Lung Cancer-Targeting Peptides with Multi-subtype Indication for Combinational Drug Delivery and Molecular Imaging

Abstract

Lung cancer is the leading cause of cancer-related death worldwide. Most targeted drugs approved for lung cancer treatment are tyrosine kinase inhibitors (TKIs) directed against EGFR or ALK, and are used mainly for adenocarcinoma. At present, there is no effective or tailored targeting agent for large cell carcinoma (LCC) or small cell lung cancer (SCLC). Therefore, we aimed to identify targeting peptides with diagnostic and therapeutic utility that possess broad subtype specificity for SCLC and non-small cell lung cancer (NSCLC). We performed phage display biopanning of H460 LCC cells to select broad-spectrum lung cancer-binding peptides, since LCC has recently been categorized as an undifferentiated tumor type within other histological subcategories of lung cancer. Three targeting phages (HPC1, HPC2, and HPC4) and their respective displayed peptides (HSP1, HSP2, and HSP4) were able to bind to both SCLC and NSCLC cell lines, as well as clinical specimens, but not to normal pneumonic tissues. In vivo optical imaging of phage homing and magnetic resonance imaging (MRI) of peptide-SPIONs revealed that HSP1 was the most favorable probe for multimodal molecular imaging. Using HSP1-SPION, the T2-weighted MR signal of H460 xenografts was decreased up to 42%. In contrast to the tight binding of HSP1 to cancer cell surfaces, HSP4 was preferentially endocytosed and intracellular drug delivery was thereby effected, significantly improving the therapeutic index of liposomal drug in vivo. Liposomal doxorubicin (LD) conjugated to HSP1, HSP2, or HSP4 had significantly greater therapeutic efficacy than non-targeting liposomal drugs in NSCLC (H460 and H1993) animal models. Combined therapy with an HSP4-conjugated stable formulation of liposomal vinorelbine (sLV) further improved median overall survival (131 vs. 84 days; P = 0.0248), even in aggressive A549 orthotopic models. Overall, these peptides have the potential to guide a wide variety of tailored theranostic agents for targeting therapeutics, non-invasive imaging, or clinical detection of SCLC and NSCLC.

Keywords: Small cell lung cancer (SCLC); liposomal drugs; magnetic resonance imaging (MRI); molecular imaging; non-small cell lung cancer (NSCLC); targeting peptides; theranostics..

Figure 1

Three novel peptides and their respective phage clones showed different binding activities to various histopathological subtypes of human lung cancer. (A) FACS data indicating that HSP1, HSP2, and HSP4 are able to bind to several human lung cancer cell lines, but not normal bronchial epithelial cells. These three peptides showed different binding patterns in SCLC and NSCLC cell lines. NSCLC includes adenocarcinoma, SCC, and LCC. FITC-conjugated Ctrl P served as a negative control. (B) Immunohistochemical staining of human NSCLC and SCLC clinical specimens using HPC1, HPC2, and HPC4 phage clones (2~5 × 10⁸ pfu/μl). Normal bronchiole was not detected by these targeting phages. The same titer of helper phage was used as negative control. The red arrow indicates plasma membrane localization of HPC1 and HPC4 in adenocarcinoma. Scale bar, 25 μm.

Figure 2

Verification of the tumor homing ability of H460-targeting phage in vivo. (A) HPC2, HPC3, and HPC4 exhibited the greatest tumor homing ability among the four phage clones of group 1, while HPC1 possessed the best ability in group 2 (n=3). Phage clones were grouped based on the presence of consensus sequences. (B) Optical imaging of whole body and dissected organs from HPC1-HL750-injected mice, as compared to those from the control phage group at 24 hr post-injection. (C) Tissue distribution of HiLyte Fluor 750-labeled phage was examined at 24 hr post-injection, and signal intensities of tumor and organs were measured using IVIS200 software. *, P<0.05; * * *, P<0.001 (n=3).

Figure 3

T2-weighted MR imaging of HSP1-, HSP2-, and HSP4-Dex-Fe₃O₄ nanoparticles in H460 xenografts. (A) SCID mice bearing similarly-sized H460 xenografts (approximately 120 mm³ in volume) were subjected to 4.7 T MRI analyses after i.v. injection with HSP1-, HSP2-, HSP4-, or Ctrl P-Dex-Fe₃O₄ nanoparticles. T2-weighted imaging was performed before injection, and at 3 hr, 6 hr, and 24 hr post-injection. Yellow arrows indicate subcutaneous tumors in the right thigh. (B) Quantitative analysis based on the T2 relative signal-to-noise ratio of the ROI. HSP1-, HSP2-, and HSP4-Dex-Fe₃O₄ nanoparticles exhibited significant targeting effect in the tumor regions as compared to Ctrl P-Dex-Fe₃O₄ nanoparticles. Data points, mean; error bars, SD; *, P<0.05; * * *, P<0.001 (n=3).

Figure 4

Visualization of the detailed tumor distribution of targeted SPIONs by T2 color mapping and histological analyses. (A) The T2-weighted MR images were subjected to pseudo-color mapping to reveal detailed signal changes in tumor tissues. Remarkable signal reduction can be seen in blue, corresponding to roughly 4/5, 3/4, and 1/4 of tumor area at 6 hr post-contrast with HSP1-, HSP2-, and HSP4-Dex-Fe₃O₄ nanoparticles, respectively. (B) Histological analyses of H460 tumor tissue specimens acquired 24 hrs after injection of HSP1-, HSP2-, HSP4-,or Ctrl P-Dex-Fe₃O₄ nanoparticles. Sections were stained with Prussian blue to detect Fe deposition, and were counterstained with Nuclear Fast Red. Targeting peptides enhanced extravasation of SPIONs out of blood vessels and deep penetration of SPIONs for cancer cell binding, while most control-SPIONs were washed out and thus rarely detected in tumor. Yellow arrows indicate blood vessels of tumor tissue. (C) Quantification of Prussian blue reaction products from the representative tumor sections. **, P<0.01; * * *, P<0.001 compared with Ctrl P-Dex-Fe₃O₄ group.

Figure 5

Endocytosis of HSP1, HSP2, and HSP4-LSRB by H460 cells, as examined by confocal microscopy. H460 cells were separately incubated with 10 μM each of HSP1, HSP2, or HSP4 peptide-conjugated LSRB at 37°C (A) or 4°C (B) for 30 min. After removal of non-bound liposomes by washing, confocal microscopy was used to examine liposomal fluorescence. Non-targeting LSRB (10 μM) was used as a control. The nuclei were stained with DAPI. Scale bar, 50 μm.

Figure 6

Targeting peptides HSP1, HSP2, and HSP4 improve the therapeutic efficacy of LD in vivo by increasing drug bioavailability. Mice bearing human LCC H460-derived xenografts with an average tumor size of (A) ~75 mm³ (n=8 in each group) or (B) ~500 mm³ (n=7 in each group) were intravenously injected with FD, LD, targeting liposomes (HSP1-LD, HSP2-LD, or HSP4-LD), or an equal volume of PBS. The administration regimens for each experiment are shown under their respective abscissae. Data points, mean tumor volumes. Error bars, SE. *, P<0.05 compared with non-targeting LD group; ***, P<0.001 compared with FD group. (C) Mice bearing human lung adenocarcinoma H1993 xenografts with an average tumor size of ~300 mm³ were intravenously injected with FD, LD, HSP1-LD, HSP2-LD, HSP4-LD (1 mg/kg, twice a week for three weeks), or an equal volume of PBS. n=7 in each group. Data points, mean tumor volumes. Error bars, SE. *, P<0.05; **, P<0.01; ***, P<0.001. All significant P values arise from comparison with the LD group. (D-E) Pharmacokinetic and pharmacodynamic analyses of LD, HSP1-LD, HSP2-LD, and HSP4-LD in H460 xenografts. At selected time points (1 hr and 24 hr) after a single dose injection (2 mg/kg), the biodistribution patterns of free form, liposomal, and targeting liposomal drug in serum (D, upper panel), tumor (D, middle panel), and normal tissues (E) were estimated by measuring doxorubicin auto-fluorescence signals. n=3 mice in each group. Auto-fluorescence signals were also detected in the nuclei of tumors (D, lower panel). *, P<0.05; **, P<0.01; ***, P<0.001; NS, no significance; Bars, mean; error bars, SD. HSP2 and HSP4 peptides selectively and significantly enhanced drug delivery to tumors and tumor nuclei. (E) Liposomal drugs have similar biodistribution patterns in normal tissues.

Figure 7

Combination therapy with HSP4-LD and HSP4-sLV in human lung LCC xenografts. (A) Cryo-TEM micrographs of HSP4-LD and HSP4-sLV. Scale bar, 100 nm. (B) Particle size distribution and zeta potential of HSP4-LD and HSP4-sLV. (C) Mice bearing H460-derived lung cancer xenografts with an average tumor size of ~200 mm³ were intravenously injected with FD/FV, LD/sLV, or HSP4-LD/HSP4-sLV at combination doses of 1 mg/kg vinorelbine and 2 mg/kg doxorubicin twice a week for four weeks, or with an equivalent volume of PBS. n=8 in each group. Data points, mean tumor volumes. Error bars, SE. *, P<0.05; **, P<0.01. (D) Body weight during treatment. a, LD+sLV compared to PBS group with P<0.05 (*). b, HSP4-LD+HSP4-sLV compared to PBS group with no significance (NS). (E) Kaplan-Meier survival curve, revealing markedly longer lifespan in mice treated with HSP4-targeted liposomal drugs as compared to other groups. (F) Median survival (in days) was significantly prolonged by HSP4-targeted LD and sLV as compared to non-targeting liposomes (*, P=0.0254).

Figure 8

HSP4-LD and HSP4-sLV combination therapy in an orthotopic model of A549 adenocarcinoma. (A) Imaging drug response of mice with luciferase-expressing A549 cell transplants to combination therapy with FD/FV, LD/sLV, or HSP4-LD/HSP4-sLV at doses of 1 mg/kg vinorelbine and 2 mg/kg doxorubicin, delivered by i.v. injection. Controls were treated with an equal volume of PBS. A total of 5 × 10⁵ cells were transplanted with Matrigel, and treatment started 5 days after cancer cell transplantation (once every two days for 16 days). n=7 in each group. (B) Luminescence signals in tumor were quantified using IVIS200 software. **, P<0.01; ***, P<0.001. (C) Body weight during the course of treatment. The overall survival rate (D) and median survival days (E) were significantly prolonged by HSP4-targeted LD and sLV, as compared to non-targeting liposomes (*, P=0.0248).