Bilirubin prodrug nano multi-drug synergistic kidney targeting delivery system and preparation method thereof

Abstract

The invention discloses a nano multi-drug synergistic targeting delivery system based on bilirubin prodrugs and a preparation method of the nano multi-drug synergistic targeting delivery system. The system is formed by self-assembly of an amphiphilic conjugate, the conjugate takes a second-generation lysine dendrimer (G2K) as a hydrophilic skeleton, and the branched end of the conjugate is covalently modified with a hydrophobic antioxidant bilirubin (Br) through a reactive oxygen species (ROS)-sensitive thioketal bond (TK) and is further coupled with a targeting ligand serine (L-serine). The delivery system can specifically recognize damaged kidney tissue, and accurate enrichment of therapeutic drugs in acute kidney injury (AKI) focuses is achieved. After entering a focus, the carrier drives the explosive release of the medicine by utilizing a dual response mechanism of TK bond breakage and bilirubin polarity overturning, and meanwhile, the endogenous anti-oxidation effect of carrier components is exerted. The delivery system provided by the invention has the characteristics of high drug loading capacity, good biocompatibility, strong targeting property and multi-drug synergistic treatment, and has a wide application prospect in the field of kidney disease treatment.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and more specifically to a bilirubin prodrug nano-multidrug targeted delivery system and its preparation method. Background Technology

[0002] Acute kidney injury (AKI) is a common critical illness characterized by a rapid deterioration in kidney function, leading to oxidative stress, mitochondrial dysfunction, inflammation, and apoptosis. Excessive accumulation of reactive oxygen species (ROS) is a key factor contributing to damage to renal tubular epithelial cells. Currently, there is a lack of specific drug interventions for AKI treatment, primarily due to low drug delivery efficiency, insufficient renal targeting, and the difficulty of modulating complex pathological mechanisms with single therapies. Bilirubin (Br), as a potent endogenous antioxidant, can significantly reduce oxidative damage by quenching free radicals and inhibiting NADPH oxidase. However, the clinical application of bilirubin is limited: firstly, it has extremely poor water solubility and easily accumulates under physiological conditions; secondly, it is rapidly cleared from the body, making it difficult to maintain effective concentrations in renal lesions. More importantly, existing bilirubin nanoparticles mostly rely on their own polarity reversal for passive release. In the high oxidative stress environment of AKI lesions, this single response mode often leads to a mismatch between drug release kinetics and pathological needs, and lacks the ability to recognize kidney-specific receptors. Furthermore, the pathological process of AKI involves synergistic effects across multiple pathways. Single delivery of bilirubin or a single therapeutic drug is insufficient to fully reverse the damage. Conventional linear polymer carriers suffer from limitations such as limited terminal functional groups, low drug loading capacity, and poor structural stability. Therefore, developing an endogenous prodrug delivery system with precise renal targeting capabilities, a dual-sensitive response triggered by ROS, and the ability to synergistically deliver multiple therapeutic drugs is of paramount clinical value for improving the treatment efficacy of AKI. Summary of the Invention

[0003] The purpose of this invention is to overcome the defects of existing technologies, such as low bioavailability of bilirubin, insufficient renal targeting, and drug release lag caused by a single response mechanism, and to provide a bilirubin prodrug nano-multidrug synergistic targeted delivery system with dual oxidative stress response capability and its preparation method. The technical solution adopted in this invention is as follows: A bilirubin prodrug-based nano-multidrug synergistic targeted delivery system, characterized in that the system comprises nanomicelles formed by the self-assembly of amphiphilic conjugates through hydrophilic-hydrophobic self-assembly, and an active drug encapsulated within the hydrophobic core of the micelles. The amphiphilic conjugate uses a second-generation lysine dendritic macromolecule (G2K) as its hydrophilic backbone, with its branched-terminal amino groups covalently linked to a reactive oxygen species (ROS)-sensitive thioketone bond (TK) and a targeting ligand, serine (L-serine), respectively; the other end of the thioketone bond is connected to hydrophobic bilirubin (Br), thereby constructing a nanocarrier with specific recognition ability for kidney injury sites and ROS-triggered dissociation ability. The construction logic and mechanism of the delivery system are as follows: The second-generation lysine dendritic macromolecule (G2K), with its highly branched topology, provides a high density of grafting sites for the thioketone bond-bilirubin (TK-Br) prodrug module and serine ligands, ensuring the stability of the carrier structure and the drug loading capacity. During the delivery stage, the system utilizes the active recognition of the highly expressed Kim-1 receptor on the surface of renal tubular epithelial cells by the surface-modified serine to achieve precise enrichment of the drug at the site of kidney injury. When the nanomicelles enter the oxidative stress microenvironment, the high level of reactive oxygen species at the lesion site triggers a dual-response degradation mechanism: on the one hand, the thioketone bond (TK) undergoes specific chemical breakage, leading to the physical disintegration of the carrier backbone; on the other hand, bilirubin (Br) in the hydrophobic core is oxidized to hydrophilic biliverdin under the action of ROS, inducing a polarity reversal within the micelles. This synergistic effect of "physical disintegration" and "polarity reversal" breaks the hydrophobic dynamic balance of the carrier, driving the encapsulated active drug to achieve explosive release at the lesion site. Meanwhile, the bilirubin fragments released by the carrier act as potent endogenous antioxidants, which can directly quench superoxide free radicals and exert a pharmacodynamic synergistic effect with the encapsulated active drugs (such as RTA408, SS-31, etc.) in inhibiting oxidative damage and protecting mitochondrial function. The preparation method of the delivery system comprises the following steps: First, using H-Lys-OMe·2HCl and Boc-Lys(Boc)-OH as raw materials, a second-generation lysine dendritic macromolecule (G2K) is prepared through amide condensation and deprotection reaction. Second, β-mercaptoethylamine is reacted with ethyl trifluoroacetate and further condensed with 2-methoxypropylene to obtain a thioketal linker (TK) with bifunctional groups. Subsequently, G2K, TK, and bilirubin are subjected to a multi-step grafting reaction under the action of a condensing agent, and serine is further coupled at the end of the backbone. The resulting amphiphilic conjugate RCTBrP is obtained through dialysis and lyophilization. Finally, the conjugate and the active drug are dissolved in an organic phase and introduced into a neutral aqueous phase system under stirring. The system self-assembles to form drug-loaded nanomicelles by utilizing hydrophilic-hydrophobic interactions. Compared with the prior art, the present invention has the following beneficial effects: 1. Achieved precise active targeted delivery: Through the specific binding of serine to the Kim-1 receptor, the biodistribution of nanoparticles in damaged kidney tissue was significantly improved, effectively reducing the systemic toxic side effects caused by traditional drug delivery methods. 2. Possesses highly efficient dual-response drug release characteristics: The TK cleavage and bilirubin polarity reversal mechanism designed in this invention endows the carrier with extremely high ROS sensitivity, overcoming the problems of slow and incomplete drug release of conventional carriers, and ensuring the instantaneous release of therapeutic drugs in the core area of ​​the lesion. 3. Achieved pharmacological synergy between carrier and drug: The carrier component of this system is itself a therapeutic molecule. The ROS scavenging effect of bilirubin during degradation combined with the multi-target intervention of the encapsulated drug can more comprehensively cover the complex pathological process of acute kidney injury and has significant clinical application potential. Attached Figure Description

[0004] The present invention will be further described below with reference to the accompanying drawings and embodiments. Figure 1 This is the 1H NMR spectrum of NH2-TK-NH2 prepared in this invention. Figure 2 This is the 1H NMR spectrum of BOC-G2K-NH2 prepared in this invention. Figure 3 This is the 1H NMR spectrum of Br-TK-G2K (RCBrP) prepared in this invention. Figure 4 This is the NMR mass spectrum of Br-TK-G2K (RCBrP) prepared in this invention. Figure 5 This is the 1H NMR spectrum of Br-TK-G2K-Ser-BOC prepared in this invention. Figure 6 This is the 1H NMR spectrum of Br-TK-G2K-Ser (RCTBrP) prepared in this invention. Figure 7 This is a transmission electron microscope image of the RCTBrP nanoparticles prepared in this invention. Figure 8 This is a hydration particle size data diagram of the RCTBrP nanoparticles prepared in this invention. Figure 9 This is a potential data diagram of the RCTBrP nanoparticles prepared in this invention. Figure 10 This is a schematic diagram of the ROS-responsive particle size change of RCTBrP nanoparticles prepared in this invention. Figure 11 This is a graph showing the biocompatibility data of the RCTBrP nanoparticles prepared in this invention after 24 hours. Figure 12 This is a graph showing the biocompatibility data of the RCTBrP nanoparticles prepared in this invention after 48 hours. Figure 13 This invention relates to an experiment on the cellular uptake and targeted blocking of RCTBrP nanoparticles prepared in this invention. Data chart. Figure 14 This is a kidney distribution diagram of RCTBrP nanoparticles prepared in this invention at different time points. Detailed Implementation

[0005] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

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Claims

1. A nano-co-targeted delivery system based on bilirubin prodrug, characterized in that, The system includes an amphiphilic conjugate and an active drug contained therein; The amphiphilic conjugate uses a second-generation lysine dendritic macromolecule (G2K) as a hydrophilic backbone, covalently linked to hydrophobic bilirubin (Br) via reactive oxygen species (ROS)-sensitive thioketone bonds (TK), and is modified with a serine (L-serine) targeting ligand on the surface of the backbone. The amphiphilic conjugates self-assemble in an aqueous environment to form nanomicelles with bilirubin as the hydrophobic core and serine as the shell.

2. The delivery system according to claim 1, characterized in that: One end of the thioketone bond (TK) is connected to the terminal amino group of G2K via an amide bond, and the other end is connected to the carboxyl group of bilirubin via an amide bond or an ester bond.

3. The delivery system according to claim 1, characterized in that: The active drug is a hydrophobic drug selected from at least one of the following: Nrf2 activator Omaveloxolone (RTA408), mitochondrial targeting peptide (SS-31), or curcumin.

4. The delivery system according to claim 1, characterized in that: The system has a ROS-triggered dual response release mechanism: S1: The thioketone bond (TK) breaks upon contact with ROS, triggering the disintegration of the nanomicelle framework; S2: The hydrophobic bilirubin (Br) is oxidized by ROS to hydrophilic biliverdin, which changes the micelle polarity and assists in drug release.

5. The delivery system according to claim 1, characterized in that: The L-serine is covalently modified on the remaining terminal amino group of G2K via its amino or carboxyl group to specifically recognize and bind to the Kim-1 receptor, a marker of kidney injury.

6. A method for preparing the delivery system according to any one of claims 1-5, characterized in that, Includes the following steps: S1: Preparation of G2K backbone: Using lysine methyl ester as the core, the amplification reaction is carried out through Boc-protected lysine, and after deprotection, the second-generation lysine dendritic macromolecule G2K is obtained. S2: Preparation of TK linkers: The intermediate containing a diamino or aminocarboxyl acetal bond is obtained by reacting β-mercaptoethylamine with ethyl trifluoroacetate, followed by condensation with 2-methoxypropene. S3: Construction of amphiphilic vector: TK linker, bilirubin and serine were grafted onto the G2K backbone sequentially via amide condensation reaction to obtain the amphiphilic conjugate RCTBrP; S4: Self-assembled drug loading: RCTBrP and active drug are dissolved in an organic solvent and added dropwise to the aqueous phase under stirring. Drug-loaded nanomicelles are formed by self-assembly through dialysis or ultrasound.

7. The preparation method according to claim 6, characterized in that: In step S3), the molar ratio of G2K, TK and bilirubin is 1 : (2-8) : (2-8).

8. The preparation method according to claim 6, characterized in that: In step S4), the mass ratio of the carrier RCTBrP to the active drug is (1-10):(1-2); the self-assembly environment is a neutral buffer system with pH 7.0-7.

4.

9. Use of the delivery system according to any one of claims 1-5 in the preparation of a medicament for treating acute kidney injury.

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