Adv. Funct. Mater.: A Novel Diagnostic–Therapeutic Platform for Triple-Negative Breast Cancer Based on Quantum-Sensing–Fused pH-Responsive Drug Delivery
Product Introduction
Equipment Parameters
I. Research Background
- Triple-negative breast cancer has a poor prognosis and lacks targeted therapies, while existing anticancer drugs suffer from significant side effects and insufficient targeting specificity.
- Nanodiamonds possess the advantages of biological inertness, facile surface functionalization, and nitrogen-vacancy (NV) color centers that enable fluorescent imaging and quantum sensing.
- The tumor microenvironment is weakly acidic (pH ≈ 6.5), making it suitable for designing pH-responsive drug delivery systems.
- Diazoxide (DZX) exhibits potential therapeutic efficacy against TNBC, yet its precise mechanisms of regulating mitochondrial reactive oxygen species (ROS) remain unclear.
II. Core Material Preparation
III. Key Performance Verification
IV. Quantum Sensing and Mechanisms of Action
Image and Text Guide
Plan 1 . pH-Responsive Nanodiamond Platform: Structure, Drug Release, and Intracellular Redox Sensing Mechanism
This figure illustrates the entire process by which diazoxide (DZX) modulates redox homeostasis in MDA-MB-231 triple-negative breast cancer cells, using a pH‑responsive fluorescent nanodiamond platform (FND‑HPG‑DMA‑DZX) in conjunction with diamond‑based quantum relaxation techniques.
a) Stepwise surface functionalization of fluorescent nanodiamonds (FNDs): hyperbranched polyglycerol (HPG) and DMA acid are sequentially grafted, followed by the final conjugation of diazine (DZX), yielding FND-HPG-DMA-DZX. b) Intracellular redox‑sensing mechanism: Upon cellular uptake, the nanocomposite accumulates in endosomes/lysosomes; the mildly acidic environment triggers cleavage of pH‑responsive linkers, releasing DZX. DZX then activates mitochondrial mitoKATP channels, enhancing mitochondrial ROS production; diamond‑based quantum relaxation spectroscopy is employed to monitor, in situ, changes in free radical levels within endosomes/lysosomes and in the cytoplasm. c) Under acidic conditions, DZX is pH‑responsively released from the DMA linker, yielding 2‑methylmaleic acid (MMA) and free DZX. d) The chemical structures of HPG, DMA, and DZX in the display system are shown.
Figure 1. Synthetic route of ND-HPG-DMA-DZX, along with FT-IR, TGA, DLS, and ζ-potential characterization.
a) Schematic of the synthesis route for pH‑sensitive ND‑HPG‑DMA‑DZX. First, hyperbranched polyglycerol (HPG) is grafted onto the nanodiamond (ND) core via ring-opening polymerization of glycidol; next, 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propanoic acid (DMA) is attached to ND‑HPG through an esterification reaction using oxalyl chloride; finally, in anhydrous DMSO, 3-((2-aminoethyl)amino)-7-chloro-4H-benzo[1,2,4]thiadiazine‑1,1-dioxide (DZX) is coupled to ND‑HPG via a ring‑opening reaction. b) Fourier-transform infrared (FT-IR) spectra of ND, NDHPG, ND‑HPG‑DMA, and ND‑HPG‑DMA‑DZX. After HPG functionalization, the C–O stretching vibration (ν, at 1080 cm⁻¹) is markedly intensified; following esterification, characteristic signals of the cyclic anhydride are clearly observed, including ν(C=O) bands at 1830 and 1760 cm⁻¹, as well as a ν(C–O–C) band at 900 cm⁻¹. Upon DZX coupling, the cyclic anhydride signals disappear, while aromatic ring signals at 1585, 1486, and 1458 cm⁻¹ become apparent, accompanied by an enhancement of the N–H bending (δ) vibration. c) TGA analysis indicates the composition of ND-HPG-DMA-DZX as follows: 10.4% HPG, 7.3% DMA, 5.9% DZX, and 76.4% ND. d) Size distribution of ND, ND-HPG, and ND-HPG-DMA in H2O as measured by DLS. e) Size and ζ-potential measurements of ND, ND-HPG, ND-HPG-DMA, and ND-HPG-DMA-DZX in H2O and PBS buffer, as determined by DLS.
Figure 2. pH‑sensitive bond cleavage mechanism, fluorescence imaging validation, and in vitro release profile of DZX
a) Schematic of the preparation process and pH‑sensitivity mechanism of ND‑HPG‑DMA‑Alexa 488 or ND‑HPG‑DMA‑DZX. AlexaFluor 488 or DZX is conjugated to ND‑HPG‑DMA via a ring-opening reaction between its amino group and the cyclic anhydride of DMA. The resulting amide–oxabicyclic structure features an amide bond (highlighted in red) that is pH‑sensitive and cleaves at pH 6.5. This cleavage releases the amine‑containing payload (AlexaFluor 488 or DZX) and generates 2‑methylmaleic acid (MMA) as a new terminal group on the ND complex. b) Fluorescence imaging of pH‑sensitive ND‑HPG‑DMA‑Alexa 488 after 4 hours of incubation in PBS at pH 7.4 (left) and pH 6.5 (right). The top panel shows the fluorescence intensity (FI) image, while the bottom panel displays the corresponding lookup table (LUT) image. Imaging was performed using a GFP/FITC bandpass excitation/emission filter set (excitation wavelength 475/28 nm, emission wavelength 525/48 nm). Scale bar = 5 μm. c) In vitro release profiles of DZX from ND-HPG-DMA-DZX in PBS at pH 6.5 and pH 7.4 over time. Data are presented as mean ± standard deviation (n = 2).
Figure 3 . Co-localization of the complex within cellular lysosomes, particle counting, and quantitative co-localization analysis
a) Example of a fluorescence confocal image of MDA-MB-231 cells stained with LysoviewBlue after incubation with FND-HPG-DMA-DZX. Scale bar for the magnified region = 5 μm. b, c) Magnified overlay images of lysosomes (stained with Lysoview 405 Blue) and FND(b) or FND‑HPG‑DMA‑DZX(c) at 4, 8, 20, 24, and 48 hours, confirming their colocalization within the endosome–lysosome compartment. Scale bar = 5 μm. d) The number of particles within each cell was counted using the 3D object counter in FIJI software. A total of 60 cells were analyzed per group. Data are presented as the mean ± standard deviation from three independent experiments, and intergroup comparisons were performed using one-way ANOVA. *p<0.05, **p<0.01, ***p<0.0001 indicate statistically significant differences, while ns denotes no significant difference. e) Quantitative colocalization analysis of FND or FND‑HPG‑DMA‑DZX with lysosomes under different incubation times. A total of 60 cells were analyzed per group. Box plots display the range of data points (from minimum to maximum), the first and third quartiles, with a line inside the box indicating the median.
Figure 4. DZX cytotoxicity, ATP levels, quantitative analysis of mitochondrial ROS, T1 Free Radical Detection and mechanism-of-action model
a) MTT assay results show the effect of free diazoxide (DZX) treatment on the metabolic activity of MDA-MB-231 cells after 24 hours. b) ATP assay results after treating MDA-MB-231 cells with 10 μg·mL⁻¹ ND‑HPG‑DMA‑DZX (equivalent to 2.75 μM DZX) for 4, 8, 20, 24, and 48 hours. c) Mitochondrial ROS levels in MDA-MB-231 cells treated with 10 μg·mL⁻¹ FND‑HPG‑DMA‑DZX at different time points; green: MitoSOX green fluorescence. d) Mitochondrial ROS were quantified in each cell based on MitoSOX green fluorescence intensity: an increase in MitoSOX fluorescence indicates elevated mitochondrial ROS production. A total of 60 cells were analyzed per group. The whiskers show the range of data points from the minimum to the maximum; the line within the box represents the median, with the upper and lower edges of the box corresponding to the first and third quartiles, respectively, and the bars indicate the minimum and maximum values. e) Using the FND–HPG–DMA–DZX method for diamond relaxation measurements, we assessed free radical levels in the endolysosomal compartment and the cytoplasm at time points of 4, 8, 20, 24, and 48 hours. An increase in T1 values indicates a reduction in free radical burden. Data were analyzed using one-way ANOVA between groups. *p<0.05, **p<0.01, ***p<0.0001 denote statistically significant differences; ns indicates no significant difference. (f) Hypothesized mechanism of intracellular redox homeostasis regulation: Under the acidic conditions of endosomes/lysosomes, DZX released from FND‑HPG‑DMA‑DZX activates mitochondrial KATP channels (①), thereby inducing mitochondrial reactive oxygen species (ROS) production. The elevated mitochondrial ROS can partially diffuse into the cytoplasm, triggering redox‑sensing signaling pathways (②), which in turn enhance antioxidant defenses and reduce the free radical burden in both the cytoplasm and lysosomes (③).
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