Nano/microvehicles for efficient delivery and (bio)sensing at the cellular level

A perspective review of recent strategies involving the use of nano/microvehicles to address the key challenges associated with delivery and (bio)sensing at the cellular level is presented.


Introduction
The ability to probe cellular activity and target therapeutics inside living cells is of considerable importance as we strive to develop tiny biosensors that monitor complex cellular events and design therapies that can modulate the processes that occur within cells. Such intracellular delivery of drugs can sharply increase the efficiency of various treatment protocols. Nanotechnology researchers have thus been aiming at developing novel nanovehicles capable of entering target cells, probing intracellular processes and ferrying their payload to sub-cellular organelles. Despite the considerable progress made in recent years in biosensing and delivery at the cellular level, current systems are still not able to address some important challenges, such as achieving ultrasensitive detection in a short time in microscale environments, and releasing functional cargoes in a quick and controlled way at specic extra-and intra-cellular locations with limited accessibility. The broad scope of operations and applications, along with the ultrasmall dimensions, accessibility and force offered by synthetic nano/ micromotors, open up intriguing possibilities for these purposes, overcoming some of the unlled gaps and unmet challenges. In this eld, apart from chemically powered nano/ micromotors propelled by toxic (e.g. H 2 O 2 ) or biofriendly (e.g. glucose, urea) fuels, fuel-free nano/micromotors, powered by various external stimuli based on magnetic, electric or ultrasonic elds, exhibit major advantages in directional motion control and have long lifetimes and excellent biocompatibility.
A tremendous effort has been made over the past decade for the implementation of strategies to design and fabricate nano/ micromotors with different functionalities. These articial nano/micromachines can be designed to accomplish challenging biomedical applications, such as biosensing and targeted drug, gene, protein and cell delivery, both at the extra-and intra-cellular levels. However, several critical challenges should be more deeply addressed, such as improving biocompatibility through smart functionalization, achieving efficient propulsion in complex biological media such as intravascular uids, achieving safe removal from the body once the mission has nished (involving the use of self-degrading nanomotors fabricated with biodegradable transient materials) and enhancing the cooperation of multiple nano/micromotors for complex tasks.
Although these unsolved critical issues should be addressed, the high performance demonstrated by nano/micromotors for José M. Pingarrón obtained his PhD in Sciences from the Complutense University of Madrid. He is a Professor of Analytical Chemistry at the Complutense University of Madrid and the head of the group, "Electroanalysis and electrochemical (bio)sensors".
His current research includes the development of nanostructured electrochemical biosensors, including enzyme electrodes, immunosensors and genosensors for the ultrasensitive detection of bacteria, low molecular weight hormones and cancer markers, as well as electrochemical arrays for multiplexed detection. He is an Associate Editor of the journal Electroanalysis and the Vicepresident of the Spanish Royal Society of Chemistry. He has authored over 350 research papers and several books and book chapters.
Joseph Wang is a Distinguished Professor, SAIC Endowed Chair and Chair of the Department of Nanoengineering at University of California, San Diego (UCSD). He also serves as the Director of the Center for Wearable Sensors (CWS) at UCSD School of Engineering. He held the Regents Professorship and Manasse Chair positions at NMSU, and served as the Director of the Center for Bioelectronics and Biosensors at Arizona State University (ASU). He received two ACS National Awards in 1999 and 2006 and 8 Honorary Professorships from Spain, Argentina, Slovenia and China. Prof. Wang is the Editor-in-Chief of Electroanalysis (Wiley). His scientic interests are concentrated in the areas of nanomachines, bioelectronics, biosensors, wearable devices and bionanotechnology. He has authored over 1000 papers that have been cited over 60 000 times (ISI H Index ¼ 119).
biosensing and targeted payload delivery at the cellular level has encouraged the scientic community to devote more effort to understanding the fundamental science behind nano/ micromotors and expanding their applications. Further developments are expected to provide intelligent, responsive and multifunctional nano/micromachines that mimic the amazing functions of natural systems for a diverse range of biomedical applications. These will allow unprecedented levels of cell manipulation and targeted intervention even at the single cell level, with the possibility of treating diseases more specically and safely. The immense progress and benets that nano/ micromotors can bring to the elds of biosensing and delivery at the cellular level, along with the potential challenges that lie ahead and the existing gaps and limitations, are discussed in the following sections.

Nano/microvehicles
Nano/microvehicles are expected to become powerful mobile biosensors 1,2 and active transport systems, holding great potential for a variety of diagnostic and therapeutic applications. The design of miniaturized and versatile nano/ microvehicles would allow access throughout the whole human body, leading to new procedures down to the cellular level, and offering localized diagnosis and treatment with greater precision and efficiency. 3,4 Such nano/micromotors with medical potential must be fabricated with non-toxic materials and optimized shapes, and must be propelled by powerful and biocompatible propulsion mechanisms.
A wide range of nano/microvehicles based on different propulsion mechanisms have been developed in recent years. 5,6 These vehicles can be classied into three different types based on their propulsion mechanism: (1) those with their own builtin propulsion powered by energy-rich molecules, (2) those with external energy sources such as magnetic or acoustic elds, and (3) systems "towed" to the site of interest by living objects with propulsion functions. In the case of autonomous systems, the engine must be equipped with some sort of navigation system to direct the nano/microvehicle to the target, while the direction of externally powered systems must be guided by an operator. 6 Another widely accepted classication divides nano/ microvehicles into only two major groups: (1) those that are chemically propelled 5 and (2) fuel-free nano/micromotors powered by biocompatible external energy sources, such as magnetism and ultrasound (US) (and sometimes optical, thermal and electrical energy). 4,7-9 Catalytic nano/ micromachines include nano/micromotors powered by external 2,10-15 or natural 16,17 fuels, and micromotors with chemotaxis behavior towards a particular substrate. 18,19 Magnetically actuated exible/helical swimmers, 20-25 USpowered nanomotors [26][27][28][29][30] and self-motile cell-based micromotors are included in the fuel-free group. 31,32 Many of these nano/micromotors show considerable promise for navigation through complex biological uids, reaching hard-to-access bodily locations, or releasing their therapeutic payloads at predetermined body destinations.
Fuel-propelled micromotors require a specic fuel and specic composition of their surface. They can drive themselves through aqueous solution using surface reactions to generate local gradients of concentration, electrical potential and gas bubbles. 33 Among the chemically powered nano/micromotors, tubular microengines are particularly attractive for biosensing applications due to their efficient bubble-induced propulsion resulting from the catalytic decomposition of fuel on their internal surface. Fuel-propelled nano/micromotors can perform different types of motion, and all of them are limited by access to fuel in the medium. The inherent lack of a dened trajectory can be solved by including a magnetic component (e.g. nickel) in their structure, thus allowing their guidance in the presence of an external magnetic eld. 34 The development of biocompatible and biodegradable nano/ micromotors, driven by natural fuel sources inherently created in biological systems, such as glucose, urea, 35-38 acid 16,17 or water, 39-43 is crucial for in vivo applications 17,44 because these systems not only circumvent the need for an external fuel, but also provide selective nano/micromotor activation only in particular environments.
Wang's group has reported the use of body fuel-powered micromotors, such as Zn or Mg-based micromotors, for in vivo drug delivery applications. Indeed, this group reported the rst example of the use of micromotors in vivo, showing the propulsion of Zn motors in the harsh acidic environment of a mouse's stomach. 16 Mg micromotors coated with a pHresponsive enteric polymer were also demonstrated to be useful for site-specic gastrointestinal (GI) delivery. 45 These micromotors safely passed through the gastric uid and were selectively activated in the GI tract of living mice, demonstrating important advantages toward localized tissue delivery. Mgbased micromotors have also been combined with a cargoloaded pH-responsive lm, demonstrating that they can autonomously and temporally neutralize gastric acid, triggering a responsive payload release in the mouse's stomach. 46 This simple but efficient motor conguration opens up future prospects in drug delivery, offering a unique rational on-board release in the presence of specic microenvironments.
Externally-powered motors, using mainly magnetic and US energies to drive their motion, are extremely interesting for in vivo biomedical applications due to their potential biocompatibility and independence of the chemical composition of the medium in which they operate. The use of magnetic elds to power nano/microswimmers has gained particular interest as magnetic elds can penetrate the human body, allowing for the wireless control of these tiny devices without harming cells and tissues. 23 A special type of magnetic helical microswimmer is articial bacterial agella (ABF), which can perform 3D navigation in a controllable fashion with micrometer precision in liquid, using low-strength rotating magnetic elds (1000 times lower than the elds used in MRI systems), translating rotational movement into translational motion in a screw-like manner. 23,47 Moreover, Felfoul et al. 48 demonstrated that harnessing swarms of microorganisms exhibiting magnetoaerotactic behavior can signicantly improve the delivery efficiency of drug nanocarriers to tumor hypoxic regions of limited accessibility.
Nanomotors propelled by acoustic forces also offer great advantages in terms of biocompatibility and cargo delivery. It has been demonstrated that US propulsion facilitates the internalization of functionalized nanowire (NW) motors into living cells and enables them to remain acoustically active, undergoing both directional motion and spinning inside the cells. 29,49 The pioneering work of Mallouk and colleagues at Penn State University demonstrated for the rst time that such a gold NW motor, powered by ultrasonic waves, was able to move inside HeLa cells without causing any damage. 49 The introduction of US-powered NW motors into living cells has opened a new door for payload delivery and (bio)sensing at the cellular level. In addition, US actuation opens up the possibility of driving and controlling nano/micromotors using deeply penetrative (yet medically safe) US to create powerful micromotors with remarkable tissue penetration properties that will be discussed in the following sections. 27,28 The use of self-motile cell-based micromotors provides great exibility regarding the type of agellated organism that can be integrated into the microbiomotor. Moreover, the motile cells employed do not need to be cultivated and are readily available, easy to handle, able to swim in highly viscous media and completely harmless. 50 Motile cells have been used as microbiomotors in connection with metal-coated polymer microhelices 31 or magnetic microtubes. 32,50 These tiny devices can also be classied according to their shape, size and material building block, which determine their propulsion mechanism and the best fabrication route for their preparation. 51 Different metal NWs, the simplest 1D structure used as nano-and micromotors, are fabricated by metal electroplating inside a membrane template (usually an anodic aluminum oxide or polycarbonate membrane). These commercially available membranes are sputtered with gold to act as a working electrode in the fabrication process, and are dissolved in the last step using acid or base solutions to release the as-prepared NWs. Janus spherical nano/micromotors, consisting of two hemispheres playing different roles (one relating to the propulsion ability, and the other relating to targeted application), are commonly fabricated by catalytic metallic (e.g. Ir, Au, Pt or Ti) thin lm deposition (by electron beam evaporation, atomic layer deposition or sputtering processes) or polymeric coating onto the surface of nano-or microbeads of different materials (e.g. SiO 2 , polystyrene or Mg) uniformly distributed on glass slides or silicon wafers. Tubular nano/ micromotors are different to those using NWs as they have tubular structures with specic inner and outer parts, both of which are suitable to be functionalized. 24 Early tubular micromotors (microtubes, microrockets or microjets) were fabricated by a rolling-up process based on the patterning of a photoresist on a substrate (e.g. Si wafer) bearing a sacricial layer. 33 The subsequent evaporation of metals at a glanced angle created a window that determined the rolling process (occurring aer etching the substrate). However, smaller sized microtubes, which are more compatible with certain applications, can be fabricated in a simplied manner by template electroplating using appropriate membranes with conical shaped pores. 52 This methodology avoids the need for a cleanroom and signicantly reduces the cost and fabrication time. It is also worth mentioning that various chemical compounds, such as polyaniline (PANI), poly(3,4-ethylenedioxythiophene) (PEDOT), polypyrrole (PPy), poly(3-aminophenylboronic acid) (PAPBA) and molecularly imprinted polymers (MIPs), can be electrodeposited allowing facile further modication. This is therefore an attractive route for fabricating versatile microtubes to be functionalized for different applications. Interestingly, all of these fabrication routes offer many alternative ways to tailor the size, shape, propulsion mechanism and nal properties of the synthesized nano/micromotors, as desired for achieving their optimum performance in the pursued applications.

Nano/microvehicles for biosensing and delivery
Affinity biosensors involving analyte-receptor interactions under static conditions have been demonstrated to achieve low detection limits. However, ultrasensitive detection in a short time remains a major challenge due to the small sample volumes and difficulty enhancing analyte-receptor interactions through enhanced mass transport in such microliter samples. The functionalities and capabilities exhibited by advanced nano/ micromotors have opened the way to new biosensing applications in previously inaccessible microscale environments. The easy functionalization of the nano/micromotor structure with specic biological or articial recognition elements, attached onto the motor surface or embedded in the motor material itself, has allowed for their implementation as dynamic biosensing systems, offering specic and fast detection of different target analytes, greatly accelerated by the efficient and fast motion of receptor-functionalized nano/micromotors through the sample, without or with minimal sample treatment. 53,54 These novel motile biosensors, in which the continuous movement of receptor-functionalized nano/micromotors around the sample increases the likelihood of target-receptor contacts, have been demonstrated to be highly selective and sensitive, eliminating time-consuming washing steps and largely simplifying and accelerating the overall biosensing procedure. In the particular case of fuel-propelled tubular microrockets, the fast motion and bubble release trigger local vortexes that promote the transport of the target analyte toward the functionalized microjet outer surface, offering the possibility to perform rapid "on-the-y" recognition events. 51,55 On the other hand, the development of a new generation of drug delivery vehicles capable of encapsulating, transporting and releasing active substances in a rapid, targeted and controlled manner is of great interest. One of the biggest challenges to face in targeted drug delivery is conquering the innate immune system of the host organism, which readily recognizes and destroys foreign material that enters the body's circulation. 6,56 Over the past decade, many nanomaterial-based systems have been developed for drug delivery applications, including liposomes, polymeric nanoparticles, dendrimers, polymersomes and nanoemulsions. 57 However, despite the signicant progress made to improve the therapeutic efficacy of such drug delivery nanosystems, important challenges, such as issues relating to off-targeting, compatibility with the immune system, low cell and tissue penetration and low therapeutic efficacy, have not yet been overcome. This demands the development of innovative technologies.
In this context, recent delivery strategies involving the use of nano/micromotors have demonstrated the integration of selfdriven and navigation capabilities, achieving not only smart drug encapsulation and release but also precise guidance and control. 13 It is important to mention at this point (as will be described in more detail later) that the clever delivery approaches proposed by Schmidt's group, using articially motorized sperm cells 31 and sperm-hybrid micromotors, 32 have demonstrated the ability to inhibit the immune response using the specic glycans on the sperm cell membrane. Although still at an early stage, these recent advances will play a major role in the future design of new micro/nanodevices with low immunogenicity that are able to bypass the immune system.
Nano/micromotors possess a high towing force that represents a major advantage for their implementation as cargo delivery platforms. Furthermore, their directionality can be controlled by the incorporation of magnetic materials in their structure, allowing for the precise motion control required for targeted drug delivery. 34 In addition, the easy functionalization of the nano/micromotor structure with specic bioreceptors has also enabled efficient modication with a great variety of cargoes and their delivery, both in extra-and intra-cellular spaces. Therefore, drug delivery nano/microvehicles promise to address several key issues for the development of more powerful therapeutics, including the evasion of immune-mediated clearance, improvements to therapeutic efficacy, enabling the combinatorial delivery of multi-cargoes, avoiding negative side effects by delivering the drug precisely at the disease-specic place, and overcoming limited access to certain areas of the body with low drug tissue distribution and cellular barrier penetration. 58,59 Taking into account the breakthrough biomedical applications of nano/micromotors demonstrated by pioneering groups in this eld 4 and the compatibility of operation demonstrated in cellular media, 51 we journey in this perspective article through recent advances in the use of nano/microvehicles for cellular biosensing and cargo delivery applications. Selected examples of such motor-based operations will be discussed, along with related challenges, envisioned opportunities and future perspectives. As will be discussed below, nano/micromotors have been applied for biosensing and delivery both in extraand intra-cellular spaces. As depicted in Fig. 1, while catalytic, magnetic and motile cell-propelled vehicles have been preferably used for extracellular applications, US-propelled nanomotors are the preferred choice for intracellular operations.

Nano/microvehicles for biosensing at the cellular level
The ability to dynamically probe intracellular processes and monitor key molecules in individual cells is the next frontier in biomedicine. As introduced above, the nano/micromotor surface can be functionalized with specic bioreceptors used for the detection and/or isolation of different biological targets under dynamic conditions at the extra-and intra-cellular levels. The continuous movement of synthetic nano/micromotors through the samples, and the dramatic modulation of mass transport by the vortex effect resulting from the locomotion of bubble-propelled microengines, signicantly enhance the interactions of the nano/micromotor sensing surfaces with the target analytes, thereby improving the recognition efficiency and offering 'on-the-y' recognition of specic biomolecular interactions. 51,60 The different biosensing strategies based on receptor-functionalized nano/micromotors are summarized in Table 1.
The developed methodologies were designed for the specic detection of relevant targets both at extra-(proteins, antigens and carbohydrates of cellular surfaces, and H 2 O 2 release to extracellular media) and intra-(mature miRNAs) cellular levels. For example, Balasubramanian et al. 11 described an immunomicromachine constructed by anti-carcinoembryonic antigen (CEA) monoclonal antibody modication of Ti/Fe/Au/Pt microrockets prepared by standard photolithography. The modied microvehicle was employed for the in vitro isolation and transport of cancer cells by means of the selective biosensing of CEA overexpressed in pancreatic cancer cells (Fig. 2a). The micromotors showed sufficient propulsive force for the efficient transport of the captured target cells even in complex biological media. The same group also reported attractive strategies to isolate bacterial and yeast cells using lectin-modied or boronic acid-based microengines with small sizes, mass produced through a low-cost membrane template electrodeposition technique. 12,61 These concepts were demonstrated for the rapid and real-time isolation of Escherichia coli using Au/Ni/PANI/Pt microtubular engines functionalized with concanavalin A and yeast cells using PAPBA/Ni/Pt microtube engines. The multifunctional capabilities of the concanavalin A-modied Au/Ni/ PANI/Pt microtubular engines were shown by performing the capture and transportation of polymeric drug carrier particles for theranostic purposes and the triggered release of the captured cargoes. All of these studies point out the great advantages offered by catalytic nano/micromotors in terms of their faster sensing capacity also at the cellular level. However, the major challenge to face for their potential in vivo application is still their need of H 2 O 2 fuel for propulsion. With this in mind, motors propelled by external stimulation, such as acoustic elds, appear to be great candidates to work in cell culture environments. The strong energy of the acoustic elds can be utilized to internalize and move nanomotors inside living cells, 49 and therefore the efficient movement of US-powered nanomotors can be exploited for intracellular sensing. An illustrative example of this strategy is the construction of a single-step nanomotor for rapid intracellular miRNA biosensing at the single cell level. 62 The concept relied on the use of US-propelled dye-labeled singlestranded DNA (ssDNA)/graphene-oxide (GO)-coated gold NWs (AuNWs), with uorescence quenched by the p-p interaction between GO and the dye-labeled ssDNA. Aer cell internalization, and in the presence of the target miRNA, the uorescence signal is recovered due to the displacement of the dye-ssDNA probe from the GO-quenching motor surface, leading to attractive intracellular "OFF-ON" uorescence switching (see Fig. 2b). The ability of the US-powered ssDNA@GOfunctionalized nanomotors to screen cancer cells was demonstrated by measuring in a few minutes the endogenous content of target miRNA-21 in two types of intact cancer cell (MCF-7 and HeLa) with signicantly different expression levels of miRNA-21. The fast cell internalization process of the nanomotors and their rapid intracellular movement under the acoustic eld led to major improvements in the sensitivity and sensing speed. The results presented demonstrated that while $60% recovery of the uorescence intensity was observed within 5 min when applying US at 9 V, 30 min were required to recover half of the uorescence under static conditions. Nano/microvehicles for delivery at the cellular level Nano/microvehicles have demonstrated considerable promise for the delivery of a wide variety of cargoes (drugs, nano-and magnetic particles, cells, oligonucleotides and proteins), both in intra-and extra-cellular spaces. Table 2 summarizes the relevant strategies for cargo delivery at the cellular level using nano/microvehicles. The methods are discussed in the following subsections by classifying them in accordance with the type of delivered cargo. Drug delivery at the cellular level using nano/microvehicles Nano/micromotors aim to deliver a drug selectively to the target tissues and cells with increased efficacy while reducing the side effects. The ability of nano/micromotors to transport therapeutic carriers to specic cellular locations such as cancer cells has been demonstrated by several groups. Wang's group 21,65   reported for the rst time the directed delivery of magnetic polymeric particles loaded with the chemotherapeutic drug doxorubicin (DOX) using magnetically driven exible nanoswimmers (Fig. 3a). These authors designed the fundamental mechanism of their fuel-free NW motors to have a cargo-towing ability, and they demonstrated the potential in vitro application using the directed delivery of drug-loaded microparticles at a speed of 10 mm s À1 through a microchannel from the pick-up zone to the release microwell containing HeLa cancer cells. Mhanna et al. 22 reported the use of functionalized helical magnetic "microvoyagers" based on liposome functionalized articial bacterial agella (ABF) to perform in vitro single-celltargeted drug delivery. The results presented demonstrated the feasibility of these microswimmers, steerable with an external magnetic eld, to deliver the hydrophilic model drug calcein to C2C12 mouse myoblasts. The uptake of the drug by the target cell was monitored using uorescence microscopy.
Wu et al. 13,66 proposed the use of self-propelled multilayer Janus capsule motors for effective drug transportation and controlled release at HeLa cancer cells. These hybrid biocatalytic Janus motors were based on hollow polyelectrolyte multilayer capsules and PtNPs 66 or catalase, 13 and were propelled by the biocatalytic decomposition of peroxide fuel. The encapsulated drug (DOX) could be rapidly released at predened sites by US 66 or NIR light 13 triggering. The same group also reported the rst example of self-propelled Janus mesoporous silica nanomotors (MSNs) with sub-100 nm diameters for drug encapsulation and intracellular delivery. 14 These Janus nanomotors employed mesoporous silica nanoparticles with chromium/Pt metallic caps and were propelled by decomposing H 2 O 2 to generate oxygen as a driving force with speeds of up to 20.2 mm s À1 . The Janus MSNs were applied in vitro for intracellular localization and the slow release of the encapsulated drug (DOX) inside HeLa cells through the catalytic hydrolysis of lipid bilayers by cellular enzymes.
The DOX delivery and NIR light-controlled release capabilities at HeLa cancer cells were also described using US-powered highly porous AuNW motors, which exhibited a large surface area and a high drug loading capacity. 26 Another approach involved the construction of biodegradable, self-propelled bovine serum albumin/poly-L-lysine (PLL/BSA) multilayer microrockets. 15 The microrockets' preparation involved a template-assisted layer-by-layer assembly of PLL/BSA layers followed by the incorporation of a heat-sensitive gelatin hydrogel, which greatly improved the encapsulation capacity, containing AuNPs, DOX and catalase (Fig. 3b). Interestingly, these protein-based microtubes can be enzymatically degraded under physiological conditions aer completing their missions.  Xu et al. 32 reported recently the use of sperm-hybrid micromotors as cargo-delivery systems for the treatment of gynecological cancers. This highly attractive strategy involves a single sperm cell serving as an active drug (DOX) carrier and as the propulsion force, taking advantage of its swimming capability. A 3D printed four-armed microtube with a nanometric iron layer, called a "tetrapod", was used to magnetically guide and mechanically release the drug-loaded sperm cell in the desired area (Fig. 3c). The use of sperm cells provides a unique ability to encapsulate hydrophilic drugs owing to their crystalline nuclei, which protect drugs from degradation by the immune response. In addition, drug transfer to the target HeLa cells and spheroids was also improved as a consequence of their somatic cell-fusion ability. Drug delivery occurs when the tubular microstructures bend upon pushing against a tumor spheroid and the sperm squeezes through the cancer cells and fuses with the cell membrane, thus minimizing toxic effects and unwanted drug accumulation in healthy tissues. This actuation mechanism makes these bio-hybrid systems unique and biocompatible vehicles for precise cargo delivery in biomedical applications.
Martel's group reported the transport of SN-38 drug-loaded nanoliposomes into tumor hypoxic regions using magnetoaerotactic motor-like bacteria, Magnetococcus marinus strain MC-1 (Fig. 3d). 48 In their natural environment, each of the MC-1 cells contains a chain of magnetic iron oxide nanocrystals which allows them to swim along local magnetic eld lines. A superior penetration depth in colorectal xenogra tumors was demonstrated compared to passive agents, demonstrating that the swarming behavior of magneto-aerotactic microorganisms can signicantly improve the delivery efficiency of drug nanocarriers.
Peng et al. 18 reported the use of nanosized so polymer stomatocytes functionalized with PtNPs, used as the engine encapsulated in the cavity of stomatocytes with DOX as the cargo and with chemotactic behavior towards H 2 O 2 gradients (created either chemically or by excreting neutrophils), as drug carrier systems. The same group recently reported the use of self-assembled biodegradable PtNP-loaded stomatocyte nanomotors, containing poly(ethylene glycol) (PEG), a semicrystalline poly(3-caprolactone) (PCL) and a glassy polymer (polystyrene, PS) for anticancer drug delivery. 19 The uptake by HeLa cancer cells and fast DOX drug release of these PEG-b-PCL and PEG-b-PS-hybrid based stomatocyte nanomotors was demonstrated during degradation under acidic conditions.
Hoop et al. 24 proposed smart multifunctional tubular nanomachines incorporating a stimuli-responsive building block for targeted drug delivery. The nanomachines consisted of a magnetic Ni nanotube for wireless magnetic propulsion, coated on the outside with Au to allow functionalization with uorescently tagged thiol-ssDNA. The inner nanotube cavity contained a pH-responsive chitosan hydrogel to carry the drug and selectively release it only in acidic environments. Methylene blue and human epithelial breast cancer cells (MDA-MB-231) were used as model drug and target cells to demonstrate the concept.
Schmidt's group 25 introduced for the rst time the term medibots, referring to dual-action biogenic hybrid micromotors with functionality both for cell microdrilling and site-directed drug release. These hybrid micromotors are plant-extracted calcied porous microneedles (40-60 mm long), coated via ebeam deposition with a magnetic Fe-Ti layer to facilitate cellular drilling by external magnetic actuation. The loading of the calcied biotubes with the anticancer drug camptothecin enabled specic drug release in the acidic environment of HeLa cancer cells.
Chen et al. recently reported a hybrid magnetoelectric nanomotor for on-site magnetically-triggered anticancer drug release in breast cancer cells (MDA-MB-231). 64 In this case, the chemotherapeutic drug paclitaxel was adsorbed onto the polydopamine-modied nanomotor surface and was efficiently released upon the application of alternating magnetic elds due to the magnetoelectric effect. Nano-and magnetic particle delivery at the cellular level using nano/microvehicles Wang's group reported the design of acoustically propelled shell-shaped nanomotors for the "on-the-move" capture and transport of multiple magnetic cargoes (3.3 mm magnetic beads), and their internalization and propulsion inside live MCF-7 cancer cells. 28 The same group also reported the rst example of an in vivo study of articial PEDOT/Zn micromotors in a living organism, showing efficient propulsion in the harsh acidic environment of mouse stomachs without additional fuel (Fig. 4a). 45 In this work, the distribution, retention, cargo delivery (using AuNPs as a model) and acute toxicity prole of these acid-driven propelled motors were evaluated via oral administration. It was demonstrated that the body of the motors gradually dissolved in gastric acid, thus autonomously releasing the carried payloads on the mouse stomach wall while self-destructing non-toxically.
US-triggered tubular microbullets are extremely promising for addressing the limited tissue penetration challenges of therapeutic particles. Acoustic droplet vaporization was employed for the propulsion of highly powerful peruorocarbon-loaded microbullets, offering directed drug delivery into diseased tissues. 67 A similar technology was reported by Soto et al. to develop acoustically-triggered microcannons capable of loading and ring nanobullets (Fig. 4b). 27 The constructed microcannons could allow the efficient loading and ring of nanoscale cargoes as nanoprojectiles, favoring the direct and deep penetration of therapeutics into tissues. Such technology is expected to be the next generation of efficient nanoscale delivery devices, capable of delivering different drug cocktails and vaccines into identied targets.

Cell delivery at the cellular level using nano/microvehicles
The Schmidt group presented a novel fertilization method involving the use of articially motorized sperm cells, where customized metal-coated polymer microhelices served as motors for transporting sperm cells with motion deciencies to help them to carry out their natural function. 31 The reported method demonstrated the active capture, transport and efficient delivery of a single live sperm cell to an oocyte cell wall.
Gene/protein delivery at the cellular level using nano/ microvehicles Gene or protein therapy implies the use of DNA or protein therapeutic substances, which are delivered into a patient's cells to treat diseases such as inherited disorders and cancers. 23,32 Fan et al. 63 demonstrated that electrically propelled protein-functionalized AuNW motors could deliver "on-the-y" cytokine tumor necrosis factor alpha (TNFa) and activated canonical nuclear factor-kappaB (NF-kB) signaling to single HeLa cancer cells.
ABFs functionalized (f-ABFs) with complexes of cationic lipids and plasmid DNA (lipoplexes) were proposed by Qiu et al. 23 for in vitro wirelessly targeted single-cell gene delivery to human embryonic kidney (HEK 293) cells. The successful in vitro transfection of the HEK 293 cells by f-ABFs, steered wirelessly by low-strength rotating magnetic elds, was demonstrated by expressing the encoding Venus protein.
The advantages of the rapid internalization and intracellular movement of US-powered nanomotors have also been exploited for intracellular oligonucleotide delivery. Wang's group described recently an effective intracellular gene silencing strategy, using acoustically-propelled AuNWs wrapped with a Rolling Circle Amplication (RCA) DNA strand, which served to anchor green uorescent protein (GFP) which was targetedly interfering with the RNA's (siGFP) payload (Fig. 5a). 29 The USpropulsion led to the fast internalization and rapid intracellular movement of the AuNWs, and hence to an accelerated siRNA delivery and silencing response. The strategy allowed 94% silencing of the GFP response in two different cell lines (HEK-293-GFP and MCF-7-GFP) aer 5 min treatment with the GFP/RCA-wrapped AuNWs. Interestingly, most of the cells remained alive aer the nanomotor treatment, even when using a high nanomotor concentration.
The same group has described recently a method based on the use of US-propelled nanomotors coated with a high pHresponsive polymer, for the rapid internalization and cytosolic  delivery of functional caspase-3 (CASP-3), with the aim of causing apoptosis in human gastric adenocarcinoma cells (Fig. 5b). 30 The nanomotor allowed 80% apoptosis of cancer cells within only 5 min, suggesting that such nanovehicles may constitute powerful tools for the cytosolic delivery of active therapeutic proteins. Compared to other apoptosis approaches, the nanomotor strategy resulted in the highest apoptosis efficiency while requiring signicantly shorter times and smaller amounts of CASP-3.
It is worth mentioning that the extraordinary capabilities demonstrated by fuel-free nano/micromotors for targeted gene or protein delivery open the door for a wide portfolio of potential applications in the elds of therapy bioengineering and cell biology in various in vitro and in vivo settings. Such intracellular delivery can thus sharply increase the efficiency of various treatment protocols.

Key challenges, envisioned opportunities and future perspectives
The discussed examples demonstrate the broad range of applications performed at the cellular level using nano/ micromotors. They constitute clear proof of the remarkable progress made in the design and construction of these tiny machines to integrate self-driven, navigation and biosensing capabilities, and perform multitasking, including drug loading, targeted transportation and autonomous or remotely-controlled release in complex environments. These capabilities make them extremely promising vehicles to be employed for biosensing and drug release applications at the cellular level. The reports described so far demonstrate that different types of nano/ micromotor can be used as suitable engines for operation in cellular environments. Moreover, at the cellular level, the biosensing applications of nano/micromotors have been much less exploited than those involving cargo delivery (mainly of chemotherapeutic drugs). Such new nanovehicles thus offer considerable promise for the monitoring of complex processes that occur within cells and for modulating such cellular processes and increasing the efficiency of various treatment protocols. While several novel intracellular applications of nanovehicles have been covered in this article, the ability to sense cellular processes and deliver payloads within the cell is still at a very early stage. Future advances in nano/micromotors will be critical to biomedical applications that rely on sensing, delivery and targeting in intracellular space.
The studies discussed above show that these smart devices, self-propelled or externally powered by catalytic, magnetic or acoustic sources, offer a myriad of mechanical movements and broad design versatility in terms of building block materials (silicon-based materials, polymers or noble and other metals), size (ranging from the nanoscale to the microscale) and shape (wires, spheres and tubes/rockets). Since each specic shape and building block material demands a given fabrication technique, determining important functioning properties apart from the vehicle motion mechanism, the design and preparation of motors devoted to a specic application requires the integration of several crucial factors. On the positive side, the large number of available possibilities to tailor the behavior and properties of the constructed engines considerably increases their potential applications.
The developed smart vehicles demonstrated outstanding capabilities in terms of speed and force, drug loading and towing capacity (easily tunable through fabrication and by changing the fuel concentration, magnetic and UV eld strengths and temperature), biocompatibility and biodegradability, wireless transport to specic hard-to-reach areas and feasibility to carry a wide variety of cargoes (drugs, genes, enzymes, cells and other relevant chemicals) and release them using biocompatible mechanisms (pH responsive, NIR irradiation and magnetic actuation). Importantly, the biosensing and delivery missions undertaken have been performed both at extra-and intra-cellular levels without compromising the viability of the target cells. While a wide variety have been applied in extracellular space, the intracellular applications have been demonstrated primarily using acoustic propelled nano/micromotors. However, it is important to mention that most of these remarkable capabilities of nano/micromotors have been demonstrated only in vitro, and potential difficulties might be encountered for in vivo applications. In addition to the limitation of using fuel-free or biocompatible fuel nanomotors, there are some serious challenges to face to achieve in vivo applications. The smart vehicles must be able to evade the immune system of the host organism and, in the specic case of liposome-based systems, destruction by the reticuloendothelial system. Drug delivery should also overcome the high interstitial pressure in late stage tumors, the endosomal escape challenge and the natural physiological barriers such as the highly acidic gastric environment. Moreover, for in vivo applications, the nano/micromotor must be readily degraded into non-toxic compounds without interference from outside through passive or built-in self-destruction mechanisms activated aer nishing their mission. 68 Novel smart materials, including biological, responsive or so materials, are therefore highly desired to provide triggered autonomous actuation and multifunctionality while avoiding irreversible malfunctions in complex physiologically relevant body systems.
Additional efforts should also be devoted towards developing functional geometries and exploring new synthesis methods for the easy, large scale, high quality, cost-efficient and environmentally friendly fabrication of nano/micromotors. The iden-tication of new energy sources for enhancing tissue penetration and achieving prolonged, biocompatible and fully autonomous operation in complex biological media such as blood (with large drag forces associated with the high viscosity of the blood cells) should also be pursued. Micromotors powered by bio-friendly fuels also require additional improvements for their practical implementation, due to the relatively short lifetimes of those using active material propellants (e.g., Mg, Zn, Al and CaCO 3 ) and the limited power and stability of enzymebased ones. In this sense, coupling synthetic nanomachines with natural biological materials can minimize these problems, apart from the desired immune evasion and biofouling occurring in complex biological uids, leading to their enhanced mobility and extended lifetime. The future biomedical operation of nano/micromachines will require individual control of the nano/micromotors wirelessly, for example by the integration of nanoscale control systems and the development and coupling with high-resolution imaging and feedback control systems for the simultaneous localization and mapping of multiple nano/micromotors in the human body.
It is worth noting that despite the long and challenging road ahead, some important steps have been already achieved to address these important issues. The stabilization of the substrates and application of appropriate coatings or preconditioning to the liposomes/cargoes have been employed in the fabrication processes of nano/micromotors to potentially address the endosome escape challenge. Moreover, highly hemo-compatible nano/microvehicles, with a negligible inuence from red blood cells on their motion, have already been developed, and more biocompatible nano/micromotors have been proposed involving coatings with different biocompatible materials, such as red blood cell membranes. Most interestingly although still scarcely explored, some authors have already reported the appropriate operation and behavior of these smart systems in in vivo environments. 45 These pioneering applications have demonstrated the capabilities of nano/micromotors for safely and rapidly yet transiently neutralizing gastric acid, and simultaneously releasing their payload without affecting the stomach function or causing adverse effects in living animals. This type of work represents an important step forward for clinical translation and paves the way for many other breakthrough in vivo applications. The limited tissue penetration has been addressed using US-powered tubular microbullets, in which the 'bullet-like' propulsion allows the vehicle to reach different depths of the tissue to perform precise nanosurgery, as well as enabling it to carry large payloads. Indeed, the latest applications in this eld have even demonstrated the use of nano/micromotors as efficient vehicles for the direct cytosolic delivery of active therapeutic proteins aer internalization in intact target cells. While progress has been made in this direction, effective intracellular delivery to the cytosol remains a challenge. Future efforts could lead to the targeting of specic cytoplasmic organelles, such as the mitochondria, through guided movement within the intracellular space. It is also worth mentioning that, although only cargo delivery applications have been considered in this review, the nearly limitless possibilities demonstrated by nano/ microvehicles to deliver a wide variety of payloads with diverse biomedical functions also open up a wide range of other opportunities for therapy, diagnostics and imaging.
Advances in this eld will likely come from the integration of the tremendous progress made in nano/micromotor research during the last few years with a variety of exciting nano/ micromotor-based strategies for the biosensing, transport and release of therapeutic agents at the cellular level, with advances in materials science, nanotechnology and molecular biology. This marriage will be essential to overcome the remaining challenges and promote the wide implementation of these smart, tiny multitasking vehicles for in vivo biosensing and cargo delivery. Furthermore, to fully realize the abilities of nano/micromotors in medicine, a closer collaboration between scientists working in the development of nano/micromachines and medical researchers will be required for designing and constructing devices able to meet the demands and needs of the medical community. Overcoming these challenges to further increase the power and functionality of nano/micromotors, together with nding effective paths to take advantage of the emerging novelties in this exciting eld toward the most relevant practical and commercial applications demanded by clinical experts, will be compulsory to ensure their utilization in diagnostics and therapy, which is still in its infancy. Therefore, substantial efforts and new innovations are still required to realize the full potential of these tiny motors in a new generation of nano/micromotors. Ideally, they should be fabricated using cutting-edge techniques, be able to mimic the natural intelligence of their biological counterparts, exhibit improved, adaptable and sustainable operation in biological media, have a deformable structure, be able to be precisely controlled, and have the ability to act both individually and collectively with synchronized coordination and self-adaptive and selfreplicating capabilities. Nevertheless, the remarkable performance already demonstrated by nano/micromotors to design problem-oriented medical devices for specic sensing or delivery functions undoubtedly represents a tremendous asset in diagnostics and therapeutics. Therefore, the accelerated translation of nano/micromotor research into practical clinical use nowadays seems more reality than science ction.

Conflicts of interest
There are no conicts to declare.