Palmitic acid‐ and cysteine‐functionalized nanoparticles overcome mucus and epithelial barrier for oral delivery of drug

Abstract Nanoparticles (NPs) used for oral administration have greatly improved drug bioavailability and therapeutic efficacy. Nevertheless, NPs are limited by biological barriers, such as gastrointestinal degradation, mucus barrier, and epithelial barrier. To solve these problems, we developed the PA‐N‐2‐HACC‐Cys NPs loaded with anti‐inflammatory hydrophobic drug curcumin (CUR) (CUR@PA‐N‐2‐HACC‐Cys NPs) by self‐assembled amphiphilic polymer, composed of the N‐2‐Hydroxypropyl trimethyl ammonium chloride chitosan (N‐2‐HACC), hydrophobic palmitic acid (PA), and cysteine (Cys). After oral administration, the CUR@PA‐N‐2‐HACC‐Cys NPs had good stability and sustained release under gastrointestinal conditions, followed by adhering to the intestine to achieve drug mucosal delivery. Additionally, the NPs could penetrate mucus and epithelial barriers to promote cellular uptake. The CUR@PA‐N‐2‐HACC‐Cys NPs could open tight junctions between cells for transepithelial transport while striking a balance between mucus interaction and diffusion through mucus. Notably, the CUR@PA‐N‐2‐HACC‐Cys NPs improved the oral bioavailability of CUR, which remarkably relieved colitis symptoms and promoted mucosal epithelial repair. Our findings proved that the CUR@PA‐N‐2‐HACC‐Cys NPs had excellent biocompatibility, could overcome mucus and epithelial barriers, and had significant application prospects for oral delivery of the hydrophobic drugs.


| INTRODUCTION
Oral administration of drugs remains the most clinically practical route of administration due to its significance in improving patient comfort and compliance. 1 However, the solubility and permeability of the drug affect the efficiency of drug absorption. 2 Nanoparticle (NP)-based drug delivery system (DDS) is developing as potential carriers to improve drug delivery and absorption. 3 NPs function not only to protect drugs from enzymatic degradation and reduce side effects, but also significantly improve the oral bioavailability and treatment efficacy of drugs. [4][5][6] NP-based DDS need to pass through two physical barriers before entering systemic circulation. The first barrier is the mucus layers secreted by goblet cells, 7 and the second is cellular uptake and transport across epithelial cells. 8 As intestinal mucus is a strong barrier against foreign pathogens, 9 it affected the efficacy of the oral delivery of NPs. Various strategies based on the hydrophilicity, shape, particle size, charge, and rigidity have been investigated to facilitate NP mucus penetration and mucoadhesive. 10 NPs with neutral and hydrophilic surface properties exhibit good mucus permeability to overcome the mucus barrier, 11 but hydrophilic/neutral surfaces also decrease the interaction with the cell membrane. 12 In contrast, positively charged NPs can increase the residence time of drugs at absorption sites and facilitate cellular uptake through electrostatic interactions with negatively charged mucins and cell membranes. 13 Notably, in addition to enhancing the mucoadhesion of NPs to prolong intestinal residence time, the balance between interaction with the mucus and diffusion through mucus should be considered to prevent the strong adhesion from removing the embedded NPs. 14 However, even if the NPs successfully pass through the mucin steric barrier, epithelial cells can act as a physical barrier to hinder the uptake of the drug-loaded NPs. 15,16 The epithelial barrier includes intestinal epithelium and cell junctions. 17 It has been reported that NPs designed with a positively charged surface could enhance the uptake by epithelial cells, 18 and hydrophobic surfaces are also preferable for efficient cellular internalization. 12 Moreover, strategies of opening the tight junction (TJ) to facilitate transepithelial transport have also been widely studied to overcome epithelial barriers. 19,20 It is challenging to develop an oral DDS that can effectively overcome mucus and epithelial barriers. In recent years, much attention has been paid to cationic chitosan due to its excellent physicochemical properties, such as good biocompatibility, biodegradability, and mucoadhesion. 21,22 Furthermore, nano-carriers developed using chitosan and its derivatives can reversibly open the epithelial TJ to promote intestinal drug absorption via transcellular and paracellular pathways. 23 The poor solubility of chitosan limits the application of chitosan. However, amino and hydroxyl groups on chitosan chains are employed as substrates for various chemical reactions, allowing them to improve the properties of the initial polymer and introduce specific properties, such as mucoadhesion, 24 antimicrobial activity, 25 and solubility. 26,27 N-2-Hydroxypropyl trimethyl ammonium chloride chitosan (N-2-HACC), a quaternized chitosan derivative, exhibits excellent aqueous solubility over a wide pH range. 28 Furthermore, due to the presence of quaternized amino groups, the N-2-HACC increases the residence time and drug concentration at absorption sites via strong electrostatic interaction with negatively charged mucins, 29 thus improving the oral bioavailability of the drug.
In addition to positively charged surfaces, hydrophobicity of material surface is also preferred for cellular internalization. 30 Amphiphilic polymers are macromolecular compounds containing hydrophilic and lipophilic segments. 31 The amphiphilic polymer containing two incompatible parts with different solubility will tend to irreversibly equilibrate via self-assembly in a selective solvent. 32 NPs based on amphiphilic polymers can facilitate transport across the epithelium due to their surface mimicking the cellular phospholipid membrane. 33 Palmitic acid (PA), a hexadecanoic saturated fatty acid, can be used as a hydrophobic chain donor to graft chitosan molecules, giving chitosan the amphiphilic structure and ability to self-assemble micelles. 34,35 In addition, many permeation studies have shown that the permeation effect of polymers is further improved by immobilizing thiol groups.
Cysteine (Cys) can significantly enhance the mucoadhesion and permeability of chitosan and its derivatives. 24,36 The excellent mucoadhesion property is mainly attributed to the covalent bonds formed between the thiol groups on Cys and the glycoproteins in the mucus, which is much stronger than the electrostatic interaction between chitosan and mucus layer. 37,38 Moreover, the thiol groups on Cys endow modified polymer with multifunctional group characteristics to achieve better drug loading. Therefore, it was possible to obtain an excellent nano-carrier with the combined effect of PA, Cys, and N-2-HACC.
Curcumin (CUR) is a naturally hydrophobic polyphenol compound with excellent antioxidant, anti-inflammatory, and anticancer activities. 39,40 Notably, CUR has a wide range of preventive properties against diseases, such as cardiovascular disease, various types of cancer, inflammation, and diabetes. 41 Nonetheless, the practical application of CUR is greatly restricted owing to its physicochemical instability, high hydrophobicity, low bioavailability, and rapid systemic elimination. 42,43 Therefore, it is vital to design an efficient delivery system to improve the bioavailability of CUR.
In the study, we aimed to develop a NP-based DDS that could effectively overcome mucus and epithelial barriers for the oral delivery of drug. The N-2-HACC could open the epithelial TJ to transport CUR on epithelial cells, and the polymerization of N-2-HACC with PA could enhance the hydrophobicity of N-2-HACC, thus contributing to cellular internalization, and endow N-2-HACC with self-assembly into micelles, encapsulating CUR. The thiolated polymer (PA-N-2-HACC-Cys) has the potential to penetrate the mucus layer. In the present study, we investigated the stability, the interaction between NPs and mucus, and transepithelial transport efficiencies in mucus-secreting cell models. The intestinal absorption in vivo and pharmacokinetics of NPs loaded with CUR after oral administration were also assessed.   (Table S1), we found that the particle size of the NPs was larger due to CUR encapsulation; and the particle size of PA-N-2-HACC-Cys NPs was smaller than that of PA-N-2-HACC NPs, which might be due to the tighter internal structure of NPs caused by the introduction of Cys. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images showed that the CUR@PA-N-2-HACC-Cys NPs were spherical, uniform in size, and well dispersed ( Figure 1b). Moreover, according to highperformance liquid chromatography (HPLC) results, the average peak area was linearly regressed with CUR concentration, and the regression equation was obtained (y = 0.6678x + 0.2871) (R 2 = 0.9999).
The encapsulation efficiency (EE) and loading capacity (LC) were calculated as 85.10 ± 1.43% and 9.41 ± 0.73% respectively by regression equation, indicating that CUR was efficiently encapsulated into the PA-N-2-HACC-Cys NPs. In addition, to investigate whether the prepared FITC-labeled polymer NPs can be used for in vitro and in vivo tracking, the in vitro leakage of FITC-labeled NPs was measured. As shown in Figure S2, the leakage was very minimal and slow, which can be used for NPs tracking in vitro.

| Stability of the CUR@PA-N-2-HACC-Cys NPs
The stability of the NPs is a prerequisite for oral administration. We investigated the stability of the CUR@PA-N-2-HACC-Cys NPs in buffers with various pH (1.2, 4.5, 6.0, 6.8, and 7.4), and these pH-varied buffers were used to simulate different environments corresponding to gastric fluid (pH 1.2), lysosomes (pH 4.5), early endosomes (pH 6.0), intestinal fluid (pH 6.8), and blood plasma (pH 7.4).  (Table 1). By comparing the correlation coefficients (r 2 ) obtained from each release kinetics model, we concluded that the first-order model was highly correlated with CUR release in SGF. However, the Korsmeyer-Peppas model was highly correlated with CUR release in SIF, referring to highest values of correlation coefficients. In addition, the major release mechanisms of the CUR@PA-N-2-HACC-Cys NPs in SGF and SIF was Super Case II transport and non-Fickian transport, respectively, according to the value of "n" diffusional exponent.

| Interaction between the PA-N-2-HACC-Cys NPs and mucus
For effective absorption into the blood capillary of intestinal villi, orally administrated NPs loaded with drugs need to overcome the mucus barrier that covers the intestinal epithelium. 50 Therefore, we investigated the interaction between the NPs loaded or unloaded with CUR and mucus by mucus-binding and mucus-penetrating assay.  37 Except for the electrostatic interaction, the formation of disulfide bonds between thiol groups of Cys and the glycoproteins of the mucus layer also played a role in mucoadhesion, which was much stronger than the electrostatic interaction between the N-2-HACC and mucus layer. 51 These results indicated that the PA-N-2-HACC-Cys NPs could prolong the residence time of CUR in the mucus-rich intestine by adhering to the mucus layer, thus achieving drug mucosal delivery.
Mucus-penetrating ability of the NPs loaded or unloaded with CUR was assessed using the transwell system. The particle size of NPs is critical to allow particles to cross the mucus, avoid rapid clearance, and ultimately enhance transmucosal transport. 14 Figure 2b shows that the percentage of particle penetration for the PA-N-2-HACC NPs, PA-N-2-HACC-Cys NPs, CUR@PA-N-2-HACC NPs, and CUR@PA-N-2-HACC-Cys NPs was 37.2 ± 0.7%, 69.7 ± 6.6%, 35.9 ± 3.3%, and 61.4 ± 4.3% at 4 h, respectively. Compared with the CUR@PA-N-2-HACC NPs, the CUR@PA-N-2-HACC-Cys NPs of smaller particle size could penetrate the mucus layer more quickly and efficiently (Table S1). Moreover, the enhanced permeation effect of PA-N-2-HACC-Cys NPs could be explained by the immobilization of thiol groups; 52 and the introduction of Cys not only enhanced the interaction between NPs and mucin but also formed disulfide bonds with mucin and attenuated the interaction between mucin, thus "diluting" mucus and enhancing the penetration ability of NPs. 53 T A B L E 1 Release constants (n) and correlation coefficient values (r 2 ) obtained by fitting curcumin release data to zero-order, first-order, Higuchi, and Korsmeyer-Peppas.

| Pharmacokinetic study
We investigated the pharmacokinetics of the free CUR, CUR@PA-N-2-HACC NPs, and CUR@PA-N-2-HACC-Cys NPs via oral administration to the SD rats (Figure 5b). For years, the unique pharmacokinetic characteristics and the mode of action of CUR remain to be of great interest. One study has been reported that only slight changes were observed in peripheral blood of patients after taking 8000 mg of curcumin daily. 58 Similarly, in another study, the concentration of CUR in peripheral circulation was at nmol/L level after receiving 3600 mg of CUR daily in patients with metastatic colon cancer. 59 In the study, the       of animals has no effect on the susceptibility to DSS-induced colitis, but male animal was more severely affected in the colon than female, 61 Hence, we have selected male mice to establish colitis models.

| Measurement of encapsulation efficiency and loading capacity
The EE and LC were measured using HPLC according to previous reports. 62,63 Briefly, the CUR@PA-N-2-HACC-Cys NPs were centrifuged at 12,000 r/min for 30 min at 4 C. The supernatant was subjected to quantification by HPLC using a Kromasil-C 18 column (250 mm Â 4.6 mm, 5 μm). The HPLC condition was set as follows: mobile phase of acetonitrile: water (containing 2% acetic acid) (52∶48, v/v); flow rate of 1.0 mL/min; detection wavelength of 426 nm. All the measurements were performed in triplicate. The EE and LC were calculated using Formulas (1) and (2)   suggest that the drug is released from NPs via Fickian diffusion, non-Fickian diffusion, Case II transport, and Super Case II transport, respectively.

| Mucus-binding assay
Mucin solution was prepared according to a previous report. 67 Mucin powder from pig stomach was dissolved in ultrapure water Binding efficiency % ð Þ¼ where dQ/dt is the flux of FITC-labeled NPs (μg/s), C 0 is the initial fluorescence intensity of NPs, and A is the membrane area (cm 2 ) of the transwell.
Then, the cells were stained with Hoechst 33342 for 10 min and washed with PBS (pH 7.4). Finally, the transwell insert containing the HT29-MTX monolayer was removed, placed onto a slide, and visualized using CLSM.

| Pharmacokinetic study
Nine male SD rats were randomly divided into three groups (n = 3).
The free CUR, CUR@PA-N-2-HACC NPs, and CUR@PA-N-2-HACC-Cys NPs were administered by gavage at a dose of 50 mg CUR per kilogram body weight. Moreover, 500 μL of blood was taken before administration, and 300 μL of blood was taken from the heart at 0.25, 0  50 mg/kg. The body weight of the mice was recorded every day, and DAI was measured to assess the severity of the colitis according to body weight loss, character of stool, and the degree of hematochezia (Table 3).
After treatment with different formulas, the mice were sacrificed after anesthesia, the colon length was measured, and the colon tissue was fixed with 10% formalin and stained with hematoxylin and eosin (H&E) for pathological examination. To count colonic goblet cells, fixed colonic tissues were also stained using Alcian blue. The slides of colon tissue were immunostained by a primary mouse ZO-1 antibody and mouse Claudin-1 antibody (Elabscience, China), and combined with a classical streptavidin-biotin-peroxidase detection system for immunohistochemical analysis.
In addition, a portion of collected colon tissue (50 mg) was homogenized with RIPA lysis buffer (Thermo Fisher, Shanghai, China) to extract total proteins. The homogenate was centrifuged at 12,000 r/ min for 15 min at 4 C. The protein content of supernatant was quantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher, Shanghai, China). The concentration of pro-inflammatory cytokines such as TNF-α and IL-1β was measured by ELISA kit (Enzyme-linked Biotechnology Co., Ltd., China).

| Statistical analysis
All the experimental data were represented as mean ± standard deviation (SD), and analyzed using one-way analysis of variance (ANOVA) (GraphPadPrism software Inc., USA). p < 0.05 was considered statistically significant.

ACKNOWLEDGMENTS
This work was partially supported by the "Pioneer" and "Leading Goose" R&D Program of Zhejiang (2022C02031) and Shanghai Yongling Biological Technology Co. Ltd.

CONFLICT OF INTEREST STATEMENT
The authors declare no competing financial interest.

PEER REVIEW
The peer review history for this article is available at https://www. webofscience.com/api/gateway/wos/peer-review/10.1002/btm2.

DATA AVAILABILITY STATEMENT
The main data supporting the results in this study are available within the paper and its supporting information. Additional data related to this work are available for research purposes from the corresponding author on reasonable request.