Preparation of spleen aminopeptide-loaded pH- responsive oral hydrogel microspheres for improving recurrent respiratory infection in children

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Scientific Reports

Aqin Wang1, Zhengbing Zhou2, Zhou Hang3, Zhengbing Lyu4, Jingchun Yang5, Lu Zhang6, Meng Wang6, Wei Liu1,7, Liqiang Zou1,7 & Jun Yu5

Direct oral intervention of spleen aminopeptide (SA) would be limited because of its varied swelling and release rate in different pH environments of digestive fluids. Herein, pH-responsive hydrogel microspheres (3.67 ± 0.04 mm) with positive SA-loaded chitosan (Cs) core and negative alginate (ALG) shell were prepared. Entrapment efficiency was 76.22% and loading rate of SA was 35.17%. Besides the hydrogen bonding, a strong core/shell structure was constructed by forming imine bond via the Schiff base reaction of amino group inCs with ketone group on SA. In vitro simulated digestion showed that the swelling rate of microspheres was 192.8% (pH 1.2), 2481.5% (pH 6.8), and 1144.3% (pH 7.4). The release rate of SA was 0.9% in simulated gastric fluid for 2 h, 6.0% in simulated intestinal fluid for 2 h and continuous release in simulated colonic fluid for 24 h with a cumulative release rate of 92.4%. After treatment, the observation group exhibited increasing levels of CD3+, CD4+, CD4+/CD8+, IgA, IgG and IgM. Total effective rate in observation group was 96.15%, being higher than that in control group (84.31%). This work provided a valuable reference for preparing drug-loaded hydrogels to improve the recurrent respiratory infection in children.

Keywords Spleen aminopeptide, pH-responsive hydrogel, Core/shell structure, Controllable release, Recurrent respiratory infection

As a commonly encountered disease in pediatric outpatient department, recurrent respiratory tract infection presented a high incidence rate for children aged 6 months to 6 years. The causes of this disease are complex, and it is clinically believed that it is mostly related to factors such as immune deficiency and malnutrition 1,2 . Clinical treatment of this disease is mainly relied on the symptomatic treatment including relieving cough, resolving phlegm and anti-infection. Children with recurrent respiratory tract infection are routinely treated with anti- infection and cough relieving, which can relieve clinical symptoms and control the development of the disease through anti-virus and antibacterial effects 3 . However, it is difficult to treat this disease because of its repeated attacks, which arouses the drug resistance. The role of drugs would decline, and the treatment effect would be affected. Therefore, the improvement of therapeutic effect by regulating and enhancing the immunity to reduce the number of disease attacks and the impact on children’s health would be desirable to treat recurrent respiratory tract infections 4,5.

1State Key Laboratory of Food Science and Resources, Nanchang University, Nanchang 330047, China. 2Shaoxing Jiawin Biotechnology Co., Ltd., Shaoxing 312300, China. 3Chongqing Hechuan District Integrated Traditional Chinese and Western Medicine Hospital, Chongqing 401520, China. 4School of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, China. 5Hangzhou Bibau Biotechnology Co., Ltd., Hangzhou 310000, China. 6Beijing Key Laboratory of the Innovative Development of Functional Staple and Nutritional Intervention for Chronic Diseases, China National Research Institute of Food and Fermentation Industries Co., Ltd., Beijing 100015, China. 7International Institute of Food Innovation, Nanchang University, Nanchang 330047, China. email: yuj@bibau.com.cn

The main raw material of spleen aminopeptide (SA) is the fresh pig or bovine spleen, from which peptides, nucleotides and amino acids are extracted. SA is a suitable adjuvant substance to treat multiple types of infectious, autoimmune and tumor diseases 6 . Studies have shown that the use of SA could increase the T lymphocyte count of tumor patients undergoing radiotherapy and chemotherapy and reduce the incidence of infection. The usage of SA to treat children with recurrent respiratory tract infection could increase lymphocyte count and relieve clinical symptoms 7 . The oral use of SA without the need for complex formulations is an existing clinical procedure in pediatric patients, as demonstrated by recent works 8,9 . Oral freeze-dried powder ofSA based anti- infection treatment of recurrent respiratory tract infection significantly improved clinical efficacy and increased the levels of IgM, IgG and IgA for children’s blood. Meanwhile, SA could enhance the number of CD4+, CD8+ and the ratio of CD4+/CD8+, and reduce the number of recurrent respiratory infections in children10.

For the administration of medicine, oral administration is a convenient, common, painless and most acceptable way. The site of drug action is gastrointestinal tract, including gastric acid, degrading enzyme, mucus layer, epithelial barrier and basement membrane. However, due to the special environment of distal part of digestive tract including different enzyme degradation and wide pH range (pH 1.2–8.3), the effective delivery and curative effect of drugs would be affected 11,12 . Stomach and small intestine are the mainly positions to release and adsorb pharmaceuticals which were taken through ordinary approaches, inducing a small amount of pharmaceuticals to reach colons. This makes focus areas unable to reach the effective drug concentration. Rectal administration can rarely make drugs reach the lesion site of colon, and problems such as uneven distribution of drugs and heterogeneity of intervention would be inevitable, leading to the unsatisfactory therapeutic effect 13,14 . The colon accounts for only 6% of the average mucosal surface area of the gastrointestinal tract. The colon (approximately area of 2 m2 and total length of 90–150 cm) presents no villi, and its epithelium is covered by a double-layered mucus composed of water, electrolytes, lipids, and glycoproteins 15 . The thickness of these colonic mucus layers varies by region, generally ranging from 400 to 600 μm. The colon has relatively low metabolic enzymes and transport proteins, which offers certain advantages in enhancing drug bioavailability 16 . For instance, simvastatin delivered to the colon via a sustained-release formulation exhibited a significantly higher oral bioavailability, which was approximately three times higher than that of immediate-release formulation 17.

The lower proteolytic activity in the colon may also be beneficial for the delivery of biological agents, such as proteins, peptides, and monoclonal antibodies. pH-dependent drug delivery methods are designed based on the fact that the pH varies in different parts of the gastrointestinal tract. Thus, the pH changes in the gastrointestinal tract for targeted drug delivery can be achieved by using intestinal polymer coatings. These coatings can be disintegrated and dissolved in response to pH changes in the gastrointestinal tract, allowing drugs to be released specifically in certain regions of the intestine 18.

Hydrogels are ideal candidates in the field of drug delivery because of their high water content, high elasticity, softness, biocompatibility and biodegradability 19 . Given their porous structure and physicochemical properties, hydrogels present a high drug loading capacity and excellent retention performance as a drug carrier 20 . Moreover, hydrogels have exhibited intelligent response characteristics, which can realize accurate drug controlled release. Hydrogels can respond to the environmental changes (e.g., pH fluctuation, redox state change and temperature difference) and trigger the drug release through the degradation or phase change mechanism 21 . Recently, hydrogels based on polysaccharides such as chitosan and sodium alginate (ALG) have being a promising choice for drug delivery carriers. Chitosan is a natural nontoxic polysaccharide, which has good biodegradability and biocompatibility and strong mucoadhesion 22,23 . Whereas, chitosan is easy to be damaged by acidic gastric environment, which makes free amino groups protonated. Therefore, chitosan usually needs a pH sensitive material to prevent it from being affected by acidic environment 24 – 26 . ALG is a natural non- toxic anionic polysaccharide, which is often used in oral drug delivery system. ALG can cross-link with Ca2+ to form hydrogel under mild conditions. Compared with single-layer hydrogel microspheres, core–shell hydrogel microspheres have exhibited multiple advantages, which can reduce the excessive release of drugs in the initial stage and improve the encapsulation efficiency 27 . Therefore, core–shell hydrogel microspheres are expected to be an ideal choice for drug delivery systems.

In this study, pH-responsive SA-loaded chitosan core/ALG shell (SA/Cs/ALG) hydrogel microspheres were prepared. By using scanning electron microscopy (SEM), stereomicroscopy, particle size analyzer, zeta potential and Fourier transform infrared spectrum (FT-IR), the morphologies, structure and surface characteristics of chitosan, SA/Cs and SA/Cs/ALG hydrogel microspheres were analyzed. The encapsulation and loading rate of SA were evaluated. pH response characteristics, swelling and drug release of microspheres under different pH conditions were investigated by simulated digestion in vitro (i.e., simulated gastric fluid, simulated intestinal fluid and simulated colonic fluid). 103 children with recurrent respiratory tract infections admitted to The First Affiliated Hospital of Nanchang University from February 2023 to January 2025 were selected to evaluate the therapeutic effect of oral SA/Cs/ALG hydrogel microspheres in the clinical treatment of recurrent respiratory tract infections of children.

Materials and methods

Materials

Spleen aminopeptide (SA, >80% of purity and the average molecular weight of SA chosen in this study was 35,000) was provided by Shaoxing Jiawin Biotechnology Co., Ltd. (Shaoxing 312300, China). Acid soluble Cs (≥ 95% of deacetylation degree, and 100–200 mpa.s of viscosity), ninhydrin, ethylene glycol monomethyl ether, sodium borohydride, sodium acetate, glacial acetic acid, ALG, and ethanol were purchased from Aladdin Scientific Corporation (China). Colonic, intestinal and gastric fluids with simulated components and concentrations were obtained from Shanghai Yuanye Biological Science and Technology Co., Ltd. All the chemicals were of analytical grade and used without further purification. Deionized (DI) water (18.2 MΩ.cm) was used in following experiments.

Synthesis of SA/Cs/ALG hydrogel microspheres

Preparation of SA/Cs hydrogel microspheres

The preparation of Cs-based hydrogel microspheres were performed according to previous reported literatures 28 – 30 . SA (500.0 mg) was dissolved in ethanol and put on a vortex instrument for 2.0 min to prepare SA ethanol solution. Cs colloidal solution (0.4%) was prepared by dissolving Cs (0.4 g) powder in acetic acid solution (1.0% and 10.0 mL) at pH 5.0. SA/Cs colloidal solution with stable combination and uniform distribution was formed after mixing SA solution with Cs colloidal solution evenly by a continuous stirring of 8 h. Then the above colloidal solution was dripped into a sodium tripolyphosphate solution (2.5%) with the assistance of an electronic digital display constant current pump (DHL-A, Chuding Instrument, Shanghai China) at a uniform speed of 5.0 rpm. The needle of the dropper was kept 2.0 cm above the liquid level. After soaking for 40.0 min, SA/Cs hydrogel microspheres with good roundness, round shape and stable structure were obtained.

Synthesis of SA/Cs/ALG hydrogel microsphere

SA/Cs hydrogel microspheres were soaked in CaCl2 solution (3.0%) for 2.0 min. ALG solution (2.0%) was added to cross-link for 40 min. Then excessive colloids were removed from hydrogel microsphere surfaces via the thorough rinsing with DI water.

Characterization

Morphological recorded was carried out using a scanning electron microscopy (SEM, Regulus 8100, Hitachi Japan). Before the SEM observation, dried samples were attached onto the surface of conductive adhesive and sprayed with a gold coating using a gold spraying instrument (JFL-160, Japan Electronics). The surface potential of each sample was measured for 3 times by a potential measuring instrument (Zetasizer Nano ZS90, Malvern Panalytical Instrumentation). Dried samples of chitosan, ALG, SA/Cs, Cs/ALG and SA/Cs/ALG were put in an agate mortar and mixed with KBr, respectively. After being tableted to obtain transparent sheets, Fourier infrared spectrometer (FTIR) was used to measure the infrared spectrum of the prepared sheet. KBr was used as a blank control, and the temperature environment was set at 25℃. The wave number was set at 4000–400 cm-1 for infrared spectrum absorption and the scanning times were 64 times.

Determination of amino acids and peptides

The concentrations of amino acids and peptides were measured using an amino acid analyzer (Hitachi L-8900) with a pre-installed ion exchange column. The flow rates of buffer and ninhydrin were 0.4 and 0.35 mL/ min, respectively. Separation and reaction column temperatures were 57℃ and 135℃, respectively. On-line derivatization was carried out with detection wavelength of 570 nm in Channel 1 and the acquisition time of 32 min. The acquisition time was 440 nm in Channel 2 (proline detection) and the acquisition time was 10 min. The injection volume is 20 μL. The mixed reference solution and test solution were measured. Specifically, the ninhydrin solution was prepared by mixing 979 ml of ethylene glycol monomethyl ether, 39 g of ninhydrin and 110 mg of sodium borohydride. Buffer solution was prepared by mixing 336 ml of DI water, 204 g of sodium acetate, 123 ml of glacial acetic acid and 401 ml of ethylene glycol monomethyl ether.

Release performance of hydrogel microspheres

Determination of encapsulation efficiency and loading capacity

Hydrogel microspheres (0.2 g) were added into absolute ethanol solution (1.0 mL), and separated using centrifugation. After repeating three times, the supernatants were combined and swirled evenly. The concentration of amino acids and peptides were measured using an amino acid analyzer (Hitachi L-8900) with a pre-installed ion exchange column. Encapsulation efficiency (%) = (1-E2/(E1 + E2)) × 100%, where E1 was the mass ofSA in hydrogel microspheres and E was the mass of free SA on the surface of hydrogel microspheres

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Hydrogel microspheres (0.2 g) were added into absolute ethanol solution (1.0 mL) and mixed with two grinding beads. Hydrogel microspheres were crushed in a high-speed tissue grinder at 60.0 Hz and 3.0 min. Then the suspension was extracted by sonicating (40.0 Hz) for 8 h. After centrifuging at 10,000 rpm for 5.0 min, collected supernatant was vortexed evenly. The concentration of amino acids and peptides were measured. Loading capacity (%) = E1/E0 × 100%, where E0 was the total mass of hydrogel microspheres. For the short-term preservation, the final hydrogel microspheres were preserved in a sterile centrifuge tube. The tube was filled with physiological saline until completely submerged, sealed and refrigerated at 2–8 °C. For the long-term preservation, the hydrogel microspheres were freeze-dried (pre-frozen at − 80 °C for 4 h and sublimated for 24 h) to obtain a solid. It was sealed and stored at − 20 °C. Before use, hydrogel microspheres were re-dissolved in PBS buffer (30 min in a 37 °C water bath).

Swelling and pH sensitivity test

The swelling capacity of SA/Cs/ALG was evaluated in simulated gastric fluid (SGF, pH 1.2), simulated intestinal fluid (SIF, pH 6.8) and simulated colonic fluid (SCF, pH 7.4). Specifically, SA/Cs/ALG hydrogel microsphere (0.01 g) was immersed into SGF, SIF and SCF (5.0 mL), respectively. The mixture was shaken at 37℃, and taken out at a predetermined time duration (0–24.0 h) by centrifuging at 120 rpm/min. The excessive DI water on the surface of microspheres was removed using a filter paper. Then the microspheres were weighted to determine the swelling ratio. All experimental data were the averages of triplicate determinations, and the relative errors of the data were < 5.0%. Swelling ratio of hydrogel microspheres was calculated as follows swelling ratio (%) = (Wt- W0)/W0 × 100%, where W0 was hydrogel microsphere weight before swelling, and Wt was hydrogel microsphere weight at different durations.

In vitro simulated SA release evaluation

The pH value and enzyme distribution in different parts of human digestive tract present the specific physiological characteristics. It is confirming that informed consent was obtained from all subjects and/or their legal guardians for experiments involving human participants (including the use of tissue samples. The pH value of SGF was maintained at around 1.2, and there were enzymes such as pepsin existed in SGF, which were responsible for the preliminary digestion of protein. The pH value of SIF was relatively neutral around 6.8, and SIF contained digestive enzymes such as trypsin and amylase to further decompose protein, carbohydrates and fat in food. SCF presented a pH value of 7.4 and contained corresponding enzymes, which were mainly responsible for the electrolyte absorption and some vitamins synthesis. A proper amount of SA/Cs/ALG hydrogel microspheres were dispersed in 100 mL of SGF, SIF and SCF respectively on a constant-temperature shaker, and vibrated at 120 rmp and 37℃ and 120 rmp. At a set time duration, 3.0 mL of samples were taken out and filtered through a 0.45 μm membrane. The same volume was supplemented to keep the release medium at the same temperature. Blank hydrogel microspheres were used as control. The same volume of anhydrous ethanol was added to all the collected samples, and diluted after a high-speed vortex for 1.0 min. Based on the standard curve of each amino acid, samples were measured in parallel for three times to obtain the concentrations of amino acids and peptides released in SGF, SIF and SCF. SA release ratio (%) = M1/M0, where M0 was the total amount of SA in samples, and M was the amount of released SA at time t

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Effectiveness of SA/Cs/ALG in clinical treatment of recurrent respiratory tract infections in children

All methods were performed in accordance with the relevant guidelines and regulations approved by the ethics committee of First Affiliated Hospital of Nanchang University (No. 2023007–064). 103 children with recurrent respiratory tract infection in First Affiliated Hospital of Nanchang University were chosen and divided into two groups by random number table method. It is confirming that informed consent was obtained from all subjects and/or their legal guardians. For control group, the ages of 51 cases with 25 males and 26 females were in the range of 1–6 years (3.57 ± 1.06 years of average value). The body mass was 8.45 ~ 31.36 kg with an average of (19.36 ± 2.81) kg. For observation group, the ages of 52 cases with 27 males and 25 females were 1–6 years (3.62 ± 1.05 years of average value). Their body masses ranged from 8.31 kg to 30.95 kg with an average of (18.79 ± 2.73) kg. No statistical difference between two groups ( P >0.05). All children’s families knew and agreed to take part into this work.

The control group was treated via the routine treatment of cough-relieving, phlegm-resolving and anti- infective drugs. Corresponding anti-infective drugs were selected for different pathogens and drug sensitivities, and cefuroxime for injection was used for intravenous drip, 30 ~ 50 mg/kg, and twice a day. Azithromycin for injection (National Medicine Zhunzi H20030269) was given by intravenous drip, 8 ~ 10 mg/kg, and once a day. Ambroxol hydrochloride injection (National Medicine Zhunzi H20183050) was used as the expectorant, 7.5 mg/ time and twice a day. Observation group was treated by combining routine therapy and oral SA/Cs/ ALG hydrogel microspheres. The drug use method, dosage and course of treatment of routine therapy were the same as those of the control group. The dosage of oral SA/Cs/ALG hydrogel microspheres was 2 mg/d. 14 days was a course of treatment, and it was taken continuously for 3 months with an interval of 14 days. The ethics committee of First Affiliated Hospital of Nanchang University (No. 2023007–064) approved the experiments. The t-test was used for the comparison of measurement data before and after treatment between the two groups. With P < 0.05 for the difference was statistically significant.

Results and discussion

Characterization of SA/Cs/ALG hydrogel microspheres

The morphologies of Cs/ALG and SA/Cs/ALG hydrogel microspheres were shown in Fig. 1 . Cs/ALG hydrogel microspheres without the addition of SA showed a milky white color and were of spherical shape with good roundness. SA/Cs/ALG hydrogel microspheres exhibited an orange-yellow color, which was the typical color of SA. The remarkable color difference in photographs clearly indicated that SA was successfully loaded into SA/Cs/ALG hydrogel microspheres. As listed in Table 1 , the particle size of chitosan microspheres without embedding SA (2.38 mm for fresh microspheres and 0.24 mm for dried ones) was smaller than that of SA/ Cs hydrogel microspheres (2.80 mm for fresh ones and 0.60 mm for dried ones). The particle size of blank Cs-

Fig. 1 . Photographs of Cs/ALG ( a ) and SA/Cs/ALG ( b ) hydrogel microspheres.

Diameter (mm)	Chitosan	SA/Cs	Cs/ALG	SA/Cs/ALG
Fresh	2.38 ± 0.05	2.80 ± 0.05	3.32 ± 0.06	4.03 ± 0.03
Dried	0.24 ± 0.03	0.60 ± 0.05	3.27 ± 0.02	3.67 ± 0.04

Table 1 . Particle sizes of hydrogel microspheres in various encapsulation states.

Fig. 2 . SEM images of the core and intact structure of gel beads at various magnifications. ( a – c ) Chitosan. ( d – f ) SA/Cs. ( g – i ) SA/Cs/ALG.

ALG hydrogel microspheres (3.32 mm for fresh ones and 3.27 mm for dried ones) was lower than that of SA/ Cs/ALG hydrogel microspheres (4.03 mm for fresh ones and 3.67 mm for dried ones). SA/Cs/ALG hydrogel microspheres were of spherical shape. The distribution of microsphere sizes fitted by Gauss function was shown in Figure S1. By counting more than 100 hydrogel microspheres, the average microsphere size of SA/Cs/ALG was 4.03 ± 0.03 mm. It is deduced that the loading of SA made the microspheres have more binding sites with Ca2+, leading to the higher concentration of Ca2+ carried by the whole microsphere. Thus, more cross-linking points were provided with sodium alginate, which affected the overall particle size. It is indicated that the core–shell structure hydrogel microspheres loaded SA successfully. Paswan et al. reported that sodium alginate/polylactic acid hydrogel microspheres loaded with curcumin also have uniform brownish yellow color, indicating the efficient loading of curcumin without obvious leaching 31.

The micromorphology and internal structure of dried chitosan, SA/Cs and SA/Cs/ALGhydrogel microspheres observed by SEM were shown in Fig. 2 . In Fig. 2 a–c with a magnification of 50, the dried blank chitosan hydrogel microspheres presented a similar morphology to wet beads including a well spherical shape and a complete surface. The protrusions on the surface of chitosan may be caused by the loss of water in oven-drying, inducing slight surface collapses. Particle size of chitosan hydrogel microspheres after drying decreased significantly, while the basic structure and morphology were not unaffected obviously. As the magnification increased to 500 and 2000, many fine folds with cavity apertures were observed on the surface ofchitosan hydrogel microspheres, which would provide possible positions for the loading of SA. SA/Cs surface was coarser, presenting larger particles than that ofchitosan (Fig. 2 d–f). SA/Cs presented a rectangular shape, which should be induced by the embedded SA. The cavities in the folds were reduced and transformed into smoother structures, which may be attributed to the addition of SA.

SA/Cs/ALG hydrogel microsphere shows a much smoother surface than SA/Cs and chitosan hydrogel microspheres at the same magnification (Fig. 2 g,h). Its structure was compact and void-free, which suggested that the shell with sodium alginate was completely coated on the surface of core, and SA was well encapsulated

in the whole hydrogel microspheres without exposure. The cross section of SA/Cs/ALG exhibited many three- dimensional porous structures on the shell of ALG (Fig. 2 i). Thus, more liquid could be stored to absorb and swell, and the interconnected pores also make the microspheres more adhesive, prolonging the retention time in the colon. The above results show that the core–shell SA/Cs/ALG hydrogel microsphere could embed SA well with a good water absorption and adhesion structure.

Zeta potentials ofchitosan, SA/Cs, Cs/ALG and SA/Cs/ALG hydrogel microspheres were shown in Fig. 3 . The potential values of these samples were 15.1 ± 1.57 mV for chitosan, 7.6 ± 1.46 mV for SA/Cs, − 39.3 ± 1.92 mV for Cs/ALG, and − 48.2 ± 2.32 mV for SA/Cs/ALG. Chitosan hydrogel microsphere was a positively charged natural polysaccharide in aqueous solution. After embedding SA, the surface potential of SA/Cs decreased to 7.6 mV, demonstrating the loading of SA in chitosan matrix. After the cross-link with sodium alginate as a coating shell, the surface potential of Cs/ALG changed to be negative. Moreover, the surface potential of SA/Cs/ALG tended to be more negative, indicating that the prepared SA/Cs/ALG hydrogel microspheres had a complete core – shell structure with a negatively charged surface. At present, an effective treatment strategy for targeting colon inflammation is to assemble negatively charged drugs and drug carriers, and then target them to the colon areas where positively charged proteins are enriched due to inflammation, thereby eliminating reactive oxygen species to alleviate inflammation 32,33 . Thus, a more negative surface of drug and drug carriers is favorable for targeting colon inflammation.

FTIR spectra ofchitosan, ALG, SA/Cs, Cs/ALG and SA/Cs/ALG hydrogel microspheres were shown in Fig. 4 . Chitosan exhibited broad peaks at 3443 and 3363 cm-1, corresponding to stretching vibration absorptions of –OH and N–H in chitosan molecules. -OH was a hydroxyl group from chitosan and crystal water. It is demonstrated the presence of hydrogen bonding between amino group and hydroxyl group in chitosan molecules. There are a large number of intramolecular hydrogen bonds in chitosan molecules. The signal of stretching vibration was distributed in a wide frequency range of infrared spectrum due to the change of strength and distance of hydrogen bonds. Absorption peaks appeared at 2913 and 2879 cm-1 should be corresponded to vibrations of methylene -CH2 and methyl -CH3, respectively. Absorption peak of carbonyl (C = O) stretching vibration of molecular lactam I band was at 1656 cm-1. Bending symmetric vibration of nitrogen–hydrogen bond (N–H) in primary amino -NH2 was at 1599 cm-1. Absorption peaks ofC-O bond and C-N bond were observed at 1381 and

Fig. 3 . Zeta potentials of chitosan, SA/Cs, Cs/ALG and SA/Cs/ALG.

Fig. 4 . FTIR spectra of chitosan, ALG, SA/Cs, Cs/ALG and SA/Cs/ALG.

1155 cm-1, respectively. Stretching vibration absorption peak of -C–O–C in chitosan pyran ring was presented at 1026 cm-1.

For ALG, broad peaks at 3437 and 2927 cm-1 were ascribed the absorption of O–H stretching vibrations in hydroxyl group and C-H, respectively. The absorption peaks at 1611 cm-1 and 1417 cm-1 were the asymmetric and symmetric vibration absorption peaks of carboxyl groups respectively. FTIR spectrum of Cs/ALG hydrogel microsphere showed a vibration absorption peak of O–H near 3451 cm-1, which was wider and stronger than that of O–H in ALG and CS. It is indicated the hydrogen bond interaction between chitosan and ALG. Moreover, the

characteristic peaks of -COO- at 1611 cm-1 in the ALG spectrum moved to 1607 cm-1 in the Cs/ALG spectrum, and this peak widened and strengthened, indicating that chitosan was introduced with some carboxyl groups, and these carboxyl groups participated in cross-linking during the formation of Cs/ALG hydrogel microspheres. It was further proved that the network macromolecular structure was formed by crosslinking Ca2+ in ALG.

FTIR spectrum of SA/Cs/ALG displayed a wider peak at 3437 cm-1 compared with that of ALG, indicating that hydrogen bonding interaction occurred among SA, chitosan and ALG. Compared with Cs/ALG, SA/Cs/ ALG presented characteristic absorption peaks of -OH connected with benzene ring at 3294 and 3123 cm-1, and of -CH2 and -CH3 at 2923 and 2839 cm-1. Peak at 1635 cm-1 was the absorption peak ofC = N imine bond, which indicated that SA was effectively embedded in the matrix of Cs/ALG. The peaks of -OH moved from 3303 and 3132 cm-1 for SA/Cs to 3294 and 3123 cm-1 for SA/Cs/ALG. Compared with the absorption peaks of SA/Cs, the peaks of -OH moved from 3303 and 3132 cm-1 to 3294 and 3123 cm-1, verifying the hydrogen bond interaction in the formation of SA/Cs and SA/Cs/ALG.

Taken together, FT-IR results demonstrated the successful loading ofSA in Cs/ALG as a core–shell structure hydrogel microspheres. SA and CS were firmly combined through the Schiff base reaction and hydrogen bond interaction. SA/Cs and ALG were integrated via the electrostatic interaction and hydrogen bond interaction.

HPLC analysis has been performed to confirm the integrity of SA post-encapsulation. The HPLC results of SA before encapsulation and released from SA/Cs/ALG hydrogel microspheres were shown in Figure S2. The peak positions for main species of amino acids in SA including aspartic acid (1), threonine (2), serine (3), glutamic acid (4), proline (5), glycine (6), alanine (7), valine (8), methionine (9), isoleucine (10), leucine (11), tyrosine (12), phenylalanine (13), lysine (14), histidine (15) and arginine (16) were consistent for SA before encapsulation and released from SA/Cs/ALG hydrogel microspheres. Therefore, this complex mixture exhibited an excellent stability during the hydrogel fabrication process.

pH-dependent swelling

Hydrogel swelling plays a crucial role in the controllable drug release. pH sensitivity of SA/Cs/ALG hydrogel microspheres was explored through evaluating their swelling behavior in different pH media. As shown in Fig. 5 , pristine size and shape of SA/Cs/ALG microspheres changed slightly before and after the digestion in SGF with pH 1.2. The swelling ratio increased slightly in the first 30 min, reaching the maximum swelling rate of 192.8%, and then remained basically constant. The sodium alginate in the shell layer was still wrapped around the core layer in a thin film.

The shell of hydrogel microspheres gradually swelled and became larger and heavier with time in the SIF solution with pH 6.8. Since the surface of SA/Cs/ALG hydrogel microspheres was composed of sodium alginate, -COOH was deprotonated under alkaline conditions to form -COO-, which enhanced the electrostatic repulsion inside the hydrogel network and resulted in a looser network structure. More space would be provide for the hydrogel to absorb water molecules to swell. The maximum swelling rate was 2481.5%, and the swelling trend began to decrease after 4 h. The shell of hydrogel microspheres began to crack and decrease the mass and swelling ratio.

The swelling ratio of SA/Cs/ALG hydrogel microspheres in the SCF with pH 7.4 increased with time in the period of 1.0–10.0 min, and the hydrogel microspheres swelled to the maximum at 10.0 min with the swelling ratio of 1144.3%. The binding force between Ca2+ and PO43- was stronger than that between Ca2+ and sodium alginate, which would lead to the break of coordination bond between Ca2+ and -COO- and accelerated the disintegration of SA/Cs/ALG hydrogel microspheres. The enzyme or microorganism in the SCF would further crack the network structure of microspheres, inducing the complete disintegration of SA/Cs/ALG hydrogel microspheres. Therefore, the swelling ratio of hydrogel microspheres in three solutions with different pH values were significantly different. This excellent pH sensitivity would enable the crack and release of SA in microspheres when entering the intestinal environment, thus achieving targeted colon delivery.

In vitro release of SA

To simulate the digestion of hydrogel microspheres in human digestive tract, a proper amount of dried SA/ Cs/ALG were placed in 100 mL solutions of SGF, SIF and SCF containing corresponding enzymes. They were uniformly vibrated in an air vibrating table at 37℃ and 120 rpm/min. As shown in Fig. 6 , different SA release rates could be achieved by different swelling degrees of hydrogel beads at different pH values. The cumulative

Fig. 5 . Swelling curves of hydrogel microspheres in different digestive fluids. ( a ) Swelling curve of SA/Cs/ALG in SGF. ( b ) Swelling curve of SA/Cs/ALG in SIF. ( c ) Swelling curve of SA/Cs/ALG in SCF.

Fig. 6 . Cumulative release profile of SA/Cs/ALG hydrogel microspheres during the digestive process.

release of SA in SGF was less than 1.0% within 2 h without obvious release. When the environment was adjusted to pH 6.8, the release of SA increased to nearly 6.0%. Under the condition of pH 7.4, the release of SA continued within 24 h, and the cumulative release rate reached 92.4%. The above results showed an obvious pH sensitivity of SA/Cs/ALG hydrogen microspheres. The cumulative drug release could increase with pH value. SA release was effectively inhibited in the simulated SIF. However, in the SCF solution which matched the pH value of colon, most SA were slowly released within 24 h, which was similar to the drug release trend in Heikal et al.'s research 34 . ALG shell acted as a protective barrier for SA/Cs to resist the external environmental stress and avoid the initial SA leakage in small intestine and stomach. This would prevent SA loss significantly before they arriving to colon, and alleviate the release of SA too fast in the early stage of digestion 35 . This pH-dependent release could effectively prevent SA from being released prematurely in the upper digestive tract and protected them from the stomach environment. SA would not be completely released before reaching the colon, thus achieving the goal of targeting the colon.

Clinical treatment of recurrent respiratory tract infections in children

Before the treatment, 1 week after treatment and 2 weeks after treatment, 5 mL of venous blood was taken from each child on an empty stomach. The levels of CD3+, CD4+ and CD8+ were measured, and the CD4+/CD8+ values were calculated. The normal values of CD3+, CD4+, CD8+ and CD4+/CD8+ were 61%-85%, 20%-40%, 19%-48% and 1.4–2.0, respectively. Before treatment, 1 week after treatment and 2 weeks after treatment, 3 mL of venous blood of each child was taken on an empty stomach, and serum levels of immunoglobulinM (IgM), immuno.globulinA (IgA) and immunoglobulinG (IgG) were measured. The normal values of IgM, IgA and IgG were 0.48–2.12 g/L, 0.71–3.35 g/L and 7.6–16.6 g/L, respectively.

The therapeutic effect was judged by following up for one year after the treatment was finished. The symptoms, condition and number of respiratory infections met the standards of normal children of the same age after one year was defined as fully cured. The obvious effect was that the number of upper respiratory tract infections in children decreased by >2/3 one year after treatment, and the course of disease was shortened. Valid was that the number of upper respiratory tract infections in children was reduced by 1/3–2/3 one year after treatment. Invalid was that the number of respiratory infections in children decreased by less than 1/3 one year after treatment,

Group	CD3+ (%)			CD4+ (%)
	Before treatment	1 week after treatment	2 week after treatment	Before treatment	1 week after treatment	2 week after treatment
Control	41.53 ± 4.26	47.62 ± 4.51	57.59 ± 5.02	21.13 ± 2.94	25.15 ± 3.01	27.59 ± 3.11
Observation	41.75 ± 4.31	50.31 ± 4.62	62.74 ± 5.21	21.52 ± 2.81	35.11 ± 3.13	37.59 ± 3.54
t value	0.260	2.989	5.107	0.688	16.465	15.219
P value	0.795	0.004	< 0.001	0.493	< 0.001	< 0.001
Group	CD8+ (%)			CD4+/CD8+
	Before treatment	1 week after treatment	2 week after treatment	Before treatment	1 week after treatment	2 week after treatment
Control	50.43 ± 2.94	47.31 ± 3.05	37.24 ± 2.94	0.84 ± 0.10	1.11 ± 0.19	1.35 ± 0.29
Observation	50.52 ± 2.96	45.24 ± 3.02	29.55 ± 3.01	0.83 ± 0.11	1.33 ± 0.20	1.53 ± 0.31
t value	0.155	3.461	13.114	0.482	5.721	3.042
P value	0.877	0.001	< 0.001	0.631	< 0.001	0.003

Table 2 . Comparison ofT lymphocyte subsets between two groups of children.

Group	IgA (g/L)			IgG (g/L)			IgM (g/L)
	Before treatment	1 week after treatment	2 week after treatment	Before treatment	1 week after treatment	2 week after treatment	Before treatment	1 week after treatment	2 week after treatment
Control	0.44 ± 0.11	0.63 ± 0.14	1.57 ± 0.35	4.18 ± 0.51	5.62 ± 1.26	7.89 ± 1.17	0.26 ± 0.05	0.59 ± 0.07	1.03 ± 0.31
Observation	0.43 ± 0.10	0.89 ± 0.19	2.06 ± 0.54	4.15 ± 0.49	6.19 ± 1.17	12.17 ± 1.09	0.27 ± 0.06	0.91 ± 0.10	1.94 ± 0.29
t value	0.483	7.894	5.453	0.304	2.380	6.939	0.918	8.780	5.388
P value	0.630	< 0.001	< 0.001	0.761	0.019	< 0.001	0.361	< 0.001	< 0.001

Table 3 . Comparison of immunoglobulin levels between two groups of children.

and the course of disease did not shorten. Total effective rate = (cured case number +obviously effective case number +effective case number)/total case number × 100%.

As listed in Table 2 , levels of T lymphocyte subsets between two groups before treatment presented no statistical difference ( P >0.05). After the treatment of 7 days, levels of CD3+, CD4+, CD8+, and CD4+/CD8+ were (50.31 ± 4.62)%, (35.11 ± 3.13)%, (45.24 ± 3.02)% and (1.33 ± 0.20), respectively. After the treatment of 14 days, these levels were (62.74 ± 5.21)%, (37.59 ± 3.54)%, (29.55 ± 3.01)% and (1.53 ± 0.31)% respectively. After the treatment of one week and two weeks, the levels of CD3+, CD4+ and CD4+/CD8+ in two groups exhibited an upward trend, while CD8+ decreased. The observation group was superior to the control group with statistical difference ( P < 0.05). Taking the levels of CD4+ as an example, the CD4+ values in the observation group were significantly higher than those in the control group (i.e., the routine therapy without taking SA-containing hydrogel microspheres). It is deduced that the immunological improvements be attributed specifically to the SA delivered by the microspheres rather than to the routine therapy.

Researchers have systematically studied the efficacy and safety of SA oral lyophilized powder in ameliorating liver injury in infants and children with human cytomegalovirus infection 8 – 10 . In their previous direct oral administration studies, the immunological data were 72.5% for CD3+ cells, 39.2% ± 6.9% for CD4+ cells, 22.0% ± 4.5% for CD8+ cells with SA at 1 day before treatment, and 70.7% for CD3+ cells, 46.2% ± 7.8% for CD4+ cells, 20.8% ± 3.9% for CD8+ cells with SA at 4 weeks after treatment. In this study, the immunological data of CD4+ cells were 41.75% ± 4.31% for observation group before treatment, 50.31% ± 4.62% at 1 week after treatment, and 62.74% ± 5.21% at 2 weeks after treatment. These values were higher than those reported in previous literatures. Thus, the necessity of administering the microspheres with the encapsulated peptide was verified.

As listed in Table 3 , levels of IgA, IgG and IgM between two groups before treatment presented no statistical difference ( P >0.05). After the treatment of 7 days, levels of IgA, IgG and IgM were (0.89 ± 0.19), (6.19 ± 1.17) and (0.91 ± 0.120) g/L, respectively. After the treatment of 14 days, these levels were (2.06 ± 0.54), (12.17 ± 1.09) and (1.94 ± 0.29) g/L, separately. After the treatment of one week and two weeks, these levels in two groups displayed an upward trend, which was higher in observation group than that in control group ( P < 0.05).

As listed in Table 4 , total effective rate of 96.15% in observation group was higher than 84.31% in control group ( P < 0.05). The above results indicated that SA/Cs/ALG hydrogel microspheres could improve the immunoglobulin level and cellular immune function to enhance the body’s immunity and promote the improvement of children’s condition. Combined use with conventional drugs could improve the cellular immune imbalance of children, reduce the number of respiratory tract infections and improve the total effective rate of treatment.

Compared with the traditional form, the improvement in the degree of immune enhancement in the microsphere delivery form was presented as following. Microspheres with drugs could exert a stronger immunomodulatory effect at the lesion site through targeted delivery and enhance the local bioavailability. Compared with traditional aluminum adjuvants, the antigens encapsulated in PLGA microspheres could be

Group	Case number	Fully cured	Obviously efective	Valid	Invalid	Efective rate (%)
Control	51	25	13	5	8	84.31
Observation	52	37	10	3	2	96.15
χ value 2	–	–	–	–	–	4.118
P value	–	–	–	–	–	0.042

Table 4 . Comparison of therapeutic effects between two groups of children.

more effectively taken up by dendritic cells and continuously stimulate the immune system through sustained- release effects. This strategy could increase the antigen-specific antibody titer by 10–100 times and significantly enhance the cellular immune responses (such as the number of IFN-γ + T cells), which was difficult to achieve with aluminum adjuvants 36 . After loading cytokines such as IL-2 or GM-CSF into microspheres, they were locally injected into the tumor site, which could maintain an effective drug concentration in the tumor microenvironment for several weeks 37 . Compared with systemic administration, this local high concentration could more effectively recruit and activate T cells and NK cells, increasing the number of tumor-infiltrating lymphocytes by 5–10 times and significantly enhancing the tumor regression rate. Therefore, microsphere delivery systems have shown great application potentials in the field of immunotherapy.

Conclusions

In summary, SA/Cs/ALG hydrogel microspheres (3.67 ± 0.04 mm) with complete appearance, compact structure, and uniform texture without crack after drying were prepared. The outer shell of microspheres was negatively charged with a surface potential of − 48.2 ± 2.32 mV. SA was successfully loaded into the core of Cs and then coated with the shell of ALG to ensure that it would not ooze out. The average entrapment efficiency was 76.22 ± 0.06%, and the loading rate of SA was 35.17 ± 1.56%. FTIR analysis demonstrated that chitosan combined with SA via the Schiff base interaction, enabling the well combination of SA in chitosan core. Chitosan and ALG combined together to form core/shell structure through the electrostatic force and hydrogen bond interaction. SA/Cs/ALG hydrogel microspheres presented a pH response characteristic in vitro swelling and drug release study, i.e., the swelling occurred and the release amount increased with the increasing pH. The drug release is slow and there is no sudden release until it is almost completely released in 24 h. ALG could protect the core loaded with SA from reaching the colon and prolong the release time. Moreover, the use of SA/Cs/ALG hydrogel microspheres in clinical treatment of children with recurrent respiratory tract infection could improve the immune function and therapeutic effect, promoting their rehabilitation.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Received: 11 September 2025; Accepted: 31 December 2025

published online:10January2026

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Acknowledgements

We acknowledged the support from the Key Research and Development Program of Jiangxi Province, China (No. 2023YFC060140X).

Author contributions

Aqin Wang: Investigation, Methodology, Data curation, Validation, Writing-original draft. Zhengbing Zhou: Conceptualization, Writing-reviewing and editing. Zhou Hang: Data curation, Writing-reviewing and editing.

Zhengbing Lyu: Resources, Writing-reviewing and editing. Jingchun Yang: Conceptualization, Writing-review-

ing and editing. Lu Zhang: Resources, Writing-reviewing and editing. Meng Wang: Resources, Writing-review-

ing and editing. Wei Liu: Resources, Writing-reviewing and editing. Liqiang Zou: Resources, Writing-reviewing

and editing. Jun Yu: Project administration, Funding acquisition, Writing-reviewing and editing.

Funding

This work was supported by the Key Research and Development Program of Jiangxi Province, China (No. 2023YFC060140X).

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval

This study was approved by the ethics committee of the First Affiliated Hospital of Nanchang University (No. 2023007-064).

Informed consent

The purpose of this study is to evaluate the immunomodulatory effect of spleen aminopeptide-containing

hydrogels on recurrent respiratory tract infections in children. The project strictly abides by ethical norms, and the legal guardians of all subjects have signed written informed consent. The main contents are as follows: 1.

Voluntary participation: The guardians have fully understood the purpose, process and potential risks ofthe

research. The guardians voluntarily agreed to the child’s participation, and know that they could quit at any

time. 2. Risks and benefits: We have known the possible risks (such as slight discomfort in blood collection and adverse reactions related to hydrogels) and potential benefits (which may improve immunity). There may be no direct personal benefits from understanding the research results. 3. Privacy protection: Personal information of

the subjects has been de-identified, and only the research team can access it with authorization. 4. Protection of children’s rights and interests: In addition to the guardian’s consent, children over 8 years old also signed the informed consent form. 5. Ethical review: This study has been approved by the Ethics Committee. It is hereby confirmed that the informed consent procedure of legal guardians of all subjects has been completed.

Additional information

Supplementary Information The online version contains supplementary material available at https://doi.org/1 0.1038/s41598-025-34890-8.

Correspondence and requests for materials should be addressed to J.Y.

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