1. Introduction
The current advancements in precision medicine have directed the research trend in biomedical engineering toward small-scale design of equipment, necessitating the use of micro/nanomanufacturing for some applications, such as disease diagnosis, surgery, disease modeling, and targeted drug delivery
[1],
[2],
[3]. This growing demand has facilitated the wide application of three-dimensional (3D) printing technologies. Traditional 3D printing is mainly utilized for macroscale manufacturing, such as printing of bone scaffolding material using selective laser sintering with good biocompatibility
[4],
[5] and corneal stroma via extrusion printing
[6]. Although traditional 3D printing has made advancements, micro/nanomanufacturing in the biomedical field is still in its nascent stage
[7]. Material selection and structure fabrication are the main challenges in micro/nanomanufacturing
[1],
[8]. Traditional biomanufacturing methods, such as extrusion printing
[9], inkjet printing
[10], stereolithography (SLA), and digital laser processing (DLP), have limitations in submicron precision
[11],
[12],
[13],
[14],
[15]. This issue can be addressed using photolithography; however, complex structures cannot be fabricated through this approach
[16].
Multiphoton polymerization (MPP) is a laser micro/nanomanufacturing technology based on light-induced polymerization reactions. Therefore, it can be used to manufacture complex structures with high precision and has micro/nano-biomedical applications, such as drug delivery and regenerative medicine
[17],
[18]. This paper comprehensively reviews the biomedical applications of MPP, particularly in precision medicine, and provides a strategy for the selection of material and design of microstructures. We hope that this review will inspire and accelerate advancements in the biomedical field. The remainder of this paper is organized as follows. Section 2 outlines the fundamentals of MPP, including the multiphoton absorption (MPA) theorem, MPP devices, and MPP process. Section 3 reviews the materials applied in the biomedical field, such as commercially available photoresists and materials composed of photoinitiators (PIs) and photopolymers. Section 4 discusses the use of MPP in different scenarios of biomedical applications and analyzes some application limitations. Section 5 summarizes the existing challenges and future perspectives on the use of MPP in precision medicine.
2. Fundamentals
The process of photon absorption can be classified into single photon absorption (
Fig. 1(a-i)) and MPA (
Fig. 1(a-ii)) based on the number of photons absorbed in a single quantum event. MPP or multiphoton lithography (MPL) involves two-photon absorption (TPA), three-photon absorption, and X-photon absorption. The concept of TPA was first proposed by Göppert-Mayer in 1931
[19],
[20]. It describes the interaction between two photons and an atom/molecule in a single quantum event, where both photons are selectively and simultaneously absorbed, providing the molecule with sufficient energy to induce transitions (
Fig. 1(a-ii)). A few decades later, in 1961, Kaiser and Garrett
[21] experimentally observed TPA. However, TPA was first applied in the manufacturing field 37 years later, in 1998
[22]; this process was known as two-photon lithography (TPL). These absorption phenomena are based on the relation between the photoresist absorption spectrum and excitation light wavelength. In Section 4, the use of MPL for micro- and nanofabrication is discussed.
MPL systems mainly comprise a femtosecond laser source and a core optical path (
Fig. 1(b)) and usually use an ultrashort pulsed femtosecond laser with a central wavelength that varies from the visible (VIS) to near-infrared (NIR) spectra depending on specific manufacturing requirements
[23]. The interaction between a laser and matter is influenced by several factors, including laser intensity, exposure duration, light wavelength, light polarization, and properties of the material being irradiated
[24].
Fig. 1(b-i) shows MPP-based micro/nanomanufacturing devices. The femtosecond laser collimates and passes through the attenuator and diffuser (i.e., expander), followed by the shutter, to expose the scanner to varying degrees of exposures. After passing through the
X- and
Y-axis scanners, the laser beam is separated based on the selected wavelength through a two-way dichroic mirror. As shown in
Fig. 1(b-ii), the NIR or VIS beam solidifies the undeveloped photoresist in the focus area of the objective lens by passing through the objective. If the laser power intensity of the focal center exceeds the threshold, the monomer in the photoresist can be cured in the presence or absence of PIs
[25]. The uncured photoresists are removed in subsequent processes. The entire printing process is observed under light-emitting diode illumination.
Fig. 1(c) shows the typical MPL process, which adopts the principles of photoinitiated free-radical polymerization
[26]. In free-radical polymerization, the PI molecules in photoresist materials absorb two (or more) photons that can be excited to produce free radicals and cations under a light source (usually NIR or VIS) with a specific wavelength and intensity. These molecules initiate the polymerization of monomers or oligomers in the central region of the focal plane and crosslink into solid polymer networks
[26]. This process continues until all the radicals react with each other via combination or disproportionation and terminate the reaction
[27],
[28]. This polymerization process can be expressed as a simple reaction as follows:
$\text { Initiation }: \mathrm{I} \xrightarrow{x \cdot h v} \mathrm{R}^{\cdot}$
$\text { Propagation : } \mathrm{R}^{\bullet}+\mathrm{M} \rightarrow \mathrm{RM}^{\bullet} \xrightarrow{\mathrm{M}} \mathrm{RMM}^{\bullet} \xrightarrow{\mathrm{M}} \mathrm{RM}_{n}^{\bullet}$
$\text { Termination : } \mathrm{RM}_{n}^{\bullet}+\mathrm{RM}_{m}^{\bullet} \rightarrow \mathrm{RM}_{n+m} \mathrm{R}$
where
is the PI,
is the monomer, and
is the active radicals,
is the number of photons absorbed by the initiation process and
≥ 2,
is the Planck’s energy equation for each adsorbed photon.
is the number of monomers contained in the polymer during propagation, and
is the number of monomers contained in the polymer during termination. Qualified PIs and photopolymers are equally important in MPP-based micro/nanomanufacturing
[28],
[29].
Table 1 [30],
[31],
[32],
[33],
[34] overviews the femtosecond laser sources used in MPL and describe their respective parameters.
3. Materials
The materials for MPL can be generally divided into three types based on their functions. First, monomer/oligomer mixtures that aggregate to form a 3D structure. Second, PIs that simultaneously absorb multiple incident photons to produce active centers and induce aggregation either by themselves or by undergoing primary or subsequent reactions with one or more additional compounds
[24],
[35],
[36]. Moreover, cross-linkers and stabilizers are occasionally added to the solution. Cross-linkers improve the printing resolution and material stiffness
[37], and stabilizers stabilize the material
[38]. The choice of solutions depends on the rest of the components. Third, photosensitizers (PSs) accelerate photoinitiation. However, PSs by themselves cannot participate directly in photoinitiated polymerization. Therefore, they are combined with PIs. Moreover, the addition of a quencher into the resin can improve feature size and resolution of 3D printing
[39].
In general, the materials should meet the following requirements:
(1) They should be completely transparent to incident light of a specific wavelength;
(2) To ensure high laser power of multiphotons and initiate polymerization, the power intensity of MPL should be higher than the ablation power threshold of PIs
[40], as shown in
Fig. 1(b-ii).
For use in biomedical applications, MPL materials must have a certain printing window to ensure stable printability. Moreover, natural materials are desirable for future applications in MPL. Materials should be designed to possess some functions, such as responsiveness to stimuli and high conductivity as well as mechanical properties, to cater to specific applications in precision medicine
[41],
[42].
3.1. Commercially available photoresists
3.1.1. IP photoresists
IP photoresists (Nanoscribe, Germany), negative tone photoresists
[43], have good mechanical properties and biocompatibility, therefore, such materials are widely used in the biomedical field, particularly as structural materials
[44]. Various IP photoresists of different types and printing characteristics are used in a wide range of biomedical applications. IP-L is an acrylic-based negative photoresist
[45] with micron-scale features and low shrinkage, which is suitable for using in bionic surfaces. IP-poly(dimethylsiloxane) (PDMS) is an elastomer used for the MPL printing of soft, flexible, and highly elastic structures, including microfluidics and microelectromechanical systems (MEMS)
[46]. IP-Q resist facilitates the high-speed fabrication of millimeter-sized devices. IP-S photoresins are acrylic-based negative photoresists with smooth surfaces for micro and mesoscale fabrication of cell scaffolds with optical-quality surface roughness and shape accuracy. IP-Dip is an acrylic-based negative photoresist, which can be used for fabricating devices with submicron features and high aspect ratios. IP-G hybrid resist is designed for oil immersion mode, enabling the submicron features and low shrinkage. IP-Visio is mainly used in life science applications, particularly for the small-scale manufacturing of multicell scaffolds with high resolution.
In general, IP photoresists can be combined with other functionalized materials for different biomedical applications. Serien and Takeuchi
[47] embedded proteinaceous networks into IP-L backbone scaffolds with high mechanical strength, providing a 3D spatially defined protein stimuli platform to mimic the 3D microenvironment and study cell behavior. In summary, IP photoresists offer possible solutions and are widely used in the biomedical field owing to their well-defined mechanical properties.
3.1.2. ORMOCER® photoresists
ORMOCER® photoresists (Fraunhofer ISC, Germany) are hybrid materials that have an inorganic (–Si–O–Si) backbone functionalized with side-chain organic groups. These organic groups (i.e., acrylates or epoxides) can participate in polymerization reactions after ultraviolet (UV)/VIS irradiation. Therefore, ORMOCER® photoresists have combined advantages of organic and inorganic materials, endowing them with good adhesion as well as high mechanical and thermal stabilities
[48]. BioORMOCER® was recently fabricated by combining these hybrid materials with bio-based and biodegradable materials, contributing toward the development of sustainable and biodegradable materials
[49]. ORMOCER® materials are optically transparent over 400–1600 nm wavelength and exhibit low optical losses in the NIR range, making them desirable candidates for TPL. Importantly, it is widely used in microneedles for drug delivery and in implants for the ear
[50] and retina
[51] owing to its stable inorganic network (i.e., low shrinkage of ∼2%).
Compared with SU-8 (IBM, USA) or IP photoresists, ORMOCER® photoresists are more suitable for tissue engineering owing to their high mechanical and thermal stabilities. BioORMOCER® also offers enhanced biodegradability, making it an ideal photoresist.
3.1.3. SZ2080TM photoresist
SZ2080
TM photoresist (IESL-FORTH, Greece) is a widely used, biocompatible, and commercially available hybrid photoresist. It is widely used in cell engineering owing to its low shrinkage and good mechanical properties and biocompatibility. For instance, Malinauskas et al.
[52] used SZ2080
TM as a cell scaffold material and determined optimal microfabrication parameters, including the average laser power, sample scanning speed, and development conditions. Maciulaitis et al.
[53], based on their previous study, used SZ2080
TM as the scaffold material to fabricate scaffolds with a 3D microstructure for the first time via ultrafast pulseddirect laser writing, and tested their biocompatibility
in vivo on rabbits. The successful outcome of that study supported the hypothesis that hexagonal-pore-shaped hybrid organic–inorganic (HOI) microstructured scaffolds combined with chondrocytes seeding can be successfully implemented for cartilage tissue engineering. Maciulaitis et al.
[54] investigated the influence of geometric characteristics, such as shape and size of holes of cellular scaffolds on cell proliferation and cartilage repair outcomes. These
in vitro experiments yielded superior results for quadrangular hole scaffolds compared with hexagonal whole scaffolds. Their findings led to the formulation of new hypotheses on the seeding of human cells on HOI scaffolds for cartilage repair.
Although SZ2080
TM has been extensively used for cell scaffold fabrication, its poor biodegradability poses a significant challenge for long-term
in vivo applications
[53],
[55].
3.1.4. SU-8 photoresist
SU-8 is a commonly used epoxy-based photoresist in life sciences, particularly in microfluidics and cell scaffolds as a structural material owing to its high thermal stability
[56],
[57]. It is also one of the most commonly used negative-type photosensitive materials for MPP
[58].
By incorporating functional materials in SU-8, it can be further adapted for various biomedical applications. Tottori et al.
[59] fabricated magnetic helical micromachines using SU-8 coated with a Ni/Ti layer via TPL and physical vapor deposition. Such micromachines are promising tools for localized drug delivery by testing the material cytotoxicity and magnetic control flexibility. Kim et al.
[60] used a similar strategy for fabricating microniches as a transporter in 3D cell culture and targeted transportation. Suter et al.
[61] presented the fabrication and controlled actuation of swimming microrobots composed of a magnetic polymer composite (MPC) containing 11 nm-diameter magnetite nanoparticles (i.e., Fe
3O
4) and SU-8. This design enabled single-cell manipulation or drug delivery in a fluidic environment. Similarly, Peters et al.
[62] manufactured twist-type actuators via TPL using magnetic polymers composed of Fe
3O
4 nanoparticles and SU-8.
In conclusion, SU-8 has high thermal stability and excellent mechanical properties, including a relatively low Young’s modulus (2–3 GPa) and high yield strength. Thus, it can be used as a structural or functional component and is particularly well-suited for the fabrication of structures with high aspect ratios. For biomedical applications, however, SU-8 will have to undergo surface modification and functionalization to enhance its adhesion to cells, proteins, and other biological materials.
3.2. Noncommercial photoresist
3.2.1. PIs
PIs are used for various purposes, ranging from reactions to the formation of structures. In two-photon initiators, the molecules absorb two photons (each having the energy of the spectral gap) simultaneously to reach an excited high-energy state and generate an active center, which triggers subsequent polymerization
[63]. These photons polymerize into a material with a 3D structure with enhanced bulk mechanical properties, such as viscosity and strength. To enhance the mechanical properties, the role of PIs in polymerization is even more important.
To improve the printability of materials, PIs must be highly sensitive to rapidly initiate subsequent reactions. The initiation efficiency of an initiator is measured in δTPA (Goeppert–Mayer (GM)). Molecules with a large δTPA are highly desirable for use as PIs. Cumpston et al.
[64] investigated the relation between PI’s molecular structure and δTPA. PIs with a large δTPA are fabricated using some common strategies, such as ① extension of conjugated π-bonds, ② increase in planarity using fused aromatic ring π–π bridge, ③ introduction of strong electronic donors and receptors to enhance the intermolecular charge transfer efficiency, and ④ introduction of functional groups with high initiation efficiency.
The aforementioned findings on TPL indicate that the fabricated polymers are also affected by free radicals and other reactive groups
[64]. PIs are eventually selected based on the processing window of materials, which is the processing range with two coordinates orthogonal to the scanning speed and printing laser power. In general, a wide range of scanning speed and power during fabrication indicates a PI with high efficiency. The biocompatibility of PIs is another crucial parameter, besides water solubility and cytotoxicity, for their application in the biomedical field. Nonionic surfactants, such as Pluronic F127 (PF127), are commonly added into conventional hydrophobic PIs to inhibit their aggregation and precipitation in water, thereby enhancing their water solubility
[65],
[66].
However, excessive nonionic surfactants can increase the cytotoxicity of PIs, which can be addressed by incorporating oil-soluble PIs into suitable supramolecular hydrophobic cavities. Thus, anionic molecules based on the macrocyclic molecules of cyclodextrin and cucurbituril carbazole were incorporated into PIs
[67],
[68],
[69],
[70],
[71] to effectively enhance their water solubility and biocompatibility.
PIs are not a mandatory component in recent MPL-based demonstrations
[25],
[72],
[73],
[74],
[75], but are indispensable in the majority of photoresists. Therefore, ensuring a balance between the efficiency and biocompatibility of PIs remains the primary focus of investigation for their use in MPL for biomedical applications.
Table 2 [63],
[70],
[76],
[77],
[78],
[79],
[80],
[81],
[82],
[83],
[84],
[85],
[86],
[87],
[88],
[89],
[90],
[91],
[92],
[93],
[94],
[95],
[96] lists the commonly used PIs in biomedical applications.
3.2.2. Photopolymers
Photopolymers can be categorized into organic, inorganic, natural, and synthetic. Herein, natural and synthetic photopolymers are discussed to gain better insights into their biocompatibility and functionality. Naturally derived photopolymers, such as collagen and gelatin, form the extracellular matrix (ECM) and affect the mechanical properties of the cellular microenvironment
[7],
[18]. Synthetic photopolymers, such as poly(ethylene glycol) (PEG), are ideal components of functional materials
[97]. Finally, smart materials, which are promising for future biomedical applications, are separately discussed.
(1)Naturally derived photopolymers. Collagen-based: Collagen forms the primary structure of proteins in the ECM of mammals, constituting 30% of their total weight
[98]. Collagen is widely distributed in the skin, bones, and other tissues, playing a crucial role in the physiologic and biochemical behaviors of various cell types. Therefore, collagen has garnered considerable attention in the biomedical field over the past few decades
[99]. However, batch-to-batch manufacturing of collagen can cause inconsistencies in their functional properties
[100]. For instance, the mechanical and biological properties of collagen may be compromised by variations in production conditions, such as temperature. Moreover, natural collagen does not readily participate in photo-crosslinking reactions, and functional groups or other materials may have to be introduced for assistance.
Biomaterials with good reproducibility and high manufacturing precision are desirable for tissue engineering applications. Tytgat et al.
[101] functionalized collagen type-I (RCPhC1) with photo-crosslinkable methacrylamide (RCPhC1-MA), norbornene (RCPhC1-NB), and thiol (RCPhC1-SH) functionalities to enable high-resolution 3D printing via TPL. However, collagen-based photopolymers have some limitations. As collagen has a wide range of sources, its properties may differ. For use in disparate biomedical applications, the source of collagen and extraction methodology must be thoroughly investigated. Second, collagen has poor mechanical properties, particularly in dry state. Consequently, processes such as photoresist development are more stringent, which limits the use of collagen-based photopolymer in certain applications.
Gelatin-based: Derived from type-I collagen, gelatin contains the arginylglycylaspartic acid sequence and promotes cell attachment and proliferation
[102],
[103]. It also has excellent biodegradability and is therefore extensively used in the biomedical field
[104],
[105],
[106]. However, it exhibits poor mechanical properties and biocompatibility
in vivo, which warrants improvement. The upper critical solution temperature of gelatin is lower than physiologic temperature
[107],
[108].
In 2000, Van den Bulcke et al.
[109] first modified gelatin hydrogels with methacrylamide (MA) to synthesize gelatin methacryloyl (GelMA). Since then, GelMA-based hydrogels have been widely used in the biomedical field owing to their tunable physical features and favorable biocompatibility. Good biocompatibility makes gelatin derivates, including GelMA-based hydrogels, another major alternative class of photo-crosslinkable biomaterials for TPL. GelMA has a major advantage over other hydrogels as its mechanical properties can be tuned based on multiple factors, such as using different PIs and adjusting photocrosslinking duration, to meet specific biomedical purposes
[110],
[111]. The mechanical properties of GelMA can also be improved by increasing the gelatin concentrations
[112]. Schuurman et al.
[113] reported that the photocrosslinking duration and elastic modulus are positively correlated. GelMA-based hydrogels have been used in drug delivery and other fields
[114] owing to its tunable properties, such as porosity, biodegradability, and swelling and mechanical properties.
Despite its advantages, GelMA has some limitations, such as low compressive strength (2–30 kPa) and shorter degradation rates (i.e., 32% and 52% after 7 and 28 days
in vivo, respectively). These limitations restrict their use in
in vivo experiments
[99],
[115].
Bovine serum albumin (BSA)-based: BSA is one of the most commonly used proteins for MPL owing to its stimulus-response properties. Kaehr and Shear
[116] recently proposed BSA fabrication strategies for submicron structures with good stimulus-response properties via MPL. Chan et al.
[117] used BSA to fabricate complex microstructures with sub-micrometer topological features. They designed BSA-based microdevices via MPL and illustrated their potential applications in the biomedical field by analyzing the biodegradation, cytocompatibility, and cell–matrix interactions of the fabricated microdevices.
However, protein-based photopolymers are mechanically unstable, and hybrid materials are used to enhance their mechanical stability. Engelhardt et al.
[85] generated microstructures using polymer–protein hybrid materials. Protein-based photopolymers, including BSA, become brittle after dehydration
[118]. Therefore, their mechanical properties must be improved before their application in the external environment of the human body. As BSA exhibits stimulus-responsiveness to light, heat, and pH, it has poor mechanical stability and may therefore be unsuitable for biomedical applications where structural stability is a requirement.
Hyaluronic acid (HA)-based: HA is one of the main components of human ECM, and thus, can be considered an ideal candidate material for fabricating scaffolds for tissue engineering. However, such natural materials typically exhibit poor mechanical properties that can be enhanced via chemical modification for use in TPL. Moreover, its biocompatibility should be considered. Kufelt et al.
[119] combined HA and poly(ethylene glycol)diacrylate (PEGDA) to develop a copolymer, which was functionalized with human epidermal growth factor to enhance its biocompatibility. Cell proliferation tests were performed based on these HA/PEGDA complexes obtained via TPL on which the scaffolds showed good biocompatibility.
Several HA-modified strategies have been studied in recent years, including HA vinyl ester (HAVE)
[120],
[121],
[122] and HA methacryloyl (HAMA)
[123]. Duan et al.
[124] further investigated the properties of TPL photoresists with HAVE precursors, which provided a highly potential alternative for fabricating high-precision cell scaffolds. Therefore, HA-based materials can be considered potential materials for future precision medicine applications.
Similar to other natural materials, HA-based photopolymers have general mechanical properties, such as low compressive strength (1–10.6 kPa). However, HAMA exhibits a high degradation rate, reaching 80% at day 20 in phosphate buffered saline (PBS)
[115]. These findings provide a broad research direction for the functionalization of HA materials.
(2)Synthetic materials. PEG-based: PEG-based hydrogels are one of the most widely used building materials in cell and tissue engineering. PEG exhibits protein and platelet rejection, thereby promoting thrombus formation and exhibiting cytotoxic effects
[125],
[126],
[127],
[128],
[129],
[130]. PEG also has superior hydrophilicity and solubility in a wide range of solvents, such as toluene and chloroform
[131],
[132]. Additionally, PEG is used extensively to increase the drug stability and retention time of biopharmaceuticals for its tendency to hinder nonspecific protein absorption
[133],
[134]. Thus, PEG-based drug carriers have been widely used in drug delivery therapy in recent years.
However, PEG is a nonbiodegradable polyether that is nonimmunogenic and nonantigenic. Thus, PEG-based materials exhibit poor performance in some chronic
in vivo biomedical applications
[135],
[136]. PEG also lacks cell adhesion sites, which restricts its long-term biomedical applications
in vivo [115].
Polyester (PE)-based: PE-based materials have been extensively used in the biomedical field owing to their tunable mechanical properties
[137]. Melissinaki et al.
[138] fabricated a polylactide (PLA)-based 3D neural tissue engineering scaffold via TPL. By attaching methacrylate groups to the PLA, resins can be readily cured under NIR irradiation. Additionally, PLA-based copolymers can be synthesized with readily tunable physical properties for various biomedical applications. Poly(ε-caprolactone) (PCL)-based microstructures have been particularly researched for fabricating high-resolution structures. Thompson et al.
[139] studied the effect of different PCL-based materials and TPL parameters on minimum laser power to obtain a high-resolution model for retina cell replacement. By increasing the molecular weight of polycaprolactone diacrylateand (PCLDA) and polycaprolactone triacrylate (PCLTA), the laser power required for polymerization increased slightly. Moreover, at all scanning speeds, regardless of its molecular weight, PCLDA polymerization required considerably higher laser intensity than PCLTA. The fidelity of PCLTA-based structures can also be tuned using different polymer concentrations, which pave the way for obtaining a high-resolution model for retina cell seeding.
In summary, polyester-based materials can be considered potential candidates for tissue engineering and other applications owing to their tunable mechanical properties and cost-effectiveness.
PDMS-based: PDMS is widely used in microfluidics owing to its transparency and elastomeric properties
[140],
[141],
[142],
[143],
[144],
[145],
[146]. To broaden the range of materials used in MPL, the mechanism and properties of PDMS-based materials have been investigated. Coenjarts and Ober
[147] first described two approaches for fabricating PDMS-based microstructures by two-photon 3D microfabrication via photohydrosilylation and radical-initiated cross-linking. A single voxel has an aspect ratio of 3–4, demonstrating the potential of this material for sophisticated microfluidic system applications. Hasegawa et al.
[148] developed a photocurable PDMS resin for manufacturing 3D micromachines via TPL, showing the potential for applications in microfluidic devices and as micromanipulation tools for biological applications. Rekštytė et al.
[149] investigated the possibility of forming 3D microstructures in PDMS doped with different PIs via MPL under different exposure conditions, achieving a higher resolution (5 μm) and throughput (720 μm
3·s
–1). They reported on the advancement in the use of PDMS-based cellular scaffolds in biomedical applications.
Despite the numerous advantages, certain characteristics of PDMS may present limitations in biomedical applications. For instance, the CH
3 group in PDMS presents a hydrophobic surface
[150],
[151],
[152], which often precludes its use in liquid biological samples
[153].
Others: Other materials, besides the mentioned photopolymers, exhibit excellent mechanical properties. For instance, Buchroithner et al.
[154] introduced two new biocompatible resin formulations, BisSR and M10, for tissue engineering
in vivo. The Young’s modulus of M10 was 40–120 MPa and increased with increasing laser intensity. The Young’s modulus of BisSR was ∼80 MPa and independent from laser writing process. Men et al.
[155] proposed TPL printable polymers with good mechanical tunability (Young’s modulus: 0.3–1.43 GPa) and good biocompatibility, showing a good potential for biodevice applications. In conclusion, MPL can be used for fabricating materials and photopolymers with excellent mechanical properties for applications, such as tissue engineering.
(3)Smart materials. Active materials or smart materials typically have properties that enable them to undergo changes in shape or size over time or in response to environmental stimuli. Smart materials are fabricated using several methods
[156],
[157], such as the functionalization of photopolymers via doping or deposition. Xia et al.
[158] doped methacrylate group-modified Fe
3O
4 nanoparticles into photoresists and fabricated remotely controlled micromachines via MPL. Tottori et al.
[59] deposited Ni/Ti thin bilayers on the surface of a helical micromachine, enabling a microrobot to be magnetically driven and obtaining enhanced surface biocompatibility.
The polymer network properties of photopolymers are also used to fabricate smart materials, such as BSA
[159],
[160],
[161]. Liquid crystalline elastomers (LCEs)
[156], smart responsive hydrogels
[162], and shape memory polymers (SMPs)
[157] are some examples of smart materials. Martella et al.
[163] prepared light-responsive liquid crystalline networks and used them for fabricating microactuators via MPL. By adjusting the amount of cross-linking agents, the deformation kinetics can be effectively controlled from the micro to macroscale. Zeng et al.
[164] prepared an LCE structure with fully reversible mechanical response to light, including ∼20% of the guides of contracted nematic liquid crystal elastic network. Martella et al.
[165] engineered a microhand controlled by optical illumination to catch microelements. This hand had a unique feature of distinguishing between particles of different color and gray levels. In addition to LCEs, SMPs are a candidate material for MPL in biomedical applications. Elliott et al.
[166] synthesized and sculpted a benzyl methacrylate-based SMP 3D structure via TPL, which exhibited nanoscale features and arbitrary 3D geometries that changed shape in response to temperature variations. These results demonstrate the benzyl methacrylate-based SMP’s potential for application in miniaturized deployable biomedical devices, such as stents for retinal vasculature.
Table 3 [123],
[139],
[147],
[148],
[149],
[167],
[168],
[169],
[170],
[171],
[172],
[173],
[174],
[175],
[176],
[177],
[178],
[179],
[180],
[181],
[182],
[124] summarizes the commonly used photopolymers in MPL.
Overall, the design of new functional and active materials for multiphoton 3D laser printing has rapidly increased in recent years, resulting in a rich variety of promising applications, particularly in biomedical applications.
4. Biomedical applications
Microminiaturization of devices provides more advanced solutions for patient diagnosis and treatment
[183]. Additionally, precision medicine has strict requirements with regard to the level of precision required in individual diagnosis and treatment. This indicates that the demand for small-scale manufacturing of biomedical applications has become an important trend. Therefore, in this review, we focus on the design of devices at micro/nanoscale. MPL enables the fabrication of micro/nanodevices that can be used in the biomedical field (e.g., therapeutic or diagnostic). Based on different scenarios of precision medicine, the devices used in the biomedical field can be classified into four types: delivery systems, microtissue modeling, surgery, and diagnosis.
4.1. Delivery systems
MPL can be used to fabricate systems for precise drug delivery and substance release, which are crucial for precision medicine. Based on the delivery target, delivery devices can be classified into two types, namely microrobots and microneedles that are mainly used for in vivo applications and in vitro transdermal delivery, respectively.
4.1.1. Drug delivery
(1) Microrobots: MPL is used to fabricate microrobots that can serve as tools for targeted
in vivo therapy due to their complex micro/nanoscale design. Therefore, MPL technology is commonly utilized in this field. Herein, we focus on the trajectory control and drug release of microrobots. Precise trajectory control can lead to precisely targeted drug delivery, which is affected by the actuation and structure of microrobots. Magnetic actuation is the most commonly used actuation method because it offers various advantages, such as contactless actuation, biological substance insensitivity, and precise trajectory control
[184],
[185],
[186]. Using this strategy, delivery carriers can be navigated into the localized trauma in the body
[187],
[188]. Several actuation strategies can be used in
in vivo delivery therapy, including light
[189], thermal stimulus
[190], chemical reaction
[191], and acoustically mediated control
[192].
Researchers have made efforts to improve the accuracy of trajectory control in
in vivo delivery therapy
[193]. Ceylan et al.
[114] developed a double-helical structure, capable of carrying objects with certain weights and swimming, which could be controlled by a rotating magnetic field. The structure degraded into a nontoxic product under the action of metalloprotease (
Fig. 2(a)). Xin et al.
[194] designed innovative conical hollow microhelices for targeted delivery with improved swimming ability and reduced lateral drift compared with straight microhelices. Xu et al.
[195] designed a sperm-hybrid micromotor for targeted drug delivery (
Fig. 2(b)). The sperm release mechanism was designed to liberate the sperm when the micromotor hit the tumor cells, allowing the sperm for swimming into the tumor and delivering the drug via sperm–cancer membrane fusion. They demonstrated that the sperm-hybrid micromotor was potentially a biocompatible platform of drug delivery therapy for diseases in females by guiding the sperm/drug to an
in vitro cultured tumor spheroid and releasing it locally.
Researchers have focused on achieving precise release time, release dose, and release frequency of drugs. Drug-release methods are used based on the application demand, where different stimulus-responsive materials and structures are designed for drug release. Stimuli responsiveness includes the response of materials to changes in pH, temperature, and other conventional physical quantities in the body environment as well as response to a specific substance, such as an enzyme
[114].
Studies on drug (or substance) release focus on two aspects: ① the structural design of drug-release carriers, including various low-resistant and multistage release structures and ② material properties, including biocompatibility and response to stimuli, such as light or pH. Lee et al.
[175] adopted the structure of pollens to attain temperature-controlled surface energy attachment characteristics that could be driven by a magnetic field for achieving drug release via pH stimulation. Song et al.
[196] employed a puffball as a structure for loading drugs (
Fig. 2(c)), which enabled controlled, graded drug release with a high drug load. Additionally, the NIR sealing layer facilitated controlled drug release and allowed for precise and controllable drug release time. In addition to drug release time and dose, multiple drug release is also crucial for precision drug delivery systems. Based on multidrug release structures, Li et al.
[197] developed a fish-inspired heterogeneous microrobot that carried acetylsalicylic acid and doxorubicin (DOX) to enhance the synergistic effect of cancer treatment.
Studies on materials are primarily focused on drug release based on stimulus-responsive materials. In simple terms, stimuli can be divided into internal (e.g., pH, temperature, redox potential, and enzymes) and external (e.g., light, electric field, and magnetic field)
[198]. A drug-carrying device can automatically respond to stimuli based on the difference between the properties of a normal physiologic environment and the tumor area in the body. Therefore, internal stimuli are considered vital. Changes in pH within the tumor region are a basis for the design of drug load structure. Ye et al.
[199] combined folic acid (FA)/GelMA and modified it with magnetic metal–organic frameworks (MOFs) to fabricate a microrobot for drug delivery against cancer cells. It degraded and released drugs in a weak acidic environment in tumor attachments. Results showed that the cancer cell inhibition rate of microrobots with FA could reach up to 93%, whereas it was only 78% in the control group without FA. For more precise drug release under pH stimulation, Xin et al.
[200] designed a shape-morphing microfish (SMMF), which combined pH response and magnetic actuation to achieve the delivery and release of DOX drugs (
Fig. 2(d)). Drug was released by opening and closing the “mouth” of the SMMF. Importantly, SMMF’s response to pH reduced from 9 in NaOH to 7.4 in PBS, which is crucial for more biomedical applications. Based on the results of their previous study, Xin et al.
[201] further improved a printing process to overcome some limitations, such as single-material printing and slow printing speed. The optimized SMMF could swim in a narrow micro-network under the control of a magnetic field. Wang et al.
[202] presented a helical MOF-based micromachine that can swim and follow complex trajectories under weak rotational magnetic fields (
Fig. 2(e)). The zeolitic imidazole framework-8 (ZIF-8) coating on the outermost part of the structure provides a solution for embedding the drug and releasing it under weak acidic conditions.
Some studies have focused on materials and heterostructures that respond to pH stimuli. Hu et al.
[203] mimicked the dynamic behaviors of botanical systems based on a pH-responsive hydrogel for 4D printing, which improved the MPL accuracy and enhanced the responsiveness of the material to external stimuli. They also recreated a cage-like structure via MPL that could grasp objects for drug delivery applications. Wei et al.
[204] fabricated a pH-responsive microstructure with reversible swelling properties and achieved a swelling ratio of 1.08–2.71 using BSA materials, thereby providing an alternative solution for drug delivery. Li et al.
[205] designed a one-shot and single-material capture structure, which could be used for multistep moldings of pH-responsive structures and multiple materials. This structure had the potential to be used in cell manipulation and drug delivery.
Drug carriers have been studied for releasing drugs in response to temperature variations. Zhou et al.
[206] developed a microrobot based on poly-
N-acryloyl glycinamide (PNAGA) that could rapidly dissolve and release drugs at 45 °C, a temperature slightly higher than the human body temperature (i.e., 37 °C). Therefore, PNAGA was considered a promising candidate for drug delivery in the human body. However, the narrow range of normal body temperatures necessitated the development of temperature-responsive drug delivery/releasing systems with high sensitivity that could rapidly release drugs around normal body temperatures (∼37 °C). Therefore, future drug delivery robots need to be further optimized in terms of material choices and structural design.
N-Isopropyl acrylamide (NIPAAm) and its modified substances are representative materials for light-responsive drug release among all the thermos/light-responsive soft materials. For instance, single-walled carbon nanotubes were incorporated as isolated fibers in a hybrid gel system of NIPAAm and
N-dimethyl acrylamide (DMAAM). They acted as molecular reservoirs to store DOX as a base and release the drug in an acidic environment
[207]. To achieve a fast response of micromachines at low actuation thresholds, Deng et al.
[208] introduced a 4D printing strategy that could be used to fabricate microdevices for drug delivery. In addition, Bozuyuk et al.
[209] fabricated a double-helical microswimmer using an external light stimulus to release a chemotherapeutic drug, DOX. The drug release was ceased by a controlled pattern of light used for controlling the release doses, further promoting the application of drug delivery in precision medicine.
To further optimize the design principle/structure of the drug-loaded microrobot, drug release was modeled. Do et al.
[169] first demonstrated that MPL parameters, such as hatching distance, slicing distance, and pore size of the carriers, can be modified to control modal drug-release time. In general, increased spacing led to a higher drug-release rate, whereas smaller spacing resulted in some occlusion, preventing media infiltration and thus resulting in reduced fluorophore release. Their study paved the way for MPL fabrication of drug delivery carriers.
In summary, microrobots fabricated via MPL are mostly in the in vitro experimental stage. In future investigations, in vivo experiments on animals or even humans should be conducted to fit practical applications.
(2) Microneedles: Microneedles have multiple applications, such as in delivery of drugs, vaccines, and bio-macromolecules; monitoring and diagnosis; disease treatment; and cosmetics
[210],
[211]. Owing to its high precision and reproducibility, MPL is used to fabricate microneedles with complex geometries; these microneedles are gradually being used in many biomedical scenarios. Microneedles have become one of the most useful tools for two-photon micro/nanofabrication in recent years. Their use in drug delivery is particularly important among other applications.
Microneedle drug delivery is particularly useful in inner ear therapy. Yu et al.
[210] and Aksit et al.
[212] used the IP-S photoresist to fabricate microneedles and used them to perform perforation experiments on the round window membrane (RWM) of guinea-pigs, demonstrating an innovative therapeutic modality for drug delivery in inner ear diseases. Chiang et al.
[213] conducted experiments on frozen human tissue and showed that two-photon printed microneedles can precisely punch holes in the human RWM. These MPL-based microneedles can potentially be used for therapeutic means, such as inner ear drug delivery. Several fabrication methods have been proposed to manufacture microneedles in small batches for further use in drug delivery therapies of the inner ear. Aksit et al.
[214] developed a hybrid additive manufacturing method using TPL and electrochemical exploiting techniques and fabricated high-precision, ultra-sharp, and gold-coated microneedles (
Fig. 2(f)). Balmert et al.
[215] combined MPL and micromolding approach to fabricate a new type of dissolving undercut microneedle arrays, which overcame the difficulty in the fabrication process via molding. Moreover, microneedles could be used for multidrug delivery and multicomponent vaccination (
Fig. 2(g)).
In addition to perforated delivery, direct transdermal delivery also uses microneedles. In this approach with strict requirements for machining precision, small holes are developed on the tip of the needle or side-open channel. Ebrahiminejad et al.
[216] combined MPL and thermal embossing for the mass production of microneedles for transdermal drug delivery (
Fig. 2(h)). To simplify microneedle fabrication, Faraji Rad et al.
[217] first fabricated prototype microneedles with open microfluidic channels via MPL (
Fig. 2(i)). Subsequently, a mold of a thermoplastic replica was used for the mass production of microneedles. They thus provided a suitable approach for the mass manufacturing of open-channel microneedles for drug delivery. However, for personalized therapy, the selection of optimal parameters for printing microneedles often involves an intensive optimization process. Faraji Rad et al.
[218] fabricated highly detailed microneedles and described the relation between MPL parameters (i.e., scanning speed, laser power, hatching, stitching, and slicing distances in
Fig. 2(j)). Moreover, they provided a detailed guideline for designing the microneedles.
In addition to the manufacturing of microneedles, some researchers combined microneedles with microfluidic devices to expand their application prospects, including biomolecular detection, which is discussed in subsequent sections.
4.1.2. Cell delivery
Along with drugs, cells are another type of therapeutic agents that require targeted delivery
[219]. Cell-based therapy has garnered attention for regenerative medicine in recent years
[220]. MPL has become a potential tool for designing micro/nanoscale cell delivery carriers for cell therapy in the past few years
[221]. Li et al.
[187] fabricated a burr-like porous spherical microrobot that carried and delivered targeted cells
in vivo, acting as a platform for regenerative medicine and cell-based therapy (
Fig. 2(k)). Moreover, the authors demonstrated that the magnetic driving capabilities and cell-carrying capacity of the microrobots were improved via simulation and
in vitro experiments. They also demonstrated the capability of microrobot to release cells at specific sites
in vivo within nude mice. These as-obtained results preliminary supported the feasibility of using magnet-driven microrobots for the targeted delivery of cells
in vivo. Jeon et al.
[222] similarly developed magnetically actuated scaffold-type microrobots as cell carriers for precise stem cell delivery and transplantation
in vitro,
ex vivo, and
in vivo (
Fig. 2(l)). The proposed design enhanced the capacity of cells. Moreover, a novel strategy enabled by rotating magnetic field propulsion improved the propulsion efficiency and controllability of cells in an
in vivo fluid environment.
However, developing
in vivo imaging technologies for tracking microrobots in animal models and fabricating 3D biodegradable microrobots remain challenging. Dong et al.
[223] proposed an integrated multifunctional microswimmer to deliver neuronal cells and stimulated their differentiation. It degraded after cell delivery inside the body (
Fig. 2(m)). They used GelMA as the substrate material and CoFe
2O
4@BiFeO
3 (CFO@BFO) core–shell magnetoelectric nanoparticles for the microswimmer, wherein GelMA served as a good basis for cell growth during delivery and the magnetoelectric nanoparticles served as magnetic motile components and induced differentiation of neuronal cells. Furthermore, micro-robotic degradation assays
in vitro and neural cell-induced differentiation experiments were performed under magnetic stimulation, which demonstrated the potential of such a design in the treatment of central nervous system (CNS) diseases. Wei et al.
[224] designed a magnetic field-driven image-guided biodegradable microrobot that could precisely deliver engineered stem cells for orthotopic liver tumors.
In vivo experiments were conducted on nude mice, during which the biodegradable microrobot released the loaded cells at the target site. After four weeks, the tumor growth was obviously inhibited, illustrating that the loaded cells were delivered effectively to the infected region. This was the first research that used degradable microrobots for the targeted delivery of therapeutic cells in vascular tissues and demonstrated their therapeutic effect in preclinical tests.
In the future, MPL will continue to be used as a tool for cell delivery, particularly targeted delivery. Long-term targeted therapeutic experiments in animals can be realized via the micro/nanomanufacturing of various functional and biomaterials, including multiple stages of driven control, cell release, and biodegradation.
4.2. Microtissue modeling
To provide more therapeutic tools for regenerative medicine, better insights must be gained into the interaction between cells or microtissues and the
in vitro environment
[225],
[226]. Based on this context, numerous studies have focused on the construction of microtissues. In contrast, MPL can be used to build complex shapes at the micro/nanoscale; therefore, it is extensively used for constructing physiologic micro/nanoscale tissues that assist disease modeling,
in vitro drug screening, and tissue repair or replacement
[227].
4.2.1. Drug screening and disease modeling
Drug discovery continues to be expensive because of the lack of translation technology for 2D cellular models. Therefore, engineered cellular systems were considered to improve the correlation between screening data and patient response in clinical practice. For precision medicine, patient/individual-specific models are required to gain deep insights into the interaction between drugs and microtissues of an individual
[227]. However, such models require personalized microstructure. Human tissues show great varieties in geometric morphologies with special features, including multiscale geometrical macro to nanoscale features. MPL can be used to construct 3D complex structures with high resolution and print
in vitro cell scaffolds or microstructures. Therefore, MPL is widely used for manufacturing microstructures and microarrays for drug screening and disease modeling.
Organ-on-chips (OoCs) are ideal tools for biomedical applications, such as disease modeling and drug and toxicity screening
[228],
[229]. OoCs fabricated via MPL are widely used in the blood–brain barrier (BBB) model, heart disease model, and other organs or tissue models. Marino et al.
[230] proposed a 3D biohybrid biomimetic BBB model at a 1:1 scale for the first time to investigate the crossing of nanomaterials for therapeutic and diagnostic brain disease applications. A model comprising connectors, junctions, and capillaries with and without boles was fabricated in IP-DiLL (Nanoscribe, Germany). In this model, porous tubular structures mimicked the structures and functions of brain microvessels. After fabricating the chip, the bEnd.3 endothelial cells were seeded around porous microcapillaries. Then, the chip was connected to an external pumping system, creating a liquid flow of ∼1 mm·s
−1, similar to the
in vitro physiologic condition. This system provided an ideal tool for investigating the BBB crossing of nanomaterials and drugs. Tricinci et al.
[231] also fabricated a 3D-printed realistic biohybrid model of the brain tumor microenvironment in a 1:1 scale that contained luminal and parenchyma compartments for high-throughput drug screening in CNS diseases (
Fig. 3(a)). In this model, the endothelial cells, hCMEC/D3, were homogeneously dispersed in the intratubular space, whereas astrocytes and U87 glioblastoma (GB) cells were cultured in the outer space. When modeling the GB microenvironment, an antibody-functionalized mutilin-loaded nanostructured lipid carrier was used to assess its impermeability to drug-loaded nanoparticles for the treatment of tumors. This study accelerated the development of novel therapeutic strategies against brain cancer and many neurodegenerative diseases
[232]. However, using MPL to generate multiple structures with feature sizes in the meso-to-milliscale range typically requires multistep printing on the order of days. Therefore, micro/nanoscale structures have been manufactured only via MPL to adopt these models.
Except for the BBB model, MPL has been used for fabricating cardiac chips. Michas et al.
[233] fabricated an integrated microfluidic system (powered by human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (hiPSC-CM)) that replicated the ventricular function on a chip (
Fig. 3(b)). The system mimicked a cardiac chamber supported by a miniaturized metamaterial scaffold, cardiac valves, and unidirectional flow against a pressure gradient. The scaffolds and valves were fabricated using IP-S and MPL.
Drug screening of liver tissue is another important aspect. Zeußel et al.
[234] used MPL to construct a scaffold that mimicked the micromorphology of a liver lobule to achieve fluid perfusion (
Fig. 3(c)). Their simulations showed that the observed shear stress, fluid velocity, and streamlines of the scaffold were comparable to the native liver lobule. The authors showed that MPL is a potential tool for
in vitro drug screening in the future. Zhang et al.
[235] fabricated microarrays in parallel using a dynamic multi-foci MPL process. They combined hologram predesigning with lens phase modulation to generate multiple femtosecond laser spots, considerably improving the efficiency of microstructure manufacturing. The built scaffolds served as an arrayed analytical platform to reveal the anticancer effects of loaded drugs.
To gain better insights into the interaction between the microenvironment and cells, many researchers fabricated cell scaffolds via MPL. Tayalia et al.
[236] first fabricated cell culture scaffolds with different lateral pores and investigated human fibrosarcoma cell line-based MPL. Barin et al.
[237] used MPL to build
in vitro glioma cell scaffolds that could retain the receptor-high replication properties of the epithelial growth factor and the cell’s microtubule-based network structure. Thus, the fabricated scaffolds could be better applied to the study of cancer cell activity and further drug screening (
Fig. 3(d)). Rengaraj et al.
[238] designed microscale scaffolds via MPL as the main structure on which they coated a bioactive film to mimic early stages development of metastatic cancer (
Fig. 3(e)). This coating comprised HA and poly-
L-lysine with controlled stiffness and were loaded with fibronectin and bone morphogenic proteins 2 and 4 (BMP2 and BMP4) as the matrix-bound proteins. They also investigated the adhesion and growth of pancreatic cancer cells (PANC1 and PAN092) on polyelectrolyte multilayers (PEM) and demonstrated that bioactive PEM can be deposited on the scaffold. Their research provided some useful evidence for longer-term studies and drug treatment to modulate cancer cells and prevent cancer growth by designing scaffolds on the microscale using MPL.
Although MPL could mimic the microstructure of some cell growth environments, the effects of cell–environment interactions (e.g., flow shear forces) remain unexplored. To further develop disease models and conducted in vitro drug screening, future studies need to focus on the functionalization of microstructure. Moreover, the in vitro flow environment must be simulated to obtain useful information on the reliability of drug screening results.
4.2.2. Tissue/cell scaffolds for cell repair/replacement
The ultimate goal of regenerative medicine is the preservation and enhancement of human tissue functions by creating their artificial substitutes
[239], for which the accurate construction of 3D human tissue models is crucial. Traditional 3D printing is performed at the macro scale and is not ideal for micro/nanomanufacturing. Therefore, MPL was used for micro/nanomanufacturing, which accelerated the use of small-scale tools, such as mini scaffolds and cell niches
[240],
[241],
[242]. These scaffold-type devices can be applied to precisely mimic the 3D cell ECM step by step, from components to structures and from spatial to temporal distributions
[243],
[244]. Moreover, protein-coated scaffolds can be used to study biocompatibility, cell migration, cell morphology, and cell mechanics
[236],
[245],
[246].
In addition to single-material cell scaffolds, heterogeneous cell scaffolds based on MPL are currently used for the investigation of ECM-induced cell growth
[247],
[248]. Richter et al.
[247] designed 3D scaffolds for cell culture via MPL using a combination of three types of resists, such as protein-repellent photoresist, protein-adhesive resist, and photoactivatable passivating resist that were functionalized with different ECM proteins (
Fig. 3(f)). They introduced a strategy to construct multi-protein functionalized 3D microenvironments, which could more accurately replicate the spatial distribution of ECM of the microtissue at the microscale. Directed self-assembly was also introduced to regenerative medicine by adding new tools, such as lockyballs. This new method produced adipose stem cells (ASCs) spheroids that enhanced regenerative effect
[249],
[250]. Ovsianikov et al.
[251] fabricated 3D scaffolds for tissue engineering using MPL and investigated the spatial resolution of PEGDA, a scaffold material, in relation to the irradiation parameters. They also investigated the relationship between different PI types and concentrations of scaffolds along with their cytotoxicity. Their study served as a guide for the fabrication of 3D scaffolds that mimicked the physical and biological properties of native cell environments.
Tremendous efforts have been invested in bone tissue construction and associated immunomodulation. Mini scaffolds fabricated via MPL are used to construct various cellular tissues, including bones. In such a design, photoresists for MPL must be biodegradable and nontoxic to cells, which considerably limit the choice of materials available for fabricating mini scaffolds
[252],
[253],
[254]. Terzaki et al.
[255] combined 3D scaffolds fabricated via MPL with self-assembling peptides targeted for calcium binding. Results demonstrated an increase in cell adhesion with increasing cell proliferation, which increased biomineralization considerably. This strategy can be potentially used for hard tissue engineering. Koroleva et al.
[256] used MPL to fabricate 3D Zr–Si scaffolds for autologous bone tissue engineering (
Fig. 3(g)). The scaffolds with different pore sizes were investigated for their mechanical properties, stem cell seeding efficiency, cell proliferation, and induction of differentiation toward osteogenic lineage. Timashev et al.
[257] fabricated PLA-based scaffolds with the Young’s modulus comparable to that of human bone. The scaffolds fabricated via PLA provided a beneficial microenvironment for the differentiation of osteogenic MSCs
in vitro and supported bone regeneration
in vitro. Mihailescu et al.
[258] used laser-assisted technology to fabricate osteoblast seeding structures for bone tissue engineering, which contained vertical microtubules arrays arranged in a triangular lattice shape. They demonstrated the efficiency of these structures in bone regeneration after the implantation of hematopoietic stem cells and osteogenic potential and offered an alternative solution for bone tissue repair. Felfel et al.
[259] fabricated multiphase hybrid scaffolds using the poly(
D,
L-lactide-
co-
ɛ-caprolactone) (PLCL) copolymer, silk-elastin-like recombinamers hydrogel, and nano-HA for bone repair applications. However, such scaffolds have a tradeoff between high resolution and high throughput, which limit their clinical applications. Weisgrab et al.
[260] proposed, for the first time, a biodegradable and biocompatible scaffold at the macroscale via MPL (
Fig. 3(h)). Nouri-Goushki et al.
[261] designed six microcolumns of different heights to observe the polarization of macrophages. These results can be potentially used for the future exploration of osteoimmunomodulatory phenomena associated with submicron topographies and are of relevance for manufacturing orthopedic implants.
In summary, MPL microtissues are important tools for investigating regenerative medicine. However, the existing microtissues cannot be used in clinical practice due to the complexity of ECM composition, geometrical structure, spatial distribution of biochemical cues, and other unknown influences.
4.3. Surgery
Microdevice systems have become a promising solution for precision surgery and have considerably extended the operational capacity of surgeons
[262]. Additionally, the laser used to create these microdevices function as a sharp scalpel during surgery. Thus, microdevices fabricated via MPP will provide a toolbox of options for delicate surgeries in the future.
4.3.1. Photodynamic therapy (PDT)
PDT is a minimally invasive technique used for multiple applications in clinical practice, particularly for the detection and treatment of cancer (e.g., prostate, breast, head and neck, skin, pancreas, and lung)
[263],
[264],
[265]. In such a therapy, tumor tissues with added PS stimulates the cells to produce reactive oxygen species (ROS) under light irradiation. The tissues undergo apoptosis, killing the targeted tumor cells or other pathogens
[265]. To improve the efficacy of PDT, three main factors should be considered: PS, light sources, and molecular oxygen
[266],
[267]. NIR light can better penetrate into biological tissues than UV/VIS light. Moreover, PSs with a high ROS generation efficiency and NIR absorption can be used for such therapy, because of which two-photon excited PDT (TPE-PDT) has been progressively used in the last decade.
In 2008, Starkey et al.
[268] reported for the first time that PDT using PSs on tumor cells can cause some degree of tumor tissue regression. This inspired researchers to design PSs with high efficiency and low toxicity, and some investigations introduced imaging methods for two-photon dynamic therapy. For instance, Guo et al.
[269] developed multimodal polymer nanoparticles (PNPs) that can be used in two-photon fluorescence imaging and TPE-PDT (
Fig. 4(a)). They, thus, proposed a promising candidate for simultaneous cellular, deep-tissue imaging and high-efficiency PDT. Moreover, Wu et al.
[80] developed a carbon dots (CDs)-based PDT system with fluorescence imaging, mitochondria targeting, and two-photon-induced aminolevulinic acid (ALA) releasing apabilities using the following steps. First, a phototriggerable material was synthesized by linking ALA to coumarin. Then, a mitochondria-targeting compound, triphenylphosphonium (TPP), and CDs were added into the derivative to create a nanosystem (CD-ALA-TPP). During PDT, the nanosystem preferentially accumulated in mitochondria to release 5-ALA, causing damage to the cancer cells under two-photon irradiation via oxidation (
Fig. 4(b)). Their study presented a novel strategy for the use of PDT with lesser side effects. Dobos et al.
[93] developed an
in vitro screening platform of TPE–PS (i.e., P2CK, Eosin Y, and porphyrin derivate) using a 3D osteosarcoma cell culture (
Fig. 4(c)). Huang et al.
[270] combined TPA fluorescence resonance energy transfer and NIR photothermal effect of unimolecular micelles to enhance the therapeutic efficiency of existing PSs (
Fig. 4(d)).
However, compared with single-photon PDT, TPE-PDT is produced with lesser ROS to efficiently kill the target tumor cells. Some PSs can, however, produce higher ROS; thus, the contradiction between the high ROS production and TPA enhancements remains unresolved, which should be addressed in future studies.
4.3.2. Micromanipulation
Micromanipulation, such as trapping and holding cells, is essential for single-cell therapy and analysis
[271],
[272],
[273]. Microdevices that can perform microoperations are expected to become an important tool for future precision surgeries.
Alapan et al.
[274] designed a shape-coded dynamic assembly of mobile micromachines that could be transported vertically along the helical thread when rotated with an applied magnetic field. They allowed for micro-object transportation and manipulation in the vertical space, providing a potential strategy for precision manipulation. Ma et al.
[161] designed complex microrobots composed of two distinct materials: relatively stiff SU-8 as the skeleton and soft pH-responsive protein as the smart muscle. These heterostructure microrobots grabbed and released micro-objects in a well-controlled manner. Hu et al.
[275] fabricated 3D shape-deformable magnetic soft structures (microactuators) using an assembly-based fabrication strategy that could be used in reconfigurable extracellular matrices for manipulating fragile micro-objects. Jia et al.
[179] fabricated 3D-printed protein-based robotic structures actuated by molecular motor assemblies, thereby proposing a novel strategy for fabricating micromanipulators, such as micro-hands and micro-arms that can grasp and wave upon activation (
Fig. 4(e)). Wang et al.
[276] developed a 3D hydrogel actuator inspired by flytraps that could be controlled by variations in pH (
Fig. 4(f)). It could grasp and release micro-objects. Additionally, different releasing strategies can be employed to precisely manipulate the releasing behavior of multiple micro-objects, enabling simultaneous or successive release processes. These findings demonstrate the potential applications of microscale soft actuators for precise manipulation in biomedical engineering. Microinjection is another important parameter for precision surgery. Yagoub et al.
[277] developed a microdevice to simplify intracytoplasmic sperm injection (ICSI), which comprised two microscale components: a pod and a garage (
Fig. 4(g)); the cell was placed in a pod docked in a garage. This study provided a strategy that can be used readily for high-throughput microinjection.
4.3.3. Cell sorting
Cell sorting is a key tool in the biomedical field, which is used to purify suspended cells from complex, heterogeneous mixtures
[278],
[279]. TPL has been applied in microfluidic systems for passive cell sorting, such as membrane-based and column-based sorting, owing to its capability to fabricate microstructures.
Wang and Papautsky
[280] designed a size-based microfluidic multimodal microparticle sorter, which improved the efficiency of sample separation and broadened the applications of inertial microfluidics in sorting complex microparticle samples. Xu et al.
[281] proposed a novel arch-like microsorter that could perform multimodal sorting of particles (i.e., high-, band-, and low-capture mode). This microsorter could be tuned flexibly and precisely in terms of front and back sorting sizes and reduced clogging for long periods of use. The performance of the microsorter was also validated by the enrichment of SUM 159 triple-negative breast tumor cells from human blood. This demonstrated its potential in many applications, such as circulating tumor cell separation and blood cell sorting. Perrucci et al.
[282] designed a membrane-based microfluidic filtration system that successfully integrated suspended microfilters obtained via TPL into a 3D-printed microfluidic structure. This system was evaluated by employing size-controlled fluorescent microparticles, thereby demonstrating its potential applications for cell sorting. Zhang et al.
[283] proposed a crossflow sorter using micropillars with different slit sizes to fractionate particles of different sizes. This type of sorter can be applied in cell sorting.
Hu et al.
[284] fabricated a tunable microfluidic device (TMFD) for the manipulation of particles and cells. The pH-responsive hydrogel in the microring array was integrated into the TMFD via TPL, which could swell and shrink in < 200 ms with pH variations. By tuning the pH, the TMFD could multifilter and trap particles of a specific size (5–10 μm;
Fig. 4(h)). Kaynak et al.
[285] fabricated a multibody mechanical system based on hydrogels that can be used in microscopic sampling via the ultrasound transduction of frequency-selective actuation. The system comprised a “μ-jet” engine, a collection chamber, and a sieve (
Fig. 4(i)). The engine transported fluid with cells once excited periodically at its resonance frequency. Then, the cells were collected in a collection chamber and filtered through the sieve to obtain the desired size range. Wang et al.
[286] fabricated a magnetically driven rotary microfilter that enabled switching between the modes of filtering and passing inside the microfluidic chips (
Fig. 4(j)). These multimode filtering functions have broadened their applications for cell sorting in complex mixtures.
4.4. Diagnosis
Microdevices are promising tools for disease diagnosis
[8],
[287],
[288],
[289], as they can selectively recognize physiologic signals or agents of targeted molecules, including cells, bacteria, proteins, and ions. These biomarkers or biosignals can be used for clinical analysis and diagnosis of cancer, infection, and other diseases.
4.4.1. Biosignal detection
Biosignal sensing and detection are vital for future precision medicine as diagnostic links, and accurate biosignal sensing is more conducive to personalized medical diagnosis
[271],
[290]. Therefore, many microarrays fabricated via MPL have been used in the biomedical field. AI-Abaddi et al.
[291] combined MPL with conventional photolithography to fabricate 3D polymer electrodes for biosignal detection (
Fig. 5(a)). Haque et al.
[292] designed bridge-on-pillars structures on a silica substrate that were 3D-printed and partially carbonized. They demonstrated the potential of a carbonized polymer electrode as a low-cost, complementary metal oxide semiconductor (CMOS)-compatible monolithic biosensor platform for disease diagnosis and treatment. However, the device was fabricated on a rigid glass or silica substrate, which hindered its chronic implantation. To address this issue, Brown et al.
[51] developed a high-aspect-ratio microelectrode array integrated on a thin-film flexible cable for neural recording in small animals, such as songbirds (
Fig. 5(b)). Thus, the combination of MPL and thin-film fabrication processes can be used to overcome some challenges, such as the miniaturization of arrays and produce structures with high aspect ratios, for precise testing. However, the lack of functions of chronic implants leads to issues related to safety and biocompatibility. Abu Shihada et al.
[293] combined MPL with thin film technology to fabricate a novel and highly customizable 3D microelecrode arrays. This microelectrode array provided unique opportunities for to study neural activity under regular or various pathological conditions (
Fig. 5(c)). In the future, if MPL-fabricated microdevices are expected to be used for biosignal sensing, particularly for implantable biosensing, the main challenge to overcome the biocompatibility of the implanted material
[1].
4.4.2. Biomarker sensing
Numerous biomarkers, such as proteins and ions, can be used for precise disease diagnosis, besides electrophysiology signals. Many studies have used MPL for fabricating diagnostic devices that can accurately detect such biomarkers. Miller et al.
[294] developed the initial microneedle ion-selective-electrode (ISE) device to measure the concentrations of physiologically relevant potassium (
Fig. 5(d)). The transducers for ISEs were tested using porous carbon and porous graphene electrodes. The results revealed that porous carbon K
+ ISEs had a detection range of 10
−5–10
−2 mol∙L
−1 with a near Nernstian slope of 57.9 mV per decade and could rapidly stabilize (∼20 s). Thus, ISEs are an attractive platform for on-body sensors to monitor potassium levels and can potentially be used for point-of-care (POC) diagnosis. Miller et al.
[295] proposed a solution for remote automated diagnostics by combining a live single-needle platform with a multifunctional tube-on-tube electrical array to detect a wide range of relevant biomarkers based on the ISE device’s design concept. Studies have also focused on detecting proteins as biomarkers. Wollhofen et al.
[296] created a 3D platform for protein assay, which comprised two types of acrylate polymers. The platform was tested for two recognition assays, including streptavidin–biotin interaction and antibody recognition of apolipoprotein A1 in high-density lipoprotein (HDL) particles. This platform has the potential to be used in on-chip flow cells for 3D multiplexed recognition assays. Trautmann et al.
[297] demonstrated a novel hybrid approach that combined microneedles with MPL and microfluidic channels using femtosecond laser irradiation and heating (
Fig. 5(e)). This microfluidic system could be used for microfluidic injection and extraction applications for POC diagnosis. Suzuki et al.
[298] fabricated hollow microneedles by mimicking mosquitos for collecting blood.
Researchers have also focused on the detection of bacterial biomarkers. Li et al.
[299] developed an enhanced biosensing platform using MPL and graphene to detect motile bacteria and wine cellar metabolites (
Fig. 5(f)). This device enabled the timely diagnosis of bacterial infections, thereby reducing complications and fatalities. Lao et al.
[300] developed a switchable self-assembly method to fabricate a 3D nanogap plasmonic structure for microfluidic surface-enhanced Raman spectroscopy (SERS) sensing (
Fig. 5(g)). They combined supercritical drying and capillary force-driven self-assembly of micropillars fabricated via MPL. The nanostructures in these microchannels enabled the sensing of anticancer drugs, demonstrating the potential for
in situ monitoring in precision medicine.
5. Conclusions, challenges, and perspectives
5.1. Conclusions
MPL is a powerful 3D laser-based micro/nanomanufacturing method that has garnered attention in several disciplines, including chemistry, materials science, biomedicine, and mechanical engineering. It can be used to mold highly complex structures with high precision, making it a valuable tool for the biomedical field. Compared with traditional biofabrication methods, such as extrusion printing, contactless methods are preferred because these methods can ensure a sterile environment during manufacturing, which is a prerequisite for biomedical applications. MPL offers superior manufacturing precision than similar light-mediated manufacturing methods, such as SLA and DLP, and can be used to fabricate complex structures at the micro/nanoscale.
In summary, the design and manufacture of diagnostic and therapeutic tools on the micro/nanoscale are desirable for precision medicine. In the past decade, two-photon nanofabrication was used in diverse applications in precision medicine, including the delivery of pharmaceuticals and living cells, modeling of microtissue for replacement and drug screening and the production of surgical tools for precision surgery and diagnostic tools, such as biosignal and biomarker sensors (
Table 4)
[301],
[302],
[294],
[303]. Among these scenarios, targeted and transdermal delivery are currently the most promising research area for future development of precision medicine as well as to optimize biomaterial structure and drug release, dose, and delivery methods via animal experiments. This approach cannot be currently applied in clinical applications due to various factors, such as PIs, photopolymers, low throughput in MPP, and optimization for practical application.
5.2. Challenges and perspectives
5.2.1. Materials: Biosafety, mechanical properties, and functionalities
Ensuring the safety of microdevices or precision medicine therapeutics is a primary and fundamental requirement in clinical applications
[304]. The safety of treatments is closely related to the biocompatibility and biodegradability of materials, particularly the biosafety of PIs and photopolymers.
Moreover, water is an essential solvent in living organisms. Water-soluble materials can interact better with the internal environment of living organisms, reducing irritation and adverse reactions to tissues. Additionally, biodegradation and metabolization can be easily achieved using body fluids. Therefore, ensuring the water solubility of PIs is crucial when designing them for use in physiologic environments. In precision medicine, efficient and biocompatible PIs are desirable for two-photon micro/nanomanufacturing to enhance fabrication efficiency. However, water-soluble chromophores and PIs often exhibit poor polymerization efficiency, making the design and synthesis of PIs a long-standing challenge in the field of MPL. Thus, the designing and synthesis of such PIs are being currently studied.
Natural materials, rather than synthetic materials, are more biocompatible with photopolymers. However, natural materials have relatively poor mechanical properties than synthetic materials. Consequently, derivatives of natural materials are being actively synthesized, which can endow the materials with good mechanical properties and biocompatibility. These derivatives are expected to be used in long-term diagnostic applications.
Various functional materials have also been thoroughly investigated, such as stimuli-responsive, superelastic, and heterogeneous materials. By developing diverse functional materials and optimizing their specific properties, such as pH-responsiveness and shrinkage rate, more feasible options can be made available for microscale tool fabrication. Furthermore, hybridized materials are used for two-photon micro/nanofabrication. Specifically, functional materials are added during fabrication to endow the fabricated material with system-specific properties, such as high magnetic drive and optical response.
In conclusion, three aspects of the synthetic design of materials must be considered: biomaterials to ensure biosafety, structural materials to match mechanical properties with targeted needs, and functional materials to provide solutions for precision medicine equipment.
5.2.2. Processing: High efficiency and resolution
Although MPL offers the advantages of high precision and complexity among the existing technologies for micro/nanofabrication, its slow printing speed limits its use for large-scale fabrication of biomedical applications. Currently, process improvements in MPL are being explored, including further improvements in manufacturing efficiency and printing accuracy. Herein, the process limitations that have been addressed in recent years are concisely overviewed, and a technical perspective on how these challenges should be addressed in the future is provided.
The fabrication process is optimized to increase the fabrication speed. Geng et al.
[305] introduced a random-access digital micromirror device scanner in MPL to achieve a high fabrication speed (22.7 kHz), without compromising on the resolution and demonstrated a strategy to perform large-scale nanomolding and construct complex structures. Saha et al.
[306] adopted a similar strategy and achieved 2–3 orders of magnitude increase in fabrication rate without compromising the sub-micrometer resolution compared with the serial MPL system. The volumetric processing rate was also > 20 mm
3∙h
−1. Ouyang et al.
[307] developed a digital holography based TPL platform that realized parallel printing with up to 2000 individually programmable laser foci to fabricate complex 3D structures with a 90 nm resolution.
In addition to improving the efficiency of MPL processing from an optical path, automated quality control and printability optimization are also gradually being applied. Lee et al.
[308] used machine learning models to accelerate the process of identifying optimal light dosage parameters and automate the detection of part quality. They proposed a process monitoring and control using videos on MPL of different parts to classify cured processes from uncured, damaged, and illuminated processes. The model achieved a classification accuracy of 95.1%. Pingali and Saha
[309] applied machine learning-based surrogate models for predicting projection TPL for face projection, which helped to select photoresist and printing parameters quickly and intelligently. Jia et al.
[310] proposed a hybrid physics-guided, data-driven modeling framework for predicting and improving the geometric accuracy in MPL using a generalizable approach in which the average prediction errors for radius and height were 5.23% and 4.66%, respectively. Furthermore, the proposed compensation strategy reduced the geometric errors from 22.19% to 3.21% for radius and from 12.18% to 4.96% for height.
Many studies have focused on improving the printing resolution. Voxel size determines the resolution of MPL because it is related to the polymerization properties as well as the photon density distribution and exposure at the laser focus. Based on these findings, Wang et al.
[311] fabricated suspended nanowires with a feature size of < 10 nm using IP-Dip photoresist under subthreshold exposure conditions. To overcome the challenge of laser power attenuation caused by height direction aberration and laser absorption, Tan et al.
[312] performed laser power compensation, which improved the accuracy of MPL. The complexity of printing was further enhanced by integrating the grayscale control
[313],
[314],
[315]. Existing investigations are focused on optimizing the printing efficiency and resolution of MPL, which are also optimized and weighed using many micro and nanofabrication methods.
For modeling of microtissues and other heterogeneous microstructures, multimaterial micro/nanoprinting must be used. Mayer et al.
[316] introduced a microfluidic system into the MPL apparatus for multimaterial 3D micro/nanomanufacturing. However, these improvements must be further validated for biomedical applications. In conclusion, the most significant challenge associated with MPL in the future will be to overcome its low throughput. To address this challenge, a potential solution is to combine DLP with MPL, which would require further integration of the DLP and MPL setup.
5.2.3. Applications: Stability and cost
Most multiphoton molded nanodevices for precision medicine are still in the pre-exploration stage from an application standpoint. Many of these devices have only been validated in vitro or in vivo for their corresponding functions. Of the four scenarios of drug delivery, disease modeling, surgery, and diagnostics, drug delivery systems are promising; however, they necessitate a more balanced approach to material and structural design. Despite their varied applications, functional materials are typically used as drug-release switches, and biomaterials must consider biodegradation characteristics for use in MPL; however, these characteristics may not be necessarily considered for tissue engineering scaffolds because they require strict and long-term biocompatibility. Moreover, drug delivery systems do not require the same level of precision as a surgical micromanipulation tool. Microtissue modeling, surgical tools, and diagnosis tools require materials with more functionality, biocompatibility, and structural design. Therefore, it is necessary to further investigate material and structural design using two-photon polymerization. The main challenges associated with the future application of multiphoton micro/nanofabrication are stability and cost. Targeted delivery devices are the most advanced in terms of application; however, due to the scarcity of animal experiments, their further development is limited. For the large-scale application of multiphoton micro/nanofabrication in a clinical setting and the optimization of such devices, extensive animal experimentation will be required. Traditional multiphoton manufacturing process have a low throughput, and animal models are expensive, which make the process costly and time-consuming. At present, many of the two-photon molded micro/nanodevices used in precision medicine are conceptual models, and their functional stability in future human models needs further verification.
Part of the cost for MPL devices result from the femtosecond laser source, which somewhat limits their upscaling. In recent years, many researchers have been working on some new mechanisms of photopolymerized micro/nano-3D printing to replace conventional MPL. Sanders et al.
[317] used a triple-state fusion upconversion mechanism for 3D printing. Notably, this technique required the use of only 4 mW of ground continuous wave excitation, thereby reducing equipment costs. Hahn et al.
[318] introduced two-step absorption in benzil, thereby replacing TPA as the fundamental photoinitiation mechanism in 3D laser nanoprinting, achieving submicron accuracy while reducing the cost of MPL. However, the printing speed was still very slow. Based on a previous study, Hahn et al.
[319] presented an approach known as light-sheet 3D laser microprinting, which combined image projection with a logical AND-type optical nonlinearity based on two-color two-step absorption. As a result, a peak printing rate of 7 × 10
6 voxels·s
−1 was achieved at a voxel volume 0.55 μm
2. To some extent, these studies have facilitated the reduction in cost and popularization of laser micro/nano-3D printing devices. However, due to stringent material requirements of biomedical applications in terms of optical properties, their application in the biomedical field remains a challenge at present. In the future, MPA and device fabrication can be facilitated using continuous laser technology, the cost of MPL application in the biomedical field can be substantially reduced.
All mentioned findings considerably expand our hypotheses on two-photon micro/nanomanufacturing for biomedical applications. Along with further exploration of material properties, structural design, and process improvement on the micro/nanoscale, many application scenarios for biomedical applications, particularly precision medicine, will be gradually realized.
CRediT authorship contribution statement
Jiarui Hu: Writing – original draft. An Ren: Data curation, Conceptualization. Weikang Lv: Data curation, Conceptualization. Abdellah Aazmi: Data curation, Conceptualization. Changwei Qin: Data curation, Conceptualization. Xinyi Liang: Data curation, Conceptualization. Xiaobin Xu: Writing – review & editing, Conceptualization. Mengfei Yu: Writing – review & editing, Conceptualization. Qi Li: Writing – review & editing, Conceptualization. Huayong Yang: Supervision, Resources. Liang Ma: Writing – review & editing, Supervision, Resources, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors would like to thank the funding from the National Natural Science Foundation of China (52275294) and the National Key Research and Development Program of China (2018YFA0703000).
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