Chrysanthemum powder (Taikang, Tai’an, Shandong, China); ovalbumin (Absin, Shanghai, China); squalane, Tween-80, Span-85 (Nanjing Weil Pharmaceutical, China); IgA, IgG antibodies (Abcam Multisciences, Hangzhou, China); CD4⁺, CD8⁺ (MultiSciences, Hangzhou, China); Tyrisin, PBS and penicillin-streptomycin (P-S) solutions (Hao Yang Biotechnology Co Ltd, Tianjin, China). Fetal bovine serum (FBS, extra grade, Lonza Scientific, Swiss). Anhydrous ethanol, acetone, concentrated hydrochloric acid (Fuyu Fine Chemical Co., Ltd., Tianjin, China); sodium hydroxide (Hengxing Chemical Reagent Manufacturing Co., Ltd., Tianjin, China); potassium hydroxide (Yongda Chemical Reagent Co., Ltd., Tianjin, China) phosphoric acid (Comio Chemical Reagent Co., Ltd., Tianjin, China), other chemicals and reagents not specified in the text belong to Analytical grade.

Female Balb/c mice (6-8 weeks) and male Japanese white rabbits (3 months) without specific pathogens were obtained from the Animal Center of Shenyang Pharmaceutical University. Animals were fed ad libitum twice daily on basal chow and water free of any drugs or contaminants for 24 hours.

The procedures for feeding and testing animals shall be governed by the Regulations on the Administration of Experimental Animals approved by The State Council of the People’s Republic of China. All animal tests in this study were approved by the Animal Ethics and Experimentation Committee of Shenyang Pharmaceutical University (SYXK2018-0009). Trained and skilled animal care personnel were involved to reduce animal suffering as much as possible.

Chrysanthemum pollen was dispersed in purified water after removing the impurities. The dispersed phase was washed repeatedly by water, 50% ethanol, 75% ethanol and anhydrous ethanol in turn until the filtrate was basically colorless. Defatted pollen was then obtained. Crude sporopollenin could be collected by filter the 85% phosphoric acid aqueous solution suspended with the defatted pollen after heat reflux with mild stirring. To obtain the dried sporopollenin refined product, crude sporopollenin was firstly washed by the following steps in order: water, NaOH (2 M), hot acetone, hydrochloric acid (2 M), acetone, ethanol and water, until the solution was colorless at each time. Finally, the refined product was collected by filtration and desiccation.

OVA was dissolved in water to make OVA solution, the Squ/OVA emulsion (w/o) was obtained by the mixture of OVA solution and squalene at ration of 1:1 by volume and emulsified with 8% Span-85 as emulsifier followed by sonication for 5 min. The Squ/OVA emulsion (w/o/w) was obtained by adding 8% Tween-80 and water for injection into Squ/OVA emulsion (w/o) followed by emulsify for 5 min. The Spo-Squ/OVA was obtained by the mixture of Squ/OVA and sporopollenin ultrasonically followed by addition of water for injection and dilution. The concentration of each group above was 1 mg/mL for OVA, 40 mg/mL for squalene and 20 mg/mL for sporopollenin.

Elemental analysis was performed by varioELcube elemental analyzer (elementar Vario EL III, Elementar, Germany), appropriate samples were weighed for testing and analysis to derive N and S elemental contents. The protein level of the samples was recorded based on the measured percentage of N with a multiplication factor of 6.25. The protein removal rate of sporopollenin was calculated relative to the protein content of defatted pollen.

OVA antigen was mixed with indocyanine green and samples were prepared as described above. Mice were anesthetized by inhalation in an appropriate amount of ether gas. The fluorescence intensity in mice nasal cavity was observed under an imaging apparatus (DPM, H2000), and the change in fluorescence intensity was observed after 0.5, 1, 2 and 4 h of administration. The antigen retention was examined quantitatively by recording the change in fluorescence intensity.

To evaluate the release of Spo-Squ/OVA, water-soluble dye was dissolved in OVA to prepare Spo-Squ/OVA, which was added to dialysis bags and the bags was dispersed in PBS buffer (pH 7.4, 37°C, 100 rpm). The dialysate was obtained at pre-determined time intervals of 0.5, 1, 2, 3, 4 h, and the drug content was measured. All data were measured in triplicate to examine the changes in drug concentration at different times.

Female Balb/c mice (6-8 weeks) were divided of 3-4 mice for each group: control group, Squ/OVA, Spo-Squ/OVA, each of the above groups was administered intranasally at a dose of 25 μL free OVA(1 μg/μL) or OVA adjuvant(1:1) for one mouse; the other Squ/OVA subcutaneously at a dose of 25 μL for one mouse. Control group was PBS administered without vaccine.

The mice were immunized twice, once every two weeks. Blood samples were obtained from the retro-orbital plexus at day 28 after immunization. The blood could stand for 1 h after the clot was fully clotted. After centrifugation (HC-2062) (5000 r/min × 10 min), serum samples were preserved at -80°C and were eventually used for investigation of IgG and IgA by ELISA. Body weight was recorded daily for 28 days, Nasal washes and mucosal sections were taken from the nasal cavity of mice on day 28. Then the mice were sacrificed by cervical dislocation, and the nasal septum, spleen and thymus samples was surgically removed on day 28.

The antigen-specific antibody titers of serum IgA and IgG, and nasal sIgA of immunized mice were measured by indirect ELISA method. The microtiter plate was coated with the OVA antigen and incubated overnight at 4°C. Then BSA was incubated at 37°C for 2 h. After washing with PBST, diluted samples, HRP-labeled detection antibodies were added to the plate in sequence. Subsequently, add TMB chromogenic solution and termination solution (2 M H₂SO₄). The absorbance at 450 nm was analyzed.

Two weeks after the second vaccination, transfer the spleen tissue to a 35 mm sterile Petri dish and add PBS containing 1 mM EDTA to the dish. Remove connective tissue or fat from the spleen tissue. Place a 70 μm cell strainer on a 50 mL sterile conical tube and rinse the strainer with medium in advance. Grind the spleen 5 times with a flattened end section with the piston of the syringe in a gentle circular motion to fully release the splenocytes. Then, splenocytes are collected and lysed with red blood cell lysate. After erythrocyte lysis, centrifuge and remove the supernatant, and wash the splenocytes with PBS. Resuspend splenocytes with 3 mL of medium. Adjust the cell concentration to 1.5×10⁶/mL and transfer to a culture plate. Then restimulated by 5 μg/mL of OVA for 24 hours. Cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD4, phycoerythrin (PE)-conjugated anti-CD8. Cells then were subjected to flow cytometry (BD, FASVerse) following the manufacturer’s instructions, and analyzed with FlowJo software (BD).

Control group, Squ/OVA, and Spo-Squ/OVA, were administrated by nasal, and Squ/OVA was injected subcutaneously. Weight of mice, diarrhea, drowsiness, hunchback or ruffled fur, nasal mucosal irritation and other physiological symptoms were observed and recorded daily.

The long-term nasal mucosal toxicity was evaluated in Japanese white rabbits were. The Spo-Squ/OVA was dosed at 20 mg/kg via the left nostril and observed the symptoms in the period of 30 min after each dose for 7 days. The number of times of various symptoms in each rabbit was recorded, and the animals were euthanized 24 h after administration. Then, the nasal mucosa was obtained to observe whether there was congestion, edema, secretion around the nostril, crusting and other phenomena, and the nasal mucosa irritation response score and irritation intensity were evaluated according to the Supplemental Table 1 .

Origin 8.0 and GraphPad Prism7 software were employed to perform statistical analysis and all the data were expressed as mean ± SD of means of 3~4 independent experiments. The one-way ANOVA or double t test (double-tail) were used to determine the statistical significance of differences, which was *P < 0.05, **P < 0.01, and ***P < 0.001.

The pollen was spherical in shape, about 28 μm particle size, and the surface was covered with 2 μm cone-shaped spine-like protrusions, which were short and thick. Three germination pores of about 2 μm in diameter could be observed and were covered by lipid membrane in a protruding shape ( Figures 1B, C ). The outermost layer of the pollen surface was covered with lipid membrane, and the middle layer was a hard sporopollenin layer, mainly composed of aliphatic organic matter, lignin (the main source of fluorescence) ( Figure 1D ),carbohydrates and a very small amount of protein (25). The interior was protein and nucleic acid-like hereditary substance. After treatment with ethanol, a depigmented pollen(DE-pollen) was obtained, the surface of the pollen began to show depressions with reduced encapsulated material. The surface substance of the germinal pore was washed away, exposing the germinal pore structure. After treatment with phosphoric acid, the hollow structure of sporopollenin was obtained. As the protein on the surface of the sporopollenin germination pore is removed, the channel connecting the inside and outside is opened, and the structure of its layers become more obvious.

The particle size of chrysanthemum pollen was 28 μm, and size of sporopollenin was about 25 μm ( Figure 2A ). These results suggest that chrysanthemum pollen and sporopollenin were more homogeneous, and the particle size was maintained after the removal of lipid film. The infrared mapping measurement results showed that the pollen infrared map had characteristic peak of phospholipid at 1600 nm Figure 2B (26), while the sporopollenin outer map lost the characteristic peak of phospholipid at 1600 nm, suggesting the complete removal of lipid film. As shown in Figure 2C , the contact Angle of Spo was 132.5° and that of Spo-Squ/OVA was 30.3°, indicating better wettability due to the presence of surfactants.

Pollen often causes allergy in the body when inhaled, and it has been pointed out that the main allergenic components were proteins and glycoproteins (27), so it was extremely important to remove the internal protein from pollen. In this study, we measured the elemental composition of pollen at each stage of treatment to affirm the dismissal of internal proteins and glycoproteins ( Figure 2D ). We focused on the content of elemental nitrogen in the pollen fraction, and elemental analysis measurements demonstrated that the content of elemental N in pollen was 7.05%, while in sporopollenin the content was 0.72%, although did not decrease to 0 due to some nitrogenous substances contained by itself ( Figure 2E ). It was suggested that the removal of protein and nucleic acid substance inside the pollen was complete. Serum IgE in mice after Spo administration was significantly lower than pollen, indicating a significant decrease in sensitization after treatment ( Supplemental Figure 1 ).

Squalene was a commonly used adjuvant in a variety of vaccines (28), previous studies have shown that making w/o/w emulsion of squalene with OVA antigen exhibited better immune effects in vivo than adsorbing the antigen on the surface of squalene emulsion (29). It was proposed to fabricate a w/o/w emulsion of squalene and antigen and load it into sporopollenin to achieve the effect of antigen protection and antigen retardation. Meanwhile, the drug would become w/o/w emulsion under the action of two surfactants, after contacting with body fluid for release, which improved the permeability of nasal mucosa. The preparation process was shown schematically in Figure 1A .

After the preliminary process screening, based on the emulsification method, oil phase type and the proportion of component, we optimized the preparation conditions. Ideally, the nanoemulsion as a vaccine adjuvant with a PDI less than 0.3 and excellent size for effective uptake of epithelium on the mucosal surface as well as distribute antigen to an APC or the lymphoid tissue (17, 30). Post-optimality, squalene and Tween-80 were employed as the emulsifiers, the ultrasonic emulsification method was used to prepare the emulsion. Squalene and OVA antigen were also made into Squ/OVA emulsion.

The particle size of Squ/OVA emulsion was 354 nm, with uniform distribution of emulsion droplets. Microscopic staining to identify the results showed that the product was w/o/w emulsion.

To evaluate the nasal immune adjuvant effect of Spo-Squ/OVA, two intranasal immunizations (IN) were performed in mice using PBS (control), Squ/OVA, Spo-Squ/OVA, the other Squ/OVA group is administered subcutaneously ( Figure 3A ). Local secretion of antigen-specific secreted IgA (sIgA) antibodies is a critical feature of the mucosal adaptive immunity, which plays the most significant role in preventing pathogens and toxins from adhering to or infecting epithelial cells and damaging the mucosal barrier (8, 31). As shown in Figure 3B , antigen immunization in the Spo-Squ/OVA induced nasal secretion of specific antibodies at a titer 3 times greater than that of control group, higher than the Squ/OVAand injection of Squ/OVA. Low IgA titers were produced when immune antigens were injected subcutaneously.

Antigens that were immunized via the nasal cavity not only can activate B cells, but also enter the circulation through blood or lymph in the upper cortex to trigger a systemic immune response (32). In this study, antigen-specific IgG antibodies were measured in serum to evaluate the effectiveness of the Spo-Squ/OVA as a nasal adjuvant on the systemic humoral immunity. As a result, the antigen immune-induced specific antibody IgG in the Spo-Squ/OVA had titers 5 times higher than control group, higher than the Squ/OVA and as same as injection of Squ/OVA. Figure 3D suggested that intranasal immunity effectively induced humoral responses. In conclusion, sporopollenin as an intranasal adjuvant and a carrier had a good effect in enhancing mucosal immunity and humoral immune response.

The antibody responses were evaluated systemically by intranasal soluble protein immunization. As shown in Figure 3C , intranasal immunization with Spo-Squ/OVA, induced very high serum IgA responses. The results indicated that the antigen-induced specific IgA antibody in the Spo-Squ/OVA, with titers that was 5 times higher than values in the control group, higher than the Squ/OVA (intranasal, subcutaneous) vaccination, suggesting that the mucosal immune pathway also elicited strong IgA generation.

To assess the antigen-specific T cell response ability of sporopollenin, CD4⁺ and CD8⁺ T cell responses were researched after immunization with OVA. As shown in Figure 3E , we used squalene as a control because it has passed clinical trials and is considered safe. The vaccine was administered subcutaneously or intranasally. Two weeks after the boost immunization, the T-cell response was analyzed using the flow cytometer (FCM) of splenocytes and re-stimulated with vaccine antigen. As Figure 3E shown, the highest CD8⁺ T cell responses were observed in Spo/OVA and Spo-Squ/OVA after intranasal immunization. In contrast, Squ/OVA (SC) cannot enhance the CD8⁺ T cell response. There was no significant difference in the response of CD4⁺ T cells induced by the adjuvant. Thus, Spo/OVA and Spo-Squ/OVA intranasal immunization were better CD8⁺ T cell inducing adjuvants compared to Squ/OVA (SC).

The Spo-Squ/OVA showed promising immunoadjuvant efficacy in eliciting systemic as well as nasal mucosal immunity. We hypothesize that its enhanced IgA production was because of prolonging residence time, delaying antigen release and increasing antigen permeability, thus providing continuous stimulation of APCs, resulting in strong activation of the IgA-secreting B cells and systemic immune responses. In order to prove this conjecture, we observed the retention time and permeability of Squ/OVA, and Spo-Squ/OVA in the nasal cavity, as well as determined their nasal mucosal wettability and released profiles, and compared the different carriers.

The result above had shown that sporopollenin was highly lipophilic with aqueous medium contact angle, oil medium contact angle, and previous studies have shown that it was a good lipophilic agent (33). In this study, the lipophilicity of Spo enabled them to be used as high-capacity carriers for loading drugs. However, the poor water wettability of sporopollenin affects its affinity and permeability of the nasal mucosa. In this study, based on a large number of experiments, we optimized the Tween-80 complex surfactant coating on the outside of sporopollenin, aqueous medium contact angle, oil medium contact angle. It was suggested that Spo-Squ/OVA could overcome the deficiency of poor wettability of sporopollenin, increase the wettability of the system nasal mucosa administration, and effectively prevent the clearance of sporopollenin by nasal mucosa.

In this study, indocyanine green was applied as fluorescent dye to illustrated the retention of antigen in mice nasal cavity. As supervised by an in vivo imaging system, the fluorescence intensity was decreased over time after immunization. As shown in Figure 4A , about 85% of PBS immunization of antigen was eliminated from the nasal cavity after 1 h and almost no fluorescent signal could be observed in the nasal cavity after 2 h. The retention of Squ/OVA into the cavum nasi was improved with a lower degree than that of Spo-Squ/OVA, where the antigen retained into the cavum nasi even after immunization for 4 h. We could reasonably infer that sporopollenin plays a critical role in prolonging the retention of antigen in the nasal cavity ( Figure 4B ).

Meanwhile, the drug was released upon contact with the nasal mucosa to generate a w/o/w emulsion ( Figure 4C ). The particle size results showed that the particle size of Figures 4D, E was 361 nm, and the emulsion droplets were uniformly distributed.

To assess the release of Spo-Squ/OVA, the release of OVA antigen was examined by adding Spo-Squ/OVA a dialysis bag (20000 D). The release profile was shown in Figure 4F and Spo-Squ/OVA revealed a slower release. The particle size measurements at different times showed that the particle size remained constant in the range of 300 nm ( Figure 4G ). The effect of complex emulsion formation on antigen retention further protracted the retention time of antigen, while the strong affinity of the complex emulsion with the nasal mucosa also increased its permeability in the nasal mucosa.

In vivo toxicity evaluation of nasal mucosal vaccine and nasal mucosal irritation of the vector was an important issue. In this study, the in vivo toxicity was evaluated administered intranasally in control group, Squ/OVA, Spo-Squ/OVA and Squ/OVA subcutaneous injection control. As shown in Figure 5B , mice immunized intranasally or subcutaneously had similar body weights to other mice, with stable weight gain within 28 days of immunization. No physiological signs including diarrhea, lethargy, hunchback or matted hair could be observed in each group either.

In addition, nasal mucosal irritation was evaluated after nasal administration of Spo-Squ/OVA. By comparing the HE images of the nasal mucosa of the two groups of mice, no irritation was observed in the nasal mucosal tissue compared to the blank group Figure 5A . By comparing the HE images of the nasal mucous membranes, no pathological characteristics caused by irritation could be observed in the nasal mucous tissue when compared to the control group.

To further investigate the long-term nasal mucosal toxicity of sporopollenin, rabbits were dosed by pipette through the left nostril 10 mg/kg of response drug for 7 days. The number of times of various symptoms in each rabbit was recorded, and the animals were euthanized 24 h after all the administration, and the nasal mucosa was dissected and removed to observe whether there was congestion, edema, secretion around the nostril, crusting and other phenomena, and the nasal mucosa irritation response score and irritation intensity were evaluated. The results indicated that this product was almost non-irritating ( Figure 5C ).