Targeted Delivery of siRNA to the Tumor

We have developed a surface-modified LPD (liposome-polycation-DNA) nanoparticle formulation by mixing cationic liposomes, a polycationic peptide and nucleic acids (mixture of DNA and siRNA) at a fixed ratio, followed by post-inserting a PEGylated lipid. This self-assembled nanoparticle formulation was around 100 nm in diameter with 90% encapsulation efficiency for siRNA. The nucleic acid was complexed with the peptide into a compact core, which was coated with two lipid bilayers. The inner lipid bilayer was stabilized by the charge-charge interaction between the cationic lipids and the compact core. Upon addition of a PEGylated lipid, the outer lipid bilayer was stripped off and the lipid anchor was inserted into the outer leaflet of the inner bilayer, resulting in approximately 10.6 mol% modification of PEG (polyethylene glycol) on the surface of the nanoparticles. The high degree of PEGylation completely shielded the charge of the nanoparticles with the zeta potential close to neutral (-5.6 ± 4.5 mV) and abolished the reticuloendothelial uptake in the isolated liver. When i.v. injected into tumor bearing mice (s.c. human lung cancer xenograft model in the nude mice), the nanoparticles delivered 70-80% injected siRNA/g into the tumor, while the normal organs only showed a moderate uptake (10-20% injected siRNA/g). After the conjugation of a targeting ligand, anisamide, at the distal end of the PEG, the intracellular delivery of siRNA into the sigma receptor expressing tumor was significantly enhanced. This led to efficient EGFR silencing, significant apoptosis induction and tumor growth inhibition at the dose of 1.2 mg siRNA/kg for three consecutive injections. The experimental murine lung metastasis model was established by i.v. injecting the mouse melanoma cells, which were stably transduced with a luciferase gene by retrovirus, into the mice. An improved metastatic tumor delivery of siRNA was discovered by using the nanoparticles. When combinatorial siRNA sequences were delivered, the oncogenes (MDM2, c-myc and VEGF) in the lung metastasis were silenced simultaneously, leading to 70-80% tumor load reduction and 30% prolongation in animal lifespan. The nanoparticle formulation showed minimal to no otoxicity in both animal models. The results promise the potential use of this formulation clinically.Doctor of Philosoph

Efficient gene silencing in metastatic tumor by siRNA formulated in surface-modified nanoparticles

We have developed a nanoparticle (NP) formulation for systemically delivering siRNA into metastatic tumors. The NP, composed of nucleic acids, a polycationic peptide and cationic liposome, was prepared in a self-assembling process. The NP was then modified by PEG-lipid containing a targeting ligand, anisamide, and thus was decorated for targeting sigma receptor expressing B16F10 tumor. The activity of the targeted NP was compared with the naked NP (no PEGylation) and non-targeted NP (no ligand). The delivery efficiency of the targeted NP was 4-fold higher than the non-targeted NP and could be competed by excess free ligand. Luciferase siRNA was used to evaluate the gene silencing activity in the B16F10 cells, which were stably transduced with a luciferase gene, in a lung metastasis model. The gene silencing activity of the targeted NP was significantly higher than the other formulations and lasted for 4 days. While confocal microscopy showed the naked NP provided no tissue selectivity and non-targeted NP was ineffective for tumor uptake, the targeted NP effectively penetrated the lung metastasis, but not the liver. It resulted in 70-80% gene silencing in the metastasis model after a single i.v. injection (150 μg siRNA/kg). This effective formulation also showed very little immunotoxicity

Efficient Oncogene Silencing and Metastasis Inhibition via Systemic Delivery of siRNA

The selective delivery of small interfering RNA (siRNA) to metastatic tumors remains a challenging task. We have developed a nanoparticle (NP) formulation composed of siRNA, a carrier DNA, a polycationic peptide, and cationic liposomes. The NP was obtained by a self-assembling process, followed by surface modification with a polyethylene glycol (PEG)-conjugated ligand, anisamide. The NP was PEGylated and a ligand was presented to target sigma receptor–expressing murine melanoma cells, B16F10. The lung metastasis model was established by intravenous (IV) injection of the B16F10 cells into C57BL/6 mice. A mixture of siRNA against MDM2, c-myc, and vascular endothelial growth factor (VEGF) co-formulated in the targeted NP caused simultaneous silencing of each of the oncogenes in the metastatic nodules. Two consecutive IV injections of siRNA in the targeted NP significantly reduced the lung metastasis (~70–80%) at a relatively low dose (0.45 mg/kg), whereas free siRNA and the nontargeted NP showed little effect. This targeted NP formulation significantly prolonged the mean survival time of the animals by 30% as compared to the untreated controls. At the therapeutic dose, the targeted NP showed little local and systemic immunotoxicity and did not decrease the body weight or damage the major organs

Anti-tumor activity of splice-switching oligonucleotides

Alternative splicing has emerged as an important target for molecular therapies. Splice-switching oligonucleotides (SSOs) modulate alternative splicing by hybridizing to pre-mRNA sequences involved in splicing and blocking access to the transcript by splicing factors. Recently, the efficacy of SSOs has been established in various animal disease models; however, the application of SSOs against cancer targets has been hindered by poor in vivo delivery of antisense therapeutics to tumor cells. The apoptotic regulator Bcl-x is alternatively spliced to express anti-apoptotic Bcl-xL and pro-apoptotic Bcl-xS. Bcl-xL is upregulated in many cancers and is associated with chemoresistance, distinguishing it as an important target for cancer therapy. We previously showed that redirection of Bcl-x pre-mRNA splicing from Bcl-xL to -xS induced apoptosis in breast and prostate cancer cells. In this study, the effect of SSO-induced Bcl-x splice-switching on metastatic melanoma was assessed in cell culture and B16F10 tumor xenografts. SSOs were delivered in vivo using lipid nanoparticles. Administration of nanoparticle with Bcl-x SSO resulted in modification of Bcl-x pre-mRNA splicing in lung metastases and reduced tumor load, while nanoparticle alone or formulated with a control SSO had no effect. Our findings demonstrate in vivo anti-tumor activity of SSOs that modulate Bcl-x pre-mRNA splicing

Hyperthermia-induced drug targeting

Introduction: Specific delivery of a drug to a target site is a major goal of drug delivery research. Using temperature-sensitive liposomes (TSLs) is one way to achieve this; the liposome acts as a protective carrier, allowing increased drug to flow through the bloodstream by minimizing clearance and non-specific uptake. On reaching microvessels within a heated tumor, the drug is released and quickly penetrates. A major advance in the field is ThermoDox® (Celsion), demonstrating significant improvements to the drug release rates and drug uptake in heated tumors (∼ 41°C). Most recently, magnetic resonance-guided focused ultrasound (MRgFUS) has been combined with TSL drug delivery to provide localized chemotherapy with simultaneous quantification of drug release within the tumor. Areas covered: In this article the field of hyperthermia-induced drug delivery is discussed, with an emphasis on the development of TSLs and their combination with hyperthermia (both mild and ablative) in cancer therapy. State-of-the-art image-guided heating technologies used with this combination strategy will also be presented, with examples of real-time monitoring of drug delivery and prediction of efficacy. Expert opinion: The specific delivery of drugs by combining hyperthermia with TSLs is showing great promise in the clinic and its potential will be even greater as the use of image-guided focused ultrasound becomes more widespread-a technique capable of penetrating deep within the body to heat a specific area with improved control. In conjunction with this, it is anticipated that multifunctional TSLs will be a major topic of study in this field.</p

Thermosensitive liposomes in cancer therapy

Thermosensitive liposomes have been a popular area of investigation for several years leading to a plentitude of scientific literature and several patents. The majority of formulations are still in the early stage of development and preclinical testing, although one formulation, ThermoDox®, is significantly more advanced. This formulation is now in clinical trials for a few different cancer indications. In this review, key patents and publications through the evolution of thermosensitive lipososomes are presented, including the use of polymers, lipids and other molecules to control the temperature sensitivity. ThermoDox® is discussed with an update on recent experiments and reports from the clinical trials. Finally, a summary of recent formulations designed to improve upon the ThermoDox® benchmark is presented, and the challenges and future directions for thermosensitive liposome technology are discussed.</p

Thermosensitive Liposomes in Cancer Therapy

Thermosensitive liposomes have been a popular area of investigation for several years leading to a plentitude of scientific literature and several patents. The majority of formulations are still in the early stage of development and preclinical testing, although one formulation, ThermoDox®, is significantly more advanced. This formulation is now in clinical trials for a few different cancer indications. In this review, key patents and publications through the evolution of thermosensitive lipososomes are presented, including the use of polymers, lipids and other molecules to control the temperature sensitivity. ThermoDox® is discussed with an update on recent experiments and reports from the clinical trials. Finally, a summary of recent formulations designed to improve upon the ThermoDox® benchmark is presented, and the challenges and future directions for thermosensitive liposome technology are discussed.</p

Development and Characterization of the Solvent-Assisted Active Loading Technology (SALT) for Liposomal Loading of Poorly Water-Soluble Compounds

A large proportion of pharmaceutical compounds exhibit poor water solubility, impacting their delivery. These compounds can be passively encapsulated in the lipid bilayer of liposomes to improve their water solubility, but the loading capacity and stability are poor, leading to burst drug leakage. The solvent-assisted active loading technology (SALT) was developed to promote active loading of poorly soluble drugs in the liposomal core to improve the encapsulation efficiency and formulation stability. By adding a small volume (~5 vol%) of a water miscible solvent to the liposomal loading mixture, we achieved complete, rapid loading of a range of poorly soluble compounds and attained a high drug-to-lipid ratio with stable drug retention. This led to improvements in the circulation half-life, tolerability, and efficacy profiles. In this mini-review, we summarize our results from three studies demonstrating that SALT is a robust and versatile platform to improve active loading of poorly water-soluble compounds. We have validated SALT as a tool for improving drug solubility, liposomal loading efficiency and retention, stability, palatability, and pharmacokinetics (PK), while retaining the ability of the compounds to exert pharmacological effects.Pharmaceutical Sciences, Faculty ofReviewedFacult