Recent Advances in Drug Delivery System Fabricated by Microfluidics for Disease Therapy

Traditional drug therapy faces challenges such as drug distribution throughout the body, rapid degradation and excretion, and extensive adverse reactions. In contrast, micro/nanoparticles can controllably deliver drugs to target sites to improve drug efficacy. Unlike traditional large-scale synthetic systems, microfluidics allows manipulation of fluids at the microscale and shows great potential in drug delivery and precision medicine. Well-designed microfluidic devices have been used to fabricate multifunctional drug carriers using stimuli-responsive materials. In this review, we first introduce the selection of materials and processing techniques for microfluidic devices. Then, various well-designed microfluidic chips are shown for the fabrication of multifunctional micro/nanoparticles as drug delivery vehicles. Finally, we describe the interaction of drugs with lymphatic vessels that are neglected in organs-on-chips. Overall, the accelerated development of microfluidics holds great potential for the clinical translation of micro/nanoparticle drug delivery systems for disease treatment.


Introduction
Microfluidic technology is a highly interdisciplinary science and technology, involving engineering, physics, chemistry, microfabrication, and other disciplines [1][2][3]. Microfluidics can process or manipulate tiny amounts (10 −9 L to 10 −8 L) of fluid within microchannels, ranging in size from tens to hundreds of micrometers. Since the heat and mass transfer processes at the microscale are scale-dependent, the fluids in microfluidic chips exhibit specific properties that are different from those at the macroscale [4]. In short, surface tension and capillary forces dominate the device, and the effects of gravity and inertial forces are negligible. The interface factors such as diffusion, surface tension and viscosity become the main factors affecting the fluid behavior [3]. In addition, the high surface area to volume ratio ensures thermal homogeneity and rapid heat transfer. These fundamental properties give rise to a wide range of advantages, including minimal use of reagents, low energy consumption, fast reaction rates, and massively parallel processes [5]. Thus, microfluidic devices are widely used in industrial and academic research.
Currently, both small drugs and macromolecular drugs require the appropriate drug delivery strategies [6]. For example, traditional small molecule drugs tend to have low solubility, which limits their bioavailability. Furthermore, the in vivo stability of protein drugs after administration is compromised by proteases, temperature and pH. Nucleic acid drugs need to be delivered into the cytoplasm to be effective. To address these challenges, it is necessary to develop appropriate drug delivery systems. Proper drug delivery allows not only on-demand delivery of active drug to target tissues or cells, but also proper control of pharmacokinetics (e.g., in vivo distribution, half-life, maximum concentration in serum, etc.) [7]. Although pharmacokinetics can be modulated by controlling the number and  [23]. Copyright 2018, Royal Society of Chemistry. (c) Fabrication process of PDMS devices by replica molding method. Reproduced with permission [29]. Copyright 2020, Multidisciplinary Digital Publishing Institute. (d) Schematic diagram of paper-based microfluidic device fabrication. Reproduced with permission [38]. Copyright 2013, Royal Society of Chemistry. (e) Schematic diagram of anisotropic wet etching and anisotropic dry etching techniques for processing glass. Reproduced with permission [40]. Copyright 2018, Wiley. (f) Schematic illustration of a typical PDMS microfluidic device fabricated by soft lithography. Reproduced with permission [42]. Copyright 2014, Springer Nature. (g) Schematic illustration of the 3D printing technique to fabricate microfluidic molds. Reproduced with permission [44]. Copyright 2021, Public Library of Science.

Drug Delivery System Fabricated by Microfluidics
Compared with a traditional drug delivery system, micro/nanoparticles can improve stability, solubility, and circulation time in vivo [49]. In order to improve the therapeutic efficacy and reduce the side effects of drugs, different kinds of micro/nanocarriers have been developed using biocompatible materials, including microgels, microcapsules, nanoliposomes, nanomicelles, and nanoemulsions [50,51]. However, conventional synthetic methods fail to obtain micro/nanocarriers with uniform size, shape and composition. Microfluidic devices offer an alternative option to address this problem. In general, the fabrication of microparticles is mainly based on the manipulation of immiscible fluids on droplet microfluidics to generate monodisperse droplets. The fabrication of nanoparticles is mainly based on the rapid mixing of multiphase fluids in microfluidics mixer. This section summarizes the preparation of micro/nanoparticles based on droplet microfluidics, centrifugal microfluidics and microfluidic mixers.

Micro/Nanogels for Drug Delivery
Hydrogels have high water content, tunable chemical and physical structure, excellent mechanical properties, and biocompatibility [52]. Micro/nanogels are three-dimensional crosslinked polymer particles with the features of hydrogels and colloidal particles [53]. Typical materials used to prepare hydrogels include natural polymers (such as alginates, chitosan, hyaluronic acid, etc.) and synthetic polymers (such as polyvinyl alcohol, polyethylene glycol, polyacrylamide, polyhydroxyethylmethacrylate, etc.). The crosslinking methods of hydrogels include ionic crosslinking, covalent crosslinking, UV curing, photoinitiation, electrostatic complexation, interfacial assembly, etc. [54]. Due to the advantages of tunable size, large surface area, abundant internal macromolecular network, and good biocompatibility, micro/nanogels can be candidates for clinical therapeutic drug delivery [55]. In addition, micro/nanogels can effectively protect the encapsulated drugs from the external environment and transport the drugs to specific tissues and/or cells through surface modification.
Alginate hydrogels have been widely used as drug carriers due to their excellent biocompatibility. It is well known that a method available for alginate hydrogels is to crosslink alginate with divalent cations such as Ca 2+ and Ba 2+ [56]. For example, Cai et al. reported a concentration-controlled microfluidic chip to synthesize DOX-encapsulated alginate nanogels (CaA@Dox) [57]. As shown in Figure 2a, alginate and Ca 2+ ions diffused into the central channel driven by a concentration gradient, forming alginate nanogels. The diameter of alginate microgels is positively related to the ratio of channel lengths and flow rate. With the increase of pH value, the carboxyl group is continuously dissociated, the hydrophilicity of sodium alginate increases, and the molecular chain is elongated. Therefore, sodium alginate has significant pH sensitivity. Sodium alginate microgels can respond to the acidic microenvironment of tumors.
In addition to microfluidic chips based on a diffusion mixing mode, centrifugal microfluidic chips are also applied to generate high-throughput centrifugal droplets [58,59]. Kim et al. utilized photo-crosslinkable methacrylate-modified hyaluronic acid microgels using a portable centrifugal microfluidic chip [60]. In the initial state, aqueous solution and oil were injected into the inlet and outlet chambers of the microchip, respectively. Due to the different specific gravity and immiscibility of oil and water, droplets were generated at the oil-water interface under the action of centrifugal force (Figure 2b). Parallel microchannels at the oil-water interface were designed to restrict the droplet size.
By exploiting the environmental sensitivity of polymer networks, hydrogels can be easily designed as stimuli-responsive carriers [61,62]. Depending on the source, stimuli can be divided into physiological environmental stimuli (i.e., tumor acidic microenvironment, inflammation or enzymes at the lesion site) and external stimuli (i.e., temperature, light or magnetic field) [63]. Poly(N-isopropylacrylamide) (PNIPAM) is a representative thermosensitive polymer [64]. PNIPAM shrinks and changes from hydrophilic state to hydrophobic state when the temperature is higher than its lower critical solution temperature (LCST) [65]. Luo et al. fabricated photosensitive microgels using PNIPAM polymers and polypyrrole (PPy) nanoparticles via a co-flow microfluidic chip (Figure 2c) [66]. PPy nanoparticles have excellent photothermal conversion properties. After the first irradiation cycle, PNIPAM microgels containing PPy nanoparticles released 45% of FITC-albumin, which was significantly higher than the control group. Among the various stimulus responses, enzyme-triggered drug release is an attractive approach [63]. Enzymatic reactions provide higher selectivity and specificity due to the specificity of the enzymes. Enzymes play an integral role in physiological processes and are involved in all biological processes. Malignant diseases or lesions lead to elevated expression of specific enzymes in certain tissues. Several natural polymers such as fibrin, collagen, gelatin, and hyaluronic acid have been used to prepare enzyme-responsive hydrogels. Hyaluronidase is overexpressed in aggressive malignancies or secreted by pathogenic bacteria at the site of infection. Busatto et al. synthesized an oil-in-water nanoemulsion to encapsulate hydrophobic drug progesterone [67]. The nanoemulsion and hyaluronic acid precursor solution were mixed in a microfluidic chip to form an oil-in-water-in-oil nanostructure (Figure 2d). Ultimately, hyaluronic acid precursors were crosslinked under ultraviolet light and formed microgels. When exposed to a concentration of 100 UI/mL of hyaluronidase, the hyaluronic acid microgels sustained drug release over two days. Another typical degradative enzyme is matrix metalloproteinase. Previous study showed that PEG microgels were prepared by using a matrix protease-degradable crosslinked peptide and dithiothreitol as the crosslinking agent [68]. The degradation rate of microgels in matrix metalloproteinase can be regulated by changing the ratio of crosslinking peptide to a dithiothreitol crosslinking agent.
Ultimately, hyaluronic acid precursors were crosslinked under ultraviolet light and formed microgels. When exposed to a concentration of 100 UI/mL of hyaluronidase, the hyaluronic acid microgels sustained drug release over two days. Another typical degradative enzyme is matrix metalloproteinase. Previous study showed that PEG microgels were prepared by using a matrix protease-degradable crosslinked peptide and dithiothreitol as the crosslinking agent [68]. The degradation rate of microgels in matrix metalloproteinase can be regulated by changing the ratio of crosslinking peptide to a dithiothreitol crosslinking agent. Overall, the micro/nanogels are able to drastically change their volume to release their internal drug in response to environmental changes. The chemical composition, size, biodegradability, morphology and surface functional properties of the micro/nanogels can be adjusted by the synthesis conditions such as monomer selection, monomer ratio, crosslinker concentration and initiator concentration. However, micro/nanogels have a homogeneous architecture, making it difficult to achieve elaborate functions. The clinical application of micro/nanogels remains challenging due to the limitations that exist during conventional batch synthesis, such as limited scalability, little control over reaction parameters, wide nanoparticle polydispersity, poor batch-to-batch reproducibility, and the need to use large amounts of chemicals and therapeutic agents. Microfluidic devices can generate discrete volumes of droplets to from micro/nanogels. Each droplet works as a microreactor, ensuring fast heat and mass transfer within a limited microvolume and enabling faster reaction kinetics. Thus, microfluidic devices precisely control the reaction parameters, such as the size and composition of droplets, to overcome constraints of conventional methods. Overall, the micro/nanogels are able to drastically change their volume to release their internal drug in response to environmental changes. The chemical composition, size, biodegradability, morphology and surface functional properties of the micro/nanogels can be adjusted by the synthesis conditions such as monomer selection, monomer ratio, crosslinker concentration and initiator concentration. However, micro/nanogels have a homogeneous architecture, making it difficult to achieve elaborate functions. The clinical application of micro/nanogels remains challenging due to the limitations that exist during conventional batch synthesis, such as limited scalability, little control over reaction parameters, wide nanoparticle polydispersity, poor batch-to-batch reproducibility, and the need to use large amounts of chemicals and therapeutic agents. Microfluidic devices can generate discrete volumes of droplets to from micro/nanogels. Each droplet works as a microreactor, ensuring fast heat and mass transfer within a limited microvolume and enabling faster reaction kinetics. Thus, microfluidic devices precisely control the reaction parameters, such as the size and composition of droplets, to overcome constraints of conventional methods.

Microcapsules for Drug Delivery
In contrast to micro/nanogels, microcapsules have a core-shell structure where the core can be solid, liquid or gas [69,70]. Microcapsules do not exhibit sudden volume changes in the same way as micro/nanogels, due to shell support [71]. The high versatility of the shell materials allows the production of microcapsules with versatile functionality, such as enhanced retention, controlled release, and stimulus responsiveness [72,73]. The well-designed microcapsule shells not only protect encapsulated drugs, but also rupture and release the internal drugs in response to external stimuli, including pH, temperature, osmotic pressure, electric fields and stress [74].
Stimuli-responsive microcapsules have been widely applied in drug delivery systems. Kim et al. prepared microcapsules based on water/oil/water (W/O/W) double emulsion droplets, showing molecular polarity and temperature-dependent permeability [75]. The intermediate oil phase contains the photocurable monomer and the phase change material dodecanol (Figure 3a). Under UV light, dodecanol remained liquid and occupied the microcapsule space without polymerization. Very different from physical voids, the permeability of microcapsules depends on molecular size and molecular polarity [76]. In this case, low polarity molecules can be dissolved in water and transferred to the other side of the shell by transferring within dodecanol. However, the transport of highly polar molecules is inhibited. Similar to other phase-change materials, dodecanol melts at temperature above its melting point. Therefore, dodecanol freezes reversibly when the temperature is below the melting point. The release of ICG from microcapsules was minimal at 4 • C for 72 h, while the in vitro release process accelerated rapidly at 37 • C. The smart design of the shell material endows the microcapsules with polarity-dependent and temperature-dependent drug release patterns, providing a smart drug release strategy.
Electric fields can also be used to control the release of active substances in microcapsules by methods including electroporation and iontophoresis [77,78]. Depending on the location of the device, drug release near the skin can be triggered by a voltage generated by an external conductive skin patch. Alternatively, a minimally invasive implantable device controlled by radio frequency can be used to control drug release [79]. The microcapsules prepared by electro-responsive materials rupture was attributed to interface deformation caused by maximum well interface stresses at the droplet/dielectric interface induced by the applied alternating electric field ( Figure 3b). During the interfacial deformation process, the fragile parts of the microcapsules were prone to defects, leading to rupture and eventually the release of internal drugs. Due to the competition between separation pressure and electrocompression, there is a critical voltage that causes microcapsules to rupture. The threshold voltages increased with the applied field frequency. In addition, high conductivity facilitated drug release at low voltage and was not limited by ion types. This capability can be extended to dual core droplets for drug co-delivery.
Preparation of temperature-responsive or pH-responsive microcapsules usually requires the addition of functional materials to the precursor solution, such as temperaturesensitive or pH-sensitive materials. However, osmolarity-triggered drug release avoids the addition of additional precursors [80]. Zhang synthesized osmotic pressure-responsive microcapsules with non-uniform shell thickness, which can be controlled by changing the flow rate ratio of the mesophase to the inner phase [81]. The thickness of the microcapsule shell varies widely, from 40 µm in the thickest part to 600 nm in the thinnest part ( Figure 3c). As a result, non-uniform microcapsules are more prone to rupture from the thinnest areas than uniform microcapsules. It is also fascinating to impart multiple stimulation responses to single microcapsules. Multifunctional responsive microcapsules with a n-hexane layer were prepared with capillary microfluidic [76]. By inducing oil layer instability, hydrophilic drugs encapsulated in microcapsules were released on demand ( Figure 3d). Ethanol solution, mechanical stress and osmotic pressure can all cause the oil layer rupture. However, the microencapsulated shell layer remained intact. For malignant diseases, multidrug codelivery strategies tend to have a significant efficacy. Encouraging progress has been made in the design of stimuli-responsive microcapsule shells based on intrinsic properties of materials. Programmable drug release can be combined with multiple stimulation sequences to achieve a combination of different release modalities, such as burst release and sustained release. Yang et al. designed a multi-stimulus-responsive microcapsule structure consisting of a pH-responsive chitosan hydrogel shell and an oil core containing curcumin and drug-loaded poly(lactic acid-ethanolic acid copolymer) (PLGA) nanoparticles ( Figure 4a) [82]. In pH solution of 1.5, the microcapsules ruptured rapidly within 60 s, which corresponds to the gastric environment. Since PLGA is semipermeable, the drug-loaded PLGA nanoparticles achieved sustained release in a neutral environment, with only 63.6% of curcumin released from the nanoparticles in 28 days. The combination of burst release and sustained release provides an effective treatment for acute diseases, with burst release alleviating the acute symptoms and sustained release maintaining the therapeutic effects. More complex Trojan-like microcapsules were prepared in a O1/W2/O3/W4/O5 quadruple microemulsion as templates for complex programmed release [83]. Functional shell materials were incorporated into the W2 and W4 phases to create capsule-in-capsule structures (Figure 4b). Chitosan, poly (ethylene glycol) diacrylate (PEGDA), and PNIPAM constituted three Trojan-like stimulus-responsive microcapsules, including CS@CS microcapsules, PEGDA@CS microcapsules, CS@PNIPAM microcapsules. The pH and temperature can stimulate the chitosan shell and the PNIPAM shell, respectively, resulting in shell rupture. Meanwhile, PEGDA has no temperature or pH response, so the encapsulated drug can be released For malignant diseases, multidrug codelivery strategies tend to have a significant efficacy. Encouraging progress has been made in the design of stimuli-responsive microcapsule shells based on intrinsic properties of materials. Programmable drug release can be combined with multiple stimulation sequences to achieve a combination of different release modalities, such as burst release and sustained release. Yang et al. designed a multi-stimulus-responsive microcapsule structure consisting of a pH-responsive chitosan hydrogel shell and an oil core containing curcumin and drug-loaded poly(lactic acidethanolic acid copolymer) (PLGA) nanoparticles (Figure 4a) [82]. In pH solution of 1.5, the microcapsules ruptured rapidly within 60 s, which corresponds to the gastric environment. Since PLGA is semipermeable, the drug-loaded PLGA nanoparticles achieved sustained release in a neutral environment, with only 63.6% of curcumin released from the nanoparticles in 28 days. The combination of burst release and sustained release provides an effective treatment for acute diseases, with burst release alleviating the acute symptoms and sustained release maintaining the therapeutic effects. More complex Trojan-like microcapsules were prepared in a O1/W2/O3/W4/O5 quadruple microemulsion as templates for complex programmed release [83]. Functional shell materials were incorporated into the W2 and W4 phases to create capsule-in-capsule structures (Figure 4b). Chitosan, poly (ethylene glycol) diacrylate (PEGDA), and PNIPAM constituted three Trojan-like stimulus-responsive microcapsules, including CS@CS microcapsules, PEGDA@CS microcapsules, CS@PNIPAM microcapsules. The pH and temperature can stimulate the chitosan shell and the PNIPAM shell, respectively, resulting in shell rupture. Meanwhile, PEGDA has no temperature or pH response, so the encapsulated drug can be released sustainably. In general, microcapsules containing two stimuli-responsive hydrogel shells are able to provide a flexible trigger mechanism for multifunctional programmed sequential release behavior.
Furthermore, monodisperse multiple emulsions can contain several smaller droplets inside [84,85]. A two-stage co-flow microfluidic device was constructed by a sequential and coaxial nested assembly of multiple glass capillaries (Figure 4c). The uniform droplets generated in the first capillary were encapsulated into larger droplets by the second capillary, forming the drop-in-drop structure [85]. The number, proportion and size of the innermost droplets can be precisely controlled by adjusting the capillary size and fluid flow rate. Higher-order multiple emulsions, such as triple, quadruple, and quintuple emulsions, can be further controllably produced by more complex nested structures.
The conventional preparation method of microcapsules is usually to make bulk emulsion first, and then form the shell of the capsule through interfacial polymerization, precipitation or coagulation. With the bulk emulsification method, it is difficult to control the size, shape and structure of the microcapsules, which greatly limits their potential clinical application. In contrast, microfluidic devices provide a new route for the construction and synthesis of microcapsules by generating emulsions. The structure of microcapsule can be precisely controlled by multiple emulsions through T-junction, flow-focusing, or co-flowing microfluidic devices. In addition, by using a microfluidic device, the drug encapsulation efficiency of the microcapsules can be greatly improved. sustainably. In general, microcapsules containing two stimuli-responsive hydrogel shells are able to provide a flexible trigger mechanism for multifunctional programmed sequential release behavior. Furthermore, monodisperse multiple emulsions can contain several smaller droplets inside [84,85]. A two-stage co-flow microfluidic device was constructed by a sequential and coaxial nested assembly of multiple glass capillaries (Figure 4c). The uniform droplets generated in the first capillary were encapsulated into larger droplets by the second capillary, forming the drop-in-drop structure [85]. The number, proportion and size of the innermost droplets can be precisely controlled by adjusting the capillary size and fluid flow rate. Higher-order multiple emulsions, such as triple, quadruple, and quintuple emulsions, can be further controllably produced by more complex nested structures.
The conventional preparation method of microcapsules is usually to make bulk emulsion first, and then form the shell of the capsule through interfacial polymerization, precipitation or coagulation. With the bulk emulsification method, it is difficult to control the size, shape and structure of the microcapsules, which greatly limits their potential clinical application. In contrast, microfluidic devices provide a new route for the construction and synthesis of microcapsules by generating emulsions. The structure of microcapsule can be precisely controlled by multiple emulsions through T-junction, flow-focusing, or co-flowing microfluidic devices. In addition, by using a microfluidic device, the drug encapsulation efficiency of the microcapsules can be greatly improved.

Lipid Nanoparticles for Drug Delivery
Benefitting from the high biocompatibility of lipid molecules, liposome and lipid nanoparticles (LNPs) are widely used as drug delivery systems. Liposomes are composed of lipid bilayers with a hollow structure, enabling them to encapsulate a variety of drugs including hydrophilic, hydrophobic, and lipophilic drugs. The fabrication methods of liposomes and LNPs can be divided into hydration method, organic solvent injection method, nanoprecipitation method and reversed-phase evaporation vesicle method [86]. Among them, the nanoprecipitation method is to dissolve lipids in organic solvents and inject them into a high-speed stirring aqueous phase to form liposome or LNPs. In general, lipids are dissolved in water-miscible organic solvents such as methanol and ethanol. Then, the two phases are mixed rapidly. In this case, the rapidly changing polarity supersaturates lipids, causing the lipids to self-assemble to form liposomes or LNPs (Figure 5a) [87].
A microfluidic mixer is a microfluidic chip that can efficiently control fluid-mixing by designing a microstructure. For flat microchannel, viscous forces dominate over inertial forces due to the low Reynolds number. The liquids exhibit laminar and uniaxial flow. Laminar liquids are mixed through molecular diffusion. This process is time-consuming, and has poor uniformity, which reduces the mixing efficiency [88]. To overcome this drawback, Zhigaltsev et al. used a staggered herringbone mixer (SHM) to fabricate sizeconstrained LNPs (Figure 5b) [89]. The asymmetrically arranged herringbone groove patterns cause the passing fluid to rotate and stretch to generate chaotic flow (Figure 5c) [90]. For a predetermined microfluidic mixer, the total flow rate and flow ratio can significantly affect the size and uniformity of LNPs. Specifically, the increase in total flow rate and flow ratio resulted in smaller particle size and higher uniformity [91].
Nucleic acids such as small interfering RNA (siRNA) and messenger RNA (mRNA), are negatively charged and easily degraded by ubiquitous RNases. The delivery of nucleic acids requires carriers to protect them from degradation and facilitate cellular uptake. However, traditional liposomes cannot achieve lysosomal escape. Cationic liposomes and LNPs have been used to achieve lysosomal escape. In microfluidic mixer chips, lipids in ethanol are rapidly mixed with siRNA in low pH (pH = 4) buffer media. LNPs are composed of ionizable lipids, helper lipids, cholesterol, and polyethylene glycol lipids. During mixing process, ionizable lipids are positively charged under acidic conditions and bind to nucleic acids through electrostatic interaction [92]. Unlike liposomes, LNPs-siRNA exhibited electron-dense core structures (Figure 5d) [93]. Benefiting from electrostatic interaction, the encapsulation efficiency of RNA was always more than 90%. In recent years, mRNA vaccines have developed rapidly. In particular, two mRNA vaccines, mRNA-1273 and BNT162b2 developed by Moderna and BioNTech/Pfizer, respectively, have received emergency authorizations for widespread clinical use against the SARS-CoV-2 virus [94][95][96].  SHM has been commercialized by Precision Nanosystems for many years. Scientific research based on this commercial chip has been widely reported. Alternative types of microfluidic mixers including microfluidic fluid dynamic flow focusing, microfluidic Tesla mixer, microfluidic bifurcating mixers and baffle mixers have also been employed for LNPs preparation [98]. In addition to LNPs, polymers soluble in water-miscible organic solvents can also be used to prepare nanoparticles by this method.

Other Micro/Nanocarriers for Drug Delivery
In addition to micro/nanogels, microcapsules, and LNPs, many exquisitely designed hierarchical micro/nanoparticles have also been used for drug delivery, including porous, Janus, and multi-compartment micro/nanoparticles. Compared with the solid ball, mesoporous spheres have a larger surface area, making it easier for them to release drugs from inside. Due to their high specific surface area and high loading capacity, porous materials are ideal candidates for drug carriers. Gu et al. fabricated spherical mesoporous colloidal photonic crystal particles (MCPCP) by selfassembly of monodisperse mesoporous silica nanoparticles (MSN) within microfluidic droplet templates (Figure 6a) [99]. The synthesized particles were tightly packed by MSN to form a highly ordered arrangement. In this case, the nanopores of MSN facilitated the loading of small molecules such as DOX, and the highly ordered macroporous structure enables the loading of macromolecular drugs such as albumin-bound paclitaxel. Furthermore, thermosensitive PNIPAM hydrogel can be perfused into the MCPCP voids to regulate drug release and prevent drug loss during transport.
Zhao et al. prepared hierarchical porous microparticles composed of PLGA and drug-loaded hollow porous MSN [100]. During PLGA curing, internal droplets leaked out SHM has been commercialized by Precision Nanosystems for many years. Scientific research based on this commercial chip has been widely reported. Alternative types of microfluidic mixers including microfluidic fluid dynamic flow focusing, microfluidic Tesla mixer, microfluidic bifurcating mixers and baffle mixers have also been employed for LNPs preparation [98]. In addition to LNPs, polymers soluble in water-miscible organic solvents can also be used to prepare nanoparticles by this method.

Other Micro/Nanocarriers for Drug Delivery
In addition to micro/nanogels, microcapsules, and LNPs, many exquisitely designed hierarchical micro/nanoparticles have also been used for drug delivery, including porous, Janus, and multi-compartment micro/nanoparticles. Compared with the solid ball, mesoporous spheres have a larger surface area, making it easier for them to release drugs from inside. Due to their high specific surface area and high loading capacity, porous materials are ideal candidates for drug carriers. Gu et al. fabricated spherical mesoporous colloidal photonic crystal particles (MCPCP) by self-assembly of monodisperse mesoporous silica nanoparticles (MSN) within microfluidic droplet templates (Figure 6a) [99]. The synthesized particles were tightly packed by MSN to form a highly ordered arrangement. In this case, the nanopores of MSN facilitated the loading of small molecules such as DOX, and the highly ordered macroporous structure enables the loading of macromolecular drugs such as albumin-bound paclitaxel. Furthermore, thermosensitive PNIPAM hydrogel can be perfused into the MCPCP voids to regulate drug release and prevent drug loss during transport.
Zhao et al. prepared hierarchical porous microparticles composed of PLGA and drug-loaded hollow porous MSN [100]. During PLGA curing, internal droplets leaked out of the double emulsion template to form microparticles with single or multiple opened structures (Figure 6b). The drug release rate was adjusted by the number of surface openings. Microparticles loaded with desferrioxamine and porcine decellularized dermal matrix formed a composite scaffold to treat partial abdominal wall defects of rat. Eight weeks after implantation, the release of desferrioxamine from the scaffold significantly accelerated neointima formation and intense collagen deposition.
Wang et al. fabricated a polymer/inorganic nanoparticle hybridized Janus-like vesicle motor without template [101]. This Janus-like vesicles motor was divided into two distinct halves, amphiphilic poly(ethylene oxide)-b-poly(styrene) (PEO-b-PS) and inorganic nanoparticles such as Pt or Au nanoparticles tethered to sulfhydryl groups (Figure 6c). Hybrid vesicles containing asymmetrically distributed Pt nanoparticles can catalyze decomposition of hydrogen peroxide (H 2 O 2 ), thereby propelling themselves into relatively oriented motion. Due to the excellent photothermal response of gold nanoparticles, hybrid vehicles were damaged by localized high temperature under NIR irradiation (808 nm). Inorganic nanoparticles were selectively incorporated into vesicles to confer versatility in vesicles.
In addition, due to the differences in the composition of Janus micro/nanoparticles, Janus micro/nanoparticles have great potential for applications in dual-stimulus responsive drug carriers. Kim et al. synthesized dual stimuli-responsive drug carriers for pH-sensitive and redox-sensitive compartments using stop-flow lithography [102]. The model drugs were released independently upon exposure to the corresponding stimuli (Figure 6d). In brief, under redox conditions, the redox-responsive compartment was completely degraded, while the other compartment remained intact. However, compartmental degradation was reversed when Janus particles were incubated under the acidic conditions. Both model drugs were effectively released under pH and redox stimulation. Micro/nanodelivery systems were summarized as in Table 1. Table 1. Stimuli-responsive strategies for micro/nanoparticles drug delivery systems.

Drug Delivery
Systems Materials Stimuli Ref.

Lymphatic Chip for Drug Delivery
The development of micro/nanoparticle delivery systems has achieved some clinical success, such as Doxil, mRNA-1273 and BNT162b2. However, the current understanding of the behavior of delivery carriers in complex physiological environments is limited, which restricts the optimal design of delivery carriers. In addition, 2D cell culture system cannot simulate complex interactions in physiological environments [143]. Therefore, the development of 3D cell culture models has attracted more and more attention. The diameter of microfluidic channels is suited for the physical dimensions of different kinds of cells. In addition to manufacturing multifunctional functional delivery carriers, microfluidic chips can also construct in vitro microphysiological systems to simulate organ-level and even organism-level functions.
Various microfluidic cell culture chips, also known as organs-on-a-chip, are designed for precision medicine [144][145][146]. However, most organs-on-a-chip often ignore the role of lymphatic drainage. The lymphatic system plays an important role in circulatory and immune systems [147]. Lymphatic vessels are composed of initial lymphatic vessels and collecting lymphatic vessels. The fundamental function of lymphatic vessels is to maintain the balance of interstitial fluid. Due to the limited transport capacity of blood vessels, interstitial fluid cannot be completely reabsorbed through the blood vessels and returned to the vascular system. The static interstitial fluid brings a pressure gradient, causing the fluid flow into the initial lymphatic vessels. However, excellent transport capacity may cause unexpected implications. For example, tumor cells are more likely to metastasize through the lymphatic system. Invasive tumor cells tend to rely more on highly permeable lymphatic systems for metastasis. Given the important role of lymphatic vessels in maintaining tissue fluids homeostasis, simultaneous construction of vascular and lymphatic systems is important for modeling cancer pathology. Tatsuya et al. used a perfusion culture method to co-culture human umbilical vein endothelial cells and lymphatic endothelial cells (LECs) in collagen gel embedded microfluidic platform with 600 µm diameter parallel channels (Figure 7a) [148]. Lymphatic vessels have significant differences in intercellular adhesion with blood vessels. The lymphatic capillaries exhibited a discontinuous buttonlike structure, while the blood vessels and the collecting lymphatic vessels showed a continuous zipper-like structure. Interspaces at the junctions of the button-like structure act as a valve to regulate the passage of immune cells, proteins and interstitial fluid. The in vitro reconstruction of lymphatic vessels structures helps to better simulate organ physiology and disease status. On a more complex microfluidic chip, blood vessels were also integrated into the tumor-lymphatic vessels model [149]. Cao et al. fabricated hollow blood vessels with an opening on both ends and lymphatic vessels with blind holes at one end by bioprinting technology (Figure 7b). The two-vessel chip has faster drug transport than a single vessel due to the accelerated drug transport in lymphatic vessels. Therefore, under DOX perfusion, the survival rate of MCF-7 cells in both vascular systems can be maintained at more than 90%. The high cell vitality was because of the timely transport of DOX by lymphatic vessels, which reduced the accumulation of DOX. This biomimetic design can simulate transport kinetics of drugs in biological tissues to some extent.
Vaccines are typically administered subcutaneously or intramuscularly, where local antigen-presenting cells (usually dendritic cells) uptake and process antigens for subsequent delivery to T cells in secondary lymphoid organs (usually lymph nodes). During the transport process, lymphatic vessels are required. Alexandra et al. investigated the 3D chemotaxis of bone marrow-derived dendritic cells (BMDCs) mediated by different Toll-like receptor adjuvants, such as MPLA (TLR4 ligand) and CpG (TLR9 ligand), in a 3D collagen matrix-based microfluidic device (Figure 7c) [150]. Free MPLA increased the chemotaxis of BMDC to chemokines CCL19 and CCL21 gradients, while free CpG was ineffective. However, CpG-encapsulated PLGA microparticles enhanced BMDC chemotaxis. Dendritic cells secreting CCL19 and LECs secreting CCL21 play an important role in the migration of dendritic cells from peripheral tissues to lymph nodes. Since vaccines require dendritic cells to present antigens to T cells in lymph nodes, modulating DC migration to lymph nodes with different adjuvants or combinations will facilitate vaccine development. Currently, the 2D cell culture models are too simple and mouse models are too complex. There is a lack of models to study the response behavior between drugs and intact tissue substructures. Ashley et al. investigated the specific localization of molecular stimuli generated by drug delivery in the B-cell or T-cell zone of lymph nodes (Figure 7d) [151]. Fluorescent dextran with two molecular weights (40 kDa and 70 kDa) reaching specific substructures of lymph nodes were studied. The T-cell zone of the lymph node retained the model drug and glucosecoupled albumin larger than the B-cell zone. The response of different substructures in the lymph nodes to external signals is variable. Investigating the differences in response of lymph node substructure regions to vaccine delivery can promote understanding of local immune responses. Lymphatic vessel models were summarized as in Table 2.

The Challenges of Microfluidic Technology
Benefiting from the rapid development of micro/nano fabrication technology, micro/nanoparticles with high dispersibility and reproducibility can be fabricated by microfluidic devices. Microfluidics faces several unavoidable challenges in the field of micro/nanoparticles synthesis. Manufacturing devices with complex geometries and configurations increases manufacturing costs and processing complexity. Despite the low cost and ease-of-preparation characteristics of capillary microfluidics, large batch-to-batch variability limits its controllability. One solution is parallelized production, which can improve production efficiency by paralleling multiple microfluidic chips. Another solution is to increase the throughput of generated micro/nanoparticles. It is also necessary to consider that the pressure in the microfluidic channel increases rapidly with the increase of the flow rate, which poses a challenge to the packaging technology of microfluidic device. In conclusion, microfluidics enables fabrication of micro/nanoparticles with specific structures and functions for drug delivery.
On the other hand, the development of the organ-on-a-chip provides an in vitro drugscreening model. The role of the lymphatic system, as mentioned previously, tends to be overlooked. In vitro construction of tissues, blood vessels and lymphatic vessels increases the complexity of the organ-on-a-chip, resulting in very low throughput. However, drug delivery behavior requires high-throughput systems to verify statistically significant studies. Microfluidic chip materials, such as PDMS, may adsorb hydrophobic small molecule drugs, making them unable to be effectively used in drug research. One of the effective methods to solve this problem is to reduce adsorption by surface modification.

Future Perspectives
Benefiting from the sophisticated control of microscale fluids, microfluidics allows the modulation of the physicochemical properties (e.g., composition, shape and surface ligands) of the micro-and nano-delivery delivery vehicles to be tuned during fabrication. Well-designed delivery vehicles can optimize drug delivery efficiency and release profiles. These well-thought-out designs help reduce adverse effects and improve outcomes during treatment.
While combating the global spread of SARS-CoV-2, the mRNA-LNP vaccine was rapidly approved and available for clinical treatment. In addition, the mRNA-LNP vaccines were fabricated on a large scale by microfluidic mixer. This has greatly encouraged people to use microfluidic technology to prepare drug delivery vehicles on a large scale. It provides valuable guidance for the preparation of micro/nano delivery vehicles using microfluidic mixing technology. In addition, this technique can be extended for the preparation of polymer nanoparticles. At present, the preparation of microparticles mainly focuses on droplet microfluidics. As mentioned previously, droplet microfluidics limits microparticles preparation throughput and batch-to-batch variability. Droplet microfluidics often requires the use of organic solutions, which will certainly limit the clinical treatments. Incomplete removal of organic solvents may be detrimental to patient health. The presence of organic solutions may also limit the delivery of bioactive drugs. For example, during encapsulation, enzyme denaturation was observed at the oil-water interface. Therefore, the large-scale production of microparticle delivery vehicles in a simple, controllable and highly biocompatible manner is still required in the future. Thermodynamically equilibrium immiscible aqueous phase solutions can be used in place of organic phase solutions. There have been some studies using all-aqueous emulsions as templates to prepare microparticles.
In vitro construction of disease models can help reduce the time it takes for drug discovery. Currently developed organs-on-chips often lack the vascular system, especially lymphatic vessels, which are often overlooked in the construction of disease models. Oversimplified models cannot simulate the physiological environment of lymphatic vessels in vivo. Considering the significant differences between animal models and human physiological states, there is still great hope in constructing rational disease models on microfluidic chips to replace animal models for drug screening. Most reported lymphatic vessel models focus on the study of lymphangiogenesis and lymphatic permeability in primary lymphatic vessels. In the future, it may be possible to introduce smooth muscle-mediated lymphatic pumping mechanisms to collect lymphatic vessels to study the behavior of active drug delivery.

Conclusions
This review first discusses the advantages and disadvantages of typical microfluidic chip materials and processing techniques. Subsequently, micro/nanoparticle drug delivery carriers, including micro/nanogels, microcapsules, LNPs, and hierarchical micro/nanoparticles, were prepared using a variety of microfluidic chips. The microfluidic chip structure design and material selection can endow the delivery vehicle with a variety of stimuli responsiveness, such as acid, pH, temperature, light, osmotic pressure, etc. Micro/nano drug delivery systems offer highly tunable drug release patterns, allowing them to incorporate multiple therapeutic modalities. In addition, we summarized the lymphatic-drug interactions that are easily overlooked in organ-on-a-chip construction. Therefore, well-designed microfluidic systems have great potential for designing effective drug development and personalized medicine. Funding: This work was financially supported by the Start-up Foundation of National Center for Nanoscience and Technology.