry 50 mL reaction flask, ginsenoside Rg3 (100 mg, 0.127 mmol) and 5 mL of anhydrous pyridine were added, and the mixture was heated and stirred in a water bath at 35 °C until completely dissolved. Separately, succinic anhydride (38.25 mg, 0.38 mmol) was dissolved in 2 mL of anhydrous pyridine and slowly added dropwise to the Rg3 solution. DMAP (3.12 mg, 0.026 mmol) was added as a catalyst. The reaction was carried out under nitrogen protection and stirred at 60 °C for 12 hours. After the reaction was completed, the solvent was removed by rotary evaporation at 40 °C to obtain an oily Rg3-COOH intermediate (succinic anhydride ring-opening linked to ginsenoside Rg3).

[0071] 2. Amide Coupling: Dissolve the above intermediate in 40 mL of MES buffer (pH 6.0). Add DMSO solution containing EDC·HCl (29.31 mg, 0.15 mmol) and NHS (17.60 mg, 0.15 mmol) sequentially, and activate the carboxyl group by stirring at room temperature for 3 hours. Separately, prepare H2N-PEG... 1000 -NH2 (63.7 mg, 0.06 mmol) was dissolved in 5 mL of MES buffer. The PEG solution was slowly added dropwise to the activated Rg3-COOH solution, and the reaction was carried out at room temperature with stirring for 12 hours.

[0072] 3. Purification: The reaction solution was transferred to a dialysis bag with a molecular weight cutoff (MWCO) of 1500 Da and dialyzed with 4 L of deionized water for 48 hours, changing the water every 6 hours. After dialysis, the solution was pre-frozen at -80°C and then freeze-dried to obtain a white powdery product, PEG. 1000 -CONH-Rg3-NH2 (denoted as Rg3-PEG) 1000 -NH2 (amide bond)).

[0073] Figure 10 Rg3-PEG 1000 -NH2 (amide bond), Rg3, NH2-PEG 1000 FT-IR comparison of Rg3-PEG-NH2 at 3414 cm⁻¹. -1 A distinct broad peak (-OH / -NH) appears at 2939 cm⁻¹. -1 The stretching vibration of the PEG backbone is -CH2-, 1735 cm⁻¹. -1 A new C=O absorption peak appears, accompanied by changes in amino-related peaks, while retaining the characteristic absorptions of Rg3 (1456, 1255, 1080 cm⁻¹). -1 This indicates that Rg3 has successfully undergone covalent coupling with aminoPEG, forming a single-terminal substituted structure.

[0074] Example 4: Bis-terminated amide bond conjugate (Rg3-CONH-PEG) 1000 -NHCO-Rg3).

[0075] Following the method of Example 3, the dosage was doubled: ginsenoside Rg3 200 mg, succinic anhydride 76.5 mg, H2N-PEG 1000 -NH2 63.7 mg, EDC·HCl 58.62 mg, NHS 35.20 mg, DMAP 6.24 mg. The reaction and purification conditions were the same as in Example 3. After purification, a white solid product Rg3-CONH-PEG was obtained. 1000 -NHCO-Rg3 (double-ended modification, denoted as Rg3-PEG) 1000 -Rg3 (amide bond)).

[0076] Figure 11 Rg3-PEG 1000 -Rg3 (amide bond), Rg3, H2N-PEG 1000 FT-IR comparison of the three (NH2, Rg3, PEG, and Rg3) at 3381 cm⁻¹. -1 It still exhibits a broad peak, 2943 / 2877 cm. -1 The characteristic absorption of PEG's -CH2- is 1737 and 1653 cm⁻¹. -1 C=O absorption and 1570 cm -1 The nearby peaks indicate that a coupling reaction has occurred. At the same time, the characteristic peaks of PEG and Rg3 coexist, and the amino signal is greatly reduced, indicating that Rg3 has been successfully grafted to both ends of the PEG backbone to form a symmetrical bi-terminal substitution structure.

[0077] Example 5: Preparation of self-assembled nanoparticles of polyethylene glycol-ginsenoside conjugate (ethanol injection method) Take the Rg3-OOC-PEG prepared in Example 11000 3 mg of -COOH was dissolved in 1 mL of anhydrous ethanol to form the organic phase. 6 mL of ultrapure water was prepared as the aqueous phase. Under an ice-water bath environment (0–4 °C) and magnetic stirring (1200 rpm), the organic phase was rapidly injected into the aqueous phase using a sterile syringe (1 mL). After injection, stirring was continued for 2 hours to allow complete self-assembly of the system. The resulting nano-suspension was transferred to a MWCO 3500 Da dialysis bag and dialyzed against 4 L of deionized water for 24 hours to remove anhydrous ethanol. After dialysis, the suspension was filtered through a 0.22 μm microporous membrane for sterilization, aliquoted, and freeze-dried to obtain a white nanoparticle powder (denoted as LP-1).

[0078] Examples 6-8 Following the same method as in Example 5, nanoparticles LP-2, LP-3, and LP-4 were prepared using the products of Examples 2, 3, and 4 as raw materials, respectively.

[0079] The nanoparticles prepared in Examples 5-8 were subjected to zeta potential and particle size tests, and the test methods are as follows: Particle size test: Take 0.5 mL of the stock solution (the liquid sample obtained by filtration of the dialysis suspension from Examples 5-8 through a 0.22 μm microporous membrane for sterilization, without freeze-drying), and dilute it to 5 mL with deionized water or PBS buffer (approximately 10-fold dilution) until the liquid is slightly bluish-white, semi-transparent, and translucent. Inject the liquid into a microparticle size cup using a disposable syringe or dropper, ensuring the liquid level is approximately 1-1.5 cm (approximately 0.5-0.6 mL), ensuring no air bubbles, and place it in the sample cell of a DLS laser particle size analyzer to detect the particle size.

[0080] Zeta potential test: Take 0.5 mL of the stock solution (the liquid sample obtained by filtration of the dialysis suspension from Examples 5-8 through a 0.22 μm microporous membrane for sterilization, without freeze-drying), and dilute it to 5 mL with deionized water or PBS buffer (approximately a 10-fold dilution) until the liquid is slightly bluish-white, semi-transparent, and translucent. Inject the liquid into the capillary electrode sample cell using a disposable syringe or dropper until the liquid level reaches the opening of the electrode plate (approximately 0.7 mL), ensuring no air bubbles and keeping the external electrode contacts dry. Place the sample cell in the DLS laser particle size analyzer to detect the zeta potential.

[0081] Figure 12 Statistical graph of zeta potential of LP-1, LP-2, LP-3, and LP-4 nanoparticles after 7 days of storage in PBS; Figure 13 The particle size distribution of LP-1, LP-2, LP-3, and LP-4 nanoparticles after 7 days of storage in PBS is shown in the following figures: LP-1: Within 7 days, the potential range is -5.85 to -8.78 mV (average approximately -7.1 mV), and the particle size range is 84.1 to 98.6 nm (average approximately 91 nm), exhibiting the smallest fluctuation range and the best stability; LP-2: The potential range within 7 days was -6.09 to -8.42 mV (average approximately -7.2 mV), and the particle size range was 85.8 to 103.8 nm (average approximately 94 nm). The overall stability was good with only slight fluctuations in the later stage. LP-3: The potential range within 7 days is -6.15~-8.92 mV (average about -7.4 mV), and the particle size range is 106.9~132.9 nm (average about 119 nm). Although there are some fluctuations, they are still maintained within a controllable range, and the stability is good. LP-4: The potential range within 7 days is -5.59 to -8.82 mV (average approximately -7.3 mV), and the particle size range is 103.4 to 124.8 nm (average approximately 116 nm). It exhibits small potential fluctuations, no drastic changes in particle size, and good stability.

[0082] Figure 14 Statistical graph of zeta potential of LP-1, LP-2, LP-3, and LP-4 nanoparticles after seven days of incubation in serum; Figure 15 The particle size distribution of LP-1, LP-2, LP-3, and LP-4 nanoparticles after seven days of incubation in serum is shown in the following figures: LP-1: Within 7 days, the potential range is -4.23 to -7.91 mV (mean approximately -5.9 mV), and the particle size range is 84.6 to 99.1 nm (mean approximately 91 nm). The potential and particle size fluctuations are minimal, with no obvious signs of aggregation or protein adsorption. It exhibits excellent stability in serum and good biocompatibility. LP-2: The potential range within 7 days was -4.62 to -7.36 mV (mean approximately -6.2 mV), and the particle size range was 86.3 to 104.3 nm (mean approximately 94 nm). The overall values ​​were stable, with only a slight increase in particle size in the later stage. No drastic changes caused by protein adsorption were observed. It showed good stability in serum and excellent biocompatibility. LP-3: The potential range within 7 days is -4.45 to -7.63 mV (mean approximately -5.9 mV), and the particle size range is 106.9 to 123.8 nm (mean approximately 116 nm). Although there are some fluctuations, they are always maintained within a controllable range. There is no sudden increase in particle size or drastic change in potential caused by protein adsorption. It has good stability in serum and has reliable biocompatibility. LP-4: The potential range within 7 days is -4.73 to -7.21 mV (mean approximately -6.2 mV), and the particle size range is 110.3 to 129.2 nm (mean approximately 119 nm). The potential fluctuation is small, and although there are slight changes in particle size, there is no abnormal aggregation. It has good stability in serum and good biocompatibility.

[0083] This invention develops a novel drug-carrier integrated delivery system capable of achieving near 100% of the theoretical drug loading capacity. Controlled release is achieved through the rational design of chemical bonds, minimizing the exposure of inactive components. An ideal drug delivery system not only needs to solve dissolution and stability issues but should also possess the ability to controllably release the parent drug at the target site or under specific physiological conditions, thus functioning as a "prodrug." Existing physical encapsulation systems often rely on the degradation or diffusion of the carrier material for drug release control, resulting in relatively simple mechanisms that are difficult to precisely regulate. In contrast, this invention covalently couples PEG with ginsenosides, essentially constructing a novel class of ginsenoside prodrugs. These covalent bonds are relatively stable in systemic circulation, protecting the active core of the saponin, but can undergo responsive cleavage in specific biological environments. Ester bonds can be hydrolyzed by esterases or in the slightly alkaline intestinal environment; while amide bonds are more stable, they can also be cleaved by proteases or in the slightly acidic environment of certain tumor tissues. This "stimulus-response" release characteristic provides the possibility of achieving specific drug activation at the lesion site, which is impossible with simple physical mixing or encapsulation techniques.

[0084] The four structural (single / double-ended, ester / amide bond) products designed in this invention not only serve as final prodrug molecules but also provide valuable chemical synthesis intermediates for subsequent more complex drug design. Their terminal active groups can be used for further coupling with targeting ligands, fluorescent probes, or other therapeutic modules to construct multifunctional integrated diagnostic and therapeutic agents, thereby greatly expanding their application boundaries. This invention not only solves the delivery problem of ginsenosides but also, through the ingenious design of its molecular structure, endows them with the potential for in vivo environment-responsive drug release and scalability as multifunctional platform intermediates, providing a new approach and technological foundation for developing next-generation, highly efficient, and intelligent ginsenoside formulations.

[0085] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A polyethylene glycol-ginsenoside covalent conjugate, characterized in that, It is formed by at least one carboxyl group in HOOC-PEG-COOH being bonded to ginsenoside via an ester bond; Alternatively, it can be formed by bonding at least one amino group in NH2-PEG-NH2 with ginsenosides via an amide bond.

2. The polyethylene glycol-ginsenoside covalent conjugate according to claim 1, characterized in that, The ginsenosides include one or more of ginsenoside Rg3, ginsenoside Rb1, and ginsenoside Rd.

3. The method for preparing the polyethylene glycol-ginsenoside covalent conjugate according to claim 1 or 2, characterized in that, This includes either method one or method two, wherein method one includes the following steps: After the first carboxyl group is activated by mixing HOOC-PEG-COOH, the first condensing agent, the first catalyst and the organic solvent, the mixture is mixed with ginsenosides and subjected to esterification reaction to obtain polyethylene glycol-ginsenoside covalent conjugates. The second method includes the following steps: Ginsenosides, succinic anhydride, a second catalyst and an organic solvent were mixed and subjected to a ring-opening esterification reaction to obtain carboxylated ginsenoside intermediates. The carboxylated ginsenoside intermediate, the second condensing agent, and the activator were mixed and activated with the second carboxyl group. Then, the mixture was mixed with NH2-PEG-NH2 to carry out an amidation reaction to obtain polyethylene glycol-ginsenoside covalent conjugate.

4. The preparation method according to claim 3, characterized in that, The first condensing agent is dicyclohexylcarbodiimide; the first catalyst is 4-dimethylaminopyridine; the molar ratio of the carboxyl group, the first condensing agent and the first catalyst in the HOOC-PEG-COOH is 1:(2.0~4.5):(0.1~0.4).

5. The preparation method according to claim 3 or 4, characterized in that, The molar ratio of HOOC-PEG-COOH to ginsenoside is 1:(2.2~8.8).

6. The preparation method according to claim 3, characterized in that, The molar ratio of the carboxylated ginsenoside intermediate, the second condensing agent, and the activator is 1:(1.1~1.5):(1.1~1.5).

7. The preparation method according to claim 3 or 6, characterized in that, The molar ratio of NH2-PEG-NH2 to the carboxylated ginsenoside intermediate is 1:(2~5).

8. A polyethylene glycol-ginsenoside self-assembled nanoparticle, characterized in that, The polyethylene glycol-ginsenoside covalent conjugate as described in claim 1 or 2, or the polyethylene glycol-ginsenoside covalent conjugate obtained by any one of the preparation methods described in claims 3 to 7, is self-assembled in an aqueous medium.

9. The method for preparing polyethylene glycol-ginsenoside self-assembled nanoparticles according to claim 8, characterized in that, Includes the following steps: A polyethylene glycol-ginsenoside covalent conjugate was mixed with a lower alcohol to obtain an organic phase; the organic phase was then injected into an aqueous phase for self-assembly to obtain polyethylene glycol-ginsenoside self-assembled nanoparticles.

10. The application of the polyethylene glycol-ginsenoside self-assembled nanoparticles of claim 8 or the polyethylene glycol-ginsenoside self-assembled nanoparticles obtained by the preparation method of claim 9 in the preparation of antitumor drugs or immunomodulatory drugs.

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