Nano-drug delivery system modified with phenolic acid or dipeptide for increasing oral absorbability and bioavailability, and use thereof

Abstract

A nano-drug delivery system modified with a phenolic acid or dipeptide for increasing the oral absorbability and bioavailability, which system is prepared from a nanocarrier and an auxiliary material. The nanocarrier is modified with a phenolic acid or dipeptide on the surface thereof as a ligand, and actively targets a receptor on the surface of intestinal epithelial cells, wherein the phenolic acid ligand is a hydroxycinnamic acid derivative, and the dipeptide ligand is a combination of a cationic amino acid and a neutral amino acid. An oral nano-drug delivery system is constructed by using a phenolic acid or dipeptide, which is an essential nutrient for a human body, as a ligand to mimic the absorption pathway of phenolic acid or dipeptide, which system significantly enhances the oral absorption efficiency of a drug by using the natural highly-efficient absorption capacity of the small intestine for nutrients.

Description

A phenolic acid or dipeptide-modified nanodelivery system for enhanced oral absorption and utilization and its application. Technical Field

[0001] This invention belongs to the field of pharmaceutical formulations, specifically referring to a nano-drug delivery system modified with phenolic acid or dipeptide to increase oral absorption and utilization, and its application. Background Technology

[0002] Nanoparticle drug delivery systems can effectively improve the solubility of poorly soluble drugs and enhance the stability of protein and peptide drugs. They can also overcome the pH and enzymatic degradation of the gastrointestinal tract and the mucus barrier to some extent, promoting oral drug absorption. Currently, they are widely used in oral drug delivery research and have achieved some progress. However, the oral absorption process still requires crossing the polarized intestinal epithelial cell barrier, and the problem of "difficult cross-mucosal drug delivery" remains. Although ligand-modified active-targeting nanoparticle drug delivery systems can specifically bind to receptors or transport proteins on the surface of small intestinal epithelial cell membranes, further improving the epithelial cell uptake efficiency of nanoparticle drug delivery systems, this is currently a hot research topic in the field of drug delivery. However, while ligand-modified nanocarriers can often significantly improve cellular uptake, their transmembrane transport performance is often unsatisfactory, ultimately leading to limited oral absorption efficiency. For example, some researchers have prepared ligand-modified nanoparticles targeting low-density lipoprotein for oral administration, which can increase cellular uptake by 2.5 times, but the transmembrane transport efficiency has not been significantly improved. Other researchers have prepared folic acid-modified targeted liposomes, which can increase cellular uptake by approximately 3.3 times; however, their pharmacological bioavailability has not been significantly improved. This demonstrates that existing research indicates that simply increasing the uptake by intestinal epithelial cells in a delivery system does not necessarily improve the transmembrane transport level of the delivery system. This is mainly because intestinal epithelial cells are polarized cells, with significant differences in protein expression and absorption characteristics between their apical and basal sides, leading to a common problem in many nanomedicine delivery systems: difficulty in cellular entry and even greater difficulty in cellular exit.

[0003] The small intestine is the primary site of nutrient absorption in the human body. Small intestinal epithelial cells extensively express various nutrient transport proteins, such as glucose transporters, amino acid transporters, and oligopeptide transporters, thus possessing a natural transport system that matches various nutrients. Utilizing the body's naturally efficient absorption of nutrients, oral nano-drug delivery systems that mimic nutrient absorption pathways hold promise for further improving oral drug absorption efficiency. However, for orally absorbed nano-drug delivery systems, cellular uptake is not the final goal. The system must also undergo complex intracellular transport pathways and basal-side exocytosis into the bloodstream to exert its therapeutic effect. A complete transmembrane transport process includes entry into cells, intracellular transport, and exocytosis, each affecting the final transport efficiency. Therefore, effectively overcoming the oral drug delivery barrier and improving transport efficiency are pressing technical challenges that need to be addressed.

[0004] Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing oral drug delivery systems, such as barriers and low transport efficiency, and to provide a nano-drug delivery system modified with phenolic acid or dipeptides to increase oral absorption and utilization.

[0006] Another objective of this invention is to provide a phenolic acid or dipeptide-modified nanodelivery system for increasing oral absorption and utilization in oral drug delivery formulations that overcome the intestinal absorption barrier.

[0007] The objective of this invention is achieved by the following technical solution: a nano-drug delivery system modified with phenolic acid or dipeptide to increase oral absorption and utilization, which is prepared from a nanocarrier and excipients. The nanocarrier is modified with phenolic acid or dipeptide as ligand on its surface and actively targets receptors on the surface of intestinal mucosal epithelial cells. The phenolic acid ligand is a hydroxycinnamic acid derivative. The dipeptide ligand is a combination of cationic amino acids and neutral amino acids.

[0008] As a preferred embodiment, the phenolic acid ligand is ferulic acid or p-coumaric acid, and the dipeptide ligand is lysine-valine or lysine-methionine.

[0009] Furthermore, the nanocarrier is at least one of polylactic acid-glycolic acid copolymer (PLGA) nanoparticles, lipid nanoparticles (LNP), solid lipid nanoparticles, polystyrene nanoparticles, polylactic acid nanoparticles, and liposomes.

[0010] The nanoparticles have a hydrophilic shell and a hydrophobic core. The hydrophilic shell is the hydrophilic end of an amphiphilic polymer, which is covalently linked to phenolic acid or dipeptide. The hydrophobic core is composed of the hydrophobic end of the amphiphilic polymer, an active ingredient, and a biocompatible carrier material. The liposomes are composed of a lipid bilayer and an inner aqueous phase. The hydrophilic active ingredient is loaded in the inner aqueous phase, and the hydrophobic active ingredient is loaded in the lipid bilayer. The surface of the liposomes is modified with phenolic acid or dipeptide ligands.

[0011] The active ingredient accounts for 0.1% to 90% of the total weight of the nanocarrier.

[0012] As one of the preferred methods, the biocompatible carrier material is selected from at least one of polylactic acid-glycolic acid copolymer (PLGA), polystyrene, polystyrene sebacic acid, polyethyleneimine, polylactic acid, polyalkyl cyanoacrylate, polyamino acids, cholesterol, fatty acids, phospholipids, sphingolipids, waxes, and fatty acid glycerides.

[0013] The active ingredient is selected from at least one of protein-peptide drugs, nucleic acid drugs, and small molecule drugs. Specifically, the protein-peptide drugs are selected from at least one of insulin, octreotide, leuprolide acetate, calcitonin, teriparatide, thymopentin, luteinizing hormone-releasing hormone, ticokine acetate, busherin, exenatide, telpoxetine, semaglutide, liraglutide, glucagon-like peptide-1, tedulglutide, glepaglutide, apraglutide, elsiglutide, dapiglutide, triptorelin acetate, leukocyte growth factor, erythrocyte growth factor, macrophage growth factor, tumor necrosis factor, epidermal growth factor, interleukin, angiogenesis inhibin, bovine serum albumin, ovalbumin, parathyroid hormone, growth hormone, somatostatin, interferon, and monoclonal antibodies. The nucleic acid drugs are selected from at least one of small interfering RNA, messenger RNA, microRNA, plasmid DNA, and antisense oligonucleotide drugs. The small molecule drugs are selected from at least one of the following: antipyretic analgesics and nonsteroidal anti-inflammatory drugs, antibacterial drugs, antitumor drugs, hormones, central nervous system drugs, peripheral nervous system drugs, circulatory system drugs, hypoglycemic drugs, and diuretics.

[0014] As a preferred embodiment, the amphiphilic polymer is at least one selected from distearylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), stearic acid polyethylene glycol carboxylic acid, and PLGA-PEG.

[0015] This invention also relates to the use of a phenolic acid or dipeptide-modified nanodelivery system for increasing oral absorption and utilization in oral drug delivery formulations for overcoming the intestinal absorption barrier.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0017] (1) This invention creatively uses phenolic acid or dipeptide, which are nutrients required by the human body, as ligands to construct an oral nano-drug delivery system that simulates the absorption pathway of phenolic acid or dipeptide. It also utilizes the small intestine's natural and efficient absorption capacity of nutrients to significantly improve the oral absorption efficiency of drugs.

[0018] (2) The present invention uses phenolic acid or dipeptide modified nanocarriers, which can be efficiently absorbed by small intestinal epithelial cells under the mediation of MCT-1 or PEPT1, respectively, which can significantly improve the oral bioavailability of drugs.

[0019] (3) This invention modifies the surface of nanocarriers with phenolic acids or dipeptides, actively targeting specific transporters on the surface of intestinal epithelial cells. This mimics the absorption of small molecule nutrients, conforms to physiological barriers, and achieves unidirectional transmucosal transport of the drug across the intestinal mucosa via "apical entry-intracellular transport-basal exit." This not only improves cellular uptake but, more importantly, also enhances the transmembrane transport efficiency of the nanocarriers. The nano-drug delivery system constructed during in vivo delivery effectively overcomes many barriers associated with oral administration, significantly improving the oral bioavailability and therapeutic effect of the loaded drug. Attached Figure Description

[0020] Figure 1 shows the synthesis and characterization of ferulic acid or p-coumaric acid modified DSPE-PEG (A represents the synthetic route of ferulic acid or p-coumaric acid modified DSPE-PEG, and B represents the 1H NMR spectrum of ferulic acid or p-coumaric acid modified DSPE-PEG).

[0021] Figure 2 shows the synthesis and characterization of DSPE-PEG modified with lysine-valine or lysine-methionine (A represents the synthetic route of DSPE-PEG modified with lysine-valine or lysine-methionine, and B represents the 1H NMR spectrum of DSPE-PEG modified with lysine-valine or lysine-methionine).

[0022] Figure 3 shows the preparation and characterization of PLGA nanoparticles loaded with liraglutide modified with phenolic acid or dipeptide (A represents the particle size, PDI, encapsulation efficiency, and drug loading of the nanoparticles, and B represents the morphology of the nanoparticles).

[0023] Figure 4 shows the cellular uptake of PLGA nanoparticles modified with phenolic acid or dipeptides.

[0024] Figure 5 shows the active targeting ability of PLGA nanoparticles modified with phenolic acid or dipeptide (A represents the active targeting ability of PLGA nanoparticles modified with phenolic acid, and B represents the active targeting ability of PLGA nanoparticles modified with dipeptide).

[0025] Figure 6 shows the transmembrane transport efficiency of PLGA nanoparticles modified with phenolic acid or dipeptides.

[0026] Figure 7 shows the in vivo pharmacokinetic study of PLGA nanoparticles modified with phenolic acid or dipeptide (A represents the plasma concentration-time curve of liraglutide, and B represents the pharmacokinetic parameters).

[0027] Figure 8 shows the in vivo pharmacodynamics of PLGA nanoparticles loaded with liraglutide modified with phenolic acid or dipeptide (A represents the blood glucose change curve over time, and B represents the area under the curve of the blood glucose change curve over time).

[0028] Figure 9 shows the preparation and characterization of phenolic acid-modified PLGA nanoparticles loaded with semaglutide (A represents particle size and zeta potential, B represents encapsulation efficiency and drug loading).

[0029] Figure 10 shows the in vivo pharmacodynamics of phenolic acid-modified PLGA nanoparticles loaded with semaglutide.

[0030] Figure 11 shows the particle size, potential, encapsulation efficiency, and drug loading of SLN modified with phenolic acid or dipeptide.

[0031] Figure 12 shows the cellular uptake of SLNs modified with phenolic acid or dipeptides.

[0032] Figure 13 shows the transmembrane transport efficiency of SLN modified with phenolic acid or dipeptide.

[0033] Figure 14 shows the in vivo pharmacodynamics of SLN modified with insulin-loaded dipeptide (A represents the blood glucose change curve over time, and B represents the area on the curve of blood glucose change over time).

[0034] Figure 15 shows the in vivo pharmacodynamics of SLN modified with dipeptide loaded with exenatide (A represents the blood glucose change curve over time, and B represents the area under the curve of the blood glucose change curve over time). Detailed Implementation

[0035] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto.

[0036] Example 1

[0037] This embodiment describes the synthesis and characterization of ferulic acid or p-coumaric acid-modified DSPE-PEG.

[0038] The synthetic route for ferulic acid or p-coumaric acid-modified DSPE-PEG is shown in Figure 1A. Ferulic acid (FA) or p-coumaric acid (CA) was dissolved in methanol with EDCI and NHS at a molar ratio of 1:5:5, and stirred at 37°C for 2 h. Then, dichloromethane containing DSPE-PEG-NH2 was added, and a small amount of triethylamine (TEA) was added dropwise to adjust the pH to weakly alkaline. The mixture was stirred at 37°C for 24 h. The reaction solution was collected, and the solvent was removed using a vacuum rotary evaporator. The product was dialyzed for 24 h (dialysis bag molecular weight 1000 Da), and finally lyophilized to obtain a white powder, i.e., FA or CA-modified DSPE-PEG (DSPE-PEG-FA, DSPE-PEG-CA). The successful synthesis of the product was verified by 1H NMR spectroscopy.

[0039] The structure of the product was confirmed using 1H NMR spectroscopy, as shown in Figure 1B. Compared to DSPE-PEG-NH 2， The product showed characteristic peaks of FA or CA at 6.0-7.5 ppm, proving that FA or CA was successfully linked to DSPE-PEG-NH2.

[0040] Example 2

[0041] This example describes the synthesis and characterization of DSPE-PEG modified with lysine-valine or lysine-methionine.

[0042] The synthetic route for lysine-valine or lysine-methionine modified DSPE-PEG is shown in Figure 2A. DSPE-PEG-NH2 and Nε-benzyloxycarbonyl-Nα-tert-butoxycarbonyl-L-lysine N-succinimide ester (Boc-Lys(Z)-OSu) were dissolved in dichloromethane at a molar ratio of 1:3. A small amount of triethylamine was added dropwise to adjust the pH to weakly alkaline. The mixture was stirred at 37°C for 24 h, and then trifluoroacetic acid (TFA) was added, with stirring continued for 6 h. Dichloromethane was removed by vacuum rotary evaporation, followed by azeotropic addition of dichloromethane to fully remove trifluoroacetic acid. The product was dissolved in 50% methanol-water and dialyzed for 24 h (dialysis bag molecular weight 1000 Da). The dialysate was lyophilized to obtain Cbz-amino-protected lysine-modified DSPE-PEG (DSPE-PEG-LYS(Z)). The structure of the product was confirmed by proton nuclear magnetic resonance spectroscopy, as shown in Figure 2B. The characteristic peak of the benzene ring in Cbz appeared at 7.13-7.48 ppm, indicating the successful synthesis of the intermediate DSPE-PEG-LYS(Z).

[0043] Intermediate DSPE-PEG-LYS(Z) and N-tert-butoxycarbonyl-L-valine N-succinimide ester (Boc-Val-OSu) or N-tert-butoxycarbonyl-L-methionine N-succinimide ester (Boc-Met-OSu) were dissolved in dichloromethane at a molar ratio of 1:3. A small amount of triethylamine was added dropwise to adjust the pH to weakly alkaline. The mixture was stirred at 37°C for 24 h, and then trifluoroacetic acid was added, with stirring continuing for 6 h. Dichloromethane was removed by vacuum rotary evaporation, and trifluoroacetic acid was removed by azeotropic reaction with dichloromethane. The evaporated product was dissolved in ethyl acetate, and palladium on carbon was added. After evacuation, sufficient hydrogen gas was introduced, and the reaction was stirred for 6 h. The product was then centrifuged to collect the supernatant, evaporated to remove the solvent, dissolved in 50% methanol-water, and finally dialyzed for 24 hours (dialysis bag molecular weight 1000 Da). The dialysate was then lyophilized to obtain lysyl-valine or lysine-methionine modified DSPE-PEG (DSPE-PEG-LYS-VAL (DSPE-PEG-LV), DSPE-PEG-LYS-MET (DSPE-PEG-LM)).

[0044] The structure of the product was confirmed using 1H NMR spectroscopy, as shown in Figure 2B, compared to DSPE-PEG-LYS(Z). ， The disappearance of the characteristic peak of Cbz in the DSPE-PEG-LV spectrum, coupled with the appearance of a methyl peak at the end of the valine side chain at 0.92 ppm, confirms the successful synthesis of DSPE-PEG-LV. Similarly, the disappearance of the characteristic peak of Cbz in the DSPE-PEG-LM spectrum, coupled with the appearance of a methyl peak at the end of the methionine side chain at 2.05 ppm, confirms the successful synthesis of DSPE-PEG-LM.

[0045] Example 3

[0046] This embodiment describes the preparation and characterization of phenolic acid or dipeptide-modified liraglutide polylactic-co-glycolic acid copolymer (PLGA) nanoparticles.

[0047] Phenolic acid or dipeptide-modified PLGA nanoparticles were prepared using a nanoprecipitation method. PLGA is an abbreviation for polylactic acid-glycolic acid copolymer. The specific procedure is as follows: PLGA, phenolic acid or dipeptide-modified DSPE-PEG, injectable soybean lecithin, and liraglutide (Lira) were prepared into 20 mg / mL DMSO stock solutions. 50 μL of PLGA, 20 μL of phenolic acid or dipeptide-modified DSPE-PEG, 20 μL of soybean lecithin, and 10 μL of liraglutide stock solution were thoroughly mixed to form the organic phase. Then, under stirring, 1 mL of ultrapure water was slowly added dropwise. After the addition was complete, stirring was continued for 5 min. DMSO was removed by ultrafiltration, and the nanoparticles were redispersed with ultrapure water to obtain phenolic acid or dipeptide-modified PLGA nanoparticles. The nanoparticle size and polydispersity index (PDI) were measured using a particle size analyzer, the nanoparticle morphology was observed using transmission electron microscopy, and the encapsulation efficiency and drug loading of the nanoparticles were detected by high-performance liquid chromatography.

[0048] Figures 3A and 3B show that the average particle size of ligand-free modified nanoparticles (PEG NPs), ferulic acid modified nanoparticles (FA NPs), coumaric acid modified nanoparticles (CA NPs), lysyl-valine modified nanoparticles (LV NPs), and lysine-methionine modified nanoparticles (LM NPs) is approximately 80 nm. They all exhibit a spherical structure and good monodispersity. The encapsulation efficiency of liraglutide is >90%, and the drug loading is >10%.

[0049] Example 4

[0050] This embodiment describes the cellular uptake of polylactic acid-glycolic acid copolymer (PLGA) nanoparticles modified with phenolic acid or dipeptides.

[0051] Following the method described in Example 3, Lira was replaced with FITC-Lira (5-fluorescein isothiocyanate-labeled Lira) to prepare fluorescently labeled PLGA nanoparticles. Caco-2 single-cell suspensions were seeded at a density of 1×10⁴ cells / well in 96-well plates and allowed to adhere for 3-4 days until cell differentiation occurred, after which cell uptake studies were conducted. Before the experiment, the cell culture medium was aspirated, and each well was rinsed twice with 100 μL of PBS. The FITC-Lira-loaded nanoparticles were diluted to 250 μg / mL (based on PLGA) with HBSS, and 100 μL of the nanoparticle-HBSS suspension was added to each well. The plates were then placed in a cell culture incubator. After 2 hours, the HBSS suspension was aspirated, and the plates were rinsed twice with PBS. Then, 100 μL of DMSO was added to each well, and the plates were shaken at 100 rpm for 10 min to lyse the cells and release the uptaken FITC-Lira. Finally, the 96-well plates were placed in a multi-functional chemiluminescence analyzer, and the fluorescence intensity at wavelengths of 485 nm and 520 nm was measured. The relative uptake was calculated using the fluorescence intensity of unmodified nanoparticles (PEG NPs) as the standard value 1.

[0052] Figure 4 shows that the cellular uptake of ferulic acid or coumaric acid-modified nanoparticles (FA NPs, CA NPs) was approximately 3.9 times that of PEG NPs, and the cellular uptake of lysine-valine or lysine-methionine-modified nanoparticles (LV NPs, LM NPs) was approximately 3.5 times that of PEG NPs, indicating that using phenolic acids or dipeptides as ligands can significantly improve the intestinal cellular uptake efficiency of nanoparticles.

[0053] Example 5

[0054] This embodiment investigates the active targeting of polylactic acid-glycolic acid copolymer (PLGA) nanoparticles modified with phenolic acid or dipeptides.

[0055] (1) Investigation on the active targeting of phenolic acid modified PLGA nanoparticles.

[0056] Caco-2 cell suspension was seeded at a density of 1×10⁴ cells / well in 96-well plates and allowed to adhere for 3-4 days. After cell differentiation, the cells were used for cellular uptake pathway studies. Before the experiment, the culture medium was aspirated, and each well was washed twice with 100 μL PBS. Using blank HBSS as a control, α-cyano-4-hydroxycinnamic acid (α-CHCA, 5 mM), an inhibitor of monocarboxylic acid transporter-1 (MCT-1), was added to each group of cells. The cells were pre-incubated in a cell culture incubator for 30 min, and then PEG NPs, FA NPs, or CA NPs loaded with FITC-Lira were added, respectively. After 2 h, the HBSS nanoparticle suspension was aspirated, and the cells were washed twice with PBS. DMSO was added to each well, and the cells were shaken at 100 rpm for 10 min to lyse the cells and release the uptaken FITC-Lira. Finally, the 96-well plates were placed in a multi-functional chemiluminescence analyzer, and the fluorescence intensity at wavelengths of 485 nm and 520 nm was measured.

[0057] Figure 5A shows that α-CHCA can significantly inhibit the uptake of FA NPs and CA NPs, but has no effect on PEG NPs. This indicates that α-CHCA has a specific uptake inhibitory effect on FA NPs and CA NPs, proving that phenolic acid modification can enable nanoparticles to actively target MCT-1 to increase the affinity between nanoparticles and cell membranes.

[0058] (2) Investigation on the active targeting of dipeptide-modified PLGA nanoparticles.

[0059] Caco-2 cell suspension was seeded at a density of 1×10⁴ cells / well in 96-well plates and allowed to adhere for 3-4 days. After cell differentiation, the cells were used for cellular uptake pathway studies. Before the experiment, the culture medium was aspirated, and each well was rinsed twice with 100 μL of PBS. Using blank HBSS as a control, glycylsarcosine (Glysar, 50 mM), the substrate of oligopeptide transporter-1 (PEPT1), was added to each group of cells. The cells were pre-incubated in a cell culture incubator for 30 min, and then PEG NPs, LV NPs, or LM NPs loaded with FITC-Lira were added, respectively. After 2 h, the HBSS nanoparticle suspension was aspirated, and the cells were rinsed twice with PBS. DMSO was added to each well, and the cells were shaken at 100 rpm for 10 min to lyse the cells and release the uptaken FITC-Lira. Finally, the 96-well plates were placed in a multifunctional chemiluminescence analyzer, and the fluorescence intensity at wavelengths of 485 nm and 520 nm was measured.

[0060] Figure 5B shows that Glysar can significantly inhibit the uptake of LV NPs and LM NPs, but has no effect on PEG NPs. This indicates that Glysar has a specific uptake inhibitory effect on LV NPs and LM NPs, proving that dipeptide modification can enable nanoparticles to actively target PEPT1 to increase the affinity between nanoparticles and cell membranes.

[0061] Example 6

[0062] This example demonstrates the transmembrane transport efficiency of polylactic acid-glycolic acid copolymer (PLGA) nanoparticles modified with phenolic acid or dipeptides.

[0063] Example 4 demonstrates that ligand-modified nanoparticle drug delivery systems can improve uptake by intestinal epithelial cells; however, cellular uptake is only the first step in transmembrane transport. Example 5 does not indicate that nanoparticles with high uptake efficiency have better transmembrane efficiency. After entering the cell, the nanoparticles undergo a complex intracellular transport process before finally delivering the drug from the basal side to the extracellular space. Example 6 aims to investigate whether nanoparticles taken up by intestinal cells possess high transmembrane transport efficiency.

[0064] Caco-2 single-cell suspensions were seeded in Transwell chambers at a density of 3-5 × 10⁴ cells / well and cultured for 14-21 days. Transmembrane resistance was measured every 2 days, and the cells were ready for experimentation when the resistance was > 600 Ω·cm². Before the experiment, the culture medium was aspirated, and each well was rinsed twice with 500 μL of HBSS, followed by equilibration with another 500 μL of HBSS for 30 min. 200 μL of FITC-Lira-loaded nanoparticles were added to chamber A (Transwell chamber), and 800 μL of blank HBSS was added to chamber B (24-well plate). The plates were then placed in a cell culture incubator. At 0 h, 0.25 h, 0.5 h, 1 h, 2 h, 3 h, and 4 h, 80 μL of sample solution was taken from chamber B, and the same volume of HBSS was added to each sample. After sampling, 80 μL of DMSO was added to the sample solution, and the plates were shaken thoroughly to destroy the nanoparticles. The fluorescence intensity was measured at wavelengths of 485 nm and 520 nm using a multifunctional chemiluminescence analyzer. A standard curve was prepared using free FITC-Lira, and the concentration of FITC-Lira in the sample solution at each time point was calculated using the standard curve. The apparent permeability coefficient (Papp) was calculated according to the following formula: Papp=(dQ / dt)×(1 / (A×C0)) (dQ / dt represents the slope of the curve of B-chamber nanoparticle content changing with time, A is the area of ​​the Transwell microporous membrane, and C0 is the initial drug concentration).

[0065] Figure 6 shows that, compared with PEG NPs, the Papp values ​​of FA NPs, CA NPs, LV NPs and LM NPs were significantly increased, about 3 times that of PEG NPs, indicating that phenolic acid or dipeptide modification can significantly improve the transmembrane transport capacity of nanoparticles, which helps them overcome the absorption barrier of intestinal epithelial cells.

[0066] Example 7

[0067] This example is an in vivo pharmacokinetic study of polylactic acid-glycolic acid copolymer (PLGA) nanoparticles modified with phenolic acid or dipeptides.

[0068] Following the method described in Example 3, Lira was replaced with CY5-labeled Lira (CY5-Lira) to prepare CY5 fluorescently labeled PLGA nanoparticles. Six-week-old male ICR mice were randomly divided into groups of five. They were fasted overnight but allowed free access to water. Mice were orally administered free CY5-Lira (Free CY5-Lira), phenolic acid- or dipeptide-modified nanoparticles loaded with CY5-Lira, orally at a dose of 5 mg / kg. Free CY5-Lira (SCFree CY5-Lira) was subcutaneously injected at a dose of 0.5 mg / kg. Blood samples were collected from the orbital sinus at 0.5, 1, 2, 4, 6, and 8 hours after administration to measure the concentration of CY5-Lira in the blood. Blood concentration-time curves were plotted, and key pharmacokinetic parameters were calculated.

[0069] As shown in Figures 7A and 7B, compared with PEG NPs, FA NPs, CA NPs, LM NPs and LV NPs significantly improved the AUC and Cmax of CY5-Lira. The oral bioavailability (Fr) of phenolic acid or dipeptide modified nanoparticles can reach more than 10%, indicating that phenolic acid or dipeptide ligand modified nanoparticles can effectively promote the oral absorption of drugs.

[0070] Example 8

[0071] This embodiment is an in vivo pharmacodynamic study of polylactic acid-glycolic acid copolymer (PLGA) nanoparticles loaded with liraglutide and modified with phenolic acid or dipeptide.

[0072] Male db / db mice were randomly divided into groups of five. They were fasted from 9:00 AM but allowed free access to water. Fasting body weight and fasting blood glucose were recorded at 2:00 PM. Subcutaneous injection of free Lira (SCFree Lira, 0.5 mg / kg) served as a positive control, while oral administration of free Lira (Oral Free Lira, 5 mg / kg) served as a negative control. Patients were also orally administered nanoparticles (5 mg / kg) modified with Lira phenolic acid or dipeptide ligands. One hour later, a glucose saline solution (0.5 g / mL, 2 g / kg) was injected intraperitoneally. Blood glucose levels were measured from the tail vein at 15, 30, 60, 90, and 120 minutes. Using fasting blood glucose as the starting point, a curve showing the change in blood glucose relative to the zero point over time was plotted, and the area under the curve (AUC) was calculated. A larger AUC indicates a greater cumulative increase in blood glucose.

[0073] Figures 8A and 8B show that neither LiraPEG NPs nor Oral Free Lira inhibited the rise in blood glucose, with the highest value increasing by approximately 6 mmol / L relative to the zero value, and no difference in AUC between the two (p > 0.05). LiraFA NPs, LiraCA NPs, LiraL-M NPs, and LiraL-V NPs significantly inhibited the rise in blood glucose. Specifically, LiraFA NPs and LiraCA NPs reduced blood glucose to below zero at 30 min, while LiraL-M NPs and LiraL-V NPs reduced it to below zero in just 15 min. Compared to the Oral Free Lira and LiraPEG NPs groups, the AUC of LiraFA NPs, LiraCA NPs, LiraL-M NPs, and LiraL-V NPs were all significantly reduced. These results indicate that, using db / db mice as a disease model, phenolic acid or dipeptide-modified nanoparticles significantly improve glucose tolerance in mice.

[0074] Example 9

[0075] This embodiment describes the preparation and characterization of phenolic acid-modified semaglutide-loaded PLGA nanoparticles.

[0076] Phenolic acid-modified PLGA nanoparticles loaded with semaglutide were prepared using a nanoprecipitation method. The specific procedures were as follows: PLGA, phenolic acid-modified DSPE-PEG, soybean lecithin for injection, and semaglutide (SEM) were prepared into 20 mg / mL DMSO stock solutions. 50 μL of PLGA, 20 μL of phenolic acid-modified DSPE-PEG, 20 μL of soybean lecithin, and 40 μL of the semaglutide stock solution were thoroughly mixed to form the organic phase. Then, under stirring, 1 mL of ultrapure water was slowly added dropwise, and stirring was continued for 5 min after the addition was complete. DMSO was removed by ultrafiltration, and the nanoparticles were redispersed with ultrapure water. The nanoparticle size and zeta potential were measured using a particle size analyzer, and the encapsulation efficiency and drug loading of the nanoparticles were detected by high-performance liquid chromatography (HPLC).

[0077] Figures 9A and 9B show that the average particle size of the semaglutide-loaded ligand-free nanoparticles (SEM@PNP), the semaglutide-loaded ferulic acid-modified nanoparticles (SEM@FNP), and the semaglutide-loaded p-coumaric acid-modified nanoparticles (SEM@CNP) is less than 100 nm. The encapsulation efficiency of semaglutide is about 50%, and the drug loading is about 20%.

[0078] Example 10

[0079] This embodiment is an in vivo pharmacodynamic study of phenolic acid-modified PLGA nanoparticles loaded with semaglutide.

[0080] Male KKay mice were fasted overnight but allowed free access to water. Fasting body weight and fasting blood glucose were measured. Different semaglutide preparations (semaglutide equivalent: 3 mg / kg) were administered orally via gavage, and semaglutide solution (semaglutide equivalent: 1 mg / kg) was administered subcutaneously. Three hours after administration, glucose solution (1.5 g / kg) was administered orally via gavage for a glucose tolerance test. Blood glucose levels were measured using a glucometer collected from the tail vein at predetermined time points (15, 30, 60, 90, 120, and 180 min). With fasting blood glucose as 100%, a blood glucose curve was plotted over time.

[0081] Figure 10 shows that compared with oral administration of free semaglutide (poSEM), the glucose tolerance of mice in the SEM@FNP and SEM@CNP groups was significantly improved. Furthermore, the glucose tolerance of phenolic acid-modified nanoparticles was further enhanced after the addition of the lysosomal escape agent chloroquine (CQ). After oral administration of the marketed semaglutide combined with 8-(2-hydroxybenzamido)octanoic acid (SNAC) (poSEM+SNAC), the blood glucose level in mice increased by 32.89±17.17% within 15 minutes, followed by a slow decrease, with a pharmacological bioavailability of 12.05±4.02%. The pharmacological bioavailability of oral SEM@CNP was comparable to that of the main component of the marketed formulation (SEM+SNAC), and was 1.63 times (7.28±4.76%) that of oral SEM@PNP. Furthermore, the addition of the lysosomal escape agent CQ further improved its pharmacological bioavailability (20.63±2.99%).

[0082] Example 11

[0083] This example describes the preparation and characterization of phenolic acid or dipeptide-modified solid lipid nanoparticles (SLNs).

[0084] Phenolic acid or dipeptide-modified SLNs were prepared using an ultrasonic double emulsification method. The specific procedures were as follows: Insulin (INS) or exenatide (EXE), and sodium cholate were dissolved in hydrochloric acid at pH 2 to obtain the inner aqueous phase; stearic acid, tripalmitic acid glyceride, phospholipids, and phenolic acid or dipeptide-modified DSPE-PEG were dissolved in an organic solvent to obtain the oil phase; and poloxamer 188 was dissolved in purified water to obtain the outer aqueous phase. First, the oil phase was added to the inner aqueous phase and ultrasonically emulsified to obtain a W / O promulgated emulsion. Then, the promulgated emulsion was added to the outer aqueous phase and ultrasonically emulsified to obtain a W / O / W double emulsion. Finally, the organic solvent was removed by rotary evaporation to obtain the phenolic acid or dipeptide-modified SLN. The nanoparticle size and polydispersity index (PDI) were measured using a particle size analyzer, and the nanoparticle encapsulation efficiency and drug loading were detected by high-performance liquid chromatography (HPLC).

[0085] Figure 11 shows that the obtained series of ligand-free SLNs loaded with INS or EXE and phenolic acid or dipeptide-modified SLNs all had particle sizes <200 nm, uniform particle size, and good monodispersity (PDI <0.3). The encapsulation efficiency of insulin was >60%, and the drug loading was >5%; the encapsulation efficiency of exenatide was >50%, and the drug loading was >3%.

[0086] Example 12

[0087] This example describes the cellular uptake of solid lipid nanoparticles (SLNs) modified with phenolic acids or dipeptides.

[0088] Following the method described in Example 9, INS or EXE was replaced with FITC-INS (fluorescein 5-isothiocyanate labeled INS) to prepare fluorescently labeled SLNs. Caco-2 single-cell suspensions were seeded at a density of 1×10⁴ cells / well in 96-well plates and allowed to adhere for 3-4 days until cell differentiation occurred, after which cell uptake studies were conducted. Before the experiment, the cell culture medium was aspirated, and each well was rinsed twice with 100 μL of PBS. The FITC-INS-loaded SLNs were diluted with HBSS and added to the 96-well plates, which were then placed in a cell culture incubator. After 2 hours, the HBSS suspension of the SLNs was aspirated, and the plates were rinsed twice with PBS. Then, DMSO was added, and the plates were shaken at 100 rpm for 10 min. Finally, the 96-well plates were placed in a multi-functional chemiluminescence analyzer, and the fluorescence intensity at wavelengths of 485 nm and 520 nm was measured. The relative uptake was calculated using the fluorescence intensity of the unmodified nanoparticles (PEG SLNs) as the standard value 1.

[0089] Figure 12 shows that the drug uptake of phenolic acid ligand modified nanoparticles (FA SLN, CA SLN) is about 1.5 times that of PEG SLN, and the drug uptake of dipeptide ligand modified nanoparticles (LM SLN, LV SLN) can reach more than 2 times that of PEG SLN.

[0090] Example 13

[0091] This example demonstrates the transmembrane transport efficiency of solid lipid nanoparticles (SLNs) modified with phenolic acid or dipeptides.

[0092] Caco-2 single-cell suspension was prepared at 3-5 × 10 4Cells were seeded at a density per well in Transwell chambers and cultured for 14-21 days. Transmembrane resistance was measured every 2 days, and the cells were ready for use when the resistance was >600 Ω·cm². Before the experiment, the culture medium was aspirated, and each well was rinsed twice with 500 μL of HBSS, followed by equilibration with another 500 μL of HBSS for 30 min. 200 μL of FITC-INS-loaded nanoparticles were added to chamber A (Transwell chamber), and 800 μL of blank HBSS was added to chamber B (24-well plate). The plate was then placed in a cell culture incubator. At 0 h, 0.25 h, 0.5 h, 1 h, 2 h, 3 h, and 4 h, 80 μL of sample solution was taken from chamber B, and the same volume of HBSS was added to each sample. After sampling, 80 μL of DMSO was added to the sample solution, and the plate was shaken thoroughly to break down the nanoparticles. The fluorescence intensity was measured at wavelengths of 485 nm and 520 nm using a multifunctional chemiluminescence analyzer. A standard curve was prepared using free FITC-INS, and the concentration of FITC-INS in the sample solution at each time point was calculated using the standard curve. The apparent permeability coefficient (Papp) was calculated according to the following formula: Papp=(dQ / dt)×(1 / (A×C0)) (dQ / dt represents the slope of the curve of B-chamber nanoparticle content changing with time, A is the area of ​​the Transwell microporous membrane, and C0 is the initial drug concentration).

[0093] Figure 13 shows that the Papp values ​​of phenolic acid and dipeptide-modified nanoparticles (FA SLN, CA SLN, LM SLN, and L-VSLN) were significantly increased, more than two-fold compared to PEG SLN. This indicates that phenolic acid or dipeptide modification can significantly improve the transmembrane transport capacity of SLN, helping it overcome the absorption barrier of intestinal epithelial cells.

[0094] Example 14

[0095] This embodiment is an in vivo pharmacodynamic study of dipeptide-modified solid lipid nanoparticles (SLNs) loaded with insulin.

[0096] Type I C57 diabetic mice were successfully constructed using the streptozotocin (STZ) method, meeting the requirement of fasting blood glucose ≥11 mmol / L. Mice were randomly divided into groups of five. Fasting was started at 9:00 AM, with free access to water. Fasting body weight and fasting blood glucose were recorded at 3:00 PM. Subcutaneous injection of free INS (SCFree INS, 5 IU / kg) served as a positive control, while oral administration of free INS (Oral Free INS, 50 IU / kg) served as a negative control. Dipeptide-modified nanoparticles carrying INS (50 IU / kg) were administered orally. Blood glucose levels were measured from the tail vein at 1, 2, 3, and 4 hours. The area on the curve (AAC) of blood glucose change was calculated; a larger area indicated a greater reduction in blood glucose.

[0097] Figures 14A and 14B show that, compared with the oral-free INS group, all mice in the INS-loaded solid lipid nanoparticle groups exhibited significantly lower blood glucose levels, indicating that solid lipid nanoparticles are a nanocarrier with a significant delivery advantage over free INS. PEG SLN reduced blood glucose to 66.3% 4 hours after oral administration, showing a relatively weak hypoglycemic effect. LV SLN and LM SLN reduced blood glucose to below 50% at 4 hours, demonstrating significantly higher hypoglycemic effects than PEG SLN. Dipeptide ligand-modified SLN significantly improved the oral delivery efficiency of insulin.

[0098] Example 15

[0099] This embodiment is an in vivo pharmacodynamic study of dipeptide-modified solid lipid nanoparticles (SLN) loaded with exenatide.

[0100] Male KKay mice were randomly divided into groups of five. They were fasted overnight, and fasting body weight and fasting blood glucose were recorded. Subcutaneous injection of free EXE (SCFree EXE, 0.1 mg / kg) served as a positive control, while oral administration of free EXE (Oral Free EXE, 1 mg / kg) served as a negative control. Nanoparticles modified with dipeptide ligands carrying EXE (1 mg / kg) were administered orally. Three hours later, mice were orally administered a glucose-containing saline solution (1.5 g / kg). Blood glucose levels were then measured via tail vein at 15, 30, 60, 90, 120, 150, and 180 minutes. Using fasting blood glucose as the starting point, a curve showing the change in blood glucose relative to zero was plotted over time, and the area under the curve (AUC) was calculated. A larger AUC indicates a greater cumulative increase in blood glucose.

[0101] Figures 15A and 15B show that, compared with the oral free EXE group, the increase in blood glucose and the AUC of mice were significantly reduced after oral administration of EXE-loaded SLN, indicating that the solid lipid nanoparticle group had better glycemic control than the oral original drug group. Specifically, the PEG SLN group showed that blood glucose levels essentially returned to their initial values ​​at 2 hours, while the dipeptide-modified SLN group showed a further decrease in blood glucose levels below the initial values ​​at 2 hours, demonstrating better glucose tolerance.

[0102] As described above, the present invention can be well implemented. It should be noted that all commonly used chemical reagents used in the above embodiments are commercially available products. Those skilled in the art can make equivalent substitutions, combinations, improvements or modifications to the present invention based on the description therein, but these will all be included within the scope of the present invention. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

Claims

1. A nano-drug delivery system modified with phenolic acid or dipeptide to enhance oral absorption and utilization, characterized in that, It is prepared from nanocarriers and excipients. The nanocarriers are modified with phenolic acid or dipeptides as ligands on the surface of the nanocarriers and actively target receptors on the surface of intestinal mucosal epithelial cells. The phenolic acid ligand is a hydroxycinnamic acid derivative. The dipeptide ligand is a combination of cationic amino acids and neutral amino acids.

2. The nano-drug delivery system modified with phenolic acid or dipeptide to enhance oral absorption and utilization according to claim 1, characterized in that, The phenolic acid ligand is ferulic acid or p-coumaric acid, and the dipeptide ligand is lysine-valine or lysine-methionine.

3. A phenolic acid or dipeptide-modified nanodelivery system for increasing oral absorption and utilization according to claim 1 or 2, characterized in that, The nanocarrier is at least one of polylactic acid-glycolic acid copolymer (PLGA) nanoparticles, lipid nanoparticles (LNP), solid lipid nanoparticles, polystyrene nanoparticles, polylactic acid nanoparticles, and liposomes.

4. The phenolic acid or dipeptide-modified nanodelivery system for increasing oral absorption and utilization according to claim 3, characterized in that, The nanoparticles have a hydrophilic shell and a hydrophobic core. The hydrophilic shell is the hydrophilic end of an amphiphilic polymer, which is covalently linked to phenolic acid or dipeptide. The hydrophobic core is composed of the hydrophobic end of the amphiphilic polymer, an active ingredient, and a biocompatible carrier material. The liposomes are composed of a lipid bilayer and an inner aqueous phase. The hydrophilic active ingredient is loaded in the inner aqueous phase, and the hydrophobic active ingredient is loaded in the lipid bilayer. The surface of the liposomes is modified with phenolic acid or dipeptide ligands.

5. A phenolic acid or dipeptide-modified nanodelivery system for increasing oral absorption and utilization according to claim 4, characterized in that, The active ingredient accounts for 0.1% to 90% of the total weight of the nanocarrier.

6. The phenolic acid or dipeptide-modified nanodelivery system for increasing oral absorption and utilization according to claim 4, characterized in that, The biocompatible carrier material is selected from at least one of polylactic acid-glycolic acid copolymer (PLGA), polystyrene, polystyrene sebacic acid, polyethyleneimine, polylactic acid, polyalkyl cyanoacrylate, polyamino acid, cholesterol, fatty acids, phospholipids, sphingolipids, waxes, and fatty acid glycerides.

7. The phenolic acid or dipeptide-modified nanodelivery system for increasing oral absorption and utilization according to claim 4, characterized in that, The active ingredient is selected from at least one of protein-peptide drugs, nucleic acid drugs, and small molecule drugs.

8. The phenolic acid or dipeptide-modified nanodelivery system for increasing oral absorption and utilization according to claim 7, characterized in that, The protein and polypeptide drugs mentioned are selected from at least one of the following: insulin, octreotide, leuprorelin acetate, calcitonin, teriparatide, thymopentin, luteinizing hormone-releasing hormone, tecoctotide acetate, busereline, exenatide, telpoxetine, semaglutide, liraglutide, glucagon-like peptide-1, tedulglutide, glepaglutide, apraglutide, elsiglutide, dapiglutide, triptorelin acetate, leukocyte growth factor, erythrocyte growth factor, macrophage growth factor, tumor necrosis factor, epidermal growth factor, interleukin, angiogenesis inhibin, bovine serum albumin, ovalbumin, parathyroid hormone, growth hormone, somatostatin, interferon, and monoclonal antibodies. The nucleic acid drug is selected from at least one of small interfering RNA, messenger RNA, micro RNA, plasmid DNA, and antisense oligonucleotide drugs; The small molecule drugs are selected from at least one of the following: antipyretic analgesics and nonsteroidal anti-inflammatory drugs, antibacterial drugs, antitumor drugs, hormones, central nervous system drugs, peripheral nervous system drugs, circulatory system drugs, hypoglycemic drugs, and diuretics.

9. A phenolic acid or dipeptide-modified nanodelivery system for increasing oral absorption and utilization according to any one of claims 4 to 8, wherein the amphiphilic polymer is at least one selected from distearate-phosphatidylethanolamine-polyethylene glycol (DSPE-PEG), stearic acid-polyethylene glycol carboxylic acid, and PLGA-PEG.

10. The use of a phenolic acid or dipeptide-modified nanodelivery system for increasing oral absorption and utilization as described in claim 1 in an oral drug delivery formulation for overcoming the intestinal absorption barrier.

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