Chemotherapy is currently one of the mainly applied methods for the treatment of malignant tumors. Nevertheless, the clinical efficacy of chemotherapy for human solid tumors is far from satisfactory since the complexity of the type and location of tumors, and the lack of selectivity of most antitumor drugs to the tumor microenvironment (TME) [1]. Nanodrug delivery systems have always been a hotspot in tumor therapy [2,3] due to the theory of the enhanced permeability and retention (EPR) effect [4], [5], [6]. However, current studies suggest that nanomedicines may not yield desired therapeutic effect in the clinical treatment of human tumors. This prompts a re-examination of nanomedicine treatment strategy for cancer. Upon reviewing numerous previous studies, it is evident that the transport mechanism of nanomedicines entry into tumors is not solely dependent on the gaps between endothelial cells, but rather on the synergistic effect of multiple mechanisms, such as the inter-endothelial gaps and the active process through endothelial cells [7]. In fact, many studies have proposed other mechanisms controlling tumor extravasation [8]. These studies emphasize that tumor-targeted drug delivery is a multifactorial issue. The complexity of the TME brings a big challenge for cancer therapy yet also offers opportunities for drug delivery with targetability [9]. Exploring the major characteristics of TME and developing new technologies and strategies that can be adapted for targeted therapy of the TME are crucial to optimize the design of antitumor drugs [10], [11], [12].

Efficiently delivering nanomedicines to the TME is a complex issue that requires consideration of both the physiological and pathological characteristics of the tumor site [13,14], such as high interstitial fluid pressure, low pH, hypoxia, abnormal vascular system, and the parameters of nanoparticles [15], including particle size, shape, charge, surface properties, material composition, and mechanical properties, as these factors would affect their interaction with biological barriers [16]. Consequently, the specific transport mechanism of nanomedicines in the TME is influenced by these factors. Certain scholars have proposed using stimuli-responsive nano-carriers and natural endogenous carriers [17,18] to deliver nanomedicines, which is regarded as the future research direction for nanomedicines in anti-tumor therapy. Additionally, there has been considerable interest in micro/nanomotors (MNMs) in the field of biomedicine due to their unique motion behavior [19], [20], [21], [22], [23], [24]. These self-propelled micro/nanomotors can sense the surrounding environment and exhibit specific spontaneous movements [25], [26], [27]. Combining the specific physiological and pathological characteristics of the TME with stimuli-responsive micro-nano motors may prove to be a promising approach for nanomedicine vehicles in anti-tumor therapy. Most of the currently reported MNMs are artificial ones with predesigned structure and morphology, tunable motility and powerful capability as biosensors [28] or cargo carriers[29]. Those artificial motors include bubble propelled motors with asymmetry structures [30,31], self-propelled motors [32], [33], [34], motors propelled by external fields such as light [35,36], electric [37], magnetic [38,39] or acoustic [40] field. While the catalyst toxicity of heavy metals, fuel source, uncertain biocompatibility and low energy conversion efficiency significantly limit their biomedical applications.

FOF1-ATPase [41] is a transmembrane rotation biomolecular motor found in various locations such as the plasma membrane of bacteria, the thylakoid membrane of chloroplasts and the inner membrane of mitochondria [42]. It has the ability to synthesize adenosine triphosphate (ATP) by utilizing transmembrane proton potential and converting chemical energy into mechanical motion with extremely high energy conversion efficiency [43,44]. FOF1-ATPase possesses favorable biocompatibility and superpotent properties compared with other artificial motors [45]. Thereby, the application of FOF1-ATPase is a possible solution to the problems of by-products and insufficient perpetual motion. The enzyme kinetics of FOF1-ATPase involves internal subunit rotation, mechanical inhibition mechanisms, substrate and product concentrations, the electrochemical potential of protons across the membrane and more [46,47]. The ‘chemical permeation hypothesis’ [48] and ‘binding change mechanism’ [49,50] provide explanations for the importance of transmembrane proton gradient and conformational changes during ATP synthesis [51]. Noji and colleagues investigated the single-molecular rotation of FOF1-ATPase driven, which is by proton motive force (PMF) and the positive ΔpH dependence of the rotational rate [52]. Current researches on the motility of FOF1-ATPase have primarily focused on understanding its single-molecule rotation mechanism [53], [54], [55], [56], [57], which contributes to its exceptional motility properties. However, there is still a lack of comprehensive understanding regarding the overall motility properties of FOF1-ATPase in specific environments. The regulation mechanisms of its motion and the limitations of existing monitoring methods have been persistent challenges, hindering practical applications and requiring immediate attention. In order to utilize FOF1-ATPase effectively in biomedical fields such as biological detection and drug delivery, it is crucial to explore its chemotaxis capabilities towards specific environments, particularly H+ ions.

In this study, FOF1-ATPase motor embedded chromatophore nanorobot (CN) was prepared and characterized. The chemotaxis of the obtained CN towards protons under different viscosities was examined in a newly designed laminar flow microfluidic system. And we also developed a TME- mimetic microchip, which was utilized to reveal the potential tumor targeting and accumulating ability of the CN in vitro. The presence of the FOF1-ATPase motors in the CN not only enhanced the endocytosis capacity in vitro, but also strongly promoted the tumor targeting and accumulating ability in vivo, suggesting it could be a promising drug delivery nanorobot with powerful self-propulsion overcoming physiological barriers and towards acidic tumor site (as illustrated in Scheme 1).