Catechol-linker and receptor-mediated site-specific delivery of bortezomib against non-small cell lung cancer

Figures

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  Figure 1. Site-specific, bio-orthogonal conjugation and pH-responsive BTZ Release.A, binding kinetics of mRBD3 and mRBD3dopa with ACE2 using MST (i) The black square indicates the curve generated by Monolith binding affinity Nano Temper Analysis 1.2.231 software, and the red dot indicates the fluorescent intensity (fraction bound) on protein-receptor interaction, and (ii) T jump plot of mRBD3 and mRBD3dopa vs. ACE2 shows how the temperature changed when the thermal activation by the IR laser was turned on, and the changes in the fluorescence signal occurred in the MST capillaries and ended within a few seconds are denoted by temperature jumps (T jumps). KD values are reported as mean ± SD using three independent protein preparations, each measured in technical triplicate. The binding curves shown are representative of three independent ligand preparations and labeling reactions. B, representative UV-vis spectra of the catechol-boronate complex measured at pH 7.4 and 5.0. C, representative CD spectrum of mRBD3, mRBD3dopa and mRBD3dopa-BTZ conjugate at pH 7.4. D, homogeneity analysis using DLS. E, quantification of cumulative BTZ release profile using UV-Vis spectroscopy (λ = 270 nm) at pH…

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  Figure 2. Functional mechanism of ester-bond stability and evaluation of selective cytotoxicity in ACE2-high A549 cells.A, representative XPS spectra showing the surface chemistry of free BTZ and the conjugate at pH 7.4 and 5.0. B, Western blot quantification of ACE2 expression (i) Protein expression levels were quantified and normalized to GAPDH, and (ii) Detection of ACE2 protein expression in A549 cells compared with HSAEC cells, Lane M: Marker, Lane 1: A549 cells and Lane 2: HSAEC cells. GAPDH was used as an internal control of protein loading. Band intensity was calculated by ImageJ and plotted using GraphPad Prism 10.1.0. Data are presented as mean ± SD from at least three independent biological replicates, each done in three technical replicates. The data points representing the mean of each independent biological replicate are overlaid. Error bars denote standard deviation across the biological replicates. Statistical analysis was made using an unpaired two-tailed t test, ∗∗∗p < 0.001. C, dose-dependent cytotoxicity of mRBD3dopa-BTZ and free BTZ in ACE2-high A549 cells, assessed by MTT assay at (i) 24 h, and (ii) 48 h. Data are presented as mean ± SD from at least three independent…

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  Figure 3. Safety, specificity and assessment of apoptosis.A, cytotoxicity of mRBD3dopa-BTZ and free BTZ in ACE2-low HSAEC cells after 48 h treatment. Data are presented as mean ± SD from at least three independent biological replicates, each done in four technical replicates. The data points representing the mean of each independent biological replicate are overlaid. Error bars denote standard deviation across the biological replicates. Statistical analysis was made using a using two-way ANOVA with multiple comparisons and Tukey’s post hoc correction, ∗∗∗∗p < 0.0001, ns = not significant. B, cytotoxicity assessment of mRBD3 and mRBD3dopa in A549 cells after (i) 24 h and (ii) 48 h treatments. Data are presented as mean ± SD from at least three independent biological replicates, each done in four technical replicates. The data points representing the mean of each independent biological replicate are overlaid. Error bars denote standard deviation across the biological replicates. Statistical analysis was made using a using one-way ANOVA with multiple comparisons and Tukey’s post hoc correction, ns = not significant compared to control. C, cytocompatibility evaluation of mRBD3dopa-BTZ and free…

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  Figure 4. Apoptotic mechanism of the ACE2-targeted mRBD3dopa-BTZ in 2D monolayer and 3D spheroid models.A, evaluation of caspase-3 and caspase-9 activities in A549 cells treated with mRBD3dopa-BTZ. Data are presented as mean ± SD from at least three independent biological replicates, each done in five technical replicates. The data points representing the mean of each independent biological replicate are overlaid. Error bars denote standard deviation across the biological replicates. Statistical analysis was made using a using two-way ANOVA (Factors: caspase type × concentration) followed by Tukey’s multiple comparisons test to compare each treatment group with the corresponding control within each caspase, ∗∗∗∗p < 0.0001. B, analysis of cell apoptosis and death in A549 and HSAEC Cells (2D monolayer) by flow cytometry. The dot plots shown are representative of three independent biological experiments. C, Nuclear condensation and fragmentation analysed by Hoechst-33342 staining. The scale bar represents 100 μm. D, cytotoxicity of mRBD3dopa-BTZ and free BTZ in 3D spheroid cultures after 48 h treatment (i) 3D A549, (ii) 3D HSAEC spheroids. Data are presented as mean ± SD from at least three…

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  Figure 5. In vivo antitumor efficacy of mRBD3dopa-BTZ in a LLC1 tumor induced C57BL/6 mice.A, representative photographs of excised lungs collected from LLC1 tumor-bearing mice following the indicated treatments after a 30-days observation period (biological replicates n = 6 mice per group). B, assessment of tumor growth inhibition in a syngeneic lung cancer model. Tumor growth was presented as the mean tumor volume (mm3) ± SEM. Tumor volume was determined by caliper measurements and calculated as length × width2/2. Error bars represent standard error of the mean. Statistical analysis was performed using a two-way repeated-measures ANOVA (treatment × time; Geisser–Greenhouse correction) followed by Tukey’s multiple comparison test for group-wise differences at each time point. C, body weight (g) of mice monitored over 30 days. Data are presented as mean ± SD (biological replicates n = 6 mice per group). Error bars represent standard deviation. Statistical analysis was made using a two-way ANOVA with multiple comparisons and Tukey’s post hoc correction, ns = not significant. D, representative H&E-stained sections of mice lung tissue after treatments: dysplastic epithelium (yellow arrow),…

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  Figure 6. Organ-protective efficacy, selectivity, and apoptotic induction of mRBD3dopa-BTZ in vivo.A, representative H&E-stained sections of mice organs collected after different treatments: in the liver-hepatocytes (black arrow), central vein (red arrow), portal triad (yellow arrow), and sinusoidal spaces (white arrow); in the kidney-glomeruli (black arrow), tubules (white arrow), and the interstitium (red arrow); and in the heart-cardiomyocytes (black arrow), myonuclei (white arrow), and intercalated disc space (red arrow). The scale bar represents 340 μm. B, representative immunohistochemical staining showing the expression of (i) CHOP, and (ii) NF-kB. The scale bar represents 100 μm. C, biodistribution of BTZ in major organs of tumor-bearing mice 12 h post-injection was quantified by ICP-MS and expressed as the percentage of total injected boron. Data represent the mean ± SD (biological replicates n = 6 mice per group), with each sample measured in technical triplicate. The data points representing the mean of each independent biological replicate are overlaid. Error bars denote standard deviation across the biological replicates. Statistical analysis was made using a two-way ANOVA…

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  Figure 7. Site-specific delivery of bio-orthogonally conjugated bortezomib against non-small cell lung cancer cells.