Preparation of resveratrol-loaded ethyl cellulose microspheres via foam-transfer and its application performances

Resveratrol (Res) has been widely used in personal-care and pharmaceutical products due to its anti-oxidant, anti-inflammatory, cardioprotective and anti-cancer properties. However, the direct application of Res as functional additives have been limited due to its insolubility and instability. In this work, Res is loaded on the biodegradable ethyl cellulose (EC) carrier to prepare highly porous EC/Res microspheres via a foam-transfer method to understand the sustained release and the degradation property. The experimental results show that when the PVA concentration = 2.5 wt%, the stirring rate = 600 rpm, the oil/water (O/W) ratio = 1:2, the polymer concentration = 1:25, the heating rate = 0.33 °C/min, and the viscosity of EC = 90 ~ 110 mpa.s, the EC/Res microspheres are obtained with a yield up to 80.70 wt% and an encapsulation efficiency of 73.89% via the batch-process; the EC/Res microspheres are further produced continuously with a higher encapsulation efficiency of 90.63% with a yield of 85.58 wt%. The EC/Res microsphere reaches a cumulative release rate up to 91.97 wt% in the phosphate buffer solution (PBS) at pH = 5.0 under 45 °C for 120 h. After the EC/Res microspheres degraded in PBS (pH = 7.4) for 45 days, the hardness decreased from 12.01 ± 0.56 MPa to 4.02 ± 0.45 MPa. This work thus provides a practical solution for Res’s application as sustained release functional additives.


Introduction
Resveratrol (Res) is a kind of natural polyphenols with strong biological activity, which is mainly extracted from grape, polygonum cuspidatum, peanut, mulberry and other plants (Svajger et al. 2012;Burns et al. 2002). Modern science has proved that Res has anti-inflammatory activity (Svajger et al. 2012), hepato-protection (Lee et al. 2012), cardio and neuro-protection (Ferreira et al. 2018), anti-carcinogenic activity (Elshaer et al. 2018), anti-aging effects (Gambini et al. 2015), obesities' and diabetes' prevention activities (Chang et al. 2016;Hussein et al. 2016). Therefore, it has been used as functional additives in cosmetics (Kanlayavattanakul et al. 2018), nutrition (Wu et al. 2011;Fibigr et al. 2016), pharmaceuticals (Yang et al. 2014;Juan et al. 2012) and other fields (Cao et al. 2021). In these works, Res has been shown to interact with a variety of cellular targets such as signaling molecules, to modulate the cell-cycle regulatory genes. In detail, Res activates a variety of transcription factors, such as inhibiting protein kinases, suppressing the expression of antiapoptotic genes and angiogenic gene products, and down-regulating the expression of inflammatory biomolecules. Additionally, enhancing the dissolution of Res through the nanoparticle engineering process has been proved to providing many benefits, including increasing its anti-oxidant and anti-inflammatory actives, and enhancing anti-atherosclerosis effects. However, due to the low dissolution ability in water and poor photostability, the direct application of Res as the functional additive has been limited (Summerlin et al. 2015;Juan et al. 2002;Signorelli et al. 2005;Soto-Valdez et al. 2011).
Many efforts have been performed to address the challenges, either by the creation of nano-Res emulsions (Davidov-Pardo et al. 2015;Huang et al. 2016;Pandita et al. 2014) or encapsulation of Res in polymer matrixes Choi et al. 2022). Oil-in-water nano-emulsion has been reported to be an ideal candidate for the encapsulation of Res, since it can create an aqueous-based matrice with the aid of natural solvents and emulsifiers, by using the highthroughput processes. Additionally, the bioavailability of Res nano-emulsion is significantly improved 1 3 Vol.: (0123456789) because it increases the dissolution of Res in the gastrointestinal tract and accelerates its absorption (Sessa et al. 2011). However, the thermodynamic stability of nano-Res emulsion is poor, which makes it unfavorable for practical storage, transport and application. Additionally, the potential toxicity of most nano-Res emulsions is not clear, which futher limits its applications (He et al. 2016).
Other than nano-emulsion systems, drug-loaded microspheres have been deployed as the most common drug delivery system, which can be fabricated either by the direct drugs' adsorption to polymer microspheres or making drug dispersion in a polymer matrix (Babazadeh et al. 2007;Oh et al. 2011). By encapsulating Res in polymer microspheres, it can not only prevent Res from the surrounding stimulations such as the temperature, pH and oxidative effects, but also achieve the controlled-release of Res (Anal et al. 2006). Therefore, many studies have been done recently to formulate Res-loaded microspheres. Sanna et al. (2015) used W/O/W double emulsion/solvent evaporation method to prepare natural Res microcapsules (MCs) by using chitosan (CS) and poly (D, L-lactic-co-glycolic acid) (PLGA) as the polymer matrixes. It was found that MCs prepared by using the two carriers had a spherical shape with an average particles size from 11 to 20 μm, and the encapsulation efficiencies of Res reached 40 ~ 52%. Additionally, Res by using CS as the carrier released 70% of Res at pH = 1.2 after 2 h; while, Res by using CS and PLGA as the carrier exhibited a significant lower release of Res of only 40%. Furthermore, all tested formulations were able to ensure a good retention and stability of Res up to 6 months. Zhou et al. (2019) used shellac resin ammonium salt (SRAS) as the wall material to encapsulate Res by using the spray drying technology to obtain SRAS/Res microcapsules. With a dosage of SRAS, Res and tween 80 in the formulation at 2 g, 0.278 g and 0.5 g, respectively, the encapsulation rate of Res in SRAS/Res microspheres reached 82.70%. The obtained SRAS/Res microcapsules showed smooth spherical structures, stable thermodynamic properties, and a higher antioxidant capacity than the pure Res. They maintained a slow release in the simulated environment of human gastric juice, but releasing rapidly in the simulated environment of human intestinal juice. Peng et al. (2010) prepared Res-loaded chitosan microspheres using vanillin as the crosslinking agent by an emulsion chemical crosslinking method. Res-loaded chitosan microspheres were obtained with a smooth surface, irregular shape, but a high encapsulation efficiency of 93.68%. It was also proved that the controlled release property was achieved, and the stabilization of Res was enhanced by Res-loaded in the crosslinked chitosan microspheres. Iselya et al. (2019) used the oilin-water emulsion/solvent evaporation method with a cosolvent of ethanol and methylene chloride to encapsulate Res into poly (lactate-glycolic acid) microspheres with a maximum drug-loading of 65 μg/mg (6.5%). By reducing Res and ethanol in the oil phase and increasing the concentration of PVA in the aqueous phase, Res-loaded poly (lactate-glycolic acid) particles with smooth surfaces were obtained. However, the drug-loaded varied 15 ~ 25 times depending on the particle size of the microsphere. Compared with the larger particle, the small particle exhibited higher drug-loading and release due to their higher surface area. Although these works encapsulated Res successfully into polymer microspheres, nearly all of them used the batch-process in the production except the spray-drying process, which is inefficient and unfriendly for industrial production. Additionally, the structures of Res-loaded microspheres reported in these works were sole solid microspheres, and the variation on the structure was limited. In order to improve the application of Res-loaded polymer microspheres as functional additives in wider fields, an efficient way is desired for the preparation of Resloaded microspheres with various structures.
In our previous work, a simple, efficient and continuous production method was developed to prepare polymer microspheres via a foam-transfer process, based on the traditional solvent evaporation method (Li et al. 2021a). With this method, evaporation of the volatile organic solvent was used to produce a large number of bubbles with the aid of a protective colloid and mechanical stirring. Oil phase droplets were entrained by bubbles and then transferred into the collector to obtain polymer microspheres. The process was further developed into a continuous production of polymer microspheres via a continuous feeding, which has been successfully applied to encapsulate oil-soluble azoxystrobin (Li et al. 2021b). Due to Res is also oil-soluble, the encapsulation of Res is also realized via foam-transfer.
Ethyl cellulose (EC) is a kind of semi-synthetic membrane forming polymer material, with good thermal stability, biocompatibility and biodegradation in vivo (Shin et al. 2018;Lin et al. 2021), which has been proved to be an excellent wall material for encapsulation of functional additives including eugenol (Simoes et al. 2020), epoxy resin (Wang et al. 2020) and curcumin (Pi et al. 2018) to produce microspheres with multi-structures (Rogers et al. 2012;Muschert et al. 2009). Therefore, in this work, ethyl cellulose is used as a carrier to prepared Resloaded EC microspheres by using the foam-transfer method. The influences of experimental conditions on the preparation of microspheres were investigated, followed by studying the sustained release property. Especially, the degradation of Res-loaded EC microsphere is quantitatively determined by using a micromanipulation technique.

Preparation of EC/Res microspheres
In a typical experiment, EC (2.0 g) and Res (0.5 g) were dissolved in DCM (50.0 g) to form oil phase, and PVA (2.5 g) was dissolved in 100.0 g pure water to serve as the aqueous phase. The aqueous phase and the oil phase were mixed in a 250 mL three-neck round bottom flask under 200 rpm stirring for 5 min; and then the temperature of the water bath was raised by 0.33 °C/min. As the oil-aqueous phase was stirred and heated, a large amount of foam entrained with oil droplets flowed out into the receiving vessel, which equipped with a 200 rpm stirring paddle and 30 °C hot water. After defoaming, the obtained Res-loaded EC microspheres were washed by using deionized water for three times to remove the dispersant PVA and the free Res, and then vacuum dried to remove DCM. The preparation process of the EC/Res microspheres was then optimized to understand the effects of the stirring rate, the dispersant concentration, the O/W ratio, the polymer concentration, the heating rate and the viscosity of EC on the properties of microspheres, and the preparation recipes, as showing in Table S1.

Continuous production of EC/Res microspheres
The oil phase (200.0 g DCM contained 2.0 g Res and 8.0 g EC) was dispersed into the aqueous phase (400 mL 2.5 wt% PVA) and they were pre-emulsified with a magnetic stirrer for 5 min. 150.0 g of the mixture was placed in a 250 mL three-necked flask. The temperature was kept 45 °C under stirring at a fixed rate of 600 rpm until foam flowed initially. The rest of the mixture was slowly poured into the reactor by using a solvent resistant pump at a flow rate of 0.04 L/min. The post-processing of EC/Res microsphere was the same as mentioned in 2.2.1. Each experiment was repeated for three times to get the average and the standard deviation of the yield, the encapsulation efficiency and the particle size.

Morphology of microspheres
An optical microscope (DM4000, Leica Microsystems, Shanghai,China) was used to observe the morphology of the prepared microspheres. Microspheres were dispersed on a microslide with deionized water and the overall image of the microsphere was obtained by using the reflected light in wet and dry conditions.
The surface morphology of the microspheres was observed via a Tescan Mira Lms scanning electron microscope (SEM). The microspheres were dried an using infrared light, and then they were dispersed on a carbon tape and coated with gold for 120 s at 40 W under vacuum.
The pore structure of the microspheres was observed by confocal laser scanning microscope (CLSM) (Nikon TiE-A1 plus). Fluorescent dye PM546 was introduced by dissolving PM546 into DCM at a concentration of 0.1 wt%, and then 0.5 g Res and 2.0 g EC were dissolved in 50.0 g 0.1 wt% PM546-introduced DCM solution to form an oil phase. The following preparation process was the same as mentioned in 2.2.1. A small amount of EC/ Res microspheres were diluted and then placed in a confocal Petri dish for infrared drying. According to the fluorescence dye PM546, 540 nm excitation wavelength was selected to observe the morphology of fluorescent microspheres (Pan et al. 2013).

Particle size and particle size distribution
200 microspheres were randomly selected from the optical microscopy images for particle size analysis by using Nano Measurer 1.2. The particle size data were further analyzed by using Origin to obtain the particle size distribution, the mean particle size and the standard error. The particle size distribution curve was drawn by Gaussian fitting.

Yield of microspheres
The yield of the EC/Res microspheres was calculated by the mass of the microspheres collected in the receiving container, as shown in Eq. (1). Three repeated experiments were performed to get the average and the standard deviation.

Encapsulation efficiency of Res-loaded EC microspheres
0.005 g of Res was dissolved in 50 mL EtOH and then the Res solution was diluted to a serious of solutions with the concentration of 2 mg/L, 4 mg/L, 8 mg/L, 16 mg/L, 30 mg/L to make the standard curve by using UV spectrophotometer at 306 nm. The encapsulation efficiency of Res in the microspheres were measured by disruption of the microspheres. In detail, 0.02 g EC/Res microspheres were weighed and then dissolved in 50 mL DCM until the microspheres were fully dissolved. Then a solution of 1 mL was dried via the reduced-pressure distillation to obtain a dry mixture, followed by soaking the mixture in 10 mL of ethanol overnight. Finally, the mixture was centrifuged for 5 min at 10,000 rpm, (1) Yield wt% = weight of microspheres in the receiving vessel theoretical weight of microspheres × 100wt% and the precipitate was removed and the supernatant was collected. The supernatant was measured by using an UV spectrophotometer at 306 nm and then the concentration of Res in the supernatant was determined according to the standard curve. Each sample was measured for three times to obtain the average and the standard deviation. The encapsulation efficiency of Res in the microsphere was calculated by Eq. (2).

Surface tension and viscosity
The interfacial tensimeter (A201, Kino Industries Co., Ltd) was used to measure the surface tension of PVA solutions under different concentrations at room temperature. The viscosity of PVA solution with different concentration was measured at room temperature by a rotary viscometer (NDJ-9S, Fangrui Instrument Co., Ltd. Shanghai, China). Each experiment was repeated for three times to calculate the average and the standard deviation.

In vitro release
The release performance of Res from EC microsphere was studied through a dynamic dialysis method. 0.1 g EC/Res microspheres were dispersed in 5 mL of PBS, which was placed in the dialysis bag. The dialysis bag was immersed in 145 mL PBS solution sealed in a 500 mL beaker. The beaker was placed in an incubator (THZ-300, ShangHai Yiheng Experimental Equipment, ShangHai, China) with a continuous shaking at 100 rpm under 37 °C away from light. At specific time intervals, 5 mL aliquot of the solution outside the dialysis bag was collected and replaced with the same volume of freshly PBS. The sustained release properties of Res formulations were evaluated by measuring the concentrations of Res dissolved in the release medium at different intervals. Specifically, Encapsulation efficiency% = weight of Res measured in microspheres theoretical weight of Res in microspheres × 100% the obtained aliquot was measured by using an UV spectrophotometer at 306 nm, and then the concentration of Res in the aliquot was determined according to the standard curve. Each measurement was repeated for three times to get the average and the standard deviation. The cumulative release rate of Res was calculated by using Eq. (3).
where Q is the cumulative release rate of Res in the microspheres, wt%; m is the total mass of Res in the microspheres, g; V 0 is the volume of the sustained release medium, mL; ρ i is the mass concentration of Res in the release medium at the release time, g/mL.
V is the volume of the slow-release medium sampled, mL.

Degradation performance measurements
Mechanical property measurement The degradation of the EC/Res microsphere was tested by measuring the mechanical properties using the micromanipulation technique. EC/Res microspheres were immersed in PBS (pH = 7.4) at 37.0 ± 0.5 °C. After a period of time, the samples were taken out and dried at 40 °C for 24 h. The mechanical properties of microspheres were then measured by using a micromanipulation rig (MF-WSF1, Changzhou, China). The diameter of each microsphere was determined by using the image under the optical microscope. In a typical experiment, a single microsphere was selected and compressed with the force applied by the probe to obtain a force-displacement curve. At least 35 microspheres were uniformly selected from each sample in the range of 20 ~ 50 μm to determine the mechanical properties of the microspheres, and the average hardness and the corresponding standard deviation were obtained. The hardness of the microspheres were obtained from the force-displacement curves (Yap et al. 2008;Luo et al. 2022). The hardness of the microsphere (H), was obtained from the slope of the linear plastic region of the force-displacement curve, in which it deviated from Hertzian behavior by using Eq. (4).
where F is the applied force, R is the diameter, and δ is the displacement.
analysis Thermogravimetric analysis (STA449F3 Jupiter) was used to study the thermodynamic property of the EC/Res microspheres to evaluate their degradation. The analysis was carried out by using high-purity nitrogen as the carrier gas in the temperature range of 25 °C ~ 500 °C, with a heating rate of 10 °C/min and gas flow rate of 20 mL/min. Three measurements were performed for each sample.

Results and discussion
The effects of experimental conditions on the properties of the EC/Res microspheres PVA concentration PVA, as a stabilizer, plays an important role in preparation of the EC/Res microspheres via the foamtransfer process, because it is not only a stabilizer to form oil droplets but also a foaming agent. Therefore, it can affect the prepared EC/Res microspheres, and the study results are shown in Figs. 1, 2 and Fig.S1. As can be seen, the obtained EC/Res microspheres were in porous structure; when the concentration of PVA ≤ 2.0 wt%, the obtained EC/Res microspheres are in poor spherical and some of them are oval; when the concentration of PVA is higher than 2.0 wt%, the spherical shape of the EC/Res microspheres became better ( Fig. 1 and Fig.S1). The particle size of the EC microspheres gradually decreases and their particle size distribution becomes narrow by increasing the concentration of PVA, which is showing in Fig. 2a, b. The yield of the EC/Res microspheres obtained from the receiving vessel increases with the concentration of PVA and then decreases (Fig. 2c). And the corresponding encapsulation efficiency shows a the similar trend (Fig. 2d). When the concentration of PVA = 2.5 wt%, a high yield of 80.70 wt% with a maximum encapsulation efficiency of 73.89% is obtained. By increasing the concentration of PVA, the surface tension of the aqueous phase decreases from 46.25 ± 2.34 mN/m to 37.45 ± 0.62 mN/m, while the viscosity increases from 4.00 ± 0.38 mpa.s to 16.33 ± 0.58 mpa.s (showing in Table S2 of the supporting information), which decreases the particle size and improves the sphere shape. The increase in PVA is also beneficial for foaming, which helps entrain more oil droplets to the receiving vessel, thus increasing the yield. For the encapsulation efficiency, when the concentration of PVA is low, the protective barrier at the interface is weak; therefore more Res dissolves in the aqueous phase, leading to a low encapsulation efficiency. When the concentration of PVA is higher, the total surface area is increasing since the particle size decreases, and more contact area leads to a higher dissolution of Res in water, causing a decrease of the encapsulation efficiency. Therefore, the PVA concentration of 2.5 wt% is selected for the following work.

Stirring rate
The agitation provides kinetic energy for the system, therefore the stirring rate also affects the properties of the as-prepared EC/Res microspheres. The EC/ Res microspheres prepared with various stirring rates are shown in Figs. 3, 4 and Fig.S2. As can be seen from Fig. 3a 1 ~ a 5 and Fig.S2, EC/Res microspheres prepared are in porous structure; when the stirring rate ≤ 400 rpm, the sphericity of EC/Res microspheres is poor and some of them are elliptic. When the stirring rate > 400 rpm, the spherical shape of EC/Res microsphere is improved. The particle size of EC microspheres gradually decreases and a narrower particle size distribution are observed with the increasing stirring rate, as shown in Fig. 4a, b. The yield of the EC/Res microspheres obtained from the receiving vessel increases at first and then decreases with the increasing stirring rate (Fig. 4c). The encapsulation rate showes a slightly decrease trend by increasing the stirring rate (Fig. 4d). When the speed is 600 rpm, the yield is highest at 80.70 wt%, and the corresponding encapsulation efficiency is 73.89%. Due to the stirring rate affectes the shear kinetic energy of the system, a larger kinetic energy is provided for the system by increasing the stirring rate, leading to a smaller particle size. Additionally, a large kinetic energy input is beneficial for foaming, which help to entrain more oil droplets to the receiving vessel to increase the yield. Conversely, if the stirring is weak, the microspheres Fig. 1 SEM images (a 1 ~ a 5 : 1.0 wt%, 1.5 wt%, 2.0 wt%, 2.5 wt%, 3.0 wt%) of the EC/Res microspheres prepared at different PVA concentrations with a large particle size and low yield are obtained. Since the particle size of the microspheres decreases with the increasing stirring rate, the encapsulation efficiency shows a slightly decrease trend because of the increase in the total surface area, in which more Res dissolves in the receiving vessel.

O/W ratio
The O/W ratio can not only influence the particle size of the as-prepared microspheres, but also affect the amount of solvent presenting in the dispersion, which further influences the yield of the microspheres. Therefore, the properties of EC/Res microspheres were studied by varying the O/W ratio, and the resutls are shown in Figs. 5, 6 and Fig.S3. As can be seen from Fig. 5a 1 ~ a 5 , Fig.S3 and Fig. 6a, b, all EC/Res microspheres prepared are in porous structure; with the decreasing O/W ratio, the particle size of the microspheres increases and the particle size distribution becomes wider. The yield of the EC microspheres increased at first and then decreases slightly with the decrease of the O/W ratio; and the corresponding encapsulation efficiency shows a similar trend (Fig. 6c , d). When the O/W ratio is 1:2, the yield is 80.70 wt%, and the encapsulation efficiency is 73.89%. By decreasing the O/W ratio, the oil phase escapes from agitation, leading to a larger particle size with a wider particle size distribution. Furthermore, due to more DCM presented in the system when the O/W ratio is high, it can help enhance the foaming process thus improving the yield. However, when the O/W ratio is extremely high, some oil droplets aggregate to decrease the yield. In the high O/W ratio range, the encapsulation efficiency is corresponded to the particle size, in which a large particle size leads to an increase of the encapsulation efficiency because of the decrease of the total contact area. When the O/W ratio is very low, DCM in the oil phase is less, making it difficult to produce a large amount of foam. The decrease on the foaming ability and transfer ability makes the oil droplets stay in the reactor for a longer time, causing a decrease of the encapsulation efficiency.

Polymer concentration
Polymer concentration affects the morphology and particle size of the microspheres, for which the results are exhibited in Figs. 7 and 8. It can be seen from Fig. 7a 1 ~ a 5 that when the polymer concentration ≤ 1:15, the sphericity of the EC/Res microspheres is poor and part of them are elliptic. When the polymer concentration > 1:15, the spherical shape of the EC/Res microspheres is improved. the EC/Res microspheres prepared are in porous structure. By decreasing the polymer concentration, the pore number decreased with larger pore dimensions from Fig. 7b 1 ~ b 5 . The particle size of the EC microspheres decreases, along with a narrower particle size distribution by decreasing the polymer concentration, as showing Fig. 8a, b. The yield of the EC/Res microspheres increases with the decreasing polymer concentration and then maintains (Fig. 8c); while the encapsulation efficiency decreases gradually ( Fig. 8d). When the concentration of polymer is 1:25, the yield is 80.70 wt%; and the highest encapsulation efficiency is obtained at 1:10, which is 83.17%. When the polymer concentration is too high, the viscosity of the oil phase was high, making it hard to disperse. Therefore, the particle size of microspheres is large with a wide particle size distribution. Furthermore, a higher polymer concentration leads to a lower foaming ability, due to the amount of DCM decreases in the oil phase, which further decreases the yield of the microspheres. The higher polymer concentration also contributed to a better encapsulation efficiency due to Res in the more viscous oil phase is not easy to migrate and then dissolve into the aqueous phase.

Heating rate
The heating rate plays an important role in the morphology and the pore structure of microspheres, and the results are displayed in Figs. 9 and 10. As can be  Fig. 9a 1 ~ a 5 , b 1 ~ b 5 , no obvious variation is observed on the spherical shape of the EC/Res microspheres. SEM images of the EC/Res microspheres shows that the EC/Res microspheres are in porous and the pores on the surface of microspheres tends to decrease as the heating rate slows down. The particle size of the EC/Res microspheres decreases, along with a narrower particle size distribution by decreasing the heating rate, as shown in Fig. 10a, b. The yield of the EC microspheres increases at first and then decreases with the decrease of the heating rate, and the corresponding encapsulation efficiency also shows a similar trend (Fig. 10c, d). When the heating rate is 0.33 °C/min, the yield is the highest at 80.70 wt%; and the encapsulation efficiency is the highest at 0.20 °C/min, which is 75.24%. A higher heating rate results in the rapid volatilization of DCM and a large population of bubbles goes through the polymer material to form porous structures along with the solidification of microspheres, leading to an expending on the volume of the microspheres and large holes on the surface and inside of the microspheres. Therefore, larger particle sizes and big holes are observed with a higher heating rate. Furthermore, the foaming ability is high and more incomplete solidified oil droplets are entrained in the collecting container, in which Res diffuses easily, resulting in a low yield and corresponding encapsulation rate of Res. When the heating rate is slow (0.10 °C/min), the foaming capacity decreases, and the oil phase is dispersed in the flask for a long time, so the particle size decreases. Additionally, more Res in oil droplets diffuses into water during the long time shearing process, resulting in the reduction of the yield and the encapsulation efficiency of the obtained EC/Res microspheres.

EC with different viscosities
The viscosity of EC has an important effect on the particle size and pore structure of the microspheres, and the investigated results are shown in Figs. 11 and 12. As can be seen from Fig. 11a 1 ~ a 6 , b 1 ~ b 6 , with the increase of the viscosity, the particle size of microspheres gradually increases. Moreover, the pore size Fig. 4 Average particle size (a), particle size distribution (b), yield (c) and encapsulation efficiency (d) of the EC/Res microspheres prepared at different stirring rates of microspheres gradually reduces, and the number of pores becomes dense until pores vanished by increasing the viscosity of EC. The particle size of the EC/ Res microspheres gradually increases and the particle size distribution becomes wider, as shown in Fig. 12a, b. The yield of the EC/Res microspheres increases at first and then decreases with the increasing EC viscosity, and the encapsulation efficiency shows a gradually increasing trend (Fig. 12c, d). When the EC viscosity is 90 ~ 110 mpa.s, the yield is 80.70 wt%. The encapsulation efficiency reaches up to 78.58% at 270 ~ 330 mpa.s. By increasing the viscosity of EC, the polymer chain length of EC increases, leading to a stronger film with increased tensile strength and elasticity. Therefore, the resistance for the evaporation of organic solvent evaporation through the wall increases, leading to smaller pore sizes on the surface of the microspheres until pore vanished. Due to the total amount of the organic solvent is constant, the number of pores increases when the volume of pores decreases, the increasing viscosity of EC leading to an increase in the number of holes on the surface of the microspheres. Moreover, with the increase of the EC viscosity, the viscosity of oil droplets increases, therefore Res is not easy to diffuse into the aqueous phase. So the encapsulation efficiency of the EC/Res microspheres increases continuously with the viscosity. However, an extremely high viscosity of EC results in a hard dispersion of the oil phase, leading to a reduced yield and a large particle size.

Continuous production of EC/Res microspheres
To verify the applicability, the EC/Res microspheres are prepared by a continuous process with the experimental conditions presented in 3.1.1 (the stirring rate of 600 rpm, the polymer concentration of 1:25, the PVA concentration of 2.5 wt% and the O/W ratio was 1:2). Finally, porous EC/Res microspheres are continuous produced (Fig. 13a, b) and the yield of the EC/Res microsphere reaches 85.58 ± 2.30 wt%, with a mean particle size of 75.13 ± 10.45 μm and an encapsulation efficiency of 90.63 ± 1.62%, which is even higher than that produced by using the batch-process in 3.1.1 (the yield was 80.70 wt%; the encapsulation ratio was 73.89%) Due to a constant temperature control at 45 °C is applied, it causes rapid volatilization of DCM and a large population of bubbles goes through the polymer material to form porous structures along with the solidification of microspheres, leading to an expending on the volume of the microspheres and large holes on the surface of the microspheres. In addition, the contact time between oil droplets and water is shorter, which increases the encapsulation efficiency of Res. Therefore, an even higher encapsulation ratio of Res is obtained in the /Res microspheres produced continuously.

In vitro release
The release performance of the EC/Res microspheres is significant for their application and it was studied by varying the pH and the temperature. Figure 14a shows the in vitro release curves of the EC/Res microspheres in PBS with different pH values, where the cumulative Res release is plotted with time (h). According to Fig. 14a, EC/Res microspheres releases Res differently under the three different pH values, in which the cumulative release of Res from EC/Res microspheres is 91.97 wt%, 64.62 wt% and 73.68 wt% in PBS at pH = 5.0, 5.8 and 7.4 respectively after 120 h. However, they all process a quick releases within the first 40 h, indicating by the large slope of these curves; and the cumulative release of Res reaches 77.40 wt%, 53.16 wt% and 52.60 wt% at pH 5.0, 5.8 and 7.4 respectively. After 36 h, the slopes reduces, indicating a slower releases of Res. The cumulative releases of Res maintains at the lowest at pH = 5.8. Either by decreasing pH or increasing pH, the cumulative releases of Res is enhanced. Especially, in acidic condition (pH = 5.0), more Res releases from the EC/Res microspheres. This is because the glycoside bonds in the EC macromolecular chain has a high stability in base, but they are easy to hydrolyze in acid, which decreases the degree of macromolecular polymerization, weakens the intermolecular force, and thus decreases the strength. Therefore, EC has excellent resistance to alkaline and weak acid. Res releases more slowly in PBS when pH = 5.8 and 7.4. Figure 14b shows the cumulative release curves of the EC/Res microspheres under different temperatures. The release curves of the EC/Res microspheres at 25 °C, 37 °C and 45 °C show a large slopes within the first 36 h with the cumulative release of Res of 17.20 wt%, 52.60 wt%, and 60.03 wt% respectively and then the release rate slows down. When the release time increases to 120 h, the cumulative releases of Res from the EC/Res microspheres reaches 30.76 wt%, 73.68 wt% and 77.91 wt% respectively at 25 °C, 37 °C and 45 °C. The cumulative release of Res from the EC/Res microspheres increases by increasing the temperature, which is due to the enhanced molecular diffusion and degradation of EC at a higher temperature.

Mechanical property measurement
The degradation behavior of the EC/Res microsphere is also significant for their application, which are displayed in Fig. 15 The degradation behavior is indicated by the mechanical properties, which is characterized by using the micromanipulation analysis rig (Luo et al. 2022). Figure 15a shows a typical force-displacement curve by compressing an EC/Res microsphere. The force-displacement data is further fitted to calculate the hardness of the EC/Res microspheres. For each sample, about 35 microspheres with different particle sizes are selected to calculate their hardness. As can be seen form Fig. 15b, by immersing EC/Res microspheres in PBS (PH = 7.4) for 45 days, the hardness gradually decreases by increasing the soaking time. The hardness decreased from 12.01 ± 0.56 MPa to 4.02 ± 0.45 MPa when soaking the EC/Res microspheres in PBS for 45 days. In summary, the mechanical data of the EC/Res microspheres before and after the degradation indicates that EC under go a significant degradation.

TGA analysis
The degradation behavior of EC/Res microsphere was further characterized by using TGA analysis by immersing them in PBS for 0, 7, 15, 30 and 45 days, which is shown in Fig. 16. The pristine EC/Res microspheres mainly lose weight at 300 °C ~ 380 °C, which is mianly due to the decomposition of the EC/Res microspheres. After the soaking in PBS, the EC/Res microspheres degrades due to the glucose groups begin to dehydrate, while glycosidic bonds, C-O bonds, C-C bonds and other chemical bonds start to break, therefore three weight loss stages are observed in the range of 50 °C ~ 100 °C, 190 °C ~ 250 °C, and 300 °C ~ 380 °C. After the soaking EC/Res microspheres in PBS for 7 days, 15 days, 30 days and 45 days, the EC/Res microspheres lose 0.99%, 2.27%, 4.85% and 7.65% weight in the range of 50 °C ~ 100 °C, which is due to the evaporation of the H 2 O molecules. The second weight loss stages in the range of 190 °C ~ 250 °C is possiblely due to the decomposition of the degradation productes of low molecular polysaccharides and few monosaccharides, and they account for 4.26%, 5.12%, 6.92% and 10.25% respectively. The third weight loss in the range of 300 °C ~ 380 °C was due to the decomposition of the residual EC. In Fig. 16a, the remaining weight left for the EC/Res microspheres soaked in PBS for 7 days, 15 days, 30 days and 45 days is mainly because that Na + and K + adsorped on the EC/Res microspheres when they are soaking in PBS. When they are initial immersed in the fresh PBS for a short time, Na + and K + may accumulate to the EC microspheres, and there is a concentration gradient on Na + and K + between microspheres and PBS. After takingout some EC/Res microspheres for the TGA analysis at different time, some acculmulated ions diffuse to PBS to maintain the equilibrium. Therefore, the shorter the soaking time, the higher the concentration of Na + and K + remained in EC/Res microspheres. therefore, more weight is remained in the TGA analysis. Comprehensively, after soaking the EC/Res microspheres in PBS, the thermal stability of EC decreases and the EC/Res microspheres degrades significantly.

Discussion
In this work, Res-loaded microspheres are prepared successfully and their release and degradation behaviors are studied. In the previous work, Res was encapsulated by chitosan (CS) and poly (D, L-lactic-co-glycolic acid) (PLGA) via a W/O/W double emulsion/solvent evaporation method, and the corresponding encapsulation efficiencies were 40 ~ 52% (Sanna et al. 2015). By the encapsulation of Res in nanoparticles coated with chitosan, an encapsulation efficiency of 51% was obtained (Pauluk et al. 2019). By using liposomes (Caddeo et al. 2008) and methoxy poly(ethylene glycol)-poly(caprolactone) nanoparticles (Shao et al. 2009) to encapsulate Res, the encapsulation efficiency of Res increased to 76.00% and 91.00% respectively. The encapsulation efficiency of Res was further increased to about 93% by preparing chitosan microspheres and vanillin-chitosan microspheres via the emulsion chemical cross-linking method (Peng et al. 2010).
The encapsulation efficiency of EC/Res microspheres prepared in our work is 73.89 ± 1.50% and 90.63 ± 1.62% respectively by using the batch-process and the continuous process. Although there is no significant improvement in the encapsulation efficiency compared with the previous research works for the batch-process, the continuous process enables a comparable high encapsulation efficiency, which is beneficial for industrial production. Furthermore, no work was conducted on the degradation behavior of these kind of microspheres, especially by using the micromanipulation technique.

Conclusion
In this study, biodegradable EC was used as the carrier to prepare EC/Res microspheres by using the improved solvent evaporation method via a foam-transfer process. The results revealed that Res was successfully loaded onto EC microspheres, and the obtained EC/ Res microspheres were in highly porous structures. The properties of the EC/Res microspheres varied with the experimental conditions. A yield of 80.70 ± 1.98 wt% for the EC/Res microspheres and an encapsulation efficiency of 73.89 ± 1.50% of Res were obtained by using the batch-process. The EC/Res microspheres were then produced continuously with an even higher yield and encapsulation efficiency of Res, which was 85.58 ± 2.30 wt% and 90.63% ± 1.62% respectively. The cumulative release rate of EC/Res microspheres in PBS at pH 5.0 under 45 °C reached 91.97 wt% within 120 h, and the mechanical property analysis showed that the EC/Res microspheres degraded significantly within 45 days, in which the hardness decreased from 12.01 ± 0.56 MPa to 4.02 ± 0.45 MPa. Compared with the previous work on the formulation of Res-loaded microspheres, the EC/Res microspheres prepared by the improved solvent evaporation method via the foam-transfer process enables a continuous production, with a relatively high yield and excellent loading efficiency, which is meaningful since it provides a practical solution for Res's application as functional additives. Funding The study was financially supported by National Natural Science Foundation of China (22068018) and Yunnan Ten Thousand Talents Plan Young & Elite Talents Project.

Data availability
The authors confirm that the data supporting the findings of this study are available within the article.

Declarations
Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethical approval and consent to participate.
No ethics approval was required in this work.

Consent for publication
All authors agree to publish the data in this work.