Theranostic biofluorescent nanocomposite for fluorescence-guided delivery of cinobufagin and apoptosis modulation in gastric carcinoma

2.1. Chemicals and measurements

All of the solvents and chemicals were of reagent-grade quality and commercially sourced. They did not require further purification. Via applying a Nicolet (Impact 410) spectrometer with KBr pellets, these complexes’ IR absorption spectra were documented from 400 to 4000 cm^-1. Through the use of a Perkin-Elmer model 240C analyzer, EA (N, H, C) was performed. Via a Bruker D8 Advance X-ray diffractometer, PXRD was measured by Cu-Kα radiation (0.15418 nm), and the X-ray tube was operated at 30 mA and 40 kV. Thermogravimetric analysis (TGA) was implemented on a Perkin Elmer thermogravimetric analyzer from RT to 700°C under N₂ atmosphere with 20 K·min^-1 heating rate.

2.2. Synthesis of I-DASA-ATPMS@CP1@CB

The synthesis procedures of CP1 and compound I have been detailed in the supporting information (Table S1). The composite material I-DASA-ATPMS@CP1@CB was synthesized via a three-step procedure. First, poly(DASA) sodium salt (500 mg, containing 3.3 mmol of carboxyl groups) was reacted with excess thionyl chloride at room temperature to convert the carboxyl groups into acyl chloride functionalities, yielding poly(acyl chloride) DASA. Without further purification, this intermediate was dissolved in a THF/methanol mixture (v/v = 1:1, 20 mL) and reacted with APTMS (100 μL, 0.5 mmol) at 70°C for 8 h, affording DASA-ATPMS. The product was collected by ether precipitation and vacuum-dried to yield a pale yellow solid (90% yield). Subsequently, compound I (300 mg, 1.0 mmol), an anthraquinone-based fluorescent amine derivative, was reacted with DASA-ATPMS (200 mg, 0.3 mmol) in dry acetonitrile (10 mL) in the presence of triethylamine (200 μL, 1.4 mmol) as a catalyst. After stirring at 70°C for 6 h, the fluorophore-functionalized macromolecule I-DASA-ATPMS was obtained by recrystallization from ethyl acetate/hexane (1:1) in 85% yield (Scheme S1). Finally, to prepare the multifunctional composite, 100 mg of I-DASA-ATPMS, 10 mg of the CP1, and 10 mg of CB were co-dispersed in 10 mL Tris-HCl buffer (pH 7.4), sonicated for 30 min, and stirred gently for 2 h. The final product, I-DASA-ATPMS@CP1@CB, was isolated by centrifugation, washed with deionized water, and lyophilized, yielding 110 mg of the composite material (Scheme S2).

2.3. Fluorescence response to AGS

Before detecting AGS, the experimental conditions for the fluorescence (FL) sensing probe were optimized, including reaction time (0-10 min), pH (3-12), and probe concentration (10-50 mg/L). Under optimal conditions, 30 mg/L of the probe solution was mixed with AGS standard solution and diluted to 2 mL with Tris-HCl buffer (pH = 10.0). Following incubation for 1 min at RT, the fluorescence spectra were documented at λ_em = 410 nm under excitation at λ_ex = 300 nm. For selectivity evaluation, the probe was exposed to various substances, including common antibiotics (ENR, PC, CLP, VAN, ERT, DIC, DOX), metal ions (K⁺, Ca²⁺, Na⁺, Mg²⁺, Zn²⁺), and other potential interferents (lactose, glucose, ascorbic acid and histidine), following the same procedure as AGS detection, to verify the probe’s high specificity for AGS.

2.4. CCK-8 assay

AGS, the human GC cell line, was provided by ATCC, USA, and regularly cultivated at 37°C and 5% CO₂ in RPMI-1640 media (Gibco) with 10% FBS (Gibco), 100 μg/mL streptomycin, as well as 100 U/mL penicillin. AGS cells were cultivated for 24 h after being injected into 6-well or 96-well plates. Control cells were treated with PBS. In the free CB group, CB solution (final concentration 25 μg/mL) was added. In the I-DASA-ATPMS@CP1@CB group, I-DASA-ATPMS@CP1@CB (final concentration 25 μg/mL) suspension was added. Each group of cells was treated for 48 hrs. Each well was spiked with 10 μL of CCK-8 reagent and inoculated at 37°C for 2 h. The absorbance at 450 nm (OD) was determined.

2.5. qRT-PCR

After 48 h of treatment, each group of cells in 6-well plates was gathered, and the extraction of total RNA was conducted with Trizol reagent (Invitrogen, USA). Via applying the miRNA 1st Strand cDNA Synthesis Kit (Vazyme, China), hsa-miR-494 cDNA was produced with 500 ng of total RNA. Then, 1 μg of total RNA was reverse transcribed into cDNA using HiScript II Q RT SuperMix (Vazyme, China). With the SYBR qPCR Master Mix (Vazyme, China), the relative levels of hsa-miR-494 and Bax were determined. Primer sequences were as follows: GAPDH-Forward: 5’-GAAGGTGAAGGTCGGAGTC-3’, GAPDH-Reverse: 5’-GAAGATGGTGATGGGATTTC-3’. Bax-Forward: 5’-TTTGCTTCAGGGTTTCATCC-3’, Bax-Reverse: 5’-TGCAAAGTAGAA AAGGGCGAC-3’. hsa-miR-494-Forward: 5’-TGAAACATACACGGGAA ACCT-3’. hsa-miR-494-Reverse: 5’-CAGTGCAGGGTCCGAGGT-3’. U6- Forward: 5’-CTCGCTTCGGCAGCACATATAC-3’. U6- Reverse: 5’-ACGCT TCACGAATTTGCGTGTC-3’.

3.1. Structural characterization of CP1

CP1’s asymmetric unit is composed of a L ligand, an isolated Co²⁺ ion, two unbound water molecules, and a deprotonated TBTA²⁻ anion (Figure 1a). The center of each Co(II) is surrounded by two O atoms of two TBTA^2- ligands (O1A, O4, symmetry code: A = 1/2 + x, 1/2 - y, -1/2 + z) and two N atoms of two different L ligands (N1, N4B, symmetry code: B = 1-x, 1-y, 1-z). Tetra-coordinated Co(II) has a twisted tetrahedral {CoN₂O₂} coordination geometry at the center with the geometric parameter τ₄ = 0.92 (in tetra-coordinated CPs, the τ₄ is between 0 and 1.00, where τ₄ = 0 indicates an ideal planar geometry and τ4 = 1 denotes a perfect tetrahedral geometry). Around the Co(II) centers, the angles of bond are 97.9(2)-117.0(3)°, and the lengths of Co-O/N are 1.890(7)-2.008(7) Å, which are equivalent to those reported in the literature for Co(II) CPs [28-30]. Each TBTA^2- ligand in CP1 employs a (κ¹-κ⁰)-(κ¹-κ⁰)-µ₂ coordination pattern, linking adjacent Co(II) ions to create a [Co(TBTA)]_n zigzag chain with a Co-Co spacing of 11.04(1) Å. The L ligand exhibits a cis-conformation with a dihedral angle of 79.39(3)° between the two benzimidazole planes that make up the whole L ligand and a N_donor···N–C_sp3⋯C_sp3 torsion angle of 112.26(8)°. L ligands connect neighboring Co(II) centers to build the 22-element [Co₂(L)₂] unit. By sharing Co(II) centers, the [Co₂(L)₂] unit further extends the 1-D [Co(TBTA)]_n chain into a 2-D layer (Figure 1b). 2-D networks can be categorized into hexagonal planar {6³} topologies (Figure 1c). With π-π stacking interactions, the 2-D (6,3) layer can be further extended into a 3-D supramolecular network (Figure 1d). Between the benzene (Cg2C: C10C–C11C–C12C–C15C–C16C–C17C) and imidazole (Cg1: N1–C9–N2–C10–C17) rings of L ligands, there is a π–π stacking interaction. The distance between the ring centers is 3.803(6) Å, the dihedral angle α is 0.8(5)°, and the sliding angles γ and β are 23.64 and 22.89°, respectively.

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Figure 1.

(a) CP1’s least building unit. (b) CP1’s 2D layer. (c) CP1’s 2D (6,3) network. (d) The π–π interactions between the neighboring layers.

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PXRD tests have been performed on the CP1 (Figure 2a) to prove the products’ phase purity. The simulated and experimental PXRD patterns’ peak locations coincide well, proving that the crystal structure accurately presents the bulk crystal products. The preferred orientation of crystal samples might be the cause of the intensity variations. For examining the CP1’s thermal stability, TGA was conducted at 25-800°C under a flow of N₂ with a 10°C·min⁻¹ heating rate (Figure 2b). The mass loss for CP1 in the 32–116°C range is around 3.89% (calculated 3.92%), and This is ascribed to the decomposition of unbound water molecules by CP1. The decomposition of the L ligand is responsible for the significant weight loss between 199 and 317°C (37.05% observed; 37.43% calculated). and that of TBTA^2- ligand is associated with weight loss at 345-547°C (obsd 51.74%; calcd 51.96%).

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Figure 2.

(a) The CP1’s PXRD patterns (b) and TGA curve.

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3.2. Structure description of compound I

The SCXRD data of compound I exhibit that it falls into the monoclinic crystal system of the Cc space group. Figure 3(a) presents that the molecular structure is composed of a benzene ring (ring C: C13-C18) and two six-membered rings (ring A: C5-C9, O2; ring B: C1-C6). Through the C8-C10 bond, the atom C8 of ring A joins an ethyl formate group (-C₁₀O₄O₃C₁₁C₁2H₆). Atom C9 of ring A joins an amino group (-N1H₂). A C-C bond from C5 and C6 is shared by rings A and B, connecting them. The O1 atom is joined to ring B by the atom C1. Through the bond of C7-C13, ring A and benzene ring C are joined. Atom C17 of Ring C joins a methoxy group (-O5C19H₃). Moreover, compound I’s adjacent molecules were further linked by intermolecular hydrogen bonding (N1-H1A.....O1: 2.018 Å) (Figure 3b) and van der Waals forces further connected to form a 3-D dense stacked structure (Figure 3c).

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Figure 3.

(a) The compound I’s molecule structure; (b) H-bonds between the neighboring molecules; (c) The compound I’s dense packing molecule structure.

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3.3. Structure characteristics of I-DASA-ATPMS@CP1@CB

To confirm the structural integrity and crystallinity of the I-DASA-ATPMS@CP1@CB composite material, a series of characterization techniques was systematically employed. As shown in Figure 4(a), the XRD pattern exhibits multiple diffraction peaks in the range of 2θ ≈ 5°-30°, particularly near 10° and 17°, indicating the formation of an ordered structure within the sensing probe. The Fourier transform infrared (FT-IR) spectrum (Figure 4b) further supports the successful assembly of the composite components. In I-DASA-ATPMS@CP1@CB, the –OH and –NH stretching vibrations originally observed at 3506.08 cm⁻¹ and 3391.94 cm⁻¹ disappear, substituted by a new peak at 3419.41 cm⁻¹, indicating the establishment of a new hydrogen bonding environment. Additionally, there is no broad -OH absorption band (2570-2650 cm^-1) relative to the -COOH group in CB, demonstrating the successful integration of CB into the composite matrix. In contrast to free H₂ATPA (1680 cm^-1), the C=O telescoping vibration at 1698 cm^-1 is blueshifted by 18 cm^-1, while the symmetric C-O telescoping vibration, which was originally located at 1236 cm^-1, is shifted to 1386 cm^-1, indicating stable coordination between Cr³⁺ ions and carboxyl groups. The scanning electron microscope (SEM) image (Figure 4c) reveals that the composite material features a clearly defined porous network morphology, which facilitates the diffusion and binding of target molecules. TGA (Figure 4d) demonstrates the material’s thermal stability, showing a three-stage decomposition process: with a total weight loss of 66.98%, the first stage, which occurs at 88.50°C, is associated with the evaporation of water and leftover solvents; the second stage, which occurs at 302.77°C, is linked to the breakdown of the organic framework; and the third stage, which occurs at 412.70°C, is associated with the breakdown of inorganic components. Nitrogen adsorption-desorption measurements (Figure 4e) indicate that the composite possesses a mesoporous structure, with a mean pore diameter of 5.26 nm, a pore volume of 0.71 cm³/g, and a Brunauer-Emmett-Teller (BET) surface area of 541.94 m²/g. These results confirm that I-DASA-ATPMS@CP1@CB was successfully constructed with a highly ordered structure, excellent thermal stability, and an ideal porous framework, providing a robust foundation for its application in selective molecular recognition and drug delivery.

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Figure 4.

Characterization of I-DASA-ATPMS@CP1@CB: (a) XRD; (b) FT-IR; (c) SEM; (d) TGA/DTG; (e) N₂ adsorption-desorption isotherms (Blue represents the thermogravimetric curve, corresponding to the left axis; yellow corresponds to the first derivative of the thermogravimetric curve, corresponding to the right axis).

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3.4. Optimization of the experimental measurements

The key factors influencing the sensing ability of I-DASA-ATPMS@CP1@CB fluorescence probe, such as reaction time, ambient pH, and probe concentration, were carefully examined for methodically assessing the potential of the probe for GC detection. Figure 5(a,b) display that the fluorescence intensity progressively increased with the probe concentration from 10 mg/L to 30 mg/L and then plateaued, indicating 30 mg/L as the optimal working concentration. Figure 5(c,d) reveal that within a pH range of 3 to 12, the fluorescence signal peaked at pH 10, suggesting that an alkaline environment favors signal enhancement. Figures 5(e, f) further explore reaction kinetics, showing that upon the addition of the target molecule CP1, fluorescence intensity sharply decreased within 1 min and then stabilized, demonstrating the probe’s “rapid response” capability. This fast, stable, and pH-tolerant fluorescence recognition is particularly critical for the early diagnosis of GC, enabling real-time detection and feedback of target molecules, thus supporting precision medicine. The finalized optimal experimental conditions were: probe concentration of 30 mg/L, pH 10, and detection time of 1 min.

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Figure 5.

Optimization of the experimental measurements (a, b) sensing probe concentration; (c, d) pH values of the system; (e, f) reaction time (Represents the fluorescence intensity at different concentrations).

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3.5. Analytical approach

Under the optimized sensing conditions, the fluorescence response of the I-DASA-ATPMS@CP1@CB probe toward varying concentrations of AGS was systematically evaluated. FRET and inner filter effects may be responsible for the steady reduction in fluorescence intensity at 410 nm when the concentration of AGS increased from 0 to 100 μM, as displayed in Figure 6(a). A strong linear relationship was established between the fluorescence quenching ratio (I₀/I) and AGS concentration, as illustrated in Figure 6(b), with a regression equation of y = 18050x + 1.0362 and an excellent correlation coefficient (R² = 0.9985). The computed LOD, which is LOD = 3σ/S (where σ is the blank standard deviation, n = 12, and S is the slope), was found to be 0.78 ng/mL on the basis of Stern-Volmer equation, which also yielded the calculated quenching constant (Ksv). These findings suggest that the probe exhibits highly sensitive detection capability for AGS.

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Figure 6.

(a) The FL spectra of the sensing probe (λ_em =410 nm) display quenching at various concentrations of AGS; (b) S-V plot of the sensing probe after being quenched by AGS.

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3.6. Selectivity, stability, and reproducibility

To ensure the specificity of the I-DASA-ATPMS@CP1@CB fluorescence probe in detecting AGS in complex biological environments, its selectivity and stability were systematically evaluated. As shown in Figures 7(a, b), common physiological interferents like Mg²⁺, K⁺, Na⁺, His, Zn²⁺, Ca²⁺, AA, glucose, and lactose had negligible influence on the probe’s fluorescence intensity, while AGS (25 ng/mL) caused significant quenching at 410 nm, with an I₀/I value exceeding 4. Similarly, among various antibiotics tested (Figures 7(c,d)), only AGS and, to a lesser extent, DOX induced notable fluorescence quenching. These findings confirm the high selectivity of the probe for AGS. The capability of the probe to reliably and selectively detect AGS in the presence of biologically relevant interferences highlights its potential for rapid and accurate GC biomarker monitoring, enabling early diagnosis and real-time therapeutic assessment. The detection performance of I-DASA-ATPMS@CP1@CB is comparable to or even superior to that of previously reported fluorescence-based sensors, as summarized in Table S2.

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Figure 7.

FL spectra and F₀/F of the sensing system with(a, b) ions and physiological substances, and (c, d) 25 ng/mL of antibiotics. DOX: Doxycycline, CLP: Chloramphenicol, ERM: Erythromycin, DIC: Dicloxacillin, PCL: Penicillin, VA: Vancomycin, ENR: Enrofloxacin, TY: Tylosin.

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3.7. pH-responsive drug release behavior

To further evaluate the therapeutic potential of the I-DASA-ATPMS@CP1@CB composite in the context of GC, in vitro drug release experiments were conducted under simulated physiological (pH 7.4) and tumor-like acidic conditions (pH 6.5 and 5.0). As shown in Figure S1, the composite demonstrated a distinct pH-dependent release profile. Specifically, the release rate of the encapsulated drug was significantly accelerated at pH 6.5, which closely mimics the extracellular environment of gastric tumors. Within 12 h, the cumulative release reached over 60%, in contrast to only 20% at neutral pH 7.4. Interestingly, although pH 5.0 represents a more acidic environment, the release rate was slightly slower than at pH 6.5. This may be attributed to possible partial collapse or protonation-induced interactions within the composite matrix at very low pH, which could hinder drug diffusion. The enhanced release behavior at mildly acidic pH aligns well with the acidic microenvironment of GC tissues, where extracellular pH typically ranges between 6.5 and 6.8 due to anaerobic metabolism and lactic acid accumulation. This feature ensures minimal premature leakage in circulation while enabling targeted, stimuli-responsive drug release at tumor sites. Such a release pattern is particularly beneficial for improving drug bioavailability and reducing systemic toxicity. Taken together, these results confirm that I-DASA-ATPMS@CP1@CB possesses pH-triggered drug release capability, making it highly suitable for GC therapy. Its release kinetics not only support environmental responsiveness but also demonstrate the potential for sustained drug delivery under tumor-relevant conditions, thereby enhancing therapeutic efficacy.

3.8. I-DASA-ATPMS@CP1@CB regulated has-miR-494 and bax

The CCK-8 assay demonstrated that I-DASA-ATPMS@CP1@CB significantly inhibited the proliferation of AGS GC cells compared with both the free CB and control groups, with a cell viability of 42.25% after 48 h of treatment, which was markedly lower than that of the free CB group (63.86%, P < 0.001; Figure 8a). qRT-PCR analysis revealed that hsa-miR-494 expression in AGS cells treated with I-DASA-ATPMS@CP1@CB was 4.07-fold higher than that in the control group, significantly greater than the 1.98-fold increase observed in the free CB group (P < 0.0001; Figure 8b). Meanwhile, the mRNA level of the apoptosis-related gene BAX increased 3.87-fold in the I-DASA-ATPMS@CP1@CB group compared with 2.13-fold in the free CB group (Figure 8c). The positive correlation between BAX and hsa-miR-494 expression suggests that the nanoplatform may promote BAX transcription through miR-494 upregulation, thereby inducing apoptosis and suppressing GC cell proliferation. Mechanistically, I-DASA-ATPMS@CP1@CB enhances apoptosis via a dual regulatory mechanism, simultaneously upregulating Bax, a key effector in the mitochondrial apoptosis pathway, and restoring the tumor-suppressive function of hsa-miR-494, which negatively regulates anti-apoptotic proteins such as survivin and BAG-1. This synergistic miRNA-protein interaction amplifies caspase-3 activation, resulting in enhanced mitochondrial membrane collapse and cell death. These findings are consistent with previous studies reporting that miR-494 sensitizes GC cells to TRAIL-induced apoptosis by targeting survivin [31] and mediates the BAG-1 signaling axis in cinobufacini-treated GC cells [32]. Collectively, these results indicate that I-DASA-ATPMS@CP1@CB exerts its anticancer efficacy through a synergistic miRNA-protein apoptotic pathway involving miR-494-mediated signaling regulation and BAX-dependent mitochondrial activation.

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Figure 8.

The effect of I-DASA-ATPMS@CP1@CB on (a) proliferation of AGS cells, (b) has-miR-494 level and (c) Bax level. **P<0.01; ***P<0.001; ****P<0.0001.

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