A vehicle-free immunomodulatory PFASs@Ga composite promotes the healing of infected wounds
Saadullah Khattak, Salim Ullah, Muhammad Tufail Yousaf, Jianliang Shen, Xiaoqun Xu, Hong-Tao Xu
TL;DR
A new vehicle-free composite material combining gallium and perfluoroalkyl substances promotes healing of infected wounds by reducing bacteria and inflammation.
Contribution
A novel vehicle-free PFASs@Ga composite is developed for immunomodulatory wound healing with antibacterial and pro-healing properties.
Findings
PFUnA@Ga shows potent antibacterial activity against MRSA and E. coli with enhanced wound closure.
The composite modulates inflammation by reducing M1 macrophages and promoting M2 macrophages and angiogenesis.
PFUnA@Ga demonstrates biocompatibility, biosafety, and superior performance compared to other PFASs@Ga composites.
Abstract
Bacterial infection and severe inflammatory responses are major barriers to successful wound healing. Drug delivery systems have shown promise in precision medicine by enhancing the targeting and protection of therapeutic agents. However, their use is limited by challenges such as biocompatibility issues, structurally complex, high manufacturing costs, suboptimal drug loading, unstable release, and premature immune clearance. Although targeted delivery aims to improve efficacy, it may increase the risk of toxicity. Consequently, conventional non-carrier drugs often remain more practical and effective. Emerging vehicle-free systems with multifunctional capabilities show promise for precise targeting, controlled release, reduced toxicity, and simplified manufacturing. Here, we rationally designed and established a vehicle-free perfluoroalkyl material (PFAS)–gallium composites (PFASs@Ga)…
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TopicsAdvanced Sensor and Energy Harvesting Materials · Wound Healing and Treatments · Nanoplatforms for cancer theranostics
Introduction
1
Many antibacterial agents have been developed in recent years to combat infectious diseases, including antibacterial peptides, metal ions, and nanomaterials [1]. Bacterial infections remain a significant clinical problem in wound healing, despite advances [2]. The United States Food and Drug Administration has recently authorized gallium nitrate (Ga(NO_3_)3) as a possible antibacterial drug for the treatment and elimination of bacterial infections [3,4]. The similarities between Ga^3+^ ions and ferric ions (Fe^3+^) can be shown from ionic radii and electronic configuration. But Ga^3+^ is not involved in the redox reactions essential for bacterial metabolism, which is a significant difference [5]. When bacteria absorb Ga^3+^ instead of Fe^3+^, it disrupts essential processes such as electron transport and DNA replication, thereby preventing bacterial growth or killing the bacteria [6].This mechanism is comparable to a Trojan horse strategy, which enables multiple attacks on the bacterial cell [7]. Furthermore, this agent has shown an enormous resistance to many multidrug-resistant (MDR) bacteria. The research findings offer new hope for preventing or eliminating harmful bacterial infections [[8], [9], [10]].
Oxygen (O_2_) is important and plays a vital role in the wound-healing process [[11], [12], [13], [14]]. Chronic diabetic wounds that are refractory and non-healing have low oxygenation due to microvascular occlusion and hypoxia, and are responsible for the chronicity of the wound [15]. Among these chronic lesions, diabetic foot ulcers are particularly challenging, and hydrogel wound dressings have emerged as promising candidates for their management, although important clinical and translational hurdles remain [16]. Hyperbaric oxygen therapy (HBOT) and topical oxygen therapy (TOT) are used universally to treat a hypoxic microenvironment [17]. However, both HBOT and TOT continue to face problems in relieving wound hypoxia. For example, the global wound-healing community has carefully studied and implemented systemic HBOT [18]. Using this approach includes a detailed balance. Patients must obtain enough O_2_, but there remains a risk of tissue hyperoxia. Clinical trials assessing its efficiency in healing hypoxic wounds have produced contradictory and often unsatisfactory outcomes [19]. TOT implies delivering oxygen to injured tissue via nonstop diffusion or pressurized systems. Its efficacy in promoting wound healing remains unreliable and somewhat notorious [20]. Devices like TWO2 and NATROX™ supply oxygen to the wound through a chamber or small tubes. Though they can be bulky, require common oxygen refills, and allow limited oxygen flow to the wound bed [21,22]. OxyBandTM, a type of TOT, is a traditional wound covering with O_2_ delivery technology. The efficiency of this may be limited by the absence of a seamless link between O_2_ delivery and the wound covering [23]. Moreover, the quick initial O_2_ release, short supply duration, and need for frequent replacements may cause minor injury and limit practical use [24]. There is a need for more convenient and effective O_2_ delivery systems that provide sustained local oxygenation at the wound site. To address the limitations of HBOT and TOT, several systems have been developed using hemoglobin (Hb), perfluorocarbons (PFCs), peroxide salts, and algae [[25], [26], [27]]. Localized oxygenation systems provide an oxygen-rich environment at the wound site while avoiding systemic hyperoxia. However, these systems may still face unmet challenges. In practice, Hb has a short time frame, poor control of O_2_ release, and serious side effects such as an increased chance of a myocardial infarction [13]. A peroxide salt is said to release an “avalanching burst” of oxygen due to a thermal runaway type reaction that occurs with water. These compounds may induce severe cytotoxicity by accumulating toxic peroxide and free radical species [28]. There is a problem with applying photosynthetic algae because it is difficult to maintain a consistent highlight dose over a long period. Furthermore, unexpected immune responses raised concerns about its biosafety [29]. Perfluorocarbons (PFCs) are chemicals that don't react much with other chemicals and are biocompatible and able to dissolve oxygen at high levels [30,31]. The solubility of O_2_ at room temperature is 40% or greater in PFC (perfluorinated chemicals), compared to 2.5% in water, plasma, and whole blood [32]. PFCs bind O_2_ and gases easily because fluorine is less polar. Also, the solubility of a gas in PFCs is proportional to the gas's pressure [33]. Perfluorodecalin (PFD), which is a member of the PFC family, has been used as an O_2_ carrier [34,35]. They are also used in ophthalmic surgery to reposition a detached retina [36]. To deliver higher concentrations of O_2_ topically during the treatment of acute ocular chemical injury, the researcher developed a perfluorodecalin supersaturated O_2_ emulsion (SSOE). In studies conducted before human testing, SSOE helps the cornea heal after injury, reduces eye inflammation, and prevents tissue damage from O_2_ deprivation [37]. O_2_ emulsion derived from PFD improves the healing process of second-degree burns in human and porcine skin [38,39]. Thus, a clinically crucial need exists to develop an O_2_ delivery process that continuously and gently oxygenates the wound without causing toxicity, and that enhances wound-healing processes, such as cellular metabolism, re-epithelialization, angiogenesis, and collagen synthesis and assembly. Here, we reported a novel antibacterial platform, PFASs@Ga composites (Fig. 1), developed by linking PFASs to Ga to enhance antibacterial activity. This new design improves the Ga effect. Also, this combination uses the unique advantages of both components to enhance the bactericidal effect. Ga strengthens the PFASs platform and adds additional antimicrobial mechanisms. Attachment of Ga to PFASs deploys a ‘Trojan horse strategy’ in which Ga interrupts bacterial iron metabolism, exerting both lateral and direct antibacterial effects. As a result, the composite's overall antimicrobial efficacy against iron-dependent bacterial pathogens is greatly increased. The release of the Ga prolongs the antimicrobial effect during treatment while avoiding side effects in neighboring tissues. Secondly, the PFASs enable sustained release of O_2_ in the wound side, which help the wound to heal fast.PFUnA@Ga exhibited significant antimicrobial activity against a range of bacteria, comparable to that of clinical antibiotics.This may have a wider use for PFUnA@Ga composites in biomedical application.Fig. 1. Schematic of the composition and application of PFASs@Ga composites for wound healing.Fig. 1
Materials and methods
2
Materials obtained from Shanghai Aladdin Biochemical Technology Co., Ltd, China, include Undecafluorohexanoic acid (PFXHxA) (C_6_HF_11_O_2_), purity greater than equal 98%, molecular weight 314.05(MW), Pentadecafluorooctanoic acid(PFOA) (C_8_HF_15_O_2_), purity greater than equal 96%, molecular weight 414.07(MW), Heneicosafluoroundecanoic Acid (PFUnA)(C_11_HF_21_O_2_), purity greater than equal 97.0%, molecular weight 564.09(MW), gallium Nitrate hydrate (Ga(NO_3_)3.xH_2_O), purity 99.9%, molecular weight 255.74(MW), N-N-Dimethylacetamide (DMA); and other chemicals are from Symbol Life Science, India. PBS (phosphate-buffered saline), DAPI (4ʹ,6-diamidino-2-phenylindole), Calcein acetoxymethyl ester (calcein-AM), and PI (propidium iodide) were obtained from Solarbio (Hangzhou, China), while SYTO9 was obtained from Thermo Fisher (Hangzhou, China). Penicillin-streptomycin, Dulbecco's modified Eagle's medium, and fetal bovine serum were supplied by Gibco (Wenzhou, China). Hopebiol (Hefei, China) provides Luria–Bertani Broth, Tryptic Soy Broth, and agar powder. RS-1 cells, L929 cells, and RAW264.7 cell-specific medium were obtained from Gibco (China). MRSA (ATCC 43,300) and E. coli (ATCC 25922). All other materials were acquired from Sigma-Aldrich (USA). Unless otherwise indicated, all other reagents and solvents were as received and classified as analytical grade of purity.
Synthesis
2.1
To make the PFASs@Ga composites, 0.1 mmol of Ga salt and 0.2 mmol of the perfluoroalkyl substances (Undecafluorohexanoic acid (PFHxA), Pentadecafluorooctanoic acid (PFOA), Heneicosafluoroundecanoic acid (PFUnA) were dissolved separately in 3 mL of distilled water and 5 mL of dimethylacetamide (DMA). A mixture of 40 μL HNO_3_ was stirred at ambient temperature for 30 min. The resulting solution was transferred into a 10 mL Pyrex tube and heated at 85 °C for 2 days. The final material was obtained after washing the product with water and DMA three times each by centrifugation at 7000 rpm for 10 min, followed by air drying.
Evaluation of physical properties/Physicochemical characterization
2.2
The samples were then analyzed using FTIR (Fourier Transform Infrared Spectroscopy) on a Bruker Spectrometer (Germany) (with ATR) from Thermo Fisher. The wavenumber range was 4000–400 cm^−1^. X-ray diffraction analysis was performed on Ga-based composite crystal structures. The JEM-1230 Transmission Electron Microscope (TEM) (Jeol, Japan) operated at 100 kV and was used to verify the PFASs@Ga composites. Before TEM analysis, the composite was suspended in ethanol, then applied to carbon-coated copper grids and dried overnight to remove the ethanol. To further investigate the structural properties of the PFASs@Ga composites, scanning electron microscopy (SEM) was performed using a SU8010 (Hitachi, Japan). The samples were covered with gold nanoparticles (less than 20 nm thick) for 30 s under vacuum to improve conductivity and imaging. Elemental analysis was performed using an Energy-Dispersive X-ray (EDX) spectrometer (S4800 FEG, Hitachi, Japan), which provides cross-sectional elemental profiles. The gallium concentration in the PFASs@Ga composites was determined using an Inductively Coupled Plasma Mass Spectrometer (ICP-MS) (Agilent 7500ce, Tokyo, Japan).
Oxygen absorption assay
2.3
To perform the O_2_-loading test, the different PFAS@Ga composites were prepared accordingly. Then, 5 mL of deionized water was added to a 20 mL penicillin bottle, which was wrapped with silicone oil. The oxygen level in the water was measured using a portable dissolved oxygen meter probe (INESA (JPB-607A, China). Subsequently, 5 mL of PFASs@Ga composite solution, at the same concentration for each composite, was injected beneath the silicone oil layer in each bottle using a syringe. The equilibrium oxygen level was then recorded. Deionized water served as the control.
Oxygen-release kinetics
2.4
PFAS@Ga composites were prepared and soaked in oxygen to evaluate their O_2_ release behavior. Deoxygenated deionized water (5 mL) was added to a 20 mL penicillin bottle sealed with silicone oil. Dissolved oxygen level was measured at exact time intervals using an oxygen electrode probe inserted just below the solution surface. Then, 5 mL of the oxygen-saturated PFASs@Ga composite solution at a known concentration was injected beneath the silicone oil layer in the bottle using a syringe. Oxygen concentration was continuously monitored throughout the process. Oxygen-saturated deionized water was used as a control.
ICP for gallium contents and release of ions
2.5
To evaluate the Ga content and release profile of PFASs@Ga composites, an Inductively Coupled Plasma-Mass Spectrometer (ICP-MS, Agilent 7500ce; Tokyo, Japan) was used. Equal-sized samples of each composite were immersed in PBS at an incubation temperature of 37 °C at 40 rpm. PBS was collected and replenished at several time points (hours) during the assay. Furthermore, the initial gallium content of the composites themselves was measured by direct ICP-MS.
Cell experiments
3
CCK-8 assay
3.1
The toxicity of the developed PFAS@Ga composites was first evaluated using a CCK-8 assay in macrophage (RAW 264.7 cells), RS1 cells, and L929 cells. The PFAS@Ga composites samples underwent sterilization with 70% ethanol, followed by three rinses with sterile PBS and exposure to UV light for 24 h. Afterwards, the samples were put in DMEM culture medium containing serum for up to 24 h. All procedures were carried out in a laminar flow hood.
The 6 × 10^3^ RAW 264.7 cells, RS1 cells, and L929 cells were then seeded in each well of a 96-well plate and incubated at 37 °C for 24 h. Later, we added the PFAS@Ga composites into each well to replace the medium. The cells were then cultured for 24 and 48 h. At these time points, each well was treated with a new medium supplemented with 10% CCK-8 and incubated for an additional 4 h. They placed the plate in the microplate reader at 450 nm for the optical density measurement. The negative control was cells not exposed to the PFAS@Ga composites.
Cell live/dead staining
3.2
The live/dead cell staining method was used to assess cell viability and visually determine cell vitality. Macrophages (RAW 264.7) cells, RS1 cells, and L929 cells were cultured in cell culture dishes at a density of 6 × 10^4^ cells per well and incubated for 12 h. The PFAS@Ga composite containing the medium was then added to the dishes. After 24 h of culture, live and dead cells were stained using the calcein-AM/PI staining kit according to the manufacturer's guidelines. Imaging was done using a Nikon confocal microscope (Japan).
In vitro antibacterial study
4
Micro-broth dilution method
4.1
Each sample was configured as a PBS solution. The bacterial concentration in Luria-Bertani (LB) medium was 10^5^ CFU/mL. Various samples were added to obtain final concentrations in 0 to 125 μg/mL in a 96-well plate. After adding the resazurin indicator to each well, the positive and negative control groups were established. All the groups were incubated at 37 °C for 12 h. When live bacteria were present, resazurin changed from blue to pink; when dead bacteria were present, it did not.
Spread plate method
4.2
A bacterial dispersion was diluted to 10^5^ CFU/mL with phosphate-buffered saline (PBS, pH 7.4). 50 μL of diluted bacterial suspension was mixed with 100 μg/mL of different materials solution after incubation for 6 h at 37 °C. The sterile PBS was used to dilute the final bacterial suspension to a suitable concentration. A total of 50 μL of the diluted bacterial suspension was spread on the solid medium and incubated overnight at 37 °C for counting the number of colonies. Each experiment was performed three times in parallel. The following Equation (1) is used to calculate the bacterial survival ratio.
NC represents the number of colonies in the control, and NS denotes the number of colonies in the sample.
Morphological observation by SEM
4.3
To observe the bacteria using SEM, they were treated with the PFASs@Ga composites and washed 3 times with PBS. Subsequently, the bacteria were fixed in 2.5% glutaraldehyde at 4 °C overnight. The bacteria were fixed and were subjected to a series of EtOH solutions (30%, 50%, 70%, 80%, 90%, and 100%). SEM support was fixed on the samples and dried under a vacuum. The acquired microorganisms were sputter-coated with gold and observed by SEM at 15.0 kV.
In vitro live/dead bacterial cell staining
4.4
After the antibacterial assay, pre-processing for SEM was performed, during which 500 μL of the bacterial suspension was incubated with the SYTO-9/PI double-stain kit in the dark for 30 min. For this process, a dyeing method was used to stain bacterial cells. Live bacterial cells were stained with green fluorescence, called SYTO-9, while dead bacterial cells were also stained with red fluorescence, called propidium iodide (PI). The CLSM was used to observe the bacterial samples placed on a glass slide. The live bacteria were stained with SYTO-9, which emits green fluorescence, while the dead bacteria were stained with PI, which emits red fluorescence.
Characterization of biofilms against MRSA and E. coli
4.5
In the crystal violet staining method, MRSA and E. coli were diluted in TSB to 10^7^ CFU/mL. After that, 1 mL of the bacterial solution was added to the 24-well plates and incubated at 37 °C for 24 h. After incubation, the plates were cleaned 3 times with PBS and allocated to different treatment groups. After treatment, the plates were washed again with PBS and dried for 30 min. The next step consisted of staining with a 5% (v/v) crystal violet solution for 1 h, followed by the addition of acetic acid (33%, v/v) after removal of the staining solution. Finally, the absorbance value was measured at 590 nm. In the fluorescence staining method, bacterial slides were first placed in a 24-well plate containing MRSA and E. coli. The same method was used to process and culture the E. coli biofilms as in the crystal violet assay. After processing, the slides were washed three times with PBS. After settling, 3 μL of a mixture of SYTO9 and PI dyes was added to the 24-well plates containing the slides, which were incubated for 20 min. A fluorescent confocal microscope was used to visualize MRSA and E. coli biofilms.
In vivo wound healing
4.6
The Animal Experiments were approved by the Animal Ethics Committee of Wenzhou Medical University (Approval No. xmsq2022-0930). The study used male BALB/C mice (6 weeks old) obtained from Beijing Vital River, China. Initially, their back hair was shaved using Veet hair removal cream. A subcutaneous model in mice was developed, and concentrated MRSA (1 × 10^7^ CFU/mL) was administered subcutaneously on the sides of the mice's backs on day −1. The mice were randomly divided into four groups: PBS, PFHxA@Ga, PFOA@Ga, and PFUnA@Ga. The PBS and PFASs mice study was carried out separately. The first group received only PBS, and the other groups received 100 μg/mL of the PFASs@Ga composites described above. Photographs of wound healing were taken on days 1, 3, 7, 14, and 21 after treatment. At the last point, the mice were euthanized, and the wound site tissues were collected for colony counts, histopathology, and immunofluorescence analysis.
Analysis of wound healing
4.7
After euthanasia, the skin around the wound was carefully cut out for histology. A 4% paraformaldehyde solution fixes the collected tissues and organs for 24 h. After tissues were detached, they underwent various staining actions to detect markers, with H&E, Masson's trichrome, CD31 (platelet endothelial cell adhesion molecule-1), CD86, and CD206 (M1 and M2 markers, respectively). Histology stains prepare slides for further study. The stained slides were viewed and photographed using a confocal microscope. Then, tissue sections were subjected to fluorescence quantification using image analysis software (ImageJ) to assess the anti-inflammatory response and wound-healing efficacy. The hearts, livers, and kidneys of the mice were also collected for H&E and Masson staining to evaluate biosafety.
Statistical analysis
4.8
All experiments, including in vitro antimicrobial assays, biofilm formation, wound size measurements, and body weight measurements, were conducted in triplicate. Statistical analyses were made using GraphPad Prism 7.04 and Origin 8.5. One-way ANOVA followed by Tukey's post hoc test was used for multiple group comparisons, while Student's t-test was applied for differences between two groups. Data are presented as mean ± standard deviation (SD). Statistical significance was indicated as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Results and discussion
5
Synthesis and characterization of Ga-based nanocomposites
5.1
Uniform PFHxA@Ga, PFOA@Ga, and PFUnA@Ga composites were successfully synthesized via a hydrothermal reaction between the corresponding perfluoroalkyl acid (0.2 mmol each) and gallium nitrate hydrate (0.1 mmol) in a deionized water/N,N-dimethylacetamide/nitric acid solvent system. After brief mixing, the solutions were incubated at 85 °C for 2 days to yield a well-defined rod-like PFASs@Ga composites (Fig. 2A). All compositions yielded rod morphologies under a synthetic protocol that allows further structural and functional studies. SEM images show the morphology of the composite. In Fig. 2B, observe that the composites are an elongated structure, measuring roughly 1 μm in length. Fig. 2C shows the PFASs@Ga composites’ TEM morphology and element mapping. The composites, PFHXA@Ga, PFPAFOA@Ga, and PFUA@Ga, have elongated structures, as shown by the scale bar (500 nm). The elemental mapping shows oxygen (O) in green, gallium (Ga) in yellow, fluorine (F) in blue, and carbon (C) in red. Elemental mapping confirmed the successful incorporation of Ga^3+^ ions and PFASs.Fig. 2. Preparation and Characterization of PFASs@Ga composites (A). Schematic Illustration of preparation of PFASs@Ga composites (B). SEM images of PFASs@Ga composites (scale bar: 500 nm) (C). TEM images and corresponding elemental mapping images of PFASs@Ga composites (scale bar: 500 nm) (D). XRD pattern of PFASs@Ga composites (E). FTIR spectra of PFASs@Ga composites (F). Ga concentration in PFASs@Ga composites (G). Release profile of gallium ions from PFASs@Ga composites in different times (hr) (H). Measurement of oxygen loading capacity of PFASs@Ga composites (I) Oxygen release kinetics of PFASs@Ga composites. Data are presented as mean ± SD (n = 3).Fig. 2
The XRD of the PFASs@Ga composites (PFHxA@Ga, PFOA@Ga, and PFUnA@Ga) is shown in Fig. 2D. The XRD designs show crystalline characteristics for the composites. It reveals that PFUnA@Ga shows sharp peaks at 2θ = (020), (110), (111), and (221), signifying crystalline character. On the other hand, PFOA@Ga presents sharp peaks that are, though, less pronounced than those of PFUnA@Ga. This indicates differences in crystallinity. The peaks of the PFHxA@Ga composite are broader and less defined, indicating a formless nature. FTIR spectra also confirm the results by highlighting the functional groups in the composites. The absorption bands assigned to Ga–O confirm the incorporation of gallium in PFUnA@Ga. The absorption bands of PFOA@Ga are similar but not identical to those of PFOA; however, their concentrations change slightly. The spectrum of PFHxA@Ga showed substantial absorption bands for the nitrate ion (NO_3_^−^) and the hydroxyl group (OH), indicating their presence (Fig. 2E). FTIR spectra of PFASs are presented in Supplementary Fig. S1.
To evaluate the release profile of Ga from PFASs@Ga composites, an Inductively Coupled Plasma-Mass Spectrometer (ICP-MS, Agilent 7500ce, Tokyo, Japan) was used. To determine the early Ga loading, the Ga content in the PFASs@Ga composites was directly measured (Fig. 2F). More uniformly sized samples of PFASs@Ga composites were immersed in Phosphate Buffered Saline (PBS) and incubated at 37 °C at 40 rpm for different time intervals, as shown in Fig. 2G. At each time point, gallium content was analyzed in the collected supernatant PBS, and then fresh PBS was added. According to ICP-MS analysis (Fig. 2F), different Ga contents were detected in PFASS@Ga composites. The PFUnA@Ga composite showed the highest Ga concentration, followed by PFOA@Ga, while PFHxA@Ga showed the lowest Ga detection. These findings indicate that the PFUnA@Ga composite has the highest Ga-loading volume, suggesting greater potential for Ga release in subsequent studies. After that, we investigated the capability of Ga-based nanocomposites to absorb and release oxygen. As depicted in Fig. 2H, the PFUnA@Ga demonstrated a significant reduction in dissolved O_2_ over the duration of the experiment, indicating a more pronounced O_2_ absorption capacity as compared to the PFHxA@Ga, PFOA@Ga, and control, which showed only minor changes. PFUnA@Ga released substantial amounts of O_2_ and maintained release over prolonged periods (6 days; Fig. 2I). Conversely, PFHxA@Ga and PFOA@Ga released much less O_2_ than the control. The findings confirm that PFUnA@Ga has a better capacity for O_2_ storage and delivery than the other composites.
In vitro cell assays
5.2
In tissue engineering, the biosafety of materials meant for versatile dressing preparation is vital. Here, we first assessed the cytocompatibility of the synthesized PFASs@Ga composites (Supplementary S2). To determine cell survival, the CCK-8 assay was performed after culturing RAW 264.7 macrophages, RS-1 cells, and L929 cells with these composites for 24 and 48 h, respectively. As shown in A and B of Fig. 3 and Supplementary S3(A), the results of the CCK-8 assay for RAW 264.7, RS-1 cells, and L929 cells treated with PFASs@Ga composites remained above 95%, indicating no cytotoxicity. After 24 h of treatment, RAW264.7, RS-1, and L929 cells were stained with a live/dead fluorescent dye. As shown in Fig. 3C and Supplementary S3(C), live cells were identified by green fluorescence (calcein-AM).Fig. 3. Cellular study conducted on PFASs@Ga composites (A). CCK-8 method assessment of cytotoxic effects exerted by PFASs@Ga composites on RAW 264.7 (B) and RS-1 cells (C). Outcomes of Calcein-AM/PI for test cells after 24 h of exposure to PFASs@Ga composites (scale bar: 50um). (D) Quantitative analysis of Live-Dead of RAW 264.7 cells (E). Quantitative analysis of Live-Dead of RS-1 cells. Data are presented as mean ± SD (n = 3).Fig. 3
In contrast, dead cells showed negligible red fluorescence (PI), indicating that all samples were well compatible during the test. Also, the cell membrane integrity was intact in all treatment groups. The quantitative results in Fig. 3D and E, and Supplementary S3(B) showed that most RAW 264.7, RS-1, and L929 cells remained viable upon treatment with the composites, with little cell death. In summary, the cell viability and morphology of PFHxA@Ga, PFOA@Ga, and PFUnA@Ga composites are observed to be high. This further proves their biocompatibility for potential biomedical applications.
Antibacterial activity
5.3
In assessing the efficacy of synthesized PFASs@Ga composites against the bacteria MRSA and E. coli. Using the microbroth dilution method, we determined the minimum inhibitory concentrations (MICs) of the PFASs@Ga composites against bacterial strains [40,41]. Figure Supplementary S4 in the Supporting Information shows that PFUnA@Ga inhibits both bacteria. PFUnA@Ga was found to be efficient at inhibiting the growth of MRSA and E. coli; the MICs were 100 μg/mL for both. The spread plate method was used to confirm the findings from the microbroth dilution method. The PFUnA@Ga found in Fig. 4A showed 'strong' inhibition against MRSA and E. coli. It showed strong antibacterial activity, with MICs of 95.08% and 99.71% against MRSA and E. coli, respectively (Fig. 4B and C). Also, time-kill curves indicated PFUnA@Ga could inhibit both bacterial species by 95% or above within 6 h (Fig. 4D, E, and 4F). PFUnA@Ga also demonstrated significant bactericidal activity against E. coli. E. coli is a Gram-negative bacterium because it possesses an outer membrane, which confers greater antibiotic resistance [42]. A 99% reduction in bacterial count was observed after 2h of incubation. Fig. 4 (G, H, I) depicts the experiment, demonstrating that PFUnA@Ga, at a MIC of 100 μg/mL, exhibited high killing efficacy against MRSA and E. coli. To assess the direct effect of PFASs@Ga composite treatment on bacterial cells, SEM was used to examine morphological changes [43]. As shown in Fig. 4J, the bacterial figures between the control, PFHxA@Ga, PFOA@Ga groups, and the PFUnA@Ga treated group were significantly different. The control, PFHxA@Ga, and PFOA@Ga groups exhibited no drastic morphological changes, with smooth, intact bacterial exteriors. In comparison, holes and wrinkled surfaces were detected in the PFUnA@Ga-treated bacteria [44]. To study the antibacterial effect in vitro, the live/dead bacterial cell staining method was used to assess bacterial viability by confocal laser scanning microscopy (CLSM) [45]. The MRSA and E. coli that emitted green fluorescence and less red fluorescence within the control groups (Fig. 4K, L, and M) had negligible therapeutic effects [46]. The red fluorescent signal was highest in the PFUnA@Ga group, indicating high bacterial death. A supermolecular system called a biofilm, composed of proteins, lipids, and sugars, can protect bacteria [47]. Deeper biofilm coloration indicates a greater antibacterial inhibition. Fig. 4 (N, O) and Supplementary S5 show the color and inhibition percentage of biofilms treated by PFASs@Ga composites. It shows that PFUnA@Ga can significantly inhibit biofilm growth. The finding shows that PFUnA@Ga has good broad-spectrum bactericidal activity. It works by interfering with the internal metabolism of the bacterium and damaging the structure of the bacterium.Fig. 4. In vitro antimicrobial properties of PFASs@Ga composites (A). Images of MRSA and E. coli colonies on agar plates following various treatments of PFUnA@Ga (B). Bacterial survival rate of MRSA (C). Bacterial survival rate of E. coli (D). Images of MRSA and E. coli colonies on agar plates following treatment of PFUnA@Ga during various times (Minutes) (E). Time-kill curve of PFASs@Ga composites of MRSA for 120 min (F). Time-kill curve of PFASs@Ga composites of E. coli for 120 min (G). Images of MRSA and E. coli colonies on agar plates following treatment of PFASs@Ga composites (100 μg/ml) (H). Bacterial survival rate of MRSA (I). Bacterial survival rate of E. coli (J). SEM image of MRSA and E. coli (K). Confocal scanning Microscopy of live/dead MRSA and E. coli (L). Quantitative statistics of live/dead of MRSA (M). Quantity statistics of live/dead of E. coli (N). Biofilm staining images of MRSA and E. coli (O). Biofilm disruption of MRSA and E. coli after treatment with PFASs@Ga composites, shown through live/dead. Data are presented as mean ± SD (n = 3).Fig. 4
In vivo evaluation of infected wound healing
5.4
To further assess the antibacterial efficacy of PFAS@Ga composites in wound healing, they were tested in a full-thickness skin defect model infected with MRSA [48]. The mice received MRSA inoculation at the wound site, followed on day 1 by treatment with PBS, PFHxA@Ga, PFOA@Ga, and PFUnA@Ga, and, secondly, separate PBS, Ga, and PFAS (PFHxA, PFOA, and PFUnA) groups were carried out, as shown in Fig. 5A. Wound healing was monitored and recorded on days 1, 3, 7, 14, and 21. Representative photographs were monitored, and wound-closure traces were analyzed. As shown in Fig. 5B and Supplementary S8(A), the corresponding group of mice developed abscesses on the first day of the post-operative study. Each abscess was characterized by substantial skin swelling and numerous white lesions on the wound surface. Similar results were observed in the remaining mice. After identifying tissue fluid and pus, it was concluded that the bacterial infection model had been successfully established [49]. The wounds treated with PFUnA@Ga after 21 days were nearly completely healed, while the PBS, PFHxA@Ga, PFOA@Ga, PFUnA@Ga, and secondly separated PBS, Ga, and PFASs groups were incompletely healed (Fig. 5C and Supplementary S8(A). Also, PFUnA@Ga showed strong antibacterial activity by inhibiting bacterial growth via sustained release of the antibacterial agent.Fig. 5. Optical images illustrating the healing process across various days **(A).**Schematic depicting the treatment pattern in the MRSA-infected mice model **(B).**Photographs documenting the progression of the wound on days,1,3,7,14, and 21 **(C).**Diagram depicting the wound area across different groups on different days **(D).**Statistical graph of residual lesion area in the process of 21 days of treatment (F). Microscopic analysis of mice wound area using H&E stain (G). Microscopic analysis of the wound area using Masson stain. Data are presented as mean ± SD (n = 6).Fig. 5
On the other hand, PFUnA@Ga rapidly absorbs wound exudate, reduces locoregional moisture, and provides a good external barrier against further bacterial invasion and contamination [50]. This double use of the present invention produced a dry, sterile microenvironment, thereby providing an excellent environment for wound healing and tissue regeneration. Quantitative analysis exposed that the wound-contraction percentages in the PBS, PFHxA@Ga, and PFOA@Ga groups were 52.79 ± 7.20% and 87.31 ± 3.28% of the early wound area (Fig. 5D). The wound areas of the separated PBS, Ga, and PFASs groups are presented in Figure Supplementary S8(B), respectively. Notably, the PFUnA@Ga group showed significantly reduced scar formation compared to other groups and attained the maximum wound healing rate of 99.62 ± 0.30% at the end of the 21-day observation period. To imagine residual bacteria in mouse wound tissues, tissues were collected and exposed to quantitative CFU analysis via homogenization and grinding [48]. PFUnA@Ga has improved antimicrobial performance compared to the PBS, PFHxA@Ga, and PFOA@Ga, as illustrated in Fig. 5E. The number of bacteria in the tissue was significantly reduced after treatment with PFUnA@Ga, attaining an impressive 91% bactericidal rate (Figure Supplementary S6). The extent of wound healing was one indicator that might not be enough to demonstrate repair in the internal subcutaneous tissues.
Furthermore, Figures Supplementary S7 and S8 (C) show negligible differences in weight change across groups, emphasizing the biosafety of the PFASs@Ga composites and the PBS, Ga, and PFASs groups. These findings indicate that PFUnA@Ga holds promise as an effective anti-infective treatment. Aligned with the concept of intelligent wound management highlighted for responsive hydrogel dressings, the PFUnA@Ga system integrates microenvironment-dependent antibacterial action, immunomodulation, and pro-angiogenic effects without requiring an additional carrier matrix [51]. Histological staining was completed to assess tissue quality in traumatic abscesses [52]. Hematoxylin and eosin (H&E) stain was performed for the pathological study of peri-traumatic tissues (Fig. 5F and G).
Moreover, immunohistochemical assays were performed to analyze the expression levels of pro-inflammatory factors (CD86 and CD206) and an angiogenic marker (CD31) in wound tissues [53]. According to Fig. 6A, the CD-86 expression levels decreased over time after PFUnA@Ga treatment compared to PBS, PFHxA@Ga, and PFOA@Ga. The anti-inflammatory effect was especially notable on Day 21 Fig. 6A. Pro-inflammatory cytokines remained at relatively high levels in PBS, PFHxA@Ga, and PFOA@Ga, indicating a chronic inflammatory response in bacterially colonized wound beds. In association with PFUnA@Ga treatment, the virtual Flo signals of these pro-inflammatory factors were notably reduced. As shown in Fig. 6B, quantitative analyses exposed that the PFUnA@Ga group exhibited a statistically significant decrease in CD-86 expression compared with PBS, PFHxA@Ga, and PFOA@Ga. Also, PFUnA@Ga treatment lowered CD86 expression by 90% relative to PBS, PFHxA@Ga, and PFOA@Ga, indicating that PFUnA@Ga promotes wound healing through an anti-infection effect.Fig. 6Immunofluorescence analysis of PFASs@Ga compounds. (A) Representative images of CD86, CD206, and CD31 immunofluorescence staining on day 2 (B). Statistical data of (B) CD86, (C) CD206, and (D) CD31 in the swelling area (E). Histological analysis of the main organs. Data are presented as mean ± SD (n = 6).Fig. 6
Furthermore, CD31, a key signaling molecule involved in regulating neovascularization [54,55], was analyzed by immunohistochemistry to evaluate new blood vessel formation and skin wound healing. Fig. 6A shows that CD31 expression was observed in the PFUnA@Ga group, which was about 2-fold higher than in PBS, PFHxA@Ga, and PFOA@Ga on day 21, indicating strong neovascularization. These results demonstrate that PFUnA@Ga not only eradicates bacteria but also modulates the local immune microenvironment and promotes neovascularization in infected wounds. Similarly, pH-responsive injectable sodium alginate/carboxymethyl chitosan hydrogels have been reported to accelerate wound healing in infected wounds through combined bacteriostasis and immunomodulation, underscoring the importance of integrating antibacterial activity with immune regulation in advanced wound dressings [56].
Also, the in vivo biosafety of PFUnA@Ga was assessed by histological analysis of main organs (heart, liver, and kidney) after 21 days of treatment. Fig. 6 (E) shows that there were no histological irregularities in any tissue sections, representing the excellent in vivo biocompatibility of PFUnA@Ga. In addition, systemic toxicity was measured using whole-blood analysis and biochemical indicators. The study quantified red blood cells (RBCs), hemoglobin (HGB), platelets (PLTs), white blood cells (WBCs), lymphocytes (Lym), and neutrophils (Neu) (Figure Supplementary S9). Results indicated no statistically significant differences between groups, and all hematological parameters were within normal limits. Based on these outcomes, the PFUnA@Ga application to bacterially infected wounds in mice eliminates bacterial infection while enhancing collagen fiber deposition at the wound site. At the same time, such treatment demonstrated significant anti-inflammatory effects and greater pro-vascularization capacities, collectively improving the wound-healing process, via a coordinated cascade of biological responses without any negative toxicity.
Conclusion
6
In summary, the PFUnA@Ga composite is a highly effective multifunctional therapeutic with sustained Ga release and enhanced oxygen delivery. It demonstrates strong, wide-ranging effects against drug-resistant bacteria such as MRSA and E. coli. PFUnA@Ga inhibits biofilm formation, which is essential for preventing chronic infections. In infected wound models treated with PFUnA@Ga, there is more rapid wound closure, lower bacterial load, and induced angiogenesis. The immunomodulatory effects lead to downregulation of pro-inflammatory M1 macrophages, upregulation of anti-inflammatory M2 macrophages, and increased tissue vascularization.
Furthermore, the safety of PFUnA@Ga for topical wound application is confirmed by its excellent cytocompatibility with skin and fibroblast cells. In short, the PFUnA@Ga composite could represent a next-generation platform with antimicrobial and wound-healing properties that synergize bactericidal, biofilm-disrupting, immunomodulatory, and tissue-regeneration to treat complex infected wounds. The promising therapy will play an important role in Future anti-infective and wound-care treatments.
CRediT authorship contribution statement
Saadullah Khattak: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Writing – original draft, Writing – review & editing. Salim Ullah: Writing – review & editing. Muhammad Tufail Yousaf: Writing – review & editing. Jianliang Shen: Conceptualization, Funding acquisition, Supervision, Writing – review & editing. Xiaoqun Xu: Writing – review & editing. Hong-Tao Xu: Funding acquisition, Project administration, Supervision, Writing – review & editing.
Declaration of competing interest
The authors declare that they have no conflict of interest.
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