Oral immunization with acidified sucralfate@N-2-HACC/CMCS NPs elicits protective immune response in guinea pigs

Oral immunization with acidified sucralfate@N-2-HACC/CMCS NPs elicits protective immune response in guinea pigs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Oral immunization with acidified sucralfate@N-2-HACC/CMCS NPs elicits protective immune response in guinea pigs Zhi Zhao, Shuai Qiao, Zheng Jin, Chunjing Zhang, Tan Hui Yin, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3841170/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Oral administration of vaccine is required to preserve the vaccine against degradation, enhance antigen absorption in the gastrointestinal tract, and trigger adaptive immune responses. Nanomaterials are an ideal delivery vector for the creation of oral vaccines, and we have previously shown that N -2-hydroxypropyl trimethyl ammonium chloride chitosan (N-2-HACC)/ N , O -carboxymethyl chitosan (CMCS) based vaccine via oral administration led to protection against Newcastle disease virus. Hence, based on the immune adjuvant activity of N-2-HACC/CMCS nanoparticles and the advantage in resisting harsh gastric conditions of sucralfate acidified (SA), we constructed an oral vaccine delivery system based on SA and N-2-HACC/CMCS nanoparticles (SA@N-2-HACC/CMCS NPs), and the NPs were formulated to incorporate BSA. The SA@N-2-HACC/CMCS NPs had a particle size of 227 ± 7.0 nm and a zeta potential of 8.43 ± 2.62 mV. The NPs displayed slow and sustained release and high stability in simulated gastric juice and intestinal fluid. RAW 264.7 could better uptake the SA@N-2-HACC/CMCS/BSA NPs. The vaccine via oral administration markedly enhanced the residence time of BSA in the intestine for more than 12 h and elicited the production of IgG and sIgA. The SA@N-2-HACC/CMCS NPs developed here for oral administration is an excellent technique for delivering antigens and provides a path of mucosal vaccine research. Vaccine delivery system chitosan derivative based nanoparticles acidified sucralfate mucosal immunity oral administration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Organisms face complex and constantly changing environments, requiring an immune system against pathogenic microorganisms [ 1 ]. The most effective strategy to manage large-scale highly pathogenic viral epidemics is with a safe and effective vaccination. The currently licensed vaccines have limitations since they require intramuscular injection, and the biosecurity of these vaccines has not been completely explored. Furthermore, several of these vaccinations were insufficiently immunogenic, suggesting that a larger dose of the immunogen may be required to provide protective immunogenicity. The mucosal immune system constitutes the first line of defence against invading pathogens [ 2 ], and mucosal immunity is effective in promoting rapid and durable immune protection compared to systemic immunity [ 3 ]. Mucosal vaccines can be classified as oral, nasal, vaginal, or rectal based on the route of administration, of which oral administration is the easiest and most ideal route of vaccine administration. Oral administration can confer antibodies by inducing both mouth and intestinal mucosal immune responses [ 4 ]. Oral immunization induces the local production of secretory IgA antibodies that are essential for the induction of a protective humoral immune response [ 5 ]. In previous studies, oral immunization with the canine distemper virus (CDV) vaccine can produce a significant immune response in mice, a finding that provides a reference for immunizing wildlife with CDV vaccines [ 6 ]. Oral immunization with the Clostridium perfringens vaccine can significantly increase the expression of specific anti-α antibodies and protect chickens from necrotic enteritis disease [ 7 ], and oral immunization with the Bacille Calmette-Guérin (BCG) and ovalbumin vaccine produce more IgG2a and improve immunity to pathogen infections [ 8 ]. Although oral immunization has many advantages compared to injected parenteral immunization, it should be highlighted that a mucosal delivery system via oral vaccination administration is essential to protect the vaccine from gastric degradation, enhance antigen absorption in the gastrointestinal tract, trigger adaptive immune responses [ 9 ], and cross the intestinal epithelial barrier [ 10 ]. As a result, there is an urgent need for innovative vaccination formulations and delivery systems that can improve efficacy and safety. Nanomaterials are an excellent vaccine delivery vector, of which polymeric nanoparticles (NPs) are a proficient platform for drug oral delivery [ 11 ]. Hence, nanotechnology-based delivery systems have great potential for the development of oral vaccines [ 12 ]. Nanocomposite materials based on chitosan have become attractive in recent years due to their biocompatibility, low toxicity, and biodegradability [ 13 ]. Our lab has synthesized N -2-Hydroxypropyl trimethyl ammonium chloride chitosan (N-2-HACC) based on the quaternized modification of chitosan, N , O -carboxymethyl chitosan (CMCS), and N-2-HACC/CMCS NPs as a nano delivery system [ 14 ]. Our studies have shown that the N-2-HACC/CMCS NPs protect loaded antigens from gastric acid degradation and increase the bioavailability of loaded drugs, and the N-2-HACC/CMCS-based vaccine via oral administration led to protection against Newcastle disease virus in chickens [ 15 , 16 ], showing that the N-2-HACC/CMCS NPs have the great potential for vaccine mucosal delivery. Sucralfate has been widely used as a mucosal protective material and for the treatment of peptic ulcers and gastrointestinal discomfort [ 17 ]. It forms a protective barrier to shield the gastric wall from ulcers or damage [ 18 ]. Experimental evidence exists to suggest that sucralfate acidified (SA) can form a thick protective film in the stomach and small intestine [ 19 ]. Thus, we hypothesized that supplementation of SA into nanomaterials would not only help to resist the complex environment of the stomach but also deliver nanoparticles to the gastrointestinal tract to prolong the residence time of nanoparticles in the intestine. To further enhance the ability of N-2-HACC/CMCS NPs to resist the complex gastrointestinal (GI) environment, based on the immune adjuvant activity of N-2-HACC/CMCS nanoparticles and the advantage in resisting harsh gastric conditions of SA, we constructed an oral vaccine delivery system based on SA and N-2-HACC/CMCS nanoparticles (SA@N-2-HACC/CMCS NPs), and the SA@N-2-HACC/CMCS/BSA NPs were formulated to incorporate bovine serum albumin (BSA). In the present study, we extended our previous work by evaluating the adhesion, antigen presenting ability, antigen sustained release and stability in the simulated GI, residence time in the intestine, and immunogenicity of the SA@N-2-HACC/CMCS NPs in a guinea pig model. Oral vaccination with the SA@N-2-HACC/CMCS/BSA NPs induces strong humoral, cell-mediated, and mucosal immunity, thus, the vaccine has great potential for delivering antigens and represents a future direction of mucosal vaccine development via oral administration. 2. Materials and methods 2.1. Animals and cell lines Female BALB/c mice and nude mice (6–8 weeks) were purchased from Changsheng Biotechnology Co., Ltd. (Benxi, Liaoning, China, Permission No. SCXK (Liao) 2020-0002), and male adult guinea pigs (300 g) were purchased from Xianglin Co., Ltd. (Harbin, Heilongjiang, China, Permission No. SCXK (Hei) 2016-002). All animals were maintained under specific pathogen-free conditions and all animal experiments were approved by the Committee of Experimental Animals of Heilongjiang University. All laboratory animal care and experiments followed the “National Research Council’s Guide for the Care and Use of Laboratory Animals’’. 293T (Human embryonic cells) was provided from the Northeast Forestry University, and RAW 264.7 (Leukemia cells in mouse macrophage) was provided from Harbin Pharmaceutical Group Biological Vaccine Co., Ltd. 293T and RAW 264.7 were cultured in DMEM/F12 medium (Gibco, NY, USA) supplemented with 10% FBS (Clark, VA, USA) and 1% antibiotics (Gibco, NY, USA). 2.2. Synthesis and characterization of sucralfate acidified Briefly, 50 mg of sucralfate was dissolved in 1 mL of 1.0 mol/L HCl, and the solution was stirred until appeared gelatinous. Then, the gelatinous was collected and freeze-dried to obtain SA. Thermogravimetry analysis (TGA) was carried out using a Discovery TGA 5500 (Waters, MA, USA). 4 mg of the SA was heated in the temperature range of 30–300 ℃ with a rate of 5 ℃/min using an N 2 flow of 20 mL/min. ZetaPALS (Brookhaven Instrument, NY, USA) was used to measure the zeta potential of the SA. 2.3. Synthesis and characterization of the SA@N-2-HACC/CMCS NPs SA@N-2-HACC/CMCS NPs were synthesized by the electrostatic adsorption method. Briefly, the N-2-HACC/CMCS NPs were synthesized according to our previous method [ 14 , 16 ], and dissolved in water to a final concentration of 1 mg/mL. Then, 50 mL of the N-2-HACC/CMCS NPs solution (1 mg/mL) was incubated with 2 mg of the SA and gently stirred for 30 min at room temperature to allow conjugation to the SA@N-2-HACC/CMCS NPs. The morphological characteristics of SA@N-2-HACC/CMCS NPs were observed using a JEM-1200EX transmission electron microscope (TEM) (Hitachi, TYK, JPN). Dynamic light scattering and zeta potential were performed on a ZetaPALS (Brookhaven Instrument, NY, USA). The content and distribution of elements on the surface of SA@N-2-HACC/CMCS NPs were characterized by energy dispersive X-ray spectroscopy (EDS) (Bruker AXS, KA, DEU). 2.4. Cytotoxicity of the SA@N-2-HACC/CMCS NPs 2.4.1. In vitro cytotoxicity RAW 264.7 was employed to investigate the cytotoxicity of SA@N-2-HACC/CMCS NPs. Briefly, RAW 264.7 was seeded into a 96-well plate at a density of 5×10 3 cells/well and cultured in DMEM/F12 (Gibco, NY, USA) supplemented with 8% fetal bovine serum (Gibco, NY, USA) and 1% penicillin-streptomycin (Gibco, NY, USA) at 37 ℃ under a humidified atmosphere containing 5% CO 2 . After 24 h, the culture medium was replaced by DMEM/F12 with different concentrations of SA@N-2-HACC/CMCS NPs (0, 15.625, 31.25, 62.5, 125, 250, 500, and 1000 µg/mL). After being cultured for 24 h, 10 µL of CCK-8 was added into each well, cell viability was measured by using CCK-8 assay (Biyuntian, Shanghai, China), and OD 450 was measured with a microplate reader (Molecular Devices, Shanghai, China). 2.4.2. In vivo cytotoxicity Before analyzing the delivery capacity in vivo , we evaluated the in vivo cytotoxicity of SA@N-2-HACC/CMCS NPs. The female BALB/c mice were orally administrated with 250 µg of the N-2-HACC/CMCS NPs, 250 µg of the SA@N-2-HACC/CMCS NPs, and saline, respectively. After 12 h, the liver, lung, and spleen of the immunized mice were collected for hematoxylin-eosin (H&E) staining. 2.5. Mucoadhesion of the SA@N-2-HACC/CMCS NPs The binding amount of mucin to the SA@N-2-HACC/CMCS NPs was determined by the interaction between the negatively charged mucin (as the mucosal component) and positively charged SA@N-2-HACC/CMCS NPs in an aqueous solution. Briefly, 0.5 mL of mucin (1 mg/mL) (Solarbio, Beijing, CHN) and 0.5 mL of the SA@N-2-HACC/CMCS NPs (4 mg/mL) were incubated at 37 ℃ for 0.5, 1, 2, 4 and 8 h, respectively, and subsequently centrifuged at 12,000 r/min for 15 min. The concentration of free mucin in the supernatant was determined at 560 nm by the periodic acid/Schiff (PAS) colorimetric method [ 20 ]. The mucin binding amount of the SA@N-2-HACC/CMCS NPs was calculated with Eq. ( 1 ). In Eq. ( 1 ), W 1 is the initial concentration of mucin used for incubation, and W 0 is the concentration of free mucin in the supernatant. $$\text{Mucin binding amount=}W1-W0$$ 1 2.6. Cellular uptake The cellular uptake of FITC-SA@N-2-HACC/CMCS NPs and FITC-N-2-HACC/CMCS NPs in RAW 264.7 was determined using the inverted fluorescence microscope (Olympus, TKY, JPN). The SA@N-2-HACC/CMCS NPs and nuclei were labeled with FITC (exhibiting green fluorescence) and DAPI (exhibiting blue fluorescence), respectively. The FITC-SA@N-2-HACC/CMCS NPs and FITC-N-2-HACC/CMCS NPs were prepared per the method described previously [ 21 ]. RAW 264.7 in the logarithmic growth phase was seeded onto the samples in a 24-well plate with cell climbing at a density of 1×10 6 cells/well and cultured overnight. The FITC-SA@N-2-HACC/CMCS NPs and FITC-N-2-HACC/CMCS NPs were added to each well at the concentration of 1 mg/mL and incubated for 1, 2, 4, 8, 12, 16, 20 and 24 h, respectively. The cells were then carefully washed with PBS (pH 7.2) to remove any excess NPs before being fixed in 500 µL of 4% paraformaldehyde for 30 min. Next, 4% paraformaldehyde was removed and 100 µL of 0.5% Triton X-100 was added for 30 min to penetrate cells. Finally, the cell climbing was stained with 50 µL of DAPI (0.5 µg/mL), and the cellular uptake of the NPs was observed using the inverted fluorescence microscope (Olympus, TKY, JPN). 2.7. Slow-release effect of the SA@N-2-HACC/CMCS NPs 2.7.1. Preparation of the SA@N-2-HACC/CMCS/BSA NPs To examine the slow-release effect of the SA@N-2-HACC/CMCS NPs, BSA was used as a model antigen. The preparation of SA@N-2-HACC/CMCS NPs loaded with BSA (SA@N-2-HACC/CMCS/BSA NPs) was as follows. First, BSA was encapsulated in N-2-HACC/CMCS NPs. The water phase was 3 mL of N-2-HACC/CMCS NPs (1.7 mg/mL), the oil phase was a combination of petroleum ether, liquid paraffin (Yongda Chemical Reagents, Tianjin, CHN) and Span-80 (Zhiyuan Chemical Reagents, Tianjin, China) and two phases were emulsified for 15 min at 600 r/min. Second, 1.5 mL of BSA at various concentrations (1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5 mg/mL) and 1 mL of the CMCS (1.3 mg/mL) were mixed in the water/oil at 600 r/min for 4 min, respectively. The production was cured at 37 ℃ for 3 h. Later, the solution was placed into a centrifuge tube and centrifuged for 30 min at 4 ℃, 12000 r/min, the supernatant was removed, the precipitation was washed four times with petroleum ether, and the N-2-HACC/CMCS/BSA NPs were produced. Finally, the SA@N-2-HACC/CMCS/BSA NPs were prepared by adding 0.1 mg of the SA in 10 mL of the N-2-HACC/CMCS/BSA NPs and gently stirring for 30 min at room temperature. The micro BCA protein assay (Thermo Scientific, Shanghai, China) was used to measure the encapsulation efficiency (EE) and loading capacity (LC) of BSA in the SA@N-2-HACC/CMCS/BSA NPs. The EE and LC are calculated using the formulas (2) and (3), where M 0 is the total weight of BSA, M 1 is the weight of BSA in the supernatant, and M N is the total weight of SA@N-2-HACC/CMCS/BSA NPs. The experiments were repeated at least three times. $$\text{E}\text{E} \left(\text{%}\right) = ({\text{M}}_{0}-{\text{M}}_{1})/{\text{M}}_{0}\times 100\text{%}$$ 2 $$\text{L}\text{C} \left(\text{%}\right) = ({\text{M}}_{0}-{\text{M}}_{1})/\left({\text{M}}_{\text{N}}\right)\times 100\text{%}$$ 3 2.7.2. In vitro release of the SA@N-2-HACC/CMCS/BSA NPs Briefly, 100 mg of the SA@N-2-HACC/CMCS/BSA NPs was resuspended in 2 mL of PBS (pH = 7.2) and placed at 37 ℃ in a shaker incubator shaking at the speed of 100 r/min. The suspension was collected at 0, 3, 6, 9, 12, 24, 36, 48, 60, and 72 h, respectively, and centrifuged at 12,000 r/min for 10 min. Then, the supernatant was harvested, and the amount of BSA was quantified by the micro BCA protein assay (Thermo Scientific, Shanghai, China). The equivalent volume of fresh PBS was replenished in the original tube for further incubation. 2.7.3. Stability assay in simulated body fluid of the SA@N-2-HACC/CMCS/BSA NPs The stability of SA@N-2-HACC/CMCS/BSA NPs was conducted in the presence of simulated gastric fluid (SGF) (pH = 1.2, Meilian Biotechnology Co., Ltd., Shanghai, China) and simulated intestinal fluid (SIF) (pH = 6.8, Meilian Biotechnology Co., Ltd., Shanghai, China). Briefly, the SA@N-2-HACC/CMCS/BSA NPs were put into 2 mL of the SGF at 37 ℃, and the supernatant samples were collected at a specified time (0, 1, and 2 h, respectively), and an equivalent volume of the SGF was replenished in the original tube for further incubation. The supernatant was separated by centrifugation at 12000 r/min for 10 min, and the content of BSA in the supernatant was evaluated using a BCA kit. After 2 h, SGF was discarded by centrifugation at 12000 r/min for 10 min and 2 mL of the SIF was added. The content of BSA in the supernatant was evaluated at specified times (3, 4, 5, 6, 7, and 8 h, respectively). 2.8. In vivo trafficking of the SA@N-2-HACC/CMCS/BSA NPs To investigate the in vivo biodistribution of the SA@N-2-HACC/CMCS/BSA NPs, cyanin 5.5 (Cy5.5) was used to label BSA. The Cy5.5-labeled BSA (BSA-Cy5.5) was prepared as described previously [ 22 ]. Female BALB/c nude mice (6–8 weeks, 20 ± 2 g) were divided into four groups: Group 1 was oral administration with the saline (negative control), Group 2 was oral administration with the BSA-Cy5.5 (100 µg), Group 3 was oral administration with the N-2-HACC/CMCS/BSA-Cy5.5 NPs (containing 100 µg of the BSA-Cy5.5), and Group 4 was oral administration with the SA@N-2-HACC/CMCS/BSA-Cy5.5 NPs (containing 100 µg of the BSA-Cy5.5). The biodistribution of SA@N-2-HACC/CMCS/BSA NPs was investigated at different times (1, 3, 6, 9, and 12 h, respectively) after administration by a small animal in vivo imaging system (Maestro CRI, MA, USA). The mice were anesthetized with 0.1 mL of 10% chloral hydrate and observed in a prone position. 2.9. Immune effect of the SA@N-2-HACC/CMCS/BSA NPs Sixty 6–8 week-old healthy male guinea pigs were fed adaptively for 1 week before the experiment. Then, the guinea pigs were divided into ten groups with 6 guinea pigs in each group: guinea pigs in Group 1 to Group 5 were injected intramuscular immunization (i.m.) with 0.2 mL of the PBS, free BSA (200 µg BSA), Seppic ISA15/BSA (containing 200 µg BSA), N-2-HACC/CMCS/BSA NPs (containing 200 µg BSA), and SA@N-2-HACC/CMCS/BSA NPs (containing 200 µg BSA), respectively; guinea pigs in Group 6 to Group 10 were immunized oral administration with 0.2 mL of PBS, free BSA (200 µg BSA), Seppic ISA15/BSA (containing 200 µg BSA), N-2-HACC/CMCS/BSA NPs (containing 200 µg BSA), and SA@N-2-HACC/CMCS/BSA NPs (containing 200 µg BSA), respectively. The booster immunization was given in the same manner at 14 days post the immunization. The blood samples were collected by heart puncture prior to priming and at 7, 14, 21, 28, 42, and 56 days post the immunization. Following coagulation, samples were incubated at 37 ℃ for 1 to 2 h and centrifuged at 3000 r/min for 10 min, and the obtained sera were stored at 80 ℃ until further analysis. The content of serum IgG, IgG1, IgG2a, IL-2, IL-4, IL-12, and IFN-γ was measured using the mouse ELISA Kit (Meilian Biotechnology Co., Ltd., Shanghai, China). The fecal pellets were also collected from each guinea pig, and diluted in PBS (pH = 7.2) up to a final concentration of 1 g dry matter/mL. The diluted fecal pellets were homogenized and centrifuged at 3000 r/min for 10 min. The supernatants were collected and stored at 80 ºC until assayed. The levels of IgA and sIgA in the fecal pellets were determined using the mouse ELISA Kit (Meilian Biotechnology Co., Ltd., Shanghai, China). 2.10. Statistical analysis The mean and standard deviation (SD) were used to represent the data. One-way analysis of variance (ANOVA) was performed to assess differences between groups, and Bonferroni's post hoc test was employed for multiple comparisons. p values less than 0.05 were deemed statistically significant, whereas p values less than 0.01 were deemed very significant. 3. Results 3.1. Characterization of the sucralfate acidified TGA curves of the sucralfate and SA were presented in Fig. 1 A. The sucralfate demonstrated low weight loss at 150–250 ℃, and SA demonstrated high weight loss at 150–250 ℃, which was likely due to the loss of lattice water. SA converted hydroxide to lattice water compared to the sucralfate, proving that the sucralfate had been successfully acidified. The zeta potential of SA was − 5.86 ± 4.24 mV (Fig. 1 B), and the negative charge could facilitate subsequent electrostatic adsorption to form NPs. 3.2. Characterization of the SA@N-2-HACC/CMCS NPs The elements on the surface of SA@N-2-HACC/CMCS NPs were S, C, H, O, and Al (Fig. 2 A), comparing the elements on the surface of N-2-HACC/CMCS NPs (Fig. 2 B), indicating the presence of the SA on the surface of N-2-HACC/CMCS NPs. The SA@N-2-HACC/CMCS NPs were spherical in nature and mono-dispersed (Fig. 2 C). The SA@N-2-HACC/CMCS NPs had a particle size of 227 ± 7.0 nm (Fig. 2 D) and a zeta potential of 8.43 ± 2.62 mV (Fig. 2 E). 3.3. Cytotoxicity of the SA@N-2-HACC/CMCS NPs When the concentration of N-2-HACC/CMCS NPs reached 1000 µg/mL, the cell viability reached 84.24 ± 8.64% (Fig. 3 A), with no significant difference compared to the control group ( p > 0.05). When the concentration of the SA@N-2-HACC/CMCS NPs reached 1000 µg/mL, the cell viability had an extremely significant difference compared with the control group ( p < 0.01), but it still was still as high as 86.01 ± 2.38% (Fig. 3 B). The higher cell viability indicated that the SA@N-2-HACC/CMCS NPs has lower cytotoxicity. The in vivo cytotoxicity of the SA@N-2-HACC/CMCS NPs showed that no significant difference in tissue integrity and cell structure was found in the N-2-HACC/CMCS NPs, SA@N-2-HACC/CMCS NPs and control groups ( p > 0.05), indicating that the SA@N-2-HACC/CMCS NPs had no cytotoxicity on tissues (Fig. 3 C). 3.4. In vitro mucoadhesive strength The amount of mucin-binding in each group increased significantly, as the incubated time went. After 8 hours of incubation, the mucin-binding value of the SA@N-2-HACC/CMCS NPs was 150.3 µg, showing a significantly higher mucin-binding value compared with that of the N-2-HACC/CMCS NPs, (72.6 µg, p < 0.01), indicating that the introduction of SA improved the binding efficiency of N-2-HACC/CMCS NPs to mucin. As a consequence, the small intestine residence time, which is an essential prerequisite for effective mucosal delivery of therapeutics, was increased. 3.5. Cellular uptake As shown in Fig. 4 , the cellular uptake of the two NPs was shown to increase with treatment time. Furthermore, the intensity of FITC fluorescence exhibited by the FITC-SA@N-2-HACC/CMCS NPs was significantly greater than that of the FITC-N-2-HACC/CMCS NPs at all time points, indicating that RAW 264.7 could better absorb the FITC-SA@N-2-HACC/CMCS NPs. 3.6. In vitro release of the SA@N-2-HACC/CMCS/BSA NPs When the concentration of BSA was 4 mg/mL, EE and LC were highest, and they were 96.81 ± 0.12% and 41.74 ± 0.05% (n = 3), respectively. Thus, 4 mg/mL of BSA was selected as the optimal concentration for the preparation of the SA@N-2-HACC/CMCS/BSA NPs. The in vitro release of BSA from the SA@N-2-HACC/CMCS/BSA NPs at PBS solution (pH 7.2) was in a sustained behavior (Fig. 5 A), the release behavior was faster at prometaphase, and 81.18 ± 4.34% of BSA was released from the SA@N-2-HACC/CMCS/BSA NPs at 72 h. A sustained release pattern could improve the duration of antigen post the immunization. 3.7. Stability of the SA@N-2-HACC/CMCS/BSA NPs The SA@N-2-HACC/CMCS/BSA NPs were tested for stability in SGF (pH 1.2) for the first 2 h and then in SIF (pH 6.8) for a further 6 h to simulate the GI environment without enzyme. As shown in Fig. 5 B, the SA@N-2-HACC/CMCS/BSA NPs exhibited sustained release, and BSA in the SA@N-2-HACC/CMCS/BSA NPs released 24.05 ± 2.41% for the first 2 h in SGF and finally released 34.88 ± 1.05% for another 6 h in SIF. The N-2-HACC/CMCS/BSA NPs showed significantly faster release in SGF, and BSA released 59.96 ± 0.85% for the first 2 h and finally released 63.09 ± 2.50% for another 6 h in SIF, indicating that the SA@N-2-HACC/CMCS/BSA NPs were mainly released in intestinal fluid and rarely in gastric fluid. 3.8. In vivo trafficking of the SA@N-2-HACC/CMCS/BSA NPs We traced the biodistribution of the SA@N-2-HACC/CMCS/BSA NPs after oral administration in nude mice by IVIS imaging (Fig. 5 C). After the oral administration for 1 h, the fluorescence of BSA was widely distributed in the mouse intestine of all administration groups. After the oral administration of free BSA for 3 h, BSA was clearly visualized, but the signal of BSA decreased and became exceedingly weak by 12 h, indicating that the free BSA was quickly cleared from the intestines. For the N-2-HACC/CMCS/BSA NPs and SA@N-2-HACC/CMCS/BSA NPs, relatively high fluorescence in the intestine was sustained for over 12 h, and the fluorescence was stronger than that of the N-2-HACC/CMCS/BSA NPs ( p < 0.01). This high accumulation ability of BSA in the intestine could be attributed to the enhanced permeability and retention effect of the SA@N-2-HACC/CMCS/BSA NPs. 3.9. Immune effect of the SA@N-2-HACC/CMCS/BSA NPs 3.9.1. SA@N-2-HACC/CMCS/BSA NPs elicit immune responses after intramuscular administration As shown in Fig. 6 A, the SA@N-2-HACC/CMCS/BSA NPs induced higher IgG antibody levels on 14 d than the other formulations post the primary immunization. The SA@N-2-HACC/CMCS/BSA NPs clearly increased IgG1 (Fig. 6 B) and IgG2a (Fig. 6 C) antibody titers compared to BSA alone or PBS ( p < 0.05), which indicated that the SA@N-2-HACC/CMCS/BSA NPs elicited more effective immune responses. Compared with the PBS and BSA alone groups, the level of IL-4 (Fig. 6 D) in the serum of guinea pigs immunized with the SA@N-2-HACC/CMCS/BSA NPs was significantly increased at 7 d post the immunization ( p < 0.05), the level of IL-2 (Fig. 6 E) and IL-12 (Fig. 6 F) was significantly increased at 14 d post the immunization ( p < 0.05), and the level of IFN-γ (Fig. 6 G) was significantly increased at 21 d post the immunization ( p < 0.05). The findings revealed that intramuscular delivery of SA@N-2-HACC/CMCS/BSA NPs caused higher cellular and humoral immune responses. 3.9.2. SA@N-2-HACC/CMCS/BSA NPs elicit immune responses after oral administration Compared with the PBS, free BSA and Seppic ISA15/BSA groups, the serum IgG (Fig. 7 A), IgG1 (Fig. 7 B) and IgG2a (Fig. 7 C) antibody titers in the SA@N-2-HACC/CMCS/BSA NPs and N-2-HACC/CMCS/BSA NPs groups were significantly higher at 14 d post the immunization ( p < 0.05), indicating that the SA@N-2-HACC/CMCS/BSA NPs and N-2-HACC/CMCS/BSA NPs produced stronger immune responses by oral administration. Compared with the PBS, BSA and Seppic ISA15/BSA groups, the production of IL-4 (Fig. 7 D) significantly increased in the guinea pigs immunized with the SA@N-2-HACC/CMCS/BSA NPs and N-2-HACC/CMCS/BSA NPs at the 7 d post the immunization ( p < 0.05), and the production of IL-2 (Fig. 7 E), IL-12 (Fig. 7 F), and IFN-γ (Fig. 7 G) significantly increased at the 14 d post the immunization ( p < 0.05). Additionally, the level of IL-12 in the guinea pigs immunized with the SA@N-2-HACC/CMCS/BSA NPs was higher at 14 d post the immunization than that of the N-2-HACC/CMCS/BSA NPs group ( p < 0.05), and extremely significantly higher than that of PBS, BSA and Seppic ISA15/BSA groups ( p < 0.05). The results showed that oral delivery of SA@N-2-HACC/CMCS/BSA NPs increased cytokine release, resulting in a stronger cellular immune response. Compared with the PBS, free BSA, and Seppic ISA15/BSA groups, the sIgA antibody titers in the fecal pellets of the guinea pigs immunized with the SA@N-2-HACC/CMCS/BSA NPs were significantly higher at 14 d post the immunization (Fig. 7 H, p < 0.05). The results indicated that the SA@N-2-HACC/CMCS/BSA NPs induced a stronger mucosal immunity response by oral administration. 4. Discussion The mucosa and intestine are exposed to large amounts of antigens, including harmful pathogenic microorganisms [ 23 ]. Thus, mucosal immunization may provide an effective means of preventing infectious agents [ 24 ]. Among the multiple routes of mucosal immunization, oral administration is more readily accepted because of its more convenient mode of immunization [ 25 ]. The emergence of nanomaterials with high biosafety and biodegradability has provided an opportunity for oral vaccines [ 26 ]. They can act as carriers to protect antigens from degradation by proteases, enhance the residence time of antigens in the gastrointestinal mucosa, and increase the number of antigens through the upper mucosal cells [ 27 , 28 ]. In this study, an antigen delivery system based on the SA and N-2-HACC/CMCS for oral administration through drinking water or feed was developed and tested in guinea pigs. Following oral administration, the first problem faced by antigens is the harsh GI environment, where antigens are exposed to host-imposed stresses, especially the low pH in the GI (pH = 1.5) [ 29 ]. Recent studies have demonstrated that a variety of nanocarriers can protect antigens from degradation in the harsh GI environment [ 30 ]. Upon overcoming the low pH environment of GI, the oral vaccine is also faced with the barrier effect of a continuously secreted mucus layer. Additionally, the antigens adhere less readily to the mucus layer and are easily washed away by the mucus layer [ 31 ]. Therefore, substances with adhesive properties can contribute to the design of a delivery system to overcome the mucus layer barrier in oral immunization and to prolong the retention of antigens in the mucosal layer. In this study, we have demonstrated that the NPs prepared using SA and chitosan derivatives are highly resistant to low pH in the GI and have good mucosal adhesion as an oral vaccine delivery system. Our findings are consistent with those of other researchers, demonstrating that the SA@N-2-HACC/CMCS/BSA NPs enhance the adhesion and antibody protection of the vaccine delivery system [ 32 , 33 ]. In addition, chitosan derivatives have a strong potential to open the tight junctions between epithelial cells, thereby improving the chance of antigen contact with immune cells and bioavailability [ 34 , 35 ]. Therefore, we consider the SA@N-2-HACC/CMCS NPs to have significant potential as an oral vaccine delivery system. As a vaccine delivery system, NPs are expected to have a good safety profile and efficient adjuvant activity [ 36 ]. The safety of SA@N-2-HACC/CMCS NPs was investigated by in vitro and in vivo cytotoxicity evaluation, and the results showed that the NPs had higher compatibility. To achieve immune function, the antigens have to reach the antigen-presenting cells (APCs), which makes the antigen-presenting role of adjuvants essential [ 37 ]. In this study, we proved that the SA@N-2-HACC/CMCS NPs could be effectively uptaken by RAW264.7. Similarly, our previous studies indicate that chitosan derivative NPs have an antigen-presenting effect [ 38 ]. Therefore, we believe the SA@N-2-HACC/CMCS NPs to be a safe delivery carrier with adjuvant activity. The release of antigens encapsulated in the nanomaterials is a key feature in the evaluation of a vaccine delivery system, as slow release increases the bioavailability of antigens and thus enhances the immune response [ 39 ]. The N-2-HACC/CMCS/BSA NPs prolonged the release time of BSA encapsulated in the N-2-HACC/CMCS NPs and achieved a sustained antigen-release effect compared to free BSA. Moreover, it was confirmed that the SA@N-2-HACC/CMCS NPs prolonged the residence time of BSA in the intestine, showing that chitosan derivative NPs have a sustained release effect, and these are consistent with those of previous findings [ 40 , 41 ]. Additionally, SA has a strong mucoadhesion in the GI [ 42 ]. Thus, we consider the SA@N-2-HACC/CMCS NPs to be a promising oral vaccine delivery system for the sustained release of antigens. IL-2, IL-12, and IFN-γ are preferentially called Th1-type cytokines, while IL-4 is generally regarded as Th2-type cytokines [ 43 ]. In the study, the SA@N-2-HACC/CMCS/BSA NPs induced the expression of IL-2, IL-12, IFN-γ, and IL-4, suggesting that the NPs can induce mixed Th1 and Th2 type immune responses. The results are consistent with the previous studies of chitosan derivatives NPs as a potential adjuvant to enhance both humoral and cellular immune responses [ 44 ]. Nanovaccine, delivered orally, induces both local and systemic immunity [ 45 ]. The SA@N-2-HACC/CMCS/BSA NPs by oral administration in guinea pigs enhanced the levels of local sIgA in the intestine and serum IgG antibody, which is consistent with the oral delivery of measles antigen-loaded chitosan NPs to enhance intestinal IgA antibody titers in mice [ 46 ]. Despite no significant increase of SA@N-2-HACC/CMCS/BSA NPs observed during the detection of cytokines and antibody levels post-immunization, compared to the N-2-HACC/CMCS/BSA NPs (but both were notably higher than the control group), the sustained release of SA@N-2-HACC/CMCS/BSA NPs in vitro and their prolonged retention in vivo were significantly superior to the group N-2-HACC/CMCS/BSA NPs. Therefore, we firmly believe that employing the SA@N-2-HACC/CMCS NPs delivery system for viral immunization will indeed further enhance the ability to combat viruses. However, at present, we lack sufficient evidence to support this viewpoint, which constitutes a limitation of this article. Moving forward, we will utilize this delivery system for viral transport in further research. 5. Conclusions Collectively, in the present study, we developed an oral vaccine delivery vector based on SA and N-2-HACC/CMCS nanoparticles (SA@N-2-HACC/CMCS NPs) and prepared BSA encapsulated in the nanocarrier. We extended our previous work by investigating the adhesion, antigen presenting ability, antigen sustained release and stability in the simulated GI, residence time in the intestine, and immunogenicity of the SA@N-2-HACC/CMCS NPs in a guinea pig model. Our results showed that the SA@N-2-HACC/CMCS/BSA NPs had good biocompatibility, stability, mucosal adhesive, adjuvant activity, and sustained release. The guinea pigs orally vaccinated with the SA@N-2-HACC/CMCS/BSA NPs exhibited significantly higher serum IgG, IgG1 and IgG2, mucosal IgA, and higher levels of IL-4, IL-2, IL-12, and IFN-γ immune responses. These findings highlighted the great potential of chitosan derivative based nanoparticle vaccine as an alternative approach that represents a future direction for developing an oral vaccine for use in livestock and poultry. Abbreviations N-2-HACC N -2-hydroxypropyl trimethyl ammonium chloride chitosan CMCS N , O -carboxymethyl chitosan SA Sucralfate acidified SA@N-2-HACC/CMCS NPs SA and N-2-HACC/CMCS nanoparticles CDV Canine distemper virus BSA Bovine serum albumin TGA Thermogravimetry analysis TEM Transmission electron microscope EDS Energy dispersive X-ray spectroscopy EE Encapsulation efficiency LC Loading capacity SGF Simulated gastric fluid SIF Simulated intestinal fluid Declarations Funding This work was supported by the “Pioneer” and “Leading Goose” R&D Program of Zhejiang (2022C02031), Zhejiang Provincial Key R&D Program of China (2021C02049), Natural Science Foundation of Heilongjiang Province (LH2020H129) and Taizhou Science and Technology Plan Project in 2023 (23gya02). Authorship contributions Kai Zhao designed the study; Shuai Qiao searched the literature and performed the experiments; Kai Zhao, Zhi Zhao, and Shuai Qiao analyzed the data; Kai Zhao and Zhi Zhao wrote the original draft; Zhi Zhao, Zheng Jin, and Chunjing Zhang organized the figure data; Kai Zhao, Zhi Zhao, Tan Hui Yin, and Chunjing Zhang revised the manuscript. All authors read and approved the final manuscript. Declaration of interest The authors declare no competing financial interest. Availability of data and materials The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate All animal protocols were approved by the Committee of Experimental Animals of Heilongjiang University. All laboratory animal care and experiments followed the “National Research Council’s Guide for the Care and Use of Laboratory Animals’’. 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Development and characterization of alginate coated low molecular weight chitosan nanoparticles as new carriers for oral vaccine delivery in mice. Carbohydr Polym. 2015;121:403–10. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3841170","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":265926022,"identity":"0c23f9b3-6137-41a8-ba95-69144e2fba01","order_by":0,"name":"Zhi Zhao","email":"","orcid":"","institution":"Taizhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhi","middleName":"","lastName":"Zhao","suffix":""},{"id":265926023,"identity":"2822684e-e291-4ad5-805d-3315c07a0cdb","order_by":1,"name":"Shuai Qiao","email":"","orcid":"","institution":"Taizhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shuai","middleName":"","lastName":"Qiao","suffix":""},{"id":265926024,"identity":"abf59b9b-47c9-456a-8a24-4360bb950fae","order_by":2,"name":"Zheng Jin","email":"","orcid":"","institution":"Taizhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zheng","middleName":"","lastName":"Jin","suffix":""},{"id":265926025,"identity":"7ad7296e-7fb6-45ba-b3e8-3371b6ea9324","order_by":3,"name":"Chunjing Zhang","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chunjing","middleName":"","lastName":"Zhang","suffix":""},{"id":265926026,"identity":"1e673d9f-5d9a-4fac-8e83-ab58106eb41a","order_by":4,"name":"Tan Hui Yin","email":"","orcid":"","institution":"Tunku Abdul Rahman University of Management and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tan","middleName":"Hui","lastName":"Yin","suffix":""},{"id":265926027,"identity":"a142b94a-375f-4ccb-8d30-6c48ef0fb847","order_by":5,"name":"Kai Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYFACHoYDDD9s6vmZmQ8+IF4LY09agmQ7W7IB0VoYGNgOJxic5zETIEqD/Izcg4cLeA7nGR9mMGNgqLGJJqiFcUZewuEZFunFZocZ0h4wHEvLbSCkhVkix+AwD48147bDDMcNGBsOE9bCBtbCxsy4uZmxTYIoLTwQLc6JG5iZ2YjTIsHzLuEwb0+ascRhNmaDBGL8It+ee/gzzw8bOf7+8x8ffKixIayFQSABiZOAQxEq4D9AlLJRMApGwSgYyQAApJM8p2oS7VgAAAAASUVORK5CYII=","orcid":"","institution":"Taizhou University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2024-01-07 02:59:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3841170/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3841170/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49414774,"identity":"10ed7c81-b5ea-476d-949e-c84c945ea525","added_by":"auto","created_at":"2024-01-10 11:42:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":13817,"visible":true,"origin":"","legend":"\u003cp\u003ePhysical properties of the sucralfate and SA. (A) TGA curve showing weight loss in the range of 50 to 300 ºC; (B) Zeta potential of the SA.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3841170/v1/c9e029894f78dae103b028a9.png"},{"id":49414467,"identity":"d2b63830-4cfa-477e-b8d2-e35719fc63e7","added_by":"auto","created_at":"2024-01-10 11:34:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":24531,"visible":true,"origin":"","legend":"\u003cp\u003eThe structural morphology and surface characteristics of the SA@N-2-HACC/CMCS NPs. (A) EDS spectrum of the SA@N-2-HACC/CMCS NPs; (B) EDS spectrum of the N-2-HACC/CMCS NPs; (C) TEM images of the SA@N-2-HACC/CMCS NPs; (D) Particle size of the SA@N-2-HACC/CMCS NPs; (E) Zeta potential of the SA@N-2-HACC/CMCS NPs.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3841170/v1/0e3396db170e8672be333140.png"},{"id":49414464,"identity":"7a5d1bf1-7276-40c8-b1c4-bcb4a85fb26f","added_by":"auto","created_at":"2024-01-10 11:34:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":110763,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity of the SA@N-2-HACC/CMCS NPs. (A) Cell viability of 293T tread with the N-2-HACC/CMCS NPs for 24 h; (B) Cell viability of 293T tread with the SA@N-2-HACC/CMCS NPs for 24 h; C: H\u0026amp;E staining.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3841170/v1/70d1f3af169380c0547f1706.png"},{"id":49414462,"identity":"122cba08-970c-4230-8c1a-a92645e6b0ea","added_by":"auto","created_at":"2024-01-10 11:34:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":55621,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of delivery efficiency for the SA@N-2-HACC/CMCS NPs. (A) Cellular uptake of the N-2-HACC/CMCS NPs into RAW 264.7; (B) Cellular uptake of the SA@N-2-HACC/CMCS NPs into RAW 264.7. Scale=60 μm.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3841170/v1/2d0a59ae3398341305e65b41.png"},{"id":49414460,"identity":"9474b4c8-adcf-4bf6-a365-2ecad7c9de29","added_by":"auto","created_at":"2024-01-10 11:34:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":27369,"visible":true,"origin":"","legend":"\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e release and \u003cem\u003ein vivo\u003c/em\u003e visualization analysis of the SA@N-2-HACC/CMCS/BSA NPs. (A) \u003cem\u003eIn vitro\u003c/em\u003e release of BSA; (B) Stability of the SA@N-2-HACC/CMCS/BSA NPs in SGF and SIF; (C) \u003cem\u003eIn vivo\u003c/em\u003e biodistribution of the SA@N-2-HACC/CMCS/BSA NPs.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3841170/v1/af6cec4d962b693c94f05131.png"},{"id":49414461,"identity":"0b94a95e-86e3-4dda-ae4f-fba3abf661d1","added_by":"auto","created_at":"2024-01-10 11:34:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":87174,"visible":true,"origin":"","legend":"\u003cp\u003eSerum IgG antibody titers, level of cytokines, and sIgA antibody titers in feces following intramuscular administration of the SA@N-2-HACC/CMCS/BSA NPs, N-2-HACC/CMCS/BSA NPs, Seppic ISA15/BSA, free BSA and PBS. (A) Serum IgG antibody titers; (B) Serum IgG1 antibody titers; (C) Serum IgG2a antibody titers; (D) Level of IL-4 in serum; (E) Level of IL-2 in serum; (F) Level of IL-12 in serum; (G) Level of IFN-γ in serum; (H) sIgA antibody titers in feces. Values represent mean±SD (n=3). Different letters indicate a significant difference (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) and similar letters indicate a non-significant difference (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05) when compared with the control group.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3841170/v1/86ec4336660450a6404c5f72.png"},{"id":49414465,"identity":"b53995ab-d9d2-4cee-a028-51c6d31387d9","added_by":"auto","created_at":"2024-01-10 11:34:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":84200,"visible":true,"origin":"","legend":"\u003cp\u003eSerum IgG antibody titers, level of cytokines, and sIgA antibody titers in feces following oral administration of the SA@N-2-HACC/CMCS/BSA NPs, N-2-HACC/CMCS/BSA NPs, Seppic ISA15/BSA, free BSA and PBS. (A) Serum IgG antibody titers; (B) Serum IgG1 antibody titers; (C) Serum IgG2a antibody titers; (D) Level of IL-4 in serum; (E) Level of IL-2 in serum; (F) Level of IL-12 in serum; (G) Level of IFN-γ in serum; (H) sIgA antibody titers in feces. Values represent mean±SD (n=3). Different letters indicate a significant difference (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) and similar letters indicate a non-significant difference (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05) when compared with the control group.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-3841170/v1/f4bc4a949fd8ca8d072250a6.png"},{"id":50232385,"identity":"687aef44-2eb7-4e5f-b502-5e0cb06ba4a3","added_by":"auto","created_at":"2024-01-26 22:07:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1310211,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3841170/v1/37502cbb-a323-4cfb-a8a7-dd7d3cf72646.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Oral immunization with acidified sucralfate@N-2-HACC/CMCS NPs elicits protective immune response in guinea pigs","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOrganisms face complex and constantly changing environments, requiring an immune system against pathogenic microorganisms [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The most effective strategy to manage large-scale highly pathogenic viral epidemics is with a safe and effective vaccination. The currently licensed vaccines have limitations since they require intramuscular injection, and the biosecurity of these vaccines has not been completely explored. Furthermore, several of these vaccinations were insufficiently immunogenic, suggesting that a larger dose of the immunogen may be required to provide protective immunogenicity. The mucosal immune system constitutes the first line of defence against invading pathogens [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and mucosal immunity is effective in promoting rapid and durable immune protection compared to systemic immunity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Mucosal vaccines can be classified as oral, nasal, vaginal, or rectal based on the route of administration, of which oral administration is the easiest and most ideal route of vaccine administration. Oral administration can confer antibodies by inducing both mouth and intestinal mucosal immune responses [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Oral immunization induces the local production of secretory IgA antibodies that are essential for the induction of a protective humoral immune response [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In previous studies, oral immunization with the canine distemper virus (CDV) vaccine can produce a significant immune response in mice, a finding that provides a reference for immunizing wildlife with CDV vaccines [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Oral immunization with the \u003cem\u003eClostridium perfringens\u003c/em\u003e vaccine can significantly increase the expression of specific anti-α antibodies and protect chickens from necrotic enteritis disease [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and oral immunization with the Bacille Calmette-Gu\u0026eacute;rin (BCG) and ovalbumin vaccine produce more IgG2a and improve immunity to pathogen infections [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Although oral immunization has many advantages compared to injected parenteral immunization, it should be highlighted that a mucosal delivery system \u003cem\u003evia\u003c/em\u003e oral vaccination administration is essential to protect the vaccine from gastric degradation, enhance antigen absorption in the gastrointestinal tract, trigger adaptive immune responses [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and cross the intestinal epithelial barrier [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. As a result, there is an urgent need for innovative vaccination formulations and delivery systems that can improve efficacy and safety.\u003c/p\u003e \u003cp\u003eNanomaterials are an excellent vaccine delivery vector, of which polymeric nanoparticles (NPs) are a proficient platform for drug oral delivery [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Hence, nanotechnology-based delivery systems have great potential for the development of oral vaccines [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Nanocomposite materials based on chitosan have become attractive in recent years due to their biocompatibility, low toxicity, and biodegradability [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Our lab has synthesized \u003cem\u003eN\u003c/em\u003e-2-Hydroxypropyl trimethyl ammonium chloride chitosan (N-2-HACC) based on the quaternized modification of chitosan, \u003cem\u003eN\u003c/em\u003e,\u003cem\u003eO\u003c/em\u003e-carboxymethyl chitosan (CMCS), and N-2-HACC/CMCS NPs as a nano delivery system [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Our studies have shown that the N-2-HACC/CMCS NPs protect loaded antigens from gastric acid degradation and increase the bioavailability of loaded drugs, and the N-2-HACC/CMCS-based vaccine \u003cem\u003evia\u003c/em\u003e oral administration led to protection against Newcastle disease virus in chickens [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], showing that the N-2-HACC/CMCS NPs have the great potential for vaccine mucosal delivery.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSucralfate has been widely used as a mucosal protective material and for the treatment of peptic ulcers and gastrointestinal discomfort [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It forms a protective barrier to shield the gastric wall from ulcers or damage [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Experimental evidence exists to suggest that sucralfate acidified (SA) can form a thick protective film in the stomach and small intestine [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Thus, we hypothesized that supplementation of SA into nanomaterials would not only help to resist the complex environment of the stomach but also deliver nanoparticles to the gastrointestinal tract to prolong the residence time of nanoparticles in the intestine.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eTo further enhance the ability of N-2-HACC/CMCS NPs to resist the complex gastrointestinal (GI) environment, based on the immune adjuvant activity of N-2-HACC/CMCS nanoparticles and the advantage in resisting harsh gastric conditions of SA, we constructed an oral vaccine delivery system based on SA and N-2-HACC/CMCS nanoparticles (SA@N-2-HACC/CMCS NPs), and the SA@N-2-HACC/CMCS/BSA NPs were formulated to incorporate bovine serum albumin (BSA). In the present study, we extended our previous work by evaluating the adhesion, antigen presenting ability, antigen sustained release and stability in the simulated GI, residence time in the intestine, and immunogenicity of the SA@N-2-HACC/CMCS NPs in a guinea pig model. Oral vaccination with the SA@N-2-HACC/CMCS/BSA NPs induces strong humoral, cell-mediated, and mucosal immunity, thus, the vaccine has great potential for delivering antigens and represents a future direction of mucosal vaccine development \u003cem\u003evia\u003c/em\u003e oral administration.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animals and cell lines\u003c/h2\u003e \u003cp\u003eFemale BALB/c mice and nude mice (6\u0026ndash;8 weeks) were purchased from Changsheng Biotechnology Co., Ltd. (Benxi, Liaoning, China, Permission No. SCXK (Liao) 2020-0002), and male adult guinea pigs (300 g) were purchased from Xianglin Co., Ltd. (Harbin, Heilongjiang, China, Permission No. SCXK (Hei) 2016-002). All animals were maintained under specific pathogen-free conditions and all animal experiments were approved by the Committee of Experimental Animals of Heilongjiang University. All laboratory animal care and experiments followed the \u0026ldquo;National Research Council\u0026rsquo;s Guide for the Care and Use of Laboratory Animals\u0026rsquo;\u0026rsquo;.\u003c/p\u003e \u003cp\u003e293T (Human embryonic cells) was provided from the Northeast Forestry University, and RAW 264.7 (Leukemia cells in mouse macrophage) was provided from Harbin Pharmaceutical Group Biological Vaccine Co., Ltd. 293T and RAW 264.7 were cultured in DMEM/F12 medium (Gibco, NY, USA) supplemented with 10% FBS (Clark, VA, USA) and 1% antibiotics (Gibco, NY, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Synthesis and characterization of sucralfate acidified\u003c/h2\u003e \u003cp\u003eBriefly, 50 mg of sucralfate was dissolved in 1 mL of 1.0 mol/L HCl, and the solution was stirred until appeared gelatinous. Then, the gelatinous was collected and freeze-dried to obtain SA. Thermogravimetry analysis (TGA) was carried out using a Discovery TGA 5500 (Waters, MA, USA). 4 mg of the SA was heated in the temperature range of 30\u0026ndash;300 ℃ with a rate of 5 ℃/min using an N\u003csub\u003e2\u003c/sub\u003e flow of 20 mL/min. ZetaPALS (Brookhaven Instrument, NY, USA) was used to measure the zeta potential of the SA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Synthesis and characterization of the SA@N-2-HACC/CMCS NPs\u003c/h2\u003e \u003cp\u003eSA@N-2-HACC/CMCS NPs were synthesized by the electrostatic adsorption method. Briefly, the N-2-HACC/CMCS NPs were synthesized according to our previous method [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and dissolved in water to a final concentration of 1 mg/mL. Then, 50 mL of the N-2-HACC/CMCS NPs solution (1 mg/mL) was incubated with 2 mg of the SA and gently stirred for 30 min at room temperature to allow conjugation to the SA@N-2-HACC/CMCS NPs. The morphological characteristics of SA@N-2-HACC/CMCS NPs were observed using a JEM-1200EX transmission electron microscope (TEM) (Hitachi, TYK, JPN). Dynamic light scattering and zeta potential were performed on a ZetaPALS (Brookhaven Instrument, NY, USA). The content and distribution of elements on the surface of SA@N-2-HACC/CMCS NPs were characterized by energy dispersive X-ray spectroscopy (EDS) (Bruker AXS, KA, DEU).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Cytotoxicity of the SA@N-2-HACC/CMCS NPs\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. \u003cem\u003eIn vitro\u003c/em\u003e cytotoxicity\u003c/h2\u003e \u003cp\u003eRAW 264.7 was employed to investigate the cytotoxicity of SA@N-2-HACC/CMCS NPs. Briefly, RAW 264.7 was seeded into a 96-well plate at a density of 5\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well and cultured in DMEM/F12 (Gibco, NY, USA) supplemented with 8% fetal bovine serum (Gibco, NY, USA) and 1% penicillin-streptomycin (Gibco, NY, USA) at 37 ℃ under a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e. After 24 h, the culture medium was replaced by DMEM/F12 with different concentrations of SA@N-2-HACC/CMCS NPs (0, 15.625, 31.25, 62.5, 125, 250, 500, and 1000 \u0026micro;g/mL). After being cultured for 24 h, 10 \u0026micro;L of CCK-8 was added into each well, cell viability was measured by using CCK-8 assay (Biyuntian, Shanghai, China), and OD\u003csub\u003e450\u003c/sub\u003e was measured with a microplate reader (Molecular Devices, Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. \u003cem\u003eIn vivo\u003c/em\u003e cytotoxicity\u003c/h2\u003e \u003cp\u003eBefore analyzing the delivery capacity \u003cem\u003ein vivo\u003c/em\u003e, we evaluated the \u003cem\u003ein vivo\u003c/em\u003e cytotoxicity of SA@N-2-HACC/CMCS NPs. The female BALB/c mice were orally administrated with 250 \u0026micro;g of the N-2-HACC/CMCS NPs, 250 \u0026micro;g of the SA@N-2-HACC/CMCS NPs, and saline, respectively. After 12 h, the liver, lung, and spleen of the immunized mice were collected for hematoxylin-eosin (H\u0026amp;E) staining.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Mucoadhesion of the SA@N-2-HACC/CMCS NPs\u003c/h2\u003e \u003cp\u003eThe binding amount of mucin to the SA@N-2-HACC/CMCS NPs was determined by the interaction between the negatively charged mucin (as the mucosal component) and positively charged SA@N-2-HACC/CMCS NPs in an aqueous solution. Briefly, 0.5 mL of mucin (1 mg/mL) (Solarbio, Beijing, CHN) and 0.5 mL of the SA@N-2-HACC/CMCS NPs (4 mg/mL) were incubated at 37 ℃ for 0.5, 1, 2, 4 and 8 h, respectively, and subsequently centrifuged at 12,000 r/min for 15 min. The concentration of free mucin in the supernatant was determined at 560 nm by the periodic acid/Schiff (PAS) colorimetric method [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The mucin binding amount of the SA@N-2-HACC/CMCS NPs was calculated with Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), \u003cem\u003eW\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e is the initial concentration of mucin used for incubation, and \u003cem\u003eW\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e is the concentration of free mucin in the supernatant.\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\text{Mucin binding amount=}W1-W0$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Cellular uptake\u003c/h2\u003e \u003cp\u003eThe cellular uptake of FITC-SA@N-2-HACC/CMCS NPs and FITC-N-2-HACC/CMCS NPs in RAW 264.7 was determined using the inverted fluorescence microscope (Olympus, TKY, JPN). The SA@N-2-HACC/CMCS NPs and nuclei were labeled with FITC (exhibiting green fluorescence) and DAPI (exhibiting blue fluorescence), respectively. The FITC-SA@N-2-HACC/CMCS NPs and FITC-N-2-HACC/CMCS NPs were prepared per the method described previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. RAW 264.7 in the logarithmic growth phase was seeded onto the samples in a 24-well plate with cell climbing at a density of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/well and cultured overnight. The FITC-SA@N-2-HACC/CMCS NPs and FITC-N-2-HACC/CMCS NPs were added to each well at the concentration of 1 mg/mL and incubated for 1, 2, 4, 8, 12, 16, 20 and 24 h, respectively. The cells were then carefully washed with PBS (pH 7.2) to remove any excess NPs before being fixed in 500 \u0026micro;L of 4% paraformaldehyde for 30 min. Next, 4% paraformaldehyde was removed and 100 \u0026micro;L of 0.5% Triton X-100 was added for 30 min to penetrate cells. Finally, the cell climbing was stained with 50 \u0026micro;L of DAPI (0.5 \u0026micro;g/mL), and the cellular uptake of the NPs was observed using the inverted fluorescence microscope (Olympus, TKY, JPN).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Slow-release effect of the SA@N-2-HACC/CMCS NPs\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1. Preparation of the SA@N-2-HACC/CMCS/BSA NPs\u003c/h2\u003e \u003cp\u003eTo examine the slow-release effect of the SA@N-2-HACC/CMCS NPs, BSA was used as a model antigen. The preparation of SA@N-2-HACC/CMCS NPs loaded with BSA (SA@N-2-HACC/CMCS/BSA NPs) was as follows. First, BSA was encapsulated in N-2-HACC/CMCS NPs. The water phase was 3 mL of N-2-HACC/CMCS NPs (1.7 mg/mL), the oil phase was a combination of petroleum ether, liquid paraffin (Yongda Chemical Reagents, Tianjin, CHN) and Span-80 (Zhiyuan Chemical Reagents, Tianjin, China) and two phases were emulsified for 15 min at 600 r/min. Second, 1.5 mL of BSA at various concentrations (1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5 mg/mL) and 1 mL of the CMCS (1.3 mg/mL) were mixed in the water/oil at 600 r/min for 4 min, respectively. The production was cured at 37 ℃ for 3 h. Later, the solution was placed into a centrifuge tube and centrifuged for 30 min at 4 ℃, 12000 r/min, the supernatant was removed, the precipitation was washed four times with petroleum ether, and the N-2-HACC/CMCS/BSA NPs were produced. Finally, the SA@N-2-HACC/CMCS/BSA NPs were prepared by adding 0.1 mg of the SA in 10 mL of the N-2-HACC/CMCS/BSA NPs and gently stirring for 30 min at room temperature. The micro BCA protein assay (Thermo Scientific, Shanghai, China) was used to measure the encapsulation efficiency (EE) and loading capacity (LC) of BSA in the SA@N-2-HACC/CMCS/BSA NPs. The EE and LC are calculated using the formulas (2) and (3), where M\u003csub\u003e0\u003c/sub\u003e is the total weight of BSA, M\u003csub\u003e1\u003c/sub\u003e is the weight of BSA in the supernatant, and M\u003csub\u003eN\u003c/sub\u003e is the total weight of SA@N-2-HACC/CMCS/BSA NPs. The experiments were repeated at least three times.\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\text{E}\\text{E} \\left(\\text{%}\\right) = ({\\text{M}}_{0}-{\\text{M}}_{1})/{\\text{M}}_{0}\\times 100\\text{%}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\text{L}\\text{C} \\left(\\text{%}\\right) = ({\\text{M}}_{0}-{\\text{M}}_{1})/\\left({\\text{M}}_{\\text{N}}\\right)\\times 100\\text{%}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2. \u003cem\u003eIn vitro\u003c/em\u003e release of the SA@N-2-HACC/CMCS/BSA NPs\u003c/h2\u003e \u003cp\u003eBriefly, 100 mg of the SA@N-2-HACC/CMCS/BSA NPs was resuspended in 2 mL of PBS (pH\u0026thinsp;=\u0026thinsp;7.2) and placed at 37 ℃ in a shaker incubator shaking at the speed of 100 r/min. The suspension was collected at 0, 3, 6, 9, 12, 24, 36, 48, 60, and 72 h, respectively, and centrifuged at 12,000 r/min for 10 min. Then, the supernatant was harvested, and the amount of BSA was quantified by the micro BCA protein assay (Thermo Scientific, Shanghai, China). The equivalent volume of fresh PBS was replenished in the original tube for further incubation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.7.3. Stability assay in simulated body fluid of the SA@N-2-HACC/CMCS/BSA NPs\u003c/h2\u003e \u003cp\u003eThe stability of SA@N-2-HACC/CMCS/BSA NPs was conducted in the presence of simulated gastric fluid (SGF) (pH\u0026thinsp;=\u0026thinsp;1.2, Meilian Biotechnology Co., Ltd., Shanghai, China) and simulated intestinal fluid (SIF) (pH\u0026thinsp;=\u0026thinsp;6.8, Meilian Biotechnology Co., Ltd., Shanghai, China). Briefly, the SA@N-2-HACC/CMCS/BSA NPs were put into 2 mL of the SGF at 37 ℃, and the supernatant samples were collected at a specified time (0, 1, and 2 h, respectively), and an equivalent volume of the SGF was replenished in the original tube for further incubation. The supernatant was separated by centrifugation at 12000 r/min for 10 min, and the content of BSA in the supernatant was evaluated using a BCA kit. After 2 h, SGF was discarded by centrifugation at 12000 r/min for 10 min and 2 mL of the SIF was added. The content of BSA in the supernatant was evaluated at specified times (3, 4, 5, 6, 7, and 8 h, respectively).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.8. In vivo trafficking of the SA@N-2-HACC/CMCS/BSA NPs\u003c/h2\u003e \u003cp\u003eTo investigate the \u003cem\u003ein vivo\u003c/em\u003e biodistribution of the SA@N-2-HACC/CMCS/BSA NPs, cyanin 5.5 (Cy5.5) was used to label BSA. The Cy5.5-labeled BSA (BSA-Cy5.5) was prepared as described previously [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Female BALB/c nude mice (6\u0026ndash;8 weeks, 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g) were divided into four groups: Group 1 was oral administration with the saline (negative control), Group 2 was oral administration with the BSA-Cy5.5 (100 \u0026micro;g), Group 3 was oral administration with the N-2-HACC/CMCS/BSA-Cy5.5 NPs (containing 100 \u0026micro;g of the BSA-Cy5.5), and Group 4 was oral administration with the SA@N-2-HACC/CMCS/BSA-Cy5.5 NPs (containing 100 \u0026micro;g of the BSA-Cy5.5). The biodistribution of SA@N-2-HACC/CMCS/BSA NPs was investigated at different times (1, 3, 6, 9, and 12 h, respectively) after administration by a small animal \u003cem\u003ein vivo\u003c/em\u003e imaging system (Maestro CRI, MA, USA). The mice were anesthetized with 0.1 mL of 10% chloral hydrate and observed in a prone position.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Immune effect of the SA@N-2-HACC/CMCS/BSA NPs\u003c/h2\u003e \u003cp\u003eSixty 6\u0026ndash;8 week-old healthy male guinea pigs were fed adaptively for 1 week before the experiment. Then, the guinea pigs were divided into ten groups with 6 guinea pigs in each group: guinea pigs in Group 1 to Group 5 were injected intramuscular immunization (i.m.) with 0.2 mL of the PBS, free BSA (200 \u0026micro;g BSA), Seppic ISA15/BSA (containing 200 \u0026micro;g BSA), N-2-HACC/CMCS/BSA NPs (containing 200 \u0026micro;g BSA), and SA@N-2-HACC/CMCS/BSA NPs (containing 200 \u0026micro;g BSA), respectively; guinea pigs in Group 6 to Group 10 were immunized oral administration with 0.2 mL of PBS, free BSA (200 \u0026micro;g BSA), Seppic ISA15/BSA (containing 200 \u0026micro;g BSA), N-2-HACC/CMCS/BSA NPs (containing 200 \u0026micro;g BSA), and SA@N-2-HACC/CMCS/BSA NPs (containing 200 \u0026micro;g BSA), respectively. The booster immunization was given in the same manner at 14 days post the immunization.\u003c/p\u003e \u003cp\u003eThe blood samples were collected by heart puncture prior to priming and at 7, 14, 21, 28, 42, and 56 days post the immunization. Following coagulation, samples were incubated at 37 ℃ for 1 to 2 h and centrifuged at 3000 r/min for 10 min, and the obtained sera were stored at 80 ℃ until further analysis. The content of serum IgG, IgG1, IgG2a, IL-2, IL-4, IL-12, and IFN-γ was measured using the mouse ELISA Kit (Meilian Biotechnology Co., Ltd., Shanghai, China).\u003c/p\u003e \u003cp\u003eThe fecal pellets were also collected from each guinea pig, and diluted in PBS (pH\u0026thinsp;=\u0026thinsp;7.2) up to a final concentration of 1 g dry matter/mL. The diluted fecal pellets were homogenized and centrifuged at 3000 r/min for 10 min. The supernatants were collected and stored at 80 \u0026ordm;C until assayed. The levels of IgA and sIgA in the fecal pellets were determined using the mouse ELISA Kit (Meilian Biotechnology Co., Ltd., Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe mean and standard deviation (SD) were used to represent the data. One-way analysis of variance (ANOVA) was performed to assess differences between groups, and Bonferroni's post hoc test was employed for multiple comparisons. \u003cem\u003ep\u003c/em\u003e values less than 0.05 were deemed statistically significant, whereas \u003cem\u003ep\u003c/em\u003e values less than 0.01 were deemed very significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characterization of the sucralfate acidified\u003c/h2\u003e \u003cp\u003eTGA curves of the sucralfate and SA were presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. The sucralfate demonstrated low weight loss at 150\u0026ndash;250 ℃, and SA demonstrated high weight loss at 150\u0026ndash;250 ℃, which was likely due to the loss of lattice water. SA converted hydroxide to lattice water compared to the sucralfate, proving that the sucralfate had been successfully acidified. The zeta potential of SA was \u0026minus;\u0026thinsp;5.86\u0026thinsp;\u0026plusmn;\u0026thinsp;4.24 mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), and the negative charge could facilitate subsequent electrostatic adsorption to form NPs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Characterization of the SA@N-2-HACC/CMCS NPs\u003c/h2\u003e \u003cp\u003eThe elements on the surface of SA@N-2-HACC/CMCS NPs were S, C, H, O, and Al (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), comparing the elements on the surface of N-2-HACC/CMCS NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), indicating the presence of the SA on the surface of N-2-HACC/CMCS NPs. The SA@N-2-HACC/CMCS NPs were spherical in nature and mono-dispersed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The SA@N-2-HACC/CMCS NPs had a particle size of 227\u0026thinsp;\u0026plusmn;\u0026thinsp;7.0 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) and a zeta potential of 8.43\u0026thinsp;\u0026plusmn;\u0026thinsp;2.62 mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Cytotoxicity of the SA@N-2-HACC/CMCS NPs\u003c/h2\u003e \u003cp\u003eWhen the concentration of N-2-HACC/CMCS NPs reached 1000 \u0026micro;g/mL, the cell viability reached 84.24\u0026thinsp;\u0026plusmn;\u0026thinsp;8.64% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), with no significant difference compared to the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). When the concentration of the SA@N-2-HACC/CMCS NPs reached 1000 \u0026micro;g/mL, the cell viability had an extremely significant difference compared with the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), but it still was still as high as 86.01\u0026thinsp;\u0026plusmn;\u0026thinsp;2.38% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The higher cell viability indicated that the SA@N-2-HACC/CMCS NPs has lower cytotoxicity.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e cytotoxicity of the SA@N-2-HACC/CMCS NPs showed that no significant difference in tissue integrity and cell structure was found in the N-2-HACC/CMCS NPs, SA@N-2-HACC/CMCS NPs and control groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating that the SA@N-2-HACC/CMCS NPs had no cytotoxicity on tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4. In vitro mucoadhesive strength\u003c/h2\u003e \u003cp\u003eThe amount of mucin-binding in each group increased significantly, as the incubated time went. After 8 hours of incubation, the mucin-binding value of the SA@N-2-HACC/CMCS NPs was 150.3 \u0026micro;g, showing a significantly higher mucin-binding value compared with that of the N-2-HACC/CMCS NPs, (72.6 \u0026micro;g, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating that the introduction of SA improved the binding efficiency of N-2-HACC/CMCS NPs to mucin. As a consequence, the small intestine residence time, which is an essential prerequisite for effective mucosal delivery of therapeutics, was increased.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Cellular uptake\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the cellular uptake of the two NPs was shown to increase with treatment time. Furthermore, the intensity of FITC fluorescence exhibited by the FITC-SA@N-2-HACC/CMCS NPs was significantly greater than that of the FITC-N-2-HACC/CMCS NPs at all time points, indicating that RAW 264.7 could better absorb the FITC-SA@N-2-HACC/CMCS NPs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.6. In vitro\u003c/em\u003e release of the SA@N-2-HACC/CMCS/BSA NPs\u003c/h2\u003e \u003cp\u003eWhen the concentration of BSA was 4 mg/mL, EE and LC were highest, and they were 96.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12% and 41.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05% (n\u0026thinsp;=\u0026thinsp;3), respectively. Thus, 4 mg/mL of BSA was selected as the optimal concentration for the preparation of the SA@N-2-HACC/CMCS/BSA NPs.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e release of BSA from the SA@N-2-HACC/CMCS/BSA NPs at PBS solution (pH 7.2) was in a sustained behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), the release behavior was faster at prometaphase, and 81.18\u0026thinsp;\u0026plusmn;\u0026thinsp;4.34% of BSA was released from the SA@N-2-HACC/CMCS/BSA NPs at 72 h. A sustained release pattern could improve the duration of antigen post the immunization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Stability of the SA@N-2-HACC/CMCS/BSA NPs\u003c/h2\u003e \u003cp\u003eThe SA@N-2-HACC/CMCS/BSA NPs were tested for stability in SGF (pH 1.2) for the first 2 h and then in SIF (pH 6.8) for a further 6 h to simulate the GI environment without enzyme. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, the SA@N-2-HACC/CMCS/BSA NPs exhibited sustained release, and BSA in the SA@N-2-HACC/CMCS/BSA NPs released 24.05\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41% for the first 2 h in SGF and finally released 34.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05% for another 6 h in SIF. The N-2-HACC/CMCS/BSA NPs showed significantly faster release in SGF, and BSA released 59.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85% for the first 2 h and finally released 63.09\u0026thinsp;\u0026plusmn;\u0026thinsp;2.50% for another 6 h in SIF, indicating that the SA@N-2-HACC/CMCS/BSA NPs were mainly released in intestinal fluid and rarely in gastric fluid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.8. \u003cem\u003eIn vivo\u003c/em\u003e trafficking of the SA@N-2-HACC/CMCS/BSA NPs\u003c/h2\u003e \u003cp\u003eWe traced the biodistribution of the SA@N-2-HACC/CMCS/BSA NPs after oral administration in nude mice by IVIS imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). After the oral administration for 1 h, the fluorescence of BSA was widely distributed in the mouse intestine of all administration groups. After the oral administration of free BSA for 3 h, BSA was clearly visualized, but the signal of BSA decreased and became exceedingly weak by 12 h, indicating that the free BSA was quickly cleared from the intestines. For the N-2-HACC/CMCS/BSA NPs and SA@N-2-HACC/CMCS/BSA NPs, relatively high fluorescence in the intestine was sustained for over 12 h, and the fluorescence was stronger than that of the N-2-HACC/CMCS/BSA NPs (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). This high accumulation ability of BSA in the intestine could be attributed to the enhanced permeability and retention effect of the SA@N-2-HACC/CMCS/BSA NPs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.9. Immune effect of the SA@N-2-HACC/CMCS/BSA NPs\u003c/h2\u003e \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e \u003ch2\u003e3.9.1. SA@N-2-HACC/CMCS/BSA NPs elicit immune responses after intramuscular administration\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, the SA@N-2-HACC/CMCS/BSA NPs induced higher IgG antibody levels on 14 d than the other formulations post the primary immunization. The SA@N-2-HACC/CMCS/BSA NPs clearly increased IgG1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) and IgG2a (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) antibody titers compared to BSA alone or PBS (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), which indicated that the SA@N-2-HACC/CMCS/BSA NPs elicited more effective immune responses.\u003c/p\u003e \u003cp\u003eCompared with the PBS and BSA alone groups, the level of IL-4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) in the serum of guinea pigs immunized with the SA@N-2-HACC/CMCS/BSA NPs was significantly increased at 7 d post the immunization (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), the level of IL-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE) and IL-12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF) was significantly increased at 14 d post the immunization (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the level of IFN-γ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG) was significantly increased at 21 d post the immunization (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The findings revealed that intramuscular delivery of SA@N-2-HACC/CMCS/BSA NPs caused higher cellular and humoral immune responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003e3.9.2. SA@N-2-HACC/CMCS/BSA NPs elicit immune responses after oral administration\u003c/h2\u003e \u003cp\u003eCompared with the PBS, free BSA and Seppic ISA15/BSA groups, the serum IgG (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), IgG1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) and IgG2a (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC) antibody titers in the SA@N-2-HACC/CMCS/BSA NPs and N-2-HACC/CMCS/BSA NPs groups were significantly higher at 14 d post the immunization (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that the SA@N-2-HACC/CMCS/BSA NPs and N-2-HACC/CMCS/BSA NPs produced stronger immune responses by oral administration.\u003c/p\u003e \u003cp\u003eCompared with the PBS, BSA and Seppic ISA15/BSA groups, the production of IL-4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD) significantly increased in the guinea pigs immunized with the SA@N-2-HACC/CMCS/BSA NPs and N-2-HACC/CMCS/BSA NPs at the 7 d post the immunization (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the production of IL-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE), IL-12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF), and IFN-γ (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG) significantly increased at the 14 d post the immunization (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, the level of IL-12 in the guinea pigs immunized with the SA@N-2-HACC/CMCS/BSA NPs was higher at 14 d post the immunization than that of the N-2-HACC/CMCS/BSA NPs group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and extremely significantly higher than that of PBS, BSA and Seppic ISA15/BSA groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The results showed that oral delivery of SA@N-2-HACC/CMCS/BSA NPs increased cytokine release, resulting in a stronger cellular immune response. Compared with the PBS, free BSA, and Seppic ISA15/BSA groups, the sIgA antibody titers in the fecal pellets of the guinea pigs immunized with the SA@N-2-HACC/CMCS/BSA NPs were significantly higher at 14 d post the immunization (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The results indicated that the SA@N-2-HACC/CMCS/BSA NPs induced a stronger mucosal immunity response by oral administration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe mucosa and intestine are exposed to large amounts of antigens, including harmful pathogenic microorganisms [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Thus, mucosal immunization may provide an effective means of preventing infectious agents [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Among the multiple routes of mucosal immunization, oral administration is more readily accepted because of its more convenient mode of immunization [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe emergence of nanomaterials with high biosafety and biodegradability has provided an opportunity for oral vaccines [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. They can act as carriers to protect antigens from degradation by proteases, enhance the residence time of antigens in the gastrointestinal mucosa, and increase the number of antigens through the upper mucosal cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In this study, an antigen delivery system based on the SA and N-2-HACC/CMCS for oral administration through drinking water or feed was developed and tested in guinea pigs.\u003c/p\u003e \u003cp\u003eFollowing oral administration, the first problem faced by antigens is the harsh GI environment, where antigens are exposed to host-imposed stresses, especially the low pH in the GI (pH\u0026thinsp;=\u0026thinsp;1.5) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Recent studies have demonstrated that a variety of nanocarriers can protect antigens from degradation in the harsh GI environment [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Upon overcoming the low pH environment of GI, the oral vaccine is also faced with the barrier effect of a continuously secreted mucus layer. Additionally, the antigens adhere less readily to the mucus layer and are easily washed away by the mucus layer [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, substances with adhesive properties can contribute to the design of a delivery system to overcome the mucus layer barrier in oral immunization and to prolong the retention of antigens in the mucosal layer. In this study, we have demonstrated that the NPs prepared using SA and chitosan derivatives are highly resistant to low pH in the GI and have good mucosal adhesion as an oral vaccine delivery system. Our findings are consistent with those of other researchers, demonstrating that the SA@N-2-HACC/CMCS/BSA NPs enhance the adhesion and antibody protection of the vaccine delivery system [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In addition, chitosan derivatives have a strong potential to open the tight junctions between epithelial cells, thereby improving the chance of antigen contact with immune cells and bioavailability [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Therefore, we consider the SA@N-2-HACC/CMCS NPs to have significant potential as an oral vaccine delivery system.\u003c/p\u003e \u003cp\u003eAs a vaccine delivery system, NPs are expected to have a good safety profile and efficient adjuvant activity [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The safety of SA@N-2-HACC/CMCS NPs was investigated by \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e cytotoxicity evaluation, and the results showed that the NPs had higher compatibility. To achieve immune function, the antigens have to reach the antigen-presenting cells (APCs), which makes the antigen-presenting role of adjuvants essential [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this study, we proved that the SA@N-2-HACC/CMCS NPs could be effectively uptaken by RAW264.7. Similarly, our previous studies indicate that chitosan derivative NPs have an antigen-presenting effect [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Therefore, we believe the SA@N-2-HACC/CMCS NPs to be a safe delivery carrier with adjuvant activity.\u003c/p\u003e \u003cp\u003eThe release of antigens encapsulated in the nanomaterials is a key feature in the evaluation of a vaccine delivery system, as slow release increases the bioavailability of antigens and thus enhances the immune response [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The N-2-HACC/CMCS/BSA NPs prolonged the release time of BSA encapsulated in the N-2-HACC/CMCS NPs and achieved a sustained antigen-release effect compared to free BSA. Moreover, it was confirmed that the SA@N-2-HACC/CMCS NPs prolonged the residence time of BSA in the intestine, showing that chitosan derivative NPs have a sustained release effect, and these are consistent with those of previous findings [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Additionally, SA has a strong mucoadhesion in the GI [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Thus, we consider the SA@N-2-HACC/CMCS NPs to be a promising oral vaccine delivery system for the sustained release of antigens.\u003c/p\u003e \u003cp\u003eIL-2, IL-12, and IFN-γ are preferentially called Th1-type cytokines, while IL-4 is generally regarded as Th2-type cytokines [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In the study, the SA@N-2-HACC/CMCS/BSA NPs induced the expression of IL-2, IL-12, IFN-γ, and IL-4, suggesting that the NPs can induce mixed Th1 and Th2 type immune responses. The results are consistent with the previous studies of chitosan derivatives NPs as a potential adjuvant to enhance both humoral and cellular immune responses [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Nanovaccine, delivered orally, induces both local and systemic immunity [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The SA@N-2-HACC/CMCS/BSA NPs by oral administration in guinea pigs enhanced the levels of local sIgA in the intestine and serum IgG antibody, which is consistent with the oral delivery of measles antigen-loaded chitosan NPs to enhance intestinal IgA antibody titers in mice [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eDespite no significant increase of SA@N-2-HACC/CMCS/BSA NPs observed during the detection of cytokines and antibody levels post-immunization, compared to the N-2-HACC/CMCS/BSA NPs (but both were notably higher than the control group), the sustained release of SA@N-2-HACC/CMCS/BSA NPs \u003cem\u003ein vitro\u003c/em\u003e and their prolonged retention \u003cem\u003ein vivo\u003c/em\u003e were significantly superior to the group N-2-HACC/CMCS/BSA NPs. Therefore, we firmly believe that employing the SA@N-2-HACC/CMCS NPs delivery system for viral immunization will indeed further enhance the ability to combat viruses. However, at present, we lack sufficient evidence to support this viewpoint, which constitutes a limitation of this article. Moving forward, we will utilize this delivery system for viral transport in further research.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eCollectively, in the present study, we developed an oral vaccine delivery vector based on SA and N-2-HACC/CMCS nanoparticles (SA@N-2-HACC/CMCS NPs) and prepared BSA encapsulated in the nanocarrier. We extended our previous work by investigating the adhesion, antigen presenting ability, antigen sustained release and stability in the simulated GI, residence time in the intestine, and immunogenicity of the SA@N-2-HACC/CMCS NPs in a guinea pig model. Our results showed that the SA@N-2-HACC/CMCS/BSA NPs had good biocompatibility, stability, mucosal adhesive, adjuvant activity, and sustained release. The guinea pigs orally vaccinated with the SA@N-2-HACC/CMCS/BSA NPs exhibited significantly higher serum IgG, IgG1 and IgG2, mucosal IgA, and higher levels of IL-4, IL-2, IL-12, and IFN-γ immune responses. These findings highlighted the great potential of chitosan derivative based nanoparticle vaccine as an alternative approach that represents a future direction for developing an oral vaccine for use in livestock and poultry.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eN-2-HACC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003cem\u003eN\u003c/em\u003e-2-hydroxypropyl trimethyl ammonium chloride chitosan\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCMCS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eO\u003c/em\u003e-carboxymethyl chitosan\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Sucralfate acidified\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSA@N-2-HACC/CMCS NPs \u0026nbsp; SA and\u0026nbsp;N-2-HACC/CMCS\u003cem\u003e\u0026nbsp;\u003c/em\u003enanoparticles\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCDV\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Canine distemper virus\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBSA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; Bovine serum albumin\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTGA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Thermogravimetry analysis\u003c/p\u003e\n\u003cp\u003eTEM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Transmission electron microscope\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEDS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Energy dispersive X-ray spectroscopy\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEE\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Encapsulation efficiency\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Loading capacity\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSGF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp; Simulated gastric fluid\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSIF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp;Simulated intestinal fluid\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the \u0026ldquo;Pioneer\u0026rdquo; and \u0026ldquo;Leading Goose\u0026rdquo; R\u0026amp;D Program of Zhejiang (2022C02031), Zhejiang Provincial Key R\u0026amp;D Program of China (2021C02049),\u0026nbsp;Natural Science Foundation of Heilongjiang Province (LH2020H129) and Taizhou Science and Technology Plan Project in 2023 (23gya02).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKai Zhao designed the study;\u0026nbsp;Shuai Qiao\u0026nbsp;searched the literature and performed the experiments; Kai Zhao,\u0026nbsp;Zhi Zhao, and Shuai Qiao\u0026nbsp;analyzed the data; Kai Zhao and\u0026nbsp;Zhi Zhao\u0026nbsp;wrote the original draft;\u0026nbsp;Zhi Zhao,\u0026nbsp;Zheng Jin, and Chunjing Zhang organized the figure data; Kai Zhao,\u0026nbsp;Zhi Zhao,\u0026nbsp;Tan Hui Yin,\u0026nbsp;and Chunjing Zhang\u0026nbsp;revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analysed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal\u0026nbsp;protocols\u0026nbsp;were approved by the Committee of Experimental Animals of Heilongjiang University. All laboratory animal care and experiments followed the \u0026ldquo;National Research Council\u0026rsquo;s Guide for the Care and Use of Laboratory Animals\u0026rsquo;\u0026rsquo;.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the co-authors were aware of this submission and approved for publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang X, Sun J, Wan L, Yang X, Lin H, Zhang Y, He X, Zhong H, Guan K, Min M, Sun Z, Yang X, Wang B, Dong M, Wei C. The \u003cem\u003eShigella\u003c/em\u003e type III secretion effector IpaH4.5 targets NLRP3 to activate inflammasome signaling. Front Cell Infect Microbiol. 2020;10:511798.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYasmeen F, Seo H, Javaid N, Kim MS, Choi S. Therapeutic interventions into innate immune diseases by means of aptamers. 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Carbohydr Polym. 2015;121:403\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Vaccine delivery system, chitosan derivative based nanoparticles, acidified sucralfate, mucosal immunity, oral administration","lastPublishedDoi":"10.21203/rs.3.rs-3841170/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3841170/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOral administration of vaccine is required to preserve the vaccine against degradation, enhance antigen absorption in the gastrointestinal tract, and trigger adaptive immune responses. Nanomaterials are an ideal delivery vector for the creation of oral vaccines, and we have previously shown that \u003cem\u003eN\u003c/em\u003e-2-hydroxypropyl trimethyl ammonium chloride chitosan (N-2-HACC)/\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eO\u003c/em\u003e-carboxymethyl chitosan (CMCS) based vaccine \u003cem\u003evia\u003c/em\u003e oral administration led to protection against Newcastle disease virus. Hence, based on the immune adjuvant activity of N-2-HACC/CMCS nanoparticles and the advantage in resisting harsh gastric conditions of sucralfate acidified (SA), we constructed an oral vaccine delivery system based on SA and N-2-HACC/CMCS nanoparticles (SA@N-2-HACC/CMCS NPs), and the NPs were formulated to incorporate BSA. The SA@N-2-HACC/CMCS NPs had a particle size of 227\u0026thinsp;\u0026plusmn;\u0026thinsp;7.0 nm and a zeta potential of 8.43\u0026thinsp;\u0026plusmn;\u0026thinsp;2.62 mV. The NPs displayed slow and sustained release and high stability in simulated gastric juice and intestinal fluid. RAW 264.7 could better uptake the SA@N-2-HACC/CMCS/BSA NPs. The vaccine \u003cem\u003evia\u003c/em\u003e oral administration markedly enhanced the residence time of BSA in the intestine for more than 12 h and elicited the production of IgG and sIgA. The SA@N-2-HACC/CMCS NPs developed here for oral administration is an excellent technique for delivering antigens and provides a path of mucosal vaccine research.\u003c/p\u003e","manuscriptTitle":"Oral immunization with acidified sucralfate@N-2-HACC/CMCS NPs elicits protective immune response in guinea pigs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-10 11:34:09","doi":"10.21203/rs.3.rs-3841170/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"419d3da8-ec82-4857-9088-93e2875be2da","owner":[],"postedDate":"January 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-26T21:59:09+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-10 11:34:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3841170","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3841170","identity":"rs-3841170","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}