Engineered photoresponsive biohybrids for tumor therapy

Abstract Engineered biohybrids have recently emerged as innovative biomimetic platforms for cancer therapeutic applications. Particularly, engineered photoresponsive biohybrids hold tremendous potential against tumors due to their intriguing biomimetic properties, photoresponsive ability, and enhanced biotherapeutic functions. In this review, the design principles of engineered photoresponsive biohybrids and their latest progresses for tumor therapy are summarized. Representative engineered photoresponsive biohybrids are highlighted including biomolecules‐associated, cell membrane‐based, eukaryotic cell‐based, bacteria‐based, and algae‐based photoresponsive biohybrids. Representative tumor therapeutic modalities of the engineered photoresponsive biohybrids are presented, including photothermal therapy, photodynamic therapy, synergistic therapy, and tumor therapy combined with tissue regeneration. Moreover, the challenges and future perspectives of these photoresponsive biohybrids for clinical practice are discussed.

convenient approach for tumor ablation for numerous cancer indications. 15 As the key components of phototherapy, a variety of photoresponsive agents (including PTA and PS) have been explored, such as noble metal nanoparticles (e.g., Au, Ag, and Pt), carbon-based nanocomposites (e.g., carbon quantum dots, carbon nanotubes, and graphene oxides), semiconductor nanocrystals (e.g., transitionmetal oxide or sulfide), organic molecules (e.g., indocyanine green [ICG], porphyrin, chlorin e6 [Ce6], metalorganic frameworks), and semiconducting polymerbased nanoparticles. 13,[16][17][18][19] Most photoresponsive agents have strong optical absorption of visible lights (400-700 nm) or NIR lights (700-1350 nm). 15 The control of light placement and the accumulation of photoresponsive agents in the targeted tumor tissues are believed to minimize off-target toxicity to surrounding tissues. 20 Unfortunately, the limited selectivity of these agents for tumor tissues necessitates the use of high doses to ensure a significant therapeutic effect, and the accumulation of the agents in non-malignant tissues may cause undesired side effects. 21 For example, most inorganic materials like noble metal and transition-metal sulfide nanoparticles are non-biodegradable and cannot be effectively excreted by the kidneys, leading to a high risk of systemic toxicity. 22,23 Although organic agents like cyanine dyes and conjugated polymers have good biocompatibility, biodegradability, and low toxicity, their clinical applications in the human body are still hampered by the potential long-term safety concerns. 16 Thus, it presents a great challenge for phototherapy to develop innovative photoresponsive platforms with negligible side effects and excellent tumor therapeutic efficacy.
Recently, photoresponsive biohybrids have gained much attention in widespread applications, including biosensors, biodevices, and biomedical engineering. 24,25 Biohybrid systems typically employ biological organisms (e.g., bacteria and algae) into artificial material systems, allowing for the design of biomimetic materials with intrinsic self-assembly and/or self-replication capabilities. 26,27 Notably, engineered photoresponsive biohybrids may be a novel and promising approach for highly efficient cancer therapy due to a range of advantages. Compared to synthetic photoresponsive materials, biomimetic biohybrids could possess enhanced treatment efficiency considering their innate biological properties, including prolonging the blood circulation time, escaping immune systems from clearance, and delivering of therapeutic drugs to the tumor targets. [28][29][30] Biohybrids further benefit from the intrinsic self-assembly and/or self-replication capabilities of living organisms. As such, the fabrication of biohybrid systems might be sustainable and easily scalable. [31][32][33] The combination of synthetic and biological components facilitates the design of biohybrid materials with multiple functions, such as tumortargeting, effective drug delivery, multimodal imaging, and synergistic tumor therapy. [34][35][36] Biohybrid systems can be tailored for biocompatibility with mammalian cells and can thus reduce side effects commonly observed with traditional materials/drugs. They further show excellent therapeutic efficacy to tumor tissues, [37][38][39] thus facilitating the desirable bench-to-clinic translation.
Therefore, research on engineered photoresponsive biohybrids as cancer therapeutic platforms has rapidly progressed in recent years. Here, we systematically summarize the design concept, engineering aspects, and therapeutic strategies of engineered photoresponsive biohybrids for cancer treatment (Figure 1). We first introduce the therapeutic potential and construction methods of such engineered biohybrids and then classify them into biomolecules-associated, cell membrane-based, eukaryotic cell-based, bacteria-based, and algae-based photosensitive biohybrids. We then summarize the range of tumor therapeutic modalities of engineered photoresponsive biohybrids, including PTT, PDT, synergistic therapy, and tumor therapy combined with tissue regeneration. Finally, we discuss the potential for future development of engineered photoresponsive biohybrids for tumor therapy. physiology (including various tumor sites, volume, stage, and metastasized tumors) and pathological complexity of solid tumors (including dysregulated metabolism, disordered vasculatures, reduced pH value, and hypoxic microenvironment). [40][41][42][43] The metabolic demands of malignant cells in tumor centers cannot be fulfilled due to insufficient availability of O 2 and nutrients, resulting in prolonged hypoxia and necrosis. Moreover, with the rapid proliferation of malignant cells, the growing tumor volume increases the distance of the tumor core to adjacent vasculature, thus decreasing O 2 and nutrient transports to the tumor centers. 44 The disordered vasculatures render the tumor core inaccessible to all molecular/nanoparticle-based therapies, because these methods primarily rely on the delivery of therapeutic agents into tumors via the enhanced permeability and retention effect. 45 This issue is a severe limitation for many standard therapeutic approaches (e.g., radiotherapy and chemotherapy) as well as recent nanoparticle-based therapeutic strategies (e.g., PTT and PDT). 40,46 Therefore, more effective and smarter therapeutic strategies are highly required for individual tumor microenvironments.
As aforementioned, current existing photoresponsive agents suffer from their poor biocompatibility/biodegradability, limited tumor-targeting and tumor-specific accumulation, short blood circulation time, and low therapeutic efficiency. 20,46,47 More recently, engineered photoresponsive biohybrids have been developed for advanced phototherapy to overcome the complex biological barriers to reach tumor regions. 20 For example, cell membrane-coated particles have been frequently used for improving circulation time, tumor-targeting, and immune stimulation in cancer treatment. [48][49][50][51][52][53][54][55] Eukaryotic cells like erythrocytes and macrophages have been used as carriers to effectively cross biological barriers and deliver photoresponsive agents to hypoxic tumor sites. 28,[56][57][58] Microorganisms, including bacteria and algal cells, can also be utilized as delivery vehicles of PS into the hypoxic tumors to improve therapeutic effects. 38,[59][60][61] Therefore, it is anticipated that engineered photoresponsive biohybrids could inherit the characteristics from their hybrid components, and provide multifunctional platforms for enhanced tumor therapy.

| Engineering strategies of photoresponsive biohybrids
Basically, engineered photoresponsive biohybrids are artificially engineered photoresponsive materials consisting of a bioactive and a structural component. The bioactive part of the biohybrid could consist of cells or bioactive molecules, and the structural part could also be of either biological or non-biological origin. At present, the engineering strategies of photoresponsive biohybrids mainly include physical and chemical engineering strategies ( Figure 2 and Table 1). Physical engineering approaches usually undergo a physical mixing or incubation process to fabricate photoresponsive biohybrids via physical adsorption or encapsulation, 4,62-69 electrostatic interaction, 3,29,34,35,39,[70][71][72] cellular uptake/ endocytosis, 30,58,[73][74][75][76][77][78][79][80] hypotonic dialysis, 56 diffusion, 37,60,81,82 or electroporation, 83 etc. For example, PS with positive surfaces could be deposited on the negatively charged cell surface via electrostatic interaction. 84 The photosensitive nanoparticles could be encapsulated within intact cells via hypotonic dialysis 56 or cellular uptake. 57,85,86 By contrast, chemical engineering approaches involve chemical bonds and covalent binding, such as covalent conjugation, 2,38,61,87-90 selfpolymerization 36,91 or biomineralization, 32,92-95 bioorthogonal reaction, 96 and so on. In one example, Tang et al. first biotinylated both ZnF16Pc-loaded ferritin (P-FRT) and red blood cells (RBCS) using biotin-X-NHS (Calbiochem) and then crosslinked them with neutravidin to conjugate P-FRT to RBCs. 96 The living materials could also be modified with the in-situ-synthesized photoresponsive agents via a self-polymerization or biomineralization process. 33,59 It is noted that both physical and chemical methods can only provide a temporary or short-term modification. Comparatively, the physical modification may be more biocompatible and friendly to host cells than chemical methods considering the

| Representative types of engineered photoresponsive biohybrids
Here, we classify the current engineered photoresponsive biohybrids based on their biological building blocks, including biomolecule-associated, cell membrane-based, eukaryotic cell-based, bacteria-based, and microalgaebased photosensitive biohybrids.

| Biomolecule-associated photoresponsive biohybrids
Biomolecules (e.g., proteins, peptides, and nucleic acids) are fundamental to all living cells, making them suitable building blocks for biohybrid materials. [98][99][100] Compared to synthetic polymers, natural biomolecules have the capacity to self-assemble, are suitable for structural templating, and are biocompatible and biodegradable. 100,101 Moreover, the biomolecules may offer fascinating benefits in tumor therapy, such as improved drug loading efficiency, specific tumor-targeting effects, controlled drug release, and powerful immune modulation. 4,52,102,103 Therefore, the development of proteins-/ peptides-/RNA-/DNA-based photoresponsive biohybrids has received increased interest for advanced tumor therapy. 3 For instance, the tripeptide Arg-Gly-Asp (RGD), a well-known tumor-homing ligand, has been used as a building block in a tumor-targeting platform. Shan et al. first fabricated tumor-targeting RGD-hepatitis B core protein virus-like particles (HBc VLP), which were then used as carriers to encapsulate the PS ICG through a disassembly/reassembly method ( Figure 3A). 3 The obtained RGD-HBc/ICG biohybrids not only maintained the original PTT/PDT effects of ICG, but also showed improved stability, long-term body retention, and tumorspecific accumulation as compared to free ICG. In another case, Zang et al. developed a size/charge/targeting changeable nano-booster (denoted as NC@Ce6) for amplifying PDT/immunotherapy ( Figure 3B). 90 The NC@Ce6 was prepared by incorporating the PS Ce6 and anti-programmed death-ligand 1 (aPDL1) into a PHresponsive nanocomplex (NC). The NC showed a reduced size and was positively charged in the presence of the acidic tumor microenvironment, which facilitated a controlled release and efficient sufficient delivery of Ce6 and aPDL1 to the tumor site.
In another example, Liu et al. designed a multifunctional theragnostic nanoplatform (MnO 2 @CaCO 3 / ICG@siRNA) by entrapping the MnO 2 core with a pHresponse cover layer of CaCO 3 and ICG, which were further functionalized with PD-L1 siRNA via electrostatic interaction for Figure 3C. 105 Under acid tumor conditions, the two inorganic materials (MnO 2 and CaCO 3 ) were decomposed into Mn and Ca ions by consuming H + ions while generating O 2 and CO 2 , which could relieve the tumor microenvironment and promote the release and diffusion of ICG. Moreover, the large amounts of enriched O 2 in local area facilitated the generation of single oxygen, resulted in enhanced PDT effects against tumor cells. By loading PD-L1 siRNA on the nanoprobes, the Mn@CaCO 3 / ICG@siRNA could inhibit tumor immune resistance for efficient immunotherapy in addition to directly killing tumor cells by PDT. Therefore, this nanoplatform may address the critical problems of poor therapeutic effects of conventional PDT and tumor immune resistance due to tumor environmental characteristics (e.g., tumor hypoxia, excessive H + ions, and immune resistance) and offer an effective strategy for synergistic PDT and immunotherapy against malignant tumors. Additionally, single-stranded DNA molecules can also be used as building blocks in photoresponsive biohybrids. In one case, Yang et al. first developed a novel DNA nanostructure via the coordination between AS1411 DNA G quadruplexes and calcium ions and then utilized the nanoscale coordination polymers to incorporate hemin and Ce6 into the G-quadruplex structure ( Figure 3D). 106 The obtained nanostructure enabled the ROS generation of Ce6 inside the cell nuclei, along with catalase-mimicking DNAzyme functions of the hemin and G-quadruplexes, thereby greatly enhancing PDT efficacy in vivo.  -5 of 20

| Cell membrane-based photoresponsive biohybrids
Cell membranes are thin semipermeable membranes composed of various biomolecules, including proteins, lipids, and carbohydrates. 109 The carbohydrates and transmembrane or membrane-anchored proteins are mainly involved in cell interfacing functionalities, while the bilayer lipid structure creates a semi-permeable barrier toward the external environment. 110 Cell membrane isolation was first achieved by Michael L.S. and colleagues in 1976 by preparing erythrocyte membranes through hypotonic lysis and isotonic resealing treatment. 111 In 2011, the Zhang group first reported the cell membrane coating technology using natural erythrocyte membranes to coat polymeric nanoparticles. 112 The resultant membrane-coated nanoparticle faithfully preserved the bilayer structures and functional surface proteins of the erythrocyte membranes, which enabled the biomimetic nanoparticle to exhibit the long circulation property from the source cells. Besides erythrocyte membranes, a variety of natural membrane-coated particles have been developed with different functions for biomedical applications. [24][25][26][113][114][115][116][117][118] For example, macrophage cell membrane-cloaked systems were demonstrated for targeted delivery of drugs to inflammatory sites and tumorous tissues 119 ; cancer cell membrane-coated particles could enhance cellular uptake, tumor-specific targeting, and immune response [48][49][50][51][52][53] ; and bacterial cell membrane-coated particles also exhibited tumor-targeting and immune stimulation features in O 2 -dependent cancer treatment. 54,55 The fabrication process of the membrane-coated particles mainly includes two steps ( Figure 4): (i) cell membranes are first isolated via hypotonic treatment, ultrasonic cell disruption or repeated freezing and thawing; and (ii) the particle cores are then coated by membranes through mechanical coextrusion, sonication, microfluidic electroporation, or electrostatic attractions, and so on. 110,113,118,[120][121][122] More recently, cell membranes have been employed as effective design strategies for biomimetic photoresponsive materials in tumor phototherapy. 6 69 Their results demonstrated that membrane coating enabled the long-term circulation of inner nanoparticles, improved their photostability and accumulation in tumors, and eventually enhanced the synergetic chemo-PDT efficacy against hypoxic tumors. Similarly, Yang et al. coated the aggregation-induced emission nanodots with mature dendritic cell membranes, which rendered the biomimetic biohybrids with a hitchhiking function for T cell-mediated cancer targeting and enhanced PTT. 124 Moreover, cytomembranes of hybrid cells, which retained the membrane proteins of different cell lines, have been used for functionalization of PS for combined tumor therapy. 49,131 Liu et al. proposed a tumor-specific immunotherapy-based nanoplatform by cloaking PS-containing metal-organic frameworks with the cytomembranes derived from dendritic cells and parent cancer cells. 131 Benefiting from the whole cancer antigens and immunological costimulatory molecules, the cytomembrane-based biohybrid exhibited ultrahigh immunotherapeutic effects against tumors compared to PDT efficacy.
RBCs (i.e., erythrocytes) are the most abundant cells in our body (over 80% of all cells) and have been widely used as drug delivery vehicles for over 30 years. 28,139,140 Benefiting from their prolonged circulation time and O 2 transport function, RBCs were used as carriers for the combined delivery of O 2 and PS in PDT ( Figure 5A). 96,141 Phototherapy has also been combined with immunotherapy by using photoresponsive immune cells. 142 In one case, Zheng et al. used tumor-targeted photoresponsive lymphocytes modified with δ-aminolevulinic acid to generate vesicle-like apoptotic bodies and synthesize anti-neoplastic drugs (protoporphyrin X, PpIX) for systematic cancer therapy ( Figure 5B). 91 Apart from the tumor-tropic immune cells, stem cells also preferentially migrate to tumor regions because of the interactions between the chemokines released from tumor tissues and the chemokine F I G U R E 4 Schematic illustrating the fabrication methods of cell membrane-coated biohybrids.
receptors expressed on the surfaces of stem cells, which can increase the tumor-targeting efficiency of therapeutic agents in stem cell-mediated delivery systems. 74,79,86,137,143 For example, Ning et al. developed photosensitive MSCs loaded with mesoporous silicacoated gold nanostars integrated with ICG as the theranostic platform for the spatiotemporal tracking of MSCs and imaging-guided PTT in treating breast cancers ( Figure 5C). 79 In this biohybrid platform, the gold nanostars were coated with a mesoporous silica shell for better photostability, which also provided large-surface areas for the subsequent integration of ICG molecules. MSCs acted as the carriers to improve the intratumoral distribution and retention of therapeutic agents, while the photoresponsive hybrid nanoparticles enabled the real-time tracking of MSCs with a high spatiotemporal resolution.
In addition, PS could be synthesized in situ within tumor cells and then exocytosed as nanoparticle-trapped vesicles with retained tumor antigens for combinatorial photo-immunotherapy ( Figure 5D). 92 The tumor-derived vesicles were further internalized by dendritic cells and secreted as dendritic cell-derived vesicles, which could not only improve the biocompatibility and immunological property of nanoparticles, but also minimize the possibility of metastasis induced by tumor-derived vesicles.
Both the whole eukaryotic cells and their membranes have been widely employed to develop biohybrid flatforms in tumor therapy. Compared with the synthetic materials, they are endogenic and are considered as much more biocompatible with multiple biofunctions originated from the parent cells. 144 For the cell membrane-coating nanoparticles, the retention of the membrane proteins makes the nanoparticles more feasible to prolong the circulation time, improve the drug accumulation in tumor tissues, and thus enhance the therapeutic efficiency for treating cancers. 68,109,122 While for whole erythrocyte cell-based biohybrids, the living vehicle cells are used to encapsulate or bind therapeutic agents and can be preferentially recruited and accumulated into the inflammatory tumor tissues. 41 2.3.4 | Bacteria-based photoresponsive biohybrids Bacteria play central roles in energy metabolism and generate important biomolecules and substrates for mammalian organisms. 146 Bacteria have been exploited in various areas of biomedical sciences and biotechnology, including bioenergy, pharmaceuticals, and bioremediation. [147][148][149][150] Several species of bacteria, including Gram-positive anaerobes (e.g., Clostridium beijerinckii and Bifidobacterium bifidum) and Gramnegative facultative anaerobes (e.g., Salmonella typhimurium), have been found to specifically colonize in tumor areas. 36,40,139,151 Such specificity can be exploited for localized drug delivery to specific tumor areas. 71,89,97,152 Bacteria-based biohybrid systems also exhibit tumortargeting functions and improved performance for either single or synergistic tumor therapy. 2,39,128,148,[152][153][154][155] To date, bacteria-based photoresponsive biohybrids have attracted great attention in phototherapy due to the good bioactivity, high drug loading efficacy, and tumor targeting capacity of the bacterial components. 2,5,29,30,38,39,59,70,71,80,84,97,156,157 For example, a biotic/abiotic cross-linked biohybrid system was developed with photosensitive ICG-loaded nanoparticles covalently attached to the surface of a genetically modified S. typhimurium strain ( Figure 6A). 38 Benefiting from the hypoxiatargeting ability, the biohybrid system exhibited excellent PTT efficacy to kill tumor cells while avoiding damage to normal tissues, indicating a bacteria-mediated therapeutic strategy for the treatment of deep-seated or large solid tumors. In another case, Fan et al. reported a bacteriamediated therapeutic system using Escherichia coli MG1655 as a vehicle to deliver TNF-α plasmids and Au nanoparticles to tumor regions ( Figure 6B). 59 The AuNPs and TNF-α plasmids were orally delivered and transported into internal microcirculation via transcytosis and then accumulated at tumor sites. Importantly, the photothermal heat generated from AuNPs could remotely activate TNF-α expression to kill tumor cells. This study suggested that bacteria-based photoresponsive biohybrids could be an effective strategy to overcome the barrier in current oral administration for tumor therapy and achieve satisfactory tumor-targeting therapeutic outcomes.

| Algae-based photoresponsive biohybrids
Microalgae are important photosynthesizers that largely contribute to the O 2 budget on Earth. [158][159][160] Recently, there has been a strong interested in using algae for biotechnological and biomedical applications, including tumor therapy. 37,81,158,161,162 Often, the algae-based biomaterials are used as light-driven O 2 generators and carriers, allowing for a sustainable delivery of O 2 to lesion sites. 34,35,62,65,149,[163][164][165][166] Additionally, hybrid photosynthetic biomaterials have been fabricated via 3D bioprinting for efficient algal cultivation in algal biotechnology and for environmental applications (e.g. coral restoration). 167,168 Such hybrid living photosynthetic biomaterials could find wide applications in biomedical engineering and environmental applications.
For the treatment of cancers, algae-based photoresponsive biohybrids are developed for phototherapy mainly because they can provide sustainable O 2 under light irradiation and thereby alleviate the hypoxic microenvironment in tumor tissues. 31,32,60,61,66,72,94,163,164,[169][170][171][172][173][174] For example, Wang et al. reported a biomineralized biohybrid algae (Algae@SiO 2 ) for alleviating tumor hypoxia and synergistic radio PDT ( Figure 6C). 174 The algae Chlorella vulgaris was modified with silica via a biomimetic silicification method. The silica shell separated the algae from the external environment, reducing their cytotoxicity while the photosynthetic activities are retained for O 2 production. Thus, the biohybrid algae could function as an effective O 2 -evolving photodynamic system to relieve the tumor hypoxia and thus improve the PDT efficacy for tumor therapy.
It is worth mentioning that the intrinsic morphology of several algae cells, for example, the helical structures of Spirulina (Sp.), could be for ideal structural templates for fabricating biohybrid materials. In one case, helical microswimmers were fabricated by coating Fe 3 O 4 nanoparticles onto the Sp. microalgae surfaces while preserving their structural features and intrinsic functionalities. 31,175 Benefiting from both the biological organic matter and the integrated magnetic component, the obtained magnetic biohybrids exhibited intrinsic autofluorescence, magnetic resonance signals, and tunable biodegradability, indicating a high potential for imaging-guided therapy. In another case, Wang et al. presented a facile method for mass production of magnetic microrobots by using the Sp. cells as structural templates ( Figure 6D). 31 In this route, (Pd@Au)@Sp was first obtained by synthesizing the core-shell-structured Pd@Au nanoparticles in Sp. templates for PTT. Subsequently, (Pd@Au)/Fe 3 O 4 @Sp was fabricated by depositing Fe 3 O 4 nanoparticles on as-(Pd@Au)@Sp for magnetic responsive ability. Moreover, chemotherapeutic doxorubicin (DOX) is further loaded to establish the (Pd@Au)/Fe 3 O 4 @Sp.-DOX platform for additional chemotherapeutic efficacy. Therefore, such biohybrids not only possessed targeted-delivery efficacy via magnetic propulsion, but also exhibited enhanced synergistic chemo-PTT capacity. This study indicated a promising and efficient algae-based responsive platform for targeted delivery, drug loading, and synergistic tumor therapy.

| Tumor PTT
In traditional PTT, tumor therapy is often facilitated by photoresponsive agents like gold nanoparticles that trigger the physical hyperthermia to ablate tumor cells. 33,62,122,124 Unfortunately, traditional photoresponsive agents have inherent shortcomings, including poor biocompatibility, low tumor targeting efficiency, short blood circulation time, and intrinsic tumor heat endurance. 50,76,93,108,176 Recently, many attempts have been made to develop engineered photoresponsive biohybrids for overcoming these 10 of 20 -WANG ET AL. limitations and improving tumor therapeutic outcomes. 30,32,36,38,59,68,83,84,169,177,178 A typical case was presented with a platelet-facilitated PTT (PLT-PTT) strategy by Rao et al., in which they used the PLTs as carriers loading PTAs for targeted delivery and enhanced the PTT effect in tumor tissues ( Figure 7A). 83 Gold nanorods (AuNRs) of about 50 nm in length and 12 nm in diameter were first synthesized and then loaded into PLTs via electroporation to obtain the AuNR-loaded PLTs (PLT-AuNRs), which preserved a good photothermal property of AuNRs ( Figure 7B). More importantly, PLT-AuNRs inherited the capabilities to evade phagocytosis and target to cancer cells from PLTs. The nanoparticles could be uptaken by the cancer cells, allowing for the selectively tumor killing efficacy under NIR irradiation ( Figure 7C). The in vivo PLT-PTT effect was demonstrated to effectively inhibit the growth of squamous cell carcinoma in mice ( Figure 7D) unique self-reinforcing characteristic of eukaryotic cellbased photosensitive biohybrids in cancer therapy. In another case, Hosseini et al. reported a gold helix phototheranostic biohybrid for image-guided targeted PTT in breast cancer. 32 They used Spirulina platensis (SP) microalga as helix structural templates for the fabrication of helical architecture of Au nanoparticles and improvement of the biosafety for clinical applications. The Au-SP biohybrid was prepared with quasi-spherical Au nanoparticles embedded throughout the SP cells. The high Xray absorbance of AuNPs and autofluorescence of SP cells were employed for dual-modal computed tomography and fluorescence imaging. In addition, the high tumor inhibition effects of Au-SP biohybrids were demonstrated both in vitro and in vivo, which indicating a promising strategy to utilize microorganism-based photoresponsive biohybrids as a theragnostic system for tumor therapy.

| Tumor PDT
Similar to PTT, PDT has been regarded as a promising approach for tumor therapy. 6,12,52,73,179 Unlike PTT, PDT uses PS to absorb light energy to generate cytotoxic ROS, for example, singlet oxygen ( 1 O 2 ), superoxide (O 2˙− ), hydroxyl radicals (·OH), and hydrogen peroxide (H 2 O 2 ), that can induce the apoptosis and/or necrosis of tumor cells. 88,106,129,165,180,181 Current PS mainly includes organic porphycenes, small-molecule dyes, gold nanoclusters, metallic nanoparticles, twodimensional materials, and quantum dots. 173,182 Despite the significant progress in PDT, the ROS production efficiency mainly suffers from (i) shortage of O 2 supply in deep-seated tumors and (ii) limited penetration depth of visible light sources. 29,66,107,151,170,171,183 To address the two issues, many strategies have been proposed including the development of engineered photoresponsive biohybrids. 38,60,61,72,127,164,172 For example, Shi group reported an NIR-driven PDT platform (named as UR-Cyan cells) by hybridizing the PS rose bengal (RB)-loaded upconversion nanoparticles (UCNPs) with photosynthetic cyanobacterial cells ( Figure 8A). 72 In this platform, the UCNPs could effectively upconvert the NIR laser into visible lights. The generated visible lights not only facilitated photosynthesis by cyanobacterial cells, but also activated RB to react with surrounding O 2 to produce 1 O 2 . The UR-Cyan cells were prepared via conjugation of UCNPs onto the surface of the cyanobacterial cells ( Figure 8B). Under NIR laser irradiation, the majority of tumor cells were killed by PDT with UR-Cyan cells ( Figure 8C). After intratumoral injection into tumor xenograft-bearing mice, the UR-Cyan cells could effectively relieve the tumor hypoxia and inhibit the tumor growth under NIR irradiation ( Figure 8D). This design offers a practical strategy to augment PDT therapeutic effects with high tissue penetration and self-supply of O 2 . In another example, Zhang group decorated UCNPs with thylakoid membranes of chloroplasts to realizing O 2 self-supply and simultaneous ROS production for hypoxic tumor therapy. 127 In this unique photosystem, the UCNPs can emit the red light upon 980 nm laser irradiation, which activated the PDT system derived from chloroplasts, facilitated O 2 generation and ROS production, and effectively eradicated the hypoxic tumors in mice. This study provides a new PDT strategy for hypoxic tumor therapy based on engineered photoresponsive biohybrids.

| Synergistic tumor therapy
Despite the significant progress of phototherapy in tumor treatment, significant limitations remain, including limited light penetration depth and low therapeutic efficacy hindered by local hypoxic microenvironments at tumor sites. 65,69,87,154,172 Specifically, absorption of visible and NIR radiation by human tissues limit phototherapy to superficial tissues, leaving deep-seated tumors or metastatic tumors untreated. Additionally, local tumor hypoxia has been shown to promote cancer cell rapid proliferation and induce drug resistance, which further increases the difficulty in cancer treatment. 34,53,106,131 Recently, engineered photo responsive biohybrids have been engineered to combine different antitumor therapeutic strategies for treating hypoxic tumors, such as synergistic PTT, PDT, chemotherapy, radiotherapy, and/ or immunotherapy. 13,31,34,35,51,64,78,80,89,90,97,103,121,128,130 For instance, Wang et al. developed an all-in-one volvox-based biohybrid microrobot (denoted as Volbot) for photosynthesis-promoted synergistic PTT/PDT ( Figure 9A). 34 This biohybrid microrobot was prepared by integrating a green algae (volvox), photodynamic agents (Ce6), and PTAs (polydopamine functionalized magnetic nanoparticles, Fe 3 O 4 @PDA) into one system. The photosynthetic algae could generate O 2 to alleviate tumor hypoxia, and photosynthesis simultaneously promoted the PDT effect of Ce6 under red light and PTT effect of Fe 3 O 4 @PDA under NIR light. In another case, Yao et al. reported a bacteria-based biohybrid therapeutic platform with tumor targeting ability for synergistic PTT and immunotherapy ( Figure 9B). 97 In this platform, the tellurium nanorods (TeNRs) were synthesized inside the E. coli Nissle 1917 (EcN) cells via facile intracellular biosynthesis. Owing to the high photothermal conversion efficiency of TeNRs along with co-stimulation by probiotic EcN as immunoadjuvants, the PTT efficacy was boosted in the immunosuppressive tumor environment, which could effectively eliminate advanced malignant tumors, prevent tumor metastasis and recurrence, and prolong survival in tumor-bearing mice.
In clinic, chemotherapy uses chemical drugs to kill widespread or metastatic tumor cells, while its efficacy is severely limited by the side effects caused by the nonspecificity and poor-selectivity to cancerous cells. 6,126,130,157 Although radiotherapy uses high-energy radiation to destroy tumor cells in confined areas, it has long-term side effects, and tumor resistance may occur because of the insufficient O 2 supply in hypoxic tumors. 35,94,163,174 In this regard, engineered photoresponsive biohybrids offer opportunities to design synergistic tumor therapeutic platforms with enhanced biosafety and antitumor performance. 169,184 For example, Pei et al. designed RBC membranes-camouflaged dimeric prodrug nanoparticles (RBC(M(TPC-PTX))) for synergistic PDT and chemotherapy ( Figure 9C). 130 In this platform, the inner 5,10,15,20-tetraphenylchlorin (TPC, a PS) and paclitaxel (PTX, a chemical drug) dimer-loaded nanoparticle core was responsible for light-triggered ROS generation for PDT and on-demand PTX release for chemotherapy, while the outer RBC membrane shell could overcome the rapid blood clearance and improve tumor accumulation, which enhanced the therapeutic efficacy and decreased the toxicity of each therapeutic agent. In another example, Zhong et al. utilized the superparamagnetic-Fe 3 O 4 -functionalized S. platensis as photosynthetic biohybrid nanoswimmer systems for tumor-targeted imaging and enhanced synergistic PDT/ radiotherapy in hypoxic solid tumors ( Figure 9D). 35 The living S. platensis not only acted as a PS due to its ROS generation ability for PDT, but also utilized as a theragnostic agent owing to the native chlorophyll for fluorescence and photoacoustic imaging. In addition, Fe 3 O 4 nanoparticles could magnetically target to the tumor site and functions as contrast agents for magnetic resonance imaging. More importantly, the living biohybrid system could generate O 2 in situ to relieve tumor hypoxia, thus enhancing the radiotherapy efficacy. This study provide a promising therapeutic platform for tumor-targeted imaging and synergistic therapy.
Taking together, all above examples suggest that the integration of synthetic and biological components enables multifunctionality that cannot be achieved in one single system. These versatile synergistic therapeutic modalities based on engineered photoresponsive biohybrids can offer innovative strategies for enhanced therapeutic performances by modulating the tumor microenvironment.

| Tumor therapy combined with tissue regeneration
Current clinical treatment strategies of solid tumors are surgical resection, chemotherapy, and radiotherapy. 3,4 However, these treatment modalities have formidable obstacles such as the critical tissue defects after the surgical resection, undesirable chemotherapy resistance and severe side effects, and low therapeutic efficacy. [5][6][7][8] Hence, it is necessary to develop a more efficient strategy to achieve both tumor therapy and tissue regeneration. [185][186][187][188][189] Engineered photoresponsive biohybrids have emerged as promising therapeutic platforms by combining the tumor phototherapy with tissue regeneration together. integrating the photosynthetic Ce6-contained cyanobacteria onto 3D-printing CaCO 3 -PCL scaffolds for enhanced PDT against osteosarcoma and simultaneous bone regeneration ( Figure 10). 82 The preparation process of the biohybrid scaffold could be divided into three steps ( Figure 10A): (i) photosynthetic Ce6-contained cyanobacteria were fabricated via internalization of Ce6 into cyanobacteria cells; (ii) 3D CaCO 3 -PCL scaffolds were prepared using 3D-printing technology; (iii) the final therapeutic platform were constructed by decorating Ce6contained cyanobacteria onto 3D CaCO 3 -PCL scaffolds. As shown in Figure 10B, the engineered biohybrid scaffolds generated O 2 upon red light irradiation (660 nm) due to photosynthesis by cyanobacteria, which subsequently activated Ce6 to produce 1 O 2 for highly effective PDT. Additionally, the generated O 2 and the gradually degrading CaCO 3 -PCL scaffolds promoted bone regeneration following tumor elimination. This study offers an insightful paradigm to utilize engineered photoresponsive biohybrids for enhanced tumor therapy and subsequent prompted tissue regeneration.

| CONCLUSION AND PERSPECTIVES
Engineered photoresponsive biohybrids are a new type of biomaterials to improve the tumor therapeutic efficacy and overcome the shortcomings in current tumor phototherapy. Here, we outlined the various biomimetic properties, augmented photosensitive abilities, and multiple biotherapeutic functions of engineered photoresponsive biohybrids. We classified the engineered biohybrids as biomolecules-associated, cell membranebased, eukaryotic cell-based, bacteria-based, and algae-F I G U R E 1 0 Engineered photoresponsive biohybrids for tumor therapy combined with tissue regeneration. (A) Schematic showing the preparation of the photosensitizer-engineered biohybrid scaffolds by integrating the photosensitive and photosynthetic Ce6-contained cyanobacteria onto 3D-printed CaCO 3 -PCL scaffolds. (B) Under 660 nm laser exposure, the engineered scaffolds could generate O 2 by photosynthesis and subsequently activate Ce6, leading to the production of reactive oxygen species (especially the singlet oxygen) for higheffective PDT against osteosarcoma. Meanwhile, the excessively generated O 2 and CaCO 3 components from the scaffolds could also prompt bone regeneration after the elimination of osteosarcoma. Reproduced with permission. 82 Copyright 2021, Elsevier. based photoresponsive biohybrids, which could be applied in various therapeutic modes, such as PTT, PDT, synergistic therapy, and tumor therapy combined with tissue regeneration. Such engineered biohybrid materials could become promising biomimetic platforms for diverse cancer therapeutic applications.
Despite many efforts on the developments of engineered photoresponsive biohybrids over the past decade, most of these photoresponsive biohybrids designed for tumor therapy are still in its infancy, and commercialization and clinical applications can be achieved. The following major challenges or obstacles should be well considered and require significant development. A key issue is the lack of simple protocols for the preparation of homogeneous functionalities. Current methods rely on complex or multi-step operations, which are difficult to replicate in clinical settings. In addition, novel highperformance PS should be exploitered instead of relying on building blocks from existing materials. Furthermore, although the biocompatibility and application potential of engineered photoresponsive biohybrids have been demonstrated for cells in laboratory settings, the longterm biosafety of these biohybrids needs to be investigated for clinical settings. We believe that the above challenges can be addressed in the near future and thus facilitate the emergence of novel biohybrid designs with better improved antitumor performance and practical cancer therapeutical applications.

ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (32201117) and the China Postdoctoral Science Foundation (2021M700141).

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.

GLOSSARY Photodynamic therapy
Photodynamic therapy (PDT) is a form of phototherapy involving light and a photosensitizing chemical substance, used in conjunction with molecular oxygen to elicit cell death.

Photothermal therapy
Photothermal therapy (PTT) refers to efforts to use electromagnetic radiation (most often in infrared wavelengths) for the treatment of various medical conditions, including cancer. This approach is an extension of photodynamic therapy, in which a photosensitizer is excited with specific band light. This activation brings the sensitizer to an excited state where it then releases vibrational energy (heat), which is what kills the targeted cells.

Photothermal conversion agents
Photothermal transduction agents (PTAs) are used in PTT to convert light energy into heat energy.

Photosensitizers
Photosensitizers are molecules, which absorb light and transfer the energy from the incident light into another nearby molecule.

Reactive oxygen species
In chemistry, reactive oxygen species (ROS) are highly reactive chemicals formed from diatomic oxygen (O 2 ). Examples of ROS include peroxides, superoxide, hydroxyl radical, singlet oxygen, and alpha-oxygen.

Engineered photoresponsive biohybrids
Artificially engineered photoresponsive materials consisting of a bioactive and a structural component. The bioactive part of the biohybrid could consist of cells or bioactive molecules, and the structural part could also be of either biological or non-biological origin.

Enhanced permeability and retention
The enhanced permeability and retention (EPR) effect is a controversial concept by which molecules of certain sizes (typically liposomes, nanoparticles, and macromolecular drugs) tend to accumulate in tumor tissue much more than they do in normal tissues.

Endocytosis
A cellular process in which substances are brought into the cell. The material to be internalized is surrounded by an area of cell membrane, which then buds off inside the cell to form a vesicle containing the ingested material.
Biomineralization A process by which living organisms produce minerals, often to harden or stiffen existing tissues.

Bioorthogonal reaction
Refers to any chemical reaction that can occur inside of living systems without interfering with native biochemical processes.

Theragnostic
A treatment strategy that combines therapeutics with diagnostics.