Natural Lycopodium clavatum spores (S-type) were purchased from Sigma-Aldrich Co., LLC (St. Louis, MO, USA). Camellia sinensis (camellia) bee pollen was purchased from Xi’an Yuenun Biological Technology Co., Ltd. (Xi’an, China). Natural Typha angustifolia (cattail) pollen grains were sieved from cattail flowers purchased from Wong Yiu Nam Medical Hall Pte. Ltd. (Singapore). Defatted Taraxacum officinale (dandelion) pollen grains were purchased from Greer Labs (Lenoir, NC, USA). All solvents and reagents were obtained from Sigma-Aldrich Co., LLC, nylon mesh was purchased from ELKO Filtering Co., LLC (Miami, FL, USA) and 50 µm Duke Standards polystyrene microspheres were purchased from Thermo Fisher Scientific Pte. Ltd. (Waltham, MA, USA).

Preparation of SDMCs

The preparation method of SDMCs from camellia, cattail and dandelion was the same as that previously reported for lycopodium with slight modifications18.

Lycopodium clavatum (Lycopodium)

Briefly, natural L. clavatum spores (100 g) were defatted by refluxing in acetone (500 mL) at 50 °C for 6 h under stirring (220 rpm). The defatted spores were then collected by vacuum filtration and air-dried for 12 h. The dried samples were then refluxed (70 °C) in an aqueous 6% (w/v) potassium hydroxide solution (500 mL) under stirring for 6 h. The samples were collected by vacuum filtration and washed with Milli-Q water (Merck Millipore, MA, Burlington, USA) (2 × 500 mL) before resuming alkaline lysis for another 6 h using a 6% (w/v) potassium hydroxide solution (500 mL). After 12 h of alkaline lysis, the SDMCs were collected by centrifugation at 4,500 rpm and washed with hot Milli-Q water (5 × 500 mL, 50 °C). After each wash, the suspension was collected by vacuum filtration. The SDMCs were then washed with hot ethanol (2 × 500 mL, 50 °C) and dried overnight under fume hood. The resultant SDMCs were subjected to acidolysis by suspension in 85% (v/v) phosphoric acid (500 mL) and stirring under gentle reflux at 70 °C for 5 h. After acidolysis, the SDMCs were collected and washed sequentially in hot water (5 × 800 mL, 50 °C), hot acetone (600 mL, 50 °C), hot 2 M hydrochloric acid (600 mL, 50 °C), hot 2 M sodium hydroxide (600 mL, 50 °C), hot water (5 × 800 mL, 50 °C), hot acetone (600 mL, 50 °C) and then hot ethanol (600 mL, 50 °C). The resulting SDMCs were finally collected by vacuum filtration. The washed SDMCs were transferred to a clean glass dish and air-dried for 12 h. Drying was completed in a vacuum oven (Memmert, Schwabach, Germany) at 60 °C for 8 h, and the dried SDMCs were then stored in a dry cabinet.

Camellia sinensis (Camellia)

Similarly, a defatting process was performed by refluxing camellia bee pollen (250 g) in hot acetone (500 mL, 50 °C) with magnetic stirring (220 rpm, 3 h). The acetone was removed by vacuum filtration, and 1 L of warm water was added to the pollen under stirring. The resulting mixture was passed through a 150 µm nylon mesh (ELKO Filtering Co., LLC) to remove any contaminants. Water was removed from the suspension via vacuum filtration. This process was then repeated for another wash cycle.

The purified pollen was refluxed again in acetone (500 mL, 50 °C), isolated by vacuum filtration, transferred to a glass dish, and air-dried under fume hood. The dry pollen powder (20 g) was resuspended in diethyl ether (250 mL) under stirring (300 rpm, 2 h) at room temperature. The process was repeated for another wash cycle using fresh diethyl ether. For the final wash, the pollen was added to diethyl ether (500 mL) and stirred (300 rpm) at room temperature overnight. The defatted pollen was isolated by vacuum filtration and left to dry under fume hood.

The defatted pollen (6 g) was refluxed in 85% (v/v) phosphoric acid (60 mL) at 70 °C for 5 h (220 rpm). The resulting SDMCs were filtered and washed sequentially with 50 mL portions of water (five times), acetone (twice), 2 M hydrochloric acid (once), 2 M sodium hydroxide (once), water (five times), acetone (once), ethanol (twice) and water (once). The samples were dried under fume hood overnight and then in a vacuum oven (60 °C, 4 h). The dried SDMCs were stored in a dry cabinet at room temperature until further characterization.

Typha angustifolia (Cattail)

Natural cattail pollen grains (10 g) were defatted by refluxing in acetone (100 mL) at 45 °C with magnetic stirring (200 rpm, 30 min). The pollen grains were then obtained by vacuum filtration and washed with acetone (50 mL). The defatted pollen grains were then dried under fume hood at room temperature for 12 h.

To isolate the SDMCs via acidolysis, the defatted pollen grains (2 g) were placed in a poly(tetrafluoroethylene) round-bottom flask containing 85% (v/v) phosphoric acid (15 mL) and refluxed at 70 °C (water bath) for 2.5 h under gentle magnetic stirring (180–200 rpm). After 2.5 h, the flask was removed from reflux and its contents were allowed to cool down to room temperature. The suspension was then diluted with deionized water (150 mL) and vacuum-filtered. The SDMCs were collected in a clean 250 mL beaker and washed with 150 mL of warm water. The warm water wash was repeated five times with vacuum filtration until the pH of the washings reached approximately 6 or 7. The resulting SDMCs were collected in a clean 250 mL beaker, and washing steps were conducted by bathing the capsules sequentially in 100 mL portions of hot acetone (twice), hot 2 M hydrochloric acid (once), hot 2 M sodium hydroxide (once), hot water (five times), hot acetone (once), hot ethanol (twice) and finally hot water (three times). During each wash, the SDMCs were stirred in a beaker to produce a homogenous mixture and to prevent the capsules from aggregating. The solvent was then removed by vacuum filtration. The final washed capsules were then transferred to a clean glass dish and air-dried at room temperature for 12 h.

Taraxacum officinale (Dandelion)

Defatted dandelion pollens (2 g) were mixed with 85% (v/v) phosphoric acid (15 mL) in a 50 mL single-neck flask fitted with a glass condenser and refluxed at 70 °C for 5 h under magnetic stirring (220 rpm). After acidolysis, the SDMCs were collected by vacuum filtration and washed sequentially with 100 mL portions of hot water (five times, 50 °C), hot acetone (twice, 50 °C), hot 2 M hydrochloric acid (once, 50 °C), hot 2 M sodium hydroxide (once, 50 °C), hot water (five times, 50 °C), hot acetone (once, 50 °C), hot ethanol (twice, 50 °C), and hot water (once, 50 °C). The final product was collected by vacuum filtration, and the washed SDMCs were then transferred to a clean glass dishand air-dried under fume hood overnight. The drying process was continued in a vacuum oven at 60 °C under 1 mbar vacuum conditions for 4 h. Finally, the dried SDMCs were stored in a dry cabinet at room temperature for further characterization.

Preparation of simulated human gastrointestinal fluids

The conditions of physiological digestion areas described in the literature39. For SGFs containing an enzyme, 0.2 wt.% NaCl and 0.32 wt.% pepsin were dissolved in deionized water, and the pH was adjusted to 2.0 by adding 0.2 M HCl to the solution. For SIFs containing an enzyme, 0.68 wt.% monobasic potassium phosphate and 1 wt.% pancreatin were dissolved in deionized water, and the pH was adjusted to 7.1 using 0.2 M NaOH and, in the case of overshoot,0.2 M HCl.

Degradation treatment of SDMCs

The SDMCs were divided into three groups that were subjected to different degradation conditions: SGF treatment, SIF treatment and a control group in which water was used as the incubation medium. For SGF and SIF treatment, the SDMCs were segmented into two 40 mg batches, each of which was loaded into a 2 mL Eppendorf tube containing 0.6 mL of SGF or SIF, mixed thoroughly by vortexing for 1 min, and incubated at 37 °C in an orbital shaker incubator (LM-450D, Yihder Technology Co., Ltd., New Taipei City, Taiwan) with circular shaking at 220 rpm. For the control group, the SDMCs (40 mg) were incubated in 0.6 mL of deionized water for 24 h under the same incubation conditions. These three batches of SDMCs were then sampled at set durations (e.g., 1 h, 24 h), isolated by centrifugation at 7,000 rpm for 15 min, washed with SGF or SIF (1 mL) for five times and deionized water (1 mL) for five times and lyophilized.


DIPA was performed using a FlowCAM VS (Fluid Imaging Technologies, Scarborough, ME, USA) equipped with a 200 μm flow cell (FC-200) and a 20x magnification lens (Olympus, Tokyo, Japan). After 0.5 mL of prerun, untreated, SGF-treated and SIF-treated SDMCs samples were primed manually into the flow cell at 2 mg mL−1. Imaging was performed at a flow rate of 0.1 mL min−1 and a camera rate of 14 frames s−1. At least 10,000 particles were counted for each measurement, and three separate measurements were performed per sample. One thousand well-focused SDMCs were selected by edge gradient ordering and manual processing for the representative images.

FTIR spectroscopy

FTIR measurement was performed with a PerkinElmer Spectrum (PerkinElmer, Seer Green, Buckinghamshire, UK) equipped with a diamond cell attenuated total reflection accessory. Reflectance infrared (IR) spectra were collected in the midinfrared region of 4,000–650 cm−1with 16 scans per measurement and 6 replicate measurements per sample.

Background spectra were collected before sample analysis and automatically subtracted from each measurement. Baseline correction was carried out using Spectrum 10 software (PerkinElmer). After baseline correction, each spectrum was standardized as previously reported. Briefly, \((x-\bar{x})/\sigma \) values were used, where x refers to the absorbance value, \(\bar{x}\) refers to the spectrum arithmetic mean, and σ refers to the spectrum standard deviation. Peak heights were measured by taking the maximum value within a given range (Table 1). The peak ratios among the various peaks were calculated to remove the potential effect of differences in sample thickness on the absolute absorbance values. Principal component analysis (PCA) was performed using Origin 2018 (OriginLab, North Hampton, MA, USA).

Table 1 Assignments of absorbance peaks in the FTIR spectra of three species of SDMCs.


Untreated and SGF/SIF-treated SDMCs samples were frozen and lyophilized overnight in a freeze dryer (Labconco, Kansas City, MO, USA) under 0.008 mbar vacuum. A small number of samples were immobilized on a sample holder with carbon tape and sputter-coated with gold to a thickness of 20 nm (20 mA, 80 s) using a JFC-1600 Auto Fine Coater (JEOL, Tokyo, Japan) to reduce charging effects during SEM imaging. FE-SEM images were taken with a JSM-7600F Schottky microscope (JEOL) at an acceleration voltage of 5.00 kV. Visual inspection of more than 50 randomly chosen SDMCs particles from all the untreated and SGF/SIF-treated SDMCs samples was performed at different magnifications (500x, 2,500x and 15,000x) to assess any morphological changes in the samples.