Development of a multifunctional polymer-based delivery system for anionic dye capture and emodin-mediated anti-inflammatory therapy in ARDS

2.1. Chemicals and measurements

All of the reagents along with chemicals are commercially available and don’t need to be further purified unless otherwise noted. Ultraviolet–visible (UV–vis) absorption spectra were measured using a Shimadzu UV-2600i spectrophotometer. The specific surface area and pore size distribution of the polymeric materials were determined by nitrogen adsorption–desorption measurements at 77 K using a Quantachrome EVO gas sorption analyzer. Prior to analysis, the samples were degassed under vacuum at 100°C for 10 h. Scanning electron microscopy (SEM) was conducted on a ZEISS Gemini 500 instrument to observe surface morphology. X-ray diffraction (XRD) patterns were acquired using a Bruker D8 Advance A25 diffractometer to assess the crystalline structure. Fourier-transform infrared (FT-IR) spectra were collected using a Nicolet IS10 spectrometer to identify characteristic functional groups. Raman spectra were recorded using a Horiba LabRAM HR Evolution spectrometer to probe molecular vibrations. Thermogravimetric analysis (TGA) was carried out on a Netzsch STA 2500 analyzer to evaluate the thermal stability of the materials.

2.2. Synthesis of compound 1 and CP1

The synthesis procedures of compound 1 and CP1 are provided in the Supporting Information. Detailed crystal characterization and descriptions can be found in Table S1 and Figures S1-S2. ¹H NMR spectra of compound 1 can be found in Figure S3.

2.3. Synthesis of CMCS-1-TMPSA@CP1@Emo

100 mg of CMCS was dissolved in 10 mL of CH₃CH₂OH and stirred at 70 °C until fully dissolved. Then, 150 mg of TMPSA and 5 mg of NaH were added, and the reaction was maintained for 8 h. The resulting mixture was dialyzed to remove small molecular impurities and freeze-dried to obtain CMCS-TMPSA. Subsequently, 100 mg of the intermediate was dissolved in 50 mL of ethanol, followed by the addition of 20 mg of compound 1, and the mixture was reacted at 70°C for 6 h to yield a light yellow solid, CMCS-1-TMPSA (Scheme S1). Finally, 50 mg of CMCS-1-TMPSA and 30 mg of CP1 were dispersed in a 1:1 ethanol/water mixture, followed by the addition of 15 mg of Emodin. After stirring in the dark for 24 h, the unbound drug was removed by centrifugation, and the product was freeze-dried to afford the final targeted drug delivery system, CMCS-1-TMPSA@CP1@Emo.

2.4. Determination of emodin loading capacity

A predetermined amount of CSCM-1-TMPSA@CP1 was dispersed in an ethanol solution of Emodin with a fixed initial concentration and stirred for 24 h at room temperature to ensure adsorption equilibrium. Subsequently, the suspension was centrifuged, and the supernatant was collected for analysis. The residual concentration of Emodin in the supernatant was determined by UV–Vis spectroscopy at 254 nm, using a standard calibration curve constructed from reference Emodin solutions. The amount of Emodin encapsulated in the composite was obtained by calculating the difference between the initial and equilibrium concentrations. The drug loading capacity (LC) and encapsulation efficiency (EE) were then evaluated according to the following equations (1, 2):

Where W_{Emo loaded}​ is the amount of Emodin encapsulated in the composite, W_carrier+Emo is the total weight of the W_{Emo loaded} carrier, and W_{Emo initial} is the total weight of Emodin added to the solution.

2.5. Cell culture and treatment

Human alveolar epithelial cells (A549, ATCC® CCL-185™) were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C under 5% CO₂. Cells between passages 15–20 were used. For experiments, cells were seeded in 96-well plates (1×10⁴ cells/well for CCK-8) or 24-well plates (2×10⁵ cells/well for ELISA). After 24 h attachment, cells were pretreated for 1 h with: (1) Emo-NPs (50 μg mL^-1), (2) blank nanoparticles (50 μg mL^-1), or (3) free emodin (10 μg mL^-1, equivalent to drug-loading in 50 μg mL^-1 Emo-NPs). Lipopolysaccharide (LPS; E. coli O55:B5, Sigma #L2880) was then added at 1 μg mL^-1 for 24 h. Experimental groups included: Control (untreated), LPS alone, LPS + Emo-NPs, LPS + blank NPs, and LPS + free emodin.

2.6. Cell viability assay

Cell viability was assessed using CCK-8 kit (Dojindo, Japan). After treatments, 10 μL CCK-8 reagent was added per 100 μL medium and incubated for 2 h at 37°C. Absorbance was measured at 450 nm using a microplate reader (BioTek, USA).

2.7. Cytokine measurement

Supernatants were collected after 24 h of LPS stimulation, centrifuged (1000 ×g, 10 min, 4°C), and analyzed for TNF-α (Cusabio, China), IL-6 (Cusabio, China), and IL-10 (Cusabio, China) according to manufacturer protocols.

3.1. Synthesis and characterization of CMCS-1-TMPSA@CP1@Emo

To investigate the structural composition and physicochemical properties of the CMCS-1-TMPSA@CP1@Emo nanocomposite, a comprehensive set of characterization techniques was employed (Figures 1a-e) and Figure S4. As shown in the FT-IR spectra (Figure 1a), CMCS-1-TMPSA exhibited characteristic absorption bands near 3410 cm⁻¹ (O–H/N–H stretching), 1650 cm⁻¹ (amide I, C=O stretching), and 1080 cm⁻¹ (Si–O–Si stretching), confirming the successful introduction of the silane coupling agent TMPSA onto the CMCS backbone. After the incorporation of CP1, new absorption peaks appeared at ∼1590 and ∼1410 cm⁻¹, corresponding to the symmetric and asymmetric stretching of carboxylate groups coordinated to metal centers, indicating the formation of MOF-like coordination networks in CMCS-1-TMPSA@CP1. Upon further loading of emodin, additional peaks emerged near 1620 cm⁻¹ and 1230 cm⁻¹, assignable to the C=C and C–O–C vibrations of the anthraquinone structure of emodin, respectively, demonstrating its successful encapsulation into the hybrid framework. The XRD pattern of CMCS-1-TMPSA@CP1@Emo (Figure 1b) revealed a broad diffraction peak centered around 2θ = 20°, indicative of an amorphous or partially disordered structure. The lack of sharp crystalline reflections suggests that the integration of CP1 and Emodin did not induce long-range ordering, which is favorable for forming interconnected porous networks and enhancing drug encapsulation flexibility. TGA analysis (Figure 1c) revealed a multi-step decomposition profile. The first weight loss of 8% occurred below 120°C, which is attributable to the release of physisorbed water and crystallization water molecules in CP1, consistent with its hydration formula. A second major weight loss was observed between 250–380°C, assigned to the decomposition of the organic ligand framework (CSCM-1-TMPSA). This was followed by an additional mass loss in the range of 380–480°C, corresponding mainly to the thermal degradation of the encapsulated emodin. Above 500°C, the mass stabilized gradually, and a final residue of 12% was retained at 700 C, which can be attributed to the thermally robust inorganic components originating from the CP1 coordination nodes. Nitrogen adsorption–desorption isotherms (Figure 1d) of CMCS-1-TMPSA@CP1@Emo displayed a typical type IV curve with a hysteresis loop, characteristic of mesoporous materials. The BET surface area was calculated to be approximately 226 m² g^-1, and the cumulative pore volume reached 0.82 cm³ g^-1. These values suggest sufficient pore accessibility and capacity for drug loading. The Barrett–Joyner–Halenda (BJH) pore size distribution (Figure 1e) showed that the majority of the pores were centered in the 3.5–30 nm range, further confirming the mesoporous nature of the carrier. Furthermore, the surface morphology and microstructural features of the composite were examined via SEM, with the corresponding images presented in Figure S5. Collectively, the above characterizations confirm the successful construction of a structurally stable, mesoporous nanocomposite with appropriate chemical functionality, high surface area, and drug-relevant pore dimensions. These features endow CMCS-1-TMPSA@CP1@Emo with significant potential for effective encapsulation and controlled release of emodin, meeting the demanding requirements of ARDS-targeted therapy.

Close

Figure 1.

Structural and physicochemical characterization of CMCS-1-TMPSA@CP1@Emo: (a) FT-IR spectra, (b) XRD pattern, (c) TGA curve, (d) N₂ adsorption–desorption isotherms; (e) BJH pore size distribution.

Export to PPT

3.2. Dye adsorption

To comprehensively evaluate the molecular recognition ability of the CMCS-1-TMPSA@CP1 composite prior to drug loading, representative anionic dyes—methyl orange (MO) and sodium fluorescein (FL)—were selected as model molecules to investigate their adsorption behavior systematically. This strategy is based on the fact that the natural product emodin primarily exists in its anionic form under neutral to mildly alkaline conditions, making these dye adsorption studies a relevant mimic for its loading behavior. Benefiting from the introduction of compound 1-modified chitosan and the abundant functional sites on the CP1 framework, the composite material exhibits excellent charge interaction capacity and structural compatibility. It is thus expected to show strong affinity for small anionic species. As shown in Figure 2(a), the UV–vis absorbance of MO decreased rapidly upon contact with CMCS-1-TMPSA@CP1 and reached equilibrium within 10 min, indicating a fast and efficient adsorption process. Specifically, the CP1 framework contains abundant carboxylate (–COO⁻) and hydroxyl (–OH) groups that promote electrostatic attraction and hydrogen bonding with anionic dyes, open metal coordination centers that provide Lewis acid–base binding sites, and π-conjugated aromatic domains that enable π–π stacking with the dye molecules. These synergistic interactions collectively accelerate adsorption and enhance affinity toward MO and FL. The adsorption kinetics (Figure 2b) conformed well to the pseudo-second-order model:qt=k₂q_e²t/(1+k₂q_et), with fitted equations q_t=42.9190(1−e^−0.4409t) (R² = 0.9952) and q_t=24.7986t/(1+0.4687t) (R² = 0.9980), indicating chemisorption as the dominant mechanism. Recyclability tests (Figure 2c) demonstrated that even after five adsorption–desorption cycles, the removal efficiency of MO remained above 95%, reflecting the excellent structural stability and reusability of the composite. Similarly, FL was used to evaluate the generality of the adsorption behavior. As shown in Figure 2(d), the absorbance of FL decreased significantly within the first 10 min, followed by a plateau. The corresponding kinetics (Figure 2e) also fitted the pseudo-second-order model, with equations qt=0.2193(1−e^−0.0302t) (R²=0.9706) and qt=0.0095t/(1+0.0405t) (R²=0.9853), further confirming the chemisorption-driven adsorption process. After five cycles, the removal efficiency of FL was still maintained at approximately 87% (Figure 2f), demonstrating the composite’s robust and repeatable adsorption performance. Building on these findings, we further explored the adsorption isotherms of CMCS-1-TMPSA@CP1 toward different dye types. As shown in Figure 3(a), in the concentration range of 0–200 mg L^-1, the composite exhibited remarkable adsorption capacities for the anionic dyes MO (181.8 mg g^-1) and FL (86.7 mg g^-1), while its adsorption capacity for the cationic dye methylene blue (MB) was negligible (0.6 mg g^-1). Notably, although MO and MB share similar molecular sizes, FL is relatively larger, indicating that the mesoporous structure of the composite facilitates the entry and adsorption of small-sized anionic dyes such as MO. Furthermore, the adsorption isotherms for MO and FL (Figures 3b and c) showed excellent agreement with the Langmuir model (R² > 0.99), indicating that the adsorption process predominantly follows a monolayer mechanism with uniformly distributed adsorption sites and no significant intermolecular interactions. CMCS-1-TMPSA@CP1 demonstrates fast adsorption kinetics, high adsorption capacity, broad applicability toward anionic dyes, and outstanding reusability. These properties not only underscore its strong affinity toward small anionic molecules via charge interactions and site-specific recognition, but also provide a solid theoretical and experimental foundation for its application in the controlled loading and targeted delivery of natural compounds such as emodin.

Close

Figure 2.

Adsorption performance of CMCS-1-TMPSA@CP1 toward anionic dyes: (a) Transient absorption spectra of MO; (b) Kinetic modeling of MO adsorption; (c) Recycling performance for MO; (d) Transient absorption spectra of FL; (e) Kinetic modeling of FL adsorption; (f) Recycling performance for FL (Bars of different colors represent the efficiency at a specific location under varying cycle counts).

Export to PPT

Close

Figure 3.

Adsorption isotherm behavior of CMCS-1-TMPSA@CP1: (a) Adsorption capacities toward different dyes; (b) Isotherm model fitting for MO (Langmuir (blue) and Freundlich (yellow) model fittings for MO adsorption); (c) Isotherm model fitting for FL (Langmuir (blue) and Freundlich (yellow) model fittings for FL adsorption).

Export to PPT

3.3. Loading of emodin

To evaluate the Emo loading capacity and recycling stability of the CSCM-1-TMPSA@CP1@Emo composite carrier, we conducted a comprehensive study of its adsorption behavior. As shown in Figure 4(a), the amount of Emo adsorbed increased rapidly within the first 100 min and gradually reached equilibrium around 400 min, achieving a maximum loading capacity of 0.24 g g^-1. This indicates that the composite carrier possesses favorable drug binding kinetics and excellent loading efficiency. To assess its reusability, five consecutive loading–release cycles were performed (Figure 4b). The results showed that even after multiple cycles, the loading capacity remained above 0.21 g/g with only a slight decline, reflecting the material’s structural stability and regeneration potential. Furthermore, the UV–vis absorption spectra in Figure 4(c) reveal the real-time adsorption of Emo from aqueous solution. The characteristic absorption peaks decreased significantly within just 5 min, further confirming the composite’s rapid and efficient binding affinity for Emo. As illustrated in Figure 4(d), the Emo release performance remained above 85% across five cycles, demonstrating excellent reproducibility and release stability. The CSCM-1-TMPSA@CP1@Emo composite exhibits outstanding adsorption kinetics, high drug loading capacity, and excellent cycling stability during the Emodin loading and release process. These findings highlight its strong potential as a drug delivery platform for the treatment of inflammatory pulmonary diseases such as ARDS.

Close

Figure 4.

(a) Emo adsorption kinetics of CMCS-1-TMPSA@CP1@Emo (Langmuir (blue) and Freundlich (yellow) model fittings for MO adsorption); (b) Emo loading capacity over five adsorption–desorption cycles; (c) UV–vis spectra showing Emo adsorption over time; (d) Emo release efficiency across five cycles (Bars of different colors represent the efficiency at a specific location under varying cycle counts).

Export to PPT

3.4. Activity test

LPS stimulation significantly compromised alveolar epithelial cell viability compared to untreated controls, inducing profound cytotoxic damage (p < 0.0001). Intervention with CMCS-1-TMPSA@CP1@Emo (Emo-NPs) completely reversed this pathological effect, restoring cellular homeostasis to levels statistically indistinguishable from healthy controls. This protective outcome significantly surpassed free emodin treatment (p < 0.01), while blank nanoparticles demonstrated no therapeutic benefit (Figure 5a). Analysis of inflammatory mediators revealed Emo-NPs potently suppressed the LPS-induced cytokine storm. Secretion of pro-inflammatory markers TNF-α (Figure 5b) and IL-6 (Figure 5c) was drastically reduced compared to injury models (p < 0.0001). Although free emodin moderately attenuated these cytokines (p < 0.0001 vs. injury group), its efficacy remained substantially inferior to nanoformulated emodin (p < 0.0001). Most notably, Emo-NPs uniquely amplified anti-inflammatory responses, elevating IL-10 production to levels significantly exceeding both injury models and free emodin treatment (p < 0.0001; Figure 5d). The collective findings establish that nano-encapsulation critically enhances emodin’s capacity to simultaneously mitigate cytotoxic damage, suppress pro-inflammatory signaling, and activate endogenous anti-inflammatory mechanisms.

Close

Figure 5.

Therapeutic effects of Emo-NPs on LPS-Injured A549 Cells. (A) Cell viability. (b) TNF-α, (c) IL-6, and (d) IL-10 levels in supernatants (ELISA). **p < 0.01, ***p < 0.001, ****p < 0.0001.

Export to PPT