Next-Generation Cancer-Specific Hybrid Theranostic Nanomaterials: MAGE-A3 NIR Persistent Luminescence Nanoparticles Conjugated to Afatinib for In Situ Suppression of Lung Adenocarcinoma Growth and Metastasis

Abstract

The rate of lung cancer has gradually increased in recent years, with an average annual increase of 15%. Afatinib (AFT) plays a key role in preventing non-small cell lung carcinoma (NSCLC) growth and spread. To increase the efficiency of drug loading and NSCLC cell tracking, near infrared-persistent luminescence nanomaterials (NIR PLNs), a silica shell-assisted synthetic route for mono-dispersal, are developed and used in the nanovehicle. After optimizing their physical and chemical properties, the NIR PLNs are able to absorb light energy and emit NIR luminescence for several hours. In this research, NIR PLNs are functionalized for drug-carrying capabilities. Effective accumulation of target drugs, such as AFT, using PLN nanomaterials can lead to unique anticancer therapeutic benefits (AFT-PLN). To minimize side effects and increase drug accumulation, nanomaterials with targeting abilities are used instead of simple drugs to inhibit the growth of tumor cells. Thus, the specific targeting aptamer, MAGE-A3 (MAp) is identified, and the PLN to increase its targeting ability (AFT-PLN@MAp) accordingly modified. The advancement of nanoscale techniques in the field of lung cancer is urgently needed; this research presents a plausible diagnostic strategy and a novel method for therapeutic administration.

Keywords: afatinib; aptamer; cancer therapy; lung cancer metastasis tracking; persistent luminescence nanoparticles.

Figure 1

A scheme of AFT‐PLN@MAp as a targeting anticancer nanoplatform. A potential strategy of AFT‐PLN@MAp is provided for diagnostic and therapeutic treatment of metastasis in lung cancer. Current therapeutic methods intending to cure the metastasis of tumor cells have major issues. This nanoplatform can be used with a MAp to target specific aptamers; PLNs are actively transported to tumor cells. PLNs embedded with AFT target drugs can effectively treat tumor cells, limiting cancer cell metastasis.

Figure 2

Basic characteristics of PLNs and the derivative PLNs. a) Schematic diagram of the synthetic AFT‐PLN@MAp procedure. This study first used mesoporous silica nanoparticles (MSN) as a material template and added inorganic metal molecules to the core of the hole to form a PLN. After washing the metal ions, the AFT drug molecules are loaded and the aptamer is modified to form AFT‐PLN@MAp. Transmission electron microscopy images of b) MSN and c) PLNs, which form a deep black dot‐like ZGOCS structure in the MSN of the original hole structure. d) Dynamic light scattering and e) zeta potential data showing the degree of dispersion and surface electrical properties of the material, respectively. The PLN has a hydration radius of approximately 125 nm and an AFT‐PLN@MAp size of approximately 225 nm. Additionally, AFT‐PLN@MAp has a suitable degree and physiological charge of dispersion and a more concentrated particle size distribution. f) X‐ray powder diffraction (XRD) data. JCPDS‐38‐1240 has a crystal phase structure consistent with the synthesized AFT‐PLN@MAp. Energy‐dispersive X‐ray spectroscopy g) mapping and h) spectrum. The elemental structure of the material was tested using Zn, Ga, Cr, and Sn. Selected area (electron) diffraction data with i) specific area and j) d‐spacing were compared with the data collected by XRD, where the (311) characteristic peak is consistent with the XRD.

Figure 3

Different compositions of PLNs can affect optical physiognomies. a) Scheme energy diagram using PLNs. Cr³⁺ and Sn⁴⁺ have the ability to delay electron emission. b) Stimulation of PLNs from under 250 to 565 nm leads to the emission of particles during UV irradiation and generates red to NIR luminescence. The lifetime results of c) ZGOCS (inset: afterglow images taken 1 min after UV irradiation) and d) ZGOC (inset: the amplified spectrum shows that the pure doping of Cr³⁺ has a long afterglow capability). The persistent luminescence signal can be detected by IVIS. The images were acquired 5 min and 10 min after UV exposure. The slice remained on top of the e) ZGOCS and ZGOC throughout the experiment. The test time was from 0.5 h to 10 h. The blue circle is a region of interest (ROI); the same regions were applied for all testing points.

Figure 4

Evaluation of MAp targeting ability. a) DNA gel of MAp analysis. The experimental group from left to right: 100 bp DNA marker, MAp, No. 45‐16 (active site, negative control), PB (52‐mer, positive control), PLN@SiO₂ (negative control), and PLN@MAp with 50 bp DNA marker. b) Expression of the MAGE‐A3 protein. Western blot results showing the amount of MAGE‐A3 protein signal in Beas2B, H1355, A549, CL1‐0, and in CL1‐5 cells. c) Flow cytometry of PLN@MAp‐treated Beas2B, A549, and CL1‐5 cell lines. The left column is the cell point distribution map, and the fluorescence of the cells can be observed in the A549 and CL1‐5 cells. There is an upward trend of luminescence in the cell lines. The filter selected for testing is PerCP‐A. d) LSCM imaging of PLN@MAp. The blue fluorescence (440 nm) is DAPI staining of the nucleus, yellow fluorescence (570 nm) is DiI staining of the cell membrane, and red luminescence (750 nm) is PLN@MAp itself releasing long‐lasting infrared light. Beas2B, A549, and CL1‐5 cell lines were tested separately (scale bar: 50 µm).

Figure 5

In vitro cell viability and toxicity analysis. a) AFT IC₅₀ testing using A549 cells. The concentration of AFT required to reach the IC₅₀ dose was approximately 243 ng mL⁻¹. b) Cytotoxicity of PLN‐MAp, AFT‐PLN, and AFT‐PLN@MAp. Cells were co‐cultured with A549 cells for 24 h. c) TUNEL assay to detect DNA breaks formed when DNA fragmentation occurs in the last phase of apoptosis. Three groups, PLN‐MAp, AFT‐PLN, and AFT‐PLN@MAp, are analyzed. Blue fluorescence (440 nm) is DAPI staining of the nucleus, and green fluorescence (520 nm) is TUNEL fluorescence (scale bar: 50 µm). d) Flow cytometric analysis of the TUNEL assay. The profile of control cells is gated to fit all different experimental groups: control, PLN‐MAp, AFT‐PLN, and AFT‐PLN@MAp. Fluorescence‐activated cell sorting profiles of cell distribution and green fluorescence protein fluorescence indicate the TUNEL⁺ percentage of cells in the assay.

Figure 6

In vivo IV injection to cure SC A549 lung tumors. a) Simulated imaging showing IV drug administration. The material was injected into the mouse through the tail vein after irradiation under the UV light source for 30 min in vitro; the luminescence intensity of PLN was observed by IVIS after 10 min. b) Testing the accumulation of AFT‐PLN and AFT‐PLN@MAp (1: heart, 2: liver, 3: spleen, 4: lung, 5: kidney, 6: tumor). The ROI value shows the luminescence intensity of the c) quantified luminescence data in different organs. This value is calculated using the IVIS ROI test. d) The treatment procedure of drug administration. First, in the IV and IT administration design, A549 and CL1‐5 tumor cells were injected and grew for 3 weeks until the tumor size was nearly 125 mm³. In the IV administration experiment, the drug was injected through the tail vein every 3 days. In the IT administration experiment, the drug was administered IT every 4 days. The tumor was removed and observed after the 5th week. e) True tumor size evaluation of the “Control,” “AFT,” “AFT‐PLN,” and “AFT‐PLN@MAp” groups. f) The weight of the mice over 5 weeks. The mouse weight increased from 19 to 25 g. g) Tumor size and h) weight of tumors isolated from mice receiving AFT, AFT‐PLN, and AFT‐PLN@MAp treatment. n = 8 (*p < 0.05 compared with the control group). H&E staining of collected tumor tissue by groups i) Control, j) AFT‐PLN, and k) AFT‐PLN@MAp. The full tumor image was obtained with a visual range zoom of 5×, and the 20× images of the tumor revealed the detailed tumor cells (scale bar: 3 µm and 200 nm).

Figure 7

In vivo IT drug delivery to cure an orthotopic CL1‐5 lung tumor. a) Schematic imaging showing IT drug administration. IT experiment: After irradiation with the UV light source for 30 min, the material was injected into the mouse intratracheally, and the luminescence intensity of the PLN was directly observed by IVIS at 10 h. b) IVIS imaging showing the PLN fluorescent signal. The PLN persistent luminescence achieved in vivo at 6 h. Furthermore, the ROI region was chosen to evaluate the intensity of PLN luminescence. c) The true tumor size evaluation by the photo. d) The luminescence of CL1‐5 cells and PLN fluorescent signal. The upper section is the luminescence of cells infected by the lentivirus. The lower section is the fluorescence of the PLN. The tumor and material locations were obtained by luminescence analyses of the IVIS. H&E staining of collected lungs from the e) control, AFT, AFT‐PLN, and AFT‐PLN@MAp groups. The tumor region was obtained with a visual range zoom of 5×, and the 20× images of the tumor revealed the detailed tumor cells (scale bar: 3 µm and 200 nm).