Advances and optimization strategies in bacteriophage therapy for treating inflammatory bowel disease

In the advancement of Inflammatory Bowel Disease (IBD) treatment, existing therapeutic methods exhibit limitations; they do not offer a complete cure for IBD and can trigger adverse side effects. Consequently, the exploration of novel therapies and multifaceted treatment strategies provides patients with a broader range of options. Within the framework of IBD, gut microbiota plays a pivotal role in disease onset through diverse mechanisms. Bacteriophages, as natural microbial regulators, demonstrate remarkable specificity by accurately identifying and eliminating specific pathogens, thus holding therapeutic promise. Although clinical trials have affirmed the safety of phage therapy, its efficacy is prone to external influences during storage and transport, which may affect its infectivity and regulatory roles within the microbiota. Improving the stability and precise dosage control of bacteriophages—ensuring robustness in storage and transport, consistent dosing, and targeted delivery to infection sites—is crucial. This review thoroughly explores the latest developments in IBD treatment and its inherent challenges, focusing on the interaction between the microbiota and bacteriophages. It highlights bacteriophages’ potential as microbiome modulators in IBD treatment, offering detailed insights into research on bacteriophage encapsulation and targeted delivery mechanisms. Particular attention is paid to the functionality of various carrier systems, especially regarding their protective properties and ability for colon-specific delivery. This review aims to provide a theoretical foundation for using bacteriophages as microbiome modulators in IBD treatment, paving the way for enhanced regulation of the intestinal microbiota.


Introduction
Inflammatory Bowel Disease (IBD) is a collection of autoimmune disorders impacting the gastrointestinal tract, influenced by the gut microbiota.Bacteriophages, viruses that infect specific bacteria, play a role in modulating these microbial communities and may offer a strategy to combat IBD-related pathogens.Although still in early stages, phage therapy shows promise, with recent advancements focusing on optimizing these treatments for IBD.Specially formulated phage therapies targeting specific pathogenic strains could minimize adverse effects on beneficial gut bacteria, yet challenges in phage delivery and specificity remain.Antibiotics, while effective, indiscriminately kill gut bacteria and contribute to dysbiosis and resistance issues (1).Traditional methods like probiotics and fecal microbiota transplantation face hurdles like poor colonization and risk of transferring harmful agents (2,3).Advances in gut microbiology have led to innovative strategies such as targeted bacteriophage delivery systems for the colon (4), using stabilizers like Eudragit FS30D, which have shown promising stability and efficacy (5).Therefore, bacteriophages are increasingly recognized as potential tools for precisely modulating the gut microbiota and addressing intestinal disorders.Bacteriophages face challenges such as high temperatures, low pH, and digestive enzymes, which can diminish their effectiveness during storage and gastrointestinal transit (6)(7)(8).Encapsulation has proven a valuable method to enhance bacteriophage stability, ensuring their viability under these conditions.Ma et al. demonstrated that encapsulating bacteriophage Felix O1 in alginate-chitosan microspheres significantly improved its survival in simulated porcine gastrointestinal environments, indicating potential for effective therapeutic delivery to the intestines (9).This paper reviews recent progress in IBD treatment, examining the interaction between the microbiome and bacteriophages, and discussing the mechanisms and potential of bacteriophage-mediated modulation of gut microbiota.It covers the influence of preparation, storage, and delivery on bacteriophage vitality and provides a summary of diverse encapsulation materials used for bacteriophage delivery.The conclusion proposes future directions for bacteriophage delivery strategies in manipulating the gut microbiota.

Treatment methods for IBD
IBD is a chronic autoimmune inflammatory condition affecting the gastrointestinal tract and extraintestinal organs.This disease encompasses various clinical and histological variations, including ulcerative colitis (UC), Crohn's disease (CD), and indeterminate colitis.The treatment strategies for IBD have continuously evolved and been explored in recent years (Table 1).

Drug therapy
Presently, therapeutic drugs for IBD encompass a range of medications, including aminosalicylates, antibiotics such as metronidazole, corticosteroids, among others (12).5-Aminosalicylate (5-ASA) salts stand out as a common pharmaceutical intervention for treating IBD, with sulfasalazine exhibiting specific alleviative effects in Crohn's disease patients.The precise mechanism through which aminosalicylate salts ameliorate IBD remains uncertain.5-ASA may possess antioxidant, anti-proliferative, or pro-apoptotic activities, allowing for localized treatment on the gastrointestinal mucosa (13)(14)(15).The potential effectiveness of 5-ASA in producing significant effects through the alleviation of mucosal inflammation has been documented (16)(17)(18).Likewise, corticosteroids prove effective in mitigating inflammatory responses, exhibiting immunomodulatory effects and finding extensive applications in treatment (19)(20)(21).Glucocorticoids are commonly employed for more severe conditions than those treated with 5-ASA (22), and their administration may be escalated when 5-ASA treatment proves ineffective (23).

Immunomodulators and biologics
Immunomodulators and biologic agents, which possess the capability to regulate the immune system, have found widespread application in alleviating symptoms and sustaining remission (24,25).Nevertheless, some patients may develop tolerance or adverse reactions to these medications.Given the pivotal role of inflammatory factors in the pathogenesis of IBD, biologic agents endowed with anti-inflammatory efficacy have emerged.Infliximab, the first anti-TNF-a drug, has demonstrated outstanding therapeutic efficacy for IBD (26,27).Simultaneous adoption of therapeutic drug monitoring (TDM) has been shown to optimize the efficacy of infliximab (28).Furthermore, adalimumab and golimumab have also been confirmed as effective treatments for IBD (29, 30).Vedolizumab, a specific antagonist against the a4/b7 integrin, inhibits the interaction between integrins and MAdCAM-1, effectively blocking the homing of immune cells to intestinal tissues and reducing mucosal inflammation in IBD patients (31).This mechanism could offer new opportunities for modulating macrophage-related processes such as mucosal healing (32).Numerous studies have demonstrated its efficacy in treating IBD; for instance, an analysis of homing receptor expression on T cells in peripheral blood and inflamed mucosa showed that treatment with vedolizumab is associated with a significant expansion of regulatory T cells in peripheral blood without a significant increase in viral infections in the IBD group (33).Therefore, as the only gut-selective biologic that specifically targets the a4b7 gastrointestinal integrin receptor, vedolizumab represents a promising therapeutic option for IBD (34).Ustekinumab, a human IgG antibody targeting the p40 subunit common to both IL-12 and IL-23, operates differently from anti-TNF therapies (35).It inhibits the biological activity of these cytokines by blocking their shared p40 subunit, impacting receptors on T cells, natural killer cells, and antigen-presenting cells (36).Monoclonal antibodies against the IL-12/23 p40 subunit have shown significant therapeutic effects in mouse models of colitis (37,38), suggesting their potential efficacy in treating related diseases.Tumor necrosis factor-like cytokine 1A (TL1A) is associated with IBD and influences the location and severity of intestinal inflammation and fibrosis, showing increased expression in inflamed intestinal mucosa (39).TL1A is closely linked to mucosal immunity, suggesting that blocking it could have potential benefits for inflammatory diseases involving the mucosal surface, such as IBD and asthma.Moreover, the TL1A-DR3 pathway has a specific association with fibrosis in Crohn's disease (40).Studies have demonstrated that TL1A antibodies effectively alleviate intestinal inflammation and reverse fibrosis to baseline levels in mouse models (41), highlighting TL1A as a promising target in IBD treatment.

Intestinal microbiota 2.3.1 Faecal microbiota transplantation
Fecal microbiota transplantation (FMT) transfers feces from healthy individuals to recipients to restore gut microbiota (42), first used to treat pseudomembranous enterocolitis (43).Increasing research links IBD to dysregulated gut microbiota (44,45), with microbial changes causing mucosal immune dysregulation and increasing susceptibility to IBD (46).FMT has proven effective for IBD treatment (47), as shown by randomized trials like Moayyedi et al., which found FMT more effective than placebos in inducing remission in active ulcerative colitis (48).Other studies, including a multicenter trial in Australia, demonstrated high-dose, multi-donor FMT's ability to induce clinical remission in active UC (49), and a trial with 73 adults showed that anaerobically prepared donor FMT was likelier to induce remission than autologous FMT (50).Recent research robustly supports FMT as a promising therapy for IBD.A key study (NCT03426683) involving 35 IBD patients, split into two groups-one receiving standardized FMT and the other traditional medications-over a year, assessed FMT's efficacy, durability, and safety, while identifying specific bacteria involved in the process.With more clinical trials underway, FMT is likely to become an established IBD treatment, providing relief for patients not responding to conventional therapies by modulating immune responses and reducing inflammation (51).Meta-analyses also support FMT's efficacy; for instance, Sudarshan et al.'s review of 53 studies noted better UC remission with more frequent FMT infusions and optimized administration (52).Another meta-analysis showed FMT as a successful treatment for CDI in IBD patients, advocating for more trials and research to confirm its effectiveness (53).

Antibiotic therapy for IBD
IBD is linked to host-microbe interactions and gut microbiome imbalances that promote the colonization of opportunistic pathogens, crucial to the disease's development (54).Certain gut bacteria exacerbate intestinal inflammation in IBD by releasing antigens or stimulatory factors, with studies indicating that microbial diversity reductions are significantly associated with IBD onset (55).Antibiotics like metronidazole and ciprofloxacin are effective in modulating the gut microbiota and treating IBD (56).A study showed that ciprofloxacin, despite its good tolerability, did not significantly differ from placebos in inducing remission in perianal Crohn's disease due to its tolerability (57).Additionally, ciprofloxacin demonstrated anti-inflammatory properties in a colitis mouse model (58).However, the broad- Long-term use of antibiotics in IBD must balance efficacy against side effects: metronidazole often has severe adverse effects (60), while ciprofloxacin is well-tolerated but expensive and can cause nausea; combined use can increase side effects significantly (61).Judicious use of antibiotics, adhering to clinical guidelines and minimizing doses and duration, can optimize benefits while reducing risks of resistance and adverse effects.

Targeting intestinal epithelial cells
The intestinal epithelial layer acts as a barrier that separates the organism from the external environment (62).Once there is a disruption in the balance of the gut barrier microbiota, uncontrolled immune reactions may erupt in the intestinal microenvironment.This imbalance can lead to unrestricted microbial growth, resulting in various diseases, including IBD (63).Deficiencies in epithelial lymphocytes, chemokine receptor expression, and pattern recognition receptors can lead to abnormal immune responses, subsequently promoting cell differentiation and increasing inflammation (64).Consequently, bacteriophage targeting of intestinal epithelial cells is being explored as a novel immunotherapeutic approach for treating IBD (65).Goŕski et al. posit that bacteriophages, beyond their antimicrobial activity, also exhibit immunomodulatory characteristics that could be beneficial in the treatment of IBD.

Treatment limitations
The majority of drugs available for IBD offer symptomatic relief rather than a complete cure.Additionally, a significant proportion of these drugs are associated with severe adverse reactions.Prolonged use of 5-ASA in IBD patients may lead to side effects such as headaches, diarrhea, nausea, and even severe complications like pneumonia, hepatitis, and myocarditis (66).Corticosteroids, while potent and rapid in treating IBD, fail to effectively maintain remission and can induce irreversible complications with long-term use, including cataracts, glaucoma, hypertension, and diabetes (67,68).While biologics broaden the therapeutic spectrum for IBD, they unfortunately bring about severe complications such as tuberculosis, fungal infections, cancer, and tumors.Beyond the serious adverse reactions of medications, the exorbitant cost of IBD treatment imposes a substantial economic burden on patients.Consequently, the development of safe, effective, and cost-efficient therapeutic drugs for clinical treatment of IBD holds paramount significance.

Role of bacteriophages in IBD
Increasing evidence in humans suggests that the microbiome plays a crucial role in the pathogenesis of IBD.For instance, clinical trial results (Clinical Trial ID: UMIN 000004123) indicate that inter-individual variations in the gut microbiota may be associated with individual differences in the risk of IBD or other diseases (69).Alterations in the gut microbiota are believed to play a pivotal role in the onset, progression, and severity of the disease.Transferring dysregulated IBD-associated microbiota to healthy mice has been shown to induce intestinal inflammation (70)(71)(72).Bacteriophages are considered a part of the gut microbiome and may influence bacterial community structure in various clinical settings.The administration of exogenous bacteriophages may represent a potential strategy for inhibiting IBD-associated pathogenic bacteria (73).Alterations in the community structure of IBD may be influenced by the prolific reproduction of bacteriophages within the intestinal bacterial ecosystem.The specific targeting of pathogenic strains by a tailored combination of bacteriophages holds potential as an eradication therapy for preventing and treating IBD, while minimizing adverse effects on the surrounding bacterial microbiome (Table 2).
Bacteriophage-mediated regulation of the gut microbiota is achieved through interactions between bacteria and phages within the colon.Acting as natural predators of bacteria, phages can disrupt or lyse host bacteria through processes such as lytic replication or lysogenic cycles.In cases of gut microbiota dysbiosis, an excess of pathogens or harmful bacteria can produce unwanted metabolites (Figure 1).These metabolites are absorbed into the systemic circulation, triggering related symptoms.Upon reaching the colon, bacteriophages can selectively kill host pathogens, reducing adverse metabolites through specific recognition and infection, while minimizing disruption to nontargeted microorganisms (Figure 1).Furthermore, the ecological niche of pathogens can be occupied and colonized by symbiotic or probiotic bacteria, restoring gut homeostasis and promoting overall health.In recent research, Lv et al. isolated a strain of Lactobacillus subspecies, Lactobacillus SF, from the feces of healthy infants and conducted a systematic probiotic evaluation.The results demonstrated that Lactobacillus SF could restore gut microbiota balance by increasing the relative abundance of bacteria capable of occupying the same ecological niche as pathogenic bacteria, thereby reducing the survival space for pathogens.This indicates that Lactobacillus SF exhibits excellent resistance to gastric fluids, colonization in the intestine, and strong antibacterial and antioxidant capabilities (84).

Personalized bacteriophage therapy
In the human microbiome, bacteriophages can be employed for targeted elimination of specific pathogens without causing harm to beneficial microbial communities (85,86).With a plethora of phages available, their precise and widespread regulatory capabilities make them an ideal tool for modulating the human microbiome (87).Bacteriophages exhibit high diversity and adaptability, enabling them to thrive in various environments and target different bacterial species (87).Tailoring bacteriophages based on the microbial characteristics of different populations allows for personalized therapeutic interventions and modulation.The low toxicity of bacteriophages in regulating the human microbiome minimizes the risk of severe immune reactions or adverse effects (88,89).Their self-replication mode enables effective treatment at low doses (90), providing a high level of safety and feasibility in utilizing bacteriophages for human microbiome modulation.In terms of infection mechanisms, bacteriophages can utilize both lytic and lysogenic pathways to infect hosts, broadening their applicability (91, 92).Co-evolution with bacterial hosts enhances their plasticity and survival rates (93, 94).Moreover, bacteriophages can serve as vectors by introducing new functionalities or silencing virulence factors to target bacterial pathogenicity.Bacteriophages possess unique characteristics that position them as precise tools for human microbiome modulation, holding tremendous potential for revolutionary breakthroughs in the regulation and treatment of the human microbiome through further research and development (Figure 2).
Viral tagging (VT) technology leverages the specificity of bacteriophages for host bacterial infection, enabling the isolation and purification of bacteriophages by marking and separating host bacteria (96).High-purity bacteriophage samples obtained through VT technology are crucial for microbial community modulation.The use of high-purity bacteriophage samples enhances the effectiveness of microbial community modulation by facilitating accurate assessment and control of the composition and functions of microbial communities.The high purity of bacteriophage samples also aids in reducing unwanted interference from host bacteria or other contaminants, thereby better achieving the desired effects of microbial community modulation.Obtaining high-purity bacteriophage samples through VT technology is a critical prelude to microbial community modulation.Research by Agarwal et al. demonstrates that polymers loaded onto bacteriophages, administered through dry powder inhalation, deposit throughout the lungs, providing active bacteriophages.This ultimately significantly reduces mouse infections and associated inflammation, successfully saving mice from mortality (97).Another study evaluated the effectiveness of bacteriophage therapy in treating methicillin-resistant Staphylococcus aureusinduced mastitis in a mouse model (98).In the mastitis mouse model, treatment with bacteriophage 4086-1 markedly inhibited the proliferation of Staphylococcus aureus in the mammary gland.Concentrations of the inflammatory markers TNF-a and IL-6 were significantly reduced, indicating the effective alleviation of the inflammatory response by bacteriophages.Using in vivo microbial cross-infection experiment; Tracking the effects of fecal viral-like particles isolated from ulcerative colitis patients and healthy controls on bacterial diversity and the severity of experimental colitis in human microbiota-associated (HMA) mice Found that several phages were transferred to HMA mice, resulting in therapy-specific changes in the gut virome; Phages as dynamic regulators of gut bacterial communities, as suggested by recent literature

Prospects and challenges in treatment
The bacterial lysis induced by lytic bacteriophages may lead to toxin contamination and the transfer of virulence genes among gut microbiota (99).In recent years, with the rapid advancements in synthetic biology and sequencing technologies, lysogenic bacteriophages have provided a feasible method for modifying bacterial genes and promoting intestinal homeostasis (100-103).During lysogeny, engineered lysogenic bacteriophages can introduce nucleic acids into the host chromosome and express carried functional genes, thereby enabling precise in situ regulation of gene expression in individual microorganisms.For example, the CRISPR/Cas9 system targeting bacterial superantigenicity and cell lysis genes has been integrated into the Staphylococcus aureus lysogenic bacteriophage, resulting in the excision of major virulence genes from the host genome.This has developed into an effective strategy to prevent toxin contamination and virulence gene transfer among gut microbiota (104).However, bacteriophagemediated therapeutic outcomes are not always effective.In an early clinical trial, despite the dose-dependent detection of T4 bacteriophages in volunteer feces after oral bacteriophage delivery, significant bacteriophage replication and a decrease in total Escherichia coli counts were not observed.Similarly, Sarker et al. found that delivered T4 bacteriophages (108 PFU) failed to proliferate on pathogenic Escherichia coli, and no improvement in diarrhea was observed.One possible cause of bacteriophage replication failure may be the presence of bacterial defense mechanisms, which inhibit bacteriophage invasion and result in A comprehensive exploration of bacteriophage-derived tools and strategies for precisely modulating the human microbiome.(A) Determining the designated target.Bacteriophages will be sourced from diverse environments using cultivation-independent techniques and replicated through cellfree systems or innovative cultivation methods.Identification of bacterial and functional targets will leverage advancements in culturing methods and multi-omics approaches.(B) Technology.The isolated phages and phage enzymes will be harnessed to modulate the human microbiota using various technologies.CRISPR/Cas9 will be transported to the target bacteria utilizing modified phage vectors.VT, viral-tagging.(C) Strategies.Moreover, to transport specific genes with diverse functions for modifying target bacteria.The ultimate objective is to reinstate a resilient microbiome in diseases associated with dysbiosis, such as inflammatory bowel disease (IBD), colorectal cancer (CRC), and so forth.[Adapted from (95)].treatment failure.It has been reported that anti-CRISPR bacteriophages can collaboratively overcome host bacteria's CRISPR resistance, rendering the host susceptible to subsequent bacteriophage infections.Therefore, delivering a mixture of bacteriophages targeting the same host (bacteriophage cocktail) could be a potential solution to increase infection likelihood.Another reason for bacteriophage replication failure may be insufficient active bacteriophage numbers in the colon (105).Indeed, bacteriophage viability is affected by various factors during storage and gastrointestinal transport, such as high temperatures and extreme pH values, leading to a significant reduction in bacteriophage titers and subsequent replication and proliferation failure (106,107).Developing bacteriophage combination-mediated IBD pathogen eradication therapy may require better strain-level bacterial target identification and addressing treatment-related challenges, such as bacteriophage delivery, off-target effects, and bacterial resistance.

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4 Optimizing formulation and encapsulation strategies for phage-based therapeutics

Ensuring the stability and activity
When developing bacteriophage therapy formulations, it is essential to understand the chemical and physical stresses that bacteriophages may undergo during preparation, processing, and storage to ensure their stability and activity.Bacteriophages consist mainly of proteins and are therefore susceptible to various factors that induce protein denaturation, such as types of organic solvents, high temperatures, pH values, ion concentrations, and interface effects.Mechanical pressures that may arise during formulation or encapsulation processes, such as shear forces during mixing, atomization during spraying, and stress during drying, also need to be carefully considered.Ensuring accurate dispersion of each bacteriophage particle is crucial during the development of formulations, although achieving complete dispersion poses significant challenges, certain techniques can achieve optimal results.Moreover, attention should be paid to controlling the morphology of bacteriophage particles to prevent aggregation or uncontrolled fusion with surrounding materials.Given the purpose and route of bacteriophage delivery, materials used to manufacture delivery vehicles should be safe, non-toxic, biocompatible, biodegradable, and non-irritating to the human body (108,109).Additionally, encapsulation materials should have good resistance to various adverse environments.Furthermore, the residence time of ingested substances in the gastrointestinal tract is limited (Figure 3).Beyond the limited transport time, even if these encapsulated bacteriophages resist adverse conditions and are transported to the colon with high vitality, they will be excreted in the feces, resulting in minimal or no release of bacteriophages into the colon.Therefore, complete release of encapsulated bacteriophages is required when they are transported in the colon (Table 3).

Enzyme/pH-responsive materials
Selection of enzymatically responsive materials capable of being degraded by specific enzymes produced by gut microbiota to release bacteriophages and pH-responsive materials that dissolve or swell under colonic pH conditions serves as ideal construction materials for achieving colonic-targeted delivery (111-114).Ahmad et al. prepared chitosan nanoparticles (CS-NP) and chitosan-phi KAZ14 bacteriophage-loaded nanoparticles (C-phi KAZ14 NP) using a simple entrapment method to effectively protect bacteriophages from gastric acid and enzymes in the chicken gastrointestinal tract.Chitosan nanoparticles exhibited potent protective effects on phi KAZ14 bacteriophages.Gel electrophoresis results demonstrated the protection of phi KAZ14 bacteriophages encapsulated in chitosan nanoparticles, while naked phi KAZ14 bacteriophages were degraded (115).In vitro release studies revealed complete inactivation of free bacteriophages under gastric conditions, while encapsulated bacteriophages maintained good vitality and steady release in the intestinal environment (9,116).Pectin remains undigested in the stomach but can be degraded by pectinases secreted by intestinal bacteria, yielding short-chain fatty acids and simultaneously releasing encapsulated substances in the colon.Dini et al. demonstrated that emulsified low methoxylated pectin as a delivery system was more effective than unemulsified pectin, alginate, or emulsified alginate microspheres.Free nonencapsulated bacteriophages exposed to an environment with a pH of 2.5 showed a 3.7-log unit reduction in titer after 10 minutes.Additionally, in the presence of 0.5 mg/ml pepsin at pH 2.5, bacteriophage titer decreased to undetectable levels within 10 minutes.When bacteriophages were encapsulated in emulsified low methoxylated pectin spheres, there was no significant The pH range, composition of digestive fluids, and transit times across different segments of the gastrointestinal tract.Encapsulation within micro-/nanoparticles or stimulus-responsive systems allows targeted triggering or sustained release at specific sites.The encapsulation of multiple emulsion drops/multiple concentric shells also extends the circulation time of bacteriophages.[Adapted from (110)].
reduction in titer after 3 hours under conditions with a pH of 2.5 and a pepsin concentration as high as 4.2 mg/ml.These results indicate that bacteriophages encapsulated in emulsified low methoxylated pectin are protected from the harsh gastric environment (117).The differences in acidity among the stomach, small intestine, and colon enable the possibility of delivering bacteriophages to target sites using pH-responsive materials (118).Alginate is a commonly used pH-responsive material for bacteriophage encapsulation.It forms insoluble alginate in gastric fluid (pH < 3.0), protecting internal bacteriophages from gastric acid and digestive enzymes.Dlamini et al. employed alginate-carrageenan microcapsules to protect genetically diverse bacteriophages of five Salmonella species from simulated gastrointestinal conditions.Their study demonstrated effective encapsulation (>95%) and maintenance of viability (>87%) of five genetically diverse bacteriophages representing three genera after exposure to simulated gastric conditions (pH 2, 3.2 mg/ml pepsin, 37°C, for 1 hour).All bacteriophages were easily released from microcapsules at pH 7.5 and exposure to simulated duodenal conditions (pH 7, 10 mg/ ml pancreatin, 37°C) (119).Eudragit S100 has been widely used as an encapsulation material for targeted bacteriophage release in the colon (5).For instance, Vinner et al. microencapsulated a Salmonella-specific bacteriophage Felix O1 in a pH-responsive polymer formulation.They incorporated the pH-responsive copolymer methacrylic acid Eudragit ® S100 (10% (w/v)) and added the biopolymer sodium alginate to the formulation.Results showed excellent protection of Felix O1 encapsulated in the formulation containing 10% (w/v) ES100 and 1% (w/v) alginate when exposed to simulated gastric fluid (SGF) (pH 1, for 2 hours).In simulated intestinal fluid (SIF), encapsulated bacteriophages previously exposed to SGF (pH 1, for 2 hours) were released as the pH increased, indicating inhibition of bacterial growth during the logarithmic growth phase (120).

Fiber and emulsion materials
Electrospinning technology is a versatile method for fiber production that does not require the use of toxic solvents or generate heat, providing a gentle encapsulation environment for bacteriophages (121, 122) (Figure 4A).For instance, Cheng et al. incorporated Escherichia coli bacteriophage T4 into poly (ϵ-caprolactone)/type I collagen (PCL-ColI) nanofibers using electrospinning to eradicate E. coli infections while establishing hemostatic functionality (123).To better maintain bacteriophage activity, R. korehei et al. pre-encapsulated T4 bacteriophages in an alginate hydrogel layer using an emulsification process and then incorporated them into fibers using electrospinning technology (124).The release of encapsulated bacteriophages from the fibers is mediated by the dissolution and/or swelling behavior of the fiber matrix (11).Thus, controlling the release rate of encapsulated bacteriophages from the fibers to the external environment can be achieved by altering the composition ratio of the composite fibers.Korehei et al. demonstrated that after injecting T4 bacteriophages into core/shell electrospun fibers prepared from poly(ethylene oxide) (PEO) and cellulose diacetate (CDA) or their mixtures, PEO-coated fibers released T4 bacteriophages immediately upon immersion in a buffer solution.The release rate of bacteriophages significantly decreased when CDA was mixed with PEO, and T4 bacteriophages were undetectable in fibers composed solely of CDA.Increasing the proportion of PEO in the fibers increased the diameter of the electrospun fibers and the viscosity of the release medium, resulting in a relatively slower release of T4 bacteriophages.The morphology of the electrospun fibers after release varied from discontinuous fibers to microexpanded fibers depending on the PEO/CDA ratio (11).
Encapsulation of bacteriophages in emulsions to form nanoparticles, followed by electrospinning, is another effective method to improve bacteriophage protection.Due to the hydrophilic nature of bacteriophages, W/O and W/O/W emulsions are commonly used for

Liposomes and hydrogel materials
Liposomes, with their core containing an aqueous phase, offer a means to incorporate and encapsulate sensitive bacteriophages.Additionally, the phospholipid bilayer membrane of liposomes can provide physical protection for the core bacteriophages (Figure 4C).Cinquerrui et al. investigated the encapsulation of two model bacteriophages, preserving their activity and estimating the yield of microfluidic encapsulated bacteriophages (79).For certain intracellular diseases, liposomes are often employed to encapsulate bacteriophages for intracellular delivery to achieve therapeutic purposes.Nieth et al. found that liposome-encapsulated bacteriophages were more effectively absorbed into eukaryotic cells compared to free bacteriophages (80).Due to their excellent biocompatibility, liposomes show immense potential in protecting bacteriophages from immune clearance by preventing phagocytic cell recognition and capture.Researchers compared the ability of bacteriophages embedded in liposomes and free bacteriophages to enter mouse peritoneal macrophages and kill intracellular Klebsiella pneumoniae.The study also compared the efficacy of liposomeencapsulated bacteriophages alone or in combination with amikacin in eradicating mature biofilms.The results demonstrated that liposome-encapsulated bacteriophages were protected, capable of entering macrophages and killing intracellular bacteria.Liposomeencapsulated bacteriophages also exhibited synergistic activity with amikacin in biofilm eradication (125).
Hydrogels can serve as physical barriers for core bacteriophages, protecting them from acidic and enzymatic degradation while prolonging their retention time during intestinal transport, making them ideal carriers for bacteriophage delivery (Figure 4D).Kopac et al. proposed an efficient PolyHIPE hydrogel system for targeted bacteriophage delivery and rapid release at the target site.T7 bacteriophages were encapsulated in low cross-linked anionic nanofibrous cellulose hydrogels, which successfully protected the bacteriophages at pH below 3.9 (stomach), while the hydrogel network dissolved completely at pH above 3.9 (duodenum), allowing bacteriophage release.The PolyHIPE scaffold protected the hydrogel from mechanical stress during transport, preventing The preparation processes and microstructures of various carriers with encapsulated phages are outlined.(A) Depicts the preparation process of electrospun fibers and the microscopic structure of phages encapsulated through the electrospinning technique.(B) Illustrates the fabrication process and microstructure of multiple emulsions containing phages.(C) Presents a schematic of liposome preparation and the microscopic structures of encapsulated phages: (i) phages encapsulated in liposomes via the thin film hydration method, and (ii) phages in liposomes through a microfluidic process.(D) Describes the preparation mechanism and microstructure of hydrogels with encapsulated phages: (i) chitosan-coated alginate hydrogel loaded with phages using the extrusion-dripping method, (ii) Eudragit S100/alginate hydrogel with phages produced through a microfluidic technique, and (iii) Eudragit S100/alginate hydrogel with phages via membrane emulsification.(E) Provides a production diagram and microstructure of dry particles with phages: (i) phages in particles through lyophilization, (ii) dried particles with phages via the electrospraying process, and (iii) solid particles containing phages through the spray drying method.[Adapted from (82)].
hydrogel collapse and accidental bacteriophage release (126).In constructing bacteriophage encapsulation carriers, a layer-by-layer assembly method was employed to coat acid-resistant polyethyleneimine and pectin onto alginate-bacteriophage hydrogels to protect bacteriophage activity during gastrointestinal transport.Hsu et al. generated alginate microbeads by dripping alginate solution into stirred calcium chloride solution, which were then coated with polyethyleneimine (PEI) and pectin.Film-coated alginate microbeads exhibited resistance to external pH changes.Increasing film thickness enhanced acid resistance.Encapsulating l bacteriophages into alginate microbeads coated with (PEI/pectin) displayed excellent in vitro acid stability compared to free l bacteriophages (127).Bacteriophage-based hydrogel-mediated delivery offers a precise strategy for modulating the expression of specific genes in individual microbes in the intestine, thereby promoting gut homeostasis and human health.The development of bacteriophage resistance is also a general consideration when delivering lytic bacteriophages (128).Therefore, the use of multiple bacteriophages (bacteriophage cocktails) may be necessary to ensure successful lytic activity and achieve the desired outcomes, broadening the host range of targeted microbes and suppressing the development of resistance (129).

Particle materials
The production methods for bacteriophage encapsulated particles mainly include freeze-drying, electrospraying, and spray-drying.Spray freeze-drying can produce controlled particle size distribution bacteriophage-loaded porous powders without subjecting bacteriophages to the high thermal stresses typically encountered in traditional spray-drying.Some protectants have been added to the formulation to enhance bacteriophage viability (Figure 4E).In Pereira et al.'s study, they developed an edible biopolymer microcapsule packaging for intestinal Salmonella, integrating lytic bacteriophage particles.For the formulation, a concentration of 2% (w/w) sodium alginate was added (130).To enhance colorectal cancer (CRC) chemotherapy effectiveness, nucleic acid bacteriophage-specific bacteriophages were conjugated with glucan nanoparticles loaded with CRC chemotherapeutic drugs to form bacteriophage-guided nanoparticles, which effectively inhibited nucleic acid bacteriophage growth and significantly extended the survival time of CRC mice (131).Additionally, in Thanki et al.'s study, combining polymers with sugars and leucine excipients also contributed to bacteriophage stability during drying (132).To precisely control bacteriophage dosage and improve oral administration convenience, these spraydried particles can be further compressed into bacteriophage tablets for colonic delivery.In Khanal et al.'s study, targeting bacteriophage PEV20 against intestinal Pseudomonas aeruginosa, bacteriophage tablets suitable for oral administration were produced using industrial-scale tablet compression and coating machines.The bacteriophage tablet produced exhibited negligible reduction in bacteriophage titer throughout the process and passed the British Pharmacopoeia tests, including friability, weight variation, disintegration, and dissolution of uncoated tablets (in 0.1 M HCl and pH 7.4 phosphate buffer).The developed formulation method can be used to produce tablets containing other bacteriophages and bacteriophage cocktails, which are effective in combating intestinal bacterial infections (133).

Conclusion and outlook
Dysbiosis of the gut microbiota is linked with inflammatory bowel disease.Recently, bacteriophage delivery has emerged as an innovative strategy, holding immense potential to alter gut microbiota composition or modulate bacterial genes through their specificity for certain host bacteria.Bacteriophages play a pivotal role in the structure and functionality of human gut microbiota, thereby affecting gastrointestinal health and disease.Given this, bacteriophages stand out as promising agents for IBD therapy, targeting pathogenic gastrointestinal bacteria.However, environmental and gastrointestinal conditions can deactivate bacteriophages, diminishing their effectiveness and presenting substantial delivery challenges.Encapsulation techniques have been employed to boost bacteriophage vitality and stability for storage and intestinal transit.Various carriers have proven effective in preserving bacteriophage stability for storage, safeguarding viability, and ensuring efficient release in the colon during gastrointestinal passage.Despite some identified limitations with certain carriers, employing a combination of methods to create composite carriers presents research opportunities to enhance bacteriophage delivery in gut microbiota modulation practically.

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(A)
Conventional phage therapy targeting gut dysbiosis associated with pathogenic microbiome.(B) Administered phages actively diminish the population of pathogenic bacteria, eliminate harmful bacterial genes, and mitigate the absorption of detrimental metabolites in situ.[Adapted from(82, 83)].

TABLE 2
Summary of phage applications in IBD treatment and modulation of gut microbiota.[Adapted from (74-77)].

TABLE 3
Overview of encapsulation materials and carriers for phage delivery.