Surface-Engineered Breviscapine Nanocrystals for Enhanced Oral Absorption and Cardioprotection in Isoprenaline-Induced Myocardial Infarction

Abstract

Purpose

Breviscapine (BRE) possesses excellent cardioprotective efficacy against myocardial infarction (MI). However, its oral application is severely hindered by BCS Class IV properties, including poor solubility and permeability, accompanied by P-glycoprotein (P-gp)-mediated drug efflux and gastrointestinal absorption barriers. Arginine-rich CPPs such as R8 can promote cellular uptake, whereas cationic surface characteristics may restrict particle mobility in gastrointestinal mucus. Conversely, hydrophilic and electro-neutral surfaces favor mucus penetration but may reduce epithelial uptake. This imposes challenges for balancing these two absorption barriers. To solve these problems, we developed surface-engineered nanocrystals with sequential barrier-crossing capability (R8-F127-VES@BRE-NCs).

Methods

We engineered multifunctional nanocrystals (R8-F127-VES@BRE-NCs) featuring a sequential barrier-crossing design: nanocrystal technology increases saturation solubility, Pluronic F127 imparts mucus-penetrating capability, R8 peptide facilitates epithelial internalization, and Vitamin E Succinate (VES) may contribute to efflux modulation.

Results

Comprehensive characterization and in vitro studies suggested that this integrated design helped address the trade-off between mucus diffusion and epithelial uptake. Pharmacokinetic studies revealed a marked enhancement in oral BRE absorption, with C_max and AUC_0-∞ increased to 185.76 ng/mL and 1418.34 ng·mL⁻¹·h (4.27- and 7.37-fold over BRE-raw). Following establishment of the isoprenaline-induced myocardial injury model, 14-day oral treatment with R8-F127-VES@BRE-NCs showed enhanced cardioprotective effects, as evidenced by reduced infarct size and improved oxidative stress-related biochemical indicators.

Conclusion

R8-F127-VES-modified nanocrystals provide a promising strategy for overcoming oral delivery barriers and enhancing the cardioprotective effects of orally administered BRE.

Graphical Abstract

Introduction

The demographic transition toward an aging population has increased the prevalence of cardiovascular disease (CVD), representing a significant global health challenge.¹ Among them, myocardial infarction (MI) remains a critical global health challenge, consistently accounting for substantial mortality and morbidity.² MI results from insufficient coronary blood flow, leading to irreversible myocardial cell damage.³ Clinically, revascularization surgery is usually the primary choice to restore the smooth blood vessels; however, the risks and high costs of the procedure cannot be overlooked.⁴ Consequently, many patients with mild disease are treated with pharmacotherapy, such as nitroglycerin and isosorbide mononitrate.⁵ Nevertheless, the long-term utility of nitroglycerin is limited by the development of tolerance, while isosorbide mononitrate often causes adverse gastrointestinal reactions.^6,7

Traditional Chinese medicine has long been used for the prevention and treatment of MI and has shown significant clinical efficacy. Erigeron breviscapus (Vant). Hand-Mazz. is an important traditional drug in China. This plant is a member of the Compositae family and is mainly distributed in southwest China. Breviscapine (BRE) is the major active component extracted from this plant,⁸ and possesses a broad range of pharmacological activities with a favorable safety profile.⁹ Recent studies on cardiovascular diseases have shown that breviscapine plays numerous cardioprotective roles, including anti-inflammatory, anti-oxidative stress, anti-apoptosis, and improvement of left ventricular remodeling.^10–12 Therefore, BRE products are widely used in the treatment of cardiovascular and cerebrovascular diseases in China, such as coronary artery disease, myocardial infarction, hypertension, hyperlipidemia and diabetic complications.¹³

Although BRE has been used in clinical practice for many years, its poor oral bioavailability has limited the development of effective oral formulations, and BRE is therefore commonly administered by injection in clinical practice. However, repeated injections are inconvenient for long-term administration, whereas oral administration offers greater convenience and may improve patient compliance. BRE has extremely low solubility (only 0.16 mg/mL) and poor lipid solubility (log P = −2.56 in PBS at pH 4.2).^14,15 Furthermore, the absolute oral bioavailability of BRE has been reported to be only approximately 0.4% in beagle dogs. In addition to poor membrane permeability, the intestinal transport of scutellarin is affected by efflux transporters, including P-gp and MRP2, which may further restrict its oral absorption.^16,17 Several formulation strategies have been explored to improve oral bioavailability of BRE, including nanosuspensions, lipid emulsions, liposomes, solid lipid nanoparticles, and other absorption-enhancing systems.^18–20 Previous oral formulations have achieved improvements in dissolution, gastrointestinal stability and intestinal permeability to varying extents. However, most of these approaches primarily addressed a single limitation, such as low solubility or limited membrane permeability.²¹ For BCS Class IV drugs such as BRE, enhanced dissolution alone may not necessarily translate into improved oral absorption, because intestinal absorption is also influenced by mucus permeation, epithelial cellular uptake and transepithelial transport, as well as transporter-mediated efflux. Accordingly, an effective oral BRE formulation should integrate dissolution enhancement with improved mucus permeation and epithelial transport, while modulating transporter-associated efflux.

Recently, many studies have demonstrated that nanosuspensions or nanocrystal suspensions (NCs) are considered one of the most attractive and promising strategies for the formulation of poorly soluble drugs. NCs, which are a colloidal dispersion of nanosized drug particles produced by an appropriate size-reduction method and stabilized by a minimal amount of suitable stabilizer,^22,23 allow for high drug loading. According to the Noyes-Whitney and Ostwald-Freundlich equations, NCs significantly increase the surface area, leading to a substantial improvement in dissolution rate.^24,25 Moreover, NCs enhance adhesion to the gut wall and spread into the intestinal water layers.²⁶ Meanwhile, the high drug loading of NCs improves patient compliance,²⁷ eliminates food effects and enhances the efficacy and safety of oral administration.²⁸

Most drugs are absorbed in the small intestine after oral administration.^29,30 This process can generally be divided into major steps for crossing the small intestinal mucosal barrier and entering systemic circulation. The initial step involves diffusing through mucus to reach the enterocyte surface, followed by cellular movement across the epithelial layer, and finally, exocytosis for systemic distribution.³¹ Beyond drug solubility and stability, several factors involving the uptake ability of intestinal epithelial cells, intestinal permeability to drugs, and multiple barriers of the digestive tract can affect the absorption of oral drugs. Among them, the intestinal barrier is considered as an important factor affecting the bioavailability of oral drugs.^32–34 Thus, developing multifunctional polymer-modified NCs may be a promising way to further increase the ability to cross the intestinal barrier and thereby enhance the oral absorption of poorly soluble drugs.

Studies have shown that nanoparticles modified with polymers possessing long hydrophilic chains, such as Pluronic^® F127, P123, or polyethylene glycol (PEG), could exhibit excellent mucus diffusion ability.³⁵ Among these, research indicates that due to the steric hindrance of Pluronic F127, it can facilitate the penetration of nanoparticles across highly viscoelastic mucus by minimizing adhesive interactions between nanoparticles and mucus constituents.³⁶ Therefore, the modification of nanocrystals with F127 enables rapid penetration of the intestinal mucus layer. In recent years, numerous studies have shown that vitamin E (VE) can inhibit drug efflux transporters. In particular, D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) has been widely reported as a polymeric material commonly used to inhibit efflux transporters.³⁷ Therefore, the surface modification of nanocrystals with F127 or a derivative of VE enhances both their cellular uptake and subsequent oral absorption. However, the hydrophilic nature of F127 makes it difficult to traverse the cell membrane.³⁸ Derivatives from the vitamin E family were chosen as the hydrophobic material to facilitate the transmembrane transport of nanocrystals while concurrently exploiting their intrinsic ability to inhibit drug efflux pumps. Vitamin E succinate (VES) is a stable derivative of vitamin E. VES is currently used in the medical, food, and cosmetic industries as a replacement for vitamin E.³⁹ Previous studies conducted by our research group demonstrated that a modified polymer (F127-VES) can be synthesized by conjugating hydrophilic Pluronic F127 with hydrophobic VES.

Cell-penetrating peptides (CPPs), often referred to as protein transduction domains, are short peptide sequences ranging from 5 to 30 amino acids with superior cellular internalization capability,⁴⁰ and they can carry macromolecules and small particles. CPPs enhance the cellular uptake of various cargoes (such as DNA, siRNA, and nanoparticles) by interacting with the negatively charged plasma membrane, significantly increasing their delivery efficiency in vitro.^41,42 Arginine oligomers, a type of CPP, have notable permeation-enhancing properties and show a strong attraction to anionic cell membranes because they contain several positively charged arginine residues, facilitating internalization.⁴³ Mitchell et al⁴⁴ showed that polymers consisting of six or more amino acids of l- or d-arginine entered cells significantly better than those composed of lysine, ornithine, and histidine of the same length. Peptides with fewer than six amino acids were not effective. R8 is a cell-penetrating peptide composed of eight arginine residues that can cross cell membranes and facilitate the entry of macromolecules into cells. Furthermore, it has been demonstrated that poly-l-arginine induces reversible opening of tight junctions between epithelial cells.⁴⁵ Additionally, R8 can be easily conjugated with hydrophobic moieties. However, the unique characteristics of CPPs, due to their polycationic groups, inevitably lead to a strong attraction to the negatively charged mucin. In other words, CPPs effectively promote agents’ cellular internalization but also significantly hinder their penetration through the mucus layer. An effective nanocarrier for oral administration should be capable of overcoming both the mucus and epithelial barriers.⁴⁶

To overcome these physical barriers, we designed an amphiphilic polymer, R8-grafted F127-VES (R8-F127-VES), and utilized it for the surface modification of NCs. Specifically, F127 is a PEO-PPO-PEO triblock copolymer, its PEO segments form a hydrated corona that reduces nonspecific interactions with mucin,⁴⁷ while the hydrophobic VES moiety provides hydrophobic anchoring to the nanocrystal surface and may contribute to the modulation of efflux-associated drug loss.⁴⁸ Here, F127-VES serves not merely as a linker, but as an amphiphilic functional backbone that provides a surface stabilizing layer, a functional corona, and a scaffold for R8 grafting, thereby constructing multifunctional nanocrystals R8-F127-VES@BRE-NCs with sequential barrier-crossing capability.

This innovative design integrates nanocrystal-mediated dissolution enhancement with a proposed three-step barrier-modulating strategy. Nanocrystal technology serves as an effective approach to enhance the solubility and dissolution rate of BRE, thereby providing the physicochemical basis for subsequent intestinal absorption. The proposed “sequential barrier-crossing” strategy then proceeds through three steps: (1) the hydrophilic PEO segments of F127 form a hydrated corona that partially reduces nonspecific interactions between cationic R8 residues and anionic mucin, thereby attenuating premature mucus adhesion during mucus diffusion. This shielding effect is not intended to completely mask R8, preserving residual cationic characteristics; (2) upon reaching the epithelial surface, the residual cationic characteristics and membrane affinity of R8 may facilitate epithelial interaction and endocytosis-associated uptake, thereby compensating for the limited passive permeability of the intestinal epithelium; and (3) the VES moiety not only contributes to nanocrystal stabilization via hydrophobic interactions with the drug surface, but also may contribute to efflux modulation, thereby further enhancing intracellular accumulation and overall drug absorption.

Collectively, this hierarchically engineered strategy, built on the F127-VES functional backbone, is hypothesized to help address the trade-off between mucus penetration and epithelial uptake, thereby improving the oral absorption of BRE and enhancing its cardioprotective effects in the isoprenaline-induced myocardial injury model. A schematic representation of the study is shown in Figure 1.

Figure 1.

Schematic illustration of multifunctional R8-F127-VES@BRE-NCs for overcoming multiple oral absorption barriers. (A) Preparation of R8-F127-VES@BRE-NCs by high-pressure homogenization. (B) Proposed barrier-modulating process during intestinal absorption, including mucus penetration, R8-associated epithelial uptake, and VES-associated efflux modulation.

Materials and Methods

Materials

Breviscapine (purity > 98%) was purchased from Nanjing King Bamboo Biological Technology Co., Ltd. (Nanjing, China). R8 was synthesized by Qiangyao Biotechnology (Shanghai, China). F127 was purchased from BASF Co., Ltd. (Ludwigshafen, Germany). Vitamin E succinate was purchased from Aladdin Industrial Corporation (Shanghai, China). TPGS was purchased from Xian Healthful Biotechnology Co., Ltd. (Xian, China). Metoprolol was purchased from AstraZeneca Pharmaceuticals LP (Waltham, MA, USA). Isoprenaline was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Porcine gastric mucin was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). DMEM high-glucose medium and DAPI were purchased from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). All other chemicals were of analytical reagent grade.

Cells and Animals

This study was approved by the Experimental Animal Ethics Committee of Jiangxi University of Chinese Medicine (approval number 20220310001) and conducted in compliance with its guidelines for the care and use of laboratory animals. All anesthesia and euthanasia procedures were performed by trained personnel in accordance with the approved institutional protocol and applicable guidelines for the care and use of laboratory animals. Rats were given a standard diet and water. The animals were kept under standard conditions (room temperature 22 °C and 50% humidity) with a regular 12: 12-hour light-dark cycle throughout the experimental period.

Synthesis of Functional Materials

Synthesis of the R8-F127-VES Conjugate

First, F127-VES was synthesized as previously reported.⁴⁹ In brief, F127, VES, DCC, and DMAP were dissolved in anhydrous DMSO at a molar ratio of 1:2.5:3:3. The reaction vessel was covered and the solution was stirred continuously at 300 rpm for 24 hours at room temperature. After the reaction was completed, the reaction mixture was transferred to a dialysis bag (molecular weight 7000 Da). Samples were dialyzed against DMSO and ultrapure water, and the dialyzate was placed at −80 °C overnight after dialysis was completed. After pre-freezing, the dialyzate was lyophilized in a freeze dryer, and F127-VES was obtained. R8, EDCI, and DMAP were dissolved in anhydrous DMSO and used together with F127-VES at a molar ratio of R8:EDCI:DMAP:F127-VES = 2:3:3:1. R8, EDCI, and DMAP were allowed to react for 2 h for activation. Subsequently, the intermediate product F127-VES obtained from the previous step was added. The reaction mixture was stirred at room temperature for 48 h. After the reaction was finished, the reaction solution was transferred to a dialysis bag (molecular weight 7000 Da). Samples were dialyzed against DMSO and ultrapure water, the dialyzate was placed at −80 °C overnight after dialysis was completed. After pre-freezing, the dialyzate was lyophilized in a freeze dryer, and R8-F127-VES was obtained. The product yield of R8-F127-VES after purification and lyophilization was 76%.

Characterization of R8-F127-VES

Samples were analyzed by Fourier transform infrared (FT-IR) spectroscopy and proton nuclear magnetic resonance (¹H-NMR) spectroscopy. FT-IR spectra of R8, F127-VES and R8-F127-VES were recorded in KBr pellets with a FT-IR spectrophotometer (Spectrum Two, Perkin-Elmer, Inc). For ¹H-NMR analysis, R8, F127-VES and R8-F127-VES were dissolved in DMSO at 25 °C and analyzed by 600 MHz NMR spectrometer (Bruker AVANCE III HD 600, Bruker, Switzerland). The formation of the polymer conjugate was qualitatively assessed by comparing the FT-IR and ¹H-NMR spectra of R8-F127-VES with those of R8 and F127-VES. The presence of characteristic spectroscopic signals consistent with R8, F127, and VES in the spectra was considered as supporting evidence for the formation of the R8-F127-VES conjugate.

Preparation of BRE-NCs Nanosuspension

The BRE-NCs nanosuspension was prepared using high-pressure homogenization. A mixture of BRE (5 mg) and stabilizer (0.5 mg TPGS) with 10 mL ultra-pure water was fully dispersed in a beaker under magnetic stirring. Then, the suspension was sheared using a high-shear homogenizer for 6 min at room temperature (FA25, Fluko Equipment Shanghai Co., Ltd., Shanghai, China). This pre-mix was passed through a high-pressure homogenizer (AH-1000D, ATS Engineering Inc., Seeker, Canada) for 20 cycles at 200 bar and 40 cycles at 800 bar.

Preparation of F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs

The F127-VES@BRE-NCs nanosuspension was prepared using high-pressure homogenization. A mixture of BRE (5 mg), stabilizer (0.5 mg TPGS), and functional material F127-VES (0.83 mg) with 10 mL ultra-pure water was fully dissolved in a beaker under magnetic stirring. Then, the mixture suspension was sheared using a high-shear homogenizer for 6 minutes at room temperature (FA25, Fluko Equipment Shanghai Co., Ltd., Shanghai, China). This pre-mix was passed through a high-pressure homogenizer (AH-1000D, ATS Engineering Inc., Seeker, Canada) for 20 cycles at 200 bar and 40 cycles at 800 bar.

R8-F127-VES@BRE-NCs nanosuspension was prepared using high-pressure homogenization. A mixture of BRE (5 mg), stabilizer (0.5 mg TPGS), and functional material R8-F127-VES (1 mg) with 10 mL ultra-pure water was fully dissolved in a beaker under magnetic stirring. Then, the suspension was sheared using a high-shear homogenizer for 6 min at room temperature (FA25, Fluko Equipment Shanghai Co., Ltd., Shanghai, China). This pre-mix was passed through a high-pressure homogenizer (AH-1000D, ATS Engineering Inc., Seeker, Canada) for 20 cycles at 200 bar and 40 cycles at 800 bar.

Solidification of Nanocrystals

It is well known that nanosuspensions have poor physical stability and often suffer from precipitation, crystal growth and particle aggregation in storage. Nevertheless, the stabilizers cannot sustain the long-term stability of nanocrystals, and liquid nanocrystals are also difficult to transport and store. The process of drying nanosuspensions into a solid form helps overcome stability challenges and streamlines further processing, including tableting or capsule filling. Then, the freeze-drying method was used to prepare the solid nanocrystals. The lyophilization conditions are as follows: First, Trehalose (5% w/v) was added as a cryoprotectant during lyophilization. Then, the BRE-NCs nanosuspension was pre-frozen at −80 °C for 24 h. The suspension was then poured into a flat container and subjected to freeze drying using a lyophilizer (SCIENTZ-10N, freeze dryer, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China).

Nanocrystal Characterization

PS, PDI and ZP Measurement

The particle size (PS), PDI, and zeta potential (ZP) of freeze-dried NCs were measured using DLS with a Malvern Zetasizer Nano-ZS using the wet method (Malvern Instruments Limited, Worcestershire, UK). Before measurement, lyophilized samples were reconstituted with distilled water. The data were analyzed using the Mie model (standard presentation: dispersant refractive index = 1.55, real particle refractive index = 1.670, imaginary particle refractive index = 0.01). All measurements were performed in triplicate.

Appearance and Morphology

The prepared formulations were inspected visually for their color and homogeneity. A scanning electron microscope (SEM) (Quanta 250, FEI, USA) was used to observe the morphology of the raw BRE, BRE-NCs, R8-F127-VES@BRE-NCs and F127-VES@BRE-NCs. The samples were coated with a thin layer of gold to increase the electrical conductivity before scanning to improve the accuracy of the measurements.

Fourier Transform-Infrared Spectroscopy (FT-IR)

Fourier transform infrared spectroscopy (FT-IR) (FT-IR Spectrometer, BRUKER IFS-55, Switzerland) was used to detect whether the initial crystalline state of powders was maintained before and after particle size reduction and freeze-drying. Prior to testing, the powdered sample was mixed and ground with potassium bromide and then compressed into tablets on an infrared tablet press. The FT-IR spectral analysis was performed within a range of 400–4000 cm⁻¹.

Powder X-Ray Diffraction (PXRD)

PXRD analysis was used to determine the crystallinity of the samples. The structures of the raw API and dry nanocrystals were characterized using a PXRD system (PANalytical, Westborough, MA, USA) with a voltage of 40 kV and a current of 25 mA, using Cu Kα radiation. The powder patterns were obtained from 0 to 60° 2θ at a scan speed of 1°/min.

Saturation Solubility

The solubility of the raw BRE and nanocrystals was measured by the saturation shake-flask method. Excess BRE-raw, BRE-NCs, F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs were dispersed in 2 mL distilled water and shaken at 100 r/min and 37 °C for 24 h in a temperature-controlled shaking bath in a QYC-200 rotary shaker (Fuma, Shanghai, China). Samples were then centrifuged at 15,000 r/min for 15 min by a refrigerated centrifuge (Sigma 2K15C refrigerated centrifuge, Sigma, England). Then, the supernatant was collected to measure the concentration of BRE using the HPLC method. The saturation solubility experiment was performed using three independently prepared test samples for each formulation (n = 3), and the results are expressed as mean ± SD.

In vitro Release Evaluation

The release characteristics of the prepared formulations were studied by using a USP II paddle method with a dissolution apparatus (ZRS-8 G, Tianjin University Wireless Factory, China). Briefly, 20 mg BRE-raw, BRE-NCs, F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs (equivalent to 20 mg of BRE) were transferred into 200 mL of 0.1% (w/v) SDS solution at (37 ± 0.5) °C and stirred at 100 rpm. Then, 1 mL samples were withdrawn at 5, 10, 15, 20, 30, 45, 60, 90, 120, and 240 min and replaced with an equal volume of blank dissolution medium. All samples were filtered through 0.22 μm membrane filters and further analyzed by HPLC. The chromatographic column used was a Titan C18 column (250 mm × 4.6 mm, 5 μm, Phenomenex Inc., Los Angeles, CA, USA). The mobile phase consisted of 45% methanol (A) and 55% 0.1% phosphoric acid solution (B). The injection volume was 10 μL, the flow rate was 1 mL·min⁻¹, and the column temperature was maintained at 30 °C. Breviscapine was detected at 335 nm using an Agilent VWD detector. The release study was performed in triplicate using independently prepared formulation samples (n = 3), and the results are expressed as mean ± SD.

Stability

To confirm that solidification technology enhances the stability of nanocrystal suspensions, stability studies of the prepared formulations were performed. In brief, the BRE-NCs, F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs were placed in a desiccator maintained at ambient conditions. Samples were withdrawn on days 1, 3, 5, 7, 15, and 30 for determination of particle size, PDI, zeta potential, and drug content to evaluate the storage stability of the prepared formulations.

In vitro Mucus Penetration Evaluation

As a natural barrier to drug absorption, the mucus layer’s dense network and negative charge, along with steric hindrance, can effectively obstruct drug diffusion and clear foreign particles such as nanoparticles. Consequently, it is necessary to investigate the interaction between the prepared formulations and mucin, as well as their diffusion in mucus, to evaluate their mucus penetration performance.

Interaction Between BRE-NCs with Mucin

Porcine gastric mucin (20 mg) was dispersed in 10 mL water and stirred overnight. Following the aforementioned procedures, the mucin solution was ready for subsequent experiments. Then, BRE-raw, BRE-NCs, F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs (equivalent to 2.4 mg/mL BRE) were added to ultrapure water (20 mL) to form dispersions. Then, 5 mL of each formulation dispersion was mixed with 1 mL mucin solution (2 mg/mL) at 37 °C for 4 h in a shaker (150 rpm). Subsequently, 100 μL of the interaction solution was collected at 0, 0.5, 1, 2, 3, 4 h, respectively. The particle size and PDI of the mucin-NCs complexes were measured using the Zetasizer Nano-ZS.

Mucus Diffusion Analysis

Furthermore, to assess the mucus-penetrating ability of the prepared formulations, a 24-well transwell system with 0.4 μm pores (Corning Incorporated, Corning, NY, USA) was used, as in our previous study.⁵⁰ Briefly, 40 µL of porcine gastric mucin (PGM) suspension was placed uniformly on the polycarbonate membrane of the transwell. Then, 600 μL of the phosphate-buffered solution (pH 7.4) was placed into the acceptor compartment, and the transwell plate was equilibrated in a constant-temperature shaker (100 rpm) at 37 °C for 30 min. After reaching equilibrium, 60 µL of BRE-raw, BRE-NCs, F127-VES@BRE-NCs, and R8-F127-VES@BRE-NCs was added to the donor chamber. Then, the transwell system was incubated at 37 °C in a shaker (50 rpm) for 3 h. Subsequently, 100 μL of the sample was collected from the acceptor chamber, and an equal volume of phosphate-buffered solution was replenished. Samples were diluted in the mobile phase and filtered through 0.22 μm membrane filters, then subjected to further HPLC analysis.

Cellular Experiments

Cell Culture

Human colon carcinoma Caco-2 cells were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd, and the cells were cultured in Roswell Park Memorial Institute 1640 Medium (RPMI 1640) containing 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) Penicillin/Streptomycin at 37°C in an atmosphere containing 5% CO₂. Caco-2 cells were cultured to 60% −70% confluence under standard culture conditions. Cells in passages 20–40 were used for all experiments.

In vitro Cytotoxicity

In vitro cytotoxicity was evaluated using the CCK-8 assay with Caco-2 cells. Caco-2 cells were seeded into 96-well plates at a density of 9×10⁴ cells/well and cultured in a 5% CO₂ incubator at 37 °C for 4 h. Subsequently, 100 μL of BRE-raw, BRE-NCs, F127-VES@BRE-NCs, or R8-F127-VES@BRE-NCs prepared in DMEM high-glucose medium (10–200 μg/mL) was added to each well. Three wells were set up for each concentration, and DMEM high-glucose medium was used as a control. After 4 h incubation, the supernatant culture medium was discarded. Then, the medium was replaced with fresh medium containing 10% CCK-8 solution and incubated for another 1 h at 37 °C. Finally, the absorbance was measured at 450 nm by a microplate reader.

Cellular Uptake and Mechanisms

Caco-2 cells were seeded in 6-well plates at a density of 4×10⁵ cells/well. After the cells reached almost 90% confluence, the original medium was discarded. The cells were then washed twice with 1 mL HBSS and incubated with BRE-raw, BRE-NCs, F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs at an equivalent BRE concentration of 200 μg/mL for 1 h. At the end of incubation, the supernatant was collected from the wells. And the wells were washed three times with cold phosphate-buffered saline (PBS) and the cells were solubilized in 50 µL solution of 0.5% triton-X 100 for 30 min. Following the addition of 200 µL of the mobile phase, the samples were analyzed by HPLC after centrifuging.

To investigate the mechanisms of these formulations entering Caco-2 cells, we conducted cellular uptake inhibition experiments. Caco-2 cells were seeded into 6-well culture plates at a density of 2×10⁵ cells/well. After the cells reached almost 90% confluence, the original medium was discarded. The cells were then washed twice with 1 mL HBSS and pretreated with selective inhibitors of different internalization pathways at 37 °C for 0.5 h, while an identical volume of PBS was added into the culture medium (DMEM) as the control group. After pretreatment, BRE-raw, BRE-NCs, F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs at a concentration of 200 μg/mL were incubated with the inhibitors for 1 h. At the end of incubation, the supernatant was collected from the wells. And the cells were washed three times with cold phosphate-buffered saline (PBS) and the cells were solubilized in 50 µL solution of 0.5% Triton-X 100 for 30 min. Following the addition of 200 µL of the mobile phase, the samples were analyzed by HPLC after centrifugation. The inhibitors used in the experiments were as follows: Chlorpromazine (100 μg/mL), Colchicine (100 μg/mL), Verapamil (100 μg/mL) and Indomethacin (100 μg/mL).

Cellular Transport and Evaluation of Caco-2 Cell Monolayer Integrity

Caco-2 cells in the logarithmic growth phase were seeded at 1 ×10⁵ cells/well in a 0.4 μm polycarbonate membrane Transwell^® 12-well plate and cultured for 21 days. The integrity of the cell monolayers was monitored by Transepithelial electrical resistance (TEER). Only when the monolayers exhibiting a stable TEER value and over 300 Ω·cm² were used for further experiments. At the beginning of the experiment, the culture medium was replaced with 0.5 mL DMEM-high glucose medium, which was added to the apical (AP) chamber and the basolateral (BL) chamber was filled with 1.5 mL DMEM-high glucose medium to replace the culture medium, then equilibrated at 37 °C for 20 min. The solution in the AP chamber was replaced with 0.5 mL BRE-raw, BRE-NCs, F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs at the concentration of 200 μg/mL. Triplicate wells were set for each sample, and the cells were then placed in the CO₂ incubator for 2 h. At predetermined time points (30, 60, 90, and 120 min), 100 μL solution in the BL chamber was collected and replenished with an equal volume of fresh high-glucose DMEM. The samples were immediately stored at −80 °C for subsequent analysis. Finally, samples were assayed using HPLC. The apparent permeability coefficient (P_app) was determined using the following equations (1):

(1)

where is the rate of permeability, A is the surface area of the filter membrane, and C₀ is the initial concentration of BRE in the apical chamber.

For the evaluation of monolayer integrity, Caco-2 cells in the logarithmic growth phase were seeded at 1 ×10⁵ cells/well in 0.4 μm polycarbonate membrane Transwell^® 12-well plate and cultured for 21 days. The integrity of the cell monolayers was monitored by TEER. Only when the monolayers exhibiting a stable TEER value and over 300 Ω • cm² were used for further experiments. Before the experiment, the culture medium was replaced with HBSS, and the cells were allowed to equilibrate. The initial TEER value was measured as a baseline. Then, the different formulations (at the same concentrations as the uptake study) were added to the apical side of the inserts. After 1 h of incubation, the TEER was measured again using a Millicell ERS-2 Voltohmmeter (Millipore).

Single-Pass Intestinal Perfusion Study

The absorption performance of the intestine was evaluated by single-pass intestinal perfusion method. Briefly, 24 Sprague-Dawley (SD) rats were divided into four groups and fasted for 12 h but had free access to water and then anesthetized with an intraperitoneal injection of 20% urethane solution (1 g/kg). Immediately, the rats were placed on a recirculating warming blanket that maintained the body temperature at 37 ± 1 °C. The abdominal cavity was opened, selected segments (about 10 ± 2 cm) were carefully exposed and segregated, including duodenum and jejunum. After being gently rinsed with normal saline, the intestinal segments were equilibrated by Krebs Ringer buffer (K-R buffer, containing 0.37 g CaCl₂, 1.40 g glucose, 0.32 g NaH₂PO₄, 0.35 g KCl, 1.37 g NaHCO₃, 7.8 g NaCl and 0.02 g MgCl₂ in 1000 mL of purified water) and then were perfused with various 10 µg/mL BRE-loaded formulations diluted by K-R buffer through the isolated segments at a flow rate of 0.2 mL/min for 2 h. At the end of the non-recovery perfusion experiment, while the rats remained under urethane anesthesia, they were euthanized by an overdose of sodium pentobarbital (150 mg/kg, i.p). Death was confirmed by the absence of respiration and heartbeat before tissue collection. The intestinal segments were then excised, and the length and diameter of each perfused intestinal segment were recorded. At the same time, the mass differences of each group of supply and receiving tubes were accurately weighed. Subsequently, the collected perfusate solution was diluted with methanol, and the mixture was centrifuged at 15,000 rpm for 15 min and the concentration of BRE in the resultant solution was determined by HPLC.

Based on the following equations, the volumes of the perfused solution influent from the intestinal segment (V_in), the volumes of the perfused solution effluent from the intestinal segment (V_out), the absorption rate constant (K_a) and apparent permeability coefficients (P_app) were calculated.

(2)

(3)

(4)

(5)

In the above equations, m_in and m_out are the masses (g) of the influent and effluent perfusate, respectively; and are the densities (g/mL) of the influent and effluent perfusate, respectively; and are the volumes (mL) of the influent and effluent perfusate, respectively; L and r are the length (cm) and radius (cm) of the perfused intestinal segment, respectively; and are the drug concentrations (g/mL) in the influent and effluent perfusate, respectively; and Q is the perfusion flow rate (0.2 mL/min).

In vivo Pharmacokinetic Study

Animals and Dosing

Healthy male Sprague-Dawley (SD) rats with SPF grade, 7 weeks old and weighing 200 ± 20 g, were purchased from Henan Skobes Biotechnology Co., Ltd., with license number SCXK (Yu) 2020–0005. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Research Council. This study was approved by the Experimental Animal Ethics Committee of Jiangxi University of Chinese Medicine, in compliance with its guidelines for the care and use of animals (approval number 20220310001). SD rats were randomized into 4 groups of 6 animals each. Animals were fasted for 12 h before experiments and given free access to water. The animal’s health status was monitored daily during the study. The BRE-raw, BRE-NCs, F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs were administered at a dose of 20 mg/kg of BRE, dispersed in distilled water before oral administration. After drug administration, approximately 0.2 mL of blood was collected at each sampling point (0.083, 0.167, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h). After each blood sampling, an equivalent volume of normal saline was administered to compensate for blood loss. Blood samples were centrifuged at 4000 rpm for 15 min, and the separated plasma was stored at −80 °C until further analysis.

Plasma Sample Preparation and Analysis

BRE concentrations were measured in plasma using LC–MS/MS. The samples were prepared by protein precipitation using methanol. Briefly, a 1.5 mL tube was filled with 100 μL plasma and 10 μL IS (internal standard, using metronidazole as the internal standard in this experiment) (5 ng/mL). Then, 290 μL methanol was added, and the mixture was vortexed for 3 min, followed by centrifugation at 15000rpm for 10 min. The plasma concentrations of BRE were determined using an AB Sciex Triple Quad 4500 (AB Sciex, Framingham, MA, USA) LC–MS/MS system. An Agilent Poroshell SB-C18 (4.6 × 100 mm, 2.7 μm) column (Agilent, Santa Clara, CA, USA) was used for separation. The mobile phase consisted of acetonitrile (A) and 0.1% formic acid (B). The flow rate was set at 0.7 mL/min, and the LC gradient was programmed according to the following time gradient elution: a linear gradient was applied by varying the proportion of B from 80% to 40% in the first 5 min; then, a linear gradient was applied by varying the proportion of B from 40% to 0% from 5 to 5.1 min, and the percentage of B was restored to 0% within 6 min. Subsequently, a linear gradient was applied by varying the proportion of B from 0% to 80% from 6 to 6.1 min, and this was maintained until the end of the run (8 min). MS/MS experiments were performed in positive-ion mode (ESI⁺) with a multiple-reaction monitoring system (MRM). The selected MRM transitions were m/z 461.1 → 287.1 for BRE and m/z 172.1 → 82.0 for metronidazole. The lower limit of quantification was 10 ng/mL.

Data Analysis

The pharmacokinetic parameters of each formulation, including the area under the concentration-time curve (AUC_0-∞), the peak concentration (C_max), time of maximum concentration (T_max), the elimination half-life (t_1/2), and the mean residence time (MRT) were calculated via PKSolver 2.0 software (China Pharmaceutical University, Jiangsu, China) with non-compartmental modeling. The data were analyzed using GraphPad Prism 8 (GraphPad Software), and the results are presented as mean ± standard deviation. Statistical analysis was conducted using one-way ANOVA. p < 0.05 and p < 0.01 were considered statistically significant.

Pharmacodynamic Studies

Animals and Establishment of Acute MI Modeling

Healthy male Sprague-Dawley (SD) rats with SPF grade, weighing 250 ± 20 g, were purchased from Sibeifu Beijing Biotechnology Co. Ltd., with license number SCXK (Jing) 2024–0001. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Research Council. This study was approved by the Experimental Animal Ethics Committee of Jiangxi University of Chinese Medicine, in compliance with its guidelines for the care and use of animals (approval number 20220310001). Isoprenaline (isoproterenol, ISO) has been widely employed to induce myocardial injury in rats and is commonly used for the experimental evaluation of cardioprotective agents. ISO administration induces marked alterations in cardiac injury markers, oxidative stress indices, and myocardial histopathology, including myocardial necrosis.⁵¹ Based on previous reports with some modifications, an isoprenaline-induced myocardial injury model was established by subcutaneous injections of isoprenaline (ISO) (105 mg/kg; once daily) for two consecutive days.⁵² Successful model induction was indicated by ST-segment elevation on electrocardiographic monitoring. After successful induction of isoprenaline-induced myocardial injury, the rats were assigned to the ISO group, positive control group, BRE-raw group, BRE-NCs group, F127-VES@BRE-NCs group, and R8-F127-VES@BRE-NCs group. Age-matched rats without ISO injection were used as the control group. The control and ISO groups were given normal saline in the same volume. The BRE-raw, BRE-NCs, F127-VES@BRE-NCs, and R8-F127-VES@BRE-NCs groups were orally administered the corresponding suspensions at an equivalent BRE dose of 20 mg/kg/day. For the positive control, metoprolol was administered at 10 mg/kg/day. Treatments were administered once daily by oral gavage for 14 consecutive days.

The Rate of Weight Change and Cardiac Index

Body weight change is an indicator of the overall health of the animal. Effective therapeutic drugs can alleviate the systemic impact of MI by attenuating myocardial injury, avoiding excessive inflammatory response, and improving cardiac function. This protective effect is reflected in the improvement of the overall health of the animal, thereby slowing or reversing the downward trend in body weight caused by MI. The body weights of rats were recorded at baseline (0 h, immediately before the first oral dose) and at 24 and 48 h thereafter. The body mass change was calculated according to the equation (6) to evaluate the overall health of the animals and recovery level of the rats in each group after MI.

The cardiac index, or the heart weight to body weight ratio, is a key morphological indicator used to evaluate heart size and the degree of cardiac hypertrophy.⁵³ Following acute myocardial injury, an intense inflammatory response leads to increased capillary permeability and tissue fluid exudation, resulting in interstitial myocardial edema, which contributes to an increase in heart weight in the short term.⁵⁴ Therefore, an elevated cardiac index directly reflects the pathological enlargement of the heart caused by myocardial edema and/or hypertrophy, making it an important quantitative measure of the severity of myocardial injury and subsequent remodeling.⁵⁵ At the end of the experiment, the rats were deeply anesthetized with sodium pentobarbital (50 mg/kg, i.p), followed by cervical dislocation as the secondary physical method to ensure death. The cardiac index was determined as the ratio of total heart weight (mg) to body weight (g). The cardiac index was calculated according to the equation (7):

(6)

(7)

In the above equations, w₁ represents the body weight measured at 24 or 48 h after the first oral administration, and w₀ represents the baseline body weight measured immediately before the first oral administration (0 h); w_h and w_b are the heart weight (mg) and body weight (g), respectively.

Biochemical Assessments

Blood samples were centrifuged at 4000 rpm for 10 min after they were allowed to stand for 1–2 h, and the upper serum was stored at −20 °C until analysis. The levels of creatine kinase (CK), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST) in serum were estimated using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

To assess oxidative stress, heart tissue was removed from the animal and quickly rinsed in ice-cold saline and homogenized in phosphate buffer (pH 7.4). The homogenates were centrifuged at 10,000 rpm for 15 min at 4 °C, and the supernatants were used for biochemical analysis. The superoxide dismutase (SOD) activity, catalase (CAT) activity and the malondialdehyde (MDA) levels were detected using commercial kits (Jiancheng Biological Engineering Institute, Nanjing, China).

Histology-H&E Staining

The heart tissues were fixed in 4% paraformaldehyde solution. After fixation, the samples were dehydrated with a graded series of alcohol and embedded in paraffin blocks. The samples were sectioned at 4 µm thickness using a microtome and placed on glass slides. The sections were stained with hematoxylin and eosin (H&E) to visualize general tissue architecture. Tissue sections were observed and photographed with a microscope (Leica MD 1000, Wetzlar, Germany).

Histology-Masson Staining

Using a Masson trichrome staining kit (Leagene DC0032, Beijing, China), the following techniques were applied to the 4-µm thick paraffin sections obtained as described above: After being baked at 65 °C for 30 min, the slides were deparaffinized using xylene and then rehydrated through a series of ethanol solutions. The sections were stained with Weigert’s hematoxylin solution for 5–10 min, followed by differentiation with acidic ethanol differentiation solution for 5 s and washing with water. Masson blue solution was applied to return the sections to blue for 3 min, and then the samples were rinsed with distilled water for 1 min. The sections were dipped in eosin for 60 s, washed with weak acid working solution for 1 min, and then submerged in aniline blue for 1–2 min. The sections were rapidly dehydrated with 95% ethanol and then with absolute ethanol three times. After xylene clearing three times, the samples were sealed with neutral gum (Solarbio, Beijing, China). The sections were examined under a microscope (Olympus CX41, Olympus).

Determination of Infarction Area

The myocardial infarction area of rat heart was determined by TTC staining. The rat hearts were briefly placed at −20 °C for 15 min to facilitate uniform sectioning and then cut into five transverse slices (2 mm) perpendicular to the long axis of the heart. All five slices from each heart were included in the quantitative analysis. The slices were exposed to 2% TTC stain for 25 min at 37 °C, and then fixed with 4% paraformaldehyde for 24 h. For each heart, the infarct size was determined using ImageJ Software (National Institutes of Health) as the percentage of the pale infarcted area relative to the total myocardial area.

Results and Discussion

Characterization of R8-F127-VES

Previously, we reported the successful synthesis of F127-VES,⁴⁹ which established the foundational methodology for its conjugation. To confirm the successful grafting of the R8 peptide onto the F127-VES backbone, FT-IR and ¹H-NMR spectroscopy were employed.

FT-IR

Figure 2A demonstrates the FT-IR spectra of the R8 peptide, F127-VES, and R8-F127-VES. R8 is an amide-bond-rich peptide; the spectrum of R8 shows a strong characteristic absorption peak at 1674 cm⁻¹ corresponding to the C=O stretching vibration of the amide bond. For R8-F127-VES, a distinct peak appeared at 1707 cm⁻¹. This peak is attributed to the carbonyl (C=O) stretching vibration associated with the succinate linkage, and its enhanced intensity suggests successful conjugation of R8 to the F127-VES polymer. A weak peak was initially present in the F127-VES at 1703 cm⁻¹, which is the characteristic peak of the ester bond in vitamin E succinate. The change in the carbonyl-related spectral feature of R8-F127-VES relative to F127-VES was consistent with the formation of the conjugate.

Figure 2.

(A) FT-IR spectra and (B) ¹H-NMR analysis of R8-F127-VES.

¹H-NMR

To further confirm the successful synthesis of the conjugate, ¹H-NMR spectroscopy was performed. Figure 2B demonstrates the ¹H-NMR spectra of the R8 peptide, F127-VES and R8-F127-VES. The spectra of the R8 and R8-F127-VES show a broad peak between δ = 7.2–7.4 ppm, which is assigned to downfield N–H protons associated with the arginine residues (amide/guanidinium). The resonant signals between δ = 7.6–7.9 ppm correspond to the repeating amide (-NH) protons within the R8 backbone. Notably, the α-proton (-CH) signal from the R8 structural unit was observed in the δ = 4.0–4.5 ppm region, a finding consistent with other studies on arginine-rich peptides and their conjugates.⁵⁶ At the same time, signals corresponding to the VES component were identified, with the methylene protons (-CH₂) at approximately 2.9 ppm and the methyl protons (-CH₃) at 2.011 ppm. The characteristic repeating units of the Pluronic F127 block copolymer were also clearly discernible, with the methylene (-CH₂) and methine (-CH) protons of the polyethylene oxide (PEO) and polypropylene oxide (PPO) blocks appearing around δ = 3.3 ppm and δ = 3.5 ppm, respectively. Both FT-IR and ¹H-NMR confirmed the successful synthesis of R8-F127-VES.

Nanocrystal Preparation and Characterization

PS, PDI and ZP

The PS, PDI, and ZP of different BRE nanocrystal formulations are summarized in Table 1. The PS of the BRE-NCs was (229.9 ± 1.320) nm. After modification with F127-VES, the PS of F127-VES@BRE-NCs increased to (271.7 ± 1.858) nm. The increase is due to the formation of a hydrated polymer layer on the nanocrystal surface. After modification with R8-F127-VES, the PS of R8-F127-VES@BRE-NCs increased to (319.2 ± 2.893) nm. The successful conjugation of R8 to the F127-VES is further supported by the increase in PS, which reflects further thickening of the surface layer. The PS of all formulations was around 300 nm, which is considered desirable for oral drug delivery, as the dissolution rate of poorly soluble drugs increases with increasing surface area.

Table 1.

The PS, PDI and ZP of Different BRE Nanocrystal Formulations (n = 3, Mean ± SD)

Formulations	Z-Average (d.nm)	PDI	Zeta Potential (mV)
BRE-NCs	229.9 ± 1.320	0.240 ± 0.013	−32.4 ± 0.346
F127-VES@BRE-NCs	271.7 ± 1.858	0.242 ± 0.007	−25.7 ± 0.153
R8-F127-VES@BRE-NCs	319.2 ± 2.893	0.236 ± 0.013	−16.1 ± 0.361

The PDI values for BRE-NCs, F127-VES@BRE-NCs, and R8-F127-VES@BRE-NCs were 0.240 ± 0.013, 0.242 ± 0.007, and 0.236 ± 0.013, respectively. PDI values were all less than 0.3, indicating a narrow and uniform particle size distribution of BRE nanocrystal formulations. It is noteworthy that the R8-F127-VES@BRE-NCs showed the lowest PDI, suggesting that modification with R8-F127-VES may have contributed to a more uniform and stable colloidal system.

The ZP of the BRE-NCs was (−32.4 ± 0.346) mV. High zeta potential leads to increased electrostatic repulsion among nanoparticles, indicating strong stability.⁵⁷ However, the strong negative charge of BRE-NCs is an unfavourable factor for oral absorption. The gastrointestinal mucus layer is rich in negatively charged mucin glycoproteins, and a highly negative surface is unfavorable for interaction with the epithelial membrane and may increase non-specific interactions in the mucus environment.⁵⁸ After modification by F127-VES, the ZP of F127-VES@BRE-NCs decreased to (−25.7 ± 0.153) mV. Decreased ZP of F127-VES@BRE-NCs was attributed to the shielding effect of the neutral hydrophilic F127 polymer chains on the surface charge. After modification by R8-F127-VES, the ZP of R8-F127-VES@BRE-NCs decreased to (−16.1 ± 0.361) mV. The significant reduction of the negative charge is a direct result of the introduction of the R8 peptide, which is a well-known cationic cell-penetrating peptide rich in positively charged arginine residues.

Appearance and Morphology

The nanocrystal preparations were first characterized by their appearance and morphology. Figure 3A and B show the appearance of the nanocrystal suspensions and their corresponding lyophilized powders. Obvious sedimentation was rapidly observed in the BRE-raw dispersion, and the supernatant became clear; this is a typical appearance for poorly soluble drugs dispersed in water. In striking contrast, all nanocrystal formulations (BRE-NCs, F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs) appeared as homogeneous and opaque yellow nanosuspensions. To improve the long-term physical stability, the nanocrystal suspensions were converted into powders via lyophilization. As shown in Figure 3B, the BRE-raw powder was aggregated and dark yellow, while all nanocrystal powders appeared as pale yellow, light, and fluffy powders. This fluffy morphology is a crucial prerequisite for rapid and complete redispersion of the nanocrystals upon rehydration.

Figure 3.

Appearance and morphology of R8-F127-VES@BRE-NCs. (A) Appearance of the suspensions: (i) BRE-raw; (ii) BRE-NCs; (iii) F127-VES@BRE-NCs; (iv) R8-F127-VES@BRE-NCs. (B) Appearance of the powders: (i) BRE-raw; (ii) BRE-NCs; (iii) F127-VES@BRE-NCs; (iv) R8-F127-VES@BRE-NCs. (C) SEM images of BRE-raw and the freeze-dried formulations: (i) BRE-raw; (ii) BRE-NCs; (iii) F127-VES@BRE-NCs; (iv) R8-F127-VES@BRE-NCs. Scale bars are indicated in the images.

The morphology of the BRE-raw and NCs formulations was examined by scanning electron microscopy (SEM). Figure 3C (i) shows the SEM image of BRE-raw, which exhibits a typical needle-like or rod-like structure. Following nanonization and lyophilization, the original morphology was completely altered in all NCs formulations. The original needle-like morphology disappeared completely. Instead, the samples presented as a porous, irregular, and flocculent morphology. The observed flocculent structure is a typical and desirable morphology for a well-formulated lyophilized nanoparticle powder and the porous structure results from water sublimation during the freeze-drying process. The flocculent and porous structure of the NCs powders is beneficial for rapid wetting and rehydration.

Fourier Transform-Infrared Spectroscopy (FT-IR)

Fourier transform infrared spectroscopy (FT-IR) was performed to evaluate whether the crystalline structure of BRE was maintained in BRE-raw, BRE-NCs, F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs (Figure 4A). The FT-IR spectrum of the BRE-raw exhibited characteristic peaks of flavonoids. A strong and broad peak at 3374 cm⁻¹ was assigned to the O-H group, and the peaks at 1604 cm⁻¹, 1563 cm⁻¹, and 1496 cm⁻¹ were assigned to C=C groups.⁵⁹ The sharp peak at 1720 cm⁻¹ was related to the C=O group. In the FT-IR spectrum of the physical mixture, all the characteristic peaks of BRE were clearly identifiable, and the characteristic peaks of the excipients were also observed. These results confirmed that no significant chemical change occurred during the mixing process. Meanwhile, although the characteristic peaks of BRE were observed, only slight peak shifts were observed, and the characteristic BRE peaks were still retained in all NC formulations, which proved that the homogenization and lyophilization processes did not affect the crystalline structure of BRE.

Figure 4.

Characteristics of R8-F127-VES@BRE-NCs. (A) Fourier transform-infrared spectroscopy. (B) Powder X-ray diffraction patterns. (C) Saturation solubility, data were expressed as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test. Compared with BRE-raw, ****P<0.0001. (D) In vitro release evaluation data were expressed as mean ± SD (n = 3).

Powder X-Ray Diffraction (PXRD)

Powder X-ray diffraction (PXRD) was employed to further characterize the crystalline structure of BRE-raw, BRE-NCs, F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs (Figure 4B). The PXRD pattern of BRE-raw showed a series of sharp, high-intensity diffraction peaks at 16.1°, 25.9° and 26.8°, which confirms its highly crystalline nature. Likewise, the PXRD pattern of trehalose (used as a lyophilization cryoprotectant) shows a series of diffraction peaks at 13.5° and 23.8°, indicating that trehalose was in a crystalline state. In the PXRD pattern of the physical mixture, all the diffraction peaks of BRE were clearly identifiable, and the characteristic peaks of trehalose were also observed. The results confirmed that hardly any chemical change occurred during the mixing process. The PXRD patterns of all NC formulations displayed diffraction peaks at 13.5°, 16.1°, 23.8°, 25.9°, and 26.8°. These results indicate that BRE remained in the crystalline state in the NC formulations, suggesting that neither the homogenization process nor freeze-drying affected its crystallinity. These results are consistent with previous reports.⁶⁰

Saturation Solubility

Solubility is a key factor affecting oral absorption. The results of the saturation solubility assay are shown in Figure 4C. BRE-raw had poor aqueous solubility, and its saturation solubility was merely (18.70 ± 0.04) μg/mL, which is one of the main limiting factors that restrict clinical application. The saturation solubility of BRE-NCs was significantly increased to (133.02 ± 0.01) μg/mL, representing a 7.11-fold improvement over BRE-raw. The reason underlying this elevation can be explained by the principles of the Ostwald-Freundlich equation: Smaller particles have higher specific surface energy and thus greater solubility than larger ones.⁶¹

After surface modification of the NCs, the saturation solubility was further increased. The saturation solubility of F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs were (145.37 ± 1.02) μg/mL and (145.34 ± 0.97) μg/mL, respectively, representing 7.77-fold and 1.09-fold increases over BRE-raw and BRE-NCs, respectively. The slightly enhanced saturation solubility was attributed to the presence of the hydrophilic Pluronic F127 on the surface. F127 is a nonionic triblock copolymer comprised of two hydrophilic end blocks of polyethylene oxide (PEO) and a central hydrophobic block of polypropylene oxide (PPO). The amphiphilic nature of F127 endows it with surfactant properties, improving the solubilization of poorly soluble drugs. It is noteworthy that, compared with F127-VES@BRE-NCs, the saturation solubility of R8-F127-VES@BRE-NCs showed a non-significant increase. This result follows expectations, as promoting epithelium penetration and enhancing cellular uptake are the major functions of the R8 peptide.

In vitro Release Evaluation

The in vitro drug release behavior of the BRE-raw and the NCs formulations was examined, as shown in Figure 4D. BRE-raw exhibited a dissolution profile characteristic of its poor water solubility. The in vitro release rate of BRE was (35.23 ± 0.03) % at 1 h, and only approximately (37.01 ± 0.11) % at 4 h. At the same time, all NCs formulations showed significantly higher drug release rates compared with BRE-raw. The in vitro release rate of BRE-NCs was (68.94 ± 0.06) % at 1 h, which was 1.96-fold higher than BRE-raw. This phenomenon can be explained by the Noyes-Whitney equation, which states that drug dissolution is directly proportional to the surface area of the drug available for dissolution. The reduced particle size provides a larger surface area.⁶²

The in vitro release rate of F127-VES@BRE-NCs was (73.41 ± 0.01) % at 1 h, which was 1.06-fold higher than that of BRE-NCs. This is attributed to the amphiphilic nature of F127, which endows it with surfactant properties, thereby improving the solubilization of BRE. The in vitro release rate of R8-F127-VES@BRE-NCs was (73.45 ± 0.04) % at 1 h, which was not significantly different from that of F127-VES@BRE-NCs. As discussed in the previous section, promoting epithelial penetration and enhancing cellular uptake are the major functions of the R8 peptide. The results of this section were in accordance with the Saturation solubility experiment.

Stability

As shown in Figure 5A–C, all NC formulations exhibited excellent physical stability. No significant change was observed in either PS or PDI for any formulation during the 30-day storage period. All PDI values of the NC formulations remained below 0.3, which suggests that no irreversible aggregation, particle fusion or Ostwald ripening occurred in the lyophilized powder upon reconstitution. Furthermore, the surface properties and chemical integrity of the NCs formulations remained stable. The ZP values of BRE-NCs, F127-VES@BRE-NCs, and R8-F127-VES@BRE-NCs remained stable around their initial values. This confirms the firm binding and stability of the surface modification layers. Meanwhile, no significant change was observed in the drug concentration of all NC formulations, indicating good chemical stability (Figure 5D).

Figure 5.

Storage stability of BRE-NCs, F127-VES@BRE-NCs, and R8-F127-VES@BRE-NCs. (A) Particle size changes of NCs formulations. (B) PDI changes of NCs formulations. (C) Zeta potential changes of NCs formulations. (D) Concentration changes of NCs formulations. All data were expressed as mean ± SD (n = 3).

Ensuring long-term stability is a key goal in drug research and development, which remains a major challenge for liquid nanosuspensions due to their physical (eg, Ostwald ripening and agglomeration) and chemical (eg, hydrolysis) instability. Thus, transformation of liquid-state nanocrystals into the solid state is necessary. Our experimental results further confirm that the solidification of NCs can provide better stability, which is a critical prerequisite for the further development of this formulation as an ideal drug delivery system for oral administration.

In vitro Mucus Penetration Evaluation

Interaction Between NC Formulations and Mucin

In order to evaluate the interaction between NCs formulations and mucin, PS and PDI measurements of the mucin-NC dispersions were conducted, and the changes in particle size and PDI of mucin-NC dispersions are shown in Figure 6A and B, respectively. The BRE-raw exhibited significant size fluctuations when mixed with mucin. This instability originates from the hydrophobicity of BRE. At the initial stage, partial disruption of the aggregates occurred as a result of stirring and hydration, leading to a temporary reduction in size. However, the inherent hydrophobicity of BRE could promote strong hydrophobic interactions with the hydrophobic domains of the mucin glycoprotein.⁶³ Consequently, unstable large aggregates were formed, resulting in an increase in particle size accompanied by a high PDI value.

Figure 6.

In vitro mucus penetration evaluation of BRE-raw and NC formulations. (A) Particle size changes and (B) PDI changes after incubation with mucin for 0–4h. Data were expressed as mean ± SD (n = 3). (C) Schematic illustration of the PGM-coated Transwell mucus barrier model. The red arrow indicates the direction of transport from the apical (donor) chamber through the PGM mucus layer and porous membrane toward the basolateral (acceptor) chamber. (D) Apparent permeability coefficients across the mucus layer. Data were expressed as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test. (compared with BRE-raw, *: p < 0.05).

Meanwhile, the PS and PDI values of BRE-NCs, F127-VES@BRE-NCs, and R8-F127-VES@BRE-NCs remained stable, which suggests that preparing BRE as nanocrystals increased its apparent solubility and thereby weakened its interaction with mucus. This stability, particularly for the R8-modified formulation, is attributed to the construction of an engineered “mucus-inert surface”. Although cationic peptides such as R8 are typically prone to electrostatic interaction with anionic mucins, the hydrophilic PEO segments of the F127 corona form a dense, neutral hydration layer. This layer effectively shields the positive charge of the inner R8 moieties through steric hindrance, thereby preventing non-specific adhesion and maintaining colloidal stability in the mucus environment.

Mucus Diffusion Analysis

Foreign particles can be trapped and cleared by the mucus covering the intestinal epithelial surface. Rapid penetration through mucus opens significant opportunities for drug absorption by epithelium.^64,65 PGM was spread on the Transwell membrane to explore the mucus permeability of the BRE-raw, BRE-NCs, F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs, and the apparent permeability coefficient (P_app) value was calculated. The schematic illustration of the mucus barrier model is shown in Figure 6C, and the results of the mucus diffusion analysis are presented in Figure 6D. The BRE-raw exhibited the lowest P_app with (5.46 ± 0.39) × 10⁻⁶ cm/s. The P_app of BRE-NCs was (6.90 ± 0.40) × 10⁻⁶ cm/s, which was slightly higher than that of BRE-raw. This result was attributed to the significantly enhanced dissolution of nanocrystals, which generated a larger concentration gradient and thereby promoted mucus diffusion. However, no significant difference was detected between BRE-raw and BRE-NCs, suggesting that unmodified nanocrystals have limited ability to penetrate the mucus.

The mucus-penetrating ability of NCs is significantly enhanced after modification with F127-VES and R8-F127-VES. The P_app of F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs was (7.50 ± 0.5) × 10⁻⁶ cm/s and (8.02 ± 0.5) × 10⁻⁶ cm/s, respectively, demonstrating a significant difference compared to the BRE-raw. The increase in P_app was due to the presence of F127, which may facilitate adsorption of NCs onto the surface through the hydrophobic PPO chain, while the PEO chains extend outward to form a mucus-inert surface coating. The mucus-inert surface coating effectively minimized the hydrophobic and electrostatic interactions that cause unmodified particles to adhere to the anionic mucin network.⁶⁶ It is noteworthy that the P_app of R8-F127-VES@BRE-NCs was the highest, although the glycosylated domains of mucins are highly negatively charged, which can lead to electrostatic interactions with, and trapping of, positively charged nanoparticles (such as R8). This result suggests that the F127 corona may reduce nonspecific interactions between R8 residues and mucin, thereby facilitating the mucus diffusion of R8-F127-VES@BRE-NCs. This efficient mucus penetration serves as the first crucial step in our sequential barrier-crossing strategy, facilitating subsequent access of the drug to the underlying epithelium.⁶⁷

Cellular Experiments

In vitro Cytotoxicity

Caco-2 cells are a widely used model for predicting intestinal drug penetration in vitro. Meanwhile, it is vital to assess the safety of the formulations. Figure 7A displays the percent viability of Caco-2 cells treated with different BRE concentrations (10, 20, 40, 60, 100, and 200 µg/mL) from BRE-raw, BRE-NCs, F127-VES@BRE-NCs, and R8-F127-VES@BRE-NCs after incubation for 4 h. The results revealed that none of the groups exhibited any cytotoxicity to Caco-2 cells during the 4 h incubation, even at a concentration of 200 µg/mL (all inhibition rates were less than 10%). There were no significant differences among BRE-raw, BRE-NCs, F127-VES@BRE-NCs, and R8-F127-VES@BRE-NCs compared to the control group. These results suggest that the formulations showed no obvious short-term cytotoxicity to Caco-2 cells under the tested 4 h exposure conditions. Further studies are warranted to establish the long-term safety profile of R8-F127-VES.

Figure 7.

(A) The effect of different concentrations of BRE-raw, BRE-NCs, F127-VES@BRE-NCs, R8-F127-VES@BRE-NCs on Caco-2 cell viability data were expressed as mean ± SD (n = 3). (B) Cellular uptake of BRE-raw, BRE-NCs, F127-VES@BRE-NCs, R8-F127-VES@BRE-NCs data were expressed as mean ± SD (n = 3). (C) Cell uptake mechanisms of BRE-raw, BRE-NCs, F127-VES@BRE-NCs, R8-F127-VES@BRE-NCs data were expressed as mean ± SD (n = 3) (I: Chlorpromazine, II: Colchicine, III: Verapamil, IV: Indomethacin) (compared with the corresponding formulation without inhibitor, &&&: p<0.001, &&&&: p<0.0001; compared to the BRE-raw, *: p<0.05, **: p<0.01, ****: p<0.0001; compared to the R8-F127-VES@BRE-NCs, ##: p<0.01, ###: p<0.001, ####: p<0.0001) (D) Schematic illustration of the Caco-2 cell monolayer Transwell model. The red arrow indicates the direction of transepithelial transport from the apical (AP) chamber across the Caco-2 cell monolayer toward the basolateral (BL) chamber. (E) The apparent permeability (P_app) of BRE-raw, BRE-NCs, F127-VES@BRE-NCs, and R8-F127-VES@BRE-NCs transport across cells data were expressed as mean ± SD (n = 3). (F) The TEER values of Caco-2 monolayers treated by NCs before and after treatment data were expressed as mean ± SD (n = 3). (G) Apparent permeability coefficient of BRE-raw, BRE-NCs, F127-VES@BRE-NCs, R8-F127-VES@BRE-NCs in duodenum and jejunum. Data were expressed as mean ± SD (n = 6) (H) Intestinal absorption rate of BRE-raw, BRE-NCs, F127-VES@BRE-NCs, R8-F127-VES@BRE-NCs in duodenum and jejunum data were expressed as mean ± SD (n = 6). (I) Blood concentration-time profiles of BRE-raw, BRE-NCs, F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs at a Dose of 20 mg/kg in SD Rats data were expressed as mean ± SD (n = 6) (compared with BRE-raw, *: p < 0.05, **: p < 0.01, ****: p < 0.0001; compared with R8-F127-VES@BRE-NCs, ###: p < 0.001, ####: p < 0.0001). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test for (B and E); two-way ANOVA followed by Dunnett’s multiple comparisons test for (C); two-way repeated-measures ANOVA for (F); and two-way ANOVA followed by Tukey’s multiple comparisons test for (G and H).

Cellular Uptake and Mechanisms

In this research, in order to further determine the effect of R8-F127-VES@BRE-NCs on enhancing BRE uptake into Caco-2 cells, a cellular uptake study was performed (Figure 7B). The cellular uptake of BRE-raw was (54.84 ± 1.07) %. The cellular uptake of BRE-NCs was significantly increased to (63.03 ± 1.24) %, which was 1.15-fold higher than the BRE-raw (p < 0.0001). This improvement is attributed to nanocrystal technology, which increases the surface area by decreasing the particle size. According to the Noyes-Whitney and Ostwald-Freundlich equations, the increased surface area contributes to enhanced saturation solubility and an accelerated dissolution rate of BRE in the aqueous microenvironment. Indeed, previous studies have shown that,⁶⁸ Caco-2 cell model studies have consistently demonstrated that the dominant pathway for nanocrystal-mediated oral absorption enhancement is accelerated dissolution rate due to increased surface area, rather than direct endocytic uptake of the particulate form.

The cellular uptake of F127-VES@BRE-NCs was significantly increased to (81.39 ± 0.74) %, which was 1.48-fold higher than the BRE-raw and 1.29-fold higher than the BRE-NCs (p < 0.0001). This suggests a change in the transport mechanism of F127-VES@BRE-NCs, involving enhanced epithelial permeability and efflux modulation. As outlined in the previous paragraphs, we propose that the improvement is due to dual effects on both the F127 and VES. F127 may increase the probability of nanoparticle contact with the epithelial surface and, as a permeability enhancer, can increase membrane fluidity and transiently regulate tight junction integrity. VES may contribute to modulation of efflux-associated drug loss.

Surface modification of nanocarriers can enhance cellular uptake. For example, Reddy et al⁶⁹ developed transferrin-decorated PLGA nanoparticles for possible targeting of transferrin receptors and demonstrated effective cellular uptake of the transferrin-coated nanoparticles in RAW264.7 cells. In contrast, octaarginine (R8) is a representative cell-penetrating peptide, and the present findings support its role in promoting cellular interaction and endocytosis-associated uptake rather than a receptor-specific internalization process. R8-F127-VES@BRE-NCs achieved the highest cellular uptake with (90.06 ± 0.36) %, which was 1.64-fold higher than the BRE-raw and represents a statistically significant difference compared to F127-VES@BRE-NCs (p < 0.0001). This enhanced cellular uptake is consistent with the intended integrated design of R8-F127-VES@BRE-NCs for addressing multiple barriers to oral drug absorption.

Specific endocytosis inhibitors were used to investigate the uptake mechanisms of the formulations in Caco-2 cells. Chlorpromazine, which blocks the assembly of clathrin at the cell membrane, was used to inhibit clathrin-mediated endocytosis (CME). Colchicine, which decreases microtubule polymerization, was used to inhibit macropinocytosis. Verapamil was used as a P-gp inhibitor. Indomethacin was used as a MRP2 inhibitor. After intervention with these inhibitors, the results are shown in Figure 7C. All inhibitors failed to significantly affect the cellular uptake of BRE-raw and BRE-NCs; this result is consistent with the hypothesis suggested by the cellular uptake results. The uptake of both BRE-raw and BRE-NCs may not substantially depend on an energy-dependent endocytic pathway, but rather may predominantly involve passive diffusion, and the increased cellular uptake of BRE-NCs may mainly result from improved solubility.

The cellular uptake of R8-F127-VES@BRE-NCs decreased significantly after intervention with chlorpromazine and colchicine, suggesting that clathrin-mediated endocytosis and macropinocytosis-associated processes may contribute to its cellular uptake. The cellular uptake of both F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs showed no significant change after treatment with verapamil. This finding is consistent with modulation of P-gp efflux in the VES-containing formulations, although complete inhibition or saturation of P-gp cannot be concluded from the present data. Indomethacin is a MRP2 inhibitor and is also a common inhibitor of cyclooxygenase (COX).⁷⁰ The results showed that the cellular uptake of both F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs exhibited a small but significant decrease. Given that inhibition of an efflux pump (such as MRP) usually increases or does not change intracellular accumulation, the decreased accumulation observed could originate from the off-target inhibition of COX-dependent endocytic pathways by indomethacin, rather than an interaction with MRP.

Cellular Transport and Evaluation of Caco-2 Cell Monolayer Integrity

The construction of the Caco-2 cell monolayer in the Transwell system is schematically illustrated in Figure 7D. The measured P_app values reflect the transport behavior of the formulations, and the results are shown in Figure 7E. The BRE-raw exhibited the lowest P_app value of (1.15 ± 0.14) × 10⁻⁵ cm/s, which was consistent with its P-gp substrate properties and poor lipid solubility characteristics.¹⁶ The P_app value of BRE-NCs was (1.53 ± 0.02) × 10⁻⁵ cm/s, which was 1.33-fold higher than that of BRE-raw. This improvement is attributed to the nanocrystal technology, which increases the solubility of BRE by decreasing the particle size. The P_app value of F127-VES@BRE-NCs was (1.62 ± 0.05) × 10⁻⁵ cm/s, which was 1.41-fold higher than that of BRE-raw. This increase may be partly attributed to VES-mediated modulation of P-gp efflux. R8-F127-VES@BRE-NCs exhibited the highest P_app value of (2.24 ± 0.16) × 10⁻⁵ cm/s, which was 1.95-fold higher than that of BRE-raw and significantly higher than that of BRE-NCs (p < 0.0001). The further increase in P_app is consistent with a contribution of R8 modification to enhanced epithelial transport. As shown in Figure 7F, the TEER values did not change significantly before and after treatment with different formulations (BRE-raw, BRE-NCs, F127-VES@BRE-NCs, and R8-F127-VES@BRE-NCs), indicating that none of the formulations disrupted the monolayer barrier.

Single-Pass Intestinal Perfusion Study

The intestinal absorption of the different BRE formulations was evaluated using the single-pass intestinal perfusion method. The absorption rate constants (K_a) and apparent permeability coefficients (P_app) obtained from the single-pass intestinal perfusion method are presented in Figure 7G and H. The P_app of BRE-raw in the jejunum was (0.66 ± 0.24) × 10⁻² cm/min, which confirms the intrinsically low intestinal permeability of BRE. Specifically, the overexpression of MRP2 and P-gp in the distal small intestine facilitates the active secretion of glucuronide conjugates such as BRE back into the lumen. Compared to BRE-raw, the absorption of BRE-NCs was significantly improved, demonstrating that the solubility of BRE was enhanced by decreasing the particle size. The P_app values of F127-VES@BRE-NCs in the duodenum and jejunum were increased to (1.80 ± 0.16) × 10⁻² cm/min and (1.82 ± 0.24) × 10⁻² cm/min, respectively. This increase may be partly attributed to VES-mediated modulation of transporter-mediated efflux. The highest absorption was observed for R8-F127-VES@BRE-NCs, and the P_app values in the duodenum and jejunum were (2.90 ± 0.45) × 10⁻² cm/min and (3.15 ± 0.51) × 10⁻² cm/min, respectively. These results further support a contribution of R8 modification to enhanced intestinal transport.

In vivo Pharmacokinetic Study

The pharmacokinetics of BRE-raw, BRE-NCs, F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs following oral administration at a dose of 20 mg/kg were investigated in SD rats. The blood concentration-time profiles of BRE-raw, BRE-NCs, F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs are shown in Figure 7I, and the pharmacokinetic parameters are shown in Table 2. The blood concentration-time profiles of BRE-raw were relatively flat, with a low C_max (43.51 ± 9.17) ng/mL and limited AUC_0-∞ (192.48 ± 53.19) ng/mL · h. This poor oral absorption is primarily attributed to the intrinsic hydrophobicity of the BRE, which severely restricts its dissolution rate in the gastrointestinal tract. The AUC_0-∞ of the BRE-NCs increased to (555.86 ± 72.16) ng/mL · h, which was 2.89-fold higher compared to the BRE-raw. These results demonstrated that the solubility of BRE was enhanced by decreasing the particle size.

Table 2.

Pharmacokinetic Parameters of BRE-Raw, BRE-NCs, F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs at a Dose of 20 mg/kg in SD Rats (n = 6, Mean ± SD)

Parameters	Unit	BRE-Raw	BRE-NCs	F127-VES @BRE-NCs	R8-F127-VES @BRE-NCs
AUC0-∞	ng/mL · h	192.483 ± 53.187	555.857 ± 72.164*	585.554 ± 184.70*	1418.344 ± 142.669***
Cmax	ng/mL	43.508 ± 9.174	76.606 ± 8.000	101.188 ± 8.260*	185.759 ± 21.496***
Tmax	h	1.000 ± 0.354	1.000 ± 0.353	1.000 ± 1.414	2.000 ± 0.707
t1/2	h	3.440 ± 0.262	7.029 ± 2.500	9.376 ± 1.933	10.372 ± 1.928
MRT0-∞	h	4.844 ± 0.517	8.977 ± 3.252	10.193 ± 3.055	10.910 ± 2.323

Notes: Compared to BRE-raw *P < 0.05, ***P < 0.001.

After modification with F127-VES, the C_max of F127-VES@BRE-NCs increased to (101.19 ± 8.26) ng/mL and the AUC_0-∞ was increased to (585.55 ± 184.70) ng/mL · h. This is attributed to the synergistic effects of F127 and VES. The hydrophilic polyethylene oxide (PEO) corona formed by F127 is known to provide steric stabilization and facilitate the diffusion of nanoparticles through the mucus barrier by minimizing mucoadhesive interactions.⁷¹ The increased oral absorption observed for F127-VES@BRE-NCs is consistent with the combined effects of the surface modification on mucus diffusion and intestinal transport, with efflux modulation potentially contributing to the observed absorption enhancement. R8-F127-VES@BRE-NCs showed the highest C_max and AUC_0–∞ among all formulations. The C_max of the R8-F127-VES@BRE-NCs was (185.759 ± 21.496) ng/mL, which was 4.27-fold higher compared to the BRE-raw and 1.84-fold higher compared to the F127-VES@BRE-NCs. The AUC_0-∞ of the R8-F127-VES@BRE-NCs was (1418.344 ± 142.669) ng/mL · h, which was 7.37-fold higher compared to the BRE-raw and 2.42-fold compared to the F127-VES@BRE-NCs. The further increases in C_max and AUC_0–∞ observed for R8-F127-VES@BRE-NCs are consistent with a contribution of R8 modification to enhanced intestinal absorption.

The pronounced increase in AUC should not be interpreted as the linear consequence of a single in vitro barrier-related parameter, but rather as the cumulative outcome of improved dissolution, mucus diffusion, epithelial interaction, and efflux modulation-associated retention during oral absorption. The relative contribution of each module was further interpreted using a stepwise formulation comparison. The comparison between BRE-raw and BRE-NCs mainly reflects the contribution of nanocrystal-mediated dissolution enhancement; that between BRE-NCs and F127-VES@BRE-NCs reflects the combined contribution of F127/VES surface modification; and that between F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs reflects the additional contribution of R8-associated epithelial interaction. The increase in AUC was most pronounced following R8 modification, suggesting that enhanced epithelial interaction and uptake contributed substantially to the overall improvement in oral exposure. Because these modules are functionally interdependent, this analysis should be interpreted as a semi-quantitative comparison rather than an exact weighting of their independent contributions. However, because these barrier-related processes were evaluated in separate experimental models rather than directly tracked in a single integrated system, their proposed sequential relationship should be regarded as a mechanistic interpretation supported by complementary evidence rather than as a directly demonstrated temporal process.

Pharmacodynamic Studies

The Rate of Weight Change and Cardiac Index

The pharmacodynamic effects of the different formulations were evaluated by the rate of weight change and cardiac index. According to Figure 8A, the weight of the control group displayed a persistent elevation during the 48-h observation period. In contrast, the weight of the ISO group decreased markedly. The observed weight loss in the ISO group may be attributed to the activation of β-adrenoceptors, leading to decreased plasma leptin levels, potentially mediated by an increase in cyclic AMP (cAMP) within the adipose tissue. Catecholamines are significant lipolytic hormones, primarily acting through β-adrenoceptors.⁷² The weight loss was effectively alleviated by oral administration of the various breviscapine formulations. Compared with the ISO group, a slight improvement in weight loss was observed in the BRE-raw group due to the poor solubility and limited absorption of the BRE. The BRE-NCs group exhibited a moderate improvement of the weight loss. The improvement in weight loss was most pronounced in the F127-VES@BRE-NCs and R8-F127-VES@BRE-NCs groups, among which the R8-F127-VES@BRE-NCs showed the greatest improvement. In addition to reflecting the systemic impact of ISO-induced myocardial injury, body weight changes also provided a general observation of short-term tolerability during the pharmacodynamic study.

Figure 8.

(A) The rate of weight change in rats data were expressed as mean ± SD (n = 6). (B) The cardiac index of different groups data were expressed as mean ± SD (n = 6). Activity of (C) CK (D) LDH (E) AST in rat serum data were expressed as mean ± SD (n = 6). Activity of (F) SOD (G) MDA (H) CAT in rat tissues data were expressed as mean ± SD (n = 6). For (B–H), statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. (compared with the control group, &&&&: p< 0.0001; compared with the ISO group, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001; compared with the R8-F127-VES@BRE-NCs group, #: p < 0.05, ###: p < 0.001, ####: p < 0.0001).

The cardiac index is usually used to evaluate the degree of hypertrophy. According to Figure 8B, the cardiac index of the control group was (2.44 ± 0.15) mg/g. In contrast, the cardiac index of the ISO group increased to (4.12 ± 0.14) mg/g. This result confirms the successful establishment of the MI model. The positive control group (Metoprolol) effectively decreased the cardiac index to (3.33 ± 0.10) mg/g, which verifies its usefulness as the positive control drug for MI. The smallest reduction in cardiac index was observed in the BRE-raw group ((3.81 ± 0.10) mg/g), followed by BRE-NCs ((3.58 ± 0.07) mg/g). After modification of F127-VES, the cardiac index of F127-VES@BRE-NCs further decreased to (3.45 ± 0.08) mg/g. Crucially, R8-F127-VES@BRE-NCs resulted in the lowest cardiac index among the BRE formulation groups, at (3.34 ± 0.14) mg/g, which suggests the most pronounced cardioprotective effect among these formulations.

Biochemical Assessments

CK, LDH, and AST are important serum cardiac biomarkers for evaluating myocardial injury. As shown in Figure 8C–E, the ISO group exhibited significantly increased serum levels of CK, LDH, and AST, which are clear indicators of myocardial injury severity. When the myocardium is deprived of oxygen and nutrients, cardiomyocytes are damaged, which may lead to disruption of the cell membrane and leakage of cardiac enzymes into the blood.⁷³ The positive control group showed a significant decrease in the serum levels of CK, LDH, and AST, demonstrating the expected cardioprotective efficacy of metoprolol in MI. Compared with the ISO group, the activities of LDH, CK, and AST in the serum of the BRE-raw, BRE-NCs, F127-VES@BRE-NCs, and R8-F127-VES@BRE-NCs groups were reduced. These results suggest a positive effect of BRE on MI, although its efficacy is limited by poor solubility. Compared to the BRE-raw group, the activities of LDH, CK, and AST in the serum of the R8-F127-VES@BRE-NCs group were significantly reduced, indicating that R8-F127-VES@BRE-NCs inhibited the abnormal elevation of serum cardiac enzymes in ISO-induced rats.

Figure 8F–H shows the oxidative stress status in myocardial tissue. Compared with the control group, the ISO group exhibited a severe imbalance in redox homeostasis, with sharply reduced SOD and CAT activities and significantly increased MDA levels. These biochemical changes confirm the successful establishment of the MI model, reflecting severe oxidative damage and the depletion of enzymatic antioxidant reserves. Compared with the ISO group, the MDA levels in the tissues of the BRE-raw, BRE-NCs, F127-VES@BRE-NCs, and R8-F127-VES@BRE-NCs groups were reduced. Notably, the R8-F127-VES@BRE-NCs group showed the strongest inhibition of lipid peroxidation, which suggests that it effectively alleviated the oxidative attack on membrane polyunsaturated fatty acids, thereby helping to maintain the membrane integrity.⁷³ At the same time, the R8-F127-VES@BRE-NCs group greatly restored the activity of SOD and CAT. This suggests that the endogenous antioxidant defense system was improved.

Histology-H&E Staining

H&E staining was conducted to observe representative histopathological changes in myocardial tissues, and the results are shown in Figure 9A. Compared with the control group, the ISO group showed disordered myofibrils, interstitial oedema, and inflammatory cell infiltration in myocardial tissue. The BRE-raw group showed disrupted myocardial fiber architecture and inflammatory cell infiltration. The BRE-NCs group showed relatively better-preserved myocardial architecture and less apparent interstitial oedema than the BRE-raw group. The F127-VES@BRE-NCs group showed relatively less apparent oedema and inflammatory cell infiltration. Among the BRE formulation-treated groups, R8-F127-VES@BRE-NCs showed relatively well-arranged myofibrils, with less apparent interstitial oedema and inflammatory cell infiltration. These H&E images are presented as representative histopathological observations rather than quantitative histological endpoints.

Figure 9.

(A) H&E staining of the (i) control group, (ii) ISO group, (iii) positive control group (metoprolol), (iv) BRE-raw group, (v) BRE-NCs group, (vi) F127-VES@BRE-NCs group, and (vii) R8-F127-VES@BRE-NCs group; scale bars: 50 μm. (B) Masson staining of the (i) control group, (ii) ISO group, (iii) positive control group (metoprolol), (iv) BRE-raw group, (v) BRE-NCs group, (vi) F127-VES@BRE-NCs group, and (vii) R8-F127-VES@BRE-NCs group; scale bars: 50 μm. (C) Infarct size of different groups data were expressed as mean ± SD (n=6). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. (compared with the control group, &&&&: p< 0.0001; compared with the ISO group, ****: p < 0.0001; compared with the R8-F127-VES@BRE-NCs group, ##: p < 0.01, ####: p < 0.0001); (D) TTC staining of different groups.

Histology-Masson Staining

Masson staining was performed to observe representative collagen staining and myocardial morphology, and the results are shown in Figure 9B. Compared with the control group, the ISO group showed more apparent blue-stained fibrous areas accompanied by disruption of myocardial architecture. The BRE-raw group also showed apparent structural disruption and blue-stained fibrous areas. The BRE-NCs group showed visually less apparent blue staining than the ISO group. The F127-VES@BRE-NCs group showed less apparent blue-stained areas and relatively better-preserved myocardial architecture. Among the BRE formulation-treated groups, R8-F127-VES@BRE-NCs showed relatively limited interstitial blue staining and a more ordered myocardial structure. These Masson staining images are presented as representative histopathological observations and were not used for quantitative comparison of collagen deposition among groups.

Determination of Infarction Area

Normal cardiomyocytes contain dehydrogenase and can therefore be stained red by TTC. After MI, infarcted cardiomyocytes lose dehydrogenase activity, making TTC staining ineffective and causing the infarcted region to appear pale.⁷⁴ After treatment of MI model rats with different BRE formulations, TTC staining was performed, and the results are shown in Figure 9C and D. Compared with the control group, the ISO group exhibited a large pale infarcted region, with an infarct area of (61.6 ± 4.33) %. These findings were consistent with successful establishment of the ISO-induced myocardial injury model. Treatment with BRE-raw resulted in a modest reduction in the pale infarct area compared to the ISO group, but it remained at a relatively high level even at (54.33 ± 5.76) %. The BRE-NCs group showed a further reduction in the pale infarct area (47.31 ± 4.84) %. The F127-VES@BRE-NCs group exhibited a marked decrease in the pale infarct area compared to the BRE-NCs, with an infarct area of (42.24 ± 4.01) %. The R8-F127-VES@BRE-NCs group significantly reduced the infarct area to (31.09 ± 4.65) %.

The pharmacodynamic results revealed that BRE-raw exhibited a limited cardioprotective effect, which may be related to the poor solubility and low permeability of BRE and its consequently limited oral exposure. The cardioprotective effect was significantly enhanced after converting BRE into nanocrystals, which may be associated with the reduction in particle size and the resulting increases in surface area, saturation solubility, and dissolution of BRE in the gastrointestinal tract. A further enhancement of the cardioprotective effect was observed with F127-VES@BRE-NCs, which was consistent with the improved mucus diffusion and intestinal transport observed for this formulation. Specifically, F127 may facilitate the diffusion of nanocrystals through the intestinal mucus barrier, while VES may contribute to the modulation of transporter-mediated efflux. Most notably, R8-F127-VES@BRE-NCs exhibited the most pronounced cardioprotective effect among the BRE formulations, with improved myocardial histopathological features and the smallest infarct area. This further improvement may be associated with R8-mediated enhancement of epithelial interaction and intestinal absorption, as supported by the cellular uptake and transport results. Nevertheless, given that experimental myocardial infarction models reproduce different aspects of the human disease, the present pharmacodynamic findings should be interpreted as evidence of cardioprotection within the context of the isoprenaline-induced myocardial injury model.⁷⁵

Conclusion

In conclusion, this study developed R8-F127-VES@BRE-NCs as surface-engineered nanocrystals intended to address multiple oral absorption barriers for BRE. The experimental results demonstrated that the formulation increased saturation solubility, accelerated dissolution, enhanced mucus diffusion, increased cellular uptake, and improved oral exposure, as reflected by AUC and C_max, compared with BRE-raw. In an isoprenaline-induced myocardial injury rat model, R8-F127-VES@BRE-NCs alleviated myocardial injury, as evidenced by reduced infarct size on TTC staining, improved serum cardiac biomarkers, attenuated oxidative stress, and ameliorated histopathological changes. Separately, the single-dose pharmacokinetic study demonstrated enhanced systemic exposure of BRE following oral administration of R8-F127-VES@BRE-NCs.

The observed improvements in oral absorption may involve the combined contributions of F127-associated mucus diffusion, R8-associated epithelial interaction and endocytosis-associated uptake, and VES-associated efflux modulation. However, the sequential relationship among these processes remains a mechanistic interpretation based on complementary evidence rather than a directly visualized process. Future studies will focus on further quantitative characterization of the polymer conjugate, validation of the formulation in complementary myocardial injury models, long-term safety of the modified polymer, and scalability of the preparation process.

Abbreviations

BRE, breviscapine; MI, myocardial infarction; BCS, biopharmaceutics classification system; VES, vitamin E succinate; CVD, cardiovascular disease; NCs, nanocrystal suspensions; PEG, polyethylene glycol; VE, vitamin E; TPGS, D-α-tocopheryl polyethylene glycol 1000 succinate; CPPs, cell-penetrating peptides; R8-F127-VES, octaarginine R8 - Pluronic F127 - vitamin E succinate; PS, particle size; ZP, zeta potential; SEM, scanning electron microscopy; FT-IR, fourier transform infrared spectroscopy; PXRD, Powder X-ray diffraction; PGM, porcine gastric mucin; RPMI 1640, roswell park memorial institute 1640 medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; DMEM, Dulbecco’s modified Eagle medium; TEER, transepithelial electrical resistance; AP, apical; BL, basolateral; P_app, apparent permeability coefficient; SD, Sprague-Dawley; K_a, absorption rate constant; MRM, multiple-reaction monitoring; AUC_(0-∞), area under the concentration-time curve from time zero to infinity; C_max, peak concentration; T_max, time of maximum concentration; t_1/2, elimination half-life; MRT, mean residence time; ISO, isoprenaline; CK, creatine kinase; LDH, lactate dehydrogenase; AST, aspartate aminotransferase; SOD, superoxide dismutase; CAT, catalase; MDA, malondialdehyde; H&E, hematoxylin and eosin; PEO, polyethylene oxide; PPO, polypropylene oxide; COX, cyclooxygenase; cAMP, cyclic AMP.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The author(s) report no conflicts of interest in this work.