Strategy to enhance lung cancer treatment by five essential elements: inhalation delivery, nanotechnology, tumor-receptor targeting, chemo- and gene therapy

Abstract

Non-Small Cell Lung Carcinoma (NSCLC), is the most common type of lung cancer (more than 80% of all cases). Small molecule Tyrosine Kinase (TK) Inhibitors acting on the Epidermal Growth Factor Receptors (EGFRs) are standard therapies for patients with NSCLC harboring EGFR-TK inhibitor-sensitizing mutations. However, fewer than 10 % of patients with NSCLC benefit from this therapy. Moreover, even the latest generation of EGFR inhibitors can cause severe systemic toxicities and are ineffective in preventing non-canonical EGFR signaling. In order to minimize and even overcome these limitations, we are proposing a novel multi-tier biotechnology treatment approach that includes: (1) suppression of all four types of EGFR-TKs by a pool of small interfering RNAs (siRNAs); (2) induction of cell death by an anticancer drug, (3) enhancing the efficiency of the treatment by the local inhalation delivery of therapeutic agents directly to the lungs (passive targeting), (4) active receptor-mediated targeting of the therapy specifically to cancer cells that in turn should minimize adverse side effects of treatment and (5) increasing the stability, solubility, and cellular penetration of siRNA and drug by using tumor targeted Nanostructured Lipid Carriers (NLC). Methods: NLCs targeted to NSCLC cells by a synthetic Luteinizing Hormone-Releasing Hormone (LHRH) decapeptide was used for the simultaneous delivery of paclitaxel (TAX) and a pool of siRNAs targeted to the four major forms of EGFR-TKs. LHRH-NLC-siRNAs-TAX nanoparticles were synthesized, characterized and tested in vitro using human lung cancer cells with different sensitivities to gefitinib (inhibitor of EGFR) and in vivo on an orthotopic NSCLC mouse model. Results: Proposed nanoparticle-based complex containing an anticancer drug, inhibitors of different types of EGFR-TKs and peptide targeted to the tumor-specific receptors (LHRH-NLC-siRNAs-TAX) demonstrated a favorable organ distribution and superior anticancer effect when compared with treatment by a single drug, inhibitor of one EGFR-TK and non-targeted therapy. Conclusions: The use of a multifunctional NLC-based delivery system substantially enhanced the efficiency of therapy for NSCLC and possibly will limit adverse side effects of the treatments. The results obtained have the potential to significantly impact the field of drug delivery and to improve the efficiency of therapy of lung and other types of cancer.

Keywords: Imaging; LHRH peptide; lung cancer cells with different resistance to gefitinib; pool of siRNAs; suppression of EGFR-TK signaling pathways.

Figure 1

Comparison of conventional and non-traditional proposed approaches for treatment of non-small cell lung carcinoma.

Figure 2

Characterization of Nanostructured Lipid Carriers (NLCs). A - Representative atomic force microscope images of NLC-siRNA complexes. B - Histogram of NLC-siRNA size distribution. C - Typical image of siRNA in agarose gel stained with ethidium bromide incubated with different amount of NLC. D - IgG secretion in vitro in supernatants of peripheral blood lymphocytes cultured with medium (control) and pool of siRNAs delivered by non-targeted and LHRH-targeted NLC. E - The human serum stability of non-condensed naked siRNA (upper panel), and the engineered siRNA nanoparticles before (middle panel) and after (lower panel) nebulization. F - Apoptosis induction in different organs in animals treated with saline (control), NLC by intravenous injection (I.V.) and by inhalation. The enrichment of histone-associated DNA fragments (mono- and oligonucleosomes) per gram tissue in different organs were measured. Values in control animals were set to unit 1, and the degree of apoptosis was expressed in relative units. F - Apoptosis induction Means ± SD are shown.

Figure 3

Cellular internalization of Nanostructured Lipid Carriers (NLC), siRNA and paclitaxel delivered by NLC (confocal microscope Leica G-STED SP8). A549 adenocarcinomic human basal epithelial (alveolar type II pneumocytes) non-small cell lung cancer (NSCLC) cells were incubated for 18 h with NLC (blue color represents near-infrared fluorescence) containing siRNA (red fluorescence) and paclitaxel (green fluorescence). Superimposition of red and blue colors gives pink color; superimposition of blue and green gives cyan color; superimposition of red and green colors gives yellow color; superimposition of red, green and blue colors gives white color.

Figure 4

Cytotoxicity of different formulations in human A549 NSCLC cells (A) and competitive inhibition of cellular cytotoxicity of LHRH-NLC-TAX complex by free LHRH peptide (B). Means ± SD are shown.

Figure 5

Cytotoxicity and apoptosis induction in human lung cancer cells with different sensitivity to gefitinib. H1781 gefitinib-insensitive (EGFR2-mutant), A549 (no EGFR-TK mutations) with moderate sensitivity to gefitinib and H3255 gefitinib sensitive (EGFR1-L858R mutant) cells were incubated within 24h with fresh media (control), gefitinib (1 μM) and NLC-siRNAs-TAX (0.3 ng/mL). A - Cytotoxicity. B - Apoptosis induction. Means ± SD are shown. *P < 0.05 when compared with control. ^†P < 0.05 when compared with gefitinib.

Figure 6

Evaluation of orthotopic model of human NSCLC in nude mice by a magnetic resonance imaging (MRI). A - Representative MRI image of a mouse with lung tumor. B-E - Computer analysis of representative MRI image of mouse lungs. Bright rich purple (orchid) color represents a healthy lung tissue, while cyan - tumor tissue (B - top; C - right; D - bottom; E - left).

Figure 7

Distribution (optical imaging, IVIS) of non-targeted NLC and tumor-targeted LHRH-NLC in mice with orthotopic model of human lung cancer. A - A representative image of mouse with orthotopic model of lung cancer (human A549 NSCLC cells transfected with luciferase were inoculated intratracheally into the lungs of nude mice); B, C - Organ distribution of fluorescently labeled by DiR NLC (I.V. and inhalation administration) and LHRH-NLC (inhalation administration) in mice with lung tumor. Ten animals were used in each experimental group. Means ± SD are shown. *P < 0.05 when compared with I.V. injection.

Figure 8

Instillation for inhalation delivery of drug and siRNA NLC-formulations.

Figure 9

Evaluation of the proposed complex approach for treatment of mice with human non-small cell lung carcinoma. A - Suppression of targeted EGFR1-TK mRNA by siRNA delivered by non-targeted and targeted NLC. B - Suppression of four EGFR-TKs by the selected pool of siRNAs delivered by targeted LHRH-NLC-siRNAs-TAX system. C - Apoptosis induction in the lung tumor. D - Changes in lung tumor volume after beginning of treatment (mice were treated on days 0, 3, 7, 11, 14, 17, 21 and 24). The progression of tumor growth was monitored using bioluminescent and magnetic resonance imaging and tumor volume was calculated using software supplied with IVIS and magnetic resonance imaging systems. 1 - Control (untreated tumor); 2 - Free non-bound TAX (I.V. administration); 3 - Non-targeted NLC-siRNAs (inhalation); 4 - Non-targeted NLC-TAX (inhalation); 5 - Non-targeted NLC-siRNAs-TAX (inhalation); 6 - tumor targeted LHRH-NLC-siRNAs-TAX (inhalation). Means ± SD are shown. *P < 0.05 when compared with control;^†P < 0.05 when compared with free TAX;^‡P < 0.05 when compare with NLC-siRNAs;⁺P < 0.05 when compared with NLC-TAX; ^×P < 0.05 when compared with NLC-siRNA-TAX.