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INTRODUCTIONLipoplex (cationic liposome-DNA complex) is formed via electrostatic interaction of anionic nucleic acids with cationic liposomes. A thin film of lipids is dried on the bottom of a glass tube and rehydrated in an aqueous solution. The resulting liposome suspension is passed through polycarbonate filters of desired pore size. This protocol also describes the preparation, physical properties, and biological activity of liposome-polycation-DNA (LPD) nanoparticles. The LPD nanoparticles contain a highly condensed DNA core surrounded by lipid bilayers with an average size of ~100 nm. The nanoparticle complex is injected into mice, and expression of the transfected DNA is monitored with an appropriate assay.

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.

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.

Colorectal cancer with peritoneal metastases is currently treated by cytoreductive surgery and locoregional chemotherapeutics. This standard treatment is associated with high morbidity, mortality, and recurrence rate. To augment the existing therapy, we developed a liposome-based delivery system containing 1,2-stearoyl-3-trimethylammonium-propane chloride (DSTAP), a cationic lipid, to localize a toll-like receptor agonist, resiquimod (R848), in the peritoneal cavity (PerC) for enhancing the immune response against cancer that had spread to the PerC. The liposomes delivered by intraperitoneal injection increased peritoneal retention of R848 by 14-fold while retarding its systemic absorption, leading to a 5-fold decreased peak plasma concentration compared to free R848 in mice. Within the PerC, the DSTAP-liposomes were found in ~40% of the dendritic cells by flow cytometry. DSTAP-R848 significantly upregulated interferon �� (IFN-��) in the peritoneal fluid by 2-fold compared to free R848, without increasing the systemic level. Combined with oxaliplatin, a cytotoxic agent inducing immunogenic cell death, DSTAP-R848 effectively inhibited the progression of CT26 murine colorectal tumor in the PerC, while the combination with free R848 only showed a mild effect. Moreover, the combination of oxaliplatin and DSTAP-R848 significantly increased infiltration of CD8+ T cells in the PerC compared to oxaliplatin combined with free R848, indicating enhanced immune response against the tumor. The results suggest that DSTAP-R848 exhibits potential in augmenting existing therapies for treating colorectal cancer with peritoneal metastases via immune activation.

Opioids account for 69,000 overdose deaths per annum worldwide and cause serious side effects. Safer analgesics are urgently needed. The endogenous opioid peptide Leu-Enkephalin (Leu-ENK) is ineffective when introduced peripherally due to poor stability and limited membrane permeability. We developed a focused library of Leu-ENK analogs containing small hydrophobic modifications. N-pivaloyl analog KK-103 showed the highest binding affinity to the delta opioid receptor (68% relative to Leu-ENK) and an extended plasma half-life of 37 h. In the murine hot-plate model, subcutaneous KK-103 showed 10-fold improved anticonception (142%MPE·h) compared to Leu-ENK (14%MPE·h). In the formalin model, KK-103 reduced the licking and biting time to ~50% relative to the vehicle group. KK-103 was shown to act through the opioid receptors in the central nervous system. In contrast to morphine, KK-103 was longer-lasting and did not induce breathing depression, physical dependence, and tolerance, showing potential as a safe and effective analgesic.