Carbon Dots-Based Nanozyme for Drug-Resistant Lung Cancer Therapy by Encapsulated Doxorubicin/siRNA Cocktail

Figures & data

Figure 1 The schematic diagram delineates how CD-PEI-DOX-siMRP1 delivers doxorubicin to tumors and antagonizes chemoresistance by hindering drug efflux through knocking down MRP1 expression.

Notes: Conversion of GSH to GSSG and subsequent ROS increase by CD-PEI oxidase and peroxidase activity further impairs MRP1 function. Collectively, CD-PEI-DOX-siMRP1 was capable of delivering drugs efficiently to tumor entities and retaining them in cells by hindering outflux of MRP1 through synergistic delivery of siRNA and perturbation of GSH -ROS balance.

Figure 2 The characteristics of CD-PEI and CD-PEI-DOX-siMRP1.

Notes: (A and B) The TEM images of CD-PEI (the inset is an auto correlation of the TEM image of the marked area using Digital Micrograph software). (C) The diameter distribution of CD-PEI. (D) The PL spectrum of CD-PEI. (E) The PL spectrum of CD-PEI-DOX-siMRP1. (F) The FTIR spectrum of CD-PEI. (G) The UV–vis spectrum of CD-PEI, CD-PEI-DOX, CD-PEI-DOX-siNC, and CD-PEI-DOX-siMRP1. (H) The release behavior of CD-PEI-DOX-siMRP1 and free DOX under different pH. (I) XPS spectrum of PEI, DOX, CD-PEI, CD-PEI-DOX, CD-PEI-DOX-siNC, and CD-PEI-DOX-siMRP1. (J) C1s, (K) N1s and (L) O1s spectra of CD-PEI-DOX-siMRP1.

Table 1 Percentage of Groups Obtained from Curve Fitting the C1s and N1s Peaks of Nanodrug

	C1s				N1s
	C-C/C=C	C-N/C-OH	C=O	O-C=O	C=C-N	N-(C)3	N-H
DOX	66.9%	11.1%	17.1%	4.9%	56.1%	26.5%	17.4%
PEI	79.2%	12.1%	4.5%	4.2%	48.8%	40.0%	11.2%
CD-PEI	65.0%	28.4%	6.6%		69.8%	30.2%
CD-PEI-DOX	45.5%	33.8%	12.8%	7.9%	65.7%	30.9%	3.4%
CD-PEI-DOX-siNC	63.9%	28.0%	3.3%	4.8%	74.0%	26.0%
CD-PEI-DOX-siMRP1	78.4%	16.4%	2.7%	2.5%	80.5%	19.5%

Figure 3 CD-PEI catalyzes the oxidation of GSH and increase of ROS in cells.

Notes: (A) The time-dependent behavior of the depletion of GSH in the presence of GSH (0.625 mM, 1.25mM and 2.5 mM) and CD-PEI (200 μg/mL) was analyzed; (B) the detection of •OH in the presence of CD-PEI and H₂O₂ using MB probe; (C) ESR spectra of •OH trapped by DMPO in the presence of CD-PEI and H₂O₂; (D) ABTS−H₂O₂ chromogenic system was used to assess the peroxidase-like activity of CD-PEI. Different solutions were tested as indicated. A full range spectrum absorbance was analyzed and shown. (E) The CLSM images of mitochondrial fragmentation in A549 cells (scale bar is 5 μm); (F) the CLSM images of ROS generation in A549 cells using DCFH-DA staining, green signals indicate ROS (scale bar is 50 μm). (G) The ROS generation in A549 cells using DCFH-DA staining measured by real-time cell monitoring system (Incucyte). (H) The schematic diagram indicated the nano-enzyme activity of CD-PEI.

Figure 4 The uptake of cells treated with indicated drugs detected by laser confocal scanning microscopy.

Notes: A549 (A) and A549/ADM (B) cells were treated with PBS (Control), DOX, CD-PEI-DOX, CD-PEI-DOX-siNC, and CD-PEI-DOX-siMRP1, respectively. Representative pictures were taken under confocal microscopes using 40X oil lens. Blue and red signals indicate nucleus and doxorubicin. CD-PEI was manifested by green signals. Signals were merged using the same field. DOX distribution in white rectangle region of the fourth panel was enlarged and reflected by densometric scale as shown in the fifth panel. (C and D) The quantitative analysis of the fluorescence intensity of DOX in A549 and A549/ADM cells was carried out by ImageJ. The scale bar is 20μm. ****p<0.0001.

Figure 5 Uptake of drugs in A549 and A549/ADM cells were determined using flow cytometry.

Notes: (A) Left panel: A549 cells were treated with the drugs for 8 h, then cells were collected and fluorescence signal of DOX was detected. Right panel: Mean fluorescence intensity of each cell was calculated and statistically analyzed. (B) Left panel: A549/ADM cells were treated with the drugs for 8 h, followed by flow cytometry analysis detecting the fluorescence of DOX. Right panel: Statistical analysis of mean fluorescence intensity of DOX in each group. ***p<0.001. ****p<0.0001. (C) The penetration of drugs was detected using A549/ADM cells. Confocal Z-stack images of cell mammospheres were obtained and shown after incubated with PBS, free DOX, CD-PEI-DOX, CD-PEI-DOX-siNC, and CD-PEI-DOX-siMRP1. The scale bar is 20 μm.

Figure 6 CD-PEI-DOX-siMRP1 inhibits the expression of MRP1 and causes inhibition of proliferation in lung cancer cells.

Notes: (A and B) Expression and quantification of MRP1 expression on A549 cells by flow cytometry. (C and D) Expression and quantification of MRP1 on A549/ADM cells by flow cytometry. (E and F) The cell viability of A549 and A549/ADM cells incubated with free DOX, CD-PEI-DOX, and CD-PEI-DOX-siMRP1 for 48 hours were analyzed by CCK8 assays. (G–I) Expression and quantification of Ki67 on A549 and A549/ADM cells by CLSM (Scale bar: 100 μm). *p<0.05, **p<0.01, ***p<0.001. ****p<0.0001.

Table 2 IC₅₀ of Free DOX, CD-PEI-DOX, CD-PEI-DOX-siMRP1 in A549 and A549/ADM Cells

IC50 (μg/mL)
A549	Free DOX	26.993
	CD-PEI-DOX	6.72
	CD-PEI-DOX-siMRP1	5.692
A549/ADM	Free DOX	130.626
	CD-PEI-DOX	18.19
	CD-PEI-DOX-siMRP1	17.4

Figure 7 Analysis of targeting and tumor-inhibitory effect of nanodrugs.

Notes: (A) PBS, Free DOX and CD-PEI-DOX, CD-PEI-DOX-siMRP1 were injected through tail vein in A549/ADM subcutaneous xenograft model. Left panel: Representative pictures were shown after the indicated treatment for different periods photographed by IVIS. Right panel: Relative immunofluorescence intensity was statistically analyzed from different groups. (B) Left panel: Organs of representative group were harvested and photographed under IVIS. Right panel: Relative fluorescence intensity of each organ was calculated and statistically analyzed. (C) Tumors were harvested from the mice of each group at the final time point, and representative tumors were aligned and shown (n = 5). (D) Tumors from each group were weighed and shown in the graph. (E) Tumor volume were recorded and plotted from each group after the indicated treatment. (F) Tumor inhibition rate was calculated from each group. PBS-treated group was used as a control. (G) Body weight of all mice was recorded and averaged in the indicated group. (H) Blood cell analysis was applied to analyze the biocompatibility of indicated drugs. ****p<0.0001.

Figure 8 The effect of different drugs on MRP1 expression and tissue integrity on mouse models.

Notes: (A) Immunostaining of MRP1 in slices from tumors obtained from each group. MRP1 was visualized with secondary antibody conjugated with Alexa Fluor-488nm fluorescence. Green signal indicates MRP1, while the nucleus was counterstained with DAPI. Scale bar: 25μm. (B) MRP1 expression was examined with IHC assays. MRP1 was stained with DAB, and the nucleus was counterstained and shown as blue signals. Representative pictures were shown. Scale bar: 50 μm. (C) H&E staining was conducted to determine the toxic effect of various drugs on organs in indicated groups. Scale bar: 50μm.