Contents
2 Application of enzyme⁃driven micro/nanomotors in disease detection and diagnosis
2.2 Molecular marker detection
3 Applications of enzyme⁃driven micro/nanomotors in disease treatment
3.2 Treatment of other diseases
4 Conclusions and outlook
1 Introduction
Inspired by the fact that natural biomolecular motors such as myosin and kinesin can convert chemical energy into mechanical energy to achieve various complex biological functions, many scientific studies have focused on the field of synthetic intelligent molecular machines and micro-and nano-motors. In recent years, with the rapid development of micro-nano technology, the field of micro-nano motor has made a breakthrough. Micro-nano motors can effectively convert various external energies (such as light, ultrasound, magnetic field, etc.) and chemical fuels into driving force, and then generate autonomous motor behavior
[1,2][3,4][5⇓~7][8⇓⇓~11]. It breaks the constraints of irregular Brownian motion and low Reynolds number on the micro-nano scale, and can cross the biological barrier to achieve rapid movement in the liquid environment through autonomous navigation
[12]. Just as macro-scale intelligent robots can perform tasks autonomously, micro-nano motors can also perform various tasks in complex environments at micro-nano scale
[13]. Due to their unique size, structure, physical and chemical properties, and diverse propulsion mechanisms, micro-and nano-motors have been widely used in many research fields
[14⇓~16]. In the field of biomedicine, micro-and nano-motors have been proved to have great potential for practical applications, ranging from targeted drug delivery, disease diagnosis and treatment, to cell sensing and imaging
[17]. Targeted drug delivery using micro-nano motors can avoid the biodegradation or excretion of therapeutic drugs, reduce the loss of drugs in blood circulation, and effectively improve drug permeability, which is more conducive to crossing various biological barriers
[18,19]. In addition, due to the ability of autonomous navigation of micro-nano motors, therapeutic drugs can reach the target directionally, which significantly improves the efficiency of drug delivery. Similarly, the cellular uptake ability of micro-nano motors makes them have certain advantages in in vivo imaging, biosensing and so on. However, there is still a long way to go to realize the practical application of micro-nano motors in organisms. This is mainly due to the fact that in vivo experiments must take into account the degradability and toxicity of micro-and nano-motors to organisms. At present, most of the micro-and nano-motors are made of materials with poor biocompatibility and non-degradability, which leads to serious rejection and lack of necessary safety when used in human body, thus limiting the practical application of micro-and nano-motors in the field of biomedicine.
To solve this problem, scientists have turned their attention to biological enzymes. Enzymes are biomolecular catalysts that accelerate chemical reactions and induce most biological movements at the cellular level. Biological enzymes are ubiquitous in organisms and are easily degraded. Therefore, micro-nano motors driven by biological enzymes have excellent biocompatibility and can reduce toxic and side effects in medical applications. They have more application prospects than traditional micro-nano motors driven by chemical fuels. At present, catalase, urease and glucose oxidase have been widely used in the construction of micro-nano motors, and great progress has been made in the application of disease treatment. Due to the unique properties of biological enzymes, enzyme-driven micro-and nano-motors also have universal commonalities. First of all, biological enzymes have strict selectivity and specificity for the substrates they act on, which makes the micro-nano motors driven by biological enzymes also selective for chemical fuels
[20]. Secondly, the biological enzymes on the enzyme-driven micro-nano motor are connected through enzyme immobilization strategies, including physical adsorption (Van der Waals force, ionic interaction, hydrogen bond and hydrophobic interaction), covalent binding of chemical bonds (such as EDC NHS, EDC crosslinking, etc.), which is one of the most significant features of enzyme-driven micro-nano motor
[21]. At the same time, because biological enzymes are produced by organisms themselves and can be naturally degraded after action, they have good biocompatibility, and micro-and nano-motors driven by biological enzymes also generally have good biocompatibility. In addition, due to the chemotactic behavior of the enzyme, the micro-nano motor can respond to the concentration gradient of the external chemical fuel, thereby controlling the movement of the micro-nano motor
[22,23]. These characteristics distinguish enzyme-driven micro-nano motors from other micro-nano motors and show superior performance. Although there have been reviews on the biological applications of micro-and nano-motors, there is no timely, comprehensive and focused review on the biomedical applications of enzyme-driven micro-and nano-motors
[24,25]. Therefore, this paper aims to focus on the applications of different enzyme-driven micro-and nano-motors in the biomedical field in recent years, including disease detection and diagnosis, drug delivery, and cancer therapy. Finally, the main challenges and future opportunities of enzyme-based micro-and nano-motors in practical biomedical applications are summarized and prospected.
2 Application of enzyme-driven micro-and nano-motors in disease detection and diagnosis
Medical imaging technology is widely used to obtain the anatomical structure and physiological data of patients in clinic, which provides strong support for disease detection. Enzyme-driven micro-nano motors have unique physical and chemical properties, good biocompatibility and autonomous movement, and can be used as imaging agents, which is expected to improve the accuracy of medical imaging technology for specific parts. At the same time, micro-and nano-motors can capture and separate biomolecules by functionalizing receptors such as DNA or RNA, which makes micro-and nano-motors have unique advantages in detecting biomolecules and are expected to be used in biomarker detection of cancer and neurological diseases.
2.1 Imaging developer
High-sensitivity analysis and imaging tools have accelerated the rapid development of medicine today. Among them, medical imaging technology is the core of accurate diagnosis, which is essential for precise surgery. It is of great significance to expand the application of micro-nano motors in the field of imaging. At present, there are many studies on the application of micro-and nano-motors in the field of in vivo imaging and tracking, for example, radiolabeled micro-and nano-motors can be tracked by positron emission tomography (PET), and gold-coated magnetic nanoparticles can be tracked by photoacoustic imaging and magnetic resonance imaging (MRI)
[26,27][28][29,30]. However, the biocompatibility of imaging agents is still not high, which limits the in vivo application of micro-nano motors to a certain extent. In order to solve this problem, enzyme-driven micro-nano motors have become an important choice for in vivo developers. Ma et al. Prepared a liquid metal nanomotor (LMs) based on Ga In Sn alloy driven by urease
[31]. The motor can exhibit good imaging characteristics through ultrasound (US) and photoacoustic (PA) techniques, thereby successfully imaging and tracking the movement of the micro-nano motor in the bladder of living mice (Fig. 1). This work provides a new idea for the future application of micro-nano motors in vivo imaging. Similarly, Mattrey et al. Designed a catalase-coated micro-nano motor and applied it to hydrogen peroxide detection and imaging studies in a rat abscess model
[32]. In this work, the micro-nano motor catalyzes the excessive hydrogen peroxide released by neutrophils at the abscess site to produce microbubbles that can be used for ultrasound imaging, which efficiently combines ultrasound sensing with the movement of the micro-nano motor to achieve accurate detection of inflammation in vivo, and has broad application prospects.
2.2 Detection of Biomolecular Markers
The effectiveness of disease treatment is closely related to the accuracy of diagnosis. However, in the field of cancer diagnosis and treatment, most solid tumors are not easily detected in early screening. In clinical practice, conventional cancer diagnosis and treatment techniques mainly include Bioanalysis of body fluids, medical imaging and tissue biopsy, but these methods often lack sensitivity and specificity
[33⇓~35]. Cancer can be effectively diagnosed by detecting tumor-related biomarkers, for example, the expression level of alpha-fetoprotein (AFP) is an important biomarker for the diagnosis of primary liver cancer, and prostate specific antigen (PSA) is regarded as an important indicator for the diagnosis of prostate cancer
[36][37]. The detection of related biomarkers can reflect the existence and growth of tumors or inflammation to a certain extent, which has important guiding significance for the early detection of diseases and the formulation of treatment plans. Therefore, this part will discuss the representative application of micro-nano motors in the detection of disease markers.
2.2.1 Detection of DNA molecules
Because the micro-nano motor has the characteristic of active motion, the speed change of the interaction between the micro-nano motor and the detection target can be used as the signal of the detection target. Zhang et al. Designed a catalase-modified DNA assembly that mimics medusae and inner fimbria muscle fibers to construct a medusa-like motor (Fig. 2A)
[38]. The motor relies on catalase to catalyze the process of H
2O
2 to produce oxygen to generate driving force, and changes the rate of movement through DNA molecular hybridization to achieve biosensing. The DNA detection method based on the medusa-like motor has the advantages of simplicity, cheapness, rapidity and high sensitivity, and has broad prospects in the detection of cancer-related biomarkers. In order to further improve the detection efficiency, Fu et al. Fabricated an efficient catalase-driven micro-nano motor by assembling multiple layers of catalase on the inner surface of polymer (3,4-ethylenedioxythiophene and sodium 4-styrenesulfonate)/gold microtubules (Fig. 2B)
[39]. In the presence of the target DNA, the sensor unit hybridizes with the target DNA, releasing multiple layers of DNA and catalase, resulting in a reduction in the speed of movement, thus having the ability to quantitatively detect DNA markers with concentrations ranging from 10 nmol·L
-1 to 1.0μmol·L
-1. The micro-and nano-motor device proposed in this work and the cyclic alternating DNA hybridization assembly technology provide a new way to fabricate efficient and multifunctional micro-and nano-motors, which will become a special tool for rapid and simple detection of biomolecules.
Similarly, Simmchen et al. Proposed an innovative catalase self-propelled nanomotor capable of performing autonomous movement, cargo transport, and target recognition (Fig. 2C)
[40]. The nanomotor is based on a mesoporous moving particle, single-stranded DNA is functionalized by attaching to one side of the particle, and when a specific oligonucleotide sequence is present in the medium, the moving motor captures and transports cargo particles functionalized with non-complementary single-stranded DNA molecules. This self-assembled hybrid nanomotor has the ability to rapidly separate and detect specific oligonucleotide chains, and will play a role in the identification and detection of cancer-related markers. In order to further improve the sensitivity of detection, Xie et al. Proposed a microprobe based on a micro-nano motor (Fig. 2D), which uses a catalase-driven poly (3,4-ethylenedioxythiophene)/Au microtubule micro-nano motor as a catalyst, relying on target-induced catalase release.The amount of biological enzyme assembled on the microtubule is reduced through DNA strand displacement hybridization, so that the purpose of slowing down the movement of the microprobe is achieved, the movement speed of the probe is negatively correlated with the target concentration, and the detection of DNA in a 0.5~10μmol·L
-1 range is realized without any washing and separation steps
[41]. The probe can be prepared in batches, has good reproducibility and stability, and the movement speed of the probe can be conveniently observed by an optical microscope.
2.2.2 Detection of protein molecules
Procalcitonin (PCT) is a protein whose plasma levels rise during severe bacterial, fungal, and parasitic infections, as well as sepsis and multiple organ failure, and reflects the activity of the systemic inflammatory response
[42][43]. The determination of serum procalcitonin has reference significance for the early diagnosis of sepsis, and can also be used to evaluate the efficacy of antibiotics in patients with sepsis. Russell et al. Achieved rapid detection of PCT by color change (Fig. 3B) through the combination of catalase-driven micro-nano motor and competitive immunoassay (Fig. 3A)
[44]. The micro-nano motor is connected with iron oxide particles, and the concentration of PCT can directly affect the amount of biological enzyme combined on the iron oxide particles, thereby indirectly affecting the movement behavior of the micro-nano motor driven by the biological enzyme and further causing the color change of the iron oxide particles. When the particle is dropped on the filter paper, it produces a colored spot. When the substrate of the enzyme is added, the micro-nano motor will push forward by itself. This process will produce a larger and lighter spot in a few seconds. This color change can be reflected by the real-time observation of the relative pixel intensity of the color point by the camera technology connected to the computer. This technique is beneficial to the early monitoring and diagnosis of septicemia, and provides a new method and approach for the rehabilitation of septicemia patients.
2.2.3 Detection of tumor cells
Detachment of tumor cells from the primary tumor mass usually signifies progression of the cancer. Therefore, detection of circulating tumor cell (CTC) content in the physiological environment is of great value in monitoring tumor progression, therapeutic efficacy of treatment regimens, cancer recurrence, and patient survival
[45]. Most of the existing detection techniques first enrich CTCs from blood and then confirm CTCs in purified samples
[46]. However, rapid and sensitive detection of circulating tumor cells remains a technical challenge due to the low abundance and fragility of CTCs in blood. Therefore, how to design a simple and reliable capture method to improve the recovery rate and detection accuracy has become the focus of researchers. The enzyme-driven self-propelled micro-nano motor provides a new way to solve this problem. Li et al. Prepared a Janus micro-nano motor for circulating tumor cell motion capture and sensing detection (Fig. 4)
[47]. Catalase is connected to one side of the Janus motor to catalyze the decomposition of hydrogen peroxide as a power source to realize random, spiral, rotary, linear and other motion behaviors; After capturing tumor cells, the micro-nano motor changed from blue to green, and its detection limit was 25 cells/mL in 1 min. In addition, even the presence of other tumor cells did not interfere with the detection of HepG2 by micro-nano motors, and the recovery rate of HepG2 cells in blood samples was more than 95%. This work demonstrates the ability of micro-nano motor to capture tumor cells, and provides a feasible strategy for real-time, rapid and sensitive detection of tumor cells and related markers in clinical medicine.
3 Application of enzyme-driven micro-and nano-motors in disease therapy
3.1 Treatment of cancer
Cancer treatment is a worldwide problem. High mortality, high recurrence rate and low treatment effectiveness make cancer the second leading cause of death in the world, which poses a serious threat to human health
[48]. At present, the traditional clinical treatment of cancer includes tumor resection surgery, radiotherapy and chemotherapy, but because the cells located in the hypoxic area of solid tumors are resistant to radiotherapy and chemotherapy, these methods are often ineffective, and have potential side effects and high cost. The main limitation in cancer treatment is how to achieve targeted drug delivery. It is difficult for anticancer drugs to penetrate tissues and lesions to reach the target site in the rapid flow of blood only by passive administration of body fluid circulation. At the same time, drugs will encounter obstacles such as mononuclear phagocytic system, renal clearance and purification, and drug competition at the tumor site
[49]. In addition, even if the drug reaches the tumor cell site, it will encounter a series of obstacles, including the heterogeneity of malignant tumors, the tortuous and irregular tumor vasculature, and the dense extracellular matrix, which will hinder the blood extravasation of drugs and the targeted transport of drugs, and only 0.7% of the drugs can really act on the effective site of tumor cells
[50]. Therefore, how to enhance the effective delivery of drugs to the tumor site has become the key to improve the efficacy of cancer treatment.
Self-propelled micro-nano motor provides a new way to solve the above problems. Micro-nano motors can provide continuous driving force for anticancer drug carriers, and can be functionalized for specific applications, so that they can cross biological barriers and reach target cells by autonomous navigation, which can greatly improve the efficiency of anticancer drug delivery. Aiming at the problem that the traditional micro-nano motor is made of non-degradable and toxic materials, the enzyme-driven micro-nano motor is driven by fuel which exists in the body and has good biocompatibility.It will not cause harm to human body, not only has the excellent characteristics of high surface area and activity of micro-nano materials, but also shows the remarkable characteristics of autonomous movement ability
[51]. Enzyme-driven micro-and nano-motors have good biocompatibility and multi-response functions, which make them have broad prospects in the field of cancer therapy. Therefore, the following part will discuss the application of micro-nano motors driven by different biological enzymes in the field of cancer therapy.
3.1.1 Application of catalase-driven micro-nano motor in tumor therapy
Hydrogen peroxide is widely involved in a variety of physiological reactions, from cytokines, insulin to AP-1 signaling to redox regulation. Hydrogen peroxide is produced by various intracellular and extracellular processes and exists in almost all aerobic metabolism
[52]. Current studies have shown that cancer cells generate oxidative stress by producing high concentrations of hydrogen peroxide and exhibiting acidic pH levels, which provide an energy source for micro-nano motors using H
2O
2 or H
+[53,54]. The inner layer of traditional micro-nano motors is usually composed of precious metals or synthetic polymers, which use catalysts such as platinum to catalyze the decomposition of H
2O
2 and produce bubbles to propel motion. Using biocompatible material catalase instead of metal catalyst to decompose hydrogen peroxide, this micro-nano motor can achieve precise targeted delivery of anticancer drugs in situ without toxic effects in vivo, showing great potential in the field of cancer treatment.
In response to the above problems, researchers at home and abroad have carried out a lot of exploration work. For example, Sanchez et al. Of the Spanish Institute of Bioengineering modified the inside of the coiled microtubules of catalase by 3-mercaptopropionic acid ligation, and prepared a new type of biocatalytic catalase-driven micro-nano motor
[55]. The nanomotor can move at a speed of 10 body length/second in a 1.5 wt%H
2O
2, which significantly enhances the motion efficiency of the nanomotor. However, the motion of the motor in this system is not flexible, and it is difficult to achieve precise control. Wilson et al. Fabricated a catalase-driven asymmetric micro-nano motor using microfluidics
[56]. They control the speed of the micro-nano motor by adjusting the surface roughness of the micro-groove, which makes the rotation and translation speed of the micro-nano motor flexible and controllable.
The above research lays a foundation for further expanding biological applications such as catalase. However, the biological barrier in vivo is also an important limitation that hinders the application of micro-and nano-motors in vivo. Since then, researchers have turned their attention to the application of enzymatic micro-and nano-motors into organisms. The first problem to be faced is how to overcome a series of obstacles in organisms, including systemic clearance, tumor accumulation, cell uptake and so on.
The size of nanoparticles is one of the important factors that determine their passage through biological barriers
[57]. Nanoparticles with a particle size of less than 200 nm are more conducive to overcoming the accumulation of cell barriers around tumors by enhancing permeability and retention effects, so Wilson et al. Prepared an ultra-small oral cell polymer nanomotor driven by catalase with a size of about 150 nm by extrusion method (Fig. 5).The motor penetrates more efficiently through a confluent monolayer of pulmonary artery endothelial cells, demonstrating enhanced penetration into the vasculature model and uptake into HeLa cells, suggesting that small-sized nanoparticles are better suited to penetrate tumor vessels
[58][59]. This micro-nano motor is expected to be successfully applied to biomedical applications, such as protein delivery, and has great potential in the field of tumor cancer therapy. Subsequently, more studies have focused on small catalase-driven micro-and nano-motors, such as the preparation of catalase-driven Janus mesoporous silica nanomotors less than 100 nm by Ma Xing
[9]. The nanomotor exhibits a 150% improvement in diffusion coefficient over the intrinsic Brownian motion at low hydrogen peroxide concentrations (below 3 wt%). Its small size and good drug loading make it expected to be used in tumor targeted therapy in vitro and in vivo in the future. Li et al. Used a complex chemical synthesis method to prepare a single-molecule nanomotor of less than 100 nm based on a bottle-brush polymer, which is driven by catalase, has tadpole-like asymmetry, can overcome Brownian motion, and at ultra-low hydrogen peroxide levels of 2mmol·L
-1(0.006%).The nanomotor showed strong directional propulsion ability in the viscous tumor microenvironment gel model, and showed excellent stability and good blood biocompatibility in the serum-containing cell culture medium, which could effectively overcome the tissue penetration barrier
[60]. These works have laid a lot of groundwork for catalase-driven micro-and nano-motors to enter biomedical applications in vivo.
Although the above work successfully achieved the control of micro-nano motor movement, enabling it to traverse biological barriers, its ability to target tumor drug delivery was not proven, so the development of biodegradable micro-nano motors that transport and release drugs in a controlled manner remains a challenging problem. He Qiang et al. Prepared a bovine serum protein/poly-L-lysine hydrochloride multilayer microtubule nanomotor (Fig. 5), which is biodegradable and can achieve targeted drug delivery. The free diffusion of hydrogen peroxide fuel from tumor cells in situ into catalase containing gelatin hydrogel leads to the catalytic production of water and oxygen bubbles.Pushing the micro-nano motor to move forward, the low melting point gelatin hydrogel melts under the irradiation of infrared light (NIR), and the encapsulated drug is rapidly released into the surrounding medium
[61]. After this series of processes, the protein-based micro-nano motor can be degraded by enzymatic reaction without causing adverse effects on the body. The use of proteins as building blocks to develop nanomotors with good biocompatibility provides a new idea for practical tumor-targeted therapy. In addition, He Qiang et al. also fabricated a catalase-driven multilayer Janus micro-nano motor for effective drug delivery (Fig. 5), which deposited a thin gold layer on one side of the polyelectrolyte layer by layer by template-assisted method, and then immobilized catalase on the other side by chemical means
[62]. This Janus micro-nano motor can move at a speed of 232 μm/s at 2% hydrogen peroxide concentration at physiological temperature. It can also encapsulate the chemotherapeutic anticancer drug doxorubicin, which is navigated by an external magnetic field to target the cell layer, and then trigger the drug release by NIR light activation, achieving efficient targeted delivery, controlled encapsulation, and remote controlled release of the anticancer drug. This work lays an important foundation for the development of multifunctional polymer micro-and nano-motors for biomedical applications.
In view of the complex biochemical environment of tumor cells, Kong et al. Developed a biocatalytic micro-nano motor assembled by succinylated β-lactoglobulin and catalase (Fig. 5) to enhance intracellular drug release
[63]. The permeability of the peroylated beta lactoglobulin is enhanced at a neutral pH value, and the H
2O
2 fuel is regulated to enter the micro-nano motor, so that the micro-nano motor is driven. At a mild acidic pH value, the succinylated beta lactoglobulin undergoes a reversible gelation process to prevent the H
2O
2 fuel from entering the micro-nano motor where the catalase is located,This work proves that micro-nano motors can use hydrogen peroxide in situ of tumor cells to respond to complex biochemical environmental stimuli according to different pH in the process of tumor diagnosis and treatment, so as to achieve precise targeted delivery of anticancer drug carriers.
Catalase has high catalytic efficiency, which is conducive to driving micro-and nano-motors to achieve rapid movement, but the use of hydrogen peroxide toxic fuel is still an obvious bottleneck in its application, and H2O2 fuel will limit the biomedical application of catalase-driven micro-and nano-motors. Although researchers have developed many enzyme-driven micro-and nano-motors that can move at low concentrations of hydrogen peroxide, the H2O2 concentrations currently used for research are still toxic to living cells, so it is necessary to continue to expand different kinds of enzyme-driven micro-and nano-motors.
3.1.2 Application of urease-driven micro-nano motor in tumor therapy
Bladder cancer is the ninth most common cancer in the world, and the current clinical treatment includes tumor resection, intravesical chemotherapy and so on
[64,65]. Due to the short residence time of drugs in the bladder, the low permeability of urothelium and the easy degradation of drugs in the bladder, as well as the passive diffusion of drug molecules in the bladder and the rapid clearance of periodic urination,Making conventional drug therapy delivery less efficient, there is a need for in situ, fuel free, bladder tissue penetrating, high drug release methods. Based on this, urease-driven micro-nano motors have become the focus of scientists.
Urease is one of the first enzymes discovered by scientists and is widely distributed in nature. Urease can decompose urea into NH3 and CO2 without bubbles. The content of urea in the bladder can reach 300 mmol·L-1, which can be used to design urease-driven micro-nano motors to achieve self-propelled mechanical force by using the concentration gradient generated by urea decomposition, so as to achieve the treatment mode of in-situ treatment in the bladder without external fuel.
In order to solve the above problems, many research groups at home and abroad have carried out a large number of studies on the preparation and performance of urease-driven motors. Ma et al. First prepared a tubular motor modified with urease, and they studied the effects of urease position and tube length on motor movement
[66]. Further, they synthesized a urease-driven Janus hollow mesoporous silica nanomotor, which can move at high speed for a long time under physiological concentration of urea
[67]. In addition, they started and stopped the movement of the motor by adding inhibitors (Ag
+ or Hg
2+) or dithiothreitol, and successfully controlled the movement direction of the motor by using a magnetic field by depositing a layer of Fe on the other side of the nanomotor. This lays an important foundation for further application of targeted therapy in vivo.
In vivo, the mucus barrier provides an important defense mechanism by limiting the penetration of pathogens, which is also a major obstacle to targeted drug delivery in cancer therapy
[68]. In order to further enhance the diffusion and permeability of micro-nano motors in tumor cells, Walker and others imitated the strategy of Helicobacter pylori to overcome the mucus barrier by producing urease, immobilized urease on the surface of magnetic micro-propellers, and in the presence of substrate urea.Urease catalytic products can increase the pH value of the environment and change the fluid properties of mucus, thereby improving the permeability of the micro-nano motor. Although the micro-nano motor is not driven by enzymes, it can effectively penetrate the mucus barrier through the functionalization of urease
[69]. To overcome the intratumoral barriers to drug delivery, Chen et al. Fabricated a self-driven Janus nanomotor based on electrospun fiber segments for intratumoral drug delivery, which was linked to urease on one side and hyaluronic acid on the other
[70]. As the urea concentration increases from the physiological concentration of 5mmol·L
-1 to 10 mmol·L
-1, the diffusivity value of this motor increases from 4.73μm
2/s to 6.24μm
2/s. By promoting local accumulation, deep tumor penetration, tumor cell capture and intracellular release of chemotherapeutic drugs, the anti-tumor efficacy of micro-nano motors can be significantly improved.
At the same time, Sanchez et al. Have successfully applied urease-driven motors in the biomedical field, especially in the fields of drug delivery for cancer treatment, targeted fragmentation, and in vivo tumor imaging
[71]. They synthesized a mesoporous silica micro-nanomotor propelled by urease for doxorubicin anticancer drug loading, release, and efficient delivery to cells (Fig. 6). Due to the large surface area of mesoporous silica, the micro-nano motor has high loading capacity, the movement initiated by urease can enhance the drug release process of the motor, and the synergistic effect of the drug and ammonia produced by urea decomposition can be more conducive to killing HeLa cells. In the presence of urea, the urease-driven micro-nano motor released four times more Dox than passive diffusion transport after 6 H. This work paves the way for the next step of in vivo drug release and cancer therapy. However, this method is still difficult to achieve drug delivery in the internal tissues of tumors. To this end, Sanchez et al. Also prepared a urease-driven nanomotor based on mesoporous silica nanoparticles for tumor tissue disruption (Fig. 6)
[72]. The outer surface of the motor contains polyethylene glycol and an anti-FGFR3 antibody, which targets bladder cancer cells in the form of 3D spheres, and the interaction between FGFR3 and the antibody inhibits the signaling pathway of fibroblast growth factor, thereby blocking the proliferation of bladder cancer cells and eventually leading to cell death. In addition, in the conventional administration process, the drug release is not controlled, which often leads to drug leakage and serious toxic side effects. To this end, they prepared a urease-propelled mesoporous silica motor to achieve precise control of drug delivery
[73]. The outer surface of the nanomotor was grafted with benzimidazole groups and sealed with urease modified with benzimidazole and cyclodextrin. The urease-driven micro-nano motor can respond to pH, and the drug can be released only under acidic conditions due to the blocking effect of the cyclodextrin modified urease. The motor fulfills some special requirements of an ideal drug delivery vehicle, improves its self-propulsion ability and its ability to sense the environment, and can deliver the payload on demand according to the predetermined stimuli of the environment, such as acidity and alkalinity.
Although micro-nano motors can precisely deliver drugs in the bladder and directly expose them to the diseased tissues to improve the therapeutic effect and minimize systemic side effects, the drugs injected into the bladder will be rapidly excreted due to regular urination.Therefore, frequent injection of drugs is often required, which is prone to infection and inflammation. The delivered drugs should adhere to the bladder mucosa quickly and remain in place during urination without interfering with the normal function of the bladder. In order to solve the above problems, Choi et al. Reported an enzyme-powered polymer micro-nano motor driven by urease, which can penetrate deeply into the mucosa of the bladder wall and stay in the bladder for a long time.The micro-nano motor is made of polydopamine hollow nanoparticles and shows high adhesion to tissues through covalent and non-covalent interactions with target tissues (Fig. 7)
[74]. This urease-driven polydopamine micro-nano motor can be a viable drug delivery vehicle for the treatment of various bladder diseases, which further improves the feasibility of urease-driven micro-nano motor in the treatment of bladder cancer.
Although the above work provides a new idea for the treatment of bladder cancer, the materials used in these motors are difficult to degrade in vivo, which can easily lead to deposition in vivo, and the urease motors with degradable materials need to be further studied. Based on this, proteins and other materials with good biocompatibility and easy to be degraded by biological enzymes in vivo have attracted the attention of scientists at home and abroad. For example, Sugai et al. Synthesized a urease-driven tubular micro-nano motor, which is connected to serum albumin on the inside
[75]. The micro-nano motor can be completely degraded when hydrolyzed in an acidic environment at pH = 3.0 for 90 min, or in a protease mixture at 37 ℃ for 120 min. This research work has achieved biodegradation through material innovation, further improving the biocompatibility of micro-nano motors in cancer therapy.
In 2021, Sanchez et al., in order to further promote the clinical application of biological enzyme-driven micro-nano motors, studied the cluster behavior of urease-driven nano motors, and tracked their behavior in vivo and in vitro through PET technology (Fig. 8)
[76]. They injected nanomotors intravenously into the bladder using a 300 mmol·L
-1 of urea aqueous solution as a carrier, and found that nanomotors could move in groups and show uniform distribution of activity and stronger fluid mixing.This intravenous route of administration has been shown to maximize drug concentration in the target organ, improve efficacy and reduce side effects, and the micro-nano motor is better suited for targeted and anticancer drug delivery.
The above work is the application of urease-driven micro-nano motors in the treatment of bladder cancer. However, for tumors in other parts of the body, urease is difficult to achieve in situ treatment, so other types of biological enzyme motors still need more exploration.
3.1.3 Application of Glucose Oxidase Driven Micro/Nano Motor in Tumor Therapy
Tumor cells escape the normal apoptotic program through an abnormal anaerobic glycolytic behavior, known as the Warburg effect, and rapidly carry out tumor metabolism and proliferation, which makes tumor cells very sensitive to intracellular glucose concentration
[77]. Therefore, by consuming glucose, the energy supply of tumor cells can be blocked to achieve the effect of cancer treatment, which is called starvation therapy
[78⇓~80].
Glucose oxidase is a ubiquitous oxidoreductase in organisms, which has good biocompatibility and biodegradability. It catalyzes the process of glucose decomposition and can effectively reduce the level of glucose in tumors.The oxygen consumption of tumor cells is increased, which can be used for hypoxia activation therapy. In addition, the production of gluconic acid enhances the acidity of tumor microenvironment, which can trigger pH-responsive drug release. In view of the excellent catalytic performance and good biocompatibility of glucose oxidase, researchers have developed a series of glucose oxidase-driven micro-nano motors to solve the problems in the field of cancer therapy.
Sanchez et al. First attempted to use glucose oxidase to fabricate self-propelled Janus nanomotors based on hollow mesoporous silica nanoparticles.The biocatalytic reaction of glucose oxidase was used to provide power, and the apparent diffusion coefficient was increased by 38%, which laid the foundation for the subsequent application of glucose oxidase-driven micro-nano motor in vivo
[81]. Then, more and more researchers have studied the micro-nano motor driven by glucose oxidase. For example, St Städler et al. Made a micro-nano motor with Janus structure, which uses polydopamine-coated silica particles as the core, and then uses poly L lysine to graft poly ethylene glycol on one side of the hemisphere.When glucose oxidase and catalase were immobilized on the other side, it was observed that the micro-nano motor showed enhanced diffusion characteristics in a glucose concentration-dependent manner.At the same time, the motor can reduce the content of hydrogen peroxide to a lower level and realize the delivery of drugs, which lays a foundation for the subsequent self-driven drug delivery of enzyme-driven micro-nano motors
[82]. In addition, in order to solve the problem of low assembly efficiency of nanomotors, Wilson et al. Proposed a new nanomotor assembly strategy, which combines biological enzymes into the stoma cell cavity through the morphological changes of polymer vesicles to form a self-assembling enzyme-driven nanomotor
[83]. By encapsulating glucose oxidase and catalase, the nanomotor can use glucose concentration gradient to achieve rapid movement. At the same time, the form of the nanomotor can effectively protect the biological enzyme in the nanomotor from being hydrolyzed by the hydrolase, which greatly enhances the ability of the enzyme-driven micro-nano motor to face complex environments when applied in vivo.
It has been shown that coupling biological enzymes with inorganic entities can greatly enhance the diffusion characteristics of micro-nano motors. St Städler et al. Prepared a micro-nano motor with glucose oxidase and inorganic platinum nanoparticles in series, and coupled the Janus micro-nano motor with inorganic biological entities, and its diffusion characteristics were significantly enhanced by 8 times
[84]. The motility system has great potential in the biomedical field and is expected to be used in targeted delivery of tumor cells. In addition, He Qiang et al. Have also studied the micro-nano motor driven by glucose oxidase. The carbonaceous nanofibers (CNF) they manufactured can be self-propelled in the presence of glucose, and the directivity can be considered in advance according to the surface wettability of the nanofibers
[85]. Hydrophilic CNF showed backward movement dominated by a local glucose gradient, whereas hydrophobic CNF showed forward movement dominated by a local gluconic acid gradient. To further improve the movement speed of the micro-nano motor, inspired by the dynamics of bacterial community, they also prepared a Janus micro-nano motor driven by glucose oxidase, which grafted polymer brushes on one side of Au nanoparticles and attached glucose oxidase on the other side
[86]. It was observed that the micro-nano motor had positive chemotaxis to the glucose gradient, and the speed could reach 120 body length ·s
-1, which greatly improved the movement speed of the micro-nano motor and realized the directional movement of the micro-nano-motor, which was beneficial to the targeted transport in tumor cells.
In targeted drug delivery, drugs modified with biological recognition elements preferentially bind to cells overexpressing specific cellular receptors. This increases the number of interactions with cells, thereby increasing retention time and cellular uptake. If biomolecular interactions can be disrupted as an external stimulus, then the selectivity of these biosensing and drug delivery methods could be further enhanced. For example, biosensor performance could be greatly improved if low-specificity interactions could be interrupted on demand. In drug delivery, drugs that can unbind healthy cells in response to external stimuli can reduce unwanted side effects. Rucinskaite et al. Proposed a strategy based on micro-nano motors to improve the binding between nanoparticles and tumor cells while reducing their interaction with healthy tissues (Fig. 9)
[87].
The nanomotor part is covered with glucose oxidase as a power source and a peptide integrin capable of overexpression, and the interaction of the peptide integrin can be controlled by fine-tuning the concentration of the enzyme substrate glucose.At the same time, the number of Janus nanomotors coated with glucose oxidase bound to cells can be controlled, so the nanomotors can selectively bind to tumor cells.Rather than in combination with healthy tissue where glucose concentrations are higher, this control mechanism facilitates the design of drug and bioimaging nanoprobes,Through the glucose concentration difference between healthy tissue and tumor microenvironment and the selective targeting of biomolecular cues, it can effectively target the delivery of anticancer drugs while minimizing unnecessary side effects on healthy tissue.
In order to further realize the in vivo application of micro-nano motors, target across the blood-brain barrier and into the central nervous system, and achieve efficient drug delivery, Joseph et al. Designed a nano-motor based on natural enzymes and complete polymer vesicles.Glucose oxidase alone or in cooperation with catalase can generate propulsion force, and polymer vesicles can be used as carriers for drug or gene delivery, which is the first time to use chemotaxis to cross biological barriers
[88]. The experimental results show that the number of enzyme-driven nanomotors entering the mouse brain parenchyma is increased by 4 times compared with non-chemotactic polymer vesicles, which provides a new direction for the design of drug delivery systems.It will play an important role in the delivery of tumor-targeted drugs in the brain and central nervous system, and is expected to be successfully applied to the field of in vivo cancer therapy.
3.1.4 Application of enzyme cascade reaction driven micro-nano motor in tumor therapy
Although the above single enzyme-driven micro-and nano-motors have shown great potential in the biomedical field, especially in cancer therapy, there are still many problems for the practical application of enzyme-driven micro-and nano-motors in vivo, such as the challenges of biocompatibility and autonomous targeted navigation and transportation. In order to further solve these problems, researchers connected multiple enzymes to the micro-nano motor at the same time, and prepared a cascade of enzymatic reactions to push the micro-nano motor. The application of different enzymes in micro-nano motors can not only improve the speed of micro-nano motors and realize the efficient movement of micro-nano motors, but also expand the application environment and scope of micro-nano-motors and realize more functions in tumor therapy.
In order to realize the motion control of micro-nano motors, Mano et al. First proposed a method of using multiple enzymes to provide power for micro-nano motors, and they prepared a micro-nano motor by using carbon fibers modified by bilirubin oxidase and glucose oxidase.One end of the fiber was modified with glucose oxidase and redox polymer I, and the other end was modified with bilirubin oxidase and redox polymer II
[89]. The motor is driven by the bioelectrochemical action of the two enzymes, and when in contact with a buffer solution of pH = 7 and a glucose concentration of 10 mmol/L, electrons are transferred from the glucose oxidase side to the bilirubin oxidase side, generating a O
2. The micro-nano motor is propelled by electron flow between the solution and oxygen. When there is an insulator between the two sides, the motor cannot move. This lays a foundation for the subsequent practical application of enzyme-driven micro-nano motors in organisms. Enzymatic cascade reactions are also often used to solve the biocompatibility problem of micro-nano motors. Glucose oxidase can catalyze the oxidation of β D glucose (C
6H
12O
6) to D glucolactone (C
6H
10O
6) and H
2O
2 in the presence of oxygen.Researchers often combine glucose oxidase with catalase. At this time, glucose is first decomposed into hydrogen peroxide by glucose oxidase, and then hydrogen peroxide is decomposed by catalase to generate driving force. In this way, the hydrogen peroxide produced by glucose decomposition will not cause harm to the body. For example, Pantarotto et al. Attached catalase and glucose oxidase to carbon nanotubes to generate and consume hydrogen peroxide in situ from the relatively stable and ubiquitous biomolecule glucose through enzymatic activation, and the synergistic action of the two enzymes produced gaseous molecular oxygen to drive the motor.This provides an attractive alternative to the direct use of hydrogen peroxide fuel, and provides a biocompatible solution for catalase-driven micro-and nano-motors, making it possible for micro-and nano-motors to be used in vivo
[90]. In addition, Abdelmohsen et al. Assembled glucose oxidase and catalase into a bowl-shaped polymer, which also realized the physiological movement of micro-nano motors in glucose solution. Due to the protection of the polymer, the coated enzyme will not be hydrolyzed by external proteases, so it can effectively complete the task of active transport, and has the advantage of good biocompatibility
[91].
After that, researchers turned their attention to the in vivo application of multi-enzyme-driven micro-nano motors. The multi-enzyme cascade reaction mechanism has better biocompatibility, and the multi-enzyme cascade can achieve multi-directional treatment of cancer, showing great advantages in the field of drug delivery and cancer treatment. Based on this, Ma Xing et al. Synthesized a dual-enzyme functionalized nanomotor based on metal-organic framework nanoparticles to enhance synergistic photodynamic therapy and starvation therapy (Fig. 10)
[92]. The nanomotor is capable of converting NIR light into blue light, which is then absorbed by TAPP to produce cytotoxic
1O
2 to achieve efficient photodynamic therapy. The modified glucose oxidase is used to catalyze the decomposition of intracellular glucose, which starves cells and produces hydrogen peroxide, thereby triggering the decomposition of H
2O
2 generated after the decomposition catalyzed by endogenous glucose oxidase to provide propulsion and increase the diffusivity of nanomotors by about 27%, thus increasing the cellular uptake of therapeutic nanomotors. Meanwhile, the generated oxygen molecules not only promote the singlet oxygen production during near-infrared light-triggered PDT, but also promote glucose oxidase-triggered glucose decomposition. Therefore, these two enzymatic cascades form the positive feedback of the whole nanomotor-based therapeutic system, greatly improving the efficacy of synergistic PDT and ST.
3.2 Treatment of other diseases
In view of the special physiological environment of tumors, based on the great potential of autonomous movement of micro-nano motors, researchers have designed a variety of intelligent micro-nano motors using catalase, urease, glucose oxidase, etc., to achieve targeted delivery of anticancer drugs and real-time detection and diagnosis of diseases. In addition to broad application prospects in cancer diagnosis and treatment, enzyme-driven micro-nano motors have great advantages in triglyceride degradation, bacterial infection, gastrointestinal diseases and so on, which will be briefly introduced below.
3.2.1 Degradation of triglyceride
In the latest research results, lipase has also been used as the engine of nanomotors and has successfully achieved effective degradation of triglycerides in vitro, which not only further expands the biocompatible enzyme library that can provide power for micro-nanomotors, but also is expected to achieve potential biomedical applications. Lipase is a biocompatible chemical with broad specificity, good stability and unique structure
[93]. In the field of enzymes, lipases present a unique catalytic mechanism with the presence of a mobile subdomain located above the active site, and depending on the environment surrounding the lipase, the lid flap on the enzyme structure can move to close or open the entrance to the catalytic site, thus hindering or allowing the free diffusion of the lipase substrate
[94]. The structure of lipase can switch between the open active or closed inactive conformation according to the movement of the cover flap, and researchers have developed lipase-driven micro-nano motors by using the unique properties of lipase, which have been applied in biomedical related fields.
Wang et al. First prepared a mesoporous silica nanomotor based on lipase modification, which can move in triglyceride solution for a long time and degrade triglyceride droplets simulating blood lipids (Fig. 11)
[95]. The researchers used lipase and the corresponding water-soluble oil substrate glycerol triacetate as a chemical fuel, and successfully achieved enhanced diffusion for about 40 min. At a low glycerol triacetate concentration of < 10 mmol/L, the diffusion coefficient increased by about 50%, and about 98% of tributyrin droplets were degraded within 50 min. The micro-nano motor system can be used as an efficient cleaner of triglyceride droplets, showing great potential in the treatment of diseases associated with triglyceride accumulation. Later, Zhang et al. Studied the motion behavior of lipase-driven micro-nano motors, and they prepared a lipase-driven micro-nano motor with a dendritic porous structure.The lipase-catalyzed decomposition of acetin into acetic acid and glycerol occurs asymmetrically on the surface of the micro-nano motor, and the concentration gradient of the decomposition products causes fluid flow around the motor, which drives the movement of the micro-nano motor by self-diffusion electrophoresis, but the movement of the nano-motor is not directional
[96]. The above results lay a foundation for further research on the application of lipase-driven micro-nano motors.
With the in-depth study of lipase-driven micro-and nano-motors, how to further improve the diffusion coefficient and motion performance of micro-and nano-motors has become the focus of researchers. Wang et al. Explored the effect of enzyme orientation on the performance of lipase-driven nanomotor by adjusting the immobilization strategy of lipase
[97]. The lipase is immobilized on the mesoporous silica nanomotor through three different interactions, namely ion adsorption, glutaraldehyde-mediated covalent bonding, and hydrophobic interaction, providing different lipase conformations and directions on the mesoporous silica nanomotor,The study shows that hydrophobic binding is the best directional strategy, and the diffusion coefficient increases by 48.4% and 95.4% compared with hydrophilic binding and fuel-free Brownian motion, respectively. The enzyme-directed strategy was shown to be a key factor in regulating and enhancing the diffusion coefficient of lipase-driven nanomotors. This work paves the way for the creation of finely tuned tools for enzyme-driven motors, thereby expanding their potential applications in the biomedical field. Sun et al. Then reported a lipase-driven micro-nano motor, which was made of poly (glycidyl methacrylate)/polystyrene particles and combined with lipase by hydrophobic interaction
[98]. Through the asymmetric distribution of lipase on Janus particles, the motor showed an enhanced diffusion behavior related to the substrate concentration, and its diffusion coefficient increased to 0.37μm
2/s at a substrate concentration of 3 mmol·L
-1, which was 61% higher, and the asymmetric structure of the motor effectively improved the enzymatic reaction and diffusion behavior.
The research of lipase-driven micro-nano motor is in the ascendant stage, and its motion mechanism and influencing factors need to be further explored, but it can be predicted that lipase will be widely used in the subsequent design and development of micro-nano motors.
3.2.2 Treatment of bacterial infection
Bacterial infections are one of the most common causes of morbidity and mortality in the world. In addition, bacterial infections can lead to cancer, which is estimated to account for 20% of all human tumors
[99]. Due to the overuse of antibiotics, there are more and more cases of antibiotic-resistant bacterial infections, and traditional treatment regimens are losing their efficacy. The ability of micro-nano motors to target drug delivery and precise release has brought new light to the treatment of this disease. In order to solve the above problems, Ma Xing et al. Proposed a liquid metal nanomotor driven by urease, which showed a significant role in antibacterial action
[31]. The main body of the nanomotor is composed of biocompatible LM nanoparticles coated with polydopamine, and urease for providing power and drug molecules such as cefixime antibiotics to be carried are connected to the surface of the polydopamine. In a uniform urea solution, the nanomotor can show chemotaxis for the transport of targeted drugs, and in addition, the nanomotor can be deformed under the irradiation of near-infrared light to complete the functional transformation from a living drug carrier to a photothermal reagent, thereby realizing the synergistic antibacterial effect of photothermal and chemotherapy. Urea fuel is extremely abundant in the bladder, so urease-driven micro-nano motors have great potential in the treatment of bladder diseases in vivo, including bacterial infections. The aforementioned LM metal-based micro-nanomotors are also expected to address the challenge of integrating multiple biomedical functions onto micro-biomedical nanorobots.
3.2.3 Treatment of Gastrointestinal Diseases
Enzyme-driven micro-and nano-motors can also play a key role in other parts of the diagnosis and treatment, such as in the gastrointestinal tract. Sanchez et al. Developed a liposome-based micro-nano motor for the treatment of gastrointestinal diseases
[100]. On the micro-nano motor, the asymmetric distribution of urease on the outer surface of the liposome is used for driving the movement of the micro-nano motor, and sodium deoxycholate is used as a marginal activator to regulate the fluidity of a lipid bilayer, so that the motility of the nano-motor containing the encapsulated urease is greatly enhanced. Sodium deoxycholate is naturally present in the gastrointestinal tract, and can effectively achieve drug delivery in the gastrointestinal tract by triggering the movement of micro-nano motors in situ. This study opens up a new way for the use of liposome-based nano motors in drug delivery and provides a new idea for the treatment of gastrointestinal diseases. Similarly, Choi et al. Also developed a urease-driven polydopamine micro-nanomotor for oral drug delivery in the treatment of gastrointestinal diseases
[101]. The nanomotor catalyzes the hydrolysis of urea to ammonia in the stomach, and the subsequent increase in pH in the stomach causes a local gel to sol transition in the mucin layer, thereby facilitating the penetration of the nanomotor into the gastric tissue layer. The in vivo test showed that the retention time of the micro-nano motor in the stomach was prolonged by 24 H, and the micro-nano motor was completely cleared within 3 d without causing any toxicity in the gastrointestinal tract (Fig. 12). The urease-driven micro-nano motor has excellent biocompatibility and can be used for the treatment of gastrointestinal cancer by encapsulating various anticancer drugs such as doxorubicin.
4 Conclusion and prospect
In this paper, the biomedical applications of enzyme-driven micro-and nano-motors are summarized in detail, especially in the diagnosis and treatment of diseases. Although enzyme-driven micro-and nano-motors have made some progress in the biomedical field, the complex biological environment still brings many difficulties and challenges to the application of enzyme-driven micro-and nano-motors in the biomedical field. For example, how to maintain the activity of biological enzymes in complex body fluids, how to overcome the influence of protein corona, and so on. Further solving these problems will be one of the development directions of enzyme-driven micro-and nano-motors in the future, and it is also a key scientific problem in this field. Therefore, there are the following problems to be solved in this field: firstly, how to effectively and controllably realize the immobilization of biological enzymes and maintain their stable catalytic activity is a challenge, which is also an important guarantee for the application of biological enzyme motors in vivo; Secondly, in complex biological fluids with low Reynolds number, precise manipulation of the motion, communication and synergy of enzyme-driven micro-and nano-motors will be an important direction of future research. In addition, large-scale precise manufacturing of micro-nano motors with complex structures and multiple functions is also a technical problem to be solved in this field.
To sum up, enzyme-driven micro-nano motors have great application prospects in the field of life medicine, but also face many scientific and technological challenges. We hope that more researchers will explore this field and provide new ideas and directions for building a "human health community".
National Natural Science Foundation of China(52073071)
National Natural Science Foundation of China(51703043)
National Key R&D Program of China(2021YFF0603500)
Natural Science Foundation of Heilongjiang Province(YQ2022E021)
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