A smart INH-loaded nanosensor for theranostic intervention in Mtb-infected macrophages

2.1. Materials and characterization

All chemical reagents and solvents were of analytical grade and used without further purification unless otherwise specified. Powder X-ray diffraction (PXRD) patterns were recorded using a Bruker D8 Advance diffractometer (Cu Kα radiation, λ = 1.5406 Å) over a 2θ range of 5°–40° with a step size of 1° min^-1 to assess the crystallinity and phase purity of the samples. Single-crystal X-ray diffraction (SCXRD) data were collected on a Bruker SMART APEX II diffractometer and refined using the SHELXTL software package to resolve the coordination configuration at the atomic level. Thermal stability was evaluated via thermogravimetric analysis (TGA) using a TA Instruments Q500 analyzer under nitrogen flow with a heating rate of 10°C min^-1 from 30 to 800°C. Specific surface area and pore size distribution were determined by nitrogen adsorption-desorption isotherms at 77 K using a Micromeritics ASAP 2460 analyzer, with prior activation at 90°C under dynamic vacuum for 12 h. Fourier-transform infrared (FT-IR) spectra were acquired on a Bruker Tensor II spectrometer in the range of 4000–400 cm⁻¹ using the KBr pellet method to confirm functional group features. Surface morphology was observed by field-emission scanning electron microscopy (FE-SEM) using a ZEISS Sigma 300 instrument at an accelerating voltage of 10 kV, revealing well-defined block-like crystal structures. Photoluminescence (PL) properties were examined using a Hitachi F-7000 fluorescence spectrophotometer at room temperature, with an excitation wavelength of 365 nm and an emission range of 400–600 nm. The results demonstrated strong and stable intrinsic fluorescence, supporting its potential application in ratiometric detection.

2.2. Synthesis of 1-HA-DPTMS@CP1@INH

Compound 1 (0.10 mmol) was conjugated to hyaluronic acid (HA, 50 mg) via esterification using Dicyclohexylcarbodiimide (DCC) (0.15 mmol) and 4-Dimethylaminopyridine (DMAP) (0.10 mmol) in anhydrous CH₃CN (10 mL) at room temperature for 24 h, yielding 1-HA after dialysis and lyophilization. Subsequently, 1-HA (50 mg) was further grafted with (3-carboxypropyl)trimethoxysilane (DPTMS, 0.15 mmol) under similar conditions to obtain 1-HA-DPTMS. The cobalt-based coordination polymer CP1 was synthesized via solvothermal reaction and isolated by centrifugation and drying. The final nanocomposite was fabricated by co-assembling 1-HA-DPTMS (10 mg) with CP1 (5 mg) in Phosphate-Buffered Saline (PBS) buffer (pH 7.4), followed by INH (1 mg) loading through incubation and adsorption equilibrium. The overall yield of 1-HA-DPTMS@CP1@INH was approximately 78%, indicating good synthesis efficiency. The resulting 1-HA-DPTMS@CP1@INH was purified by centrifugation and lyophilized for further use (Scheme S1).

2.3. Cell culture

RAW 264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) high-glucose medium (Gibco, USA) supplemented with 10% fetal bovine serum (Hyclone, USA) and 1% penicillin-streptomycin (Sigma-Aldrich, USA) at 37°C in a humidified atmosphere containing 5% CO₂. Cells in the logarithmic growth phase were seeded into 6-well plates (Corning, USA) at a density of 1 × 10⁶ cells/well and incubated for 24 h to allow cell attachment. The Mycobacterium tuberculosis H37Rv standard strain used in this study was obtained from the American Type Culture Collection (ATCC 27294, USA). The strain was authenticated by 16S rRNA gene sequencing and tested for purity by Ziehl–Neelsen acid-fast staining prior to use. For infection, RAW 264.7 cells were exposed to M. tuberculosis H37Rv at a multiplicity of infection (MOI) of 10. After 4 h of incubation, the supernatant was removed, and the cells were washed three times with sterile PBS (Gibco, USA) to eliminate extracellular bacteria. Fresh DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was then added for subsequent culture and experimental assays.

2.4. Cell grouping and intervention

Blank control group: normally cultured RAW 264.7 cells without any treatment and not infected with Mycobacterium tuberculosis. Model control group: RAW 264.7 cells only infected with Mycobacterium tuberculosis. 1-HA-DPTMS@CP1@INH group: RAW 264.7 cells infected with Mycobacterium tuberculosis were added with nanoparticles loaded with INH (Sigma-Aldrich, USA) at a specific concentration (50 μg mL^-1, with the drug content corresponding to INH concentration). Empty nanoparticle control group: RAW 264.7 cells infected with Mycobacterium tuberculosis were added with empty nanoparticles (without INH loading) at the same concentration as the nanoparticle group to exclude the influence of the nanoparticle carrier itself.

2.5. Cell viability detection

After 48 h of cell intervention, 10 μL of CCK-8 solution (Dojindo, Japan) was added to each well, and incubation was continued for 1-4 h. The absorbance (OD value) of each well was measured at 450 nm with a microplate reader (Bio-Rad, USA).

2.6. Detection of CatK and MMP9 expression in cells

Cells were collected, total RNA was extracted using Trizol reagent (Invitrogen, USA), and RNA was reverse-transcribed into cDNA according to the instructions of the reverse transcription kit (Takara, Japan). Using cDNA as a template, specific primers for CatK and MMP9 were designed to perform a qRT-PCR reaction (Applied Biosystems, USA).

2.7. Detection of cytokine secretion

Cell culture supernatants were collected, and the secretion levels of cytokines related to OATB inflammation, such as TNF-α, IL-6, and IL-1β, were detected according to the instructions of the ELISA kit (Multi Sciences, China) to evaluate the effect of 1-HA-DPTMS@CP1@INH on the cellular inflammatory response.

3.1. Structural characterization of 1-HA-DPTMS@CP1@INH

The structures of compound 1 and CP1 were determined by SCXRD, with detailed crystallographic data and structural information provided in Figures S1 and S2. To systematically verify the structural composition, physicochemical properties, and potential of 1-HA-DPTMS@CP1@INH as a drug delivery carrier, a series of characterizations, including FTIR, XRD, TGA, and Brunauer-Emmett-Teller (BET) surface area and porosity analysis, were conducted (Figure 1). As shown in Figure 1(a), the FTIR spectrum of 1-HA-DPTMS@CP1@INH exhibited new characteristic absorption peaks at 1675 cm⁻¹ (C=O stretching vibration) and 1623 cm⁻¹ (–NH–C=O amide bond), confirming the successful incorporation of INH via non-covalent interactions. Additionally, peaks at 1043 cm⁻¹ and 801 cm⁻¹ correspond to the Si–O–Si skeletal vibrations, indicating successful grafting of DPTMS and the formation of an organosilicon framework.

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Figure 1.

Characterization of 1-HA-DPTMS@CP1@INH: (a) FTIR spectra, (b) XRD patterns, (c) TGA curve, (d) N₂ adsorption–desorption isotherms, and (e) Pore size distribution curve.

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XRD analysis (Figure 1b) further confirmed that while 1-HA-DPTMS displayed distinct crystalline peaks, these peaks were significantly diminished or broadened after the incorporation of CP1 and INH, indicating a partial transition to an amorphous state. This structural change is beneficial for the homogeneous dispersion of drug molecules. The TGA curve (Figure 1c) revealed two major weight loss stages within the 200–500°C range, with a total weight loss of approximately 72%, attributable to the decomposition of the organic shell, ligands, and drug components. This confirms the successful loading of functional components and reasonable thermal stability.

Furthermore, the nitrogen adsorption–desorption isotherm (Figure 1d) exhibited a typical type II curve with a pronounced hysteresis loop, indicating a mesoporous structure. The BET-specific surface area was calculated to be 228.6 m² g^-1, with a pore volume of 0.42 cm³ g^-1 and an average pore diameter of approximately 6.8 nm. The corresponding pore size distribution curve (Figure 1e) revealed a dominant and narrow peak centered around 6–8 nm, further confirming the uniform mesoporous nature of the 1-HA-DPTMS@CP1 framework. Such a well-defined porous architecture facilitates efficient guest molecule diffusion and provides abundant active sites for INH loading. As shown in Supplementary Figure S3, the surface of the 1-HA-DPTMS@CP1@INH composite exhibits a typical granular distribution and a rough texture, with irregularly attached layers and sheet-like structures observed in some regions. These morphological features indicate significant structural changes after composite assembly, which contribute to the formation of a hierarchically porous framework with increased specific surface area and uniform pore distribution. Such a structure facilitates efficient drug encapsulation and pH-responsive release, allowing sustained delivery of INH within the physiological environment. Additionally, the incorporation of HA not only improves the biocompatibility and colloidal stability of the nanosystem but also provides CD44 receptor-mediated targeting capability, enhancing selective uptake by macrophages in inflammatory tissues. Furthermore, the well-defined coordination polymer backbone and the presence of cobalt centers enable stable fluorescence quenching behavior, which is essential for the sensitive detection of INH molecules. Collectively, these structural attributes endow 1-HA-DPTMS@CP1@INH with dual drug delivery and sensing capabilities, confirming its rational design and potential as a multifunctional theranostic nanoplatform.

3.2. Fluorescence sensing performance of 1-HA-DPTMS@CP1 toward INH

To evaluate the sensing performance of the constructed nanoplatform for anti-tuberculosis drug detection, its fluorescence response behavior was systematically investigated. As shown in Figure 2, the synthesized 1-HA-DPTMS@CP1 material exhibited a prominent fluorescence "turn-off" response toward INH, displaying a concentration-dependent quenching effect. With increasing INH concentration (0–180 μL), the fluorescence intensity of 1-HA-DPTMS@CP1 significantly decreased (Figure 2a), indicating strong PL quenching upon INH addition. The quenching efficiency approached 90% at high concentrations (Figure 2b), suggesting a high sensitivity of the system toward INH detection.

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Figure 2.

(a) Fluorescence spectra of 1-HA-DPTMS@CP1 with increasing INH concentrations. (b) Quenching efficiency vs. INH concentration. (c) Linear plot of fluorescence intensity vs. INH concentration.

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Further quantitative analysis revealed a good linear correlation between fluorescence intensity and INH concentration in the range of 0–1.0 mM, with a correlation coefficient of R² = 0.9871 (Figure 2c). The limit of detection (LOD) was calculated using the formula LOD = 3σ/k, where σ is the standard deviation of blank measurements, and k is the slope of the linear regression. The low LOD value and excellent linearity confirm the material's high selectivity and sensitivity toward INH. These results not only demonstrate the effective interaction between INH and the hybrid nanocarrier but also highlight the promising application of this system in tuberculosis-related therapeutic monitoring and biomedical diagnostics. The fluorescence quenching mechanism is mainly attributed to a static quenching process involving the formation of non-fluorescent complexes between the pyridine and hydrazide groups of INH and the cobalt centers within the CP1 framework. This coordination interaction facilitates photoinduced electron transfer (PET) from the excited fluorophore to the INH–metal complex, resulting in efficient fluorescence suppression.

3.3. Selectivity evaluation and anti-interference performance

To further assess the selective recognition ability and practical applicability of 1-HA-DPTMS@CP1 toward INH, fluorescence response experiments were conducted in the presence of physiologically relevant interferents commonly found in the urine or inflammatory exudates of OATB patients, including NaCl, KCl, NH₄Cl, glucose (Glu), dopamine (DOP), and uric acid (UA). These species were chosen based on their potential to interfere with sensing via ionic strength alteration, hydrogen bonding, π–π stacking, or redox interactions. In each test, 1-HA-DPTMS@CP1 (0.1 mg mL^-1) was dispersed in PBS buffer (pH 7.4), and 1 mM of each interferent was added individually. Fluorescence spectra were recorded after 30 min incubation at room temperature (λ_ex = 365 nm, λ_em = 400–600 nm). Additionally, mixed groups containing each interferent and INH (1 mM) were tested to evaluate anti-interference performance. As shown in Figure 3(a–f), none of the interferents significantly affected the fluorescence signal, while the introduction of INH consistently triggered notable fluorescence quenching. These results demonstrate the excellent specificity and anti-interference capacity of 1-HA-DPTMS@CP1, highlighting its reliability for INH detection in complex biological environments.

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Figure 3.

Selective fluorescence response of 1-HA-DPTMS@CP1 (0.1 mg mL^-1) toward INH (1 mM) in the presence of various potential interfering substances. (a–f) represent the fluorescence spectra of the probe after the addition of 1 mM of (a) NaCl, (b) KCl, (c) NH₄Cl, (d) glucose (Glu), (e) DOP, and (f) UA, followed by the subsequent addition of INH (1 mM).

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3.4. Reusability and quenching mechanism of 1-HA-DPTMS@CP1 toward INH

Based on the remarkable fluorescence quenching capability of the 1-HA-DPTMS@CP1 nanomaterial toward INH, its reusability and quenching mechanism were systematically evaluated to verify its practical feasibility as a chemical sensor. As shown in Figure 4(a), cyclic detection experiments demonstrated that 1-HA-DPTMS@CP1 maintained consistent fluorescence intensity over five consecutive INH detection and washing cycles, with each cycle exhibiting efficient fluorescence quenching in the presence of INH, confirming its excellent stability and recyclability.

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Figure 4.

(a) Reusability of 1-HA-DPTMS@CP1 over five ON/OFF detection cycles with and without INH (1 mM); (b) Stern–Volmer plot of fluorescence intensity ratio (I₀/I) versus INH concentration.

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To further investigate the quenching mechanism, the Stern–Volmer equation was applied to analyze the effect of varying INH concentrations on fluorescence intensity (Figure 4b). The results revealed a strong linear relationship (R² = 0.996) between the fluorescence intensity ratio (I₀/I) and INH concentration, yielding a Stern–Volmer constant (K_SV) of 550.82 M⁻¹, indicating a classical dynamic quenching mechanism. These findings demonstrate that 1-HA-DPTMS@CP1 enables efficient, stable, and reversible recognition of INH, highlighting its strong potential for sensing applications.

3.5. In vitro cell viability assay

The biological effects of different treatments on cell viability, osteolytic enzyme expression, and inflammatory cytokine secretion were systematically evaluated ( Figure 5). As shown in Figure 5a, CCK-8 assays demonstrated that after 48 h of incubation, cells treated with INH-loaded nanoparticles (INH-NPs, 1-HA-DPTMS@CP1@INH) exhibited significantly higher viability than those in the model group (****P < 0.0001), while no statistically significant difference was observed relative to the control group, indicating minimal cytotoxicity. In contrast, treatment with blank nanoparticles did not lead to a significant improvement in cell viability compared with the model group. Quantitative real-time PCR analysis further revealed that the expression levels of the osteolytic-related genes CatK and MMP9 were markedly upregulated in the model group. Notably, INH-NPs treatment significantly downregulated the expression of both genes, reducing their levels toward those observed in the control group ( Figures 5b and 5c, ****P < 0.0001). By comparison, the blank-NPs group showed no statistically significant regulatory effect, indicating that the suppression of osteolytic gene expression was primarily attributable to INH delivery rather than the nanocarrier itself. Consistent with these transcriptional results, ELISA assays showed that the secretion of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β was substantially elevated in the model group, whereas treatment with INH-NPs significantly attenuated cytokine release to levels approaching those of the control group ( Figures 5d–f, ****P < 0.0001). In contrast, blank nanoparticles failed to induce comparable anti-inflammatory effects. Collectively, these findings indicate that INH-loaded nanoparticles effectively preserve cellular viability while suppressing osteolytic enzyme expression and inflammatory responses, thereby providing mechanistic support for their therapeutic potential in OATB-associated pathological conditions.

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Figure 5.

Effects of different treatments on cell viability, osteolytic-related gene expression, and inflammatory cytokine secretion in RAW 264.7 macrophages. (a) Cell viability assessed by CCK-8 assay after 48 h of treatment. (b) Relative mRNA expression levels of CatK and MMP9 determined by quantitative real-time PCR (qRT-PCR). (c–f) Secretion levels of pro-inflammatory cytokines TNF-α (d), IL-6 (e), and IL-1β (f) measured in culture supernatants by ELISA. Data are presented as mean ± SD (n ≥ 3). (Statistical significance is indicated as ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.)

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3.6. Revised drug release behavior section

The in vitro drug release behavior of 1-HA-DPTMS@CP1@INH was systematically evaluated under different pH conditions (7.4, 6.5, and 5.0) to mimic the physiological and OATB microenvironments. As shown in Figure 6, the cumulative release of INH exhibited distinct pH dependence. At neutral pH 7.4, only about 28% of the loaded drug was released within 80 h, indicating strong structural stability and minimal premature leakage under physiological conditions. In contrast, the release rate increased markedly at mildly acidic pH 6.5 (42% after 80 h) and reached 72% at pH 5.0, reflecting the acidic microenvironment of M. tuberculosis-infected macrophages and granulomatous lesions.

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Figure 6.

pH-dependent cumulative release profiles of INH from 1-HA-DPTMS@CP1@INH at 37°C under different conditions (pH 7.4, 6.5, and 5.0).

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The accelerated release at lower pH can be attributed to protonation of the coordination bonds between INH and the cobalt centers within the CP1 framework, along with acid-triggered hydrolysis of the HA–silane network. Such pH-responsive release behavior enables the nanoplatform to remain stable during systemic circulation but rapidly liberate INH after endocytosis into infected macrophages, where phagosomal pH typically decreases to 5.0–6.0. This smart release mechanism ensures high local drug concentration at infection sites, enhancing bactericidal efficacy while minimizing systemic toxicity. Together with its high BET surface area (228.6 m² g^-1) and mesoporous structure (average pore diameter = 6.8 nm, Figure 1d–e), the nanoplatform provides abundant diffusion channels for drug adsorption and pH-triggered release, validating its potential for precision therapy in OATB.