Lyoprotectant Optimization for the Freeze-Drying of Receptor-Targeted Trojan Horse Liposomes for Plasmid DNA Delivery.

Trojan horse liposomes (THLs) are a form of ligand-targeted nanomedicine, where a plasmid DNA is encapsulated in the interior of a 100-150 nm pegylated liposome, and the tips of a fraction of the surface pegylated strands are covalently linked to a receptor-specific monoclonal antibody (MAb) via a thio-ether linkage. The goal of this work was to develop a lyophilization methodology that enables retention of the structure and function of the THLs following the freeze-drying/hydration process. THL fusion and leakage of plasmid DNA were observed with several lyoprotectants, including trehalose, hyaluronic acid, γ-cyclodextrin, or sulfobutylether-ß-cyclodextrin. However, the use of hydroxypropyl-γ-cyclodextrin, at a 40:1 wt/wt ratio relative to the THL phospholipid, eliminated liposome fusion and produced high retention of encapsulated plasmid DNA and THL-mediated gene expression after lyophilization followed by hydration. The freeze-dried THL cake was amorphous without cavitation, and the diameters and functional properties of the THLs were preserved following hydration of cakes stored for at least six months. Intravenous administration of the hydrated freeze-dried THLs in the Rhesus monkey demonstrated the safety of the formulation. Blood plasmid DNA was measured with a quantitative polymerase chain reaction method, which enabled a pharmacokinetics analysis of the blood clearance of the THL-encapsulated plasmid DNA in the primate. The work shows that optimization of the lyoprotectant enables long-term storage of the MAb-targeted DNA encapsulated liposomes in the freeze-dried state.

Silencing Receptor EphA2 Enhanced Sensitivity to Lipoplatin™ in Lung Tumor and MPM Cells.

Receptor EphA2 is overexpressed in lung cancer and malignant pleural mesothelioma (MPM) which promote tumorogenesis. Lipoplatin™, a new liposomal cisplatin formulation, is used against resistant tumors. Use of cisplatin-based drugs leads to unacceptable toxicities. To improve the effectiveness of Lipoplatin, enhancing the cellular sensitivity of lung tumor and MPM cells is critical. Therefore, we targeted receptor EphA2 by silencing interference RNA (siRNA) and treated tumor cells with Lipoplatin. The combined effects of siRNA-EphA2 and Lipoplatin were determined. We report that silencing EphA2 significantly enhanced the cellular sensitivity of lung tumor and MPM cells to Lipoplatin and maybe a potential therapy for lung cancer.

Plasmid DNA gene therapy of the Niemann-Pick C1 mouse with transferrin receptor-targeted Trojan horse liposomes.

Niemann-Pick C1 (NPC1) is a lysosomal cholesterol storage disorder, that severely affects the brain, and is caused by mutations in the NPC1 gene, which encodes an intracellular membrane transporter of non-esterified cholesterol. Therapeutic options for NPC1 are few, and classical enzyme replacement therapy with the recombinant protein is not possible as the NPC1 gene product is an insoluble membrane protein, which increases the need for development of gene therapy for NPC1. While viral based gene therapy is under development, it is important to investigate alternative approaches to brain gene therapy without viral vectors. The present work develops a plasmid DNA approach to gene therapy of NPC1 using Trojan horse liposomes (THLs), wherein the plasmid DNA is encapsulated in 100 nm pegylated liposomes, which are targeted to organs with a monoclonal antibody against the mouse transferrin receptor. THLs were encapsulated with a 8.0 kb plasmid DNA encoding the 3.9 kb human NPC1 open reading frame, under the influence of a 1.5 kb platelet derived growth factor B (PDGFB) promoter. THLs were administered weekly beginning at 6-7 weeks in the NPC1-/- null mouse, and delivery of the plasmid DNA, and NPC1 mRNA expression in brain, spleen, and liver were confirmed by quantitative PCR. THL treatment reduced tissue inclusion bodies in brain, and peripheral organs, but did not prolong lifespan in these mice. The work suggests that early treatment after birth may be required to reverse this disease model with NPC1 gene replacement therapy.

EphA2 targeted intratumoral therapy for non-small cell lung cancer using albumin mesospheres.

Lung cancer, primarily non-small cell lung cancer (NSCLC), is the leading cause of cancer mortality and the prognosis of patients with advanced or metastatic NSCLC is poor. Despite significant advances in diagnosis and treatment, little improvement has been seen in NSCLC mortality. Recently, Intratumoral Chemotherapy, a direct local delivery of chemotherapeutic drugs, has shown promise in clinical studies. However, toxicity and high dosage of chemotherapeutic agents used for treatment are a limitation. Moreover, these drugs damage indiscriminately, cancerous as well as normal tissues. Thus, a novel therapeutic strategy that targets only malignant tissue sparing normal tissue becomes an urgent issue. Ephrin receptor-A2 (EphA2), a new biomarker, is over-expressed in NSCLC, but not on normal epithelial cells. Receptor EphA2 is a cell surface protein, which upon binding to its ligand EphrinA1 undergo phosphorylation and degradation which attenuates NSCLC growth. Targeting the tumor, sparing the normal tissue and enhancing the therapeutic effects of ligand proteins are the goal of this project. Thus a novel method, intratumoral EphA2 targeted therapy, has been developed to target the oncogenic receptors on tumor tissue by using albumin mesosphere (AMS) conjugated ephrinA1 in mice bearing NSCLC tumors.

Nanoparticle-based targeted gene therapy for lung cancer.

Despite striking insights on lung cancer progression, and cutting-edge therapeutic approaches the survival of patients with lung cancer, remains poor. In recent years, targeted gene therapy with nanoparticles is one of the most rapidly evolving and extensive areas of research for lung cancer. The major goal of targeted gene therapy is to bring forward a safe and efficient treatment to cancer patients via specifically targeting and deterring cancer cells in the body. To achieve high therapeutic efficacy of gene delivery, various carriers have been engineered and developed to provide protection to the genetic materials and efficient delivery to targeted cancer cells. Nanoparticles play an important role in the area of drug delivery and have been widely applied in cancer treatments for the purposes of controlled release and cancer cell targeting. Nanoparticles composed of artificial polymers, proteins, polysaccharides and lipids have been developed for the delivery of therapeutic deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences to target cancer. In addition, the effectiveness of cancer targeting has been enhanced by surface modification or conjugation with biomolecules on the surface of nanoparticles. In this review article we provide an overview on the latest developments in nanoparticle-based targeted gene therapy for lung cancers. Firstly, we outline the conventional therapies and discuss strategies for targeted gene therapy using nanoparticles. Secondly, we provide the most representative and recent researches in lung cancers including malignant pleural mesothelioma, mainly focusing on the application of Polymeric, Lipid-based, and Metal-based nanoparticles. Finally, we discuss current achievements and future challenges.

MicroRNA-302b targets Mcl-1 and inhibits cell proliferation and induces apoptosis in malignant pleural mesothelioma cells.

MicroRNAs belonging to the miR-302 family are emerging as key players in the control of cell growth, and maintaining pluripotency during cell fate determination and differentiation in embryonic stem cells. However, the mechanisms whereby ephA2/ephirnA1 signaling regulates miR-302b expression and attenuates malignant pleural mesothelioma (MPM) cell growth are not known. Our study identified a novel mechanism of ephrin-A1 mediated anti-oncogenic signaling in MPM. Ephrin-A1 treatment up regulates miR-302b expression in MPM cells and attenuates cell proliferation and tumorsphere formation via repression of myeloid cell leukemia-1 (Mcl-1). The expression of miR-302b was analyzed by qPCR, the expression of Mcl-1 was analyzed by RT-PCR, immuno-blotting and Immunofluorescence staining. To confirm that ephrin-A1 regulates the expression of Mcl-1 mRNA through miR-302b up regulation, cells were transfected with and without miR-302b and miR-302b inhibitor prior to ephrinA1 treatment. The cell proliferation and tumorsphere formation was measured by WST-1 and matrigel assays respectively. In addition, to confirm the binding of miR-302b to the 3'UTR of Mcl-1 Luciferase assay was performed. Ephrin-A1 treatment induced several fold increases of miR-302b expression in MM cells. In ephrin-A1 treated MM cells, Mcl-1 expression was significantly down regulated when compared to control. Moreover, ephrin-A1 activation significantly inhibited MM cell proliferation and tumorsphere growth. Furthermore, ephrinA1 and miR-302b induced apoptosis in MM cells. The present data suggests that ephrin-A1 induces the expression of miR-302b in MM cells which targets Mcl-1 thereby inhibits MM tumorsphere growth by inducing apoptosis.

Cisplatin loaded albumin mesospheres for lung cancer treatment.

The low solubility of cisplatin in aqueous solution limits the treatment effectiveness and the application of cisplatin in various kinds of drug-eluting devices. Although cisplatin has a high solubility in Dimethyl sulfoxide (DMSO), the toxicity of cisplatin can be greatly reduced while dissolved in DMSO. In this study, the solid powder of cisplatin-loaded albumin mesospheres (CDDP/DMSO-AMS), in a size range of 1 to 10 µm, were post-loaded with cisplatin and showed high cisplatin content (16% w/w) and effective cytotoxicity to lung cancer cells. Cisplatin were efficiently absorbed into the albumin mesospheres (AMS) in DMSO and, most importantly, the toxicity of cisplatin was remained at 100% after the loading process. This CDDP/DMSO-AMS was designed for the intratumoral injection through the bronchoscopic catheter or dry powder inhalation (DPI) due to its high stability in air or in solution. This CDDP/DMSO-AMS showed a fast cisplatin release within 24 hours. In the in vitro study, CDDP/DMSO-AMS showed high effectiveness on killing the lung cancer cells including the non-small cell lung cancer (NCL-H23 and A549), malignant mesothelioma (CRL-2081) and the mouse lung carcinoma (Lewis lung carcinoma) cell lines. The albumin based mesospheres provide an ideal loading matrix for cisplatin and other metal-based drugs due to the high swelling degree and fast uptake rate in the organic solvents with high polarity. In addition, to investigate the effects of polysaccharides, such as chitosan and chondroitin, on enhancing loading efficiency and lasting cytotoxicity of cisplatin, the polysaccharide-modified albumin mesospheres were synthesized and loaded with cisplatin in this study.

Arginine-conjugated albumin microspheres inhibits proliferation and migration in lung cancer cells.

Arginine is one of the essential amino acid involved in numerous biosynthetic pathways that significantly influence tumor growth. It has been demonstrated that arginine is effective to inhibit proliferation of cancer cells when an appropriate dose is applied. Generally, induction of cell death requires high concentration of arginine while low concentration of arginine facilitates cell proliferation. In addition to the apoptosis induced by metabolism of arginine, it has also been reported that in an ideal solution environment, arginine may assemble into arginine clusters to kill cancer cells. Therefore, to make the arginine an effective anticancer agent, arginine/albumin microspheres were designed and synthesized to provide a localized high concentration of arginine on tumor sites. In addition, the arginine/albumin mesospheres (AAMS) are also expected to provide an arginine-rich surface on microspheres, which is similar to the arginine cluster, to effectively inhibit tumor growth. In this study, the AAMS were synthesized through a water/organic solvent emulsion system and the surface properties were characterized. The in vitro effects of AAMS on A549, CRL-2081, MAK9 lung cancer cells (LCC) proliferation, migration, and tumor growth were determined. The expression of oncogenic protein EphA2 and transcription factor slug was also determined. AAMS significantly inhibited the cell proliferation, cell migration and tumor growth in all the three LCC, while same concentration of free arginine promoted the LCC tumor growth and migration. Our studies indicate that the synthesized AAMS has a more effective inhibiting effect on proliferation, migration and tumor growth of LCC than freely released arginine. The expression of EphA2 receptor mRNA was significantly decreased when compared to control cells. In addition the mRNA expression of transcription factor slug was also inhibited by AAMS suggesting that AAMS affects the expression of EphA2 and slug and may regulate LCC proliferation and migration. These data suggests that the AAMS can be an ideal delivery vehicle for therapeutic interventions against LCCs.

Targeted delivery of let-7a microRNA encapsulated ephrin-A1 conjugated liposomal nanoparticles inhibit tumor growth in lung cancer.

MicroRNAs (miRs) are small noncoding RNA sequences that negatively regulate the expression of target genes by posttranscriptional repression. miRs are dysregulated in various diseases, including cancer. let-7a miR, an antioncogenic miR, is downregulated in lung cancers. Our earlier studies demonstrated that let-7a miR inhibits tumor growth in malignant pleural mesothelioma (MPM) and could be a potential therapeutic against lung cancer. EphA2 (ephrin type-A receptor 2) tyrosine kinase is overexpressed in most cancer cells, including MPM and non-small-cell lung cancer (NSCLC) cells. Ephrin-A1, a specific ligand of the EphA2 receptor, inhibits cell proliferation and migration. In this study, to enhance the delivery of miR, the miRs were encapsulated in the DOTAP (N-[1-(2.3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium)/Cholesterol/DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[cyanur(polyethylene glycol)-2000])-PEG (polyethylene glycol)-cyanur liposomal nanoparticles (LNP) and ephrin-A1 was conjugated on the surface of LNP to target receptor EphA2 on lung cancer cells. The LNP with an average diameter of 100 nm showed high stability, low cytotoxicity, and high loading efficiency of precursor let-7a miR and ephrin-A1. The ephrin-A1 conjugated LNP (ephrin-A1-LNP) and let-7a miR encapsulated LNP (miR-LNP) showed improved transfection efficiency against MPM and NSCLC. The effectiveness of targeted delivery of let-7a miR encapsulated ephrin-A1 conjugated LNP (miR-ephrin-A1-LNP) was determined on MPM and NSCLC tumor growth in vitro. miR-ephrin-A1-LNP significantly increased the delivery of let-7a miR in lung cancer cells when compared with free let-7a miR. In addition, the expression of target gene Ras was significantly repressed following miR-ephrin-A1-LNP treatment. Furthermore, the miR-ephrin-A1-LNP complex significantly inhibited MPM and NSCLC proliferation, migration, and tumor growth. Our results demonstrate that the engineered miR-ephrin-A1-LNP complex is an effective carrier for the targeted delivery of small RNA molecules to lung cancer cells. This could be a potential therapeutic approach against tumors overexpressing the EphA2 receptor.

Targeted lung cancer therapy using ephrinA1-loaded albumin microspheres.

OBJECTIVES: EphrinA1, the ligand of EphA2 receptor tyrosine kinase, has been proven to suppress the growth of tumours. The aim of this study was to conjugate ephrinA1 on the surface of albumin microspheres and investigate the non-small cell lung carcinoma growth and migration in vitro. METHODS: Bovine serum albumin microspheres were designed and synthesized using a natural polymer albumin by emulsification chemical cross-linking. EphrinA1 was then conjugated on the surface of microspheres by imine formation. The microspheres conjugated with ephrinA1 (ephrinA1-MS) were characterized for particle size, surface morphology, loading efficiency and stability in vitro. The ephrinA1-MS were labelled with fluorescein isothiocyanate to determine phagocytosis. In addition, the effects of ephrinA1-MS on A549 cell growth and migration were determined. KEY FINDINGS: Albumin microspheres exhibited low toxicity for A549 cells (above 90% cell viability). More than 80% of microspheres were phagocytosed within 2 h of incubation. EphrinA1-MS decreased the expression of focal adhesion kinase more effectively than recombinant ephrinA1 alone. Furthermore, ephrinA1-MS showed significant inhibition of non-small cell lung cancer migration when compared with resting cells. EphrinA1-MS attenuated the growth of tumour colonies in matrigels. CONCLUSIONS: The developed ephrinA1-MS may serve as potential carriers for targeted delivery of the tumour suppressive protein ephrinA1, with minimal cytotoxic effects and greater antitumour therapeutic efficacy against non-small cell lung cancer.