Modified probiotics and the related combinatorial therapeutics

Modified probiotics and the related combinatorial therapeutics

PDF

Luo Zhao^a^,^b, Mengya Niu^a^,^b, Zilin Ma^a^,^b, Fengyun He^a^,^b, Xinxin Liu^a^,^b, Xunwei Gong^a^,^b, Zhanfei Chai^a^,^b, Ziqing Wang^a^,^b, Qianhua Feng^a^,^b^,^*, Lei Wang^a^,^b^,^c^,^d^,^*

Acta Pharmaceutica Sinica B | 2025, 15(5) : 2431 - 2453

Less

Acta Pharmaceutica Sinica B | 2025, 15(5): 2431-2453

• REVIEW •

Modified probiotics and the related combinatorial therapeutics

Full

Luo Zhao^a^,^b, Mengya Niu^a^,^b, Zilin Ma^a^,^b, Fengyun He^a^,^b, Xinxin Liu^a^,^b, Xunwei Gong^a^,^b, Zhanfei Chai^a^,^b, Ziqing Wang^a^,^b, Qianhua Feng^a^,^b^,^*, Lei Wang^a^,^b^,^c^,^d^,^*

Affiliations

^aSchool of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China

^bHenan Key Laboratory of Nanomedicine for Targeting Diagnosis and Treatment, Zhengzhou 450001, China

^cLuoyang Central Hospital Affiliated to Zhengzhou University, Luoyang 471009, China

^dTumor Immunity and Biomaterials Advanced Medical Center, Zhengzhou University, Luoyang 471009, China

About Author:

E-mail addresses: fengqianhua@zzu.edu.cn (Qianhua Feng),

wanglei1@zzu.edu.cn (Lei Wang).

^†

These authors made equal contributions to this work.

Author contributions

Lei Wang, Qianhua Feng and Luo Zhao chose the topic and designed the outline for this review. Luo Zhao and Mengya Niu wrote the manuscript. Zilin Ma and Fengyun He drew the figures and designed the tables. Xinxin Liu, Xunwei Gong, Zhanfei Chai and Ziqing Wang revised the manuscript. All of the authors have read and approved the final manuscript.

doi: 10.1016/j.apsb.2025.03.021

Outline

Abstract

Less

Probiotics have shown excellent application prospects in preventing and treating many diseases. However, their sensitivity to the harsh environment in vivo always leads to a massive loss of viability and insufficient therapeutic effect. Fortunately, modified probiotics have emerged and provide multiple possibilities for their use in various diseases. Modification not only endows probiotics with extra capacity to resist severe environments but also gives them exogenous characteristics, such as prolonged retention time and improved therapeutic effects. Modified probiotics could combine with other therapies, which has opened up new avenues to enhance the efficacy of probiotic-based therapy. In this review, we have summarized the current physicochemical and biological modification strategies of probiotics. In addition, the progress of research on probiotic-based combination therapy has also been extensively reviewed, which contributes to the enhanced delivery of probiotics or other active constituents and provides new ideas for disease treatment, bioimaging, and diagnosis.

Key words

Probiotics / Physicochemical modification / Biomedical modification / Combinatorial therapeutics

Cite this Article

Luo Zhao, Mengya Niu, Zilin Ma, Fengyun He, Xinxin Liu, Xunwei Gong, Zhanfei Chai, Ziqing Wang, Qianhua Feng, Lei Wang. Modified probiotics and the related combinatorial therapeutics[J]. Acta Pharmaceutica Sinica B, 2025 , 15 (5) : 2431 -2453 . DOI: 10.1016/j.apsb.2025.03.021

Full Text

Less

1.　Introduction

Less

Probiotics are defined as live microorganisms that, when given in adequate amounts, confer a health benefit on the host¹. The probiotics market is projected to reach USD 105.7 billion by 2029, growing atan 8.2% CAGR from USD 71.2 billion in 2024². Many clinical trials have shown that probiotics are pivotal in human health, disease prevention, and treatment. Traditional probiotics, such as strains of Lactobacillus and Bifidobacterium, have shown efficacy in treating diseases like irritable bowel syndrome (IBS)³, inflammatory bowel disease (IBD)⁴, and certain infections^5,6. Furthermore, probiotics also contribute to tumor prevention and treatment by enhancing immunity, inhibiting carcinogens, promoting beneficial metabolites, suppressing harmful gut bacteria, and altering the tumor microbiome^7,8.

Even though probiotics have shown many exciting achievements, the clinical translation of probiotic-based therapy remains challenging⁹. This is primarily due to their high sensitivity to environmental changes. For instance, probiotics taken orally may face low colonization and survival rates, whereas probiotics injected intravenously are subject to clearance by the circulatory system¹⁰. As a result, using probiotics alone may not always yield the desired therapeutic outcomes.

Recent advancements have focused on developing modified probiotics to overcome these limitations. Modifications are currently divided into physicochemical and biological categories¹¹. After modification, probiotics can be equipped with molecules like medicine and antibodies or protective coatings, such as hydrogel networks and nanoparticles^12,13. The methods above could protect probiotics and endow them with new functions simultaneously.

With the help of various material modifications, probiotics may acquire increased resistance to the harsh gastrointestinal environment, greater oral absorption efficiency, improved ability to adhere to mucosal surfaces, and enhanced colonization of the intestines^14,15. Those new functions enable the integration of probiotics with other therapeutic approaches for treatment. Combined therapy leverages the advantages of probiotics to deliver therapeutic agents directly to specific disease sites while also optimizing the therapeutic efficiency of probiotics themselves. In addition, integrating probiotics with other therapeutic approaches has opened up new avenues for disease imaging¹⁶, diagnosis¹⁷, and treatment¹⁸. Combining probiotics with photosensitizers and immunoactivators forms an immunoreactive nanosurface on the probiotic. This hybrid system provoked robust anti-tumor immunity, and the combination therapy significantly inhibited tumor growth and extended the survival of animals. Moreover, genetic engineering has been widely used to create therapeutic agents in various probiotics^19,20. Gene editing probiotics like in vivo “therapeutic factories” could produce edited biotherapeutics precisely at disease lesions for improved therapeutic efficacy^21-23. These synergistic approaches can potentiate the therapeutic effects of probiotics, offering a multifaceted strategy to tackle complex diseases.

This review article discusses physicochemical and biological modification methods based on various surface structures on probiotic cells. After introducing new functions, we then outline the use of the modified probiotics in combination therapy and other applications in detail (as shown in Fig. 1). We anticipate that this review article will foster the development of innovative strategies for creating advanced probiotic-based therapies through modification.

2.　Probiotics modification

Less

Probiotic modification involves altering or coating the surface of probiotics using physical, chemical, or biological techniques to introduce new functionalities distinct from their original properties. In probiotics, surface structures and characteristics are critical for cell communication and interactions with surrounding environments²⁴. Elements such as surface charge, chemical groups, proteins, and antigens significantly influence key physiological functions, including adhesion, proliferation, and differentiation. Modifying the surface of probiotics can also trigger specific physiological signals and responses.

The primary aim of probiotic modification is to reduce potential side effects while enhancing their functionality and broadening their applications by refining surface characteristics and facilitating beneficial interface interactions. As our understanding of probiotic surface properties deepens and interdisciplinary research advances, various physicochemical and biological methods have emerged as effective approaches for modifying probiotic surfaces.

2.1.　 Physicochemical modification

The surface of probiotics mainly comprises cell walls and exterior structures, such as flagella and pili. Probiotics’ multilayer cell wall structure comprises polysaccharides, proteins, and lipids. Those components are rich in diverse functional groups (e.g., hydroxyl, carboxyl, amine, and free thiol groups), offering modifiable reaction sites for chemical modification on the surface of probiotics^25,26. Multiple chemical modification methods have been developed based on these structural characteristics. Many materials that contain amino or carboxyl groups can be modified into probiotics with the aid of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC)/NHydroxysuccinimide (NHS), those can bond with probiotics together. Additionally, functional polymers can be treated using bioorthogonal chemistry to enable the spatiotemporal delivery of bacteria by a click reaction with the target location²⁷. There are different modification methods for individual probiotics and multiple probiotics. For individual probiotics, materials form as coating or conjugators on the surface of probiotics. As for multiple probiotics, encapsulating is the primary method of modification. The physicochemical modification examples are listed in Table 1^29-63.

2.1.1.　 Single-cell modification

The individual probiotic coating is a single-cell nano-encapsulation technology in which a coating method imparts non-inborn or natural external features to live cells. Because of the complete coverage of a coating on the surface of probiotics, individually coated probiotics typically exhibit enhanced bioavailability, a controlled release profile, and improved pharmaceutical effects in comparison with uncoated probiotics²⁸.

2.1.1.1.　 Surface coating on single cell

Metal-polyphenol network coating: Polyphenols are natural plant chemicals abundant in fruits, vegetables, and cereals. Polyphenolic biomolecules share the same structural characteristics: the phenolic hydroxyl groups. The abundance of phenolic hydroxyl groups provides polyphenols with firm adherence to various surfaces.

Interestingly, metal ions are usually introduced to the surface of biomaterials via phenolic hydroxyl groups, forming metal-phenolic networks (MPNs). Based on their above features, polyphenols can be used to coat probiotics. Through the self-assembly of metal and polyphenol, Fe-TA coated probiotics (Lactobacillus reuteri, L. reuteri@FeTA) were then placed into an injectable hydrogel made of carboxylated CS and oxidized hyaluronic acid to create Gel/L@FeTA. To expedite wound healing, the hydrogel efficiently enhanced collagen deposition surrounding the wound-regulated inflammatory markers and increased the expression of angiogenesis-related proteins⁶⁴. Based on the Fe-TA prebiotic “armor,” Xie et al.³⁰ constructed the EcN@Fe-TA@mGN to form a dynamic barrier, which enhanced the EcN colonization in the intestine. Furthermore, the combination of carboxymethylated β-glucan with EcN was found to have a synergistic effect in regulating the gut microbiota and stimulating the production of short-chain fatty acids (SCFAs). In a related study, Pan et al.²⁹ employed a single-cell coating strategy using TA and trivalent ferric ions, referred to as “nano-armor”, to shield bacteria from the effects of six clinically significant antibiotics.

Apart from the typically utilized Fe³⁺, the metal calcium ion Ca²⁺ can also chelate with TA. As shown in Fig. 2A, Yang et al.³¹ created a super probiotic (EcN@TA-Ca²⁺@Mucin) layer-by-layer coat of EcN with tannins and mucins. It was additionally demonstrated that mucin increased probiotic colonization and development of probiotics in the mucus layer through interaction with mucus, providing better resilience to the challenging gastrointestinal environment and robust adherence to the intestine. Furthermore, through the scavenging of reactive oxygen species (ROS), the EcN@TA-Ca²⁺@Mucin complex had the potential to significantly reduce inflammation, alleviate the adverse consequences of bacterial translocation in inflammatory bowel disease, and enhance the diversity and abundance of intestinal microbiota.

Polymer coating: There are two main polymer categories: natural and synthetic. Due to their biocompatibility and simple formulation parameters, polymers are widely used as drug-delivery vehicles⁶⁵. In the case of probiotics, polymers can be used as a coating to protect them, and functionalized polymers can enhance the targeted therapeutic effect of probiotics and increase probiotic colonization at the target site.

To alleviate intestinal inflammation, Zhou et al.³⁴ engineered the oral probiotic EcN (EcN-pE) to overexpress catalase and superoxide dismutase, which are utilized to remove ROS from inflammatory areas. To enhance the bioavailability of EcN-pE in the gastrointestinal tract, EcN-pE was coated with natural polysaccharides CS and alginate (ALG) by a layer-by-layer electrostatic self-assembly strategy, and the CS/ALG-coated EcN-pE (EcN-pE(C/A)₂) was effective in alleviating inflammation and repairing colonic epithelial barriers in different chemically induced IBD mouse models.

Polydopamine (PDA), a natural polymer, has a variety of applications in the delivery of living cells. Due to the powerful adhesive capabilities of its catechol moiety, PDA has been widely used as a coating layer on the surface of various materials. Moreover, the polymerization reaction of dopamine can take place in cell-friendly settings. PDA can be deposited on the cell surface via covalent bonds (e.g., Michael additions and Schiff base reactions), π–π accumulations, or hydrogen bonds^66,67. Chen et al.⁶⁷ attempted to functionalize individual yeast cells with PDA and discovered that yeast cells with PDA coating maintained their viability, which was regulated by the thickness of the shell during the cell cycle. As shown in Fig. 2B, Pan et al.⁶⁸ modified the surface of probiotics with a co-deposited coat of PDA and CS. The surface-modified probiotics improved the enrichment of the microbial community in the intestine more than the original probiotics. Furthermore, the surface coating with a protective layer and a navigational function could protect the probiotics against gastric fluid and bile acid and enhance the targeting ability. The excellent surface coating characteristics are conducive to improving the treatment of intestinal inflammation.

With the mucosal adhesion function, catecholamine-based polymers that could adhere to mucous membranes emerged in recent years. Liu et al.³³ utilized the oxidative self-polymerization of norepinephrine (NE) on the surface of probiotic EcN to form a poly norepinephrine (pNE) membrane. Then, hyaluronic acid (HA) conjugated poly(propylene sulfide) was self-assembled into HPN nanoparticles with ROS scavenging ability. These were further modified onto the NE-EcN to form HPN-NE-EcN. Based on the pNE coating helping EcN to colonize in the intestine, this system exerted an excellent therapeutic effect on IBD. Notably, the NE layer-coating strategy effectively encapsulated living EcN cells for cytoprotection, while the NE layer coating and HPN conjugation did not impact EcN's growth and proliferation. Furthermore, the HPN nanoparticles can self-degrade after fully reacting with ROS as the physicochemical transformation of PPS shifts from hydrophobic to hydrophilic, thus enhancing the safety profile of this approach.

In addition to natural polymers, the commercial polymer Eudragit plays a massive role in delivering probiotics⁶⁹. Due to the pH-dependent properties of the polymer eutectic, pH-responsive probiotic delivery systems can be prepared. Liu et al.⁷⁰ reported a method of bilayer encapsulation involving tannic acid (TA) and Eudragit L100 to coat probiotics, aiming at overcoming the obstacles of oral drug delivery. TA and Eudragit L100 layers coated EcN showed robust resistance to the harsh external environment of the gastrointestinal tract. In addition, the pH-responsive degradation of the external Eudragit L100 layer endowed EcN with a selective intestine-targeted effect, and the strong adhesion capacity of the TA layer prolonged the retention time of EcN without affecting viability, leading to superior prophylactic and therapeutic efficacy.

Lipid coating: Apart from polymer coating, many more established packaging materials are used in pharmacies to coat probiotics. As shown in Fig. 2C, Cao et al.³⁵ attempted to prepare a phospholipid molecular layer as a coating material on the surface of a probiotic by self-assembly, and the presence of calcium ions condition helped the supramolecular self-assembly of dopamine hydrochloride (DOPA) on the negatively charged surface of the probiotic. Cholesterol was used to stabilize the self-assembled lipid membrane further. The raw materials used were FDA-approved phospholipid molecules (widely found in cell membranes), which were favorable for subsequent clinical trials and translation. Taking EcN, for instance, after donning a lipid membrane, the lipid membrane-coated bacteria (LCB) can withstand harsh external conditions, such as strong acids, bases, antibiotics, and alcohols with different solubilities. In another research, the same supramolecular critical self-assembly technique was employed by Zhang et al.⁷¹ to modify LGG. Balsalazide (Bal) as the 5-ASA prodrug was planted in the phospholipid molecule layer, which chemically altered the phospholipid molecule 1-hexadecanoyl-sn-glycero-3-phosphocholine (LPC). The lipid prodrug LPC-balsalazide (LPC-Bal) helped lactobacillus rhamnosus GG (LGG) resist digestion juices from the stomach and intestine to reach the colon target lesion. Once in the colon, an azo-reductase enzyme sheared the lipid layer to release the UC therapeutic drug 5-ASA. This inhibited colonic inflammation, improved the pathological environment, and created a favorable microenvironment for LGG colonization. In addition, LGG can successfully colonize the intestine and then control the intestinal microbiota to improve ulcerative colitis (UC) lesions.

Liposomes are another type of material that is used to coat probiotics in addition to lipids. Liposomes are a subgroup of lipid NPs, usually composed of phospholipids, which can form unilamellar and multilamellar vesicular structures. This enables the delivery of hydrophilic, hydrophobic, and lipophilic drugs via liposomes. Liposomes, which could self-assemble on the surface of probiotics, are a more substantial coating than simple lipid coatings. By self-assembling on the surface of LCB, Zhou et al.³⁶ used liposomes to enclose probiotics with a liposomal coat. The liposome coating had a thickness of about 100 nm, which could offer LCB good protection and increase the colonization efficiency in the intestine. In treating Parkinson's disease (PD), LCB can self-regulate the production of γ-aminobutyric acid at a constant concentration and maintain a long half-life.

In addition to oral drug delivery, the lipid-coating engineered bacteria EcN BL21 was also used for intravenous drug delivery to treat tumors. Although oncolytic bacteria have made recent preclinical strides in cancer therapy, dose-limiting toxicity has long been a problem for clinical applications. Feng et al.⁷² developed a straightforward but effective technique to create charge-reversible lipid membrane-coated bacteria (CRLCB). Those bacteria could switch surface charges in neutral or acidic solutions, which leads to mild interactions with negatively charged normal cells. These characteristics of bacteria enhanced their compatibility with blood cells and the immune system. Furthermore, compared to wild-type strains, lipid-encapsulated bacteria have longer blood circulation lifetimes and lower rates of tissue capture. Therefore, engineered bacteria exhibit improved tumor specificity and anti-tumor efficacy even at low doses.

Mineral coating: Mineralization, a natural process whereby organisms create a hard exterior to protect and support soft tissues, is essential for the survival of biological systems⁷³. Although oral microecological agents effectively modulate the intestinal microbiome, they are constantly subjected to multiple aggressions from the oral cavity to the intestinal tract.

As shown in Fig. 2D, inspired by the protective mechanism of mineralization, Geng et al.³⁷ developed an electrostatic interaction-mediated biointerfacial mineralization, which produces super-resistant and self-removable coatings on bacterial surfaces. Using the next-generation probiotic Bacteroides fragilis (BF839) as a model bacterium, polyvinyl–pyrrolidone (PVP) was used as a stabilizer to bind with negatively charged bacteria. Then, PVP on the bacteria surface was composited with calcium ions and sodium carbonate, respectively. The absorbed calcium ions guided the multiphase nucleation and deposition of calcium carbonate on the surface of the BF839 cells to form a mineral coating. The coating gave the bacteria strong resistance to manufacturing-related oxygen exposure, ultraviolet (UV) radiation, and 75% ethanol. After oral administration, the mineral coating could actively neutralize stomach acid and release the encapsulated bacteria through a spontaneous and rapid dual catabolic reaction. Besides neutralizing acid, the released calcium ions also promoted bile acid aggregation, allowing bacteria to survive in both gastric and bile acid environments. Further supported by the therapeutic efficacy of the coated bacteria on colitis mice, biointerfacial mineralization provides a versatile platform for developing next-generation active oral biotherapeutics. In addition, they explored the versatility of interface mineralization to coat EcN. To confirm the self-removability of the coating in vivo, the genetically engineered mCherry expressing EcN was coated with the calcein-labeled mineral layer and then orally administrated to mice. The luminal contents were extracted from the stomach and intestine post-administration. Notably, the calcein signal decreased, but the mCherry signal increased from 0 to 5 h, suggesting that demineralization of the coating occurred and protected the released EcN by neutralizing intragastric pH.

Other coating materials: Besides the mentioned materials, numerous other coating materials can coat probiotics using chemical or physical forces. The two-dimensional SiH@TPGS-PEI coating could protect probiotics against damage from aggressive gastric juices in an acidic gastric environment. Moreover, SiH@TPGS-PEI also decomposed naturally when it reacted with water to create hydrogen. In response to a neutral or mildly alkaline intestinal environment, probiotics release anti-inflammatory hydrogen to treat colitis³⁸. Most prior attempts at surface coating probiotics usually possessed the function of protecting probiotics; however, they lacked additional therapeutic benefits. Hou et al.⁷⁴ outlined a strategy inspired by silk cocoons’ potent therapeutic and protective properties that involve synergistically coating probiotic bacteria with therapeutic nano coating to enhance biotherapeutics. Silk fibroin could self-assemble onto the bacterial surface by switching from a random coil to β-sheet conformation. Thanks to its innate pharmaceutical activity and protective barrier role, silk fibroin endowed the coated probiotic with improved survival against gastric insults and enhanced therapeutic effect on colitis. Peng et al.⁴¹ used the polysaccharide structure of alginate to chelate with calcium ions to produce a colloidal framework on probiotic EcN. At the same time, the anti-inflammatory drug 5-ASA was encapsulated in the alginate-coated EcN (EcN@Alg) to form 5-ASA loaded EcN@Alg (EcN/5-ASA@Alg). With the protection effect of alginate-based coating, the drug-loaded probiotics could resist the gastric environment. Upon arriving in the intestine, 5-ASA is released along with the disintegration of the coating. This drug delivery system protects drug-loaded probiotics from gastric juices after oral administration and enhances the therapeutic efficacy against colitis by increasing the abundance and diversity of the microbiota, alleviating inflammation and repairing the intestinal barrier.

Surface coating probiotics with various materials can enhance their stability, survivability, and therapeutic efficacy in the gastrointestinal tract. Each method has unique benefits and drawbacks. The choice of coating material should consider the specific application, the environment in which the probiotics will function, and the desired therapeutic effects. Optimizing these methods can improve the efficacy and stability of probiotic formulations, ultimately enhancing health outcomes in various conditions. The comparison of the coating methods above is listed in Table 2 as follows.

2.1.1.2.　 Surface conjugation on single cell

Apart from coating the whole bacteria, conjugation is another way to modify the probiotic. There are two types of conjugation methods: non-covalent conjugation and covalent conjugation⁷⁵. Covalent conjugation and non-covalent conjugation are two distinct binding methods in chemistry. Covalent conjugation involves the formation of a chemical bond between two or more atoms through the sharing of electron pairs. On the other hand, non-covalent conjugation refers to weak interactions between atoms or molecules, such as electrostatic forces, van der Waals forces, or hydrogen bonds. Non-covalent conjugation methods include electrostatic interactions, in situ deposition, and lipid-insertion methods, which are simple and easy to operate on the surface of probiotics. Nonetheless, covalent conjugation methods, such as metabolic labeling and click reaction, are more stable than non-covalent conjugation. Surface conjugation of probiotics is a simple yet efficient strategy, which not only gives probiotics the extra ability to resist external environmental threats but also endows them with exogenous qualities that are neither innate nor naturally attainable in a controlled manner⁷⁶.

Non-covalent conjugation: Various lipid nanoparticles are frequently used to modify probiotics with organic substances. For instance, Akolpoglu et al.⁷⁷ combined magnetic nanoparticles with nanoliposomes (NLs) containing photothermal agents and chemotherapeutic molecules to create ICG-DOX NLs on EcN, with an integration efficiency of about 90%. As shown in Fig. 3A, this biohybrid microrobot was constructed by attaching the synthetic materials, streptavidin magnetic materials and nanoparticles (mNPs), and biotinylated NLs to EcN via non-covalent interactions. Compared with the control group, the bacterial biohybrids, when applied to cancer cells in a 3D tumor sphere model, led to a high mortality rate, which was further elevated under NIR radiation. The bacterial biohybrids outperformed the previously described EcN-based micro-robots because they retained their original motility, which could move through biological matrices and colonize in tumor spheroids when exposed to magnetic fields. They could also release drug molecules on demand in response to near-infrared light stimulation. These bacterial biohybrids provided a versatile micro-robotic platform for guided locomotion in three-dimensional biological networks and provided stimulus-responsive therapies for a variety of medical applications.

Additionally, paclitaxel (PTX) and the glucose transporter 1 (GLUT1) inhibitor BAY-876 were added to human serum albumin to create a nanomedicine (HPB), which was then electrostatically grafted onto the probiotic EcN to create EcN@HPB. Due to the targeted effect of EcN, this engineered biohybrid significantly increased the ability of HPB nanomedicine in the tumor site. The released BAY-876 inhibitor subsequently prevented the internalization of glucose by tumor cells after EcN competitively depleted glucose. In summary, the decreased glucose bioavailability in tumor cells activated and promoted macropinocytosis in an adenosine monophosphate (AMP)-activated protein kinase (AMPK)-dependent manner, increasing cellular HPB uptake and improving the therapeutic effectiveness of PTX⁴³.

Metallic materials such as metal–organic frameworks (MOFs) are typically used to modify probiotics. As shown in Fig. 3B, Han et al.⁴⁴ created E@Fe-DOX by depositing adriamycin (DOX) loaded iron-polyphenol (Fe-DOX) nanoparticles on E. hallii, which can be specifically targeted to hypoxic tumor regions, to release both DOX and Fe³⁺ for immunogenic cell death (ICD). The mapping images of C, N, and Fe elements indicated that Fe-DOX nanoparticles were located on the surface of the probiotic. Lactate is abundantly expressed in the tumor microenvironment (TME), and E. hallii could change lactate into butyrate. E. hallii prevented the polarization of pre-tumor M2-like macrophages and enhanced the activity of cytotoxic T cells by continuously converting intratumoral lactate to butyrate. Subsequently, E@Fe-DOX promoted the development of tertiary lymphoid structures (TLS), a site of immune cell aggregation to boost ICD-induced anti-tumor immunity. In the colon cancer mice model, the combination therapy of E@Fe-DOX and α-PD-1showed a prolonged mice survival rate compared to the PBS group.

Wei et al.⁷⁸ constructed resiquimod (R848) loaded poly-lactic-co-glycolic acid (PLGA) nanoparticles (defined as PR848) and DOX-loaded PLGA nanoparticles (defined as PDOX) by using the emulsification solvent evaporation method, respectively. Then, electrostatically, PR848 nanoparticles were linked to non-pathogenic EcN MG1655 to form Ec-PR848. After intravenous injection (i.v.), Ec-PR848 could target hypoxic tumors and polarize M2 macrophages to M1 macrophages to stimulate the immune system's anti-tumor responses. Meanwhile, the intratumorally injected PDOX triggered ICD to encourage dendritic cells (DCs) maturation as well as antigen presentation and to activate cytotoxic T lymphocytes (CTLs) for the weakening of tumor immunosuppression, which in turn activated the anti-tumor immune response and killed tumor cells.

Covalent conjugation: Cao et al.⁴⁶ reported an artificially enzyme-modified platform based on the probiotic Bifidobacterium longum (BL) to treat IBD (Fig. 3C). This platform used an iron single-atom catalyst (Fe SA) nanomaterial with atomically dispersed active metal centers to mimic the natural antioxidant defense system effectively and replace anti-inflammatory clinical medications. Then, boronic acid–poly(ethylene glycol) (C18-PEG-B)-modified Fe SA (defined as B-SA) was conjugated onto BL via click reaction to obtain the probiotics-artificial enzymes system of BL@B-SA50. This single-atom catalyst artificial enzymes can imitate the antioxidant enzymatic activities of superoxide dismutase (SOD) and catalase (CAT) to scavenge superoxide radicals (O₂^‒) and hydrogen peroxide (H₂O₂) and act as antioxidant biomolecules to scavenge hydroxyl radicals (–OH), thereby delivering a potent and quick relief of inflammatory symptoms. Additionally, this synthetic enzyme may serve as a probiotic guardian to shield probiotics in capsules from oxidative damage due to high levels of ROS in inflamed tissues and quickly restore intestinal barrier function and the balance of the intestinal microecosystem.

Due to insufficient intratumoral delivery of antitumor agents, the chemotherapy of pancreatic ductal adenocarcinoma (PDAC) is severely hindered. To alleviate this problem, Han et al.⁴⁵ employed an improved chemotherapy for pancreatic cancer (Fig. 3D). More chemotherapeutic drugs were delivered to the tumor site via the gut–pancreas axis by using a probiotic spore-based oral medication delivery method. Commercial Clostridium butyricum (C. butyricum) spores were covalently grafted with the powerful cytotoxic drug gemcitabine-loaded mesoporous silica nanoparticles (MGEM). The result was that hybridized SPORE-MGEM significantly increased intra-tumor drug accumulation and improved anti-tumor efficacy via gut-pancreas axis-guided delivery.

2.1.2.　 Multiple cells encapsulation

Single-cell probiotics are modified primarily for surface coating, whereas multiple-cell probiotics are modified for bulk encapsulation to protect probiotic cells. Typically, the encapsulated probiotics are given orally or in situ. However, before the probiotics enter the target organ, they should navigate a complex environment that includes digestive fluid and a variety of enzymes⁷⁹. Therefore, selecting coating materials is necessary. Natural polysaccharides, including CS⁸⁰, ALG⁸¹⁸², and HA⁸³, have recently become widely employed. These natural and biocompatible materials can be used as coating components to encapsulate probiotics and form diverse dosage forms.

2.1.2.1.　 Hydrogel

The biocompatible hydrogels with a unique 3D cross-linked network structure allow them to hold large amounts of water and bodily fluids to avoid disintegration. Usually, one or more organic or inorganic polymers link together through covalent bonds or non-covalent bonds (e.g., electrostatic interactions, hydrophobic interactions, and hydrogen bonds) to form the hydrogels. Polysaccharides (e.g., ALG, CS, HA) and protein (gelatin and whey protein, etc.) are the most frequently used structural components of hydrogel matrix, respectively as physical barrier and buffer, which endow hydrogel with the expansion, water-absorbing quality and pressure tolerance. The existing oral hydrogels often take advantage of differences in pH, chemical composition (e.g., stomach acid, bile salts), or digestive enzymes between the stomach and intestinal to pre-determine the timing of the disruption of probiotic–hydrogel interaction, allowing hydrogels to pass through the stomach and deliver probiotics to intestinal⁸⁴.

Yang et al.⁸⁵ created an oral probiotic delivery system based on calcium tungstate microgel (CTM) by cross-linking calcium ions with ALG. In Fig. 4A, CTM deliberately disrupted the ecological niche of the excessively expanded Enterobacteriaceae in colitis to promote probiotic colonization. Additionally, the calprotectin, as the calcium-binding protein that is abundantly expressed in colitis, efficiently removed calcium from CTM to release tungsten, which dislodged molybdenum in the molybdenum enzyme to inhibit Enterobacteriaceae. For the encapsulation and targeted distribution of probiotics, a ROS-responsive HA hydrogel was created by Huang et al.⁸⁶ The methacrylated HA and thioredoxin were cross-linked biologically to obtain HA-LR. LR, a model probiotic encapsulated in the hydrogel, showed a significantly better survival rate under simulated digestive conditions. The negatively charged hydrogel helped probiotics preferentially adhere to inflammatory areas. The thioredoxin link in the hydrogel was selectively broken by excess ROS produced by inflamed colonic mucosa, accompanied by hydrogel disintegration and localized probiotic release. Additionally, the hydrogel had a sufficient capacity to scavenge ROS and shield HT-29 cells from oxidative injury.

Besides treating digestive diseases, probiotics are often crucial in treating different illnesses. One example is the introduction of probiotics for the treatment of vaginitis. Although molecular therapeutics are frequently used to treat Candida vaginitis, the high recurrence rate hindered their application, and these methods destroy vaginal tissues and exacerbate the vaginal microbiota's imbalance. In Fig. 4B, Wei et al.⁸⁷ combined the rGO@FeS₂ nano-enzymes [reduced graphene oxide (rGO)] with Lactobacillus (a probiotic that generates lactic acid and H₂O₂) to create a responsive hydrogel-rGO@FeS₂/Lactobacillus@HA (FeLab). FeLab significantly reduced bacterial growth in mice with Candida vaginitis while causing bare harm to vaginal mucosal cells and encouraged the healing of the mucosa. Additionally, with an upsurge in the proportion of the thick-walled bacterial phylum (particularly Lactobacillus) and a decline in Aspergillus, the vaginal microbiota was remodeled, lessening the likelihood of inflammation returning.

2.1.2.2.　 Microspheres and microcapsules

Apart from the probiotic-based hydrogels, hydrogels can also form microspheres. Probiotics or functional food molecules are encapsulated in hydrogel microspheres, a unique emulsion-based delivery medium with good biocompatibility and controlled release features. The probiotic-based hydrogel microsphere therapy is efficient and secure for treating intestinal illnesses. As shown in Fig. 4C, Wang et al.⁸⁸ formed a novel nitric oxide (NO)-responsive Poly-γ-glutamic acid hydrogel microcapsules (NRPM) to encapsulate Lactobacillus. Microspheres with wide-ranging diameters (100–600 μm), high cell densities, and homogeneity (6.0 × 10⁸ cells/mL) were required to allow accurate downstream evaluation and application. The modified probiotics displayed high survivability in simulated gastric (89.67%) and intestinal (93.67%) fluid environments because of the cytoprotective impact of NRPM, whereas the data of free cells were 0% and 61.60%, respectively. Additionally, in vitro and in vivo research suggested that microspheres responded to NO stimulation and released probiotics fleetly, maintaining the intestinal barrier and regulating the intestinal flora balance.

In the study of Cheng et al.⁸⁹, layer-by-layer electrostatic droplet self-assembly of alginate and protamine was used to create enzyme-triggered fusion-like microcapsules. This study demonstrated that the probiotics could be shielded from acid and bile acid by the fusion structure of two fisetin layers in alginate microcapsules, which could then be broken down layer by layer by trypsin to regulate bacterial release, aid in bacterial adhesion, and effectively colonize the proximal colon and distal ileum.

2.1.2.3.　 Nanofiber

In 2006, Salalha et al.⁹⁰ attempted to encapsulate bacteria (Escherichia coli, Staphylococcus albus) in electrospun nanofibers. The most widely used technique for creating nanofibers is electrospinning⁷⁰. Electrospun nanofibers have a high specific surface area and porosity, which can preserve the bioactivity of the encapsulated compounds and facilitate the release of bioactive molecules.

Nanofiber-encapsulated probiotics have attracted more attention recently⁹¹. Ajalloueian et al.⁵⁷ created a nanofiber to shield the probiotic LGG using electrospinning technology. As shown in Fig. 4D, Pullulan, positioned between the inner and outer layers of PLGA, protected the three-layer electrostatically spun nanofiber. LGG delivered by the multilayer structure could survive intestinal transport and recover activity in all areas of the intestine.

In Fig. 4E, Grilc et al.⁵⁸ incorporated spores from two different Bacillus strains, individually or in combination, into hydrophilic polyethylene oxide (PEO) and composite PEO/ALG nanofibers. The nanofiber mats had a significant load of viable spores (>7 log CFU/mg), and the vitality of the spores was maintained throughout electrospinning after six months of storage at room temperature. Spores were rapidly expelled from PEO nanofibers, but the ALG in them prolonged their release. The produced nanofiber contained genotyped novel prospective probiotic strains with simultaneous immunomodulatory and antibacterial activities that were demonstrated to be a promising tool for local therapy of biofilm-associated illnesses, including periodontitis⁵⁸.

Treatment for microbiological disruptions of the female genitourinary system often involves the usage of antibiotics. Probiotics are increasingly being used as an adjuvant and alternative to antibiotic therapy, which calls for the development of innovative delivery methods for vaginal use. Minooei et al.⁹² developed a novel, quick-dissolving platform that employed electrospun PEO fibers to deliver Lactobacillus acidophilus (LAC) and the antibiotic metronidazole. The vaginal colonization of probiotics encapsulated in PEO fibers achieved the same level as free LAC.

Conventional physical modification strategies are generally straightforward and safe, largely because they do not require additional toxic chemical agents. Multiple cells encapsulation, which can protect the probiotics from the harsh gastrointestinal environments, often realizes the bulk encapsulation of probiotics⁹³. However, this approach has some limitations, including low encapsulated efficiency, early release of probiotics, and a time-consuming process.

Compared with the conventional multiple cells encapsulation, the single-cell coating can overcome the issues for probiotic delivery and provides some unique features, such as resistance to the in vivo gastric environment, improved colonization, and a potential therapeutic effect in treating related diseases. However, the single-cell coating of probiotics still has some drawbacks. For instance, the layer-by-layer self-assembly technique is cumbersome and time-consuming, which hinders its large-scale application⁹⁴. Overall, enhancing the coating efficiency of probiotics and increasing the resistance of coatings to gastric juice and bile acids will undoubtedly promote the biomedical use of probiotics.

Therefore, chemically modifying probiotics is an effective approach for single-cell modification, which can protect them from harsh environments, thereby enhancing their survival rate. Additionally, chemical modification can increase the colonization rate of probiotics in the intestine, extend their intestinal retention time, and improve treatment outcomes for related diseases. Controlled release of probiotics can also be achieved after chemical modification by incorporating stimuli–responsive properties. Furthermore, chemical conjugation can introduce additional functional substances to probiotics, endowing them with enhanced functions to improve therapeutic efficacy and broaden their applications. However, there is still room for improvement in chemical engineering strategies, such as developing more biocompatible materials and mild reaction conditions that do not compromise bacterial cell viability and reducing the use of chemical reagents.

2.2.　 Biomedical modification

Synthetic biology and microbiology advancements have led to significant progress in creating genetically modified probiotics for various biomedical purposes. Biological engineering methods are preferred over physicochemical modifications because they are more compatible and secure for probiotics due to their limited use of external chemicals and covalent bonds. While physicochemical techniques can modify a wide range of bacteria by targeting common functional groups on their surfaces and giving them various external properties, biological engineering techniques can introduce lasting functions⁹⁵.

2.2.1.　 Genetic engineering

Traditional methods make it challenging to generate enough therapeutically active proteins and peptides required to treat endocrine and cancerous illnesses, which restricts their widespread application. The unique characteristics of probiotics, such as safety, lesion targeting, tissue retention ability, gene editing, and high proliferation rates, have shown significant application potential in treatment, biological imaging, and diagnosis^96,97. Due to the rapid development of synthetic biology, researchers are now focusing on engineering probiotics as bioactive factories to express various therapeutic components in situ. It is worth noting that various types of recombinant proteins have been effectively expressed since the development of engineering bacteria. For example, the first recombinant protein product—human insulin, was introduced in 1982, sparking researchers’ enthusiasm for using genetic engineering to create recombinant protein therapies⁹⁸. So far, numerous recombinant protein medications have already hit the market, which has had significant positive economic and societal effects.

As shown in Fig. 5A, phenylketonuria is a metabolic disorder characterized by high levels of phenylalanine (Phe) in the blood. Isabella et al.⁹⁹ genetically modified EcN 1917 to overexpress Phe metabolic enzymes, significantly reducing circulating Phe during intestinal administration. Probiotics are genetically modified to express a range of antibodies to elicit an immune response and defend against particular bacterial or viral infections. For instance, in Fig. 5B, Chowdhury et al.²¹ engineered a non-pathogenic Escherichia coli strain to specifically lyse within the tumor microenvironment and release an encoded nanobody antagonist of CD47 (CD47nb), which further increased the activation of tumor-infiltrating T cells and stimulated rapid tumor regression in a syngeneic tumor model in mice. Additionally, there is growing evidence linking IBD to intestinal microbiome^22,23. Engineering probiotics have been utilized to provide anti-inflammatory chemicals in treating Crohn's disease (CD), UC, and IBD. As shown in Fig. 5C, Praveschotinunt et al.¹⁰⁰ genetically modified EcN to create fibrous matrices that promote gut epithelial integrity in situ. In addition to EcN, many other probiotics are also used for genetic engineering, such as Lactobacillus, which was engineered to continuously deliver glucagon-like peptide-1 (GLP-1) in the intestine to treat memory disorders induced by lipopolysaccharide (LPS) and improve spatial learning in mice⁵⁹. In addition, L. lactis was edited to produce anti-inflammatory cytokine interleukin-10 (IL-10), which can effectively relieve colitis symptoms in mice⁶⁰. By converting Lactobacillus casei into a toxoid-toxin that expresses and secretes Clostridium perfringens, Gao et al.⁶¹ created a vaccination against Clostridium perfringens infection, providing a more theoretical and practical basis for expanding the application of engineered probiotics.

Genetically engineered probiotics have great potential as highly targeted, self-sustaining therapeutic agents that adapt and respond dynamically to the body's needs, creating in situ therapeutic responses based on specific biological signals. For instance, these engineered probiotics could be designed to sense particular biomarkers associated with disease states, such as inflammation in the case of IBD, and respond by producing tailored therapeutic molecules (like anti-inflammatory cytokines) only when needed, enhancing both efficacy and safety. Additionally, combining genetically engineered probiotics with immune-modulating functionalities presents a promising area for cancer immunotherapy, as probiotics could be engineered to enhance immune activity directly within tumor sites, which could personalize cancer treatment while minimizing systemic side effects.

2.2.2.　 Biofilm encapsulation

Microorganisms are often surrounded by a thick, dense extracellular polymeric matrix that they secrete, primarily composed of polysaccharides, proteins, and extracellular DNA. This complex structure, known as a biofilm, enables microorganisms to thrive in extreme conditions and resist various physical and chemical threats, including environmental removal, displacement by physical forces, exposure to antibiotics, and attacks by the host immune system. Inspired by the robust resistance of biofilms to harsh environments, probiotics that can form biofilms or are coated with biofilms gain improved adhesion and resilience in transplanted gut microbiota. There are two categories of biofilm origins: biofilm sourced from other organisms and biofilm generated by probiotics.

The YM and EcN were physically extruded through a porous polycarbonate membrane with a diameter of about 1 μm to create YM-encapsulated EcN (EcN@YM). Due to the high expression of β-glucan on YM, the Dectin-1 receptor on the microfilmed cells (M-cells) in the intestinal epithelium may selectively attach to it and internalize EcN@YM after oral delivery. M-cells transferred the absorbed live bacteria to lymphoid follicles, which could strengthen the mucosal immune response. In the gut, there were noticeably high levels of secretory immunoglobulin A (sIgA), CD11c⁺ DCs, CD4⁺ T-cells, and IgA⁺ B-cells. In particular, pathogens such as Salmonella, EcN, and Shigella were largely repressed, while commensal bacteria were successfully kept in the infected gut due to the improved immunity⁶². Finally, this yeast membrane-camouflaged probiotic substantially reduced intestinal barrier disruption in the Salmonella-infected mouse and intestinal manipulation models.

Other academics have been interested in the possibility of using probiotic spores as a natural biofilm to protect probiotics in addition to using yeast membrane biomimicry. As shown in Fig. 6A, by in vitro mechanical extrusion, Song et al.¹⁰¹ created nanomaterials from the probiotic spore shell, which then acted as “armor” to encapsulate probiotics. In addition to enhancing the natural biological activity and intestinal colonization capacity of probiotics, probiotics enclosed in spore coat nanomaterials could maintain the homeostasis of intestinal commensal flora and repair the integrity of the intestinal mucosal barrier. The probiotics were able to reduce the symptoms of colitis greatly and showed success in preventing colitis-related cancers.

In Fig. 6B, Wang et al.¹⁰² developed a self-coating strategy with biofilms produced by probiotics themselves. Bacillus subtilis (BS) can generate enormous amounts of extracellular polysaccharides and proteins such as TasA and BslA, creating a biofilm when BS grows on glycerol glutamate (Msgg) plates. Polysaccharides connect the probiotics, BslA creates a hydrophobic membrane, and TasA self-assembles into fibers, adhering to the cell wall. For the production of solid biofilms, the minimal salts Msgg culture plate serves as an attachment site. The membrane was homogenized to create individual biofilm-encapsulated BS (BCBS). Encapsulated BCBS showed a 125-fold increase in biological activity and a 17-fold increase in colonization capability compared to unencapsulated BCBS in vivo investigations on a pig model. BCBS also showed an increased decolonization effect in a mouse model of Staphylococcus aureus (SA) infection.

Similarly, incubation with biocompatible dextran microspheres (DM) could stimulate the biofilm produced by Lr. Additionally, to promote the creation of biofilms, these microspheres might be filled with advantageous ingredients such as sucrose or maltose. As a preventative measure and a form of therapy, a single dose of Lr in its biofilm stage lessens the severity and frequency of experimental C. difficile infections⁶³.

Despite significant advancements in probiotic modification, several challenges remain unresolved. First, physically modified probiotics suffer from low encapsulation efficiency and premature release¹⁰³. Second, chemically modified probiotics face high costs, environmental concerns, potential toxicity to probiotics, and risks to human health. For genetically engineered probiotics, limited genetic engineering platforms and incomplete genomic information for some strains pose challenges to their broader application. To overcome these issues, new materials should be explored for probiotic modification, such as probiotic spores, fungal cell walls, microalgae shells, and other advanced artificial approaches like microfluidics and bioprinting. Furthermore, it is crucial to gather more genomic information on other probiotics and develop efficient and reliable genetic manipulation techniques for different probiotic species to obtain diverse functional probiotics with enhanced intestinal delivery efficiency.

3.　Modified probiotics in combinatorial therapeutics

Less

Probiotic therapies have shown great potential in disease treatment. However, some disorders, like severe diarrhea, inflammatory bowel disease, and colon cancer, cannot be treated with the use of probiotics alone. The characteristics of probiotics, including rapid growth ability, anaerobic tendency, and immunomodulatory function, make them suitable as drug delivery vehicles. As a result, probiotic-based drug delivery has attracted much attention from scholars. Furthermore, combining probiotics with other therapies can lessen single therapies’ adverse effects while enhancing and amplifying the therapeutic effectiveness. Probiotic-based combination therapy offers a new course of treatment for gastrointestinal diseases or other diseases that benefit from probiotic therapy¹⁰⁴.

3.1.　 Combination of modified probiotics and pharmacotherapy

To achieve optimal therapeutic effects, the majority of anti-IBD drugs must reach the colon at high concentrations. However, 5-ASA and its prodrugs are easily absorbed and eliminated in the upper gastrointestinal tract following oral administration, implying that drug accumulation in the colon is restricted¹⁰⁵. Moreover, most nanomedicines cannot penetrate the dense tumor mesenchyme due to the disordered vascular system and high tumor mesenchymal pressure, and only a few nanomedicines reach the tumor site^106-109. Therefore, improving the effect of drug targeting and bioavailability remains a significant challenge. Probiotic therapies show enormous promise in improving the combined efficacy. Combining probiotics with drugs or drug-loaded nanoparticles has demonstrated outstanding anti-tumor efficacy^110,111.

The complex and variable gastrointestinal microenvironment leads to suboptimal colonization efficiency of most probiotics, limiting their wide application in disease treatment^112,113. Zhang et al.⁷¹ assembled the anti-UC prodrug Bal onto liposomal LPC to form a prodrug nanomolecule LPC-Bal to enhance probiotic colonization while maintaining bacterial activity. Then, LPC-Bal was modified onto the surface of LGG to obtain a prodrug-encapsulated LGG, abbreviated as LBL. The LBL effectively increased the colonization efficiency of LGG, improved the pathological microenvironment, reshaped the gut microbiota composition, and exhibited outstanding anti-UC properties (Fig. 7A). In addition, several studies have shown that both parthenogenetic anaerobic bacteria, such as Clostridium difficile, and probiotics, such as Bi and EcN, possess the intrinsic tumor-targeted ability due to their instinctive tendency towards hypoxic and eutrophic tumor microenvironment. Therefore, precise delivery of drugs to the tumor region can be achieved by modifying drug-carrying nanocarriers on the bacterial surface. The Bi was covalently modified with DOX-loaded CaP/SiO2 nanoparticles (DNPs) to construct the DNPs@Bi for tumor targeting therapy by He et al. (Fig. 7B). On the one hand, pH-responsive DOX release could induce chemotherapy and immunogenic death in the immunosuppressive tumor microenvironment (ITME). On the other hand, tumor-targeted Bi was able to significantly enhance tumor-associated antigen presentation from B16F10 cells to DCs via recombinant connexin 43 (Cx43)-dependent gap junctions¹¹⁴.

Controlled release of drugs within the target site is challenging to achieve, particularly in fluctuating microenvironments like tumors or inflamed tissue. However, researchers have identified probiotic spore (PS) as a potential carrier for controlled drug release. PS is a dormant life form with low water content and high resistance to harsh environments such as high temperatures, acids and alkalis, ultraviolet rays, and a variety of chemicals¹¹⁵. PS can resist extremely harsh gastric environments. Moreover, after reaching the intestine, PS absorbs water and sheds hydrophobic spore coat proteins to regerminate into probiotics and colonize the intestine^116,117. After colonization, Bacillus initiates multiple biological actions, such as regulating intestinal flora, secreting therapeutic metabolites (e.g., SCFAs), and enhancing innate and adapted immunity¹¹⁸. Inspired by this natural process, researchers have organically combined probiotic spores with drugs or drug-loaded nanoparticles through hydrophobic interaction, electrostatic adsorption, hydrogen bonding, etc. This method significantly achieves the natural-spore-germination-based drug controlled release profile and improved drug bioavailability. As shown in Fig. 7C, Song et al.¹¹⁹ developed an oral nanoparticle generator where the spores were loaded with chemotherapy drugs (doxorubicin, sorafenib, DOX/SOR) and modified with deoxycholic acid (DA). On the one hand, the nanoparticle generator protected the loaded drugs from the harsh intestinal environment. On the other hand, the autonomously generated DOX/SOR/Spore-DA nanoparticles could effectively penetrate epithelial cells, thereby increasing drug release efficiency on the basal outer side. The in vitro and in vivo studies have shown that the nanogenerator autonomously generated many nanoparticles in the intestine, providing a new strategy for cancer treatment. In Fig. 7D, Zheng et al.¹²⁰ chemically modified prebiotic Dextran (Dex) onto the commercial C. butyricum spores to obtain Spores-Dex through a host-guest chemical reaction. The results indicated that Spores-Dex could specifically accumulate in tumor sites after oral administration. Dextran can be fermented by bacteria such as C. butyricum and produce short-chain fatty acids, significantly increasing the overall richness of the microbiota. This work reveals the possibility of using highly safe strategies to regulate gut microbiota and provides promising avenues for treating various gastrointestinal diseases. In addition, probiotics/spores and drugs/drug-loaded nanoparticles can also be encapsulated by hydrogels or microspheres to promote the colonization of probiotics and control the release of drugs through conditional responsive release^41,121.

There are also clinical examples of directly combining drugs with probiotics, leading to significant breakthroughs in disease treatment. Recently, a phase 1 clinical trial (NCT03829111) showed that when patients with metastatic renal cell carcinoma (mRCC) received treatment with nivolumab + ipilimumab, combined with the probiotic oral drug CBM588, the median progression-free survival (PFS) was greatly improved, with a median PFS extension from 2.5 to 12.7 months, an increase of nearly 400%¹²². For the first time, it has been demonstrated that oral probiotic drugs can improve the gut microbiota homeostasis of cancer patients and enhance immune therapy response. Immune checkpoint inhibitors, such as CTLA-4 and PD-1, are powerful tumor immunotherapy agents. However, their clinical use can disrupt the immune balance, causing side effects like diarrhea, colitis, and even severe autoimmune reactions. Sun et al.¹²³ revealed that Bifidobacterium optimize the gut microbiota composition and enhance the function and metabolism of intestinal regulatory T cells (Tregs) under CTLA-4 blockade, effectively reducing intestinal inflammation caused by anti-CTLA-4 antibodies.

3.2.　 Combination of modified probiotics and phototherapy

Photodynamic therapy (PDT) as an effective cancer treatment is being pursued with enthusiasm¹²⁴. The TME responsive photosensitizers (PSs) are necessary for the tumor-targeted precise PDT. However, many obstacles hinder the widespread application of PS-based therapies, such as the limited tumor penetration and retention, rapid weakening of the photodynamic effect, and the need for multiple irradiations for combination therapies^125,126. Since probiotics tend to flow into the hypoxic and immunosuppressive TME, probiotic-based tumor-targeted therapeutic systems have been developed in recent years.

As shown in Fig. 8A, Guo and colleagues¹²⁷ used surface modification and gene expression to construct a monochromatic radiation-based ternary system that combined photoacoustic and photothermal techniques within BL21 cells. They explored its application in photoacoustic imaging-guided synergistic photothermal therapy in tumors. Indocyanine green and PDA nanoparticles were co-deposited on the surface of the melanin-expressing bacterium BL21 by in situ polymerization. The integrated BL21 could produce stable triple photoacoustic and photothermal effects after monochromatic irradiation, owing to the adequate absorption capability of melanin, indocyanine green, and PDA at 808 nm. This opened up a fresh possibility for the creation of novel photosensitizers. Intratumoral injection of the ternary photosensitizer combined with BL21 showed a considerable improvement in tumor regression and extended survival in colon cancer and breast cancer-bearing mice.

Yang et al.¹²⁸ coupled Lactobacillus acidophilus (LA) with 2D CoCuMo layered-double-hydroxide (LDH) nanosheets (LA&LDH) for tumor-targeted precise NIR-II photodynamic treatment. Following tail vein injection, LA induced low pH (5.4), and the LA metabolites produced glutathione (GSH) in TME may etch 2D CoCuMo-LDH nanosheets. When exposed to 1270 nm laser irradiation, the phase transition of the CoCuMo-LDH nanosheets from crystalline to amorphous significantly increased their ROS generation activity and achieved complete tumor elimination effectively. This work showed probiotics could be employed as a tumor-targeting platform for effective and precise NIR-II PDT.

Shi et al.¹²⁹ combined D-alanine (d-Ala) as the metabolite of C. butyricum with aggregation-induced emission (AIE) photosensitizer TPApy to create an engineered C. butyricum to cure melanoma. Once injected into melanoma, the modified C. butyricum increased only in the anaerobic region, enhanced the tumor immune microenvironment, and destroyed the hypoxia region. Following that, the relatively high oxygen level in the peripheral zone triggered the death of C. butyricum. Under light irradiation, the PSs on the bacteria exerted a photodynamic impact in the oxygen-rich area, further eliminating melanoma residues.

Exploring novel probiotics to regulate the gut microbiota holds significant therapeutic promise because the discovery of the gut–brain axis has demonstrated the capacity of gut microbial metabolites to impact brain function. In PD, a common neurological illness, dopaminergic neurons in the substantia nigra deteriorate, which results in both motor and non-motor symptoms. PD patients frequently have gastrointestinal problems. Gut microbiota and their metabolites, such as SCFAs and gamma-aminobutyric acid (GABA), communicate with the brain through many ways (endocrine pathway, immune system, nerve pathway), which is discovered with the elucidation of the gut–brain axis. As a result, probiotics that reshape gut microbiota will be crucial in treating PD.

Pan et al.¹³⁰ developed three blue-light-responsive probiotics as orally live biotherapeutics. The three engineered Lactobacillus strains were designed to produce GABA, granulocyte colony-stimulating factor (GCSF), or GLP1 upon exposure to blue light to modulate anxiety disorders, PD, or neural signaling pathways, respectively. The micro-nano system targeted the small intestine and promoted the abundance of exogenous L.lactis in the intestines. This achieved accurate control of mental processes such as anxiety, PD, and vagal nerve stimulation.

In addition to creating probiotics that respond to blue light, Zhang et al.¹³¹ also created an EcN 1917 strain (ROEN) with a red light-responsive controlled drug release profile. ROEN enclosed in pH-sensitive alginate hydrogel microcapsules (AMCs) was given orally to target the intestines. After oral administration, ROEN released Exendin-4 under optogenetic control in the PD mouse model. The released Exendin-4 fused with the anti-neonatal Fc receptor affibody, which endowed ROEN with the function of regulating the gut–brain axis. This red-light optogenetic probiotic offered a novel drug delivery and gut–brain axis regulatory platform.

3.3.　 Combination of modified probiotics and sonodynamic therapy

There have been several research on using ultrasound (US) in detecting and treating diseases since light has a limited ability to penetrate human tissues, whereas ultrasound has a high penetration rate. Sonodynamic therapy (SDT) is a low-frequency ultrasound-mediated, non-invasive physical stimulation therapy with high biocompatibility. Hematoporphyrin monomethyl ether (HMME) has been approved for clinical use in treating tumors. Still, its low molecular weight, as well as poor tumor enrichment and retention, severely limit the performance of HMME-based sonodynamic therapy. In addition, due to the immunosuppressive nature of tumor tissues, the immunogenic anti-tumor efficacy of SDT is low¹³². In contrast, probiotics can accumulate and retain in hypoxic tumor cores. It has been shown that Bifidobacterium preferentially accumulates in tumors when it enters the bloodstream. This is likely because the hypoxic microenvironment is a tropism, and the immunosuppressive circumstances encourage bacterial colonization and growth¹³³.

In Fig. 8B, Lu et al.¹³⁴ grafted the sonosensitizer HMME onto the specialized anaerobic probiotic Bifidobacterium longum (BiL) to create HMME@BiL, which achieved the tumor-targeted HMME delivery with the help of the anaerobic tendency of BiL. This improved the accumulation of sonosensitizers in tumor location, resulting in a practical SDT effect. Simultaneously, the STING agonist SR717 promoted anti-tumor immunity by encouraging the tumor infiltration of cytotoxic T-cells and NK cells and releasing associated cytokines, making sonoimmunotherapy (SIT) possible. In the CT26 colorectal cancer metastasis model, the experimental results showed that combination treatment of HMME@BiL + US + SR717 could successfully inhibit primary tumors and reduce the course of the disease.

However, many modified probiotics can release payloads outside the tumor site after injection, potentially harming healthy tissues. H. Abedi et al.¹³⁵ created a focused ultrasound-assisted controlled administration of therapeutic microbes to overcome this constraint. Focused ultrasound is one form of energy that can be applied non-invasively to specific anatomical regions, including solid tumors. The research developed genetically engineered EcN that express temperature-sensitive gene regulatory switches and tumor-suppressive nanobodies. The engineered EcN expressed nanobodies to kill tumor cells when the tumor site was heated by focused ultrasound. By introducing temperature-dependent genes and nano-antibody genes, the scientists created bacterial strains that manufactured tumor-suppressing nanoantibodies when heated to a trigger temperature of 42–43 °C. These strains did not induce anti-tumor nano-antibodies when put into the body since the usual body temperature is 37 °C. Instead, they developed invisibly within the tumor up until a heat source outside of it raised them to the trigger temperature. This technology provided a pivotal tool for the spatiotemporal targeting of potent probiotics therapies in multifarious biological and clinical scenarios.

3.4.　 Combination of modified probiotics and magnetotherapy

Although engineered probiotics are adaptable, their unpredictability in vitro and inaccurate in vivo localization are drawbacks. Therefore, techniques that enable the controlled long-term localization of exogenous probiotic agents in vivo are required. The magnetic field is an ideal control mechanism since it can freely penetrate biological tissues. However, magnetic fields, like the GI tract, are challenging to apply with enough strength to directly manipulate magnetically tagged cells in deep tissue. As shown in Fig. 8C, Buss et al.¹³⁶ attempted the cellular localization assisted by magnetic particles (CLAMP) method. When given orally and combined with an applied magnetic field, a composite biomagnetic material made of probiotics (EcN BL21 and EcN 1917) and microscale magnetic particles (carboxyl-functionalized superparamagnetic iron oxide particles) allowed probiotics to be captured and retained in the gastrointestinal tract of mice. This technology enhanced the accumulation and colonization of probiotics at targeting locations. This cellular localization provided external physical control to an important developing class of microbial therapies.

3.5.　 Combination of modified probiotics and immunotherapy

Immunotherapy, as a powerful clinical strategy for treating cancer, has some drawbacks, such as the relatively low response rate and severe side effects, such as autoimmunity and nonspecific inflammation¹³⁷. Either of these will severely limit immunotherapies’ efficiency¹³⁸. Many delivery materials appeared to enhance the immune response and weaken the side effects. Various nanoparticles and even T cells are used to convey therapeutics to target the tumor area, reduce systemic immune toxicity, and decrease the accumulation of non-target sites¹³⁹. Probiotics, especially those anaerobic probiotic bacteria, have a particular characteristic: the natural tendency to hypoxic tumors, making them excellent candidates for transmitting immunotherapies. As a result, the combination of probiotics and immunotherapy emerged.

Li et al.¹³⁹ reported an approach of decorating probiotic EcN with triple immune nanoactivators to develop tumor resident living immunotherapeutics. The tumor-specific antigen OVA and immune checkpoint inhibitor α-programmed cell death protein (PD) 1 were conjugated to PDA nanoparticles. Then, these conjugators were attached to the bacterial surface via in situ precipitation polymerization of dopamine. Such decorated probiotics show spatiotemporal tumor retention and proliferation-dependent drug release. In addition to acting as a connector, PDA's photothermal effect could switch tumor-associated macrophages M2 to a pro-inflammatory M1 phenotype. The OVA antigens stimulated the DCs and initiated immune responses specific to the tumor. At the same time, the anchored immune checkpoint inhibitor α-PD-1blocked immune checkpoints and activated CTLs. This research created a promising platform to combine probiotics with immunotherapy.

In Fig. 8D, Liu et al.¹⁴⁰ coated EcN with a hybrid immunoreactive nanosurface using the same method: one-step in situ polymerization with the polymerization of dopamine. Specifically, the complex of a checkpoint-blocking antibody and a virus-specific antigen covalently conjugated to PDA nanoparticles were deposited on the probiotic EcN, termed EcN-αPD1-S1. Given the natural tendency to hypoxic tumors of EcN, the coated probiotic enabled sustained release and improved exposure of carried αPD1 and S1 protein for long-acting stimulation of immune cells.

3.6.　 Other applications for bioimaging and diagnosis

Probiotics can colonize in vivo biointerfaces, particularly the oral mucosa, reproductive tract, and gastrointestinal tract, and target specific lesion sites such as tumors. Under their inherent features, such as mobility, editability, and targeting, probiotics integrated with imageable agents are frequently used as living probes for bioimaging and disease diagnosis^17,141. As the primary parts of mammalian microbiomes, both Gram-positive and Gram-negative bacteria can colonize various biological interfaces in vivo and impact human health and even the course of diseases¹⁴².

As shown in Fig. 9A, Jiang et al.¹⁴³ developed a genetically engineered probiotic strain EcN 1917 by coexpressing the genes firefly luciferase (Fluc) and luciferin-regenerating enzyme (LRE) to produce robust, consecutive, and red-shifted bioluminescence for bacteria-tracking both in vitro and in vivo. This study provided an optical in vivo tumor-targeting system for investigating bacteria-associated cancer therapy.

The intelligent whole-cell biosensors constructed via synthetic biology have been extensively studied recently. In Fig. 9B, Zou et al.¹⁴⁴ constructed an intelligent probiotic (i-ROBOT) to diagnose, record, and ameliorate IBD. The main component of i-ROBOT was probiotic EcN 1917, which could respond to different levels of the inflammatory marker thiosulfate by stimulating a base-editing system to produce a heritable genomic DNA sequence and generate a colorimetric signal simultaneously. The adjustable release of the immunomodulator Acanthocheilonema viteae cystatin (AvCystatin) was also driven by fluctuations in thiosulfate. After oral administration of i-ROBOT in colitis mice, molecular recording signals were generated in processed fecal and colon samples, and the disease was ameliorated effectively. i-ROBOT offered a promising platform for gastrointestinal disorders and other metabolic diseases.

Although numerous studies have highlighted the crucial role of modified probiotics in combination with other treatments for various diseases and in bioimaging and diagnosis, their biosafety remains a significant concern, especially that of modified probiotics. Modification may alter the natural behavior of probiotics, enabling them to more easily adhere to epithelial cells or resist normal clearance processes, thereby increasing the risk of unintended colonization or pathogenicity. Some modified probiotic strains may transfer genes to gut microbes, potentially contributing to antibiotic resistance or other health risks^145,146. For populations with HIV, cancer, or individuals undergoing organ transplants or receiving immunosuppressive drugs, the weakened immune response can reduce the body's ability to contain and eliminate live microbial agents, increasing the risk of infections. Immunocompromised individuals may be more susceptible to infections such as bacteremia (bacterial infections in the bloodstream) or sepsis if the probiotic strains translocate from the gut into the bloodstream. Cases of sepsis associated with Lactobacillus and other common probiotic strains have been reported in severely immunocompromised patients¹⁴⁷.

In light of these risks, it is essential that studies on probiotics—especially modified ones—include robust safety assessments tailored to vulnerable populations. Regulatory guidelines could also mandate testing protocols that better predict the safety of probiotics in immunocompromised individuals, helping to balance the benefits and risks across different groups.

4.　Modified probiotics in clinical trials

Less

Although numerous fundamental studies that employ modified probiotics to solve the above problems have been reported, moving the physicochemical modified probiotics or biomedical modified probiotics from the laboratory to the clinic remains challenging. This may be attributed to the incomplete understanding of specific key properties of probiotics and the lack of comprehensive clinical trials. Since there are barely any clinical studies about applying physicochemical-modified probiotics, we listed the clinical studies on genetic engineering probiotics here in Table 3.

5.　Conclusions and outlooks

Less

Due to their innate qualities, probiotics present enormous potential for biomedical applications, including disease treatment, bioimaging, and diagnosis. However, their high sensitivity to environmental changes hinders the application. Various modification technologies are being developed to convey and protect probiotics from serious situations while generating significant therapeutic effects and lowering side effects. Moreover, combining probiotics and other therapeutics further amplifies the therapeutic effect, making it come to the fore.

Several clinical studies confirmed that probiotics are irreplaceable in treating specific diseases¹⁴⁸. Due to obstacles in modified probiotics’ stability, efficacy, and safety, translating them into clinical applications remains challenging. The variability and complexity of the gastrointestinal environment affect the stability, colonization and therapeutic efficacy of modified probiotics. In addition, ensuring that probiotics deliver therapeutic agents precisely to targeted sites, such as inflamed tissues or tumors, while maintaining bioavailability is a critical yet challenging aspect of clinical application. Then, the systemic administration of bacteria vector at the wrong dose might lead to unwanted adverse cardiovascular complications. Addressing these challenges is crucial for the effective clinical translation of modified probiotics, and we should focus research on improving stability, targeting precision, and establishing clear regulatory pathways.

This article provides an overview of probiotic modification strategies and introduces an innovative perspective by proposing the combinatorial therapeutics of modified probiotics with other therapeutic approaches. With constantly emerging fundamental studies in this new interdisciplinary research field, modified probiotics will open new avenues for fundamental studies and medical applications.

References

Less

Suez

, Zmora

, Segal

, Elinav

. The pros, cons, and many unknowns of probiotics. Nat Med 2019;25:716—29.

Probiotics Industry worth $105.7 billion by 2029. Available from: https://www.marketsandmarkets.com/PressReleases/Probiotics.asp. (Accessed 20/March/2025).

Spiller

. Probiotics: an ideal anti-inflammatory treatment for IBS?. Gastroenterology 2005;128:783—5.

Jakubczyk

, Leszczyńska

, Górska

. The effectiveness of probiotics in the treatment of inflammatory bowel disease (IBD)—a critical review. Nutrients 2020;12:1973.

Koh

, Hwang

, Lee

, De Sotto

, Lee

JWJ

, Lee

, et al. Engineering probiotics to inhibit Clostridioides difficile infection by dynamic regulation of intestinal metabolism. Nat Commun 2022;13:3834.

Tao

, Zhang

, Wei

, Maniura-Weber

, Li

, Ren

. An injectable living hydrogel with embedded probiotics as a novel strategy for combating multifaceted pathogen wound infections. Adv Healthc Mater 2024;13:e2400921.

Schmitt

, Greten

. The inflammatory pathogenesis of colorectal cancer. Nat Rev Immunol 2021;21:653—67.

White

, Sears

. The microbial landscape of colorectal cancer. Nat Rev Microbiol 2023;22:240—54.

McCoubrey

, Elbadawi

, Basit

. Current clinical translation of microbiome medicines. Trends Pharmacol Sci 2022;43:281—92.

10.

Yao

, Xie

, Du

, McClements

, Xiao

, Li

. Progress in microencapsulation of probiotics: a review. Compr Rev Food Sci Food Saf 2020;19:857—74.

11.

Beveridge

, Graham

. Surface layers of bacteria. Microbiol Rev 1991;55:684—705.

12.

, Wang

, Liu

, Rawding

, Bu

, Hong

, et al. Chemically and biologically engineered bacteria-based delivery systems for emerging diagnosis and advanced therapy. Adv Mater 2021;33:e2102580.

13.

Lin

, Wu

, Zhang

, Chen

, Guo

, Chen

, et al. Surface-modified bacteria: synthesis, functionalization and biomedical applications. Chem Soc Rev 2023;52:6617—43.

14.

Dos Santos

, Carvalho

, Meneguin

, Sábio

, Gremião

MPD

, Chorilli

. Oral delivery of micro/nanoparticulate systems based on natural polysaccharides for intestinal diseases therapy: challenges, advances and future perspectives. J Control Release 2021;334:353—66.

15.

Khare

, Palakurthi

, Shah

, Palakurthi

, Khare

. Natural product-based nanomedicine in treatment of inflammatory bowel disease. Int J Mol Sci 2020;21:3956.

16.

Zhang

, Liang

, Zhang

, Wang

, Zhou

, et al. Oral carbon dot nanozymes with red fluorescence and superoxide dismutase-like activity for colitis bioimaging and inflammation management. Chem Eng J 2024;493:152842.

17.

Chen

, Chen

, Shi

. In vivo bio-safety evaluations and diagnostic/therapeutic applications of chemically designed mesoporous silica nanoparticles. Adv Mater 2013;25:3144—76.

18.

Liu

, Gao

, Li

, Niu

, Liu

, Shen

, et al. Melanin nanoparticle-modified probiotics for targeted synergistic therapy of ulcerative colitis. ACS Appl Mater Inter 2024;16:31950—65.

19.

Lim

, Song

. Development of bacteria as diagnostics and therapeutics by genetic engineering. J Microbiol 2019;57:637—43.

20.

Wang

, Kazmierczak

, Eisenstark

. Strains, Mechanism, and perspective:salmonella-based cancer therapy. Int J Microbiol 2016;2016:5678702.

21.

Chowdhury

, Castro

, Coker

, Hinchliffe

, Arpaia

, Danino

. Programmable bacteria induce durable tumor regression and systemic antitumor immunity. Nat Med 2019;25:1057—63.

22.

Cryan

, O’Riordan

, Cowan

CSM

, Sandhu

, Bastiaanssen

TFS

, Boehme

, et al. The microbiota—gut—brain axis. Physiol Rev 2019;99:1877—2013.

23.

Tang

WHW

, Kitai

, Hazen

. Gut microbiota in cardiovascular health and disease. Circ Res 2017;120:1183—96.

24.

Tuson

, Weibel

. Bacteria—surface interactions. Soft Matter 2013;9:4368—80.

25.

Zhao

, Zhao

, Zhuang

, Lo

, Baker

MAB

, Lo

, et al. Frequent pauses in Escherichia coli flagella elongation revealed by single cell real-time fluorescence imaging. Nat Commun 2018;9:1885.

26.

Guo

, Ding

, Zhou

. Harnessing bacteria for tumor therapy: current advances and challenges. Chin Chem Lett 2024;35:108557.

27.

Monteiro

, Chafsey

, Leroy

, Chambon

, Hébraud

, Livrelli

, et al. Differential biotin labelling of the cell envelope proteins in lipopolysaccharidic diderm bacteria: exploring the proteosurfaceome of Escherichia coli using sulfo-NHS-SS-biotin and sulfo-NHS-PEG4-bismannose-SS-biotin. J Proteomics 2018;181:16—23.

28.

Centurion

, Basit

, Liu

, Gaisford

, Rahim

, Kalantar-Zadeh

. Nanoencapsulation for probiotic delivery. ACS Nano 2021;15:18653—60.

29.

Pan

, Gong

, Wang

, Shang

, He

, Catania

, et al. A single-cell nanocoating of probiotics for enhanced amelioration of antibiotic-associated diarrhea. Nat Commun 2022;13:2117.

30.

Xie

, Ji

, Liu

, Wan

, Zhang

, Xiong

, et al. Modified prebiotic-based “shield” armed probiotics with enhanced resistance of gastrointestinal stresses and prolonged intestinal retention for synergistic alleviation of colitis. ACS Nano 2023;17:14775—91.

31.

Yang

, Yang

, Ye

, Zhang

, Nie

, Cheng

, et al. Physiologically inspired mucin coated nissle 1917 enhances biotherapy by regulating the pathological microenvironment to improve intestinal colonization. Acs Nano 2022;16:4041—58.

32.

Pan

, Li

, Hou

, Lin

, Wang

, Pang

, et al. Polymerization-mediated multifunctionalization of living cells for enhanced cell-based therapy. Adv Mater 2021;33:e2007379.

33.

Liu

Jun

, Wang

, Heelan

, Chen

, Li

, Hu

. Mucoadhesive probiotic backpacks with ROS nanoscavengers enhance the bacteriotherapy for inflammatory bowel diseases. Sci Adv 2022;8:eabp8798.

34.

Zhou

, Li

, Chen

, Li

, Chen

, Dong

, et al. Programmable probiotics modulate inflammation and gut microbiota for inflammatory bowel disease treatment after effective oral delivery. Nat Commun 2022;13:3432.

35.

Cao

, Wang

, Pang

, Cheng

, Liu

. Biointerfacial self-assembly generates lipid membrane coated bacteria for enhanced oral delivery and treatment. Nat Commun 2019;10:5783.

36.

Zhou

, Guo

, Li

, Peng

, Wang

, et al. Nanoarmour-shielded single-cell factory for bacteriotherapy of Parkinson’s disease. J Control Release 2021;338:742—53.

37.

Geng

, Wang

, Wu

, Cao

. Biointerface mineralization generates ultraresistant gut microbes as oral biotherapeutics. Sci Adv 2023;9:eade0997.

38.

Zhu

, You

, Chen

, Xu

, Yue

, Ma

, et al. Inorganic nanosheet-shielded probiotics: a self-adaptable oral delivery system for intestinal disease treatment. Nano Lett 2023;23:4683—92.

39.

Huang

, Liang

, Yang

, Li

, Niu

, Fan

, et al. Stimulation-responsive mucoadhesive probiotics for inflammatory bowel disease treatment by scavenging reactive oxygen species and regulating gut microbiota. Biomaterials 2023;301:122274.

40.

Yang

, Zhang

, Yang

, Peng

, Ge

, Tan

, et al. An oral “Super probiotics” with versatile self-assembly adventitia for enhanced intestinal colonization by autonomous regulating the pathological microenvironment. Chem Eng J 2022;446:137204.

41.

Peng

, Feng

, Yang

, Nie

, Yu

, Ding

, et al. Gastrointestinal microenvironment responsive nanoencapsulation of probiotics and drugs for synergistic therapy of intestinal diseases. ACS Nano 2023;17:14718—30.

42.

Yao

, Lu

, Zhang

, Xie

, Han

, Zhang

, et al. Improved functionality of Ligilactobacillus salivarius Li01 in alleviating colonic inflammation by layer-by-layer microencapsulation. NPJ Biofilms Microbiomes 2021;7:58.

43.

Wang

, Chen

, Luo

, Ji

, Han

, Song

, et al. Interference of glucose bioavailability of tumor by engineered biohybrids for potentiating targeting and uptake of antitumor nanodrugs. Nano Lett 2022;22:8735—43.

44.

Han

, Zhang

, An

, Wang

, Qiao

, Zeng

, et al. Metabolic regulation of tumor microenvironment with biohybrid bacterial bioreactor for enhanced cancer chemo-immunotherapy. Adv Funct Mater 2023;33:2302728.

45.

Han

, Chen

, Fu

, Cheng

, Zhang

. Probiotic spore-based oral drug delivery system for enhancing pancreatic cancer chemotherapy by gut—pancreas-axis-guided delivery. Nano Lett 2022;22:8608—17.

46.

Cao

, Jin

, Gao

, Ding

, Wen

, Qian

, et al. Artificial-enzymes-armed Bifidobacterium longum probiotics for alleviating intestinal inflammation and microbiota dysbiosis. Nat Nanotechnol 2023;18:617—27.

47.

Kim

, Hlaing

, Lee

, Saparbayeva

, Kim

, Hwang

, et al. Exfoliated bentonite/alginate nanocomposite hydrogel enhances intestinal delivery of probiotics by resistance to gastric pH and on-demand disintegration. Carbohydr Polym 2021;272:118462.

48.

, Yang

, Ma

, Lu

SJJ

, Liu

, Pan

, et al. Hydrogel-encapsulated engineered microbial consortium as a photoautotrophic “living material” for promoting skin wound healing. ACS Appl Mater Inter 2023;15:6536—47.

49.

Yang

, Han

, Chen

, Li

, Yu

, Qu

, et al. Novel probiotic-bound oxidized Bletilla striata polysaccharide-chitosan composite hydrogel. Mater Sci Eng C Mater Biol Appl 2020;117:111265.

50.

Xiao

, Lu

, Liu

, Kong

, Bai

, Mu

, et al. Encapsulation of Lactobacillus rhamnosus in hyaluronic acid-based hydrogel for pathogen-targeted delivery to ameliorate enteritis. ACS Appl Mater Inter 2020;12:36967—77.

51.

Liu

, Liu

, Li

, Hu

, Zhao

, Zhang

, et al. Improved gastric acid resistance and adhesive colonization of probiotics by mucoadhesive and intestinal targeted Konjac glucomannan microspheres. Adv Funct Mater 2020;30:2001157.

52.

Chen

, Li

, Cao

, Yan

, Zhang

. Hierarchy-assembled dual probiotics system ameliorates cholestatic drug-induced liver injury via gut—liver axis modulation. Adv Sci 2022;9:2200986.

53.

Cheng

, Liu

, Xie

, Li

, Ma

, Xue

. A Colon-targeted oral probiotics delivery system using an enzyme-triggered fuse-like microcapsule. Adv Healthc Mater 2021;10:e2001953.

54.

Peñalva

, Martínez-López

, Gamazo

, Gonzalez-Navarro

, González-Ferrero

, Virto-Resano

, et al. Encapsulation of Lactobacillus plantarum in casein-chitosan microparticles facilitates the arrival to the colon and develops an immunomodulatory effect. Food Hydrocoll 2023;136:108213.

55.

Tan

, Mahotra

, Chan

, Loo

SCJ

. In situ alginate crosslinking during spray-drying of lactobacilli probiotics promotes gastrointestinal-targeted delivery. Carbohydr Polym 2022;286:119279.

56.

Zhang

, Liu

, Fang

, Chang

, Ren

, Cheng

, et al. Construction of probiotic double-layered multinucleated microcapsules based on sulfhydryl-modified carboxymethyl cellulose sodium for increased intestinal adhesion of probiotics and therapy for intestinal inflammation induced by Escherichia coli O157:H7. ACS Appl Mater Inter 2023;15:18569—89.

57.

Ajalloueian

, Guerra

, Bahl

, Torp

, Hwu

, Licht

, et al. Multi-layer PLGA-pullulan-PLGA electrospun nanofibers for probiotic delivery. Food Hydrocoll 2022;123:107112.

58.

Grilc

, Zidar

, Kocbek

, Rijavec

, Colja

, Lapanje

, et al. Nanofibers with genotyped Bacillus strains exhibiting antibacterial and immunomodulatory activity. J Control Release 2023;355:371—84.

59.

Chen

, Tian

, Huang

, Zhao

, Wang

, Xia

, et al. Engineered commensal bacteria prevent systemic inflammation-induced memory impairment and amyloidogenesis via producing GLP-1. Appl Microbiol Biotechnol 2018;102:7565—75.

60.

Steidler

, Hans

, Schotte

, Neirynck

, Obermeier

, Falk

, et al. Treatment of murine colitis by secreting interleukin-10. Science 2000;289:1352—5.

61.

Gao

, Ma

, Wang

, Bai

, Jia

, Feng

, et al. Oral immunization of mice with a probiotic constitutively expressing the α-toxoid induces protective immunity against α-toxin. Virulence 2019;10:166—79.

62.

Lin

, Mukherjee

, Li

, Hou

, Pan

, Liu

. Mucosal immunity-mediated modulation of the gut microbiome by oral delivery of probiotics into Peyer’s patches. Sci Adv 2021;7:eabf0677.

63.

Shelby

, Janzow

, Mashburn-Warren

, Galley

, Tengberg

, Navarro

, et al. A novel probiotic therapeutic in a murine model of Clostridioides difficile colitis. Gut Microbes 2020;12:1814119.

64.

Zhou

, Zou

, Xu

, Han

, Xiang

, Guo

, et al. Metal-phenolic self-assembly shielded probiotics in hydrogel reinforced wound healing with antibiotic treatment. Mater Horiz 2023;10:3114—23.

65.

Mitchell

, Billingsley

, Haley

, Wechsler

, Peppas

, Langer

. Engineering precision nanoparticles for drug delivery. Nat Rev Drug Discov 2021;20:101—24.

66.

Chen

, Wang

, Qin

, Zhang

, Sun

, et al. Bacteria-driven hypoxia targeting for combined biotherapy and photothermal therapy. ACS Nano 2018;12:5995—6005.

67.

Chen

, Guo

, Zhu

, Qiao

, Zhang

, Sun

. Combination of bacterial-photothermal therapy with an anti-pd-1 peptide depot for enhanced immunity against advanced cancer. Adv Funct Mater 2020;30:1906623.

68.

Pan

, Li

, Hou

, Lin

, Wang

, Pang

, et al. Polymerization-mediated multifunctionalization of living cells for enhanced cell-based therapy. Adv Mater 2021;33:2007379.

69.

Dong

, Sahle

, Lohan

, Saeidpour

, Albrecht

, Teutloff

, et al. pH-sensitive Eudragit^® L 100 nanoparticles promote cutaneous penetration and drug release on the skin. J Control Release 2019;295:214—22.

70.

Liu

, Li

, Wang

, Ding

, Lee

, Hu

. Biomaterials coating for on-demand bacteria delivery: selective release, adhesion, and detachment. Nano Today 2021;41:101291.

71.

Zhang

, Zhu

, Zhong

, Xu

, Lang

, Chen

, et al. Prodrug integrated envelope on probiotics to enhance target therapy for ulcerative colitis. Adv Sci 2022;10:e2205422.

72.

Feng

, Liu

, Pan

, Jin

, Wu

, Chen

, et al. Acid-directed electrostatic self-assembly generates charge-reversible bacteria for enhanced tumor targeting and low tissue trapping. ACS Appl Mater Inter 2022;14:36411—24.

73.

Luz

, Mano

. Mineralized structures in nature: examples and inspirations for the design of new composite materials and biomaterials. Compos Sci Technol 2010;70:1777—88.

74.

Hou

, Li

, Cao

, Lin

, Pan

, Pang

, et al. Decorating bacteria with a therapeutic nanocoating for synergistically enhanced biotherapy. Small 2021;17:e2101810.

75.

Lahav-Mankovski

, Prasad

, Oppenheimer-Low

, Raviv

, Dadosh

, Unger

, et al. Decorating bacteria with self-assembled synthetic receptors. Nat Commun 2020;11:1299.

76.

, Liu

. Decorated bacteria and the application in drug delivery. Adv Drug Deliv Rev 2022;188:114443.

77.

Akolpoglu

, Alapan

, Dogan

, Baltaci

, Yasa

, Aybar Tural

, et al. Magnetically steerable bacterial microrobots moving in 3D biological matrices for stimuli-responsive cargo delivery. Sci Adv 2022;8:eabo6163.

78.

Wei

, Pan

, Yuan

, Shao

, Wang

, Guo

, et al. Polarization of tumor-associated macrophages by nanoparticle-loaded Escherichia coli combined with immunogenic cell death for cancer immunotherapy. Nano Lett 2021;21:4231—40.

79.

Trush

, Poluektova

, Beniashvilli

, Shifrin

, Poluektov

, Ivashkin

. The evolution of human probiotics: challenges and prospects. Probiotics Antimicrob Proteins 2020;12:1291—9.

80.

Hoang

, Jo

, Phan

, Park

, Oh

, et al. Dual pH-/thermo-responsive chitosan-based hydrogels prepared using “click” chemistry for colon-targeted drug delivery applications. Carbohydr Polym 2021;260:117812.

81.

Yousefi

, Khanniri

, Shadnoush

, Khorshidian

, Mortazavian

. Development, characterization and in vitro antioxidant activity of chitosan-coated alginate microcapsules entrapping Viola odorata Linn. extract. Int J Biol Macromol 2020;163:44—54.

82.

Phan

VHG

, Mathiyalagan

, Nguyen

, Tran

, Murugesan

, Ho

, et al. Ionically cross-linked alginate-chitosan core-shell hydrogel beads for oral delivery of insulin. Int J Biol Macromol 2022;222:262—71.

83.

, Wei

, Liu

, Du

, Zhang

, Wang

, et al. Regulating type H vessel formation and bone metabolism via bone-targeting oral micro/nano-hydrogel microspheres to prevent bone loss. Adv Sci 2023;10:2207381.

84.

Chen

, Zhang

, Wu

, Yao

, Cha

, Gao

. Reductase-labile peptidic supramolecular hydrogels aided in oral delivery of probiotics. ACS Appl Mater Inter 2023;15:31214—23.

85.

Yang

, Peng

, Tan

, Ge

, Xie

, Zhou

, et al. Calcium tungstate microgel enhances the delivery and colonization of probiotics during colitis via intestinal ecological niche occupancy. ACS Cent Sci 2023;9:1327—41.

86.

Huang

, Wang

, Kong

, Wang

, Li

, Zhang

, et al. ROS-responsive hyaluronic acid hydrogel for targeted delivery of probiotics to relieve colitis. Int J Biol Macromol 2022;222:1476—86.

87.

Wei

, Liu

, Wang

, Zhou

, Zhao

, Zhou

, et al. A probiotic nanozyme hydrogel regulates vaginal microenvironment for Candida vaginitis therapy. Sci Adv 2023;9:eadg0949.

88.

Wang

, Guo

, zhang

, He

, Yang

, Chen

, et al. Poly-γ-glutamic acid microgel-encapsulated probiotics with gastric acid resistance and smart inflammatory factor targeted delivery performance to ameliorate colitis. Adv Funct Mater 2022;32:2113034.

89.

Cheng

, Liu

, Xie

, Li

, Ma

, Xue

. A colon-targeted oral probiotics delivery system using an enzyme-triggered fuse-like microcapsule. Adv Healthc Mater 2021;10:e2001953.

90.

Salalha

, Kuhn

, Dror

, Zussman

. Encapsulation of bacteria and viruses in electrospun nanofibres. Nanotechnology 2006;17:4675—81.

91.

, Ma

, Wang

, Liu

, Gu

, Qian

, et al. Preparation of pectin-based nanofibers encapsulating Lactobacillus rhamnosus 1.0320 by electrospinning. Food Hydrocoll 2022;124:107216.

92.

Minooei

, Gilbert

, Zhang

, Sarah NeCamp

, Mahmoud

, Kyser

, et al. Rapid-dissolving electrospun nanofibers for intra-vaginal antibiotic or probiotic delivery. Eur J Pharm Biopharm 2023;190:81—93.

93.

Giordano

, Maresca

, Mauriello

. Microencapsulation and sonication: a multiplephysical approach to attenuate the probiotic ATCC 393. Heliyon 2023;9:e23144.

94.

Qiu

, Jing

, Han

, Zhang

, Ba

, Xu

, et al. Enhanced gut microbiota delivery of a model probiotic (Faecalibacterium prausnitzii): layer-by-layer encapsulation using riboflavin-conjugated sodium alginate and glycol chitosan. Food Hydrocoll 2024;154:110055.

95.

Liu

, Xiao

. Engineered extracellular vesicles-like biomimetic nanoparticles as an emerging platform for targeted cancer therapy. J Nanobiotechnology 2023;21:287.

96.

Bourdeau

, Lee-Gosselin

, Lakshmanan

, Farhadi

, Kumar

, Nety

, et al. Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. Nature 2018;553:86—90.

97.

Min

, Nguyen

, Kim

, Hong

, Choy

. Quantitative bioluminescence imaging of tumor-targeting bacteria in living animals. Nat Protoc 2008;3:629—36.

98.

Goeddel

, Kleid

, Bolivar

, Heyneker

, Yansura

, Crea

, et al. Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc Natl Acad Sci USA 1979;76:106—10.

99.

Isabella

, Ha

, Castillo

, Lubkowicz

, Rowe

, Millet

, et al. Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria. Nat Biotechnol 2018;36:857—64.

100.

Praveschotinunt

, Duraj-Thatte

, Gelfat

, Bahl

, Chou

, Joshi

. Engineered E. coli Nissle 1917 for the delivery of matrix-tethered therapeutic domains to the gut. Nat Commun 2019;10:5580.

101.

Song

, Zhao

, Zheng

, Wang

, Gao

, Feng

, et al. A bioinspired versatile spore coat nanomaterial for oral probiotics delivery. Adv Funct Mater 2021;31:2104994.

102.

Wang

, Cao

, Zhang

, Meng

, Ming

, Liu

. Bioinspired oral delivery of gut microbiota by self-coating with biofilms. Sci Adv 2020;6:eabb1952.

103.

, Sun

, Yang

ZHY

, Gao

, Ran

, Xu

, et al. Rational design of self-assembled cationic porphyrin-based nanoparticles for efficient photodynamic inactivation of bacteria. ACS Appl Mater Inter 2020;12:54378—86.

104.

, Zhang

, Liu

, Chai

, Diao

, Ma

, et al. Probiotics formulation and cancer nanovaccines show synergistic effect in immunotherapy and prevention of colon cancer. iScience 2023;26:107167.

105.

Zhao

, Zhang

, Mao

, Ping

, Zhang

, Lin

, et al. Discovery of a colon-targeted Azo prodrug of tofacitinib through the establishment of colon-specific delivery systems constructed by 5-ASA-PABA-MAC and 5-ASA-PABA-diamine for the treatment of ulcerative colitis. J Med Chem 2022;65:4926—48.

106.

, Wang

, Wientjes

, Au

. Delivery of nanomedicines to extracellular and intracellular compartments of a solid tumor. Adv Drug Deliv Rev 2012;64:29—39.

107.

, Chu

, Wei

, Zhang

, Qi

, Lu

, et al. Platelet-membrane-coated nanoparticles enable vascular disrupting agent combining anti-angiogenic drug for improved tumor vessel impairment. Nano Lett 2021;21:2588—95.

108.

Zhao

, Yang

, Wang

, Zhao

, Zhang

, Liu

, et al. Penetration cascade of size switchable nanosystem in desmoplastic stroma for improved pancreatic cancer therapy. ACS Nano 2021;15:14149—61.

109.

, Aimetti

, Langer

, Gu

. Bioresponsive materials. Nat Rev Mater 2016;2:16075.

110.

, Jiang

, Zheng

, Shao

, Liu

, Huang

, et al. Oral delivery of bacteria: basic principles and biomedical applications. J Control Release 2020;327:801—33.

111.

Cao

, Liu

. Bacteria and bacterial derivatives as drug carriers for cancer therapy. J Control Release 2020;326:396—407.

112.

Anselmo

, McHugh

, Webster

, Langer

, Jaklenec

. Layer-by-layer encapsulation of probiotics for delivery to the microbiome. Adv Mater 2016;28:9486—90.

113.

Abbasi

, Rad

, Ghasempour

, Sabahi

, Kafil

, Hasannezhad

, et al. The biological activities of postbiotics in gastrointestinal disorders. Crit Rev Food Sci Nutr 2022;62:5983—6004.

114.

, Wang

, Gou

, Lu

, Liu

, Wang

, et al. Enhanced immunogenic cell death and antigen presentation via engineered bifidobacterium bifidum to boost chemo-immunotherapy. ACS Nano 2023;17:9953—71.

115.

McKenney

, Driks

, Eichenberger

. The Bacillus subtilis endospore: assembly and functions of the multilayered coat. Nat Rev Microbiol 2013;11:33—44.

116.

Song

, Anselmo

, Huang

. Nanotechnology intervention of the microbiome for cancer therapy. Nat Nanotechnol 2019;14:1093—103.

117.

, Narayan

, Castaing

, Tian

, Subramaniam

, Ramamurthi

. A versatile nano display platform from bacterial spore coat proteins. Nat Commun 2015;6:6777.

118.

Sanders

, Merenstein

, Reid

, Gibson

, Rastall

. Probiotics and prebiotics in intestinal health and disease: from biology to the clinic. Nat Rev Gastroenterol Hepatol 2019;16:605—16.

119.

Song

, Zheng

, Jia

, Zhao

, Feng

, Zhang

, et al. A probiotic spore-based oral autonomous nanoparticles generator for cancer therapy. Adv Mater 2019;31:e1903793.

120.

Zheng

, Li

, An

, Xie

, Han

, Xu

, et al. Prebiotics-encapsulated probiotic spores regulate gut microbiota and suppress colon cancer. Adv Mater 2020;32:2004529.

121.

Chen

, Qiao

, Cao

, Han

, Zeng

, Zhang

. Spore germinator-loaded polysaccharide microspheres ameliorate colonic inflammation and tumorigenesis through remodeling gut microenvironment. Mater Today 2023;63:32—49.

122.

Dizman

, Meza

, Bergerot

, Alcantara

, Dorff

, Lyou

, et al. Nivolumab plus ipilimumab with or without live bacterial supplementation in metastatic renal cell carcinoma: a randomized phase 1 trial. Nat Med 2022;28:704—12.

123.

Sun

, Luo

, Liang

, Yin

, Guo

, Rush

, et al. Bifidobacterium alters the gut microbiota and modulates the functional metabolism of T regulatory cells in the context of immune checkpoint blockade. Proc Natl Acad Sci USA 2020;117:27509—15.

124.

, Wang

, Wu

, Tong

, Zhao

, Li

, et al. Dual-responsive supramolecular photodynamic nanomedicine with activatable immunomodulation for enhanced antitumor therapy. Acta Pharm Sin B 2024;14:765—80.

125.

Jia

, Liu

, Wang

, Hu

, Yu

, Zhou

, et al. Dual-responsive nanoparticles with transformable shape and reversible charge for amplified chemo-photodynamic therapy of breast cancer. Acta Pharm Sin B 2022;12:3354—66.

126.

Zhang

, Zhang

, Hu

, Hao

, Quan

, Ren

, et al. Engineering a porphyrin COFs encapsulated by hyaluronic acid tumor-targeted nanoplatform for sequential chemo-photodynamic multimodal tumor therapy. Int J Biol Macromol 2024;279:135328.

127.

Guo

, Cao

, Li

, Fu

, Lin

, Wang

, et al. Integrating bacteria with a ternary combination of photosensitizers for monochromatic irradiation-mediated photoacoustic imaging-guided synergistic photothermal therapy. ACS Nano 2023;17:5059—71.

128.

Yang

, Hu

, Bian

, Meng

, Yu

, Li

, et al. Coupling probiotics with 2D CoCuMo-LDH nanosheets as a tumor-microenvironment-responsive platform for precise NIR-II photodynamic therapy. Adv Mater 2023;35:2211205.

129.

Shi

, Liu

, Li

, Wu

, Gao

, et al. Living bacteria-based immuno-photodynamic therapy: metabolic labeling of Clostridium butyricum for eradicating malignant melanoma. Adv Sci 2022;9:e2105807.

130.

Pan

, Sun

, Cui

, Ma

, Yang

, Liu

, et al. Light-sensitive Lactococcus lactis for microbe—gut—brain axis regulating via upconversion optogenetic micro-nano system. ACS Nano 2022;16:6049—63.

131.

Zhang

, Pang

, Sun

, Liu

, Pan

, Zhang

, et al. A red light-controlled probiotic bio-system for in-situ gut—brain axis regulation. Biomaterials 2023;294:122005.

132.

Corbet

, Feron

. Tumour acidosis: from the passenger to the driver’s seat. Nat Rev Cancer 2017;17:577—93.

133.

Sivan

, Corrales

, Hubert

, Williams

, Aquino-Michaels

, Earley

, et al. Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy. Science 2015;350:1084—9.

134.

, Wang

, An

, Huo

, Xu

, et al. Probiotic engineering and targeted sonoimmuno-therapy augmented by STING agonist. Adv Sci 2022;9:e2201711.

135.

Abedi

, Yao

, Mittelstein

, Bar-Zion

, Swift

, Lee-Gosselin

, et al. Ultrasound-controllable engineered bacteria for cancer immunotherapy. Nat Commun 2022;13:1585.

136.

Buss

, Ramesh

, English

, Lee-Gosselin

, Shapiro

. Spatial control of probiotic bacteria in the gastrointestinal tract assisted by magnetic particles. Adv Mater 2021;33:e2007473.

137.

Yang

, Wang

, Song

, Li

, Lei

, Li

, et al. Metal coordination nanotheranostics mediated by nucleoside metabolic inhibitors potentiate STING pathway activation for cancer metalloimmunotherapy. J Control Release 2024;370:354—66.

138.

Goldberg

. Improving cancer immunotherapy through nanotechnology. Nat Rev Cancer 2019;19:587—602.

139.

Riley

, June

, Langer

, Mitchell

. Delivery technologies for cancer immunotherapy. Nat Rev Drug Discov 2019;18:175—96.

140.

Liu

, Zhang

, Wang

, Yang

, Cao

, Wang

, et al. Dressing bacteria with a hybrid immunoactive nanosurface to elicit dual anticancer and antiviral immunity. Adv Mater 2023;35:e2210949.

141.

Liu

, Luo

, Pang

, Zhang

, Ruan

, Wu

, et al. Advances of nanoparticles as drug delivery systems for disease diagnosis and treatment. Chin Chem Lett 2023;34:107518.

142.

Lin

, Du

, Song

, Wang

, Yang

. Imaging commensal microbiota and pathogenic bacteria in the gut. Acc Chem Res 2021;54:2076—87.

143.

Jiang

, Yang

, Li

, Zhao

, Sun

, Müller

, et al. Bacteria-based live vehicle for in vivo bioluminescence imaging. Anal Chem 2021;93:15687—95.

144.

Zou

, Du

, Fang

, Zhou

, Ye

. Biomarker-responsive engineered probiotic diagnoses, records, and ameliorates inflammatory bowel disease in mice. Cell Host Microbe 2023;31:199—212.e5.

145.

Montassier

, Valdés-Mas

, Batard

, Zmora

, Dori-Bachash

, Suez

, et al. Probiotics impact the antibiotic resistance gene reservoir along the human GI tract in a person-specific and antibiotic-dependent manner. Nat Microbiol 2021;6:1043—54.

146.

Fatahi-Bafghi

, Naseri

, Alizehi

. Genome analysis of probiotic bacteria for antibiotic resistance genes. Antonie Van Leeuwenhoek 2022;115:375—89.

147.

Kunz

, Fairchok

, Noel

. Lactobacillus sepsis associated with probiotic therapy. Pediatrics 2005;116:517—8.

148.

Charbonneau

, Isabella

, Li

, Kurtz

. Developing a new class of engineered live bacterial therapeutics to treat human diseases. Nat Commun 2020;11:1738.

Appendix

Less

Year 2025 volume 15 Issue 5

PDF

Cite this Article

BibTeX

Article Info

doi: 10.1016/j.apsb.2025.03.021

Receive Date：2024-08-21
Online Date：2026-09-17

Article Data

Affiliations

History

Received：2024-08-21
Revised：2025-02-20
Accepted：2025-02-28

Affiliations

^aSchool of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China

^bHenan Key Laboratory of Nanomedicine for Targeting Diagnosis and Treatment, Zhengzhou 450001, China

^cLuoyang Central Hospital Affiliated to Zhengzhou University, Luoyang 471009, China

^dTumor Immunity and Biomaterials Advanced Medical Center, Zhengzhou University, Luoyang 471009, China

Corresponding:

^* Corresponding authors.

References

Share

https://castjournals.cast.org.cn/joweb/apsb/EN/10.1016/j.apsb.2025.03.021

Share to

Scan QR to access full text

Cite this article

BibTeX

Citations

表12种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

关闭全屏

BibTeX
EndNote
RefWorks
TxT

Table 1 Modification of probiotics.

Modification type			Probiotic	Modified material	Bond/modification method	Ref.
Physicochemical modification	Surface coating on single cell	Metal-phenolic	EcN	Fe³⁺, TA	Hydrophobic interaction, electrostatic interaction,Hydrogen bonding	29
			EcN	Fe³⁺, TA, mGN	Hydrogen bonding	30
			EcN	Ca²⁺, TA, mucin	Hydrogen bonding,Disulfide bonding,Hydrophobic interaction	31
		Polymer coating	EcN	PDA, CS	Covalent or hydrogen bonds	32
			EcN	pNE, HPN	Oxidative self-polymerization	33
			EcN	CS, ALG	Electrostatic self-assemble	34
		Lipid coating	EcN	DOPA, cholesterol	Biointerfacial supramolecular self-assembly	35
			LCB	Liposomes,	Self-assembly	36
		Mineral coating	BF839	CaCO₃, PVP	Electrostatic interaction–mediated biointerface mineralization	37
		Other coating materials	EcN	SiH@TPGS-PEI	Electrostatic interaction	38
			Lac	DOPA, PA, HA	Amide bonding	39
			EcN	TA, F68	Self-assemble	40
			EcN	5-ASA, Ca²⁺,ALG	Electrostatic interactions,Ionic bonds	41
			Li01	CS, ALG	Electrostatic attraction	42
	Surface conjugation on single cell	Non-covalent	EcN	PTX, HPB	Hydrophobic interaction, electrostatic interaction	43
			E. hallii	Fe-DOX	Supramolecular self-assemble	44
		Covalent	C. butyricum	MGEM	Amide bond	45
			BL	Fe SA	Click reaction	46
	Multiple cells encapsulation	Hydrogel	LGG	Bentonite/ALG nanocomposite hydrogels	Electrostatic interactions, van der waals interactions	47
			L. Lactis	CS, PEGDA	Amide bond	48
			LP	BSP, CS	Schiff-base reaction	49
			L. rhamnosus	Thiolated HA	Disulfide bonds	50
		Microspheres and microcapsules	NZ9000	sOKGM	COO⁻−Fe³⁺ coordination, disulfide bonds	51
			LDB, LGG	Calcium ALG microsphere, CS, Eudragit L100-55	Electrostatic interactions	52
			E. coli MG1655	ALG, protamine	Electrostatic interactions	53
			L. plantarum	Casein-CS	Ionic bonds,Schiff-base reaction	54
			L. plantarum,L. paracasei	Ca²⁺, ALG,Sucrose	Ionic bonds	55
			FS2-3, SN15-2	CMC–SH	Disulfide bonds	56
		Nanofiber	LGG	Pullulan nanofibers, electrospun PLGA	Electrostatic interactions	57
			Bacillus	PEO, PEO/ALG	Electrostatic interactions	58
Biological modification	Genetic engineering		Lactobacillus	–	Gene editing	59
			L. lactis	–	Gene editing	60
			Lactobacillus casei	–	Gene editing	61
	Biofilm encapsulation		EcN	YM	Physical extrusion	62
			Bi	Biofilm produced by Lr	–	⁶³

ALG, alginate; Bacillus, Bacillus strains; BF839, Bacteroides fragilis; Bi, Bifidobacterium bifidum; BL, Bifidobacterium longum; BSP, Bletilla striata polysaccharide; Ca²⁺, calcium ions; CaCO₃, calcium carbonate; C. butyricum, Clostridium butyricum; CMC-SH, thiolated carboxymethyl cellulose; CS, chitosan; DOPA, dopamine hydrochloride; DOX, doxorubicin; EcN, Escherichia coli Nissle; E.coli MG1655, Escherichia coli MG1655; E. hallii, Eubacterium hallii; Fe³⁺, ferric ions; Fe-DOX, DOX-loaded iron-polyphenol nanoparticles; Fe SA, iron single-atom catalyst; FS2-3, Bifidobacterium adolescentis FS2-3; F68, poloxamer 188; HA, hyaluronic acid; HPB, BAY-876 bound human serum albumin nanodrugs; IBD, inflammatory bowel disease; Lac, Lactobacillus acidophilus; LDB, Lactobacillus delbrueckii subsp. Bulgaricus; LGG, Lactobacillus rhamnosus GG; Li01, Ligilactobacillus salivarius Li01; L. lactis, Lactococcus lactis; L. paracasei, Lactobacillus paracasei; LP, Lactobacillus plantarum; L. plantarum, Lactobacillus plantarum; Lr, Lactobacillus royale; L. rhamnosus, Lactobacillus rhamnosus; MGEM, gemcitabine-loaded mesoporous silicon nanoparticles; mGN, carboxymethylated β-glucan; NE, norepinephrine; NZ9000, Lactococcus lactis NZ9000; PA, phenylboric acid; PEGDA, poly(ethylene glycol) dimethacrylate; PEO, polyethylene oxide; PLGA, poly-lactic-co-glycolic acid; PVP, polyvinyl-pyrrolidone; PTX, paclitaxel; SiH@TPGS-PEI, copolymer-modified two-dimensional H-silicene nanomaterial; SN15-2, Bacillus subtilis SN15-2; TA, tannic acid; YM, yeast membrane; 5-ASA, 5-aminosalicylic acid; ‒, not applicable.

Table 2 Comparison of surface coating methods on single-cell.

Method	Advantage	Disadvantage
Metal-polyphenol network coating	1)Strong adhesion in the gastrointestinal tract.2)Enhances probiotic stability.3)Regulates inflammatory responses.4)Provides a protective “armor” against antibiotics.	1)Complex synthesis and application processes.2)Potential toxicity concerns with metal ions if not properly controlled.
Polymer coating	1)Biocompatible and easy to formulate.2)Enhance bioavailability and stability of probiotics.3)pH-responsive coatings enable targeted release in the intestine.	1)Time-consuming layer-by-layer techniques requiring careful optimization.2)Synthetic polymers raise concerns about biocompatibility and biodegradability.
Lipid coating	1)Shield probiotics from acids and bile.2)Capable of encapsulating both hydrophilic and hydrophobic compounds.3)Enhance compatibility with bodily fluids using the charge-reversible lipid membranes.	1)Complicated manufacturing processes2)Increased costs.3)Potential instability during storage
Mineral coating	1)Mimics natural mineralization processes.2)Enhances survivability in acidic and bile-rich environments.3)Effective probiotic release with self-removable coatings.	1)Specific conditions that may limit scalability.2)Difficulties in controlling the thickness and uniformity of the coating.
Other coating materials	1)Silk fibroin offer inherent pharmaceutical activity and protection.2)Alginate effectively encapsulates therapeutic agents and protects probiotics in the gastric environment.3)Some coatings provide additional therapeutic benefits.	1)Complex and time-consuming2)Therapeutic efficacy depending on the specific material and method.

Table 3 Examples of genetic engineering probiotics in clinical trials.

Engineered probiotic	Disease	Company/university	Stage	ClinicalTrials.gov identifier
B. longum to deliver the plasmid for IL-12 expression	Solid tumors	IQVIA	Phase 1	NCT04025307
EcN 1917 to target STING-activation	Solid tumors	Synlogic	Phase 1	NCT04167137
EcN 1917 to metabolize ammonia	Cirrhosis and hepatic insufficiency	Synlogic	Phase 1/2	NCT03447730
L. lactis to secrete human Trefoil factor 1	Oral mucositis in patients with head and neck cancer	Actobio therapeutics/Oragenics	Phase 2	NCT03234465
L. lactis to deliver human proinsulin and hIL-10	Type 1 diabetes	Actobio therapeutics	Phase 2a	NCT03751007
EcN 1917 to metabolize phenylalanine	Phenylketonuria	Synlogic	Phase 3	NCT03516487

Figure 1 Schematic illustration of the modification of probiotics and the related combinatorial therapeutics.

Figure 2 Metal-polyphenol network, polymer, lipid and mineral coated probiotics. (A) TA- Ca²⁺ and mucin-coated EcN. Reprinted with the permission from Ref. 31. Copyright © 2022, American Chemical Society. (B) The surface of probiotics was coated with a co-deposited coat of polydopamine and chitosan (CS). Reprinted with the permission from Ref. 68. Copyright © 2021, Wiley-VCH. (C) Scheme illustrating the preparation of the lipid membrane-encapsulated EcN. Reprinted with the permission from Ref. 35. Copyright © 2019, Nature Publishing Group. (D) Schematic illustration of biointerface mineralization of BF839 and confocal images of mCherry-expressing EcN coated with calcein-labeled mineral layer. Scale bar = 10 μm. Reprinted with the permission from Ref. 37. Copyright © 2023, AAAS.

Figure 3 Non-covalent and covalent conjugation of probiotics. (A) The construction of bacterial biohybrids carrying mNPs and NLs and the cancer cell killing efficiency. Reprinted with the permission from Ref. 77. Copyright © 2022, AAAS. (B) The construction, element mapping and treatment efficiency of E@Fe-DOX. Reprinted with the permission from Ref. 44. Copyright © 2023, Wiley-VCH. (C) The construction of BL@B-SA50. Reprinted with the permission from Ref. 46. Copyright © 2023, Springer Nature. (D) The construction, whole-body distribution and treatment efficiency of biohybrid system SPORE-MGEM. Reprinted with the permission from Ref. 45. Copyright © 2022, American Chemical Society.

Figure 4 Hydrogel, microspheres and microcapsules, and nanofiber encapsulated probiotics. (A) The construction of an oral probiotic delivery system based on CTM. Reprinted with the permission from Ref. 85. Copyright © 2023, American Chemical Society. (B) The construction of a responsive hydrogel FeLab. Reprinted with the permission from Ref. 87. Copyright © 2023, AAAS. (C) Schematic illustration of intestinal-targeted NRPM using a pH-responsive gel microsphere. Reprinted with the permission from Ref. 88. Copyright © 2022, Wiley-VCH. (D) Electrostatic spinning is used to create a nanofiber to shield the probiotic LGG. Reprinted with the permission from Ref. 57. Copyright © 2022, Elsevier. (E) The SEM of PEO and composite PEO/ALG nanofibers. Scale bar = 1 μm. Reprinted with the permission from Ref. 58. Copyright © 2023, Elsevier.

Figure 5 Engineering probiotics as bioactive factories. (A) Engineered EcN to express genes encoding Phe-metabolizing enzymes. Reprinted with the permission from Ref. 99. Copyright © 2022, AAAS. (B) Engineered EcN is constitutively expressing CD47 nanobody to induce anti-tumor immunity. Reprinted with the permission from Ref. 21. Copyright © 2022, AAAS. (C) Engineered EcN for IBD treatment by curli production via self-assembly of chimeric CsgA proteins. Reprinted with the permission from Ref. 100. Copyright © 2019, Springer Nature.

Figure 6 Biofilm encapsulated probiotic. (A) Schematic illustration of the preparation of CN-coated probiotics, and the mechanism for treating colitis. Reprinted with the permission from Ref. 101. Copyright © 2021, Wiley-VCH. (B) Design of biofilm self-coating strategy for the delivery of BS and characterization of the probiotic after biofilm self-coating. Scale bar = 1 μm. Reprinted with the permission from Ref. 102. Copyright © 2020, AAAS.

Figure 7 Combination of modified probiotics and pharmacotherapy. (A) Prodrug integrated envelope on probiotics to enhance target therapy for ulcerative colitis. Reprinted with the permission from Ref. 71. Copyright © 2023, Wiley-VCH. (B) Enhanced immunogenic cell death and antigen presentation via engineered Bifidobacterium bifidum to boost chemo-immunotherapy. Reprinted with the permission from Ref. 114. Copyright © 2023, American Chemical Society. (C) A probiotic spore-based oral autonomous nanoparticles generator for cancer therapy. Reprinted with the permission from Ref. 119. Copyright © 2019, Wiley-VCH. (D) Prebiotics-encapsulated probiotic spores regulate gut microbiota and suppress colon cancer. Reprinted with the permission from Ref. 120. Copyright © 2020, Wiley-VCH.

Figure 8 Combination of probiotics with phototherapy, sonodynamic therapy, magnetotherapy and immunotherapy. (A) Optoacoustic imaging-guided synergistic photothermal therapy. Reprinted with the permission from Ref. 127. Copyright © 2023, Wiley-VCH. (B) Probiotic-microbe-based SIT against cancer with STING agonist. Reprinted with the permission from Ref. 134. Copyright © 2022, Wiley-VCH. (C) Spatial control of probiotic in the gastrointestinal tract assisted by magnetic particles. Reprinted with the permission from Ref. 136. Copyright © 2021, Wiley-VCH. (D) Dressing bacteria with a hybrid immunoreactive nanosurface to elicit dual anticancer and antiviral immunity. Reprinted with the permission from Ref. 140. Copyright © 2023, Wiley-VCH.

Figure 9 Probiotic-based bioimaging and diagnosis. (A) EcN was engineered to contain the core module Fluc-LRE. Reprinted with the permission from Ref. 143. Copyright © 2021, American Chemical Society. (B) Schematic diagram of the components and sensory pathways of i-ROBOT. Reprinted with the permission from Ref. 144. Copyright © 2023, Elsevier.

Articles: Latest Articles; Most Read; Collections

Updates: Events; News; Multimedia

About: About Us

Contact

No. 86 Xueyuan South Road, Haidian District, Beijing

100081

010-62199257

qkjq@cast.org.cn

Copyright © 2025 China Association for Science and Technology. All rights reserved. For all open access content, the relevant licensing terms apply.
Sponsored by the Office of the Leading Group for Cybersecurity and Informatization of CAST, and supported by Science and Technology Review Publishing House