A bilirubin-modified dendritic nanomedicine carrier, its preparation method and application

Abstract

This invention discloses a bilirubin-modified dendritic nanomedicine carrier, its preparation method, and its applications, belonging to the field of pharmaceutical formulation technology. The bilirubin-modified dendritic nanomedicine carrier is synthesized by an amidation reaction between dendritic macromolecules and bilirubin. It exhibits relatively small particle size, good water solubility, high stability, and a large number of amine functional groups on its surface, making it suitable for delivering small-molecule hydrophobic drugs. This invention also discloses a nanomedicine composed of the aforementioned bilirubin-modified dendritic nanomedicine carrier and a small-molecule hydrophobic drug, with the small-molecule hydrophobic drug loaded within the hydrophobic cavity of the bilirubin-modified dendritic nanomedicine carrier. This nanomedicine exhibits extremely low cytotoxicity, good biocompatibility, and high antioxidant properties, showing broad application prospects in the preparation of drugs for the treatment and / or prevention of retinal vascular diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, specifically relating to a bilirubin-modified dendritic nanomedicine carrier, its preparation method, and its application. Background Technology

[0002] Posterior segment eye diseases, including age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity, are a rapidly growing cause of visual impairment worldwide. Current treatments for posterior segment eye diseases primarily involve intravitreal injections of medication to prevent disease progression, but these methods are costly and require repeated visits. Nanotechnology offers a promising platform for targeted drug delivery to the eye, with the potential to overcome anatomical and physiological barriers to provide a safe, effective, and sustainable treatment modality.

[0003] Dendritic macromolecules are large molecules with a dendritic structure, formed by the linear linkage of oligomers through repeated branching units. They typically consist of a core, a polymer backbone, and side chains of branching units, and are highly branched monodisperse polymers. The chemical structure, molecular size, molecular mass, distribution, and shape of dendritic macromolecules are controllable. Furthermore, their internal hydrophobic cavity structure contributes to their excellent biocompatibility and biosafety, playing a crucial role in the biomedical field and showing promise in the treatment of posterior segment ocular diseases. However, as the branching generation increases, the number of terminal amino groups in dendritic macromolecules increases, leading to stronger cationicity. This can cause cell membrane rupture and even apoptosis, resulting in cytotoxicity. Additionally, ordinary dendritic macromolecules exhibit poor stability and lack targeting specificity.

[0004] In the prior art, modifying dendritic macromolecules with functional groups to reduce their cytotoxicity and / or increase their targeting is a feasible technical solution. For example, Chinese patent document CN107596385A discloses a tumor-targeting and environmental pH-responsive drug-controlled release nanocarrier. In this invention, the dendritic macromolecular nanopolymer material polyamide-amine PAMAM is used as the main structure, and amphiphilic block copolymer methoxy polyethylene glycol (PEG)-polyaspartic acid (PLA) is connected. Doxorubicin (DOX) is covalently conjugated to the hydrophobic segment of the amphiphilic block copolymer arm and connected through pH-sensitive hydrazone bonds, so that the system has the characteristics of intelligent drug release in vivo, and the drug release can be controlled by the in vivo pH value.

[0005] Chinese patent document CN108888774A discloses a triptolide-dendritic macromolecular conjugate. This conjugate consists of a central dendritic polyamide-amine type dendritic organic polymer nanocarrier, polyethylene glycol, a surface-targeting ligand, and triptolide. Specifically, the amino groups on the surface of PAMAM are partially carboxylated with succinic anhydride to obtain a PAMAM-COOH derivative. After further activation, it is PEGylated to obtain a PAMAM-PEG-COOH derivative. After further activation, it is linked with a surface-targeting ligand to obtain a multifunctional PAMAM derivative. The triptolide is covalently complexed with this multifunctional PAMAM derivative to obtain the conjugate. This conjugate is mainly used to treat colon cancer, liver cancer, breast cancer, etc.

[0006] However, the above research mainly focuses on the development of targeted cancer drugs, and there is still much room for development in the preparation of drugs for the diagnosis and treatment of ophthalmic diseases. Summary of the Invention

[0007] This invention provides a bilirubin-modified dendritic nanoparticle drug carrier with relatively small particle size, good water solubility, high stability, and good antioxidant properties; the surface contains a large number of amine functional groups with a positive charge, which is beneficial for targeting retinal cells; the interior has abundant hydrophobic cavities, which can be used to deliver small molecule hydrophobic drugs; and it has extremely low cytotoxicity and good biocompatibility.

[0008] The specific technical solution adopted is as follows:

[0009] A bilirubin-modified dendritic nanomedicine carrier is synthesized by an amidation reaction of dendritic macromolecules and bilirubin. The dendritic macromolecules include third-generation polyamide-amines, third- and fifth-generation polyamide-amines, fourth-generation polyamide-amines, fourth- and fifth-generation polyamide-amines, fifth- and fifth-generation polyamide-amines, sixth- and sixth-generation polyamide-amines, seventh-generation polyamide-amines, eighth- and ninth-generation polyamide-amines, tenth-generation polyamide-amines, second-generation polylysine, or third-generation polylysine.

[0010] The zeta potential results showed that the bilirubin-modified dendritic nanomedicine carrier was positively charged, while the outer side of the retinal pigment epithelial cell membrane was negatively charged. This is beneficial for the bilirubin-modified dendritic nanomedicine carrier to target the retinal pigment epithelial cell membrane.

[0011] Preferably, the dendritic macromolecule is a fourth-generation polyamide-amine, and the molar ratio of the dendritic macromolecule to bilirubin undergoing the amidation reaction is 1:5-50.

[0012] Further preferred, the molar ratio of the fourth-generation dendritic macromolecular polyamide-amine to bilirubin undergoing the amidation reaction is 1:20. Characterization by 1H NMR spectroscopy shows that the molecular weight of the carrier synthesized under this feed ratio is more stable.

[0013] The present invention also provides a method for preparing the bilirubin-modified dendritic nanomedicine carrier, comprising the following steps:

[0014] (1) Dissolve dendritic macromolecules in an organic solvent to obtain a dendritic macromolecule solution; dissolve bilirubin in an EDC / NHS solution and stir to obtain a bilirubin solution;

[0015] (2) The dendritic macromolecule solution was added dropwise to the bilirubin solution under constant pressure, stirred in the dark, and dialyzed to obtain the bilirubin-modified dendritic nanomedicine carrier.

[0016] Dropping under constant pressure allows for a more complete reaction, resulting in a product with a more stable molecular weight and more uniform particle size.

[0017] Preferably, in step (1), the organic solvent is dimethyl sulfoxide or N,N-dimethylformamide.

[0018] Preferably, in step (2), the reaction time is 6-48 hours with stirring in the dark. The reaction in the dark can prevent bilirubin from decomposing and deteriorating when exposed to light.

[0019] The present invention also provides a nanomedicine comprising a bilirubin-modified dendritic nanomedicine carrier and a small molecule hydrophobic drug, wherein the small molecule hydrophobic drug is loaded within the hydrophobic cavity of the bilirubin-modified dendritic nanomedicine carrier; wherein the small molecule hydrophobic drug includes at least one of the following: lotus leaf alkaloid, paclitaxel, resveratrol, quercetin, dihydroquercetin, curcumin, melatonin, triptolide, dihydroartemisinin, epicatechin, naringenin, malvatin, rapamycin, cyclosporine, ethyl caffeate, crocin, zinc protoporphyrin, ferrostatin-1, dexamethasone, triamcinolone, and anthocyanins.

[0020] Experimental results have shown that the nanomedicine exhibits extremely low cytotoxicity, good biocompatibility, and high antioxidant properties, making it a promising candidate for the preparation of drugs for the treatment and / or prevention of retinal vascular diseases.

[0021] Preferably, the small-molecule hydrophobic drug is lotus leaf alkaloid. Lotus leaf alkaloid is a morphine-like alkaloid with the potential effect of resisting ferroptosis, which has been shown to be involved in the pathogenesis of retinal degenerative diseases such as age-related macular degeneration.

[0022] Preferably, the drug loading of the nanomedicine is 20wt%-80wt%.

[0023] Preferably, the organic solution of the small molecule hydrophobic drug is added dropwise to the bilirubin-modified dendritic nanoparticle carrier solution under constant pressure, and the mixture is stirred, rotary evaporated, and centrifuged to obtain the nanoparticle.

[0024] Preferably, the mass ratio of bilirubin-modified dendritic nanomedicine carrier to small molecule hydrophobic drug is 1:0.2-10.

[0025] In a further preferred embodiment, when the small molecule hydrophobic drug is lotus leaf alkaloid and the dendritic macromolecule is a fourth-generation polyamide-amine, the mass ratio of the bilirubin-modified dendritic nanoparticle drug carrier to the small molecule hydrophobic drug is 1:2.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] In this invention, the bilirubin-modified dendritic nanoparticle carrier has a relatively small particle size, good water solubility, and high stability, exhibiting excellent cellular uptake. This overcomes the shortcomings of most common small-molecule hydrophobic drugs, which often have poor water solubility. Its surface contains a large number of amine functional groups, making it a targeted drug release carrier with extremely low cytotoxicity and good biocompatibility. Furthermore, the nanomedicines prepared using this bilirubin-modified dendritic nanoparticle carrier can achieve the loading of different drugs, enabling targeted treatment of various fundus diseases and resulting in stronger therapeutic effects. Attached Figure Description

[0028] Figure 1 This is a TEM image of the PAM-Bili carrier in Example 1.

[0029] Figure 2 This is a TEM image of Nuci@PAM-Bili in Example 5.

[0030] Figure 3 The diagram shows the hydrodynamic diameters of the PAMAM and PAM-Bili carriers in Example 1 and the Nuci@PAM-Bili carrier in Example 5.

[0031] Figure 4 The images show the 1H NMR spectra of the PAM-Bili supports synthesized in Examples 1-4.

[0032] Figure 5 The images show the 1H NMR spectra of PAMAM, Bilirubin, and PAM-Bili carriers in Example 1.

[0033] Figure 6 The image shows the MALDI-TOF mass spectra of PAMAM and PAM-Bili carriers in Example 1.

[0034] Figure 7The images show the UV spectra of PAMAM, Bilirubin, PAM-Bili carrier in Example 1 and Nuciiferine and Nuci@PAM-Bili in Example 5.

[0035] Figure 8 The chart shows the drug loading rate and encapsulation efficiency of Nuci@PAM-Bili prepared in Examples 5-8.

[0036] Figure 9 The graph shows the DPPH antioxidant capacity test results of the PAM-Bili carrier in Example 1 and the Nuci@PAM-Bili in Example 5 at different concentrations.

[0037] Figure 10 The graph shows the cytotoxicity test results of the PAM-Bili vector in Example 1 and the Nuci@PAM-Bili in Example 5 at different concentrations, where A represents the PAM-Bili vector and B represents the Nuci@PAM-Bili.

[0038] Figure 11 To verify the test results of the PAM-Bili vector in Example 1 and the Nuci@PAM-Bili in Example 5 protecting ARPE-19 cells from sodium iodate-induced cell death through lactate dehydrogenase and CCK-8 experiments, A is the result of the lactate dehydrogenase experiment and B is the result of the CCK-8 experiment. Detailed Implementation

[0039] The present invention will be further illustrated below with reference to the embodiments and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Operating methods not specifically specified in the following embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0040] Example 1: Preparation of PAM-Bili carrier

[0041] Methanol was removed by evaporating 1 mL of a fourth-generation polyamide-amine dendritic macromolecule (PAMAM) methanol solution (purchased from Merck Life Sciences, catalog number: 412449-10G) using a rotary evaporator to obtain a fourth-generation polyamide-amine dendritic macromolecule solid product. This solid product was dissolved in 10 mL of dimethyl sulfoxide (DMSO) and stirred thoroughly at room temperature to obtain a polyamide-amine solution with a mass concentration of approximately 8.13 g / L. 66.9 mg of bilirubin was dissolved in an EDC / NHS dimethyl sulfoxide solution (40.14 mg NHS, 65.56 mg EDC, 10 mL DMSO) and activated at room temperature for 2 hours to obtain a bilirubin solution with a mass concentration of approximately 6.69 g / L. Thus, the PAM-Bili carrier was synthesized at a molar ratio of bilirubin to polyamide-amine of 20:1.

[0042] Using a constant-pressure dropping funnel, the polyamide-amine solution was slowly added dropwise to the bilirubin solution under stirring at room temperature. The mixture was stirred at room temperature for 24 hours in the dark to obtain a mixed solution. The mixed solution was dialyzed at room temperature for 7 days using a dialysis bag (3500mW, MD44). On the first and second days, the dialysate was DMSO to remove ungrafted bilirubin from the system. The dialysate was changed every 2 hours. On the third day, the dialysate was a mixture of DMSO and distilled water. The dialysate was changed every 2 hours. From the fourth to the seventh day, the dialysate was changed to distilled water. Dialysis was performed continuously for 4 days. After the dialysis was completed, an aqueous solution of bilirubin-modified polyamide-amine (PAM-Bili carrier) was obtained.

[0043] The PAM-Bili carrier aqueous solution was frozen at -80℃ for 12 hours, and then freeze-dried in a freeze dryer for 24 hours to obtain a spongy reddish-brown solid, which is the fresh freeze-dried PAM-Bili carrier powder.

[0044] Example 2

[0045] The only difference between this embodiment and Example 1 is that, in the preparation process of the PAM-Bili carrier, the molar ratio of bilirubin to polyamide-amine is 5:1.

[0046] Example 3

[0047] The only difference between this embodiment and Example 1 is that, in the preparation process of the PAM-Bili carrier, the molar ratio of bilirubin to polyamide-amine is 10:1.

[0048] Example 4

[0049] The only difference between this embodiment and Example 1 is that, in the preparation process of the PAM-Bili carrier, the molar ratio of bilirubin to polyamide-amine is 15:1.

[0050] Example 5: Preparation of Nuci@PAM-Bili

[0051] 113.75 mg of the PAM-Bili carrier lyophilized powder prepared in Example 1 was dissolved in distilled water to obtain an aqueous solution of the PAM-Bili carrier. 227.50 mg of nuciferine was dissolved in methanol solution. After complete dissolution, the solution was transferred to a constant pressure dropping funnel. The methanol solution of nuciferine was added dropwise to the aqueous solution of the PAM-Bili carrier under constant pressure. The mixture was stirred at room temperature for 24 hours. Methanol in the system was removed by rotary evaporation. The product was further centrifuged at 4°C (5000 rpm, 15 min). The supernatant was taken, which is the aqueous solution of the nanomedicine Nuci@PAM-Bili.

[0052] The tested nanomedicine Nuci@PAM-Bili had a drug loading of 58.8 wt%.

[0053] Example 6

[0054] The only difference between this embodiment and Example 5 is that, in the preparation of Nuci@PAM-Bili, the mass ratio of bilirubin-modified polyamide-amine to small molecule hydrophobic drug is 2:1.

[0055] The tested nanomedicine Nuci@PAM-Bili had a drug loading of 28.6 wt%.

[0056] Example 7

[0057] The only difference between this embodiment and Example 5 is that, in the preparation of Nuci@PAM-Bili, the mass ratio of bilirubin-modified polyamide-amine to small molecule hydrophobic drug is 1:1.

[0058] The tested nanomedicine Nuci@PAM-Bili had a drug loading of 40.5 wt%.

[0059] Example 8

[0060] The only difference between this embodiment and Example 5 is that, in the preparation of Nuci@PAM-Bili, the mass ratio of bilirubin-modified polyamide-amine to small molecule hydrophobic drug is 1:4.

[0061] The tested nanomedicine Nuci@PAM-Bili had a drug loading of 62.9 wt%.

[0062] Sample Analysis

[0063] The products prepared in the examples were characterized and their performance was tested. The results are shown below:

[0064] Figure 1This is a TEM image of the PAM-Bili carrier in Example 1. Figure 2 The image shown is a TEM image of Nuci@PAM-Bili in Example 5, which shows that Nuci@PAM-Bili has a dendritic structure.

[0065] Figure 3 The figures show the hydrodynamic diameters of the PAMAM and PAM-Bili carriers in Example 1 and the Nuci@PAM-Bili carrier in Example 5, revealing that their particle sizes are all at the nanometer level. The particle size of Nuci@PAM-Bili is smaller than that of PAM-Bili, possibly because the small-molecule hydrophobic drug lotus leaf alkaloid encapsulated in the dendritic cavity has an attractive effect on the dendritic cavity structure.

[0066] Figure 4 The images show the 1H NMR spectra of the PAM-Bili carriers synthesized in Examples 1-4. It can be seen that when the molar ratio of polyamide-amine to bilirubin in the amidation reaction is 1:20, the characteristic peaks of both bilirubin and polyamide-amine are quite obvious.

[0067] Figure 5 The images show the 1H NMR spectra of PAMAM, Bilirubin, and the PAM-Bili support in Example 1, demonstrating the successful synthesis of the PAM-Bili support.

[0068] Figure 6 The image shows the MALDI-TOF mass spectra of PAMAM and the PAM-Bili carrier in Example 1. The molecular weight of PAMAM is 13939.69, and the molecular weight of the PAM-Bili carrier is 18803.87. Calculations indicate that approximately nine bilirubin molecules were successfully conjugated to PAMAM.

[0069] Figure 7 The images show the UV spectra of PAMAM, Bilirubin, and the PAM-Bili carrier in Example 1, and Nuci@PAM-Bili in Example 5. Characteristic peaks of PAMAM and Bilirubin appear in the PAM-Bili carrier, and characteristic peaks of the PAM-Bili carrier and Nuci@PAM-Bili appear in the Nuci@PAM-Bili carrier, proving the successful synthesis of the nanocarrier.

[0070] Figure 8 The chart shows the drug loading rate and encapsulation efficiency of Nuci@PAM-Bili prepared in Examples 5-8. It can be seen that the optimal feed ratio (by mass) of PAM-Bili carrier to Nuciiferine is 1:2, which yields a drug loading rate of 69.0% and an encapsulation efficiency of 58.8%, achieving the best results.

[0071] Figure 9The graph shows the DPPH antioxidant capacity test results of the PAM-Bili carrier in Example 1 and the Nuci@PAM-Bili in Example 5 at different concentrations. It can be seen that the DPPH scavenging rate of Nuci@PAM-Bili loaded with lotus leaf alkaloids is significantly greater than that of the PAM-Bili carrier.

[0072] Figure 10 The graph shows the cytotoxicity test results of the PAM-Bili vector in Example 1 and the Nuci@PAM-Bili in Example 5 at different concentrations. In the graph, A represents the PAM-Bili vector and B represents Nuci@PAM-Bili. It can be seen that the PAM-Bili vector and Nuci@PAM-Bili have extremely low cytotoxicity to cells at different concentrations.

[0073] Figure 11 To verify the effectiveness of PAM-Bili vector and Nuci@PAM-Bili in protecting ARPE-19 cells from sodium iodate-induced cell death using lactate dehydrogenase and CCK-8 assays, Figure A shows the results of the lactate dehydrogenase assay, and Figure B shows the results of the CCK-8 assay. It can be seen that both PAM-Bili vector and Nuci@PAM-Bili can effectively protect ARPE-19 cells, and their effects are superior to those of the drugs alone, namely bilirubin and lotus leaf alkaloid.

[0074] The embodiments described above provide a detailed explanation of the technical solutions 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, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A bilirubin-modified dendritic nanomedicine carrier, characterized in that, It is synthesized by an amidation reaction of dendritic macromolecules and bilirubin, wherein the dendritic macromolecules are fourth-generation polyamide-amines.

2. The bilirubin-modified dendritic nanomedicine carrier according to claim 1, characterized in that, The molar ratio of dendritic macromolecules to bilirubin undergoing amidation reaction is 1:5-50.

3. The method for preparing bilirubin-modified dendritic nanomedicine carriers according to claim 1 or 2, characterized in that, Includes the following steps: (1) Dissolve dendritic macromolecules in an organic solvent to obtain a dendritic macromolecule solution; Dissolve bilirubin in EDC / NHS solution and stir to obtain bilirubin solution; (2) The dendritic macromolecule solution was added dropwise to the bilirubin solution under constant pressure, stirred in the dark, and dialyzed to obtain the bilirubin-modified dendritic nanomedicine carrier.

4. The method for preparing the bilirubin-modified dendritic nanomedicine carrier according to claim 3, characterized in that, In step (1), the organic solvent is dimethyl sulfoxide or N,N-dimethylformamide.

5. The method for preparing the bilirubin-modified dendritic nanomedicine carrier according to claim 3, characterized in that, In step (2), the reaction time is 6-48 hours with stirring in the dark.

6. A nanomedicine, characterized in that, The drug is composed of a bilirubin-modified dendritic nanoparticle drug carrier as described in claim 1 or 2 and a small molecule hydrophobic drug, wherein the small molecule hydrophobic drug is loaded into the hydrophobic cavity of the bilirubin-modified dendritic nanoparticle drug carrier; the small molecule hydrophobic drug is lotus leaf alkaloid.

7. The nanomedicine according to claim 6, characterized in that, The drug loading of the nanomedicine is 20wt%-80wt%.

8. The nanomedicine according to claim 6, characterized in that, An organic solution of a small-molecule hydrophobic drug was added dropwise to the bilirubin-modified dendritic nanoparticle carrier solution under constant pressure. The mixture was stirred, rotary evaporated, and centrifuged to obtain the nanoparticle.

9. The nanomedicine according to claim 6, characterized in that, The mass ratio of bilirubin-modified dendritic nanomedicine carriers to small molecule hydrophobic drugs is 1:0.2-10.

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