Smart bionanomaterials for treatment and diagnosis of inflammatory bowel disease

3.1.1 Definition and properties

Hydrogels are three-dimensional networks of hydrophilic polymers that can swell in water and retain a large amount of water while maintaining the structure due to chemical or physical cross-linking of individual polymer chains [50]. Due to their high water absorption capacity, porosity, flexibility, soft structure, controlled release, and similarity to living tissues, hydrogels are suitable for various biomedical applications such as drug delivery, tissue engineering, and wound dressing. These properties can be adjusted by changing the polymer type, its concentration, and reaction conditions [51,52]. The reported disadvantages of hydrogels are their inhomogeneous mechanical strength, difficulty in shaping them in predesigned geometries, and difficulty in loading hydrophobic drugs [51,53].

3.1.2 Active ingredients and their release

Active ingredients encapsulated in smart hydrogels include anti-inflammatory compounds such as 5-aminosalicylic acid (5-ASA), HA, and dexamethasone. Additionally, probiotic bacteria, such as Lactobacillus reuteri and Lactobacillus acidophilus, have been included in the smart hydrogel formulation against IBD (Table 1). Smart hydrogels involved in the treatment and diagnosis of IBD respond to external stimuli such as pH, temperature, enzymes, and ROS.

Temperature changes can affect the volume of hydrogels prepared from thermally sensitive polymers, thereby controlling drug release and bioavailability. Different formulations of hydrogels have been composed of (1,3)-(1,6) β-glucan and poly (N-isopropylacrylamide) and loaded with the anti-inflammatory drug 5-ASA. Increasing β-glucan content resulted in a higher swelling ratio and drug-loading capacity. The optimized composition released almost 90% of the drug within 1 h at 37 °C [54]. Similarly, 5-ASA was loaded into a pH-sensitive hydrogel composed of poly(amidoamine) and chitosan. The hydrogel protected the drug in the acidic environment of the stomach and enabled its release at colon pH. The hydrogel delivered the drug in its ionized state, which increased its solubility and allowed the drug to be localized at the site of inflammation [55]. The targeted delivery of 5-ASA was proposed by Saboktakin et al. who encapsulated the drug in a chitosan-dextran sulfate hydrogel [56]. A colon-targeted, pH-sensitive core–shell hydrogel with a core of thiolated-HA hydrogel and shell of alginate hydrogel was developed. HA has anti-inflammatory properties but is susceptible to degradation in the stomach. The use of calcium alginate hydrogel as a shell-protected HA resulted in anti-inflammatory effects and alterations in gut microbiota in mice with colitis. The alginate shell remained stable in artificial gastric fluids (pH 1) but slowly lost its thickness as the pH increased [57].

Enzymes, such as esterases and matrix metalloproteinases, are overexpressed in the colonic lumen and tissue of IBD patients and can be used as biomarkers [58]. The development of an enzyme-responsive hydrogel that can be degraded by IBD-specific enzymes will enable targeted delivery. For example, encapsulation of the corticosteroid drug dexamethasone in hydrogel microfibers generated from ascorbyl palmitate resulted in preferential adhesion to inflamed tissue in vitro and a significant reduction in inflammation in mice with colitis, observed as reduction of the colon weight, myeloperoxidase activity, and TNF-α expression. The dexamethasone-loaded hydrogel was stable for 16 days in phosphate-buffered saline (PBS) at 37°C, and the addition of esterase resulted in a rapid and dose-dependent release of the drug [59].

Smart hydrogels can also be used for the delivery of probiotics. The ROS-responsive hydrogel loaded with L. reuteri was designed for targeted delivery to the inflamed colon. Methacrylated HA crosslinked with thiolated thioketal allowed up to 95% encapsulation and 90% survival rate of the probiotic. Thioketal linkages can be cleaved in the presence of excessive ROS, resulting in the degradation of the hydrogel and the release of the probiotic. Approximately 90% of L. reuteri was released after incubation with 10 mM H₂O₂ for 60 min. Moreover, the hydrogel scavenged excessive ROS, protected HT-29 cells, and improved the survival rate of mice with colitis [60]. NO can be used as an external stimulus for the local delivery of probiotics to the colon. NO responsive hydrogel microcapsules loaded with probiotic L. acidophilus were fabricated with poly-γ-glutamic acid. Hydrogels treated with nitroferricyanide(iii) dihydrate released 67.9% of the probiotic, whereas only 4.6% of the probiotic was released without nitroferricyanide(iii) dihydrate. In vivo studies in a mouse model of colitis confirmed immunomodulatory effects by improving the disease activity index. In addition, the system improved the microbiota balance by increasing the proportion of beneficial bacterial genera such as Turicibacter, Acidaminococcaceae, Negativicutes, and Phascolarctobacterium [61].

3.1.3 Composition and manufacturing

Hydrogels are synthesized from natural or synthetic polymers [62,63]. Most smart hydrogels for the treatment of IBD are synthesized from natural polymers such as ascorbyl palmitate, chitosan, HA, poly-γ-glutamic acid, and β-glucan, while poly (N-isopropyl acrylamide) is an example of synthetic polymer. Natural polymers have better biocompatibility and biodegradability but lower stability and mechanical strength compared to synthetic polymers [52].

Crosslinking of the polymer chains can be physical, chemical, or a combination of both (Figure 2). The structure of the hydrogel is influenced by various factors, including their composition, crosslinking, and encapsulated ingredient. The chitosan-dextran sulfate hydrogel has a solid and consistent structure, and the incorporation of 5-ASA resulted in a smooth surface and compact structure [56]. Conversely, HA hydrogels are characterized by a porous structure, and encapsulation of L. reuteri did not have a significant influence on the hydrogel structure [60].

Figure 2

Synthesis methods of hydrogels, their interaction, and encapsulation of active ingredient. The image was created with Biorender.com.

Physically crosslinked hydrogels are prepared by intermolecular reversible interactions such as hydrophobic/hydrophilic interactions, hydrogen, and ionic bonds [64]. These types of hydrogels are simpler because gelation does not require crosslinking molecules, but they lack mechanical strength and stability. In regard to smart physically crosslinked hydrogels in IBD, ascorbyl palmitate has been frequently used as a polymer due to its self-assembly into a gel. Ascorbyl palmitate hydrogel is prepared by dissolving ascorbyl palmitate in heated dimethyl sulfoxide and water, followed by cooling [59].

Chemically crosslinked hydrogels have stronger linkages between polymer chains due to covalent bonds between the polymer and the crosslinker [65]. Crosslinker is a bi-reactive molecule that forms bridges between the chains of polymers, such as chitosan, HA, poly-γ-glutamic acid, β-glucan, and poly (N-isopropyl acrylamide). Chitosan is a natural polymer that can self-crosslink; alternatively, crosslinking can be assisted by adding glutaraldehyde. Carbonyl groups of glutaraldehyde covalently bind with the amino groups of chitosan or other polymers making gel more rigid and stable [66]. Glutaraldehyde-saturated toluene was used to crosslink chitosan with dextran sulfate [56] and polyamidoamine [55] to form a smart hydrogel for colon delivery of 5-ASA.

Likewise, HA and alginate are natural polymers that are used to synthesize smart hydrogels for colon targeting. An aqueous solution of thiolated HA and sodium alginate was injected through a gas shearing device where homogeneous droplets were generated. The addition of silver nitrate and calcium nitrate provided Ag⁺ and Ca²⁺ for crosslinking of HA and sodium alginate, respectively [57]. Another crosslinker for the formation of smart HA hydrogel is thioketal, which is sensitive to ROS. Methacrylated HA was mixed with a probiotic suspension, followed by the addition of thioketal dissolved in PBS. The mixture was incubated at 37°C for gelation [60]. Methacrylated β-glucan was used as a linker for poly (N-isopropyl acrylamide) to synthesize a temperature-responsive hydrogel. The hydrogel was synthesized at room temperature with redox polymerization using tetramethylethylenediamine and potassium persulfate as redox pair [54].

Microfluidic technology is an advanced tool for smart hydrogel preparation, as it enables precise particle manipulation and mixing of liquids [67]. Droplet microfluidic technology was used for the synthesis and encapsulation of probiotics in poly-γ-glutamic acid hydrogel, using N,N-(2-amino-1,4-phenylene) diacryl amide as a linker. A microfluidic chip was designed to merge three channels into a single channel, thereby allowing uniform distribution of probiotics and polymer. Droplets of the mixture of probiotics and polymer were obtained under shear stress with perfluorinated oil. The droplets were solidified by irradiation with visible light, resulting in smart hydrogels uniformly loaded with probiotic bacteria [61].

3.2.1 Definition and properties

Nanoparticles are a broad class of materials with dimensions below 100 nm. Based on their physical and chemical characteristics, nanoparticles can be classified into carbon, metal, ceramic, semiconductor, polymeric, and lipid nanoparticles [68,69]. Smart nanoparticles for IBD are mainly polymeric. They respond to different physical, chemical, and biological stimuli, such as ROS, pH, ligands, magnetic field, and gut microbiota, and thus provide controlled release. They can protect the drug by encapsulation, reduce adverse effects, and target specific cells [70]. They are also easy to prepare and have good mucoadhesive and biocompatible properties. On the other hand, their use is limited by chemical stability and particle aggregation [71,72]. Compared to polymeric nanoparticles, lipid nanoparticles are more affordable, can be easily scaled up, are biodegradable, and have excellent biocompatibility. However, they are limited by their lower loading capacity due to the lower solubility of the drugs [73]. Inorganic nanoparticles are characterized by good optical and magnetic properties, making them suitable for targeting IBD. However, their main limitations are long-term toxicity and burst drug release. These can be partially overcome by physical or chemical modification of the outer surface [71].

3.2.2 Active ingredients and their release

Smart nanoparticles deliver anti-inflammatory agents to the colon or regulate microbiota homeostasis. Examples of smart nanoparticles are summarized in Table 2. The majority of the smart nanoparticles effectively localize in the inflamed area of the intestines and act by inhibiting the proinflammatory cytokines in vitro and in vivo. Nanoparticles from polyphenol tannic acid and polyethylene glycol (PEG) were used to encapsulate infliximab and protect it from degradation in the digestive tract. The hydroxyl groups of tannic acid are easily oxidized by ROS in the inflamed colon, leading to the cleavage of the hydrogen bond between tannic acid and PEG and the release of the antibody at the site of increased oxidative stress. Oral administration of antibody-loaded nanoparticles resulted in the accumulation of infliximab in the inflamed area and reduced the levels of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 [74]. Another approach to lower the levels of proinflammatory cytokines is the oral administration of small interfering RNA (siRNA) against TNF-α encapsulated in thioketal nanoparticles. The nanoparticles were prepared from poly-(1,4-phenylene acetone dimethylene thioketal), which degrades in the presence of ROS. Oral administration of nanoparticles improved the clinical symptoms of induced colitis in mice by improving the intestinal epithelium, increasing weight, and reducing neutrophil levels [75]. Redox-dependent targeting of IBD by nanoparticles has also been studied for HA [76], tempol [77], budesonide [78], and magnolol [79].

Several pH-sensitive nanoparticles have been developed for the local treatment of IBD. Curcumin was encapsulated in polylactide-co-glycolic acid (PLGA) and polymethacrylate polymer nanoparticles. The release of the drug was pH dependent, with acidic pH (1.2 and 4.5) limiting the release rate to <15%, and pH 7.2 triggering a rapid release. The nanoparticles enabled better permeation through Caco-2 cell monolayers and significantly reduced TNF-α secretion in murine models [80]. PLGA and Eudragit S100 nanoparticles were used for the encapsulation of budesonide. The release of the drug was pH dependent, with a rapid release in a simulated colon environment (pH 7.4). The nanoparticles showed significant therapeutic effects in a rat colitis model [81]. Similarly, PLGA and Eudragit FS30D were used for the synthesis of nanoparticles loaded with the immunosuppressive drug cyclosporine A, which enabled pH-dependent release and improved the health status of mice with colitis [82]. Eudragit S100 was also used to coat inorganic mesoporous silica nanoparticles, resulting in pH-sensitive nanoparticles suitable for colonic delivery of prednisolone and budesonide. Upon encountering the pH above 7, high percentage of prednisolone and budesonide (71 and 60%, respectively) were released [83].

The upregulation of proinflammatory proteins in IBD provides molecular stimuli for the development of proinflammatory protein-sensitive nanoparticles. For example, folate receptors are proinflammatory proteins that are overexpressed during inflammation. PLGA nanoparticles conjugated with folic acid and loaded with resveratrol demonstrated targeted anti-inflammatory properties by reducing neutrophil and lymphocyte accumulation in rats as a result of binding of conjugated nanoparticles to cells that overexpressed folate receptor [84]. Lipid nanoparticles loaded with TNF-α siRNA and coated on the surface with rat IgG2a antibody against Ly6C showed 94.2% knockdown in vitro and reduced the TNF-α protein levels in the colon of mice by approximately threefold [85]. In a similar fashion, IL-10-modified messenger RNA (mRNA) was encapsulated in lipid nanoparticles and coated with a Ly6C antibody. In mouse models of induced colitis, the nanoparticles recognized Ly6C receptors on inflammatory leukocytes and delivered the modified mRNA encoding IL-10, resulting in increased level of IL-10 and decreased levels of TNF-α and IL-6 in mice models of colitis [86].

Targeted delivery of oral probiotics with nanoparticles was achieved by labeling Escherichia coli with streptavidin-coated superparamagnetic iron oxide nanoparticles, allowing colonization of specific regions of the gut by applying an external magnetic field. Under the influence of magnetic arrays worn by mice, labeled E. coli were localized and retained in distinct regions of the gastrointestinal tract. The application of magnetic field enabled localization and retention of 17.0 ± 1.5% of bacteria in the small intestine 9 h post-gavage, whereas only 0.6 ± 0.5% of bacteria were detected in the small intestine without a magnetic field [87]. Conversely, certain nanoparticles can be degraded by the gut microbiota and serve as nutrients. These nanoparticles can alter the composition of the microbiota and significantly increase the levels of butyrate-producing bacteria, which can downregulate the production of proinflammatory cytokines. Oral administration of small carbon nanotubes and graphene oxide flakes to mice for 28 days resulted in gut microbiota alterations, with increased levels of butyrate-producing bacteria and increased levels of intestinal butyrate [88]. Similar results were obtained with fullerenol nanoparticles administered orally to mice for 4 weeks. These nanoparticles were also degraded by the microbiota, and the number of butyrate-producing bacteria increased after 7–28 days of treatment [89]. Selenium nanoparticles at specific doses also increased the levels of butyrate-producing bacteria from the genera Lactobacillus and Faecalibacterium in chickens and served as an effective selenium supplement [90]. Similarly, curcumin nanoparticles increased the abundance of butyrate-producing bacteria from Clostridium cluster IV, Clostridium subcluster XIVa, and Clostridium cluster XI, possibly by improving curcumin absorption and by inducing mucosal immune cells with regulatory properties [91].

3.2.3 Composition and manufacturing

Smart nanoparticles for the treatment and diagnosis of IBD are most commonly prepared from polymers and lipids using dialysis or emulsification (Figure 3).

Figure 3

Schematic representation of the preparation process of nanoparticles with dialysis and emulsion methods (solvent evaporation and single emulsion). The image was created with Biorender.com.

When using dialysis, the active ingredient and polymer are dissolved in water-miscible organic solvents and placed in a dialysis tube. The organic solvent diffuses out of the dialysis tube, allowing aggregation of the polymer due to the increase in concentration and decrease in solubility. The suspension is freeze dried, resulting in the formation of nanoparticles [92]. Dialysis against water is used to prepare PEG- [74], HA- [76], β-cyclodextrin- [77], and butyrate-based polymeric [79] smart nanoparticles loaded with different anti-inflammatory agents. Dialysis with microfluidic technology was used to synthesize smart lipid nanoparticles for delivering siRNA [85] and modified mRNA [86]. A mixture of lipids (MC3, DSPC, cholesterol, DMG-PEG, and DSPE-PEG) in ethanol was mixed with siRNA or modified mRNA in acetate buffer. The mixture was injected into a microfluidic mixing device and dialyzed against PBS. The nanoparticles were then coated with recombinant scFv onto which rat IgG2a antibody was attached via the Fc domain [85,86].

When using emulsification, the drug and polymer are dissolved in a volatile organic solvent that forms the oil phase, followed by the addition of water with a stabilizer to form oil in water emulsion [93]. The solvent is then evaporated using high temperature, vacuum, or continuous stirring, resulting in precipitation of the polymer and formation of nanoparticles. The size of the nanoparticles can be controlled by varying the parameters of evaporation [94]. The nanoparticles are collected with ultracentrifugation and can be lyophilized to enhance the shelf life [93]. This method was used to synthesize thioketal nanoparticles composed of poly-(1,4-phenyleneacetone dimethylene thioketal) [75], pH-sensitive PLGA with Eudragit FS30D [82], and PLGA with Eudragit S100 [80,81], as well as ligand-sensitive PLGA [84] nanoparticles.

Other chemical reactions such as alkaline reaction [89] and chemical reduction [90] are also used in the synthesis of smart inorganic nanoparticles. Chemical synthesis was used to manufacture mesoporous silica nanoparticles by mixing hexadecyltrimethylammonium bromide, NaOH, and tetraethylorthosilicate in deionized water for 2 h. The suspension was vacuum-filtered and the particles were dried overnight. Then, the particles were mixed with Eudragit S100 and prednisolone or budesonide in methanol which was then evaporated [83].

3.3.1 Definition and properties

Smart nanofibers are polymeric fibrous materials with nanometer diameters, high surface-to-volume ratios, low weight, and high, controllable porosity. They are produced by electrospinning [95]. Smart nanofibers can deliver small molecular drugs [96,97,98], as well as larger biological drugs, such as proteins, nucleic acids, microorganisms, and even stem cells [98,99,100,101,102]. Like other smart bionanomaterials, smart nanofibers can respond to external stimuli and enable targeted and controlled drug delivery. The disadvantages of electrospinning include extended manufacturing time and low production efficiency, but these can be circumvented by using multiple jets and nozzle-less electrospinning [103]. Another drawback is a high voltage, which can decrease the stability of biological drugs, such as proteins, nucleic acids, and cells. However, the addition of lyoprotectants can alleviate this challenge, allowing the ingredient to be preserved for a longer period of time [98,99,101].

3.3.2 Active ingredients and their release

In IBD, smart nanofibers were developed for the delivery of anti-inflammatory drugs such as prednisolone, diclofenac sodium, and budesonide, as well as probiotic bacteria such as Lactobacillus plantarum and Lactobacillus rhamnosus (Table 3).

Chitin nanofibers, surface deacetylated, and reinforced with a sulfobutyl ether β-cyclodextrin gel, were developed for the controlled release of poorly water-soluble prednisolone. The full content of the drug was released slowly over 12 h irrespective of the pH (1.2 or 6.8), enabling colon delivery. The in vivo use of nanofibers in mice showed the anti-inflammatory activity of the material by preserving the length of the colon, reducing symptoms of colitis and histopathological changes in the colon tissue [104]. Polyvinylpyrrolidone nanofibers coated with shellac were produced with coaxial electrospinning. Shellac is not soluble at acidic pH but dissolves at neutral pH. These properties enable the targeted release of diclofenac sodium with a release rate of only 7.1% at pH 2 and rapid release at pH 7.4, making this a promising formulation against IBD [105]. Coaxial electrospinning was also used to encapsulate budesonide in ethyl cellulose nanofibers coated with Eudragit S100. The drug showed a pH-dependent release profile, with the highest drug release at pH 7.4, corresponding to colonic conditions. When budesonide was encapsulated in core–shell nanofibers, the mean peak concentration in the colon of rats was approximately 2.6 times higher than when the drug was encapsulated in single-layer fibers [106].

Sodium alginate is a pH-responsive polysaccharide that can be used to synthesize pH-responsive nanofibers. Alginate was used as a shell for polyvinyl alcohol (PVA) nanofibers loaded with the probiotic bacterium L. plantarum. Encapsulated probiotics in the double-layered nanofibers retained their viability after exposure to acidic conditions compared to the nonencapsulated bacteria. Probiotic bacterial proliferation was observed at higher pH, suggesting that this system could be used to deliver probiotics to the colon [107]. Similarly, L. plantarum was encapsulated in coaxial electrospun nanofibers with polylactic acid (PLA) and fructooligosaccharides (FOS) as the shell material. The survival rate of probiotics at simulated intestinal pH was 72%. PLA is resistant to acid, which allows for the protection of probiotics and targeted release in the colon. FOS remained in the culture media and may have further promoted bacterial growth by acting as a prebiotic [108]. L. rhamnosus was encapsulated in Eudragit S100 and PVA/pectin nanofibers using coaxial electrospinning. Encapsulation of bacteria resulted in 90.07 and 91.96% survivability in simulated gastric and intestinal fluids, respectively [109]. Similarly to coaxial electrospinning, multilayer nanofibers composed of different layers that were spun sequentially showed promising results as probiotic delivery system. L. rhamnosus encapsulated in pullulan nanofibers sandwiched between two PLGA mats provided better retention of viability compared to spray-dried bacteria and bacteria encapsulated in single-layer nanofibers. In addition, sandwiched bacteria demonstrated better tolerance to low gastric pH, while enabling effective colonization of the jejunum and cecum of mice three days after administration [110].

3.3.3 Composition and manufacturing

Smart nanofibers are produced by conventional or coaxial electrospinning (Figure 4). Conventional electrospinning is a method that applies high electric voltage to a droplet of a polymer solution such as polyethylene oxide, PLA, and polystyrene [111]. The voltage overcomes the surface tension of the polymer droplet, resulting in a “whipping” motion, solvent evaporation, and nanofiber formation [95]. The final product is a solid thin, soft, and flexible film. Unlike the other delivery systems, encapsulation of active ingredients, especially bacteria, has a significant influence on the morphology of the nanofibers. Upgraded method of electrospinning is coaxial electrospinning, in which two components (core and shell) are electrospun simultaneously by using an additional syringe pump and a coaxial nozzle. A core polymer flows through the inner nozzle, while a shell polymer flows through the outer nozzle. Similar to the conventional method, a high electrostatic field is applied, resulting in the formation of two-layered nanofibers [112,113].

Figure 4

Illustration of conventional and coaxial electrospinning and synthesis of mono and double-layered nanofibers. The image was created with Biorender.com.

Coaxial electrospinning was used to prepare smart nanofibers loaded with diclofenac sodium. A mixed solution of 35% (w/v) polyvinylpyrrolidone and 5% (w/v) diclofenac sodium was used as the core fluid, while 10% (w/v) of shellac solution was used as the sheath fluid [105]. In a similar fashion, budesonide was encapsulated in ethyl cellulose nanofibers as the core, while Eudragit S100 was used for the shell layer [106]. Eudragit S100 was also used as a shell layer to protect the viability of the probiotic bacterium L. rhamnosus. Bacterial suspension was added to a 9:1 mixture of 12% (w/v) PVA and 11% (w/v) pectin as the core layer [109]. Another probiotic encapsulated in core–shell smart nanofibers was L. plantarum. Bacterial suspension (10¹⁰ CFU/mL) was mixed with 6% (w/v) PVA as core solution and an 8:2 mixture of 8% (w/v) PVA and 2% sodium alginate as shell solution [107]. L. plantarum was also encapsulated in another smart core and shell nanofiber formulation, using 8% (w/v) PLA and 2.5% (w/v) FOS as shell solution and 1% (v/v) L. plantarum in MRS growth medium as core solution [108].

3.4.1 Definition and properties

Smart hybrid bionanomaterials combine two or more bionanomaterials to provide better efficacy and fewer side effects of delivered drugs [114]. Assembly of two or more bionanomaterials can improve their characteristics in delivering active ingredients to the inflamed intestine, thereby improving the treatment and/or diagnosis of IBD. Most smart hybrid bionanomaterials combine nanoparticles and hydrogels. They respond to external stimuli such as biomolecules, pH, ROS, and magnetic field.

3.4.2 Active ingredients and their release

Smart hybrid systems can deliver active ingredients into the colon or sample the microbiota to aid in the diagnosis of IBD (Table 4).

Cluster of differentiation 98 (CD98) is a glycoprotein antigen overexpressed on the surface of colonic epithelial cells and macrophages in IBD. A hybrid oral delivery system with nanoparticles in hydrogel was designed to target CD98. PEG-urocanic acid-modified chitosan nanoparticles coated with single-chain CD98 antibodies on their surface and loaded with CD98 siRNA were encapsulated in a chitosan/alginate hydrogel. The modified nanoparticles interacted with CD98 and reduced its levels in vitro and in mice with colitis; moreover, they also reduced the expression of TNF-α, IL-6, and IL-12 [115]. Targeted delivery of siRNA against TNF-α was achieved by encapsulation in a hybrid material that was responsive to changes in pH and intestinal enzymes. siRNA was encapsulated in polycationic 2-(diethylamino)ethyl methacrylate nanogels, which were further encapsulated in an enzymatically degradable poly(methacrylic acid-co-N-vinyl-2-pyrrolidone) hydrogel. At intestinal pH, the hydrogel swelled and enabled the infusion of intestinal fluid and degradation of the gel by intestinal enzymes such as trypsin. The released nanogels loaded with siRNA TNF-α were absorbed by RAW 264.7 macrophages and downregulated the secretion of TNF-α [116].

Naturally occurring tripeptide KPV can alleviate colon inflammation. Its encapsulation in HA-embedded PLGA nanoparticles resulted in an immunosuppressive response following administration to mice with colitis. Moreover, when these nanoparticles were encapsulated in a chitosan/alginate hydrogel, they prevented mucosal damage and downregulated TNF-α even more effectively. The hydrogel degrades at colonic pH, suggesting that the system acts in the colonic lumen and not in other sections of the gastrointestinal tract. In addition, HA can bind to the CD44 receptor, which is abundant on the surface of epithelial cells and macrophages in IBD-affected colon tissues [117].

Selenoprotein, the main in vivo form of selenium, plays a critical role in inflammation and immune cell activation. To increase the in situ production of selenoprotein, selenium nanoparticles were modified with HA and encapsulated in a protective shell of a calcium alginate hydrogel. The hybrid system protected mice with colitis by decreasing epithelial damage and downregulating the expression of proinflammatory cytokines TNF-α, IL-6, and IL-1β. The hybrid system of nanoparticles and hydrogels resulted in a greater downregulation of cytokines than the conventional system using only nanoparticles. The authors also observed that the system increased the relative abundance of Verrucomicrobiota, Firmicutes, and Bacteroides [118].

Anti-inflammatory 6-gingerol is one of the most bioactive compounds in ginger. Its encapsulation in medium-chain triglycerides and beeswax nanoemulsions coated with chitosan and octenyl succinic anhydride-modified resistant starch resulted in pH-responsive microcapsules that mitigated colitis symptoms in mice. In vitro and in vivo studies have confirmed the colon targeting and anti-inflammatory effects of the hybrid material by regulating antioxidant/anti-inflammatory pathways and increasing the Firmicutes/Bacteroidetes ratio [119].

Olsalazine is a prodrug that is not absorbed in the intestines but breaks down into two 5-ASA molecules by the colon bacterial azoreductases. To improve the colon retention, hybrid delivery system composed of olsalazine-containing copper nanoneedles and inulin hydrogel was designed. The hybrid system demonstrated pH-responsive behavior, facilitating targeted release at the colonic pH of 7.4, and enhanced bio-adhesion due to its macroporous structure. In the in vivo studies on mice, reduced proinflammatory cytokines, neutrophil infiltration, and epithelial damage in the colon were observed. In addition, the prebiotic properties of inulin within the hybrid system improved the intestinal microbiota composition by decreasing the abundance of harmful microorganisms (Proteobacteria, Romboutsia, and Enterococcus) and increasing the abundance of butyrate-producing bacteria, particularly Lachnospiraceae [120].

Hydrogel microspheres were produced from sodium alginate and PVA, and loaded with L. plantarum. To decrease the pore size and enable controlled release, sodium alginate, and PVA were crosslinked with ROS-sensitive 1,4-phenylenediboronic acid. The in vitro release rate of L. plantarum depended on the H₂O₂ concentration. In the presence of 0.1 mM H₂O₂, the release rate was over 73%, whereas in the presence of 0.05 and 0 mM H₂O₂ the release rate decreased to 56 and 37%, respectively. Regarding safety, hydrogel microspheres showed no toxicity in MRC-5 and L-929 cell models [121]. Probiotic was also delivered in a hybrid delivery system composed of ROS-reactive nanoparticles conjugated to the surface of EcN and encapsulated in a polynorepinephrine layer. Conjugated nanoparticles were fabricated from HA and polypropylene sulfide, allowing them to scavenge ROS. Encapsulation of EcN in the polynorepinephrine layer not only protected the probiotic from the external environment but also enabled better adhesion to the gut mucosa which extended the retention time in the gut and enabled better histopathological score. The hybrid system was effective in the prevention and treatment of colitis, as well as in balancing the disturbed gut microbiota in mice by increasing the abundance of beneficial bacteria (Muribaculaceae, Prevotellaceae_UCG-001) and decreasing the abundance of harmful bacteria (Desulfovibrionaceae) [122].

3.4.3 Composition and manufacturing

The most common smart hybrid bionanomaterials for treatment of IBD are combinations of hydrogels and nanoparticles. Poly(propylene sulfide) and HA were assembled into nanoparticles by linking them with ROS linker ethylenediamine. The mixture was dialyzed against water/methanol and lyophilized. The nanoparticles were conjugated to the surface of E. coli Nissle 1917 (EcN) and encapsulated in polynorepinephrine by mixing the bacteria and polymer [122].

The nanoemulsion method was used to synthesize nanoparticles loaded with 6-gingerol. The lipid phase consisted of medium-chain triglyceride, beeswax, and 6-gingerol, while the aqueous phase consisted of 3% (w/v) Tween 80 in water. The two phases were mixed, homogenized, sonicated, and added dropwise to a chitosan solution and then to octenyl succinic anhydride-modified resistant starch solution, which was in the end spray dried [119]. The double emulsion solvent evaporation method was used to synthesize PLGA nanoparticles loaded with the tripeptide KPV. The tripeptide (water phase) was mixed with PLGA (oil phase) forming the first emulsion. The emulsion was then sonicated and mixed with the second aqueous phase composed of PVA and chitosan which further stabilized the nanoparticles. In addition, the chitosan allowed the conjugation of HA through the formation of amide bonds. Finally, the nanoparticles were encapsulated in an alginate and chitosan hydrogel allowing delivery to the colonic lumen. The hybrid system exhibited a larger size than that of the individual nanoparticles [117].

Polycationic 2-(diethylamino)ethyl methacrylate gels were chemically crosslinked with tetraethylene glycol dimethacrylate. The gels were then mixed with TNF-α siRNA in PBS (pH 5.5), forming an electrostatic complex. After encapsulation, the gel was mixed with a poly(methacrylic acid-co-N-vinyl-2-pyrrolidone) hydrogel crosslinked with GRRRGK peptide and further lyophilized and milled [116]. A similar method was used for encapsulation of probiotic bacteria. Sodium alginate and PVA solutions were mixed with L. plantarum suspension and ultrasonicated to achieve a uniform distribution of bacteria. The mixture was then added dropwise into CaCl₂ solution (100 mM), allowing the Ca²⁺ ions to crosslink the polymers. The hydrogel was then immersed in a gelatin solution (3% (w/w)) and stirred, forming the hybrid material [121].

Synthesis of copper/olsalazine nanoneedles was done by the addition of CuCl₂ solution into olsalazine sodium, followed by heating the mixture at different temperatures. The precipitates were then centrifuged and washed with water. The resulting suspension was mixed with inulin, heated at 70°C, and subsequently left to cool at room temperature overnight [120].

3.5.1 Definition and properties

Unlike inanimate smart bionanomaterials, smart probiotics are live organisms, i.e., genetically engineered safe bacteria. Analogous to smart bionanomaterials, they can respond to external stimuli and deliver active ingredients with diagnostic and therapeutic potential in IBD. This is usually achieved by the inducible expression of the recombinant proteins with anti-inflammatory activities.

3.5.2 Active ingredients and their release

Probiotics can modify the gut microbiota and interact with the immune system at various levels. Their immunomodulatory properties have been associated with the alleviation of IBD symptoms and regulation of gut microbial homeostasis [17,123,124]. These effects can be further upgraded by the delivery of diagnostic and therapeutic recombinant proteins (Table 5).

Bacteriocin nisin is a food preservative and component of the nisin inducible expression system [125]. The addition of nisin to nisin-inducible species triggers the synthesis of recombinant therapeutic proteins. Nisin-responsive smart probiotics have been developed to exert their anti-inflammatory effects by neutralizing proinflammatory cytokines, producing anti-inflammatory substances, or decreasing oxidative stress. Specific TNF-α-binding affibodies were successfully displayed on the surface of the nisin-sensitive Lactococcus lactis [126,127,128], and engineered bacteria were tested in vitro, ex vivo, and in vivo. Binding of IL-6 by engineered, nisin-responsive Lc. lactis was successfully performed in vitro, and the bacteria neutralized up to 94% of the cytokines in a cell model [129]. IL-23-binding proteins secreted by the recombinant Lc. lactis following nisin induction were successful IL-23 blockers [130]. Superoxide dismutase (SOD) is an enzyme that converts ROS into molecular oxygen and hydrogen peroxide, thus providing a protective function. SOD administration can neutralize ROS and prevent disease progression. Two recombinant lactic acid bacteria (Lc. lactis and L. plantarum) expressing SOD following nisin induction reduced the number of ulcerations and the macroscopic damage score in experimental rats with colitis. In contrast, colonic infusion of bovine SOD resulted in poorer therapeutic outcomes, which confirms the added value of smart probiotics [131].

In addition to nisin, other substances have been tested for the controlled induction of recombinant protein synthesis for the treatment of IBD. EcN was engineered to express SOD and catalase, following induction with isopropyl-β-d-thiogalactoside. The strain was coated in chitosan and sodium alginate biofilm and showed a high superoxide scavenging rate (94%). The strain also improved the IBD symptoms in mice and increased the abundance of microbes that are important for maintaining homeostasis in the intestine, namely Lachnospiraceae_NK4A136 and Odoribacter [132]. EcN was also engineered to synthesize ketone body (R)-3-hydroxybutyrate. The biosynthetic genes were inserted into the genome of EcN under the control of low oxygen inducible promoters, which are suitable for anaerobic conditions in the gut. Engineered EcN produced 0.6 g/L (R)-3-hydroxybutyrate, colonized the gut of mice, and promoted the growth of Akkermansia spp. to over 31% of the microbiota, while with the nonengineered EcN, the proportion of Akkermansia spp. was only 2%. The final outcome of the study of engineered EcN in this mouse model was an improvement in IBD symptoms [133].

Expression of the anti-inflammatory cytokine IL-10 in Lc. lactis under the control of the groESL operon promoter was induced by stresses such as heat shock, UV radiation, and changes in pH [134]. EcN was engineered to express granulocyte macrophage-colony stimulating factor (GM‐CSF) under the control of the NO-responsive promoter hmp. The engineered strain exhibited anti-inflammatory properties in an in vitro CD model [135]. The NO-responsive promoter pNorV, the YebF secretion sequence, and the anti-inflammatory interferon lambda 1 (IFNL1) gene were inserted into the genome of EcN. When exposed to NO, the engineered EcN demonstrated anti-inflammatory and protective properties in vitro. It reduced the concentration of the proinflammatory cytokines such as IL-4, IL-5, IL-13, and IL-33, improved the tight junctions, and decreased the transcription level of inducible NO synthase by 84.1% in the inflamed primary intestinal epithelial cells [136]. Bacillus subtilis was engineered to synthesize IL-1 receptor antagonists and autolyze upon sensing unfavorable conditions in the colon of mice. IL-1 receptor antagonist was detected both in the intestinal washings and in the serum after intracolonic administration of the strain. Furthermore, the anti-inflammatory properties of engineered B. subtilis were demonstrated by improving the survival rate of mice after the administration of a lethal dose of bacterial lipopolysaccharide [137]. Bacteroides ovatus was engineered to produce TGF-β. The gene was cloned downstream of the xylanase promoter that was activated in the presence of xylan. Engineered bacteria, together with xylan, were administered to mice, resulting in improved stool consistency and reduced rectal bleeding. Inflammation was reduced by 38% compared with that in untreated mice [138]. Similarly, the xylan sensitive Bc. ovatus was engineered to produce keratinocyte growth factor-2 (KGF-2), a molecule beneficial for reducing IBD symptoms. Administration of engineered bacteria and xylan to mice with colitis resulted in a significant reduction in inflammation and healing of the epithelium [139]. Another advantage of the xylan-dependent system is the potential prebiotic activity of xylan. This approach consists of the simultaneous administration of probiotics and prebiotics with potential synergistic activity.

3.5.3 Engineering of smart probiotics

A variety of gene editing techniques are implemented in the engineering of smart probiotics [140]. Smart probiotics for the treatment and diagnosis of IBD are prepared using recombinant DNA technology in which a heterologous DNA fragment is inserted into a plasmid vector. This is then transformed into bacteria using an appropriate technique, such as electroporation (Figure 5) [141]. Engineered plasmids often contain genes that enable retention of the plasmid and selection of transformants (e.g., antibiotic resistance genes) [141]. Plasmid-introduced recombinant genes can be inserted into the bacterial chromosome or expressed from the plasmid, whereby the expression is driven by constitutive or inducible promoters that are triggered by external stimuli [142]. Chromosomal integration of DNA allows for better genetic regulation, gene conservation, stability, and lower metabolic burden [143].

Figure 5

Engineering of smart probiotics and synthesis of recombinant proteins. The image was created with Biorender.com.

The development of smart probiotics for the treatment of IBD has focused on the food-grade bacteria Lc. lactis and EcN, which are amenable to the genetic manipulation. Other species from the genera Lactobacillus (old nomenclature), Bifidobacterium, and Bacteroides have also been engineered for the treatment of IBD. Lc. lactis have been engineered with plasmids containing nisin and groESL inducible promoters and transformed with electroporation [126,127,128,129,130,131,134]. Standard cloning methods have also been used to engineer EcN [132,133,135]. To transform EcN and Bacillus subtilis, electroporation and simpler heat shock method were used [137]. Alternative modes of transformation are conjugation and transduction. In conjugation, bacteria share plasmid DNA through conjugative pili [144], whereas in transduction the DNA sharing is mediated by a bacteriophage [145]. Bacteroides ovatus was engineered by conjugation to produce TGF-β [138] and KGF-2 [139]. Conjugation with phage λ Red recombinase was used to insert pNorV-YebF-IFNL1 into the EcN genome [136].