Size-tailored and acid-degradable polyvinyl alcohol microgels for inhalation therapy of bacterial pneumonia†

Xiang Zhou‡ ^a, Jingjing Zhou‡^a, Lanlan Wang^a, Bingbing Zhao*^a, Yukun Ma^a, Ni Zhang^a, Wei Chen*^ab and Dechun Huang*^ab
aDepartment of Pharmaceutical Engineering, School of Engineering, China Pharmaceutical University, Nanjing 210009, China. E-mail: zhaobb@cpu.edu.cn; w.chen@cpu.edu.cn; cpuhdc@cpu.edu.cn
^bEngineering Research Center for Smart Pharmaceutical Manufacturing Technologies, Ministry of Education, School of Engineering, China Pharmaceutical University, Nanjing 211198, China

First published on 6th August 2024

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1. Introduction

Pulmonary infection, ranking as the third leading cause of global mortality, is instigated by infections with ubiquitous pathogens. Failure to promptly detect and treat infections may result in the development of systemic infections, which contribute to an annual mortality rate exceeding 4 million.^1–4 Currently, the clinical management of lung infections primarily relies on oral or intravenous administration of antibiotics, which is associated with drawbacks, such as delayed absorption, limited patient adherence, and vulnerability to first-pass effect, consequently leading to restricted bioavailability.^5–8 Furthermore, systemic drug administration necessitates the utilization of elevated dosages in order to attain desired therapeutic outcomes, thereby engendering both drug resistance and adverse effects.^5,9,10 Consequently, it becomes imperative to discern and implement efficacious treatment strategies that can effectively mitigate these prevailing challenges.

Inhalation therapy has gained widespread attraction in the treatment of respiratory diseases owing to its capacity for targeted delivery of antibiotics to the infection site, thus enhancing therapeutic efficacy at reduced dosages.^11–14 Inhaled formulations such as tobramycin solution and tobramycin powder have received FDA approval for clinical use to improve the treatment of pulmonary respiratory diseases caused by Pseudomonas aeruginosa infections.^15,16 Nevertheless, numerous biological and physical barriers within the lung pose challenges in achieving precise administration of therapeutic agents to the lesion site.^1,17–19 It has been demonstrated that the deposition location is primarily determined by the size of inhaled drug particles. The upper respiratory tract typically captures particles larger than 5 μm in diameter through entrainment, while particles smaller than 0.5 μm are easily expelled by respiration. Particles ranging from 1 to 5 μm in diameter effectively penetrate deeper into the lungs due to inertial impaction and deposition.^20–22 However, the application of inhalable drug formulations faces challenges due to their nano-micro scale dimensions, which result in easy exhalation upon inhalation and potential nanotoxicity caused by alveolar retention.^23–25 Consequently, microparticles with a suitable size have garnered significant attention due to their capacity to efficiently traverse mucosal barriers and achieve deep deposition in the lungs.

As the delivery systems for inhalation administration, various polymeric materials such as poly(lactic-co-glycolic acid), chitosan, polyethylene glycol, polyvinyl alcohol (PVA) and their derivatives have been developed and applied.^26–29 Among these materials, PVA stands out as an FDA-approved pharmaceutical material with anti-mucus adherence properties, making it highly versatile in enhancing drug penetration through mucus.^30–32 However, the lack of precise control over the targeted drug release often results in a compromised efficacy of inhalation therapies. To improve the effectiveness of antibiotics, it is imperative to develop delivery systems that can respond to the microenvironment of infection and release therapeutic agents based on specific signals at the site of infection.^33–35 Considering the acidic microenvironment in the infected lung region, pH-responsive delivery systems have been extensively investigated for treating lung diseases.^6,36 Building upon this premise, delivery systems with appropriate sizes were designed to achieve therapeutic objectives by traversing the mucus layer and depositing in the lungs, where the antibiotic release is triggered by the micro-acidic environment at the infection site.

In this project, we developed a size-tailored, acid-degradable antimicrobial PVA microgel for the treatment of bacterial infections in the lung, in which vinyl ether methacrylate (VEMA) functionalized PVA (PVA_VEMA) formed nano/micro aggregates upon elevated temperature, followed by UV-exposure and further loading of the antibiotic drug piperacillin (PIP) through hydrophobic interaction to prepare PVA drug-loaded microgels with varying dimensions (Scheme 1). After inhalation, microgels effectively penetrated the mucosal barrier and exhibited efficient accumulation in the middle and deep lung regions. Subsequently, the acetal linkage formed by vinyl ether and hydroxyl groups on the PVA_VEMA microgel was susceptible to degradation under weak acidic conditions, thus facilitating the controlled release of PIP in the pulmonary infection environment. This innovative approach demonstrated remarkable therapeutic efficacy against lung infections and would hold great potential for developing a controlled delivery microsystem specifically tailored for inhalation therapy.

2. Experimental section

2.1. Materials

Piperacillin (PIP, Shanghai yuanye Biotechnology, 95%), aldehyde (Aladdin, 99.5%), cyanine5 (Cy5, Ruisi Biological), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, Energy Chemical, 99%), N-hydroxy-succinimide (NHS, Energy Chemical, 99%), succinic anhydride (SA, Energy Chemical, 99%), ethyleneglycol vinyl ether (Aldrich, 97%), methacryloyl chloride (Aldrich, 97%), triethylamine (Et₃N, Acros, 99%), p-toluene-sulfonic acid monohydrate (PTSA, Sigma-Aldrich, 98%), 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (I2959, Aldrich, 98%), polyvinyl alcohol (PVA, M_w = 15000, Acros), isoflurane (Hengfengqiang Biotechnology, Jiangsu), paraformaldehyde (Sangon Biotech, 4%), pGEN-luxCDABE (Baosai Biotechnology, Hangzhou), enzyme-linked immunosorbent assay (ELISA) (tumor necrosis factor-α (TNF-α), Multi sciences) was used as received.

2.2. Synthesis of VEMA-functionalized PVA (PVA_VEMA)

The synthesis of vinyl ether methacrylate (VEMA) was similar to that of vinyl ether acrylate (VEA).³⁷ In brief, ethylene glycol vinyl ether (150 mL, 1.73 mol) and Et₃N (310 mL, 4.2 mol) were added to a solution of dichloromethane (1.2 L), followed by dropwise addition of methacryloyl chloride (168 mL, 1.73 mol) under a nitrogen atmosphere in an ice bath with stirring at room temperature for 8 hours. The resulting reaction mixture was subjected to three extractions with saturated aqueous sodium carbonate solution. After drying the organic phase with anhydrous sodium sulfate and subsequent filtration and concentration, the final product VEMA was obtained as a white oily liquid through vacuum distillation.

For the synthesis of PVA_VEMA, VEMA (2 mL, 0.014 mmol) was added to a 30 mL solution of PVA (1.0 g, 0.067 mmol) in DMSO with PTSA catalyst (120 mg, 0.631 mmol) under stirring. After 6 h, the reaction was terminated by the addition of Et₃N (132 μL, 0.596 mmol). Subsequently, the PVA_VEMA was purified under extensive dialysis (MWCO: 3500) in methyl alcohol for 24 h to remove unreacted VEMA and the catalyst, followed by rotation evaporation and precipitation in diethyl ether to obtain the product.

2.3. Preparation of G@PIP and Cy5-G@PIP

The photoinitiator of I2959 (0.025, 0.25, 0.5, 0.75 mg mL⁻¹) was added to PVA_VEMA aqueous solutions at concentrations of 0.5, 5, 10, 15 mg mL⁻¹, respectively, and the solution was stirred for 2 min at 40 °C under a nitrogen atmosphere to facilitate PVA_VEMA aggregates. The aggregates were then exposed to UV light for 10 min to obtain PVA_VEMA microgels with varying sizes of PVA_VEMA Gel-0.2 (G-0.2, size: 0.2 μm), PVA_VEMA Gel-1.6 (G-1.6, size: 1.6 μm), PVA_VEMA Gel-3.5 (G-3.5, size: 3.5 μm) and PVA_VEMA Gel-7.8 (G-7.8, size: 7.8 μm), respectively. For the preparation of drug-loaded microgels, the PIP dissolved in ethanol was mixed with different sizes of PVA_VEMA microgel, which were then subjected to alternating ultrasound and vortexing for 30 seconds each and repeated three times to obtain a drug-loaded microgels G-0.2@PIP, G-1.6@PIP, G-3.5@PIP and G-7.8@PIP, respectively. The synthesis of Cy5-G@PIP was conducted according to a previously established methodology.^38,39 PVA_VEMA-COOH was synthesized by reacting PVA_VEMA with succinic anhydride in a 1:5 molar ratio, using triethylamine as a catalyst, allowing the reaction to proceed overnight. The product was then dialyzed in methanol and subsequently precipitated using ether. Carboxylated G@PIP of various sizes was prepared utilizing the same UV cross-linking protocol as previously described. These G@PIP were then conjugated with Cy5-NH₂ in the presence of EDC and NHS as catalysts. The final product, Cy5-G@PIP, was obtained after dialysis. The particles were characterized via dynamic light scattering (DLS) and scanning electron microscopy (SEM, Hitachi S4800). Furthermore, the Cy5-labeled microgel was observed by using an inverted fluorescence microscope (Leica/Olympus, DMI3000 B/IX73).

2.4. pH-degradation of PVA_VEMA microgel

To investigate the degradation product acetaldehyde from PVA_VEMA microgels, an equivalent quantity of PVA_VEMA microgels was centrifuged at 13000 rpm for 10 min to remove the supernatant. The microgels were then mixed with 1 mL of pH 7.4 and pH 6.6 buffer solutions and incubated in a 37 °C water bath with thorough stirring. At specific time intervals (0, 12, 24, 48, 72, and 120 h), the samples were centrifuged at 13000 rpm for 10 min, and the supernatants were analyzed by using gas chromatography. Calibration curves were generated using aqueous solutions of acetaldehyde at various concentrations to quantify the acetaldehyde in the samples.

2.5. In vitro antibacterial assay

E. coli was cultured in 5.0 mL of Luria–Bertani (LB) liquid medium containing 5.0 μL of ampicillin (100 mg mL⁻¹) for 24 h. The bacteria concentration was determined using the spread plate method and then concentrated to 1.0 × 10⁹ CFU mL⁻¹ as a stock bacterial liquid. Initially, the minimum inhibitory concentration for 50% bacterial growth inhibition (MIC₅₀) and the minimum bactericidal concentration (MBC) of PIP were determined. The 50 μL of the stock bacterial solution was added to 10 mL of LB liquid medium (pH 7.4) containing 10 μL ampicillin to prepare the diluted bacterial solution. Subsequently, 1 mL of diluted bacterial solution was put into a 24-well plate with a series of concentrations of PIP (0, 0.15, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 mg mL⁻¹) and incubated at 37 °C for 18 h. Afterwards, the G@PIP with varying dimensions containing MIC₅₀, MBC and 2-fold MBC (2MBC) of PIP were measured for antimicrobial activity under pH 6.6 and pH 7.4 conditions in vitro. In a 24-well plate, 1 mL of diluted bacterial solution (pH 6.6 and pH 7.4) was incubated with PBS, G-0.2@PIP, G-1.6@PIP, G-3.5@PIP and G-7.8@PIP, and then placed on a shaking table at 37 °C and 120 rpm min⁻¹ for 24 h. Following incubation, 20 μL of the bacterial solution was diluted 1.0 × 10⁵ times and spread onto agar plates, which were then incubated at 37 °C for 18 h to observe the microbial growth. Each microgel group contained MIC₅₀, MBC and 2MBC concentrations of PIP.

2.6. In vivo G@PIP lung distribution and live imaging

C57BL/6 mice (male, 27 ± 2 g) were purchased from Hangzhou Ziyuan Experimental Animal Technology Co. (Nanjing, China). All the animal experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals approved by the China Pharmaceutical University. To evaluate the biodistribution and pulmonary accumulation of G@PIP with various sizes via nebulized inhalation in vivo. The G@PIP of varying dimensions were marked with the fluorescent dye Cy5 and administered through nebulized inhalation using an HY-QSEOI (Yuansenkade Biotechnology, Beijing) device. At predetermined time points (0.5, 1, 2, 4, 8, and 12 h), mice were anesthetized with isoflurane gas and subjected fluorescent images using an in vivo imaging system (620 nm excitation, 670 nm emission). After the final imaging time point, the mice were euthanized, and lung tissues were harvested for frozen sections. The fluorescence intensity in the lung tissues of each group was quantified using Living Image software.

2.7. Antibacterial activity in vivo

To establish the mouse pneumonia model, an Amp-resistant strain transformed with the pGEN-luxCDABE plasmid (E. coli-lux, 10 μL, 2.0 × 10⁹ CFU mL⁻¹) was precisely injected into the right lung parenchyma of the mice. Subsequently, PBS, PIP, G-0.2@PIP, G-1.6@PIP, G-3.5@PIP, and G-7.8@PIP were administered into C57BL/6 mice via a nebulizing device at 12, 24, and 36 h intervals. Live imaging was recorded at predetermined intervals of 0, 12, 36, 72, 96, and 120 h to monitor the bacterial status within the mouse lungs. In addition, following 120 h of drug administration, the mice were euthanized, various organs (heart, liver, spleen, lung, and kidney) were harvested for bioluminescence imaging to visualize bacterial infection. Furthermore, to quantify the residual bacterial population within the lung tissue post-drug administration, lung tissues were rinsed with sterile pre-cooled saline, dried, weighed, and homogenized in a 10-fold saline solution. Subsequently, 50 μL of the 10-fold diluted homogenate was inoculated into 5 mL of LB liquid medium supplemented with 5 μL of ampicillin and incubate at 37 °C for 24 h. After culture, bacterial colony counts were estimated by measuring the optical density at OD₆₀₀ using a SpectraMax i3× Multi-Mode Microplate Reader.

For the evaluation of post-treatment lung inflammatory factors, bronchoalveolar lavage fluid (BALF) was collected by flushing the bronchus thrice with 200 μL of chilled sterile water through an endotracheal tube. The supernatant was isolated by centrifugation at 4000 rpm for 10 minutes at 4 °C. The concentration of tumor necrosis factor-α (TNF-α) in the supernatant was quantified using an ELISA kit. Additionally, the lungs and other organs underwent fixation in 4% paraformaldehyde, followed by dehydration and embedding in paraffin. Tissue sections of 4 mm thickness were stained with hematoxylin and eosin (H&E) for histological examination.

2.8. Statistical analysis

All results were shown as mean ± standard deviation (SD). Biological replicates (n = 3–5) were performed in all experiments. One-way Anova was used to measure differences for more than two groups. All statistical analyses were evaluated by using a GraphPad Prism (8.0). *P < 0.1, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

3. Results and discussion

3.1. Preparation and characterization of G@PIP

PVA_VEMA were synthesized via an acetal reaction between the vinyl ether group and hydroxyl in the presence of PTSA (Fig. S1A, ESI†). The functionality of VEMA was quantified to be 0.37% by using ¹H NMR analysis, based on the integral ratio of the double bond signal from VEMA at δ 5.65–6.10 ppm and the methyl group signal from PVA at δ 1.85–2.05 ppm (Fig. S1B and C, ESI†). The hydrophobic modification of PVA-grafted VEMA units reduced both intra- and intermolecular hydrogen bonding forces of the adjacent OH groups, thereby imparting PVA_VEMA with thermal transformation capacity. As shown in Fig. 1A and Fig. S2 (ESI†), PVA_VEMA existed as unimers with an average particle size ranging from 9 to 18 nm at temperatures below 15 °C and formed aggregates when the temperatures increased to 20 °C. In addition, the critical solution temperature range of various PVA_VEMA concentrations was observed between 15 and 20 °C. These findings suggest that the phase transition temperature is independent of PVA_VEMA concentration but is potentially associated with its degree of substitution.³⁷ The photoinitiator I2959 catalyzed the double bond in PVA_VEMA, initiating radical polymerization upon exposure to UV light and resulting in the formation of microgels. While the cross-linked PVA_VEMA microgel transformed into aggregates at a low critical temperature (5 °C), it no longer existed as monomers. In contrast, non-crosslinked PVA_VEMA exhibited reversible thermal responsiveness under low-temperature conditions (Fig. S3, ESI†). Additionally, the size of the cross-linked PVA_VEMA microgels exhibited a positive correlation with the concentration of cross-linking. At 40 °C, the particle sizes of the microgels resulting from UV-crosslinking PVA_VEMA solutions at concentrations of 0.5, 5, 10 and 15 mg mL⁻¹ were measured at 0.2, 1.6, 3.5 and 7.8 μm, respectively (Fig. 1B). Moreover, minimal variation in size was observed for PVA_VEMA microgels after loading with the hydrophobic antibiotic PIP (Fig. S4, ESI†). Scanning electron micrographs (SEM) revealed the microscopic morphology of the spherical microgels with different sizes formed following crosslinking at different PVA_VEMA concentrations (Fig. 1C). Furthermore, the static plots of microgel solutions after crosslinking with varying PVA_VEMA concentrations were observed, as shown in Fig. 1D, the G-0.2 group with smallest size showed no deposition after settling, whereas the larger size G-7.8 exhibited significant deposition, further confirming a positive correlation between microgel size and PVA_VEMA concentration.

3.2. pH degradation of PVA_VEMA microgel

According to previous studies, under an acidic microenvironment, the acetal linker connecting the VEMA and PVA backbones can undergo degradation to yield natural PVA, poly(hydroxyethyl acrylate) (PHEA) and acetaldehyde, without generating any toxic by-products.^37–39 Therefore, we investigated the acetaldehyde content in the degradation product of PVA_VEMA microgels under simulated lung infection microenvironmental pH conditions (pH 6.6) and physiological pH conditions (pH 7.4) conditions to determine whether the microgels can be effectively degraded under a slightly acidic microenvironment. Gas chromatography analysis, as shown in Fig. S5A (ESI†), revealed peak retention times of 3.8 min for acetaldehyde and 4.2 min for the photoinitiator I2959. Moreover, under pH 6.6 conditions, the degradation of acetaldehyde by PVA_VEMA microgels progressively increased over time, reaching a concentration of 8.7 ng mL⁻¹ after 120 h, whereas at pH 7.4, the degradation remained relatively stable with only a concentration of 1.3 ng mL⁻¹ after 120 h (Fig. S5B, ESI†). The observation suggests that the PVA_VEMA microgels demonstrate instability under weakly acidic conditions, which present a favorable characteristic for the safe delivery of drug-loaded microgels to the lungs via inhalation. This feature prevents abrupt release and enables efficient degradation in the pH 6.6 lung microenvironment, thereby facilitating targeted drug delivery.

3.3. In vitro antibacterial assay

E. coli, one of the main pathogens of pulmonary infections, was chosen as the model organism for investigating the antimicrobial efficacy of G@PIP. The antibacterial potency of PIP was assessed through MIC₅₀ and MBC assays conducted in LB medium. The MIC₅₀ and MBC values of PIP against E. coli-lux were determined as 0.9 mg mL⁻¹ and 1.8 mg mL⁻¹, respectively (Fig. S6, ESI†). Subsequently, PVA_VEMA microgels of varying dimensions loaded with MIC₅₀, MBC and 2MBC concentrations of PIP were evaluated for in vitro antibacterial activity at pH 6.6 and pH 7.4. As shown in Fig. 2A, G@PIP (PIP: MIC₅₀) exhibited negligible antimicrobial activity at pH 7.4 across different dimensions; however, they all displayed weak antimicrobial activity at pH 6.6 due to the degradation of G@PIP and subsequent release of minor amounts of PIP. The antimicrobial activity of G@PIP (PIP: MBC) exhibited an enhanced effect at pH 7.4 and pH 6.6 (Fig. S7, ESI†). Conversely, G-0.2@PIP, G-1.6@PIP, G-3.5@PIP and G-7.8@PIP (PIP: 2MBC) displayed inhibition efficiencies of 37%, 46%, 52%, and 46% at pH 7.4, respectively, while achieving complete bacterial inhibition at pH 6.6 (Fig. 2B and C). These findings suggest that all the different dimensions of G@PIP can undergo degradation in a slightly acidic environment (pH 6.6), leading to the effective release of PIP for eradicating bacteria.

3.4. In vivo lung distribution and live imaging

The impact of G@PIP size on pulmonary drug delivery and lung deposition was investigated by monitoring the fluorescence signal in vivo after nebulized inhalation of Cy5-functionalized G@PIP into mouse lungs. Fig. 3A illustrates the fluorescence signals of G@PIP with varying dimensions in the pharynx and lungs after 30 min of nebulized inhalation. Notably, the G-7.8@PIP group exhibited the highest fluorescence signals in the pharynx, surpassing those of the G-0.2@PIP, G-1.6@PIP, and G-3.5@PIP groups by 5.5-fold, 3.3-fold, and 1.7-fold, respectively (Fig. 3B). In contrast, the G-3.5@PIP group displayed the most robust fluorescence signal in the lung region, which was 8.9, 2.1, and 2.7 times stronger than that of the G-0.2@PIP, G-1.6@PIP and G-7.8@PIP groups, respectively (Fig. 3C). These results revealed that the G-3.5@PIP group exhibits the most effective and prolonged pulmonary deposition. This outcome was attributed to the smaller particle sizes of the G-0.2@PIP and G-1.6@PIP groups, which were more susceptible to exhalation and clearance from the lung compared to the G-3.5@PIP group. On the other hand, the larger particle size of the G-7.8@PIP group resulted in tracheal retention and deposition in the pharynx, preventing efficient transport from the lung. Literature reports indicate that particles exceeding 5 μm in diameter typically deposit in the upper respiratory tract via entrainment, whereas particles ranging from 1 to 5 μm in diameter are effectively transported deeper into the lungs through inertial impaction and deposition.^20–23 Consequently, the G-3.5@PIP group accumulated prominently in the lungs post-inhalation, underwent acid degradation, and released antibiotics, thereby improving the therapeutic efficacy against pneumonia. Meanwhile, fluorescent results from frozen sectioning revealed minimal fluorescent staining in the control and G-0.2@PIP groups, weak fluorescent staining in the G-1.6@PIP and G-7.8@PIP groups, and stronger fluorescent staining in the G-3.5@PIP group, are consistent with the trend of fluorescent staining in vivo (Fig. 3D). These results confirmed the remarkable deposition effect of G-3.5@PIP, rendering it highly suitable for pulmonary inhalation therapy.

3.5. In vivo anti-infection activities

E. coli stands out as a prominent pathogen linked to respiratory infections and pneumonia.^3,40 To visualize the distribution of the bacteria, a pneumonia model was developed by utilizing an Amp-resistant strain such as E. coli-lux. The optimal dose of E. coli-lux was determined to be 10 μL (2.0 × 10⁹ CFU mL⁻¹) (Fig. S8A and B, ESI†). The animal model of E. coli-lux lung infection was established via lung puncture, and subjects were subjected to varying dimensions of G@PIP administered at specific time intervals (Fig. 4A). The therapeutic efficacy of G@PIP was assessed through in vivo bioluminescence signal detection in mice. Notably, bioluminescence signals remained robust in the lungs of mice from the PIP, G-0.2@PIP, and G-7.8@PIP groups even after 120 h of treatment, as shown in Fig. 4B and C. This observation was attributed to the tendency of G-0.2@PIP to escape with the airflow due to its smaller particle size, whereas G-7.8@PIP tended to be trapped by the trachea owing to its larger particle size, impeding its reach to lung tissues for effective PIP release. Meanwhile, both the G-1.6@PIP and G-3.5@PIP groups demonstrated favorable lung deposition; however, the G-1.6@PIP group was less efficacious compared to the G-3.5@PIP group, with fluorescent signals nearly disappearing after 96 h of treatment. This result may be ascribed to the more favorable size of G-3.5@PIP for lung deposition, whereas the G-1.6@PIP fraction may be exhaled.⁴¹ Similarly, bioluminescence signal intensity in isolated organs of mice after 120 h of treatment further corroborated the superior therapeutic effect of the G-3.5@PIP group (Fig. 4D). Additionally, changes in the body weight of mice provided additional evidence of the therapeutic efficacy of G@PIP with varying dimensions. Weight reduction correlates with heightened disease severity, with rapid weight decline during the pre-infectious phase serving as a crucial indicator of deteriorating animal condition following pathogen infection.⁴² In particular, compared to other groups wherein mice experienced a decline in body weight, the G-3.5@PIP group exhibited a significant and gradual increase in body weight post-treatment, indicative of favorable therapeutic efficacy (Fig. 4E).

The severity of lung infection in mice was assessed by quantifying residual bacterial counts, which were detected through culturing lung tissue homogenates and measuring their OD₆₀₀ values. As illustrated in Fig. 4F, the OD₆₀₀ values of the G-3.5@PIP group exhibited a significant decrease compared to other groups, indicating that nebulized inhalation of G-3.5@PIP effectively eradicated E. coli-lux by degrading and releasing PIP within the acidic microenvironment of the lung. Furthermore, the levels of TNF-α, a representative inflammatory cytokine, were measured in lung lavage fluid to further investigate the interaction between G@PIP and the immune system in pneumonia-afflicted mice.^43,44 Following inhalation treatment, TNF-α levels decreased to varying degrees in the PIP, G-3.5@PIP and G-1.6@PIP groups compared with the Control group. Notably, the G-3.5@PIP group showed superior anti-inflammatory effect attributed to its enhanced deposition efficacy within the lungs. Interestingly, TNF-α levels were abnormally elevated in the G-0.2@PIP and G-7.8@PIP groups surpassing those observed in the control group (Fig. 4G). This abnormal increase may be attributed to the inhalation of therapeutically ineffective microgels, exacerbating discomfort in pneumonic mice and consequently worsening their condition.

3.6. Histological analysis

To evaluate the therapeutic efficacy of various microgels, mice were euthanized 120 h post-bacterial infection, and major organs were subsequently isolated for histopathological examination using H&E staining. As depicted in Fig. 5A, inhalation treatment with G-3.5@PIP notably ameliorated lung histopathology compared to the control group. This improvement was evidenced by a significant reduction in inflammatory cell infiltration, lesion size, and alveolar airspace along with enhanced alveolar septal thickening, indicating considerable therapeutic effectiveness. Furthermore, the H&E staining of vital organs including the heart, liver, spleen, and kidney revealed no significant tissue damage, inflammatory response, cellular degeneration, or necrosis (Fig. 5B), suggesting minimal systemic toxicity. These findings further confirmed that the G-3.5@PIP as a promising biologically safe inhalation material for targeted pulmonary drug delivery against bacterial lung infections.

4. Conclusions

In conclusion, the robust construction of PVA-based microgels was successfully achieved through UV irradiation of thermo-triggered PVA_VEMA aggregates. The sizes of microgels could be adjusted and optimized with the polymer concentrations during the cross-linking process for efficient pulmonary deposition in inhalation therapy. Hydrophobic interaction facilitated efficient loading of PIP onto microgels, thereby conferring potent antimicrobial properties against E. coli. After nebulization inhalation, the medium-sized particles of microgels (∼3.5 μm) exhibited superior lung deposition and underwent degradation due to the hydrolysis of the acetal-containing network, thus liberating antibiotic PIP and effectively mitigating E. coli infections and associated inflammation. These PVA-based microgels offer a versatile and efficient approach for the direct fabrication of a targeted micro-drug delivery system, specifically tailored for inhalation therapy to effectively combat pneumonia-associated infections.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article or its ESI.†

Conflicts of interest

The authors have no conflicts of interest to declare.

Acknowledgements

This work was financially supported by the National Key Research and Development Program of China (2022YFF1100600), the National Natural Science Foundation of China (NSFC 52173294), the Demonstration Project of Modern Agricultural Machinery Equipment &Technology in Jiangsu Province (NJ2022-11), the Excellent Teaching Team of the “Blue Project” in Jiangsu Province, the Jiangsu Funding Program for Excellent Postdoctoral Talent (2023ZB585), the Fundamental Research Funds for the Central Universities (2632023FY03).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4tb01224k

‡ These authors contributed equally to this work.

This journal is © The Royal Society of Chemistry 2024