Pickering emulsion: From controllable fabrication to biomedical application

Pickering emulsion, stabilized by solid particles, possesses low toxicity, high stability, large surface area as well as good biocompatibility in comparison with traditional molecular surfactant‐stabilized emulsion. As such, Pickering emulsion has received much attention in numerous fields including the chemical industry, food industry, agriculture, and biomedicine. In this review, we summarize the recent progress of Pickering emulsion from controllable fabrication to biomedical applications. Firstly, we elaborate on the influence of solid‐particle surfactant on the fabrication of Pickering emulsions and their corresponding preparation technologies. Secondly, we focus on the biomedical applications of Pickering emulsion, including antitumor treatment, antiviral infection, biosensing and biocatalysis as well as wound healing. Finally, we further introduce the proof‐of‐concept of Pickering emulsion and comment on the prospects of this emerging field.


| INTRODUCTION
Pickering emulsion was first explored by Ramsden and Pickering in the early 1900s 1,2 In the study, they found that sulfate particles enabled to adsorb onto the oil-water interface and block the coalescence of droplets, allowing the formation of a stable emulsion. In general, there are three components in the fabrication of Pickering emulsion, including solid particles, water phase, and oil phase. Compared with the traditional emulsion stabilized by molecule surfactants, Pickering emulsion can form a core/ shell structure and exhibit high stability due to a more efficient reduction of interfacial energy. Also, solid particles strongly bind to the oil-water interface and can provide a large specific surface area, which is beneficial to molecular transport, diffusion and even reaction in the oilwater interface. In biomedical applications, Pickering emulsion also possesses low toxicity and good biocompatibility compared with traditional surfactant-stabilized emulsion. For example, Porta et al. reported that silicabased Pickering emulsions served as implantable drug carriers for controllable drug delivery. 3 Retinoic acid used as a model drug was entrapped in the Pickering emulsion to study the development and tail regeneration of zebrafish. The retinoic acid-loaded Pickering emulsion showed excellent biocompatibility without inflammation at the injection site of zebrafish. Thanks to the rapid developments of material science and technology, more and more innovative solid particles with tunable properties, such as stimuli-responsive particles, have been designed to prepare multifunctional Pickering emulsions. For example, Low et al. reported a responsive Fe 3 O 4 @cellulose nanocrystal-stabilized Pickering emulsion containing curcumin, into which the release of curcumin was observed to be 14.59 � 3.66% and 53.30 � 5.08% without and with an external magnetic field, respectively. Thus, the magnetically responsive Pickering emulsion could greatly inhibit the proliferation of human colon cancer cells due to the efficiently magnetically responsive drug accumulation and release into the tumor tissues. 4 Zheng et al. designed conductive Ti 3 C 2 T x nanoflakes-stabilized Pickering emulsion. 5 The self-assembly of Ti 3 C 2 T x nanoflakes at the oil-water interface formed a segregated network, enabling to effectively reduce the electrical percolation threshold. The conductive Pickering emulsion exhibited excellent potential in tissue engineering, biosensors, electromagnetic interference shielding, and so on. To date, Pickering emulsion has become a new research hotspot in the application of biomedicine, such as antitumor treatment, antiviral infection, biosensing, biocatalysis and wound healing ( Figure 1). Several studies have been reviewed on Pickering emulsions and their applications a few years ago. [6][7][8][9][10][11] However, few reviews have described the structural advantages of Pickering emulsions in the biomedical applications, such as flexibility and fluidity. Also, based on the features of different diseases, the classification discussion of Pickering emulsion on different disease treatment has not been well summarized.
In this review, we summarize the recent progress of Pickering emulsion from controllable fabrication to biomedical applications. Firstly, we summarize the effect of solid particles with different properties (such as wettability, size, shapes, roughness, and so on) on the fabrication of Pickering emulsions. And also, we discuss the different preparation technologies of Pickering emulsion. Finally, we categorize and summarize the biomedical applications of Pickering emulsion in recent years. Through these discussions, we further describe the current challenges and prospects of Pickering emulsion in the field of biomedicine.

| The wettability of solid particles determines the type of Pickering emulsion
The surface wettability of solid particles is a crucial factor that dominates the stability and the types of Pickering emulsions, usually characterized by the contact angle θ of solid particles. 12 In Equation (1), γ αβ represents the tension of the interface, R represents the radius of a single spherical particle and E represents the interfacial energy required to remove the solid particles from the oil-water interface.
As shown in Figure 2, solid particle with hydrophilicity (θ < 90°) can easily form an oil-in-water (O/W) emulsion, whereas solid particles with hydrophobicity (θ > 90°) tend to form a water-in-oil (W/O) emulsion. When θ is close to 90°, the particles possess the maximum desorption energy at the oil-water interface, according to Equation (1), and a more stable Pickering emulsion is generally formed. And when θ approaches 0°o r 180°, the solid particles are approaching super hydrophilicity or super hydrophobicity, making these solid particles difficult to completely stabilize the interface.

| The effect of the size of solid particles on the fabrication of Pickering emulsion
The size of solid particles is a critical factor that affects the size of Pickering emulsion. Researchers have shown that a variety of amphiphilic solid particles can successfully be used to formulate Pickering emulsions, which may be either inorganic or organic particles. [13][14][15][16][17][18] Thus, it is critical to investigate the size effect of various solid particles on the fabrication of Pickering emulsions. In general, the size of Pickering emulsion is reduced as the size of the solid particles decreases. Kim et al. used silica nanoparticles with different sizes (5,12,25, and 80 nm) to prepare Pickering emulsion. 13 They showed that the obtained Pickering emulsions with smaller size and higher apparent viscosity were formed as the size of solid particles decreased. This was because the adsorption kinetics of larger particles were slow, leading to a high adsorption barrier and a less effective package at the interface. Binks and Lumsdon developed Pickering emulsions stabilized by polystyrene latex particles. The average size of the obtained Pickering emulsion (35-75 μm) increased initially with increasing particle size and then remained constant. 14 Besides, Qi et al. used poly (D,L-latctic-co-glycolic acid) (PLGA) particles with different sizes (330, 620, and 1150 nm) to fabricate Pickering emulsion. 15 In comparison, PLGA particles with size of 330 nm exhibited higher interfacial activity, allowing to prepare a more stable Pickering emulsion because smaller-sized PLGA particles were more efficient to form a dense layer at the interface to hinder the coalescence of droplets. In addition, the size of solid particles from natural sources has been confirmed to exert the same role in the fabrication of Pickering emulsions. Li et al. found that Pickering emulsion could not be prepared by large-sized potato starch granules (52.1 μm), while Pickering emulsion could be formed by using small-sized rice starch granules (5.2 μm). 16 The obtained Pickering emulsions could remain stable against coalescence for at least several months.

| The effect of concentration of solid particles on the fabrication of Pickering emulsion
The concentration of solid particles is another factor that affects the size and stability of Pickering emulsion. [19][20][21][22] Jessica et al. reported the effect of the concentration of silica particles on the stability of the Pickering emulsion. 19 They found that a Pickering emulsion could be formed at 0.3 wt.% silica particles, however the obtained Pickering emulsion could flocculate together within a few days. In comparison, when they increased the silica particle concentration above 1 wt.%, a stable Pickering emulsion could be formed. Varanasi et al. studied the general rule of Pickering emulsion fabricated by sulfated cotton cellulose nanocrystals (CNC). 21 They found that the Pickering emulsion could hardly be formed when the concentration of CNC ranged from 0.1 to 0.5 wt.%, while the Pickering emulsion presented partial stability at 1-2 wt.% concentration. When the concentration of CNC further increased to 3-5 wt.%, the stable Pickering emulsion was prepared. Moreover, several studies analyzed the rheology properties of CNC to explain the stability of CNC-based Pickering emulsions. Li et al. found that the rheological behavior of the CNC was strongly dependent on their concentration and thus proposed the existing strong hydrogen bonding between CNC and water molecules that enabled to affect the stability of CNC-based Pickering emulsion. 23 Xu et al. observed that CNC could efficiently aggregate at the oilwater interface as a result of the increase in their concentration so that they could be trapped by their neighbors through electrostatic interactions. 24 In addition, Paunov et al. developed a thermodynamic model for the adsorption of charged particles at the oil-water interface, into which they derived the equations regarding the respective adsorption isotherms of particles. They observed charged solid particles had a higher adsorption energy at high concentration. 25 At high concentration, negativelycharged CNC aggregates (containing sulfate groups) had higher adsorption energy that reduced the repulsion force between CNC and oil-water interface to stabilize the emulsions.

| The effect of shape of solid particles on the fabrication of Pickering emulsion
The shape of solid particles is also a factor that affects the size and stability of Pickering emulsion. In most cases, sphere particles are usually used as a stabilizer to fabricate Pickering emulsions. In recent years, owing to the rapid development of non-spherical particles, more and more particles with various shapes, including worms, 22 fiber, 26,27 cubes, 28 peanuts, 28 ellipsoids, 29 mushrooms, 30 and rods, 31 have been applied to prepare Pickering emulsion. However, the stabilization mechanism of these nonspherical particles has not been fully elucidated. Fujii et al. studied the effect of different shapes of hydroxyapatite (HAP) nanoparticles on the size and stability of Pickering emulsion. 26 They synthesized HAP nanoparticles with spherical, rod-shaped, fiber-shaped morphologies via a wet chemical method. These HAP nanoparticles could successfully stabilize methyl myristate-in-water Pickering emulsion for at least 6 months. The size of the Pickering emulsion increased in order from the spherical HAP, fibershaped HAP to rod-shaped HAP. In their study, compared with non-spherical HAP particles, spherical particles had the largest surface area, enabling to stabilize larger oilwater interface areas. In another work, it was reported that rod-like-shaped CNCs were flexible enough to bend at the oil-water interface, giving rise to high deformationmediated capillary interactions responsible for the stability of Pickering emulsion. 31 Madivala et al. developed two types of non-spherical particles-prolate ellipsoid polystyrene latex particles with aspect ratios ranging from 1 to 9 and spindle hematite particles with aspect ratios ranging from 1 to 6. They found when the aspect ratio of the hematite particles exceeded 4.6, a stable Pickering emulsion could be formed while a stable Pickering emulsion was already obtained from polystyrene particles with lower aspect ratios (3.0) compared to the hematite particles. The results demonstrated that a stable Pickering emulsion could be obtained for non-spherical particles with a sufficient aspect ratio. It was explained that the suitable aspect ratio could lead to the increased coverage in the oil-water interface and the occurrence of shape-induced capillary interactions. 32 Collectively, different shaped-solid particles possibly present distinctive mechanism on the fabrication of Pickering emulsion, thus needing to be deeply investigated.

| The effect of surface charge of solid particles on the fabrication of Pickering emulsion
The surface charge of solid particles usually affects the stability of Pickering emulsions. Different reaction conditions can influence the electrostatic repulsion among charged solid particles, such as pH value and salt concentration. Ridel et al. observed that the stability of Pickering emulsion prepared by charged silica nanoparticles could be ensured by decreasing the pH value from 9 to 3 due to the reduced electrostatic repulsion. 33 Luo et al. conducted the interfacial assembly of Pickering emulsion stabilized by gold nanoparticles functionalized with stoichiometric ion-pairs. 34 They could control the electrostatic interactions by adjusting the aqueous pH value. The particles at the oil-water interface maintained stability at pH ≤10, while the particles could be partially dispersed in the aqueous phase at 10 < pH < 11.4 and fully dispersed at pH ≥11.4. The mechanism of these particles desorbing from Pickering emulsion was due to electrostatic repulsion between the particles. In addition, a previous report showed that salts were well known for screening the electrostatic repulsion on the surface of CNC. 35 Varanasi et al. studied the effect of NaCl or CaCl 2 addition on the stability of Pickering emulsion stabilized by CNC. 20 They demonstrated that the adsorption of ions and ion pairs within the CNC surface could shield the surface charge of CNC and reduce the electrostatic repulsion, which facilitated the CNC to migrate and adsorb onto the oil-water interface in the presence of Na + and Ca 2+ ions. Collectively, the stability of Pickering emulsion with charged solid particles can be governed by the surface charge in the presence of electrolytes or adjusting pH value.

| The effect of surface roughness of solid particles on the fabrication of Pickering emulsion
The surface roughness of solid particles can affect the stability of Pickering emulsions. It was reported that surface roughness appreciably lowered particle emulsifying power, enabling to further affect the stability of Pickering emulsions. Vignati et al. prepared Pickering emulsions stabilized by monodisperse and fluorescent silica colloids with a smooth or a rough surface. It suggested the surface roughness could have a negative impact on the stability of Pickering emulsion. 36 However, the results on the surface roughness of solid particles were not absolute for the stability of Pickering emulsion. 37 San-Miguel and Behrens indicated that particle surface roughness could be beneficial to the stability of emulsions as long as wetting occurred homogeneously, whereas a loss of stability for emulsion when wetting occurred heterogeneously. Collectively, to conclude a general rule on particle roughness, in-depth studies of Pickering emulsions require investigating.

PREPARING PICKERING EMULSION
Several technologies, such as rotor-stator homogenization, high-pressure homogenization, ultrasonic emulsification, membrane emulsification, and microfluidic techniques, have been applied to fabricate the Pickering emulsion. 9 The comparison of preparation methods of Pickering emulsion is summarized in Table 1.

| Rotor-stator homogenization
A rotor-stator homogenizer, consisting of a rotor with blades and a stator with openings, is considered to be one of the most popular methods to prepare Pickering emulsion ( Figure 3A). As the rotor rotates, the liquids were drawn in and then accelerated tangentially, finally fed radially through the slots. 38 The size of the Pickering emulsion was mainly dominated by the shear force generated between the rotor and the stator. In general, the size of the obtained Pickering emulsion is inversely proportional to the rotation speed and homogenization time. [39][40][41] The homogenization speeds mostly varied from 5000 to 30,000 rpm, while the emulsification time were in the range from several seconds to a few minutes. Benefiting from a variety of advantages including low operating costs, ease of setting up and using, and timesaving, the rotor-stator homogenization had been extensively used for the preparation of Pickering emulsion in laboratory and industrial scales. 42 However, there were still many drawbacks during the process of rotor-stator homogenization. Owing to the frictional forces, the temperature was likely to increase, resulting in the destabilization of the temperature-sensitive Pickering emulsion during the preparation process. Moreover, the broad size distribution of the obtained Pickering emulsion was not desired for many applications. And also, the high shear rate might make some fragile aggregates and particles deformed or destabilized. 43,44

| High-pressure homogenization
High-pressure homogenization is another common method used for preparing Pickering emulsion ( Figure 3B). The high-pressure homogenizer usually consists of a high-pressure pump and a homogenizing nozzle.

Preparation method Advantage Disadvantage References
Rotor-stator homogenization Generally, the primary emulsion with a larger size was obtained using a rotor-stator or a vortex mixer. Then, the primary Pickering emulsion could be broken to form the smaller size emulsion under a high-pressure pump. 45 In addition, the size of Pickering emulsion could be controlled easily by tuning the homogenizer pressure as well as the number of homogenizer cycles during the process of homogenization. 46,47 In general, the size of the Pickering emulsion decreases with the increase in the homogenizer pressure and cycles. A large volume of samples could be obtained continuously and repeatably via high-pressure homogenization. Also, it was easy to obtain nanosized-emulsion. However, the drawback of this method is the high running cost resulting from the high energy consumption. The minimum volume needed was usually at least tens of milliliters, and the micro/nanoparticles might induce the abrasion of the equipment during the process of homogenization, especially with high pressure. On the other hand, the high shear pressure could deform or even disrupt the particle emulsifier, leading to a broad droplet size distribution, which was similar to rotor-stator homogenization. 48-50

| Ultrasonic emulsification
The most common ultrasonic device for preparing Pickering emulsion is the use of an ultrasonic probe. The ultrasonic probe transmits energy to the liquids, leading to the emulsification by acoustic cavitation and ultrasonic force ( Figure 3C). The major parameters, such as the ultrasound frequency, power, and homogenization time had an influence on the size of Pickering emulsion. 51 The homogenization process was easy and rapid. 52 However, during the preparation process of Pickering emulsions, there are the risks of particle deformation or disruption similar to rotor-stator homogenization and high-pressure homogenization. Also, the obtained Pickering emulsion exhibited a broad size distribution.

| Membrane emulsification
There are two types of membrane emulsification methods, including direct membrane emulsification and premix membrane emulsification. Direct membrane emulsification is a commonly used membrane emulsification technology ( Figure 3D). In direct membrane emulsification, the Pickering emulisons are prepared by pressing or injecting a pure dispersed phase into a continuous phase through a microporous membrane. In the premix membrane emulsification, the pre-mix emulsion is firstly formed, which is then pressed through a microporous membrane. Various emulsification technologies based on the principle of direct membrane emulsification have been developed, including cross-flow membrane emulsification, 48 rotating membrane emulsification, 53 stirred-cell membrane emulsification, 50,54 and vibrating membrane emulsification. 55 Membrane emulsification presents many advantages over the previous three preparation technologies. First of all, membrane emulsification had a low shear force and nearly no risk of particle breakage, thus a stable Pickering emulsion could be formed. 48 Secondly, the Pickering emulsion produced by membrane emulsification exhibited uniform size and good polydispersity. 50,54 Furthermore, membrane emulsifying equipment is low cost because of low energy consumption. 50 However, there were still some shortcomings in this technology, for example, it was only suitable for low-viscosity systems. 56,57

| Microfluidic emulsification
Microfluidic devices are also used to prepare Pickering emulsion by microchannel emulsification. As shown in Figure 3E, in a cross-flowing device, when the dispersion phase was extruded in microchannel, the solid particles in the continuous phase enabled to adsorbed on the surface of the droplts immediately, resulting in the formation of Pickering emulsion. Compared with conventional emulsion stabilized by surfactants, microfluidic technique can rapidly prepare the Pickering emulsion. The size of the Pickering emulsion can be regulated by changing the liquid flow rate or microchannel geometry. 50,54,[58][59][60] Because the microchannels have uniform channel size, the uniform Pickering emulsion could be formed. However, microfluidic technique is more efficient to prepare the Pickering emulsion with large sizes, usually more than several microns. It remains a great challenge to fabricate nano-sized Pickering emulsion.

| THE APPLICATIONS OF PICKERING EMULSION IN BIOMEDICINE
As is well known, biomaterials can be designed to mimic the structure and function of living systems, facilitating them to effectively treat diseases. [61][62][63] Pickering emulsions have great potential in the biomimetic field because of their excellent viscoelasticity and flexibility. 64 Pickering emulsions employed in biomedicine mainly focus on developing their innovative biological function and borading their biomedical potential by using diverse solid particles as a stabilizer. These particles used for stabilizing Pickering emulsion are mainly divided into inorganic and organic particles. The most studied inorganic particles include silica particles, 65 HAP particles, 66 and magnesium hydroxide particles. 67 Organic particles included polymer particles 64,68-70 and natural particles derived from natural sources (such as starch, 71 cyclodextrin, 72 and cellulose 73 ).
Herein, we will introduce the biomedical applications of Pickering emulsion in recent years, mainly including disease treatment, bioimaging and biosensing, biocatalysis, and so on.

| The application of Pickering emulsion in tumor treatment
Due to high loading capacity, low toxicity and good biocompatability, Pickering emulsion as a drug delivery system presents a great potential for tumor treatment. Pickering emulsions loaded with therapeutic agents are employed to treat tumors, including chemotherapy, immunotherapy, and interventional therapy as well as combination treatments.

| Chemotherapy or combination therapy
Chemotherapy is the most common treatment strategy for cancers. Chen et al. fabricated Pickering emulsion using self-assembling deformable poly(N-isopropylacrylamide)based nanogels at oil-water interface ( Figure 4A). 74 Interestingly, the nanogels-stabilized micron-sized Pickering emulsion could further be compressed into nano-sized emulsion under ultrasonication due to ultrasonication-triggered dehydration of the nanogels. The nano-sized emulsion loading with paclitaxel significantly extended blood circulation in vivo ( Figure 4B), greatly enhancing the accumulation of paclitaxel and thereby exhibiting excellent anti-tumor effect in vivo ( Figure 4C). Other documents still confirmed that soft particles could significantly decrease oil-water interfacial tension, which was beneficial to stabilize Pickering emulsion well. [75][76][77] In addition, it is a feasible strategy to improve tumor therapeutic efficacy of Pickering emulsion by enhancing tumorspecific ability or tumor microenviroment-responsive ability. Shang et al. employed cRGD and cystamine to cross-link with poly (N-isopropyl acrylamide-co-acrylic acid) nanoparticles by chemical coupling method.

PAN ET AL.
Among them, cRGD served as a tumor targeting ligand and cystamine served as a controlled release agent responsive to tumor microenvironment. The cRGD and cystamine modified poly (N-isopropyl acrylamide-co-acrylic acid) nanoparticles could be used to fabricate Pickering emulsion, achieving tumor microenvironment glutathioneresponsive controlled release and targeting delivery for melanoma. 78 A combination of chemotherapy and immunotherapy is a promising strategy to enhance tumor treatment efficacy in recent years. Due to the core/shell structure of the Pickering emulsion, it can simulateously load therapeutic agents with hydrophilicity and hydrophobicity for combination tumor therapy. Jia et al. constructed a Pickering emulsion loaded with chemotherapeutic drugs and immunosuppressors (doxorubicin [DOX] and PD-1/PD-L1 inhibitor HY19991). The nanogels loaded with DOX severed as a stabilizer to fabricate Pickering emulsions into which oil-soluble HY was encapsulated in the inner oil phase. Thus, DOX could be released from nanogels and entered into tumor cell nucleus, while HY19991 was released from the inside of Pickering emulsion and entered into tumor cell cytoplasm. 79 In addition, Pickering emulsion can develop drug-controlled release ability by modulating the size and packing density of colloidal particles. [80][81][82] Hu et al. designed a Pickering emulsion stabilized by galactose functionalized hydroxyethyl starch-polycaprolactone (Gal-HES-PCL) nanoparticles loaded with DOX and indocyanine green (ICG), into which DOX could freely release from the carrier to enter the nucleus and exert a therapeutic effect, while free ICG stayed on the emulsion to perform excellent imaging ability and photothermal effects. 83 Thus, colloidal particles usually serve as templates for fabrication of Pickering

| Immunotherapy
Pickering emulsions are also easy to deliver immune checkpoint inhibitors for immunotherapy treatment of tumors. 86 Tselikas et al. designed a PLGA nanoparticleengineered Pickering emulsion loaded with anti-CTLA4, which could slowly and continuously release anti-CTLA4 for 3 weeks and could effectively cure mice in a syngeneic immunocompetent preclinical tumor model. 87 In addition, it was reported that antibodies mixed with oil could reduce the risk of treatment-induced side toxicity to further promote the clinical use of Pickering emulsion. 88 Notably, the Pickering emulsion was able to monitor drug distribution in real-time when radiopaque ethiodized oil with imaging ability served as the oil phase. 89 Vaccines are one of the most effective ways to prevent cancer. To enhance the immune response, adjuvants are usually added to vaccines in the practical applications. However, it is difficult to functionalize modification and load multi-level immunostimulating components for traditional emulsion stabilized by surfactants. 90 Also, most particle adjuvants were rigid structures, which were difficult to mimic the flexibility and fluidity of pathogens as they entered cells. 91 Pickering emulsion can overcome the adjuvant's shortcomings and exert good vaccine adjuvant effects due to its unqiue struture advantage. Xia et al. developed a Pickering emulsion stabilized by PLGA nanoparticles, which provided a high surface area benificial to antigen loading and elastic deformation ( Figure 5A). 64 Due to the good flexibility and fluidity, the Pickering emulsion could mimic the dense and repeated arrangement of surface antigens of pathogens ( Figure 5B). In particular, when interacted with antigen presenting cells, the Pickering emulsion could present the force-dependent deformation and increase the contact area; meanwhile, the antigen in the contact area could flow dynamically and activate the immune recognition ( Figure 5C). It was detected the Pickering emulsion could effectively trigger the humoral and cellular adaptive responses ( Figure 5D,E). Compared with commercial adjuvants and conventional emulsion stabilized by surfactant, the Pickering emulsion significantly reduced the tumor volume of mice in EG7 lymphoma ( Figure 5F), indicating its broad clinical application potential. This work showed that Pickering emulsion could mimic the structure of natural antigens (cancer cells, pathogenic bacteria, etc.) to enhance antigen presentation, which provided a new idea for the design of antitumor vaccine adjuvants.

| Interventional therapy
Lipiodol is a contrast agent widely used in the clinical interventional treatment of hepatocellular carcinoma owing to its preferential accumulation in hepatic cancer tissue and its good X-ray imaging capability. 92 However, due to its low mechanical strength, free lipiodol is prone to cause vascular recanalization. 93 Traditional emulsions using lipiodol as the oil phase lack enough stability to resist the shock of blood while Pickering emulsion can address this issue. Li et al. designed a temperaturesensitive Pickering emulsion using a double-layer nanogel as a stabilizer. The temperature-sensitive Pickering emulsion firstly exhibited favorable flowability at sol phase at 25°C beneficial to diffuse into peripheral tumor arteries and then presented the highest yield stress at unflowable gel phase above 37°C, enabling to resist blood flushing and effectively accumulate in tumor tissues ( Figure 6A,B). 94 On the 21st day after lipiodol embolism, the tumor (the black shadow) reappeared ( Figure 6C), indicating that the embolized artery was recanalized again. In comparison, there was no black shadow in the DSA image of the Pickering emulsion embolism. Also, the tumor volume of Pickering emulsion group was smaller than that of the free lipiodol group (Figure 6D), suggesting Pickering emulsion showed a long-term embolism effect. Furthermore, it was proved that the embolism ability of Pickering emulsion using lipiodol as the oil phase was better than that of solid embolism agents. 95 In addition to blocking the tumor blood vessels, embolism agents could be designed to deliver drugs. However, due to poor stability of traditional emulsions, embolism agents-based emulsion easily led to rapid drug release thereby having great side effects and reducing the drug accumulation in the tumor tissue. Frederic Deschamps et al. developed Pickering emulsion stabilized by PLGA nanoparticles enabling slow release of oxaliplatin and causing decreased drug concentration in normal tissues at 1 h with Pickering emulsion but higher ratio between tumor and left liver at 24 h. 96

| The application of Pickering emulsion in antiviral infection
Since the outbreak of the novel coronavirus in 2019, it is of great importance to develop a vaccine adjuvant platform that can respond quickly and scale up production on the premise of safety standards. Peng et al. prepared alumstablized Pickering emulsion and then loaded the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), which had a rough surface (similar to the pathogenic body), exhibiting a higher cell affinity (beneficial to the efficient endocytosis of immune cells) ( Figure 7A). 97 Their positive surface charge could induce "lysosome escape" to trigger the cytosolic delivery and cross-presentation of antigens ( Figure 7B), synergistically enhancing the humoral and cellular immune response of the vaccine ( Figure 7C,D). A similar design was applied to vaccines for the H9N2 influenza virus. Zhang et al. developed positively-charged particles loaded with H9N2 antigens to form Pickering emulsions. Compared with the commercial alum adjuvant, this Pickering emulsion vaccine also generated a strong humoral and cellular immune response. 98 In summary, Pickering emulsion, as a rapid response platform, amplifies the therapeutic effect in response to sudden outbreaks. Therefore, as a universal vaccine adjuvant "chassis," Pickering emulsion is expected to respond quickly critical moments by changing the ingredients of the formula.

| The application of Pickering emulsion in biosensing
Conductive composite materials are necessary components for biosensors. The manufacture of conductive composite materials usually required traditional toxic solvents and long post-process time. 99 Instead of traditional emulsion, Pickering emulsion with low toxicity is a better choice. As shown in Figure 8A, the research team at Yale University designed a self-coagulating conductive Pickering emulsion, which was printed on the polymer substrate and accompanied by the evaporation of ethanol. Then, the colloid relaxed on the substrate and the thin polymer resin quickly polymerized. 100 As a result, the polymer substrate spontaneously possessed the surface with the injected conductive composite material. The method did not require any additional heating or pressure treatment steps and reduced solvent toxicity. Meanwhile, the self-coagulating conductive Pickering emulsion was certified for the validity of wearable biosensors ( Figure 8B,C). Moreover, the strong specific surface area of the colloid makes them suitable to act as a stabilizer for biosensing Pickering emulsions. Ling et al. explored a plasma gel composed of silver nanocubes as a threedimensional SERS platform based on Pickering emulsion for submicron toxin detection. Compared to the traditional suspension platform, the SERS sensitivity of Pickering emulsion platform was more than 3000 times higher, and its accuracy could reach the submilligram molecular level. 101 Kim et al. designed a graphene quantum dotstabilized Pickering emulsion. The modified graphene quantum dots had a high luminescence intensity, which provided great applications in fluorescence sensors and imaging. 102 Ye et al. prepared Pickering emulsion with molecular selectivity and fluorescence response through copper-catalyzed click reaction, and it had potential application in biosensing. 103 Rezwan et al. simultaneously introduced magnetic iron oxide and fluorescent silica nanoparticles into a single submicron colloid, which was tailor-made for biosensing and bioimaging. 104

| The application of Pickering emulsion in biocatalysis
Although two-phase catalysis is a kind of green biocatalysis, the reaction rate of this system is limited by the organic/water interface area. 105 The high surface area of the Pickering emulsion interface is suitable for biocatalysis reaction. Sun et al. reported Pickering emulsion interfacial biocatalysis platform based on robust and recyclable enzyme-polymer conjugates that served as both catalytic sites and stabilizers at the interface of Pickering emulsions. 106 As shown in Figure 9A, the Pickering emulsionbased interface biocatalysis platform was prepared by using a conjugate formed by benzaldehyde lyase (BAL) and poly(N-isopropyl acrylamide) as a stabilizer. The mild conjugation process preserved the structure of the enzyme ( Figure 9B). Interestingly, the stability of BAL was increased after conjugating with poly(N-isopropyl acrylamide) ( Figure 9C). The enzyme-polymer conjugates on the Pickering emulsion interface significantly increased the interface area and the catalytic performance was 270 times of improvement compared with the two-phase system without emulsification ( Figure 9D). Furthermore, excellent catalytic results were also obtained by replacing the enzyme with glucose oxidase (Figure 9E), indicating that the Pickering emulsion interface biocatalysis platform was applicable to a wide range of enzymes and reactions. Although the Pickering emulsion interface biocatalysis platform significantly improved the catalytic effect, long- term exposure to the natural enzyme at the oil-water interface could still affect the structure of the enzyme. 107,108 Accordingly, Qu et al. developed a Pickering emulsion interface biocatalyst platform stabilized by the artificial enzyme particle. Compared with the natural enzyme, the artificial enzyme-stabilized biocatalysis platform showed higher stability, which had more than 20 cycles of recyclability and high storage stability of more than 30 days. It was worth noting that their catalytic activity loss was negligible. 109 In addition, their research team further designed a Pickering emulsion stabilized by near-infrared/visible light-tunable interface-active nanomaterials, which could easily realize the product recovery, biocatalyst and emulsifier recovery. 110

| The application of Pickering emulsion in wound healing
Pickering emulsion is expected as a drug carrier to provide a new direction for wound healing treatment due to its low toxicity and good stability. As shown in Figure 10A, Wang et al. explored Pickering emulsion composite hydrogel stabilized by carboxymethyl chitosan-sodium alginate nanoparticles as drug carrier for wound healing treatment. 111 The mixture of curcumin-coated emulsion and poloxamer407 solution showed a good antibacterial effect ( Figure 10B), wound healing ability ( Figure 10C), cell proliferation ability ( Figure 10D) and angiogenesis ability ( Figure 10E). This might be attributed to the strong permeability of Pickering emulsion beneficial to the improvement of drug delivery efficiency. Although there was no significant difference in wound healing rates between the curcumin group and the Pickering emulsion group after 14 days, the latter promoted high storage in the stratum corneum and slowly released the drug to deeper layers of the skin. In addition, other compoents of Pickering emulsion also exerted therapetuic effect. 112,113 Bao et al. developed a Pickering emulsion composed of chitosan nanoparticles, tea tree oil and curcumin. The tea tree oil contained in the Pickering emulsion could be sustained to release and further strengthen its antibacterial activity toward several bacteria. Besides, it could be loaded with hydrophobic curcumin to achieve synergistic healing effects. In particular, chitosan nanoparticles could provide high viscosity facilitating Pickering emulsion spraying by the shear thinning behavior. The Pickering emulsion could recover to the high viscosity after spraying onto the wounds, leading to a long residence time and avoiding the irritation and toxicity. This sprayed Pickering emulsion exhibited promising prospects for wound healing. 114

Pickering emulsion
Besides, Pickering emulsion presents a promising prospect in other biomedical fields, such as bioimaging, bioseparation and topical protection, and so on. Leon et al. prepared reduced graphene oxide nanosheets to stabilize Pickering bubble through the amalgamation in the presence of perfluorocarbon gas. The obtained Pickering bubble could not only be used as an ultrasound contrast agent but also introduce photoacoustic properties, thus enabling us to present dual-modality ultrasound and photoacoustic imaging agent. 115 In the field of bioseparation, Sun et al. prepared bovine hemoglobin colloidal particles to stabilize the Pickering emulsion. Also, bovine hemoglobin colloidal particles played three roles simultaneously, including emulsifier, template protein and sacrificial material for further exposing recognition sites. 116 After dopamine polymerized, the template proteins were removed, forming the bovine hemoglobin-imprinted polymers based on Pickering emulsions-hydrogels (Hydro-MIPs). The prepared Hydro-MIPs possessed enhanced rebinding kinetics, and the maximum rebinding capacity to bovine hemoglobin was 3.97 times higher than that of the non-imprinted polymers. In addition, Pickering emulsion had better skin permeation and sustained release capacity compared to the surfactant-stabilized emulsion. Thus, it could be used as an excellent skin detergent, 117 and a skin sunscreen cream. 118 Salerno et al. used Fuller's earth and silica particles to prepare Pickering emulsions and the results demonstrated that both Pickering emulsions significantly increased the skin decontamination efficiency compared to aqueous suspension. 117 Marto et al. reported Pickering emulsion stabilized by physical UV filters, presented a powerful protection against UVB-induced damage in human keratinocyte cells. 118

| CONCLUSION
In this review, we give a comprehensive guide to the recent progress of Pickering emulsions and their applications in biomedicine. With the rapid development of materials science and biomedicine, a large number of researchers have focused on the development of stable Pickering emulsion. In particular, a variety of novel particles have emerged, for example, stimuli-responsive particles, which can present the "switch" effect by triggering external environment changes (such as pH, light, magnetic field, and temperature) to meet complex microenvironments in vivo. This innovative Pickering emulsion has promoted the development of materials science and biomedicine.
Although promising progress in Pickering emulsion has been made in recent years, there are still great challenges and opportunities in the near future. Several issues need to be solved for Pickering emulsion in the field of biomedicine. Firstly, the size of the current Pickering emulsion remains relatively large, usually more than several microns suitable to oral or topical administration, and thus greatly limit the biomedical application range. Accordingly, it is encouraged to develop various administration routes for Pickering emulsion based drug delivery systems, such as intravenous injection or intraperitoneal injection. To fabricate nano-scaled Pickering emulsions, active smaller-sized nanoparticles should be developed. However, the self-assembly of these smaller-sized nanoparticles at the nanoscale is still challenging. Using amphiphilic Janus particles may be a promising choice for the fabrication of stable nano-scaled Pickering emulsion. Combined with the advantage of high interfacial adsorption capacity of spherical particles and the amphiphilicity of molecular surfactant, Janus particles exhibit excellent interfacial activities in stabilizing Pickering emulsion. Secondly, the natural properties of Pickering emulsions are not well investigated, such as their viscoelasticity and flexibility. Back to the living system, the natural biological particles, such as cells and their derivatives, are essentially soft matter with excellent viscoelasticity, which is critical for them to traverse various physiologic barriers for efficient cargo delivery. Although Pickering emulsion provides a bio-inspired soft delivery system, the current studies of Pickering emulsions mainly focus on developing their high loading capacity and low toxicity, but ignore their role of the viscoelasticity in drug delivery. Therefore, comprehensively investigating the role of viscoelasticity would extend the in-depth application of Pickering emulsion in the drug delivery, especially for the treatment of challenging diseases. For example, owing to the existing blood-brain barrier/blood-brain tumor barrier (BBB/ BBTB), solid nanoparticle-based drug delivery systems remain a great challenge for glioblastoma chemotherapy. In general, large size of particles hinders them from efficiently bypassing physiologic barriers. If micro-scaled Pickering emulsion enables to cross BBB/BBTB by virtue of its intrinsic viscoelasticity, it will greatly extend the applicable scope of Pickering emulsion in disease treatment. With the help of its structural advantage, Pickering emulsion undergoes elastic deformation enabling to increase its contact areas with cells or tissues, thereby amplifying its biological effect to achieve excellent biomimetic potential. Finally, microenvironment responsive-Pickering emulsion is expected to develop and cope with complex microenvironments in vivo beneficial to precisely treat diseases. Due to wide exploration of the multifunctional solid particles, microenvironment responsive-Pickering emulsions would be achieved using these multifunctional solid particles.