Reaction‐controlled microgel‐lipase co‐stabilized compartmentalized emulsion for recyclable biodiesel production

Pickering emulsions have been widely used for biphasic catalysis in the past decade. However, it remains a great challenge to achieve simple product collection and enzyme recovery. Poly(N‐isopropylacrylamide) (PNIPAM)‐based microgels can endow Pickering emulsions with stimuli‐responsiveness, while most microgel‐stabilized emulsions are oil‐in‐water (O/W) type and not ideal for interfacial catalysis. Besides, altering temperature or pH value for demulsification is time‐ and energy‐consuming and may cause irreversible deactivation of enzymes. In this work, inverse water‐in‐oil (W/O) Pickering emulsions were formed using hexanoic acid‐swollen microgels as the sole emulsifiers. When lipase was added in the water phase, stable oil‐in‐water‐in‐oil (O/W/O) Pickering double emulsions could be formed through one‐step emulsification, owing to the synergistic effect of the hydrophobic microgels and hydrophilic lipase at the interface. Compared with other biphasic systems, such double emulsion systems represent a desirable platform for highly efficient biodiesel production because of the ultra‐high interfacial areas and fast mass transport between two phases. More importantly, the switchable transition between hydrophobicity/hydrophilicity of microgels is controlled by the catalytic reaction. Therefore, double emulsions demulsify spontaneously when substrates are used up without the need for energy input or loss of enzymatic activity, enabling the facile collection of products and demonstrating the excellent recyclability of the biphasic catalysis system.


INTRODUCTION
Microgels, as soft colloids, can swell or shrink in a solvent and spontaneously adsorb at the oil-water interface without any external energy input. [1] Owing to their distinct physicochemical properties, microgels inherit the advantages of polymers and particles and have attracted considerable research interest. [2,3] Poly(N-isopropylacrylamide) (PNIPAM)-based microgels have been extensively investigated in recent decades, [4][5][6][7] and applied in delivery systems, microreactors, and emulsion stabilization. [8][9][10][11][12][13] Compared with conventional Pickering emulsions that are stabilized by rigid particles, microgels are more desirable emulsifiers for stabilizing Pickering emulsions owing to their deformable and stimuli-responsive properties. [14][15][16][17][18] Notably, oil-in-water (O/W) emulsions are typically stabilized using PNIPAM-based microgels owing to their intrinsic hydrophilicity. However, certain applications such as biphasic catalysis and the fabrication of advanced materials necessitate the use of water-in-oil (W/O) emulsions. [9,19,20] Although O/W Pickering emulsions stabilized by stimuli-responsive microgels can be used for interfacial catalysis, [21,22] the emulsion must be demulsified after catalytic reaction to collect the products in the oil phase and recycle the enzyme in the water phase. This phase separation process requires frequent changes in the temperature or pH value of the surrounding environments, which may lead to irreversible structural damage and inactivation of enzymes, resulting in unrecyclable enzyme-mediated interfacial catalysis.
To overcome the challenges of using microgels in interfacial bio-catalysis, stable W/O emulsions must be prepared. [23] By altering the comonomers of microgels, hydrophobic microgels were obtained that can be used for stabilizing W/O Pickering emulsions. [24,25] Moreover, binary particle systems consisting of hydrophilic microgels and hydrophobic silica particles have been developed to prepare W/O emulsions, which can then be used for interfacial biocatalysis. [26,27] Recently, Jiang et al. reported that hybrid microgels with tunable hydrophobicity can be fabricated by integrating hydrophobic silica particles on the surface of hydrophilic PNIPAM microgels to achieve the phase transition between O/W and W/O emulsions. [28] However, in these systems, the enzymes are either trapped inside the microgel networks or encapsulated by water droplets, resulting in a complex and time-consuming immobilization process or limited catalytic activity.
As an important enzyme, lipase plays a main role in multiple biological and chemical reactions, such as fat hydrolysis and biodiesel production. [29,30] Since lipase is soluble in water and the substances are dissolved in oil, the hydrolysis or esterification process mainly occurs at the oil-water interface. Therefore, a large interfacial area between the aqueous and organic phases must be ensured to enhance the efficiency of biphasic enzymatic reactions. [31][32][33] The reaction area can be increased using two main strategies: 1) obtaining smaller emulsion droplets by using surfactants or polymers as the stabilizer; [34][35][36] and 2) constructing emulsions with a multicompartment structure inside larger droplets (termed double emulsions). [37,38] Notably, although surfactants can decrease the interfacial energy and provide sufficient interfacial area during emulsification, the presence of the surface-active molecules may influence the lipase activity and lead to the desorption of the lipase from the interface through competitive adsorption. [39,40] To facilitate the biphasic bio-catalysis, lipase must be anchored at the interface while preventing any loss of catalytic activity. [41] On the other hand, double emulsions have been widely prepared using surfactants, polymers, and particles. Two types of stabilizers with opposite wettability should typically be used to stabilize both W/O and O/W interface simultaneously. Therefore, the selection of stabilizers is more important when preparing double emulsions than when preparing single emulsions. Additionally, the products and enzymes exist in immiscible phases that must be separated after the reaction to realize recycling of the catalytic system, while the controlled phase separation of double emulsions without enzyme deactivation is challenging.
Considering these aspects, this study was aimed at establishing a simple and feasible strategy for preparing compartmentalized emulsions co-stabilized by poly(Nisopropylacrylamide-co-4-vinylpyridine) (PNIPAM-co-4VP) microgels and lipases in two immiscible phases. To realize emulsion compartmentalization, we first dispersed lyophilized microgels in the oil phase and used polar additives (hexanoic acid) for the in situ hydrophobization of microgels to induce emulsion inversion from conventional O/W to W/O type. In the presence of water-soluble lipase in the aqueous phase, the compartmentalized oil-in-water-inoil (O/W/O) Pickering double emulsions could be generated by one-step manual shaking or low-speed vortexing. The facile generation was attributed to the simultaneous adsorption and assembly of the lipase and microgels at the oil-water interfaces. The double emulsions with high interfacial areas enabled the rapid diffusion and transport of substances, thus promising efficient interfacial esterification. The microgels recovered their hydrophilicity after the polar additives were consumed. Consequently, PNIPAM-co-4VP microgels transferred from the interface to the aqueous phase, leading to the spontaneous phase separation of the emulsion. In this manner, the products and lipase could be directly collected from the oil and water phases, respectively. Therefore, polar additive-swollen microgels can realize the reactioncontrolled interfacial bio-catalysis, which can facilitate large-scale and sustainable biodiesel production.

RESULTS AND DISCUSSION
PNIPAM-co-4VP microgels were synthesized through conventional precipitation polymerization in aqueous dispersions. The morphology and particle size of microgels in dry and water-swollen states were investigated. As shown in Figure 1A,B and Figure S1, the mean hydrodynamic diameter of PNIPNM-co-4VP microgels is about 240 nm, nearly twice that of microgels in the dry state, indicating that microgel are swollen in the aqueous solution and the dehydration leads to significant volume shrinkage. As microgels typically show a core-corona profile, we further visualized the morphology of PNIPAM-co-4VP microgels on a substrate. The height profile of a single microgel on the silicon wafer shows that the maximum height of PNIPAM-co-4VP microgel in the dry state is around 17 nm and the height gradually decreases toward microgel periphery until it is a thin layer consisting of tangled polymer chains ( Figure 1C). The change in height profile indicates that PNIPAM-co-4VP microgels are soft and deformable due to the collapse of polymer matrix when drying on the substrate. Initially, both lipase and PNIPAM-co-4VP microgels only formed O/W emulsions when dispersed in the water phase ( Figure 1D,F). As shown in the corresponding confocal laser scanning microscopy (CLSM) images, the PNIPAM-co-4VP microgels adsorbed on the droplet surface to form a physical barrier against droplet coalescence and constructed a particle bridge between adjacent droplets to form three-dimentional gel networks in the continuous phases, thereby enhancing the emulsion stability. Subsequently, we investigated the influence of hexanoic acid on the emulsion morphology and appearance. As shown in Figure 1E,G, the addition of hexanoic acid in the oil phase did not considerably influence the emulsion type and microgel distribution. In addition, the lipase formed wrinkles on the droplet surface regardless of the presence or absence of polar substances, owing to the high surface coverage and packing density ( Figure S2). [42] Previously, we observed that a certain concentration of octanol can promote the swelling and improve the hydrophobicity of poly(N-isopropylacrylamide-co-methacrylic acid) (PNIPAM-co-MAA) microgels in the oil phase, thereby facilitating the formation of stable inverse W/O emulsions. [43] In contrast to those in the case of the O/W emulsions stabilized solely by hydrophilic microgels (Figure 1F,G) The emulsion type could be reversed from typical O/W to W/O when PNIPAM-co-4VP microgels were pre-swollen by hexanoic acid in the oil phase. In addition, the in situ modified microgels appeared to more readily adsorb at the droplet surfaces and form more densely packed interfacial layers, potentially because of the enhanced interfacial activity and higher hydrophobicity of the hexanoic acid-swollen microgels ( Figure 2B). Surprisingly, further cooperation of lipase in the water phase led to the generation of smaller oil droplets within the larger water droplets of these inverse W/O emulsions. As shown in Figure 2C, the double emulsion contained inner oil droplets with different scales ranging from one to dozens of microns. The corresponding CLSM image shows that the formation of double emulsion droplets could be explained by the synergistic adsorption and uneven distribution of the hydrophobic microgels and hydrophilic lipase at the droplet surfaces. The different wettability and the size of the two building blocks (microgels and lipase) indicates that the surface of the larger W/O emulsion droplets was dominated by the hydrophobic PNIPAM-co-4VP microgels, whereas the inner smaller O/W emulsion droplets were mainly stabilized by the hydrophilic lipase, as illustrated in Figure 2A.
To clarify the stabilization mechanism of inverse O/W/O Pickering double emulsions, the three-phase contact angle and dynamic interfacial tension were measured. The dynamic interfacial tension indicates that lipase exhibited a satisfactory interfacial activity and could spontaneously adsorb at the oil-water interface to decrease the interfacial tension ( Figure 3A). The addition of hexanoic acid appeared to reduce only the initial interfacial tension through the solvent effect ( Figure S3), and it did not promote the adsorption of lipase. In contrast, the hexanoic acid-swollen PNIPAM-co-4VP microgels can significantly reduce the interfacial tension within a few seconds ( Figure 3B), leading to rapid interfacial tension equilibrium (approximately 5 mN/m). This rapid equilibrum process was indicative of the fast diffusion toward the interface and the excellent interfacial activity of the hexanoic acid-swollen microgels. Further addition of the lipase solution led to only minor changes in the adsorption kinetics curves, probably because of the much higher interfacial activity of the hexanoic acid swollen-microgels compared to that of lipase. Specifically, the microgels first adsorbed over the droplet surfaces, and then the lipase in the water phase filled the gaps between the neighboring microgels, thereby further decreasing the interfacial tension. Furthermore, the ζ-potential values and hydrodynamic diameters of the microgels, lipase, and their complex indicates no strong electrostatic interaction between them ( Figure S4). In other words, the presence of lipase did not facilitate the adsorption or diffusion of microgels from the oil phase to the interface.
As shown in Figure 3C and Figure S5, lipase exhibited excellent hydrophilicity in both air and oil mediums. In contrast, the contact angle of a water drop on the hexanoic acid-swollen PNIPAM-co-4VP microgel layers was approximately 20 and 40 • higher in the air and oil, respectively, F I G U R E 2 (A) Schematic of formation O/W/O Pickering double emulsion by the combination of the in situ modified PNIPAM-co-4VP microgels in the oil phase and lipase in the water phase. Appearance, optical microscopy images, and corresponding CLSM images of (B) inverse W/O emulsion solely stabilized by 0.5 wt% modified PNIPAM-co-4VP microgels in the oil phase, and (C) O/W/O Pickering double emulsion co-stabilized by 0.5 wt% modified PNIPAM-co-4VP microgels in the oil phase and 2 wt% lipase in the water phase than those of the original microgels. The water contact angle of the modified microgels remained relatively constant during the measurement, which demonstrates the enhanced affinity of hexanoic acid-swollen microgels toward the oil phase ( Figure 3D). These results indicate that the presence of hexanoic acid can effectively enhance the hydrophobicity of microgels, resulting in the formation of stable W/O emulsions. Figure 3E shows the interfacial dilatational moduli of a drop surface laden with the assembled microgels or lipase. The PNIPAM-co-4VP microgels (Sample 1) formed a weak interfacial film with low viscoelasticity (|E*| ∼ 2 mN/m) owing to the low surface coverage and electrostatic repulsion between the in-plane microgels ( Figure S5b). Although the lipase was less interfacial active than the microgels, the viscoelasticity of the lipase-laden interface (Sample 2) was considerably higher, with a complex modulus of approximately 123 mN/m. This strong viscoelasticity of the lipase-laden interface has been reported as a feature of the unique interfacial activity of lipase. [44] The introduction of hexanoic acid into the biphasic system caused a reduction of dilatational moduli, potentially because of the increased miscibility of the polar and apolar phases that resulted in a lower interfacial energy. Moreover, the lipase was expected to undergo conformational rearrangements at the interface in the presence of polar substances. Worth noting that the presence of lipase can enhance the dilatational viscoelasticity of the drop surface laden with hexanoic acid-swollen microgels. Specifically, the addition of 0.1 and 0.5 wt% lipase in the biphasic system containing 0.1 wt% microgels in the oil phase led to a significant increase in |E*| from 2.022 mN/m to 4.568 and 10.608 mN/m, respectively.
The appearance of pendant drops during volume reduction and expansion is shown in Figure 4F,G. Unlike the hexanoic acid-swollen PNIPAM-co-4VP microgels (Sample 1), wrinkles were clearly visible on the lipase-laden drop surfaces when a small compressive force was applied (Samples 2 and 3). The presence of the wrinkles indicates the formation of a highly elastic layer with a high surface coverage of lipase, which led to the buckling and jamming of the adsorption layer during deformation. Furthermore, the interface of the microgel-lipase mixture exhibited a stronger viscoelastic response than sole microgel-laden interface. In the case of the strengthened surface, wrinkles were formed on the drop surface, and the drop exhibited a high deformability under compression (Sample 4). Evidence for these phenomena is provided by the corresponding Lissajous plots presented in Figure 4A-E, which show distinctive features of these interfaces. The Lissajous plot of PNIPAM-co-4VP microgel-laden interface ( Figure 4A) is overall symmetric, and only a small surface pressure can be observed in both compression (lower part of the cycle) and expansion processes (upper part of the cycle) owing to the low surface coverage of microgels. In contrast, the plot of the lipaseladen interface ( Figure 4D,E) exhibits obvious asymmetry, likely caused by the structural rearrangement of the lipase during interface deformation. The results suggest the occurrence of strain hardening with a stronger elastic response in the expansion at even a small deformation (5%). Compared to the feature of the expansion, the wider cycle in the lower part of the plot indicates the increased viscous response of the interface during compression.
In terms of the interface laden with microgel-lipase mixture, the higher surface pressure during deformation indicates that compared with the interface covered by swollen microgels ( Figure 4A), the presence of lipase endowed the complex interface with a stronger viscoelastic response ( Figure 4B,C) and a higher dilatational modulus ( Figure 3E). The improved viscoelasticity of the microgel-lipase complex interface is illustrated in Figure 4H. Because of the core-corona profile and electrostatic repulsion between the positively charged PNIPAM-co-4VP microgels, the microgels at the interface were sparsely distributed, with free space between adjacent microgels. Therefore, the formed drop surface exhibited low coverage and weak viscoelasticity, and no wrinkles appeared during deformation ( Figure 4G). After lipase was introduced in the biphasic system, the surface-active lipase with a small size could adsorb onto and fill the confined spaces between microgels, thereby improving the overall surface coverage ratio. Moreover, lipase could form non-covalent physical interactions with microgels. Consequently, the microgellipase mixture buckled and jammed together and potentially underwent attractive capillary interactions during deformation, leading to higher interfacial viscoelasticity and better stability of both the interface and as-prepared emulsions.
In recent years, microgel-stabilized emulsions have been prepared for reversible catalysis owing to their responsiveness. [21,22] However, the regulation of pH values and temperature may adversely influence the catalytic reactions, for instance, in the form of inactivation of the sensitive enzymes. In addition, the demulsification of stable emulsions requires additional processing steps, which is energy-consuming and unfavorable for industrial production. Therefore, a green strategy for achieving efficient and recyclable bio-catalysis must be identified.
In this study, we observed that hexanoic acid molecules can enhance the hydrophobicity of PNIPAM-co-4VP microgels and promote the formation of stable inverse W/O Pickering emulsions ( Figure 2B). If the microgels are initially swollen by alcohol and organic acid, they can be expected to not only function as emulsion stabilizers but also facilitate biphasic interfacial catalysis. Furthermore, with the conversion of two polar reactants (hexanoic acid and 1-hexanol) to the non-polar product (hexyl hexanoate), the hydrophobicity and interfacial activity of the microgels are expected to decrease, allowing the rapid demulsification of the biphasic catalysis system and the recycling of enzymes. To verify these hypotheses, the esterification of 1-hexanol with hexanoic acid was conducted to evaluate the catalytic performance of the inverse Pickering emulsion systems. As shown in Figure 5B, an O/W/O Pickering double emulsion was created, with numerous small oil droplets encapsulated within larger water droplets when emulsifying the oil phase containing a high concentration (2 M) of polar substances and PNIPAM-co-4VP microgels with the lipase solution. The versatility of this strategy was also investigated by using another type of microgels (PNIPAM-co-MAA) with different sizes and surface properties ( Figure S6). It was found that the emulsion stabilized by PNIPAM-co-MAA microgels displayed a similar complex inner structure ( Figure 5E).
Interestingly, the emulsion stabilized by PNIPAM-co-4VP microgels became unstable and demulsification sponta-neously occurred after approximately 40 min of reaction time ( Figure 5C). The stabilization and demulsification mechanisms are illustrated in Figure 5A. Based on the aforementioned result (Figure 2C), the unique double emulsion morphology was considered to be the effect of the costabilization of the hydrophobic microgels and hydrophilic lipase in the presence of polar substances at high concentrations. In the initial stage of the reaction, the inner O/W interface was mainly occupied by hydrophilic lipase, and the outer W/O interface was mainly laden with the hydrophobically modified microgels. After consuming 1-hexanol and hexanoic acid, the PNIPAM-co-4VP microgels gradually recovered their hydrophilicity and subsequently transferred from the interface to the aqueous phase, which led to phase separation ( Figure 5C). Therefore, the product solution could be easily separated and collected after the reaction. The residual water phase containing free lipase could be recycled and re-emulsified with a fresh substance solution for recyclable catalysis ( Figure 5D).
The double emulsion co-stabilized by PNIPAM-co-MAA microgels and lipase was considerably more stable than the biphasic catalysis system stabilized by PNIPAM-co-4VP  Figure 5E,F indicates that the morphology of the double emulsions after the reaction was similar to that before the reaction. In addition, the corresponding CLSM images show that the PNIPAMco-MAA microgels and lipase remained tightly adsorbed at the interface of both W/O and O/W even at low concentrations of polar additives, thereby enhancing the stability of the double emulsion ( Figure 5H). Although demulsification was not achieved, the biphasic catalysis system containing PNIPAM-co-MAA microgels could be recycled by removing the upper oil layer and adding a fresh substrate solution after gravitational sedimentation ( Figure 5G).
The catalytic performances of the O/W/O Pickering double emulsions co-stabilized by microgels and lipase and those of the other three biphasic catalysis systems are demonstrated in Figure 6A. The catalytic rates and conversion efficiencies of the two types of microgel-stabilized emulsions were similar, and both PNIPAM-co-4VP and PNIPAM-co-MAA microgels endowed the double emulsions with a high initial reaction rate, especially in the first 30 min. The catalysis was nearly complete with approximately 90% conversion after 50 min, whereas only 20% of the conversion was achieved in the biphasic system containing free lipase in the water phase. As shown in Figure 6B, the similarity in the catalysis performances of the microgel-stabilized double emulsions was attributable to the different size distributions of the inner oil droplets. In other words, the oil droplets co-stabilized by PNIPAM-co-4VP microgels and lipase had a uniform size of approximately 3 μm, whereas the PNIPAM-co-MAA microgels and lipase resulted in inner droplets with wide ranging sizes. In addition to the larger oil droplets generated because of the larger PNIPAM-co-MAA microgels at the interface, smaller droplets (1-2 μm) stabilized by lipase were present, which provided additional interfacial areas for catalysis and enhanced the catalysis efficiency.
To investigate the effect of the soft microgels on the catalytic performance in biphasic systems, a Pickering emulsion stabilized by hydrophobic rigid silica particles with the size (∼240 nm) similar to that of the PNIPAM-co-4VP microgels was prepared. Interestingly, we observed that the  Figure S7), although the initial reaction rate of the as-prepared emulsion was only half of that of the emulsions stabilized by microgels ( Figure 6A). Considering the interfacial activity of lipase, we attempted to emulsify the oil and water phases containing free lipase. The result indicates that W/O emulsions could not be formed, as they underwent rapid phase separation in a few seconds owing to the high oil-water ratio (W:O = 1:4) and intrinsic hydrophilicity of lipase. However, some small oil droplets could be observed in the water phase after emulsification. Although these small droplets rapidly coalesced to form larger droplets after formation, the final O/W emulsion was stable with droplet sizes between 10 and 20 μm during catalysis ( Figure S8). As only a small fraction of the oil phase was emulsified, the lipase-stabilized emulsion exhibited a limited improvement in catalytic performance compared with the biphasic system containing free lipase, and both systems had a final conversion level of less than 60% ( Figure  S9). On the other hand, the initial reaction rate (first 10 min) of the microgel-stabilized emulsions was more than twice those of the silica particle-stabilized Pickering emulsion and lipase-stabilized emulsion, and approximately 11 times higher than that of the free lipase system ( Figure 6A).
The high reaction rate and high conversion of substances in the systems containing microgels were contributable to the high interfacial areas offered by the double emulsions and multiple catalytic sites at the interface owing to the presence of lipase. In addition, "semi-open" microgels at the interface provided channels for rapid mass diffusion during the reaction and attracted more polar substances onto the interface because of hydrogen bonding, thus facilitating the catalytic reaction. Subsequently, we measured the specific activity of lipase in different systems. Figure 6C shows that the emulsion system stabilized by PNIPAM-co-4VP microgels exhibited the highest specific activity (1344 U mg −1 ), considerably higher than that of the emulsion stabilized by silica particles (623 U mg −1 ). In addition, the specific activity of lipase after emulsification (523 U mg −1 ) was higher than that of the free lipase in the water phase (112 U mg −1 ). Furthermore, we evaluated the recyclability of the Pickering double emulsion system stabilized by PNIPAM-co-4VP microgels by repeating the catalytic reaction 10 times. The cyclic catalysis did not require time-and energy-consuming processes such as highspeed centrifugation or temperature variations. Therefore, enzymatic activity was well retained ( Figure 6D), suggesting the excellent recyclability of the proposed reaction-controlled interfacial bio-catalysis system.

CONCLUSION
In summary, we developed a facile strategy to prepare O/W/O Pickering double emulsions by the synergistic combination of hydrophobic microgels and hydrophilic lipase in two immiscible liquids. Hexanoic acid-swollen microgels exhibited an enhanced surface hydrophobicity, which facilitated the formation of stable inverse W/O Pickering emulsions. Lipase functioned as a special stabilizer for double emulsions and provided multiple reaction sites for interfacial catalysis. Compared with those of the free lipase in the water phase or conventional Pickering emulsions stabilized by rigid particles, the double emulsion systems containing microgels and lipase at the interfaces exhibited a considerably higher reaction rate, catalytic efficiency, enzymatic activity, and final conversion for esterification between hexanoic acid and 1-hexanol because of the ultra-high interfacial areas and rapid transport of substances. Because the concentration of polar additives could control the wettability of PNIPAMco-4VP microgels, the double emulsion was spontaneously demulsified after the catalytic reaction. Subsequently, the products could be simply collected, and the double emulsions could be recycled multiple times for catalysis without the loss of enzymatic activity. Since microgel is capable of immobilizing other enzymes, we envisage that such double emulsion systems containing microgels and lipase can be used for large-scale industrial production of biodiesel and provide a desirable platform for potential cascade catalysis.

A U T H O R C O N T R I B U T I O N S
Xin Guan: conceptualization, methodology, investigation, formal analysis, and writing-original draft. Zhili Wan: formal analysis, writing-review and editing, funding acquisition, and supervision. To Ngai: conceptualization, writing-review and editing, project administration, funding acquisition, and supervision.

A C K N O W L E D G M E N T S
The authors gratefully acknowledge the financial support from the Hong Kong Special Administration Region (HKSAR) General Research Fund (CUHK14304619, 2130642) of The Chinese University of Hong Kong, the National Natural Science Foundation of China (32172347), and the Natural Science Foundation of Guangdong Province (2021A1515011000).

C O N F L I C T O F I N T E R E S T
The authors declare no conflict of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.