microorganism-integrated microbiotic nanomedicine

Nano Today 32 (2020) 100854

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Review

Nanomaterials/microorganism-integrated microbiotic nanomedicine Minfeng Huo a,b,1 , Liying Wang c,1 , Yu Chen a,b,∗ , Jianlin Shi a,b,∗ a State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050, PR China b Centre of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing, 100049, PR China c Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, 200072, PR China

a r t i c l e

i n f o

Article history: Received 18 August 2019 Received in revised form 16 January 2020 Accepted 14 February 2020 Keywords: Microorganisms Nanomedicine Biohybrids Tumor-therapy

a b s t r a c t Based on the great advances of the nanomaterials and nanotechnology in the last decade, nanomedicine combining the nanomaterials and nanobiotechnology has been attracting intensive attentions worldwide owing to its bright prospects in generating numerous theranostic modalities against various pathological lesions. Nevertheless, synthetic nanomaterials-based nanomedicine is now facing critical challenges of less effective targeting and insufficient accumulations at pathological sites, raising the critical issues of exaggerated therapeutic dosages and the consequent risks of biosafety. Alternatively, natural microorganisms such as bacteria and viruses have evolved to be capable of targeting certain types of cells effectively by specific mechanisms such as tropism, which enable them to serve as natural vesicles for targeted cargo delivery. By the hybridization of the nanomaterials and microorganisms, researchers nowadays can build versatile microbial nanohybrids for novel and promising nanomedical therapeutics. In this review, we will start from the fundamentals of the physiochemical properties of the microorganisms, and summarize the most recent progress in the basic construction methodologies of microbial nanohybrids and their distinct therapeutic performances for various diseases. The perspectives, promising research frontiers and remaining challenges in this interdisciplinary area will be comprehensively outlooked to provide insights towards the “swallowing the surgeon” envisions. © 2020 Published by Elsevier Ltd.

Contents Definition of “microbiotic nanomedicine” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Fundamentals of microbial nanohybrids integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Bacterial nanohybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Physical attachments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Chemical propagation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Biological reconstruction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Viral nanohybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Chemical conjugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Biotemplation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Fungal hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Microbial nanohybrids for cancer therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Principles of nanohybrids for tumor therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Bacterial nanohybrids for tumor therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Hyperthermia therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Chemodynamic therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

∗ Corresponding authors at: State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050, PR China. E-mail addresses: [email protected] (Y. Chen), [email protected] (J. Shi). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.nantod.2020.100854 1748-0132/© 2020 Published by Elsevier Ltd.

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Immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Viral nanohybrids for tumor therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Drug/nanomedicine delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Molecular imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Oncolytic viral hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Fungal nanohybrids for tumor therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Microalga-based nanohybrids for tumor therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Microbial derivatives for anti-infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 CRediT authorship contribution statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Definition of “microbiotic nanomedicine” Medication is a perpetual topic ever since the emergence of human beings. In ancient times, our ancestors learned and acquired skills to use some of the herbal medications to control bleeding, improve wound healing and cure some of the other diseases. These experiences constitute the earliest pharmacology in human history. When it comes to modern medication, the advances of organic synthesis, chemical biology and the outburst of the industrial revolutions have significantly accelerated the explorations and discovery of advanced pharmaceutics and treatment methodologies against various lesions. By now, modern medications have been capable of eliminating several types of diseases such as smallpox and polio permanently, and effectively curing a broad spectrum of common sicknesses. However, enormous difficulties are still present in controlling a majority of pathological abnormalities that are aggressively malignant, frequently reoccurring and typically intractable, such as cancer, bacterial infection, cardiovascular diseases, etc. The past decades have witnessed the rapid emergence and evolution of nanoscience and nanotechnology [1–3]. Scientists have created fundamental theories, practical methodologies, and versatile material platforms in diverse disciplines, especially nanomedicine – the applications of nanotechnology to diagnose, treat or reverse the process of a wide range of medical challenges, such as serious pathological diseases [4,5]. With great endeavors being devoted, numerous nanomedical platforms and exquisite technologies have been developed and employed to combat these diseases from prophylaxis to diagnosis, therapeutics, and prognosis [6–14]. Typically, traditional tumor therapy involves the application of non-selective cytotoxic chemotherapeutics (e.g., doxorubicin (DOX), cisplatin, etc.) or harmful ionization radiations, which cause severe adverse impacts against human beings [15,16]. While in fundamental research regarding the nanomaterials and nanotechnology-based tumor therapy, numerous nanosized drug delivery platforms have been developed for enhanced therapeutic efficacy and mitigated harmful side effects [17–19]. Furthermore, by taking advantages of the characteristic energy-conversion properties of the nanomaterials, versatile therapeutics including photo-based nanotherapy (photodynamic [20–22], photo-hyperthermia [23–25]), ultrasound-based nanotherapy (sonodynamic, high-intensity focused ultrasound) [26–28], radiation-based nanotherapy (radiodynamic) [29–31] and most recently evolved catalysis-based nanotherapy (i.e., nanocatalytic medicine [32–34]) have been achieved, probably under the guidance of highly spatiotemporal-resolved bio-imaging monitoring, such as positron emission tomography (PET) [35,36], computed tomography (CT) [37,38], magnetic resonance imaging (MRI) [39,40] and photoacoustic imaging (PA) [41,42], etc. The advantage of nanotherapies over the traditional cancer therapeutics lies in the effective and selective damages against the lesion tissues while

minimizing the harmful adverse effects to the normal tissues and organs. Especially, the catalytic nanomedicine, which has been presented most recently by our group, is mainly devoted to lesions (e.g., tumors)-specific therapy without significant adverse effects by catalyzing the intra-tumoral chemical reactions to generate therapeutic toxins in-situ, in response to the endogenous tumoral microenvironment and/or focused exogenous stimuli, and no negligible reactions will be initiated in the normal tissues. Excitingly, a number of these therapeutic nanoplatforms and methodologies have been recently transferred into clinics, improving the life qualities of human beings in significant ways (Fig. 1a). Despite the prominent advantages that researchers have approached in the frontiers of nanomedicine, most of the nanotherapeutics are still in their primary stage [43–46]. One of the major obstacles toward clinical translations are the sophisticated physiological barriers (e.g., blood-brain barrier (BBB) [47,48], heart barrier and placental barrier [49,50]) and complicated in vivo immunosystems that are used to defend our bodies against the invasions of foreign substances such as pathogens in normal circumstances [51,52]. These barriers and systems could effectively obstruct a wide range of medicines administrated through patient-friendly approaches (e.g., oral administration, intravenous injection and intramuscular injection), thereby deteriorating the drug performance. Specifically, in nanomaterials-based brain disease treatment, BBB traverse is the most intractable issue to realize the brain drug delivery as the endothelial cells of the capillary wall will actively decline the medicine to access into the brain [53,54]. In tumor therapeutics, insufficient tumor accumulation of the nanomedicine further limits the multimodal therapeutic performance [55]. Technically, the general idea to conquer this problem in modern nanomedicine is through targeting decoration and vectorization design. These strategies include the surface modification of specifically targeting molecules and/or moieties such as proteins and peptides (e.g., angiogenin, TAT) [56,57], magnetic guidance [19,58] as well as homologous cell membrane coating [59,60]. Nevertheless, the inconsistent spatiotemporal distributions of these targeting interactions can significantly reduce the targeting efficiency and eventually lead to undesired medicine distributions, attenuated therapeutic performance and the possibility of underlying bio-toxicities. Therefore, revolutionary strategies are in urgent demand to tackle the pathological targeting issues during therapeutics based on nanomedicines. Microorganisms have been reprogramming the world and sustainably providing practical solutions for agriculture, industry and public health [61,62]. Standing on the shoulders of giants, it is perceived that “microorganism” is a collective term for all organisms that cannot be seen with naked eyes by human beings. This contains almost all prokaryotes including unicellular bacteria, archaea, and microalgae, some typical eukaryotes such as fungi and protists, as well as non-cellular microorganisms like viruses. As pathogens, bacteria and viruses have evolved to develop strong

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Fig. 1. a, Developments of medications in human history. b, Schematic illustrations of the pros and cons of nanomedicine and microorganism-based medication. The hybridization of nanomaterials and microorganism may initiate the next generation of microbiotic nanomedicine featuring targeted therapeutics and guaranteed biosafety.

natural tropism to infect certain types of cells or tissues of animals/humans effectively. The remarkable resemblance of such a tropism to drug delivery expectation has aroused great interest among researchers to employ these pathogenic microbes as the effective dosing tool during medical treatments [63]. For a century, there have been numerous reports on the employment of microbes for the treatments of certain diseases such as cancer and smallpox, receiving satisfied therapeutic performances, which, unfortunately, are accompanied by severe infections [64–66]. Although attenuation and deactivation strategies have been employed to reduce the infections extensively, such a risky therapeutic approach is blamed to merely serve microbes with monotonous therapeutic modality to cure cancer with much diversity. Eventually, the public enthusiasm for microbiotic therapy was dissipated by the emergence and developments of radiotherapy and chemotherapy from the beginning of the 20th century. Nevertheless, a question remains: is it possible to take advantage of microbiotic therapy while minimizing its diverse undesired effects such as infection by implementing other emerging approaches? Such an effort then catalyzes the emergence of

the combination of microbiotic therapy with advanced therapeutic modalities. It is clear that numbers of the microorganisms could actively provide natural tropism for pathological targeting [67,68] while nanomedicine-based multimodal therapeutic performance is appealing in the last decade [43]. Therefore, the integration of microbes and nanomaterials into a hybridized nanoplatform may prospectively enable the renascence of microbiotic therapy of prominent therapeutic performance with adequately enhanced vectorizations of nanomedicine at pathological sites and substantially reduced side effects of microbes, generating an attractive superadditive therapeutic effect. Herein, “microbiotic nanomedicine” is defined as the application of nanomaterialshybridized microorganisms in the theranostics of a variety of pathological diseases. Such a microbiotic nanomedicine demonstrates the advances co-contributed by the nanomaterials and nanotechnology-based medical treatment modalities, as well as the chemical and biological fundamentals of the microorganisms. Based on a great variety of nanomaterials and the microorganism, the diverse combinative functionalities including therapeutics,

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molecular imaging, and biodetections, could render sophisticated methodologies regarding the improvement of human health (Fig. 1b). In the past decades, great progress on microbiotic nanomedicine has been made in the field of nanomedicine. In this review, we will summarize the design and fabrication methodologies and the corresponding therapeutic performances of the microbe-nanomaterial “nanohybrids” in a comprehensive manner as far as we can from the viewpoint of materials-microbe interactions. Pathogenic or non-pathogenic bacteria, viruses and other microbes such as yeast and microalgae, are reviewed as model microbiotic units to fabricate the nanohybrids with nanomaterials. Subsequently, promising applications of nanohybrids in the treatments of cancer, bacterial infection and viral infection, are overviewed extensively. Finally, the underlying potentials and possible strategies of the nanohybrids for improved medical performances, and their future applications will be outlooked.

ating integral nanohybrids for possible nanomedical applications [81,82]. Depending on the biological viability of the microorganism, the encapsulation interaction can be further classified. Several kinds of the microorganism (e.g., virus) could passively endocytose or encapsulate nanomaterials with specific surface chemistry and/or morphology, forming an integrated living biohybrid with appealing functionalities [83,84]. However, due to the biological heterogeneity, the endocytosed nanomaterials may not be able to reach a satisfying loading amount in this case, thereby decreasing the interests of research. In most of the cases, the intracellular contents of the microorganism can be extracted through chemical treatment or biological engineering methods, creating abundant spaces for the encapsulations of drugs, small molecules, and nanomaterials into such non-living microorganisms upon physical infusion. Although the biological activity has been lost, the natural tropism activity originated from the surface pathogens are well preserved, guaranteeing the vectorized delivery with much-enhanced biocompatibility.

Fundamentals of microbial nanohybrids integration

Bacterial nanohybrids

Microbial nanohybrids is herein defined as the conjugates between microorganisms and nanomaterials through specific physiochemical interactions. By the construction of the nanohybrids, the biologically active microbes could be served as a novel smart drug delivery carrier, heading to the target region under varied stimulation signals such as chemotaxis [69] or magnetotaxis [70,71]. The hybridized nanomaterials could then be delivered via a hitch-hike travel along with the microorganism to the targets. Besides, when a specific destination is reached, multiple therapeutic modalities could be implemented based on the properties of the nanomaterials, generating dominant therapeutic outcomes at local lesion sites. Furthermore, the nanohybrid integration may significantly benefit the critical interactions between each other, including the possible mechanistic conduction [72,73] and chemical attractions [74,75]. This will potentially generate underlying synergetic effects of the microbiotic nanohybrids during the nanomedical applications. The fabrication methodologies of microorganism-based nanohybrids are substantially dependent on the fundamental understandings and manipulations of the interactions between microorganisms and nanomaterials [76,77]. For most microbial nanohybrids, the interactions could be generally categorized into (i) physical or chemical conjugations of nanomaterials to the extracellular space (ECS), and (ii) nanomaterial encapsulations into the intracellular space of the microorganism (Fig. 2). Depending on the relative spatiotemporal distribution of the nanomaterials to the microorganism, these microbial nanohybrids are endowed with diversified application potentials including targeted drug delivery and theranostics. The physical/chemical conjugation methodologies enable nanomaterials to hybridize with the desired microorganism through electrostatic attractions or chemical bindings in the ECS [78]. In this case, the surface chemistry of nanomaterials and the ECS of the microorganism are of substantial significance. Due to the presence of ionization equilibrium of the surface species, nanomaterials can be electrostatically charged, providing basal attractions towards specific microorganisms with complementary charge properties and chemical status [79]. In addition, dangling surface functional groups or engineered/expressed molecules/proteins on the ECS of the microorganism also provide reactive chemical conjugation sites for specific nanomaterials [80]. Such facile and effective conjugation processes have triggered numerous investigations on fabrications and practical applications of microbiotic nanohybrids. While upon encapsulation, the intracellular space of the microorganism is preferentially occupied by nanomaterials, cre-

Bacteria are unicellular prokaryotic biological cells with a great variety of species featuring diversified dimensions (from hundred nanometers to few micrometers) and shapes (e.g., coccus, bacillus, spirillum, and vibrio). Bacterial species can be extremely distinct from each other as they have evolved to adapt to various types of environments for millions of years. In general, judging by their cellular structural discrepancies, bacteria have been classified explicitly into gram-positive and gram-negative bacteria by the traditional Gram staining methodology [85]. The ECS of a bacterial cell is thereby categorized accordingly. A typical gram-positive bacteria cell generally contains a thick peptidoglycan cell wall outside the plasma membrane, while for gram-negative bacteria, in addition to a much thinner peptidoglycan cell wall than that on grampositive one, an additional outer membrane is coated outside [86]. For the intracellular space of a bacterial cell, the cytoplasm typically holds the nucleic acids and proteins inside a multi-component cytoskeleton [87]. Some of the bacterial cells may also harbor the protein-bound organelles [88] such as carboxysome [89] and thylakoid, that functions in carbon fixation and photosynthesis respectively [90]. Bacterial nanohybrids are the most frequently reported biohybrids in the literature and have offered plenty of opportunities in the frontier of photocatalysis [91–93]. According to the interactions between the nanomaterials and the microorganism, the fabrication fundamentals in bacterial nanohybrids mainly lie on three methodologies: physical attachments, chemical propagations, and biological engineering. Physical attachments The electrostatic interaction between the bacterial cells and nanomaterials enables the bacterial nanohybrid formation by physical attachments. Typically, in a neutral medium, the ECS of gram-positive bacterial cells is strongly negatively charged due to the characteristic existence of teichoic acid (TA) molecules, in comparison to the mildly negatively gram-negative ones. These TA molecules could facilitate stable electrostatic interactions with positively charged nanomaterials such as cetyltrimethyl ammonium bromide (CTAB)-terminated nanorods (Fig. 3a–b) [94] and poly(llysine)-coated Au nanoparticles (Fig. 3c–d) [95]. Nevertheless, for mildly-negatively-charged gram-negative bacterial cells, direct conjugations of the nanomaterials may conceivably lead to the easy disintegration of the nanohybrids. Therefore, layer-by-layer selfassembled polyelectrolytes-mediated indirect conjugations of the nanomaterials could be achieved. Within the assembled polyelectrolyte layers, nanomaterials (e.g., Fe3 O4 magnetic nanoparticles,

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Fig. 2. Fabrication methodologies of microbial nanohybrids including physical attachments, chemical conjugations, and biological reconstructions.

Fig. 3. SEM images of nanorods and nanospheres-deposited Bacillus cereus (a-b) (Adapted from Ref. [94] with permission of American Chemical Society, Copyright 2005), Au nanoparticles-deposited Bacillus cereus (c-d) (Adapted from Ref. [95] with permission of WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Copyright 2005), assembled nanoparticles-conjugated Salmonella (e) (Adapted from Ref. [97] with permission of American Chemical Society, Copyright 2015), nanoparticles-loaded Listeria monocytogenes (f-g) (Adapted from Ref. [98] with permission of Nature Publishing Group, Copyright 2007), CdS quantum dots-decorated Moorella thermoacetica (h-i) (Adapted from Ref. [74] with permission of American Association for the Advancement of Science, Copyright 2016). TEM image of MOF monolayer coated on Moorella thermoacetica (j) (Adapted from Ref. [102]), Au nanoparticles-conjugated E. coli (k) (Adapted from Ref. [112] with permission of American Chemical Society, Copyright 2018), Au NPs-loaded E. coli bacterial membrane (l). Inset in (l): enlarged Au NPs-loaded E. coli bacterial membrane particle (Adapted from Ref. [117] with permission of American Chemical Society, Copyright 2015). SEM image of molecules-encapsulated Lactobacillus demi-bacteria (m), Inset, TEM image of the molecules encapsulated Lactobacillus demi-bacteria (Adapted from Ref. [116] with permission of Wiley-VCH Verlag GmbH & Co. KGaA, Copyright 2017).

MNPs) and chemotherapeutic drugs (e.g., doxorubicin) with diversified electrostatic properties could be encapsulated, realizing large hybridization amounts with great promises (Fig. 3e) [96,97]. Technically, physical attachment is the most facile methodology

to fabricate bacterial nanohybrids without difficulties. However, these bacterial hybrids may not feature sufficient physiological biostability when delivered into the comprehensive pathological environment in vivo.

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Chemical propagation From the material points of view, the conjugation linkages between nanomaterials and bacterial cells may not be strong enough by physical attachments where the intrinsic physicochemical properties of the bacterial cells are not fully exploited. Chemical propagation of the residual functional groups on the peptidoglycan structure of bacterial cells is another effective approach to fabricate nanohybrids based on bacterial cells (Fig. 3f–g) [98]. For instance, by exploiting the intrinsic primary amino groups on the bacterial cell membrane, carboxyl-functionalized nanoliposomes have been successfully conjugated onto Magnetococcus marinus strain MC-1 bacteria through carbodiimide crosslinkers, with which the carboxyl groups can be propagated and sensitized to react with the amides on MC-1 [99]. In another condition, in the presence of cysteine and transition metal ions precursors, a number of the bacterial species (e.g., E. coli, M. thermoacetica) are capable of synthesizing metal sulfide nanoparticles via in situ biomineralization (Fig. 3h–i) [74]. This approach strongly relies on the coordination affinity of the transition metal ions to the chalcogenide compounds during the synthesis and has successfully realized the fabrication of bacterial nanohybrids with CdS [100] and NiSx [101] nanoparticles with adequate homogeneity and yields. In addition to the hybridization with quasi-zero-dimension nanoparticles, two-dimensional metalorganic framework (MOF) monolayers have been conjugated via the coordination between the TA molecules and zirconium clusters in MOF (Fig. 3j) [102], and the formulated bacterial nanohybrids may feature the enhanced artificial photosynthesis and reduced cytosolic damages of the byproduct (reactive oxygen species) against the cells. The specific characters of bacterial antigen originated from the unique O-antigen conformations in lipopolysaccharides (LPS) also provide explicit chemical conjugation sites for nanomaterials [98,103]. This requires the design and preparation of the corresponding complementary bacterial monoclonal antibody (mAb). Most of the mAb contains abundant primary amides that are specifically reactive towards carboxyl or N-hydroxysuccinimide esters (NHS)-functionalized nanomaterials. In addition, upon mAb biotinylation, the bacteria-conjugated mAb could offer further conjugation sites with streptavidin or avidin decorated nanomaterials by avidin-biotin interaction, which is the most stable non-covalent interaction ever found in nature [104]. The abundant chemical propagation approaches enable diversified fabrications of nanohybrids thereafter. The present approach has been well demonstrated with remarkably high specificity and efficacy, rendering the preparation of bacterial nanohybrids with a great variety. Most recently, researchers have proposed another chemical propagation approach – copper-free click chemistry, to synthesize the bacterial nanohybrids. Specifically, such click chemistry is realized by the cycloaddition reaction between cycloalkyne and azides [105]. Nanomaterials of interests are initially functionalized with cycloalkyne-containing DBCO through the chemical conjugation of DBCO-based PEG derivatives (e.g., DBCO-PEG-NH2 ). For bacterial cells of interests, labeling of 2-azido-2-deoxy-d-glucose onto the bacteria results in the exposure of azide groups, which is highly reactive towards DBCO by copper-free click reaction [106]. The formulated bacterial hybrids are highly stable for further in vitro and in vivo application. Biological reconstruction Biological reconstruction of the bacterial nanohybrids is typically divided into two steps, genome recombination of the microbes and the hybridization with the nanomaterials. For decades, researchers have been able to reconstruct the genome of a bacterial strain through a plasmid introduction [107,108]. In a typical synthetic biological process, bacterial strains could be engineered with the introduction of polyhistidine-tag (His-tag)

expressing gene plasmid, rendering the polyhistidine expressions on ECS of the bacterial cells [109,110]. The His-tag thus provides intensive conjugation interactions towards positively charged Ni3+ and Co3+ coordinated nitrilotriacetic acid-functionalized nanomaterials such as gold nanoparticles and quantum dots [111]. Similar hybridization strategies can be achieved by the introductions of Spy-tag/SpyCatcher and other functionalized conjugation pairs (Fig. 3k) [112,113]. Although these methods can help to construct the bacterial nanohybrids with great generality, key technologies to manipulate the recombinant strains are required. The encapsulation methodologies to build bacterial nanohybrids also require the genome modification process. Prior to the nanomaterials encapsulation, the intracellular contents of bacterial cells are to be extracted, typically by the induction of gene E into the genome of a bacterial cell, during which the diffusions of both bacterial inner and outer membranes could be observed, leading to the complete extraction of the cytoplasm [81,114,115]. Another approach to realize the intracellular content extraction is biomineralization, as purposed by Ma and co-workers. After condition optimizations, they have prepared porous demi-bacterial with intact pathogenic morphology by a hydrothermal process and enabled the encapsulation of vaccines and adjuvants for tumor therapy. Upon engineering, although the biological activity has been deprived, these non-living pathogenic bacterial cells could still accomplish satisfactory nanomaterials delivery (Fig. 3m) [116]. Bacterial outer membrane vesicles (OMVs) are also important sources of bacterial-derived drug delivery systems. These OMVs can be isolated by a differentiated centrifugation and concentration process. These OMVs offer a facile approach in the effective delivery of nanoparticles of tens of nanometers in size to prepare the bacterial nanohybrids with high yield (Fig. 3l) [117]. Viral nanohybrids The virus is another family of microorganisms on the nanometer scale. A freestanding virus is supposed to be in the form of a particle, termed as “virions”, typically comprised of the genetic material (DNA or RNA molecules) with a protein coat (capsid) [118]. For some viral species, an additional lipid membrane is enveloped to maintain the shape and geometry of the virion. The viral capsid and envelope offer the external space of a virus and maintain major biological functions of protection, delivery of genome, as well as the interactions between the hosts [119]. To facilitate the controlled fabrications of viral nanohybrids, physiochemical properties of the viral capsid and viral envelope have been exploited. Different from the ECS of bacterial cells, viral capsid is made of numerous oligomer protein subunits with a variety of structure (e.g., icosahedral, prolate and helical), thereby enabling abundant residual chemical bonding to nucleic acids, peptides, small molecules, and nanomaterials via a number of surface groups such as carboxyl, amide, thiol, aniline and phenol groups [120,121]. In addition, the flexible viral capsid architecture also benefits the conjugation and encapsulation of the cargos into the interior of the viruses. Recent years have marked the advances in the fabrication of viral nanohybrids toward promising applications [122]. Chemical conjugation Direct conjugation of the viral capsid to nanomaterials has been the most facile methodology to prepare viral nanohybrids with high efficacy. Taking adenovirus as a typical paradigm, the lysate residue of adenovirus was initially coated with abundant maleimide groups by reacting to the surface amide bonds with sulfosuccinimidyl (4-N-maleimidomethyl)cyclohexane-1-carboxylate crosslinkers. The exposed sulfosuccinimidyl could then bind to thiolated manganese-doped iron oxide, yielding the integrated biohybrids that are rather stable in physiochemical conditions [123].

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Similar methods have been explored for the bioconjugation of gold nanoparticles [124] and quantum dots [125] onto MS2 bacteriophage capsid and Q␤ bacteriophage [126]. In fact, the residual functional groups on viral capsids could be effectively propagated through organic chemistry, endowing viruses with greater possibility for viral bioconjugation with chemotherapeutic drugs (Fig. 4a) [127], chromophores [128,129], proteins [130–132] and imaging contrast agents (Fig. 4b) [133] and nanomaterials (Fig. 4c–f) [123,125,134–137] at both the exterior and interior efficiently. In most cases, such incorporation could be made simply by mechanical mixing. The strong enough electrostatic interactions and abundant chemical bonding environment of the viruses provide critical biostability for the nanomedicines/nanomaterials that are conjugated and/or encapsulated, enabling exquisite cargo delivery. In several extended paradigms, amino acids on viral capsid were modified by genome recombination for enhanced bioconjugations. With the methods such as the introduction of cysteine or the expressions of polyhistidine tags, nanomaterials such as gold nanoparticles [138], QDs [139] and Fe3 O4 NPs [140] can be conjugated to form the viral nanohybrids.

cles via electrostatic interaction mediated by a poly(allylamine hydrochloride) (PAH) middle layer in between [158]. The S. cerevisiae-based nanohybrids could thereby favor the energyefficient production of the shikimic acid metabolite sustainably (Fig. 6). Featuring high biocompatibility, yeasts could be chemically engineered into empty cells (yeast capsule (YC), consisted of glucan, also termed as glucan particles, GP) by the treatments of alkaline, acidic and organic solvents [159], in which the cytoplasm of the yeast cell, as well as the polysaccharides on the cell wall, could be completely removed, facilitating the cargo encapsulations such as peptides, RNA, small molecules and nanomaterials [159]. In an excellent report, Aouadi and colleagues demonstrated that the encapsulation of siRNA into chemically extracted yeast capsules could effectively silence the genes in mouse macrophages, leading to the suppression of the systematic inflammation [160]. This report demonstrates macrophage-mediated guest transport, providing possibilities to employ yeast capsules for drug delivery against inflammatory diseases and even cancer [161].

Biotemplation Featuring the typical symmetric structure of the viral capsid, a number of viruses can serve as biotemplates for nanoparticles’ in situ chemical assemblies, which could be achieved by the incubation of virus templates in solutions containing desired metal precursor or functionalized nanoparticles. Several typical examples for such virus-directed biotemplations include the assembly of Co3 O4 and Au-Co3 O4 nanowires on M13 virus (Fig. 4g) [141], Pt NPs on tobacco mosaic virus (TMV) virus (Fig. 4h) [142] and Fe3 O4 NPs on M13 virus (Fig. 4i) [143]. The successful templating also relies on the physical and chemical interactions between viral capsid and assembled precursors and nanoparticles. Upon proper engineering of the viruses and nanomaterials, the viral nanohybrids could exert appealing applications in diverse fields.

Microbial nanohybrids for cancer therapeutics

Fungal hybrids Yeasts are unicellular eukaryotic microorganisms that belong to the fungus kingdom. By the well-established fermentation process, yeasts have been extensively applied in baking and alcoholic beverages. Due to the easy cultivation and genome modifications, Saccharomyces cerevisiae (S. cerevisiae), as the most important yeast species, has been widely investigated in genetics and cell biology [144–146]. The S. cerevisiae cells generally share an ellipsoid-like architecture (approximately 3−4 ␮m in diameter) containing nucleus, mitochondrion, and vacuole, surrounded by a characteristic negatively charged chitin cell wall [147,148]. Earlier attempts to fabricate the fungal hybrids mainly includes the in situ synthesis of the nanomaterials such as quantum dots [149,150] and metal oxide nanoparticles [151–153]. Upon the addition of cadmium and selenium precursors, the yeast cells could actively reduce the selenium precursors by the endogenous reductases at the subcellular level, forming Se-Cys complexes for cadmium ion combination. The in situ biosynthesis of CdSe quantum dots were further confirmed by the fluorescence images at yeast sites, validating a facile, and energy-saving and cost-effective approach to prepare quantum dots (Fig. 5a) [149]. While in another paradigm, chemical coating of the quantum dots (Fig. 5b) [154] or functionalized SiO2 shells (Fig. 5c) [155–157] onto yeast cells through the surface chemical bonding has been achieved recently. Furthermore, in a most recently published report, biologically engineered S. cerevisiae was hybridized with biocompatible polyphenol groups-functionalized InP semiconductor nanoparti-

Cancer has been regarded as a kind of genetic disease caused by a series of genetic mutations including the pro-oncogenes, tumor suppressor genes, and the DNA repair genes [162]. Upon carcinogenesis, cells proliferate exaggeratedly without regular apoptosis when a certain kind of cellular function is destructed. As the genetic mutations develop, these mal-functioned, unlimitedly proliferating cells will aggregate to form carcinoma. Corresponding signal transductions are thereby altered from normal cells substantially, dominating the cell fates of tumor cells [163,164]. With the highest morbidity and mortality, cancer has become one of the most critical problems in the public health of human beings. Cancer has been bringing substantial physiological sufferings, ecological burdens as well as psychological trauma to cancer patients for quite long time periods. Fortunately, continuous progress has been made by generations of scientists and researchers all over the world during recent decades. The physiological complexity of tumor has built intractable obstacles opposing therapeutics. It is conceived that traditional mono-modality therapy may be unable to provide ultimate solutions during the combat against cancer. In the explorations of pathogenic bacteria and viruses for tumor therapy, significant unbalanced therapeutic consequences and biotoxicity problems will result. In a general sense, a biocompatible dose of these pathogens may lead to attenuated tumor therapeutic effect, gaining the risks of tumor metastasis and multiple tumor reoccurrence. While fierce eradication by high amounts of these pathogens could inevitably cause infections towards healthy tissues and necessitates additional administrations of antibacterial and antiviruses agents. Furthermore, significant differences among individuals gain additional endeavors to the researchers who are exploring tumor bacteriotherapy and/or virotherapy. To solve this controversial issue, a combination of somewhat well-controlled bacteriotherapy/virotherapy and enhanced biocompatible nanomedical therapies is of promising perspectives for tumor treatments. Recent advances in nanofabrications shed light on the application of microbiotic nanohybrids, with which the demand to employ severely infectious living microorganisms can be eliminated, resulting in much reduced biotoxicity concerns. In addition, nanomaterials could be effectively targeted towards the tumor foci based on the natural tropism property and exert the multimodal therapeutics with high efficacy.

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Fig. 4. TEM images of Phenanthriplatin-loaded Tobacco Mosaic Virus (a) (Adapted from Ref. [127] with permission of American Chemical Society, Copyright 2016), polymersencapsulated P22 viral capsid (b) (Adapted from Ref. [133] with permission of Macmillan Publishers Limited, Copyright 2012), manganese-doped magnetism-engineered iron oxide NPs-conjugated adenoviruses (c) (Adapted from Ref. [123] with permission of Wiley-VCH Verlag GmbH & Co. KGaA, Copyright 2007), 10 nm Au NPs-assembled inside MS2 viral capsid (d) (Adapted from Ref. [136] with permission of American Chemical Society, Copyright 2013), iron oxide NPs-assembled Brome mosaic virus (e) (Adapted from Ref. [137] with permission of American Chemical Society, Copyright 2011), quantum dots-encapsulated Brome mosaic virus (f) (Adapted from Ref. [125] with permission of American Chemical Society, Copyright 2006), Co3 O4 nanowires-assembled M13 virus (g) (Adapted from Ref. [141] with permission of American Association for the Advancement of Science, Copyright 2006), Pt NPs-conjugated Tobacco Mosaic Virus (h) (Adapted form Ref. [142] with permission of Nature Publishing Group, Copyright 2006) and Fe3 O4 MNP-assembled M13 virus (i) (Adapted from Ref. [143] with permission of Nature Publishing Group, Copyright 2012).

Fig. 5. a, Mechanism of the in situ synthesis of CdSe quantum dots based on yeast cells. b, Fluorescence profiles of the CdSe QDs-yeast hybrids (Adapted from Ref. [149] with permission of Wiley-VCH Verlag GmbH & Co. KGaA, Copyright 2009). c, Synthetic mechanism of chemical conjugation of QDs toward yeast cells. d, Confocal image of QD585-yeast hybrids (Adapted from Ref. [154] with permission of Royal Society of Chemistry, Copyright 2014). e, TEM image of silica-coated yeast cells. f, Confocal image of Rhodamine-linked streptavidin-functionalized yeast@SiO2 (Adapted from Ref. [155] with permission of Wiley-VCH Verlag GmbH & Co. KGaA, Copyright 2011).

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Fig. 6. a, Schematic illustration for the preparation of InP hybridized S. cerevisiae cells. b, Shikimic acid production performance of the yeast nanohybrids with or without light irradiation. c, TEM images of the microstructure of InP hybridized S. cerevisiae cells (Adapted from Ref. [158] with permission of American Association for the Advancement of Science, Copyright 2018).

Principles of nanohybrids for tumor therapeutics The complicated in vivo tumor physiological environment in the human body have called for the dedicated fabrication and functionalization of microbiotic nanohybrids. The primary consideration in nanohybrid fabrication would be their biocompatibility and biotoxicity which determines their clinical potentials. Since most of the pathogenic microbes are severely infectious, strategies such as attenuation and deactivation are needed prior to the hybridizations with nanomaterials. In addition, biocompatible nanomaterials are introduced to guarantee the non-toxicity of the overall nanohybrids. In such nanohybrids, the therapeutic effects are dominantly contributed by the integrated nanomaterials, while the low-infectious or non-pathogenic microbes play roles of both carriers and targeting units, for well-balanced therapeutic outcomes. These nanohybrids may synergize the multimodal therapeutics of the nanomaterials and special biological functionalities of the microbes with satisfied biocompatibility. The second concern comes from the integrity of the nanohybrids. Upon systematic administration during in vivo evaluations, the nanohybrids would travel through inside the vascular system of high salinity and abundant proteins before they could finally reach the targeted destination (e.g., tumor foci). The nanohybrids may disintegrate if they are not firmly conjugated, resulting in the separate microbes and nanoparticles, dysfunctioning the therapeutic and diagnostic potentials. Based on this consideration, nanohybrid fabrication by strong physical attachments, chemical conjugations as well as biological reconstructions are highly preferred for the nanohybrid constructions with required stability and integrity. Furthermore, the nanomaterials-microbes interactions specifically raise the consideration of the conjugation efficiency and distribution homogeneity. The homogeneity of the nanomaterials’ distribution within/around a microbe may subtly affect the

responses of nanohybrids to external stimulation such as magnetic field, electric field, laser irradiation, and ultrasound stimulation. Together, with the high load amount and homogenous distribution of the nanomaterials, such microbiotic nanohybrids would offer an expected therapeutic perspective for tumor treatments. One of the most critical considerations is the synergetic functionalities of the fabricated microbiotic nanohybrids, which determine the ultimate antitumor outcomes based on tumor targeting, tumor penetration and tumor therapeutic performances in a synergetic way. In both scientific research and clinical practices, tumor targeting is the most important factor to guarantee the tumor accumulation of nanomedicine. For microorganisms, several pathogenic bacteria have been demonstrated to intrinsically possess remarkable tumor accumulation performance. Other non-severely pathogenic bacteria may travel under the guidance by the applied external fields, rendering the controlled motion and delivery of the nanohybrids. As for viruses, few of them feature intrinsic tumor-targeting tropism. However, by means of the genome recombinant, numbers of the viral genera (e.g., adenovirus, reovirus and herpes simplex) have been successfully engineered to preferentially infect and kill tumor cells by oncolysis. These oncolytic viruses have shown satisfactory antitumor performance in specific types of cancer such as skin melanoma, head and neck tumors. Effectual clinical trials have been demonstrated. A few in vivo pharmacokinetic investigations of other microbes such as yeasts and microalgae have demonstrated the least tumor pathological targeting performances. However, the integration of these microbes with nanomaterials is still appealing in oral administration modalities. Another issue in this consideration to be solved is the deep tumor penetration. On accounts of the aggressive growth of the tumor xenografts with chaotic vasculature, most of the chemotherapeutics and nanomedicines are not capable of penetrating through

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the deep-seated interior of the xenografts, which deteriorates the overall therapeutic outcomes and could even induce severe tumor metastasis. In this context, the passive delivery of the most common nanomaterials along with the bloodstream would benefit no deep penetration. Researchers have recently developed self-propelled Janus nanoparticles (e.g., Pt-containing Fe3 O4 nanocubes) for stimuli-responsive locomotion favoring their tumor-specific accumulation. However, such chemical-reaction based locomotion only results in accelerated motion of nanoparticles therefore it is random and non-directional. While microbes are biologically active, they could actively penetrate deep inside the tumors upon the application of external stimulated chemical and magnetic signals. In addition, some of the microbes are equipped with flagellum, a whip-like appendage that could provide the motive engine to gain better penetration. With that, large malignant tumors could be expected to be completely eradicated with better prognosis by the employment of such biologically active microbial nanohybrids. Considering the therapeutic perspectives of microbial nanohybrids, synergistic therapeutic effects between microorganisms and nanomaterials are conceived to regress malignant tumors with multimodalities. Regarding the nanomedicine side, several modalities are extensively explored and studied to induce tumor cell death. The effective intratumoral generation or accumulation of reactive oxygen species (ROS) is one of the effective methods to cause tumor cell apoptosis. Approaches to elevate the levels of ROS include Fenton reaction-based nanocatalytic medicine, radiotherapy, and photodynamic therapy, with the applications of certain types of nanomaterials, e.g., Fenton-catalytic nanoreagent, radioactive nanomaterials, and semiconductors. Highly intensive external stimulation (e.g., heat, ultrasonic and radiation) could also lead to irreversible tumor cell death. With the microorganism counterparts, tumor immunotherapy is a most recently emerging modality for cancer treatment, in which immune cells are effectively activated to eliminate cancer cells. For instance, injection of Salmonella bacteria is supposed to stimulate the nearby immunosystem to excrete tumor necroptosis factor (TNF) and other cytokines to destruct the tumor cells. Other pathogenic microbes may able to regulate the tumor immune microenvironment, benefiting the overall the synergetic therapy with nanohybrids. These microbiotic nanohybrids may offer much diversified and effective therapeutic possibilities for tumor treatments. Bacterial nanohybrids for tumor therapeutics In the middle of the 19th century and early 20th century, a few physicians and doctors realized the tumor therapeutic potentials using bacterial cells. In 1868, pioneer clinical observations based on pathogenic bacteria was initially reported by physician W. Busch, who surprisingly found significant tumor reduction of his sarcoma patients after infection of Streptococcus pyrogens [64]. Inspired by this observation, physician W. B. Coley elaborately purified and injected Serratia marcescens into the tumors of four of his sarcoma patients. Although two of the patients died of severe infection later, the other two patients, fortunately, gain satisfactory tumor suppression outcomes. Coley then improved the receipt through heat attenuation of the bacterial cells (known as Coley’s toxins) [64,65] and succeeded in several tumor treatment cases thereafter. Although Coley’s toxins suffer from unstable therapeutic consequences, the controversial attempts have aroused numerous studies on the possible mechanistic insights of bacterial-based tumor therapy. In the middle of the 20th century, researchers found that several genera of obligate anaerobe (e.g., Clostridium [165,166], Bifidobacterium [167]) showed specific distribution to tumor region rather than other to normoxic organs upon injection into the human body. This phenomenon was explained by the hypoxia-targeting nature of these injected bacterial spores

toward tumors with low oxygen tensions. In addition, based on the abundant tumor vasculature entrapment, chemotaxis guidance and immune escape properties, effective tumor targeting and accumulation behavior were observed for a few facultative anaerobes (e.g., Salmonella, Escherichia) as well. These studies raise the interests of researchers to seek better tumor therapeutics based on bacteria. The emergence and development of nanoscience and nanotechnology have provided fundamental approaches to manipulate or engineer bacteria with nanomaterials on the cellular level, making bacterial nanohybrids the most extensively studied nanomedicine in tumor therapeutics in the last decades. In exploiting the natural tropism of the bacterial cells, proteins and drugs could achieve much higher accumulations inside the tumor than those without the bacterial cells, maximizing the molecular imaging and chemotherapeutic results. Upon proper functionalization, nanomaterials could be hybridized with the cells to generate multiple therapeutic modalities against tumors. Furthermore, the synergetic immune effect induced by the bacterial nanohybrids have received extensive attention, promising the clinical translation based on the designed bacterial nanohybrids Drug delivery Pioneer research on the fabrication of bacterial nanohybrids was accomplished by Akin and co-workers [98]. They innovatively coated the bacterium Listeria monocytogenes with fluorescein and bioluminescent gene-conjugated nanoparticles via streptavidinbiotin interactions. The synthetic nanohybrids can be effectively internalized into cells and prevent the entrapment via phagocytosis by the release of the bacterium toxin (listeriolysin O). Bright fluorescence of the fluorescein and the bioluminescence of the expressed proteins could be observed both in vitro and in vivo, implying the fluorescence-guided therapeutic potentials (Fig. 7). This prototypical bacterial nanohybrids promise the possibility of bacteria-based nanoparticle delivery. The applications could be further extended to the employment of other therapeutic entities, such as peptides, antibodies, and small molecular drugs. In another report, similarly through streptavidin-biotin interactions, quasi-monodisperse double emulsions with fluorescence dyes being encapsulated inside were hybridized onto the motile E. coli bacterium. With an exquisitely designed transwell membrane experiment, these nanohybrids were proved to be able to swim across the membrane and realize dye delivery with a variety of choices. This work clearly implicates the feasibility of targeted transport and delivery by the application of nanohybrids [168]. Expectedly, E. coli and Salmonella-based nanohybrids have been proved to show significant targeting performance towards tumors owing to their native tropism. In clinics, magnetic guidance is of substantial significances to benefit the therapy. Therefore, such a tropism feature could be further enhanced and regulated by integrating Fe3 O4 MNPs, with which magnetic-directed vectorizations and magnetic resonance imaging could be realized. A feasible paradigm has been demonstrated in polyelectrolyte multilayer (PEM) microstructures-conjugated E. coli-based nanohybrids [96]. During the layer-by-layer (LbL) PEM fabrication, chemotherapeutic DOX, and Fe3 O4 MNPs were encapsulated inside. The formulated nanohybrids were found to perform both chemotaxes (along with ␣-methyl-dl-aspartate) and magnetic guidance towards the tumor cells, achieving the dual-targeting (bacteria-directing and magnetic-driven) drug delivery and release in the in vitro investigations (Fig. 8) [96]. Cyanobacteria is a type of photoautotrophic bacteria produced by a photosynthesis process, containing branches of species. Spirulina platensis (S. platensis) is a subspecies of cyanobacterium Spirulina, with a typical helical structure. Zhang’s groups have reported the preparation of magnetite nanostructured porous hollow microhelices by depositing the metal precursors (typically Fe3+

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Fig. 7. a, Schematic illustration of multiple nanoparticles-conjugated bacterial nanohybrids through biotinylation and surface-antigen interactions. b, Merged confocal images of bacteria cell (blue) with Texas red nanoparticles (red) and FITC nanoparticles (green). c, The simulated height profile according to the merged confocal image. d, Confocal images of the bacterial nanohybrids-internalized cells. e, Bioluminescence profile of luciferase gene expressions of mice treated with PBS (for animal 1-3) and microbots (animal 4-6) (Adapted from Ref. [98] with permission of Nature Publishing Group, Copyright 2007).

Fig. 8. a, Schematic illustration of the conjugated PEM-MNP on E. coli bacterium. b, Thickness profile of PEM upon the sequential coating of the polyelectrolyte layers. c, TEM image of a PEM-MNP-E.coli nanohybrid cell. d, SEM image of a PEM-MNP-E.coli nanohybrid cell. e, DOX releasing profiles of PEM-MNP-E.coli nanohybrids under different pH conditions. Swimming profiles of the nanohybrids in the absence (f) and presence (g) of the magnetic guidance (Adapted from Ref. [96] with permission of American Chemical Society, Copyright 2017).

and Fe2+ ) towards S. platensis in a solution, followed by subsequent annealing and particle reduction. The superparamagnetic microhelices feature interesting swimming properties upon the exposure to a low-strength rotating magnetic field (i.e., a forward velocity of 64.75 ␮m s−1 upon 10 Hz rotating magnetic frequency). Besides, steady cargo (Au nanorods and RhB dye) delivery performance of the nanohybrids has been demonstrated. With excellent cytocompatibility, the synthetic spirulina-based microhelices have been regarded as a potent, versatile nanohybrid for in vivo biomedical applications (Fig. 9a-b) [169]. In a later report, the authors further developed the Fe3 O4 -S. platensis biohybrid for tumor visualization. The bimodal imaging performances of the biohybrids (bacterial autofluorescence and magnetic resonance contrast imaging) have endowed the biohybrid with an attractive precise tumor positioning function (Fig. 9c–e) [170].

Biomagnetization is an improved magnetic guidance modality in bacterial nanohybrids. Such performance involves the application of biologically active and magnetically taxis bacteria for nanomedicine delivery. Magnetococcus marinus, for instance, is a magneto-taxis species of alphaproteobacteria due to the existence of the magnetosomes inside the cells formed by biomineralization. With these magnetosomes, the strains can adapt their orientations along with the Earth’s geomagnetic field [171]. Such a magnetictaxis property can be exploited by applying an external magnetic field for their motion control. In an interesting report, the Magnetococcus marinus strain MC-1 has been manifested to feature deep penetration property, as revealed by the strong fluorescence of MC-1 at a deeper solid tumor region compared to the weak fluorescence of biologically inactive microspheres (Fig. 10a). Such a deep penetration profile of the bacteria then endows the nanohybrid with intriguing drug delivery performance. Through

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Fig. 9. a, FESEM image of Au nanorods-loaded S. platensis nanohybrids. b, Time course Rhodamine B fluorescence decay profile of RhB-loaded S. platensis nanohybrids revealing the corresponding cargo release profile (Adapted from Ref. [169] with permission of Wiley-VCH Verlag GmbH & Co. KGaA, Copyright 2015). c, Schematic illustration of the preparation of Fe3 O4 NPs-S. platensis nanohybrid. d, Schematic illustration of the in vivo magnetic-guided nanohybrids accumulation. e, Cross-section MR images of a rat’s stomach collected in 5 min and 12 min post-injection of nanohybrids per os (Adapted from Ref. [170] with permission of American Association for the Advancement of Science, Copyright 2017).

Fig. 10. a, TEM image of MC-1 cells identifying the magnetosomes inside. b, SEM image of MC-1 cells hybridized with SN-38 drug-loaded liposomes. c, Confocal images of the slices at varying distances away from the injection site demonstrating the MC-1 penetration and accumulation behavior. Microsphere group was employed as a control to eliminate the shape effect of the penetration. d, Confocal images showing the bacterial population of the dissected tumor slices at varying distances along with the injection site (Adapted from Ref. [99] with permission of Nature Publishing Group, Copyright 2016).

carbodiimide chemistry, CN-38 drug-loaded liposomes-hybridized MC-1 cells were demonstrated to have preserved the dominant penetration profiles towards the hypoxic regions of solid tumors under the external magnetic field guidance by tumor vectorization (Fig. 10b–d) [99]. Photoacoustic (PA) imaging is an ultrasound-based molecular imaging approach on the basis of the photoacoustic effect. In as early as 2014, biogenic gas vesicles (GVs) derived from Halobacteria NRC-1 and Anabaena flos-aquae, two typical species of bacteria and microalgae, have demonstrated great potentials in photoacoustic imaging [172]. GVs are protein-shelled and gas-filled vesicles with diverse dimensions that are extracted from the microbes. Under different ultrasound frequencies, the GVs suspensions generated dose-dependent PA signals while the collapsed GVs suspension only showed background signals in comparison (Fig. 11). The present paradigm offers biocompatible and promising molecular imaging reporters for high-performance PA imaging both in vitro and in vivo [172]. Biologically active bacteria-based tumor vectorization is the most attractive factor in tumor therapeutics as the therapeutic results substantially depend on the drug or nanomaterials accumulation and penetration. Besides the natural tropism toward tumors, magnetic nanoparticle hybridizations result in the highly efficient magnetic targeting, while the employment of magneto-taxis bacte-

ria could further enhance such a guidance performance. Together, these paradigms demonstrate the nanohybrids’ motion enabled tumor vectorization and promoted drug delivery, paving the fundamentals for multimodal tumor theranostics.

Hyperthermia therapy Photothermal therapy is one of the most minimal invasive nanomedical therapy based on the photothermal conversion effect of the nanomaterials upon laser irradiation [173–175]. Destructive hyperthermia damages may induce necrosis of the tumor cells effectively. However, considering the relatively low EPR effect, insufficient nanomaterials accumulations may lead to limited hyperthermia outcome. In exploiting the natural tropism property of bacterial cells, photothermal therapeutic performance based on nanohybrids could be maximized due to the enhanced nanomaterials accumulation at tumor foci. This strategy can guarantee the hyperthermia outcome and therapeutic efficacy effectively. Under such advantages, Chen and colleagues have innovatively coated the anaerobe Salmonella strain VNP20009 with polydopamine through a single-step in situ self-polymerization method, forming photothermal-active bacterial nanohybrids pDAVNP (Fig. 12a-b) [176]. When the anaerobe pDA-VNP hybrids were injected through tail-vein during in vivo experiments, the anaerobe pDA-VNP hybrids could actively travel to the hypoxic

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Fig. 11. a, Schematic illustration of GV revealing the dimensions and the contents. b-c, TEM images of gas vesicles derived from Halo (b) and Ana (c) bacterial species. Left panel, intact GVs, right panel, collapsed GVs. d-e, PA signals of GVs suspensions at different concentrations under ultrasound frequencies at 4.8 MHz, 8.6 MHz and 17 MHz (Adapted from Ref. [172] with permission of Nature Publishing Group, Copyright 2014).

Fig. 12. a, Schematic illustration of the combination of photothermal therapy and biotherapy using pDA-VNP nanohybrids. b, SEM image of pDA-VNP nanohybrids. c, Photothermal performance of pDA-VNP nanohybrids with varied concentrations under the irradiation of 808 nm laser. d, Bacterial hybrid distribution in tumor and different major organs in 4 days post-treatment. e-f, TNF-␣ and IL-4 levels in mice serum in control and different experiment groups sampled on day 1, 4, 8 and 20 (Adapted from Ref. [176] with permission of American Chemical Society, Copyright 2018).

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Fig. 13. a, Schematic illustration of the biomineralization approach to synthesize Au NPs-coated temperature-programmable bacteria. b-c, TEM images of TPB@Au at low magnification (b) and high magnification (c). d, Photothermal profiles of TPB@Au at varied concentrations under the irradiation of 808 nm laser. e, Western-blotting diagrams of the expressed proteins of tumor tissues in control and different treatment groups. f, In vivo tumor development profiles of mice in control and varied treatment groups (Adapted from Ref. [178] with permission of American Chemical Society, Copyright 2018).

tumor region (Fig. 12d). Upon NIR laser irradiation, a prominent tumor localized temperature increase could be observed (Fig. 12c). In addition, the fragmentations of the tumor cells were then believed to further promote the immune responses in tumor foci, as evidenced by the increased concentration of both TNF-␣ and IL-4 cytokines (Fig. 12e–f). This work provides a synergetic paradigm of photothermal-based and hypoxic-targeting biotherapy for B16F10 tumor xenografts eradications [176]. Combining with the recently translated cancer immunotherapy facilitated by the PD-1 blockade approach, Chen and co-workers demonstrated the pDA-VNP hybrids-enabled and integrated PTT therapy and the immunotherapy by anti-PD-1 peptide depot, AUNP-12. With a sustained peptide releasing profile, malignant tumors were demonstrated to be immunopermissive within a board period, leading to prominent destruction [177]. In another recent report, Fan and coworkers designed an advanced bacterial therapeutic nanohybrids based on recombinant thermal-sensitive programmable bacteria E. coli MG1655 strain [178]. With the successful gold nanoparticle hybridization and TNF-␣ expressing plasmid transduction, such bacteria could express and then excrete the therapeutic protein TNF-␣ upon localized heat generation by Au NPs upon NIR irradiation. In addition, the orally administrated bacterial nanohybrids could be traversed into the internal microcirculation system by M cells transcytosis. The bacterial cells then effectively accumulate inside the tumor and generate the therapeutic effect upon local laser irradiation (Fig. 13). This paradigm highlights the combination of nanomedicine and bacteria recombinant technology during tumor therapeutics [178]. The administration methodologies of the bacterial nanohybrids have attracted the attention of numbers of researchers. The accumulation and therapeutic results of two administration modalities were compared. It has been found that instead of the injection of direct cargo-carrying anaerobes, the antibody-conjugated nanoparticles could effectively target the bacterial spores administrated separately, thereby realizing the successful retention and deep penetration of the nanohybrids. Imparted by the upcon-

version properties of the conjugated NaYF4 nanoparticles and photothermal performance of Au nanorods, these biohybrids have been proved to achieve satisfactory tumor eradication outcome upon the near-infrared laser irradiation [179]. In an improved paradigm, a combination strategy of photothermal therapy and chemotherapy using the bacterial nanohybrids was also reported. Specifically, Wang and co-workers deposited the Pd@Au nanoparticles onto Fe3 O4 NPs-coated Spirulina biotemplates, followed by subsequent encapsulation of doxorubicin chemodrugs. The constructed (Pd@Au)/Fe3 O4 @Sp nanohybrids featured strong responses under the presence of magnetic fields, realizing the effective drug release. Synergetic photothermal and chemotherapeutic performance of the fabricated bacterial nanohybrids has been confirmed on the cellular level [180]. Magnetic hyperthermia is another way to generate localized hyperthermia for tumor destruction with the help of the magnetic field. In a recent report, magnetotactic bacteria Magnetospirillum gryphiswaldense has been shown to present high therapeutic potential against tumors via the magnetic hyperthermia. The intracellular biomineralized magnetic nanoparticles enabled a temperature increase by ca. 12.5 ◦ C of the bacteria suspension upon the application of an external magnetic field (600 Oe). In addition, these bacterial cells can be easily internalized into the human lung tumor A549 cells, making these bacterial cells particularly feasible for effective tumor magnetic hyperthermia therapeutics [181]. Owning to the bacterial tropism and magnetic guidance of the nanohybrids, tumor-accumulated nanomaterials-based hyperthermia therapy is quite attractive as it will generate tumor eradication outcomes. The combination of the photothermal performance with other therapeutic modalities based on bacterial cells is also highly appealing in future research. Chemodynamic therapy Chemodynamic therapy (CDT) is a recently proposed tumor therapeutic modality based on the chemical radical generating reactions [33,34]. Typically, CDT can be realized by intravenous

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delivery of Fenton nanocatalysts into the tumor region, where the nanocatalysts could catalyze the Fenton reaction in the presence of H2 O2 under tumorous mild acidic microenvironment. To further enhance the CDT efficacy, several useful strategies have been proposed and demonstrated, including the concentration elevation of the chemical reactants such as H2 O2 , and the enhancement of the catalytic activities of the nanocatalysts. However, observations have indicated that even with these particular strategies, tumor accumulation of the Fenton catalysts is still the primary determining factor during chemodynamic therapeutics. In a very recent report, Fan and colleagues designed genetically modified E. coli bacterial cells through synthetic biology approach, upon which the E. coli bacterial cells could actively express the respiratory chain enzyme II (NDH-2) [182]. This enzyme is mainly responsible for the electron transfer from NADH to oxygen, leading to the overproduction of H2 O2 [183,184] and subsequently enhanced the intracellular level of H2 O2 (Fig. 14a–b). Furthermore, the genetically modified E. coli (Ec-pE) were conjugated with Fe3 O4 nanoparticles as the Fenton catalyst by surface carbodiimide chemistry. The formulated nanohybrids could then achieve excellent Fenton chemistry in the presence of self-supplemented H2 O2 , producing large quantities of hydroxyl radicals (Fig. 14c). Based on the successful immune evasion and native tropism of the engineered nanohybrids, significant tumor suppression could be effectively achieved (Fig. 14d) [182]. Immunotherapy Bacterial nanohybrids could also be applied in vaccination therapeutics. In a typical paradigm, vascular endothelial growth factor receptor 2 (VEGFR2) encoding DNA nanovaccine was coated onto the attenuated Salmonella bacterium by electrostatic interaction, inducing the substantial immune response (T-cell activation and cytokine production) at tumor-site by oral administration of the microbiotic nanohybrids [97]. This study demonstrates a highly attractive bacteria-based vectorized immunotherapeutic strategy for cancer treatment. In a recent report, tumor antigen- and adjuvant-accommodated hollow porous bacterial pathogens were established and used for tumor therapeutics and vaccinations [116]. The authors hydrothermally treated and engineered the Bacillus bacterium as carriers for cytidine-phosphate-guanosine (CpG) and Ovalbumin (OVA) molecules, resembling bacterial pathogens to enhance antigen delivery/presentation and immune activations. Upon the optimized hydrothermal process, the intracellular content of Bacillus was eliminated while the extracellular structure was transformed into porous nanoarchitecture to form a drug delivery nanohybrid (Fig. 15a–b). With the enhanced vaccination properties, robust cellular and humoral responses could be activated. Effective tumor therapy, antimetastasis and satisfied tumor prophylaxis can be achieved by the applications of the nanohybrids (Fig. 15c-d) [116]. Viral nanohybrids for tumor therapeutics The mid-1950s has marked the emergence of the potentials of virus-based therapy, as cancer patients who had been vaccinated recently showed signs of tumor suppressions, probably due to the productions of interferon and tumor necrosis factors [185]. The phenomenon has attracted the attention of researchers to develop therapeutic functionalities of viruses against the tumor. One of the discoveries is contributed by oncolytic viruses (OV). These OVs can effectively infect and kill cancer cells by oncolysis with high selectivity. In addition, they could also stimulate the hostantitumor immune mechanism to participate in combat. Clinically approved OVs include herpes simplex virus and Oncorine (H101). Other contributions of virus-based tumor therapy are the developments of viral gene therapy and viral immunotherapy based

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on the genome recombination technology. In exploiting the gene delivery and infection functions, tumor suppressor genes (e.g., anti-angiogenesis genes) recombinant viruses could be employed to destruct tumors. As for the viral immunotherapy, viruses are genetically engineered to express specific antigens (e.g., cancer antigens) that could activate the host immune system. Based on this approach, the immune responses could be properly enhanced, inducing considerable cancer-killing performance. When viral functionalities are combined with nanoparticulate therapeutics, effective synergetic therapeutic efficacy could be obtained. As instructed in the basics of the material, due to the presence of loosening proteinous architecture exposing abundant surface functional groups, the virus is a promising candidate to form viral nanohybrids at the positions of both inside the viral interior and outside the viral capsid. With the intact crystalline symmetry, viruses are also potent in the synthesis and assembly of certain kinds of nanomaterials in a predetermined patterned way, offering specifically functionalized nanomaterials in the research areas such as electrocatalysis, photocatalysts, and nanomedicine. It is well-known that virus is the severely infectious pathogens which can biologically and exaggeratedly replicate themselves inside the hosts of a variety of life forms including human, animals, plants and even bacteria. Upon infection, viruses could attach to the surfaces of the host cells and inject their genetic materials inside. The genetic DNA and/or RNA could subsequently invade the cell nucleus and dominate the host cell fate. Exploiting the nutrients (typically carbon, nitrogen and phosphate) inside the host cell, the genetic materials replicate and assemble to form many more virions, disrupting the host cells and heading for the next infection cycle. Natural tropism and genetic materials invasion are two critical processes for viral infection. With the help of the natural tropism process, the viruses could be engineered as a fundamental drug delivery system for cargo delivery, such an active cell targeting performance may significantly enhance the local accumulations of drugs and nanoparticles, probably resulting in enhanced therapeutic performance. On the other hand, the recombinant genetic invasion is one of the curing modality based on gene therapy. When these viruses are hybridized with the nanomaterials, concentrated nanomedicines could be effectively produced, triggering the extensive interest for the controlled fabrications of specific viral nanohybrids. This section summarizes the recent progress of the virus-based nanohybrids in the promising application of tumor imaging and therapy. Drug/nanomedicine delivery As a biologically active drug delivery system, viruses with different three-dimensional architectures have offered plenty of choices for guest molecules encapsulation and delivery. Featuring no infection issues and high plasma circulation time, the plant virus, TMV is the most popular species for potential biological applications in tumor therapy [186,187]. To realize the drug delivery potentials using TMV, Anna and colleagues have facilely infused Phenanthriplatin, a platinum-based chemodrugs, into the TMV interior through the electrostatic interactions to fabricate the viral nanohybrids (Fig. 16a–b). The as-synthesized PhenPt-TMV nanohybrids exhibited acidity enhanced drug release, thereby favorable for the self-triggered tumor therapeutics (Fig. 16c). During the in vivo experiment, the authors observed significantly promoted viral nanohybrid accumulations at tumor sites compared to the maldistribution in liver and kidney, achieving the dominant tumor suppression of MDA-MB-231 xenografts (Fig. 16d–e) [127]. Later efforts demonstrated that the TMV nanoparticles can serve as delivery systems for a variety of chemodrugs [188] as well as the target designations to elevate the tumor accumulation efficiency [189]. Bacteriophage MS2 is an isometric non-enveloped virus that belongs to the genus Lentivirus, and has been extensively engi-

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Fig. 14. a, Schematic illustration of the therapeutic mechanism of Fe3 O4 nanoparticles-conjugated genetically engineered E. coli for tumor Fenton therapeutics. b, TEM image of the bacterial cells indicating the successful conjugation. c, ESR spectra of Fe(II) and Fe3 O4 nanoparticles in the absence or presence of H2 O2 revealing the hydroxyl radical production by Fenton reaction. d, in vivo tumor growth curves of Ec-pE + Ec-pE@MNP- and their material counterparts-injected tumor-bearing mice (Adapted from Ref. [182] with permission of Wiley-VCH Verlag GmbH & Co. KGaA, Copyright 2019).

Fig. 15. a, Preparation scheme of antigen- and CpG-encapsulated demi-bacteria. b, Confocal images of bacterial cells revealing the successful encapsulations of OVA and CpG. c-d, Expression levels of SIINFEKL-MHC1 and CD40 on dendritic cells when incubated with different material counterparts. e, Tumor growth curves of the nanohybrids- and their material counterparts-administrated tumor-bearing mice (Adapted from Ref. [116] with permission of Wiley-VCH Verlag GmbH & Co. KGaA, Copyright 2017).

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Fig. 16. a, Material design and drug encapsulations of PhenPt-TMV viral nanohybrids. b, TEM images of PhenPt-TMV viral nanohybrids. c, pH-dependent drug release profiles of PhenPt-TMV. d, Ex vivo fluorescence profiles of major organs and tumor dissected from tumor-bearing PhenPt-TMV administrated mice. e, Tumor growth curves of PhenPt-TMV-based tumor therapeutics (Adapted from Ref. [127] with permission of American Chemical Society, Copyright 2016).

neered as viral nanohybrids for tumor diagnosis and therapeutics as well. Nicholas and colleagues performed dual-surface engineering of the MS2 virus for constructing photosensitizer-encapsulated viral nanohybrids [190]. On one hand, a maleimide groupsfunctionalized photodynamic nanoagent porphyrin was employed to bind the interior cysteine-87 coat protein via sulfonamide interactions. On the other hand, phenylamine residues seated outside the viral capsid were exploited to conjugate the DNA aptamer via oxidative coupling reactions (Fig. 17a–b). Thanks to the aptamer targeting conjugation, the overall DNA-aptamer functionalized porphyrin-encapsulated MS2 nanohybrids were developed to selectively accumulate into the Jurkat leukemia T cells rather than erythrocytes (Fig. 17c–d). Upon 415 nm laser irradiation (Soret band of porphyrin), the photodynamic performance of the viral nanohybrids effectively led to the death of Jurkat cells rather than the U266 control cell line, as confirmed by the flow cytometry analysis (Fig. 17e–h). The dual functionalizations of the viral capsid endowed the nanohybrids with multi-functionalities (impressive targeting and therapeutic consequences) and high biocompatibilities [190]. In another dual-surface engineered MS2 nanohybrids paradigm, a variety of cargos such as quantum dots, chemodrugs, ricin toxin A-chain could be effectively conjugated into pac site RNA with proper conjugation crosslinkers [135]. These pac site-modified cargoes could then trigger in vitro self-assembly with siRNA into the interior space of the MS2 virus. Furthermore, these engineered viruses could then be modified with an HCC targeting peptide (SP94) and a fusogenic peptide (H5WYG), forming multicargo-encapsulated viral nanohybrids (Fig. 18a). As revealed by the confocal microscopic image, the respective fluorescence of the cargo-integrated nanohybrids could be observed (Fig. 18b). Based on the targeting profiles, the cytosolic distribution of the viral nanohybrids into Hep3B cells rather than the hepatocytes could be confirmed, demonstrating the effective targeting performance (Fig. 18c–d). This work demonstrates the multivalent conjugations of the MS2 virus using a variety of nanomaterials, chemotherapeutic drugs as well as the targeting peptides,

realizing versatile tumor targeting and drug delivery functions [135]. Recently, Benjamin and colleagues found that the bacteriophage Q␤ virions could be suitable for nanoparticle in situ synthesis, particularly the gold nanoparticles. This is possibly attributed to the abundant disulfide bonds located in the protein secondary structure on the viral capsid, offering the adsorption sites for the in situ reductions of gold ions. In addition, the viral nanohybrids feature a laser-triggered drug release performance, validating the laserresponsive cell-killing effect in vitro (Fig. 19) [191]. Molecular imaging M13 filamentous bacteriophage is an E. coli infecting rod-shaped virus and has been frequently explored for enhanced tumor diagnosis. In previous works, M13 virus was genetically engineered with multiple glutamic acid residues [192] and secreted protein acidic and rich in cysteine (SPARC)-binding peptide (termed as SBP) [193], respectively by colleagues of Belcher and Kelly, rendering the M13 virus overall negatively charged and a SPARC-overexpressing tumor-targeting performance. In a representative work in 2012, these authors corporately assembled Fe3 O4 MNPs with the engineered M13 virus in a filamentous orientation to form nanohybrids (M13-SBP-MNP) (Fig. 20a–b) with superior MR imaging performance (Fig. 20c) [143]. Selective tumor-targeting performance of the nanohybrids was confirmed among the SPARC-overexpressing tumor cell line (C4-2B) and non-SPARC-expressing tumor cell line (DU145). According to the MR images of in vivo tumor-bearing mice, substantially enhanced T2 -weighted contrasts signals could be observed for C4-2B tumors in 24 h post-injection of the nanohybrids, while for DU145 tumors, no magnetic resonance signals could be observed (Fig. 20d–e) [143]. In addition to the M13 virus used for the targeted Fe3 O4 MNPs MR imaging, the M13-SBP virus was also employed to hybridize with single-walled carbon nanotubes (SWNTs) for NIR-II fluorescence-based in vivo deep, disseminated tumor visualization (Fig. 21a) [194]. With enhanced NIR-II fluorescence photostability, the SBP-M13-SWNTs nanohybrids were observed to enable

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Fig. 17. a, Schematic illustration of the dual-surface engineering of the MS2 virus. b, UV–vis spectra of porphyrin-encapsulated MS2 viral nanohybrids. Inset, TEM image of the MS2 viral nanohybrid. c, Percentages of dead cells when Jurkat or U266 cells were treated under different conditions. d, Optical image revealing the selective accumulation of viral nanohybrids into Jurkat cells rather than erythrocyte. e-h, Flow cytometry results of relative fluorescence intensities of FITC-stained apoptotic Jurkat cells (e) and U266 cells (f) and PI-stained compromised Jurkat cells (g) and U266 cells (h) (Adapted from Ref. [190] with permission of American Chemical Society, Copyright 2010).

sensitive in vitro detection for OVCAR8 ovarian cancer cells (SPARCoverexpressing) of as few as 104 cells (Fig. 21b–c). This performance favors the in vivo non-invasive tumor detection with high fluorescence intensity and adequate signal-to-noise ratio, as compared to SBP-M13 virus-based AlexaFluo750 NIR-I dye and FITC visible dye, further benefiting the fluorescence-guided tumor surgical resections (Fig. 21d). As evidenced by a greater number of excised sub-millimeter tumor nodes under SBP-M13-SWNTs imaging guidance and bright tumor-targeting fluorescence, promising clinical perspectives of SBP-M13-SWNTs nanohybrids could be expected, compared to the unguided counterparts [194]. The proteinous architecture of virus also enables the augmented molecular imaging performance. In a representative report, high relaxivity MRI contrast agents Gd-diethylene triaminopenta acetic acid (Gd-DTPA) was hybridized with the MS2 virus through surface chemical conjugation (500 Gd chelates into one viral capsid). Due to the increased coordination number of water molecules and exchange rates, the viral-Gd hybrids demonstrated a T1 relaxivity of as high as 7200 mM−1 s−1 per viral nanoparticles at 1.5 T,

three orders of magnitude enhancement compared to a single Gdchelate moiety (5.2 mM−1 s−1 ) [195]. In another report, by the genetic introduction of cysteine at position 87 of the MS2 viral capsid, the Gd hydroxypyridinone (Gd-HOPO)-based MRI relaxivity of the nanohybrid could be similarly enhanced to 6876 mM−1 s−1 per unit, on account of the strengthened attachment of the chelates as a linker onto the viral capsids [196]. Recent designs of contrast agents and their engineering into/onto other viruses such as TMV, P22 virus and adenovirus have also been reported [197–199]. From the clinical point of view, enhanced MRI performance favors the imaging quality improvement during the therapeutics of diverse pathological abnormalities. Oncolytic viral hybrids Although the OVs can infect cancer cells effectively, most of the OVs did not present tumor-targeting property without genetic modification. An alternative method to impart the tumor-targeting performance to these OVs is cell membrane coating. Specifically, in a most recently published article, Lv and co-workers have coated

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Fig. 18. a, Schematic illustration of multi-cargo-encapsulated viral nanohybrids. b, In vitro confocal images of various substances revealing the successful endocytosis of the nanohybrids loaded with different fluorescence substances. c-d, Confocal images of Hep3B cells (c) and hepatocytes (d) revealing the selective accumulation of the viral nanohybrids towards Hep3B tumor cells (Adapted from Ref. [135] with permission of American Chemical Society, Copyright 2011).

Fig. 19. a, Preparation route of Au NPs-decorated, DOX-encapsulated Q␤ bacteriophage nanohybrids. b-c, illustrator (b) and TEM image (c) of the viral nanohybrids. d, UV–vis absorbance of DOX encapsulated nanohybrids before or after the laser irradiation. e, Confocal images of laser or non-laser irradiation of the viral hybrids revealing the laser-triggered DOX release among cells (Adapted from Ref. [191] with permission of American Chemical Society, Copyright 2018).

the genetically engineered red blood cell membrane onto the OVs, endowing the viruses with tumor-cell targeting performance when administrated by vein. The as-prepared oncolytic adenovirus@bioengineered cell membrane nanovesicles (OA@BCMNs) viral hybrids could preserve the original infectivity and replication capacity of the viruses, thereby inducing the prominent cytotoxicities against cancer cells specifically rather than normal tissue cells (Fig. 22) [200]. Besides red blood cell membrane decorations, oncolytic adenovirus coated by tumor cell membranes were also prepared to form tumor-targeting ExtraCRAd viral hybrids for cancer immunotherapy. It should be noted that the tumor-specific immunities were significantly elevated by the intravenous injection of the hybrids, and ultimately leading to prominent tumor destruction [201].

Fungal nanohybrids for tumor therapeutics Recently, inflammatory and tumor sites targeting performances of yeast cells (YCs) after oral administrations have been reported [202,203]. In a specific design, QDs, Fe3 O4 MNPs, anti-inflammatory drug indomethacin (IND) and anti-tumor drug paclitaxel (PTX) were successfully encapsulated into YCs via electrostatic forces (Fig. 23a). Indicated by the immunological fluorescence profiles of QDs-YCs and the MRI profiles of Fe3 O4 MNPs-YCs, the remote targeting capability of YCs from the oral garage to distant pathological sites has been confirmed (Fig. 23b–c). With the delivery of PTX or co-delivery of IND and PTX, such yeastbased nanohybrids could induce substantial tumor suppression consequences with high biocompatibilities (Fig. 23d). This work

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Fig. 20. a-b, Schematics (a) and TEM image (b) of SPPTGIN-conjugated magnetic nanoparticles-assembled M13 viral nanohybrids. c, T2 relaxation profiles of M13 viral nanohybrids in DU145 and C4-2B tumor cells. d-e, MR images of C4-2B (d) and DU145 tumors (e) in mice before and after the injection of M13 viral nanohybrids (Adapted from Ref. [143] with permission of Nature Publishing Group, Copyright 2012).

Fig. 21. a, Schematic illustration of SWNT-assembled M13 viral hybrids for tumor-site NIR fluorescence imaging. b, Fluorescence profiles of the M13 viral hybrids in the presence of different amounts of tumor cells. c, Fluorescence stability profiles of M13 viral hybrids and FITC. d, Comparison of the in vivo fluorescence profiles between SWNT-M13 viral hybrids and other fluorescein counterparts. (Adapted from Ref. [194]).

implicates and validates the macrophage recognition functionality of YCs and provides a wide application promise against inflammation and tumor diseases [203]. In another paradigm, tumor-targeted delivery of cisplatin precursor-encapsulated YCs has been realized most recently by patient-friendly oral administration. Researchers found that better biocompatibility and biosafety could be achieved when compared to the intravenous injection modality of free cisplatin, advancing the possible

drug administration strategies mediated by the synthetic YCs [204]. Microalga-based nanohybrids for tumor therapeutics Microalga is a kind of photosynthetic microscopic algae cells that live within water columns including freshwater and marine system, and one of the most critical types of microorganisms to

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Fig. 22. a, Schematic illustration of red blood cell membrane nanovesicles-coated oncolytic adenovirus for tumor oncolysis. b-c, illustration (b) and TEM image (c) of OA@BCMNs hybrids. d, Optical images of cells upon treatments with PBS, OA, BCMNs, and OA@BCMNs. e, Tumor accumulation profiles of OA and OA@BCMNs-preS1. f, In vivo tumor development profile of mice treated with OA@BCMNs-PreS1 and their material counterparts (Adapted from Ref. [200] with permission of American Chemical Society, Copyright 2019).

other lives on earth since their photosynthesis process has counted 50 % of the total atmospheric oxygen production. These algal cells are generally 2−10 ␮m in dimension depending on different types of species, containing mitochondrion, chloroplast, thylakoids, nucleus and other organelles within the cytoplasm. Some species in algae such as Chlamydomonas, contain flagella that could endow the cells with locomotion properties [205,206]. Compared to bacteria and viruses, microalga features the least natural tropism since they are generally non-invasive to other types of cells, which ensures their relatively high biocompatibility for most of the microalga species. The promising potential for the employment of microalgae as a component of medicine is their oxygen-productive photosynthesis and locomotion properties, which could be an effective combination approach to improve the medical performances in wound healing, and treatments for cancer, and a certain type of other oxygen-tension related diseases. Chlamydomonas species have been extensively studied for nanomaterials hybridization. A Chlamydomonas cell is protected by the cell wall composed of hydroxyproline-rich glycoproteins, rending the cell wall with abundant free carboxyl groups (−COOH) and hydroxyl groups (−OH) that could facilitate the electrostatic hybridization towards positively charged nanomaterials (Fig. 24a) [207]. Chemical conjugations via carbodiimide chemistry can also be realized (Fig. 24b) [208]. In a typical paradigm, magnetic Fe3 O4 nanoparticles-conjugated amine-functionalized polystyrene (PS) beads were coated by a cationic polyelectrolyte PDDA, yielding

a positively charged magnetic microbeads. With a gentle coincubation, the magnetic microbeads could physically attach onto the cell wall of the microalga, as confirmed by the TEM image (Fig. 24c) [209]. Diatom is a large group of brown microalgae worldwide, with a distinct cell wall structure composed of the biogenic, microporous and hydrated silicon dioxide framework, called frustule. It has been employed in domains of bio-photonics [210], nanofabrication and drug delivery [211]. The porous frustule offers numerous adsorption sites for the physical interactions with chemodrugs, gold nanoparticles [212] and etc., directly forming the hostguest microalgal nanohybrids without difficulties. In a typical physical attachment manner, Todd and co-workers directly encapsulated the functionalized iron oxide nanoparticles onto the surface of diatom, granting the superior magnetic responses of diatom cells. With the magnet targeting, the hybridized diatom cells could be effectively accumulated into the tumor region, generating significant MRI signals and autofluorescence at the tumor site [213]. Another approach for the diatom-based nanohybrids is the chemical hybridization. Dopamine-functionalized Fe3 O4 nanoparticles could be hybridized onto the frustule of Aulacoseira sp. (a major diatom species), which endows the hybridized diatom cells with magnetic guidance drug delivery performance (Fig. 24d–e) [214]. Toster and co-workers found that the PVPcoated diatoms could regiospecifically grow and assemble with Au nanoparticles under varied pH conditions (Fig. 24f) [212]. Alter-

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Fig. 23. a, Preparation route of yeast nanohybrids by chemical treatments and electrostatic conjugation. b, Schematic illustration of the tumor accumulation of the yeast capsule after oral administration. c, Fluorescence image of indomethacin (IND) encapsulated yeast capsule. d, In vivo tumor growth curve by yeast capsules therapeutics (Adapted from Ref. [203] with permission of American Chemical Society, Copyright 2017).

Fig. 24. a, Scheme of microbeads-conjugated Chlamydomonas reinhardtii (Adapted from Ref. [207] with permission of American Chemical Society, Copyright 2018). b, Brightfield image of bead-conjugated Chlamydomonas reinhardtii (Adapted from Ref. [208] with permission of National Academy of Sciences, Copyright 2005). c, SEM image of polystyrene-conjugated Chlamydomonas reinhardtii (Adapted from Ref. [209] with permission of Wiley-VCH Verlag GmbH & Co. KGaA, Copyright 2018). d, SEM image of Fe3 O4 MNPs conjugated diatom. Inset, e, zoom-in view of the SEM image (Adapted from Ref. [214] with permission of Royal Society of Chemistry, Copyright 2010). f, TEM image of Au NPs-loaded around the pores of the diatom (Adapted from Ref. [212] with permission of American Chemical Society, Copyright 2009). g1 -g2 , TEM image of DNA-modified 13 nm Au NPs-coated diatom (Adapted from Ref. [215] with permission of Wiley-VCH Verlag GmbH & Co. KGaA, Copyright 2004).

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natively, such a nanoparticle assembly could also be made on the DNA-functionalized diatom cells (Fig. 24g) [215]. For example, Delasoie and co-workers successfully adopted the strategy to simultaneously decorate the diatom cells with Vitamin B12 as a tumor-targeting vector and encapsulate the anticancer drugs (e.g., 5-FU, cisplatin) inside the frustule [216]. Recently, researchers from Losic’s group have biomineralized the diatom cell using a facile magnesiothermic reduction process to give silicon nanoparticles. The doxorubicin-loaded SiNPs showed significantly enhanced cytotoxicity against the cancer cells when compared to equivalent free DOX [217]. Microbial derivatives for anti-infections Pathogenic bacteria and viruses can cause severe infections against human beings and can lead to inflammation and death as exacerbate [218,219]. For bacterial infections, since the primary discovery of streptomycin and penicillin as antibiotic agents, the combat between the antibiotic advancement and drug resistance evolution of bacteria has been everlasting [220–222]. On the front of the nanomedicine development, potent oxidizers (e.g., reactive oxygen species) [223,224] and violent external mechanical forces induced by nanomaterials (e.g., MoS2 nanosheets, Au NPs) could be employed in effective bacteria destruction [225–228]. However, a severe bacterial infection will lead to the intense colonization and subsequent formation of bacterial biofilms that can prevent the penetration of nanomaterials and protect themselves from the external forces (e.g., UV irradiation, hyperthermia). Therefore, the design of the bacterial biofilm-permeable antibacterial systems with effective stimulations of native immune responses is highly appealing for complete bacteria eradication [229,230]. The excreted outer membrane vesicles (OMVs) from the bacterial cell are promising microbial-derivated materials to construct biomimetic nanohybrids due to the presence of inherited immunogenic antigens. The OMVs can be coated onto antibacterial Au NPs to form immunogenic active biohybrids to serve for Neisseria meningitides eradication (Fig. 25a) [117]. The E. coli-derived OMVs can be isolated by differentiated centrifugation with proper filtrations. The formulated nanohybrids (Fig. 25b–c) could induce rapid activation and maturation of dendritic cells. In addition, the elevated local concentrations of IFN-␥ and IL-17 manifests that the OMV-based nanoparticulate biohybrids have acquired specifically enhanced immune response for promising bacterial vaccination treatment (Fig. 25d–f) [117]. This methodology may create a universal antibacterial approach in combination with the effective stimulations of native immunity. In addition, other non-living derivative nanohybrids have also been reported. In a sophisticated design, researchers prepared a toxin-adsorbing biocompatible nanosponge by fusing the red blood cell membrane onto the poly(lactic-co-glycolic acid) (PLGA) nanoparticles. With the prominent toxin adsorption capacity, the formulated nanosponge was regarded as the potential detoxification platform for the prevention and treatment of infections caused by injuries and diseases [231]. A later demonstration of this strategy focused on the elimination of the Zika virus, in which the Zika virus-infected host-cell membrane was integrated FDA-approved gelatin nanoparticles by forming a nanoparticle@cell membrane formulation. During the following in vivo investigations using murine models, such injected nanohybrids were demonstrated to attenuate the Zika virus-induced inflammatory response, thereby improving the overall survival rates [232]. Collectively, the coated cell membranes in these paradigms typically serve as the binding or targeting sites to the microorganisms or their biological toxins, while the nanoparticulate core guarantees the physiological stability and prolonged circulations of the nanohybrids in vivo. These

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paradigms provide insightful ideas for the design and application of potent in vivo detoxifiers against microorganisms. Conclusions and outlook Human history has witnessed the primitive trials of using microbes for medical treatments. Although these pioneer attempts have received controversial comments, their precious experiences have been inspiring and encouraging researchers to develop sophisticated nanomedicines nowadays. With the recent advances of nanomedicine, a variety of therapeutic modalities and even synergistic multimodalities have been explored for disease treatments with distinct characteristics. Yet in most of the practical cases, less effective passive targeting and limited accumulations within the disease foci have hindered the overall progress of the clinical translation process. The assembly and integration of microbes and nanomaterials in a single architecture have endowed these hybridized nanoplatforms with unanticipated excellent properties in nanomedicine including the native tropic targeting, intensified accumulation, and enhanced immune responses. Considering the highly promising and probably unprecedented therapeutic prospectives, the present review initially summarized the design and fabrication methodologies for bacteria, virus, microalgae, and yeast-based nanohybrids in a material point of view, on the basis of the physical, chemical and biological understanding of the architectures of corresponding microbes. Next, with distinct advantages, these nanohybrids have been overviewed of their excellent performances and the most recent progress on tumor imaging and therapeutics. Typical paradigms of the antitumoral nanohybrids have been discussed in an extensive but concise manner, thus to comprehensively outline the development status and multi-facet efforts dedicated by numerous researchers. Finally, besides the tumor therapeutic-based nanomedicine, the nanohybrids have also been revealed on their encouraging performances in domains such as bacterial infection, ischemic heart diseases, molecular imaging as well as tissue engineering, implicating a broad spectrum of applicable pathologic models in which the nanohybrids can play roles. Nevertheless, limitations and challenges still remain the rational designs and applications of these nanohybrids for even more promising prospects. Furthermore, with the unprecedented developments of nanoscience and biotechnology, further advances and possible future clinic translations are to be addressed in detail, in the great hope to realize Richard Feynman’s perspective envisions – “swallowing the surgeon” (Fig. 26). (1) The current practices of the nanohybrids mainly focus on microbe-based drug delivery including the traditional chemotherapeutic medicines (e.g., DOX and PTX) and nanomaterials, which mainly enable single therapeutic modalities. With the emergence of various nanomaterials-based therapeutic strategies including nanocatalytic therapy, PDT, RDT, etc., the nanohybrids could accomplish diverse therapeutic functions effectively against tumors and other pathological diseases based on the native targeting and accumulation features of microbes. The intrinsic vulnerability of the microbes upon the exposures to external physical fields, such as intense NIR photons, high-energy radiation, and even sono-mechanics, could lead to the attenuation of the microbes, reducing their risks of infection against human bodies. (2) The major functionalities of the microbes are the tropic targeting, active attenuation and immune response stimulation for numbers of pathogenic targets. These precious functions could be engineered into many versatile platforms through the applications of synthetic biology, by which desired imaging proteins

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M. Huo, L. Wang, Y. Chen et al. / Nano Today 32 (2020) 100854

Fig. 25. a, Schematic illustration of AuNPs-encapsulated bacterial OMVs for bacterial vaccination. b, TEM image of the AuNPs-encapsulated bacterial OMV. c, Hydrodynamic sizes of OMVs before and after AuNPs encapsulation. d-e, INF-␥ level (d) and IL-17 level (e) of the bacterial suspension under different treatments. f, CD11 level of the bacterial suspension under different treatment (Adapted from Ref. [117] with permission of American Chemical Society, Copyright 2015).

Fig. 26. Potential research practices for microbiotic nanomedicine – From fundamental research to clinical translation.

M. Huo, L. Wang, Y. Chen et al. / Nano Today 32 (2020) 100854

(3)

(4)

(5)

(6)

(e.g., green fluorescence proteins), targeting proteins (e.g., RGD peptides) and therapeutic proteins (e.g., IFN-␥, TNF-␣) could be integrated into, promoting the better diagnosis, therapeutics, and prognosis. The fabrication of nanohybrids involves multi-purpose manipulations by various physical, chemical or biological decorations, which, however, may increase the risk of microbes’ deactivation during the preparation, largely reducing the general yield for homogeneous nanohybrids. In the massive production, the fabrication conditions need to be further simplified, optimized and standardized for clinical trials and applications. The biosafety concerns come from the biocompatibility issues of both microbes and nanomaterials. For microbes, proper attenuation strategy is necessary for a number of severely infectious pathogens. The biodegradation and clearance profiles of microbes after the therapeutic process has been rarely reported, which is a critical period needed to be considered in clinical trials. Nevertheless, nanomaterials that are to be hybridized with microbes should be evaluated of their biocompatibilities comprehensively both in vitro and in vivo in advance to guarantee the overall biosafety of the nanohybrids. Few examples have been reported on the oral administration strategy of nanohybrids, though painless and convenient, most probably due to the presence of complicated biological barriers from the alimentary system to the vascular system and the consequent extremely low drug efficacy. Therefore, great efforts should be made to find the most critical factors of these biological barriers of specific pathological diseases to be treated, and more importantly, the effective approaches overcome or circumvent them, to achieve efficient accumulations in the pathological sites and the resultant therapeutic outcomes. The ultimate goal for “swallowing the surgeon” envision, also lies on the on-line and full-range monitoring and manipulation of these nanohybrids with instant, accurate and intelligence feedbacks. This would further require the elaborate integration with microelectronics to facilitate the remote tracking and control, which largely depends on the fast and accurate response of microbes to the external commands by well-designed chemical and biological engineering during their travel and penetration in biological systems, accumulation and therapeutic actions at the lesions, and the suicidal decomposition and/or excretions.

CRediT authorship contribution statement Minfeng Huo: Conceptualization, Writing - original draft. Liying Wang: Writing - original draft, Funding acquisition. Yu Chen: Conceptualization, Writing - review & editing, Supervision, Funding acquisition. Jianlin Shi: Conceptualization, Writing - review & editing, Supervision, Funding acquisition.

Declaration of Competing Interest The authors declare no conflict of interest.

Acknowledgments We greatly acknowledge the financial support from the National Key Research and Development Program of China (grant no. 2016YFA0203700), National Natural Science Foundation of China (grant no. 51722211, 21835007, and 51672303), Program of Shanghai Academic Research Leader (grant no. 18XD1404300) and China Postdoctoral Science Foundation (grant no. 2019TQ0231, 2019M661634).

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M. Huo, L. Wang, Y. Chen et al. / Nano Today 32 (2020) 100854

Liying Wang received her Ph.D. degree at SICCAS in 2019. She is currently a post-doctoral fellow with Prof. Huixiong Xu at Shanghai Tenth People’s Hospital, Tongji University. Her research includes the structural design and functionalizations of mesoporous silica and novel biomaterials for nanomedical applications.

Prof. Yu Chen received his Bachelor’s degree at Nanjing Tech University and Ph.D. at SICCAS. He is now a full professor at SICCAS. His research focuses on Materdicine (interdiscipline of materials and medicine), which includes the design, synthesis and biomedical applications of zero-dimensional mesoporous biomaterials (mesoporous silica/ organosilica), two-dimensional biomaterials (graphene, metal oxides, TMDCs, MXenes and Xenes) and 3D-printing scaffolds, including nanocarriers for drug/gene delivery, molecular probes for molecular imaging, and nanoagents for nanocatalytic therapy,

energy-conversion nanotherapy and in situ localized disease therapy. He has published more than 180 scientific papers in the Materdicine field with a total citation of more than 13,000 times (hindex: 61). He was recognized as a Highly Cited Researcher for 2018 and 2019 by Clarivate Analytics, Web of Science. Prof. Jianlin Shi received his Bachelor’s degree from Nanjing Tech University in 1983, obtained his Ph.D. degree in 1989 at SICCAS, and has been working at the institute since then. He had once worked on the processing science of advanced ceramics, solid-state sintering theory of advanced ceramics, and high-temperature reliability of structural ceramics from 1983 to 1998. Presently his main research interest includes the structural design and synthesis of mesoporous materials and mesostructured nanocomposites, and the catalytic and biomedical performances of the materials for applications in environmental protection and nanomedicine. He was elected as an academician of the Chinese Academy of Science in 2019. More than 480 papers have been published, and the publications were cited by others for more than 35,000 times with an H-index of 108. He has been presented as highly cited researcher in 2018 and 2019 by Clarivate Analytics, Web of Science.