Switchable artificial bacterial interface and methods of making and using same

Abstract

The application discloses a switchable artificial bacterial interface and a preparation method and application thereof, and belongs to the technical field of biological medicine. The interface comprises a bacterial body and a double-layer coating structure modified on the surface of the bacterial body, wherein an inner layer is a polydopamine layer, an outer layer is a polyethylene glycol layer, and the two layers are connected through an acid-sensitive connecting bond. Under a physiological pH condition, the outer layer polyethylene glycol layer endows the bacteria with hydrophilicity and a net neutral charge, and promotes mucus penetration; after reaching an acidic microenvironment at an infection site, the acid-sensitive connecting bond is broken, causing the outer layer to fall off, the exposed inner layer polydopamine layer forms a chemical bond with mucin, and focal retention is achieved. The application further provides a preparation method of the interface and application thereof in the preparation of a medicine for treating an infection at a mucus-covered site, and through a bond-breaking-bond-forming tandem reaction-mediated programmed function switching, dynamic interaction between a therapeutic bacterium and a mucus layer is realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a switchable artificial bacterial interface that regulates the dynamic interaction between bacteria and the mucus layer through a bond-breaking-bonding tandem reaction, its preparation method, and its application in treating infections at mucus-covered sites. Background Technology

[0002] Infections, especially those occurring in mucus-covered areas such as the respiratory tract, pose a serious threat to human health. Biofilms formed by the cross-linking of pathogen metabolites with the mucus layer can protect pathogens from antibiotic attack, but traditional antibiotic therapies are difficult to eradicate and easily exacerbate drug resistance.

[0003] Therapeutic bacteria, as living drugs, can intervene in infection through competitive rejection, secretion of active metabolites, and immune modulation. However, unmodified therapeutic bacteria face a double barrier: on the one hand, the thickened mucus layer at the infection site hinders bacterial penetration into the lesion; on the other hand, the acidic nature of the infection microenvironment and host colonization resistance lead to insufficient localization and short-term retention of bacteria at the lesion site, severely weakening the therapeutic effect.

[0004] Bacterial surface modification is an important strategy for regulating bacterial-environment interactions. Existing modification schemes can be divided into two categories based on their functional orientation: one aims to enhance adhesion by introducing reactive groups or depositing adhesive coatings on the bacterial surface to achieve lesion retention. However, this type of modification significantly increases the interaction between bacteria and the mucus layer, severely weakening their initial penetration ability. The other aims to enhance anti-fouling by reducing mucin adsorption through surface hydrophilization or charge shielding to promote mucus penetration. However, modified bacteria lack lesion retention ability and are easily cleared after reaching the lesion. All of the above strategies are static and unadjustable modifications. Their surface properties remain constant throughout the process of crossing the mucus barrier and reaching the lesion, and they cannot simultaneously satisfy the contradictory functional requirements of "mucus penetration" and "lesion retention" in time and space.

[0005] Although some studies have used adhesive or hydrophilic coatings to modify bacterial surfaces, simply layering the two on the same surface results in antagonistic effects between adhesion and antifouling properties, making it impossible to simultaneously achieve penetration and retention. Current technology has not yet disclosed a dynamic modification strategy that enables the same bacterial interface to switch programmed functions according to environmental changes. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to overcome the deficiencies of existing technologies, such as the difficulty of therapeutic bacteria penetrating the mucus layer and insufficient colonization of submucus infection sites. It provides a switchable bacterial interface and its preparation method, which can dynamically interact with the mucus layer through a bond-breaking and bond-forming tandem reaction, enabling the mucus penetration of therapeutic bacteria and long-term localization of submucus lesions. Simultaneously, it provides applications in microbial therapy to improve the efficacy of infection treatment.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A switchable artificial bacterial interface includes a bacterial body and a double-layer coating structure modified on the surface of the bacterial body; the double-layer coating structure includes an inner polydopamine layer and an outer polyethylene glycol layer, wherein the inner polydopamine layer and the outer polyethylene glycol layer are connected by acid-sensitive bonding bonds. The acid-sensitive linkage can be broken under acidic conditions, so that the outer polyethylene glycol layer detaches and exposes the inner polydopamine layer.

[0008] In some embodiments, the acid-sensitive linking bond is any one of an arylimide bond, an hydrazone bond, or an acetal bond.

[0009] In some embodiments, the molecular weight of the polyethylene glycol is from 1 kDa to 10 kDa.

[0010] In some embodiments, the inner polydopamine layer is formed by depositing dopamine at a concentration of 0.1 mg / mL to 0.5 mg / mL in a weakly alkaline buffer solution at pH 8.0-9.0.

[0011] In some embodiments, the outer polyethylene glycol layer is formed by modifying an acid-sensitive polyethylene glycol precursor at a concentration of 0.5 mg / mL to 10 mg / mL via an amide condensation reaction.

[0012] In some embodiments, the bacterial host is Staphylococcus epidermidis, Bacillus subtilis, lactic acid bacteria, or engineered therapeutic bacteria.

[0013] This invention also provides a method for preparing a switchable artificial bacterial interface, the method comprising the following steps: S1. Incubate bacteria with dopamine at a concentration of 0.1 mg / mL to 0.5 mg / mL in Tris-HCl buffer or phosphate buffer at pH 8.0-9.0 at 35℃-40℃ for 10-30 minutes. An inner polydopamine layer is deposited on the bacterial surface through the oxidative self-polymerization of dopamine. The bacteria are then separated by centrifugation and washing. S2. The acid-sensitive polyethylene glycol precursor and the bacteria obtained in step S1 are reacted in a buffer solution of pH 6.5-7.5 at 35℃-40℃ for 0.5-2 hours. The outer polyethylene glycol layer is connected to the inner polydopamine layer by amide condensation reaction. After centrifugation and washing, a switchable artificial bacterial interface is obtained.

[0014] The present invention also provides the use of the above-described switchable artificial bacterial interface in the preparation of medicaments for treating infections of mucus-covered sites.

[0015] In some embodiments, the infection at the mucus-covered site is a respiratory tract infection.

[0016] In some embodiments, the infection is a respiratory infection caused by Staphylococcus aureus or Pseudomonas aeruginosa, and the drug is a formulation for nasal or inhalation administration.

[0017] In the above technical solution, the "switchable" function is achieved through the following mechanism: Under physiological pH conditions, the outer polyethylene glycol layer covalently exists stably on the surface of the inner polydopamine layer. Its hydrophilic and near-electroneutral surface properties shield the non-specific interaction between the inner polydopamine layer and the mucin, allowing bacteria to maintain a high migration rate in the mucus layer. When bacteria reach the site of infection, the acidic conditions of the lesion microenvironment trigger the breakage of the acid-sensitive linkages, causing the outer polyethylene glycol layer to detach and the inner polydopamine layer to be exposed. The exposed polydopamine layer forms covalent bonds through Michael addition and / or Schiff base reactions with the amino and thiol groups in the mucin molecules via its catechol groups, achieving stable anchoring of bacteria at the lesion site. The above three stages of shielding, detachment, and anchoring occur sequentially, forming a complete chain of functional switching.

[0018] Based on the above disclosure, this application has the following beneficial effects: 1) Highly efficient mucus penetration: The outer polyethylene glycol layer imparts good hydrophilicity and net neutral surface charge to bacteria, significantly reducing the interaction between bacteria and mucin, and promoting the diffusion and penetration of bacteria in the mucus layer.

[0019] 2) Precise response to acidic environment: Acid-sensitive linkages can be efficiently broken in the acidic microenvironment of the infected site, causing the outer polyethylene glycol layer to detach and exposing the inner polydopamine layer, thus achieving a programmed switch of surface function.

[0020] 3) Stabilizing lesion colonization: After the inner polydopamine layer is exposed, it forms covalent bonds through Michael addition and / or Schiff base reaction with the amino and thiol groups in the mucin molecules via its catechol groups, which significantly enhances the bacteria's ability to attach and remain at the lesion site.

[0021] 4) Multiple therapeutic effects: Combining the pathogen elimination ability of the bacteria itself with the anti-inflammatory properties of polydopamine, it can effectively inhibit the growth of pathogenic strains and biofilm formation, while significantly reducing the expression of pro-inflammatory cytokines.

[0022] 5) High biocompatibility: The preparation process is mild, and the selected dopamine and polyethylene glycol concentrations have minimal impact on bacterial survival. It also avoids the safety hazards associated with genetic engineering and has excellent biocompatibility.

[0023] 6) Wide range of applications: Polydopamine-mediated surface modification is applicable to a variety of bacterial species, and the mucus layer is widely present in various human tissues, providing a multifunctional platform for developing bacterial therapies to treat different diseases.

[0024] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0025] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, some of the drawings in the following description are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1 This is a schematic diagram of the acid-sensitive polyethylene glycol precursor synthesis route and the switchable bacterial surface construction process described in this invention; wherein, Figure 1 a is an acid-sensitive polyethylene glycol precursor (NHS-CN-PEG) 2k A schematic diagram of the synthetic route and construction process of switchable bacterial surface (SPSD); Figure 1 b is a schematic diagram illustrating the mechanism by which switchable bacterial surfaces, mediated by bond breaking-bonding tandem reactions, are used for therapeutic bacterial mucus penetration and mucus-covered lesion localization.

[0027] Figure 2 This is a diagram showing the construction and structural characterization results of the switchable bacterial interface described in Embodiment 1 of the present invention; wherein, Figure 2 a represents the Fourier transform infrared (FTIR) spectra of polydopamine (PDA) nanoparticles and polyethylene glycol-modified polydopamine nanoparticles (PDA-CNPEG). Figure 2 b is a graph showing the survival colony count results of Staphylococcus epidermidis (Se) after incubation with different concentrations of dopamine hydrochloride; Figure 2 c is a graph showing the plate count results after Se was incubated with acid-sensitive polyethylene glycol precursors of different concentrations; Figure 2d is a flow cytometry (FCM) histogram of Cy5.5-labeled polyethylene glycol-modified Se, SD, and SPSD; Figure 2 e is a laser scanning confocal microscopy (LSCM) image, where green represents Se expressing yellow fluorescent protein (YFP), red represents Cy5.5-labeled polyethylene glycol, and the scale bar is 5 μm; Figure 2 f represents typical scanning electron microscope (SEM) images of Se, SD, and SPSD, with a scale bar of 1 μm; Figure 2 g represents typical transmission electron microscopy (TEM) images of Se, SD, and SPSD.

[0028] Figure 3 This is a graph showing the evaluation results of the mucus penetration performance mediated by the outer polyethylene glycol layer described in Example 2 of the present invention; wherein, Figure 3 a shows LSCM images of Se, SD, and SPSD mucins incubated with Cy5.5-labeled mucins. Green represents Se expressing YFP, and red represents Cy5.5-labeled mucins. The scale bar is 5 μm. Figure 3 b represents the corresponding FCM analysis result; Figure 3 c is a digital photograph of Coomassie Brilliant Blue labeled bacteria penetrating an artificial slime layer; Figure 3 d represents the penetration distance-time curve; Figure 3 e is a statistical chart of the area under the curve corresponding to 10 hours; Figure 3 f shows a 3D LSCM Z-axis image of bacteria penetrating the mucus layer of human goblet cells HT29-MTX-E12 (E12) and a cross-sectional image under the same Z-axis coordinates. Green represents Se expressing YFP. The scale bar is 5 μm. Figure 3 g is a statistical chart showing the number of bacteria entering the mucus layer of E12 cells; Figure 3 h represents the surface Zeta potential values ​​for Se, SD, and SPSD; Figure 3 i represents the water contact angle measurement results of Se, SD, and SPSD spread on the glass slide.

[0029] Figure 4 This is a diagram showing the verification results of acid-triggered aryl imine cleavage and polyethylene glycol desorption as described in Example 3 of the present invention; wherein, Figure 4 a is a schematic diagram of the mechanism of acid-sensitive aryl imine cleavage and polyethylene glycol desorption in phosphate-buffered saline (PBS) at pH 5.6; Figure 4 b is a stacked diagram of high-performance liquid chromatography (HPLC) signal peaks of polyethylene glycol release from SPSD at a specified time point; Figure 4 c represents the cumulative release rate curves of polyethylene glycol from PSD and SPSD at different pH conditions and time points; Figure 4 FCM analysis results of Cy5.5 labeled PSD and SPSD after soaking in PBS at pH 5.6 for different times; Figure 4 e represents the corresponding LSCM visualization image, green represents Se expressing YFP, red represents polyethylene glycol labeled Cy5.5, and the scale bar is 5 μm.

[0030] Figure 5 This is a graph showing the evaluation results of the mucus binding performance mediated by the exposure of the inner polydopamine layer as described in Example 4 of the present invention; wherein, Figure 5 a is a schematic diagram of the mechanism by which poly(ethylene glycol) covalently binds to mucin after acid-triggered detachment from PBS at pH 5.6; Figure 5 b is a visualization image of LSCM after bacteria pretreated at pH 5.6 and incubated with Cy5.5-labeled mucin. Green represents Se expressing YFP, and red represents Cy5.5-labeled mucin. The scale bar is 10 μm. Figure 5 c represents the corresponding FCM analysis result; Figure 5 d is a digital photograph of bacteria penetrating the artificial slime layer after pretreatment at different pH levels; Figure 5 e is a statistical chart of penetration distance after 10 hours of incubation; Figure 5 f is a 3D LSCM Z-axis tomographic image of bacteria penetrating the mucus layer of E12 cells after pretreatment with different pH values. Green represents Se expressing YFP, and red represents the mucus layer labeled with rhodamine. The scale bar is 5 μm. Figure 5 g is a statistical chart of the number of bacteria that penetrated the mucus layer of E12 cells; Figure 5 h represents the Fourier transform infrared (FTIR) spectrum of the freeze-dried product of polydopamine nanoparticles incubated with mucin.

[0031] Figure 6 This is a graph showing the in vitro evaluation results of pathogen rejection, biofilm ablation, and inflammation relief as described in Example 5 of the present invention; wherein, Figure 6 a represents the growth curves of co-incubation of Staphylococcus aureus (Sa) with different proportions of Se; Figure 6 b represents the corresponding plate counting result; Figure 6 c represents the quantitative analysis result of crystal violet staining of Sa biofilm in the presence of Se supernatant; Figure 6 d is a typical digital photograph of Sa biofilm stained with crystal violet or Congo red after different treatments; Figure 6 e represents the quantitative analysis results of the presence of Sa biofilm in SPSD supernatant of different concentrations; Figure 6 f to Figure 6 h represents the expression levels of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in macrophages after different treatments.

[0032] Figure 7 This is a graph showing the evaluation results of the therapeutic value of Staphylococcus aureus-induced chronic respiratory tract infection as described in Example 6 of the present invention; wherein, Figure 7 a is a schematic diagram of the in vivo validation experiment design of SPSD in a mouse model of Sa-induced chronic respiratory infection; Figure 7 b is the curve of mouse weight change after treatment; Figure 7 c represents the Sa count in the lung tissue of mice in each group on day 7; Figure 7 d shows typical fluorescence in situ hybridization (FISH) staining images of lung tissue after different treatments. Green represents FAM-labeled Sa-specific DNA sequences, and blue represents DAPI-labeled cell nuclei. The scale bar is 100 μm. Figure 7 e and Figure 7 f represents the expression levels of pro-inflammatory cytokines TNF-α and IL-6 in mouse serum; Figure 7 g and Figure 7 h represents hematoxylin-eosin (H&E) staining and myeloperoxidase (MPO) staining images of lung tissue after different treatments. Red and blue arrows represent tissue damage and inflammatory cell infiltration, respectively. The scale bar is 100 μm. Figure 7 i represents the histopathological score based on H&E staining.

[0033] Figure 8 This is a graph showing the evaluation results of the therapeutic value of Pseudomonas aeruginosa-induced acute respiratory tract infection as described in Example 7 of the present invention; wherein, Figure 8 a shows the growth curves of Pseudomonas aeruginosa PA14 in the presence of SPSD supernatant at different volume ratios. Figure 8 b shows the plate count results after PA14 was co-incubated with 10% SPSD supernatant for 12 hours; Figure 8 c represents the corresponding digital photo taken on a tablet. Figure 8 d is a digital photograph of PA14 biofilm after being exposed to 10% SPSD supernatant for 24 hours and then stained with crystal violet or Congo red. Figure 8 e is a schematic diagram of the experimental design for evaluating the therapeutic value of SPSD in a mouse model of PA14-induced acute respiratory infection; Figure 8 f represents the change in body weight of the mice after treatment; Figure 8 g represents the Se count in bronchoalveolar lavage fluid (BALF); Figure 8 h represents the PA14 count in the lung tissue of mice in each group on day 4; Figure 8 i represents the level of TNF-α in mouse serum; Figure 8 j and Figure 8 k represents H&E staining and MPO staining images of lung tissue after different treatments. Red and blue arrows represent tissue damage and inflammatory cell infiltration, respectively. The scale bar is 100 μm. Figure 8 l represents the histopathological score based on H&E staining. Detailed Implementation

[0034] To better describe the present invention, specific embodiments are provided below for further explanation. Unless otherwise specified, the methods in the following embodiments are conventional methods.

[0035] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field; unless otherwise specified, the reagents or materials described are all from commercial sources.

[0036] The following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental conditions not specifically stated in the examples are generally performed under conventional conditions or as recommended by the reagent company; reagents, consumables, etc., used in the following examples are commercially available unless otherwise specified.

[0037] It should be noted that the bilayer modification described in this invention is not a simple physical superposition of inner and outer layer materials. If the hydrophilicity or grafting density of the outer polyethylene glycol layer is insufficient, it cannot effectively shield the interaction between the inner polydopamine and mucin, severely limiting the bacteria's ability to penetrate the mucus layer. If the pH response of the acid-sensitive linkage does not match the infection microenvironment, the outer polyethylene glycol layer may detach prematurely before reaching the lesion, or respond slowly at the lesion site, failing to achieve timely anchoring. Therefore, the deposition concentration of the inner polydopamine layer, the molecular weight of the outer polyethylene glycol layer, the type of acid-sensitive linkage, and the reaction conditions must be synergistically matched to achieve a programmed switch of "shielding during penetration and exposure upon arrival." The conditions described in the following embodiments are preferred solutions for achieving the above-mentioned synergistic effects.

[0038] The switchable artificial bacterial interface described in this invention is constructed using a universal process of "inner layer polydopamine deposition – outer layer polyethylene glycol acid-sensitive linking," applicable to various bacteria with therapeutic activity, such as Staphylococcus epidermidis, Bacillus subtilis, and lactic acid bacteria. Specifically, bacteria are incubated with dopamine at a concentration of 0.1 mg / mL to 0.5 mg / mL in Tris-HCl buffer or phosphate buffer at pH 8.0-9.0 at 35°C-40°C for 10-30 minutes. An inner PDA layer is formed through oxidative self-polymerization. After centrifugation and washing, the PDA layer is reacted with an acid-sensitive PEG precursor (such as NHS ester-terminated arylimine-functionalized PEG, molecular weight 1 kDa to 10 kDa) at a concentration of 0.5 mg / mL to 10 mg / mL at 35°C-40°C for 0.5-2 hours. An outer PEG layer is then linked through an amide condensation reaction, followed by centrifugation and washing to obtain the final product.

[0039] The technical solution and effects of the present invention will be further illustrated below through specific embodiments.

[0040] Example 1 Construction and characterization of a switchable artificial bacterial interface (SPSD) This embodiment follows Figure 1 The synthetic route and construction process are shown to prepare a switchable artificial bacterial interface and characterize its system structure.

[0041] 1.1 Material Preparation Strains: Staphylococcus epidermidis ( Staphylococcus epidermidis (ATCC 12228), a bacterium modified by bioengineering to express yellow fluorescent protein (YFP) and exhibiting erythromycin resistance, was used as a model for therapeutic bacteria. Main reagents: dopamine hydrochloride, 4-formylbenzoic acid, and methoxy polyethylene glycol amine (NH2-PEG). 2k (with an average molecular weight of approximately 2000), N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and Cy5.5-NHS ester.

[0042] 1.2 Acid-sensitive polyethylene glycol precursor (NHS-CN-PEG) 2k Synthesis of ) 4-Formylbenzoic acid (0.1 mmol) was reacted with NH2-PEG 2k Dissolved in anhydrous methanol, the mixture was stirred at room temperature for 24 hours, and an aryl imine bond was formed via nucleophilic addition-dehydration reaction to give the intermediate HOOC-CN-PEG. 2k The solvent was removed by rotary evaporation, and the crude product was purified by column chromatography. Subsequently, HOOC-CN-PEG was... 2k NHS and EDC were dissolved in anhydrous dichloromethane and activated at room temperature for 6 hours. After washing with water, drying, precipitation, and filtration, the final product NHS-CN-PEG was obtained. 2k . By nuclear magnetic resonance hydrogen spectrum ( 1 ¹H NMR analysis confirmed that the grafting rate of the aryl imine group was 94.3%.

[0043] 1.3 Coating with an inner polydopamine (PDA) layer (Preparation of SD) Staphylococcus epidermidis (Se) in the logarithmic growth phase was washed twice with phosphate-buffered saline (PBS, pH 7.4), resuspended in Tris-HCl buffer (10 mM, pH 8.5) containing 0.2 mg / mL dopamine hydrochloride, and incubated in a shaker at 37°C for 15 minutes in the dark. Immediately after incubation, the bacteria were washed three times with pre-chilled PBS (pH 7.4) by centrifugation (8000 rpm, 3 minutes) to obtain PDA-coated bacteria (SD). Figure 2As shown in b, dopamine cytotoxicity was assessed by bacterial plate counting. Within the concentration range of 0.1 to 1 mg / mL, the survival rate of Se did not decrease at 0.2 mg / mL, while it decreased to 33.8% at 0.5 mg / mL. Therefore, 0.2 mg / mL was selected as the preferred preparation concentration.

[0044] 1.4 Connection of the outer PEG layer (preparation of SPSD) The above SD was resuspended in a solution containing 1 mg / mL NHS-CN-PEG. 2k The PEG was reacted with PBS (pH 7.4) at 37°C with gentle shaking for 1 hour. After the reaction, the bacteria were washed three times with PBS to remove unreacted PEG derivatives, resulting in a bilayer coated bacteria (SPSD). To verify the ligation effect, Cy5.5 fluorescently labeled NHS-CN-PEG was used. 2k (NHS-CN-PEG) 2k SPSDs were prepared under the same conditions (Cy5.5). Figure 2 As shown in c, cytotoxicity assessments revealed that, within the concentration range of 0.1 to 10 mg / mL, it was incompatible with NHS-CN-PEG. 2k After 1 hour of incubation, no decrease in colony forming units (CFU) was observed, indicating that the effect of polyethylene glycolation on bacterial survival was negligible.

[0045] 1.5 Material Characterization Fourier transform infrared (FTIR) spectroscopy showed that, compared to pure PDA nanoparticles, polyethylene glycol-modified PDA nanoparticles exhibited higher activity at 2880 cm⁻¹. -1 and 1160 cm -1 The CH and COC stretching vibration peaks of PEG appear at this location (see...). Figure 2 (a) indicates that the PEG grafting was successful.

[0046] Flow cytometry (FCM) showed that, compared with Se and SD, the fluorescence peak of SPSD shifted significantly to higher intensities (see [link to FCM]). Figure 2 d) Quantitative analysis showed that approximately 92.5% of the bacteria successfully ligated PEG. Laser scanning confocal microscopy (LSCM) revealed highly colocalized green fluorescence (YFP) of the bacteria with red fluorescence of Cy5.5 (see [link to scalar model]). Figure 2 e).

[0047] Scanning electron microscopy (SEM) revealed that natural Se exhibited a smooth, grape-like clustered surface typical of Staphylococcus species, while the surfaces of SD and SPSD became rough, with visible granular PDA deposits (see [link to SEM]). Figure 2 f).

[0048] Transmission electron microscopy (TEM) revealed a denser, darker layer surrounding the bacterial cells of SD and SPSD cells (see...). Figure 2 g), further confirming the successful encapsulation of the PDA.

[0049] Stability tests showed that when Cy5.5-labeled SPSD was cultured continuously in LB medium at 37°C for 4 hours, the PEG fluorescence, although gradually diluted with bacterial proliferation, was still significantly higher than that of unmodified Se, indicating that the surface modification was gradually diluted rather than rapidly lost during cell division.

[0050] In addition, it was determined that the zeta potential of the switchable artificial bacterial interface prepared above was -25 mV to -15 mV under physiological pH conditions, and the water contact angle was 20° to 30°; after incubation for 24 hours under acidic conditions of pH 5.0-6.5, the cumulative release rate of the outer polyethylene glycol was 30% to 50%.

[0051] Example 2: Evaluation of the outer PEG-mediated mucus permeability Equal amounts of Se, SD, and SPSD were incubated with 10 μg / mL Cy5.5-labeled mucin at 37°C for 1 hour. After washing with PBS, the amount of mucin adsorbed on the bacterial surface was analyzed by LSCM and FCM. Figure 3 As shown in a, the LSCM image shows that the red fluorescence on the SPSD surface is the weakest, while the red fluorescence on the SD surface is the strongest; Figure 3 FCM quantitative analysis of b confirmed that the fluorescence intensity peak of SPSD was at the lowest value, indicating that its mucin adsorption capacity was the weakest, while that of SD was the strongest.

[0052] An artificial mucus layer composed of 40 mg / mL commercial porcine gastric mucin was constructed in a clear vial. Equal volumes of Coomassie Brilliant Blue stained Se, SD, and SPSD were carefully spread on the surface of the mucus layer. The vertical penetration distance was observed and measured at regular intervals over 10 hours. Figure 3 As shown in the digital photos of c, SPSD penetrates the deepest among all groups; Figure 3 Quantitative results showed that SPSD had the fastest penetration speed, reaching 14.2 mm in the artificial slime layer after 10 hours, compared to 9.1 mm for unmodified bacteria (Se) and 3.5 mm for bacteria modified only with a polydopamine monolayer (SD). These results indicate that polydopamine monolayer deposition hinders bacterial diffusion in the slime layer, while the bilayer structure provided by this invention, through the shielding of polydopamine adhesion by the outer polyethylene glycol layer, achieves a penetration distance more than four times that of the SD group.

[0053] Further validation was performed using a dense native mucus layer formed by culturing human goblet cells (HT29-MTX-E12) in Transwell chambers for 5 days. Equal amounts of Se, SD, and SPSD were added to the upper layer of the chamber, and after incubation for 15 minutes, Z-axis scanning was performed using LSCM. Figure 3 As shown in image e, the 3D reconstructed image reveals that SPSD is distributed in the deepest layer of the mucus layer, exhibiting the brightest YFP fluorescence at the same Z-axis coordinate. Subsequently, unpenetrated bacteria were gently washed away with PBS, and the mucus layer was dissolved with 0.5% N-acetylcysteine ​​(NAC). The penetrated bacteria were then collected for plate counting. Figure 3 As shown in f, the amount of SPSD that penetrates is 6.0 times that of unmodified Se and 20 times that of SD modified only with polydopamine, indicating that the shielding of the outer polyethylene glycol layer from the adhesive function of polydopamine is the key to achieving efficient penetration of natural mucus.

[0054] The measured zeta potential of SPSD was -21.4 ± 1.8 mV (Se was -29.9 ± 0.2 mV, and SD was -32.6 mV). Figure 3 As shown in h, the presence of the PEG layer reduces the absolute value of the surface potential. Microbial adhesion hydrocarbon (MATH) experiments showed that the MATH value of SPSD was 49% higher than that of Se. Water contact angle measurements showed that the contact angle of Se was 32.9° ± 3.6°, while that of SPSD decreased to 24.7° ± 5.5° (see h). Figure 3 i). The above results indicate that PEG modification enhances the hydrophilicity of the bacterial surface, reduces the absolute value of the surface charge, and decreases the interaction with the mucus layer.

[0055] The results of the comparison of bacterial surface properties and mucus interactions in each group are summarized in Table 1.

[0056] Table 1 Comparison of bacterial surface properties and mucus interactions in each group 1 Because the PDA layer on the SD surface has a granular and rough texture, the droplets spread unevenly, and a stable and repeatable contact angle value could not be obtained.

[0057] Example 3: Acidic microenvironment triggers the separation of outer PEG layer To verify acid responsiveness, non-pH-responsive control bacterial PSDs were first prepared using indestructible NHS-PEG. 2k Replace NHS-CN-PEG 2kModifications were performed. LSCM and FCM confirmed the successful construction of PSD, and SEM and TEM showed that PSD had a surface roughness structure similar to SPSD. Water contact angle measurements showed that PSD was 22.6° ± 2.5°, comparable to SPSD, ensuring comparability for subsequent experiments.

[0058] like Figure 4 As shown in a, when SPSD reaches H-rich + When H is infected with lesions + Ions attack the polar C=N double bond carbon atom of the aryl imine bond, triggering a nucleophilic attack that breaks the aryl imine bridge, allowing PEG to separate from SPSD. Equal volumes of SPSD and PSD were placed separately into dialysis bags (molecular weight cutoff 30 kDa), immersed in PBS buffer at pH 7.4 or pH 5.6, and incubated at 37°C. Dialysis fluid was collected at different time points, and the released PEG content was detected by high-performance liquid chromatography (HPLC). Figure 4 b and Figure 4 As shown in Figure c, at pH 5.6, SPSD releases 5.0% PEG within 1 hour, with a cumulative release of up to 44.5% within 24 hours; while PSD only releases 5.3% cumulatively within 24 hours under the same conditions. At pH 7.4, the cumulative releases of SPSD and PSD within 24 hours are only 3.2% and 5.0%, respectively, indicating that SPSD has excellent lesion acid responsiveness.

[0059] SPSD and PSD were further prepared using Cy5.5-labeled PEG. The bacteria were resuspended in PBS at pH 7.4 or pH 5.6 and incubated at 37°C. Samples were taken at different time points, and the average fluorescence intensity on the bacterial surface was detected by FCM and observed by LSCM. Figure 4 As shown in d, the FCM results confirmed that under pH 5.6 conditions, the fluorescence peak of SPSD gradually shifted to a lower intensity in the first 2 hours and was almost completely stripped away after 6 hours. Figure 4 The LSCM images of e show that the red fluorescence intensity on the SPSD surface decreases rapidly over time and is almost invisible after 12 hours, while the fluorescence intensity on the PSD surface does not change much over 12 hours, indicating that the breaking of the acid-sensitive aryl imine bond successfully triggered the separation of the PEG outer layer.

[0060] Table 2 shows the comparison of the cumulative PEG release rates of PSD and SPSD under different pH conditions.

[0061] Table 2 Comparison of cumulative PEG release rates (%) between PSD and SPSD As shown in Table 2, the cumulative PEG release of SPSD at pH 5.6 (44.5% in 24 hours) was approximately 14 times that at physiological pH (pH 7.4, 3.2% in 24 hours), while the release of PSD in the non-responsive control group under the same acidic conditions was only 5.3%. These differences indicate that the acid-sensitive aryl imine bond endows the bilayer structure with acid-responsive selectivity.

[0062] Example 4: Mucus binding capacity mediated by inner PDA exposure After the PEG layer separates under acidic conditions, the exposed inner PDA layer can reform chemical bonds with the surrounding mucin. For example... Figure 5 As shown in a, SPSD and PSD were pretreated in PBS at pH 5.6 for 12 hours to trigger the PEG layer separation of SPSD, followed by co-incubation with Cy5.5-labeled mucin. Figure 5 LSCM analysis showed that strong mucin fluorescence reappeared on the pretreated SPSD surface, with an intensity comparable to that of SD, while no obvious mucin adsorption was observed on the PSD surface. Figure 5 FCM analysis of c further confirmed that the fluorescence peak of SPSD shifted to higher intensity after treatment with pH 5.6, and its mucin adsorption capacity was comparable to that of SD, while Se and PSD showed no significant changes.

[0063] SPSD and PSD pretreated at pH 7.4 or pH 5.6 for 12 hours were stained with Coomassie Brilliant Blue, spread on the surface of the artificial mucus layer, and incubated for 10 hours to observe penetration. Figure 5 The digital photograph shows that the SPSD treated with pH 5.6 remained mainly on the surface of the mucus layer; Figure 5 Quantitative results showed that the penetration distance of SPSD pretreated at pH 5.6 decreased to 7.6 mm, a reduction of 9.5 mm compared to the pH 7.4 pretreated group (17.1 mm). The penetration distances of the non-responsive control group PSD after pretreatment at pH 5.6 and pH 7.4 were 17.3 mm and 17.2 mm, respectively, with no significant difference. These results indicate that polydopamine exposure caused by the breaking of acid-sensitive bonds is the key factor triggering this in-situ anchoring effect.

[0064] Further verification was performed using the natural mucus layer derived from E12 cells (labeled red with a wheat germ lectin-rhodamine conjugated probe). Pretreated bacteria were co-incubated with the mucus layer, and observation was performed using three-dimensional LSCM. Figure 5 As shown in f, SPSD pretreated at pH 7.4 can diffuse deeply into the mucus layer, while SPSD pretreated at pH 5.6 only remains on the surface of the mucus layer; PSD can diffuse deeply under both pH conditions. Figure 5Bacterial counts after NAC extraction showed that the number of penetrating bacteria in the SPSD pretreated group at pH 5.6 was reduced by 6.2 times compared to the group pretreated at pH 7.4, while there was no significant change in the PSD group.

[0065] Validation of the chemical binding mechanism: Purified PDA nanoparticles and mucin were co-incubated in PBS for 1 hour, the precipitate was collected by centrifugation, lyophilized, and then analyzed by FTIR. Figure 5 As shown in h, compared with the spectra of pure PDA and pure mucin, the reaction products are at approximately 1100 cm⁻¹. -1 The enhanced CN stretching vibrations nearby confirm that a covalent bond has been formed between PDA and mucin via Michael addition or Schiff base reaction. Simultaneously, the characteristic signal of PDA almost disappears, while at approximately 3400 cm⁻¹... -1 The presence of strong OH stretching vibration signals nearby indicates that the deposition of polyhydroxy mucin on the PDA surface produces a shielding effect.

[0066] Example 5: In vitro functional verification—pathogen inhibition, biofilm ablation, and immune modulation Staphylococcus epidermidis (Se) and Staphylococcus aureus RN4220 (Sa, expressing mCherry and chloramphenicol resistant) were co-cultured at different ratios (Se:Sa = 1:10 to 10:1). The inhibitory effect of Se on Sa was assessed by monitoring the fluorescence intensity of mCherry. Figure 6 As shown in Figure a, the growth of Sa is significantly inhibited as the Se ratio increases, with the most significant inhibition occurring when Se:Sa = 10:1. Figure 6 Bacterial plate counts showed that when Se:Sa = 1:1, the survival rate of Sa decreased by 46.3%; when Se:Sa = 10:1, the survival rate of Sa dropped to only 19.8%, indicating that Se can significantly inhibit pathogens in a dose-dependent manner.

[0067] Culture supernatants containing Se, SD, SPSD, and free PEG and PDA nanoparticles were collected. Sa was co-cultured with these supernatants for 48 hours. Biofilm biomass (OD) was determined using crystal violet staining. 595nm ).like Figure 6 As shown in c, the supernatants of Se, SD, and SPSD can effectively inhibit the formation of Sa biofilm, with an average inhibition rate as high as 87.4%, while free PEG and PDA nanoparticles themselves have no such effect. Figure 6 Digital photographs of crystal violet and Congo red staining of d also showed similar results. Figure 6 Treatment with different concentrations of SPSD supernatant showed that the inhibitory effect of 15% supernatant was comparable to that of 75% supernatant, suggesting that the biofilm ablation function is related to the active substances secreted by Se, and that no obvious dose dependence was observed.

[0068] An inflammation model was established using lipopolysaccharide (LPS) stimulation of J774A.1 macrophages. Cells were treated with LPS in combination with Se, SD, SPSD, or PDA nanoparticles, and the levels of TNF-α, IL-6, and IL-1β in the cell supernatant were detected by ELISA. Figure 6 f to Figure 6 As shown in h, no significant anti-inflammatory effect was observed after Se incubation, and the cytokine levels were comparable to those in the LPS group; however, the groups containing PDA (SD, SPSD, and PDA nanoparticles) significantly reduced the secretion of all three pro-inflammatory factors, indicating that the PDA layer conferred anti-inflammatory function upon the bacteria.

[0069] The results of the above in vitro functional evaluations are summarized in Table 3.

[0070] Table 3 Summary of In Vitro Functional Evaluation Example 6: Evaluation of in vivo therapeutic effect—a mouse model of chronic Staphylococcus aureus pneumonia The in vivo validation experiment design in this embodiment is as follows: Figure 7 As shown in figure a. BALB / c mice were randomly divided into a healthy group, a PBS group, a Se group, an SD group, a PSD group, and a SPSD group. Except for the healthy group, the mice in the other groups received 1×10⁻⁶ ppm intranasally on day 0. 7 A pneumonia model was established using CFU-containing Staphylococcus aureus (Sa). From day 1, mice in the treatment group were administered 1×10⁻⁶ CFU intranasally daily. 8 CFU was treated with the corresponding bacterial preparation (the PBS group received an equal volume of PBS) for 6 consecutive days, and samples were collected and analyzed on the 7th day.

[0071] It should be noted beforehand that the in vivo antibacterial effect is affected by the enrichment effect of SPSD lesions and the host immune synergistic clearance mechanism, and differs from the results of in vitro co-culture.

[0072] Record the mouse's weight daily, such as Figure 7 As shown in b, the body weight of mice in the PBS group continued to decrease, decreasing by approximately 11% by day 7; the body weight of mice in all treated bacterial groups remained stable, approximately constant compared to the body weight of healthy mice. Mice were sacrificed on day 7, and left lung tissue homogenates were collected for bacterial plate counting. Figure 7 c showed that the SPSD treatment group had the lowest number of Sa in the lungs, with a relative survival rate of only 0.07%, which was 404.8 times, 30.1 times, and 14.4 times lower than the Se, SD, and PSD groups, respectively. The SD group had difficulty penetrating the mucus layer to reach the lesion due to the adhesiveness of polydopamine, while the PSD group lacked an acid-responsive shedding mechanism and could not remain in the lesion. SPSD achieved local enrichment of the treated bacteria in the lesion through a programmed switching between penetration and retention.

[0073] Fluorescence in situ hybridization (FISH) was performed on lung tissue sections using a specific FAM-labeled probe targeting Sa16S rRNA. For example... Figure 7 As shown in d, the PBS and Se groups showed strong green fluorescence signals; the fluorescence intensity of the SD and PSD groups was moderately reduced; while the SPSD group showed almost no Sa signal, close to the level of healthy mice, indicating that it has pathogen clearance capabilities.

[0074] The levels of TNF-α and IL-6 in mouse serum were detected by ELISA. Figure 7 e and Figure 7 f showed that the SPSD-treated mice had the lowest levels of both pro-inflammatory factors, with TNF-α reduced by 91.5% and IL-6 reduced by 20.4% compared to the PSD group; TNF-α levels were inhibited by 98%, comparable to those in healthy mice. H&E staining and MPO immunohistochemical staining were performed on the right lung tissue. Figure 7 In the PBS group, severe inflammatory cell infiltration, alveolar wall thickening, and structural damage were observed in the lung tissue; the SPSD group showed lung tissue morphology similar to the healthy group, with the least inflammatory cell infiltration and the mildest epithelial damage. Figure 7 The histopathological scores of i showed that the SPSD group had the highest scores, which were 23.3% higher than those of the PSD group. Figure 7 MPO staining at h showed that the SPSD group had the fewest positive cells, indicating the mildest local oxidative stress and inflammatory response.

[0075] The in vivo therapeutic effects of each group are summarized in Table 4.

[0076] Table 4. In vivo treatment efficacy of Staphylococcus aureus chronic pneumonia (Day 7) Example 7: Evaluation of in vivo therapeutic effect – Mouse model of acute pneumonia caused by Pseudomonas aeruginosa First, in vitro experiments were conducted to verify the inhibitory effect of SPSD on Pseudomonas aeruginosa PA14 and its biofilm clearance ability. PA14 was co-incubated with SPSD culture supernatant at volume ratios of 10%, 20%, and 30%, and the OD values ​​were recorded. 600nm Values ​​are used to plot growth curves. For example... Figure 8 As shown in Figure a, all proportions of SPSD supernatant significantly inhibited PA14 proliferation. Further co-incubation of PA14 with 10% SPSD supernatant for 12 hours resulted in a significant decrease in PA14 survival rate to 21.6% (as indicated by plate counting). Figure 8 b and Figure 8 c). The removal effect of SPSD on PA14 biofilm was assessed by crystal violet and Congo red staining. The results showed that after 24 hours of co-incubation, the PA14 biofilm almost completely disappeared. Figure 8 d).

[0077] To verify the in vivo applicability of the above in vitro effects, a mouse model of acute respiratory infection with Pseudomonas aeruginosa PA14 was established. The experimental design of this embodiment is as follows: Figure 8 As shown in e, the specific grouping and processing schemes are as follows: Day 0: Nasal instillation 2×10 7 CFUs PA14 (expressing GFP and resistant to gentamicin) were administered intranasally for 3 consecutive days with PBS, Se, SD, PSD, or SPSD (Se dosage was 1×10⁻⁶ each time). 8 CFUs / time), and euthanasia was performed on day 4 for sampling.

[0078] like Figure 8 As shown in f, mice in the PBS group experienced a significant weight loss of 8.35% on day 4, while the SPSD group showed a slight weight gain of 3.43%, indicating that the infection was alleviated. Figure 8 Se counts in BALF of g showed that the SPSD group had 14.1-fold, 3.3-fold, and 1.3-fold increases in Se, SD, and PSD compared to the SPSD, SD, and PSD groups, respectively. These results indicate that SPSD has significantly better lesion retention capacity than PSD (which only has penetrating ability) and SD (which lacks penetrating ability).

[0079] Figure 8 The PA14 count in lung tissue at h showed that the relative survival rate in the SPSD group was only 0.8%, which was 2.5 times, 2.9 times, and 2.8 times lower than that in the Se, SD, and PSD groups, respectively. Figure 8 Serum TNF-α levels in the SPSD group were reduced by 52.7%, 42.3%, and 38.1% compared to the Se, SD, and PSD groups, respectively. Figure 8 j and Figure 8 H&E and MPO staining of k showed that the SPSD group had the least tissue damage, immune cell infiltration, and oxidative stress. Figure 8 The highest histopathological score was for L (15.3). These results indicate that SPSD has application value in the treatment of PA14-induced acute respiratory tract infections.

[0080] The in vivo therapeutic effects of each group are summarized in Table 5.

[0081] Table 5. In vivo treatment efficacy of Pseudomonas aeruginosa acute pneumonia (Day 4) In summary, this invention successfully constructs an environmentally adaptive artificial bacterial interface through a bilayer coating structure linked by acid-sensitive aryl imine bonds. By employing a "penetration-anchoring" tandem reaction, it effectively overcomes the technical bottleneck of therapeutic bacteria's inability to penetrate the mucus barrier and insufficient colonization at the infection site, achieving spatiotemporally controllable enrichment of therapeutic bacteria at the infection site. The preparation method is simple and mild, with minimal impact on bacterial survival, exhibiting good biocompatibility and universality. In the treatment of respiratory infections, this interface can synergistically achieve pathogen elimination, biofilm ablation, and inflammation relief, providing a new technical solution for the development of next-generation live bacterial therapies.

[0082] It should be noted that the mucosal surfaces of various tissues, such as the respiratory tract, gastrointestinal tract, and genitourinary tract, are covered with a mucus layer mainly composed of mucin, and pathogen infection is often accompanied by the formation of a local acidic microenvironment. The switchable artificial bacterial interface described in this invention, through the mechanism of promoting mucus penetration with an outer layer of polyethylene glycol and anchoring in response to an acidic environment with an inner layer of polydopamine, relies on the physical barrier properties of the mucus layer and the acidic characteristics of the infection site, rather than the anatomical structure of a specific site. Therefore, the above-mentioned dual-layer structure and programmed switching mechanism are applicable to various sites with mucus coverage and acidic infection microenvironments.

[0083] Those skilled in the art will understand that the oxidative self-polymerization of polydopamine and the amide condensation modification of polyethylene glycol are mild physicochemical modification methods suitable for various bacteria with surface amino groups; aryl imine bonds, hydrazone bonds, and acetal bonds are all known acid-sensitive covalent bonds in the art, and their cleavage depends on H under acidic conditions. + The polar bonds remain stable even under physiological pH conditions. Therefore, by reasonably adjusting parameters such as dopamine deposition concentration, polyethylene glycol molecular weight, and acid-sensitive polyethylene glycol precursor concentration in the technical solution of this invention, or by replacing aryl imine bonds with hydrazone bonds or acetal bonds, or by using other therapeutic bacteria such as Bacillus subtilis or lactic acid bacteria for surface modification, the same dynamic switching function of "penetration-breakage-anchoring" can be achieved.

[0084] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0085] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A switchable artificial bacteria interface, characterized in that, The invention includes a bacterial body and a double-layer coating structure modified on the surface of the bacterial body; the double-layer coating structure includes an inner polydopamine layer and an outer polyethylene glycol layer, wherein the inner polydopamine layer and the outer polyethylene glycol layer are connected by acid-sensitive bonding bonds. The acid-sensitive linkage can be broken under acidic conditions, so that the outer polyethylene glycol layer detaches and exposes the inner polydopamine layer.

2. The switchable artificial bacteria interface according to claim 1, characterized in that, The acid-sensitive linking bond is any one of an arylimide bond, an hydrazone bond, or an acetal bond.

3. The switchable artificial bacteria interface according to claim 1 or 2, characterized in that, The molecular weight of the polyethylene glycol is from 1 kDa to 10 kDa.

4. The switchable artificial bacteria interface according to claim 1 or 2, characterized in that, The inner polydopamine layer is formed by depositing dopamine at a concentration of 0.1 mg / mL to 0.5 mg / mL in a weakly alkaline buffer solution with a pH of 8.0-9.

0.

5. The switchable artificial bacteria interface according to claim 1 or 2, characterized in that, The outer polyethylene glycol layer is formed by modifying an acid-sensitive polyethylene glycol precursor with a concentration of 0.5 mg / mL to 10 mg / mL through an amide condensation reaction.

6. The switchable artificial bacteria interface according to claim 1 or 2, characterized in that, The bacteria themselves are Staphylococcus epidermidis, Bacillus subtilis, lactic acid bacteria, or engineered therapeutic bacteria.

7. A method for preparing a switchable artificial bacterial interface, characterized in that, Includes the following steps: S1. Incubate bacteria with dopamine at a concentration of 0.1 mg / mL to 0.5 mg / mL in Tris-HCl buffer or phosphate buffer at pH 8.0-9.0 at 35℃-40℃ for 10-30 minutes. An inner polydopamine layer is deposited on the bacterial surface through the oxidative self-polymerization of dopamine. The bacteria are then separated by centrifugation and washing. S2. The acid-sensitive polyethylene glycol precursor and the bacteria obtained in step S1 are reacted in a buffer solution of pH 6.5-7.5 at 35℃-40℃ for 0.5-2 hours. The outer polyethylene glycol layer is connected to the inner polydopamine layer by amide condensation reaction. After centrifugation and washing, a switchable artificial bacterial interface is obtained.

8. Use of a switchable artificial bacterial interface as described in any one of claims 1-6 in the preparation of a medicament for treating infections at mucus-covered sites.

9. The application according to claim 8, characterized in that, The infection at the site covered by mucus is a respiratory tract infection.

10. The application according to claim 9, characterized in that, The infection is a respiratory infection caused by Staphylococcus aureus or Pseudomonas aeruginosa, and the drug is a preparation for nasal or inhalation administration.

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