pDNA-lipoplexes engrafted with flagellin-related peptide induce potent immunity and anti-tumour effects.

Complexes of cationic lipids and DNA (lipoplexes) are widely used for non-viral gene delivery and DNA vaccine development, but cationic lipids are toxic and promote non-specific interactions with cells, leading to poor efficacy. Near-neutral lipoplexes, on the other hand, can obviate toxicity, but a convenient means to target them to specific cells such as dendritic cells (DCs) has been lacking. Here, we show that a His-tagged flagellin-derived peptide (denoted 9Flg), previously reported to promote binding of liposomal antigen to TLR5-expressing cells, can be used to target near-neutral pDNA-lipoplexes incorporating the chelator lipid NTA(3)-DTDA (3(nitrilotriacetic acid)-ditetradecylamine) to DCs and other antigen-presenting cells (APCs). Thus, we show that pDNA-lipoplexes engrafted with 9Flg target pDNA to APCs in vitro and in vivo. Following i.v. administration, radiolabelled 9Flg-lipoplexes exhibited increased accumulation in spleen, lung and liver. Vaccination of C57BL/6 mice with 9Flg-lipoplexes containing either pcDNA3.1-SIIN (pSIIN) or a Kunjin virus replicon-based vector (pKUN), each encoding the epitope OVA(257-264) (SIINFEKL), induced Ag-specific T cell priming, and elicited strong cellular immunity as reflected by a marked increase in the number of Ag-responsive IFN-γ-producing CD8(+) T cells. Importantly, compared to i.m. injection of these SIINFEKL-encoding pDNAs in naked form, the i.v. administration of pSIIN or pKUN in 9Flg-lipoplexes to C57BL/6 mice induced a significantly more potent anti-tumour response in the B16-OVA melanoma tumour model. The targeting of near-neutral 9Flg-lipoplexes bearing pDNA encoding tumour antigens to TLR5 on APCs, therefore, is a powerful approach for developing more effective DNA vaccines and immunotherapies.

Effective tumor targeting and enhanced anti-tumor effect of liposomes engrafted with peptides specific for tumor lymphatics and vasculature.

The use of liposomes to target drugs to tumors represents an attractive therapeutic strategy, especially when used with convenient targeting moieties such as peptides. Here we explored several peptides for their ability to target liposomes to tumors. The metal chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA(3)-DTDA) was incorporated into liposomes to enable the engraftment of His-tagged peptides containing targeting motifs specific for tumor vasculature markers VEGFR-1 (p39-Flt-1) and neuropilin-1 (p24-NRP-1), or a motif known to accumulate in hypoxic areas of tumors (p47-LyP-1). Peptide-engrafted liposomes were examined for their biodistribution and anti-tumor effects after i.v. administration. Our results show that radiolabelled liposomes engrafted with either p24-NRP-1 or p47-LyP-1 and then injected into mice bearing subcutaneous B16-F1 tumors, show increased accumulation in the tumor. For p24-NRP-1-liposomes, tumor targeting was significantly increased when the stabilizing lipid phosphatidylethanolamine polyethylene glycol-750 (PE-PEG(750)) was used instead of PE-PEG(2000) in the liposome lipid mixture. Importantly, compared to the controls, p24-NRP-1 liposomes containing 10 mol% PE-PEG(750) and loaded with doxorubicin significantly inhibited the rate of tumor growth in the tumor-bearing mice. Our findings demonstrate that the use of drug-containing liposomes incorporating NTA(3)-DTDA and engrafted with NRP-1 targeting peptide is a convenient strategy to enhance the therapeutic effect of non-targeted doxorubicin.

Increasing the antitumor efficacy of doxorubicin-loaded liposomes with peptides anchored via a chelator lipid.

The therapeutic efficacy of anticancer drugs like doxorubicin can be significantly increased by their incorporation into liposomes, but an ability to actively target the drug-containing liposomes to tumors could well provide an even greater curative effect. In this work, a commercial preparation of doxorubicin-loaded liposomes (Caelyx) was modified by incorporation of the metal chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA(3)-DTDA) to enable engraftment of histidine-tagged targeting molecules. Our results show that when engrafted with p15-RGR, a His-tagged peptide containing a sequence purported to bind platelet-derived growth factor receptor ß (PDGFRß), NTA(3)-DTDA-containing Caelyx (3NTA-Caelyx) can be targeted to NIH-3T3 cells in vitro, leading to increased cytotoxicity compared with non-targeted 3NTA-Caelyx. PDGFRß is known to be expressed on pericytes in the tumor vasculature; however, when radiolabeled p15-RGR liposomes were administered to mice bearing subcutaneous B16-F1 tumors, minimal accumulation into tumors was observed. In contrast, an alternative targeting peptide, p46-RGD, was found to actively direct liposomes to tumors (4.7 %ID/g). Importantly, when injected into tumor-bearing mice, p46-RGD-engrafted 3NTA-Caelyx significantly decreased the tumor growth rate compared with controls. These results indicate that the incorporation of NTA(3)-DTDA into liposomal drugs could represent a simple modification to the drug to allow engraftment of targeting molecules and to increase its efficacy.

Targeting of plasmid DNA-lipoplexes to cells with molecules anchored via a metal chelator lipid.

BACKGROUND: The ability to deliver plasmid DNA (pDNA) to specific cells in vivo is crucial for achieving efficient targeted transfection with nonviral vectors. We previously used stealth liposomes containing the chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA(3)-DTDA) to target delivery of antigen and cytokines to immune cells in vivo. In the present study, we utilized liposomes containing NTA(3)-DTDA and the ionizable aminolipid 1,2-dioleoyl-3-dimethyl-ammonium-propane (DODAP) to incorporate pDNA into complexes for targeting to cells. METHODS: Liposomes containing DODAP, NTA(3)-DTDA and helper lipids were acidified (pH 5.5) and mixed with pDNA to form complexes. These lipoplexes were neutralized and engrafted with His-tagged molecules for targeting to extracellular receptors. Targeted transfection efficiency was assessed using the enhanced green fluorescent protein reporter gene. RESULTS: Initial transfections of HEK-293 cells using a His-tagged peptide (T2) related to the Arg-rich motif of HIV-1 TAT protein resulted in a low transfection efficiency (<2.5%). Optimization of the lipid formulation and use of an endosome-destabilizing peptide and inhibitor of DNase II, increased transfection approximately 20-fold. These lipoplexes are approximately 250 nm in diameter, and transfection efficiencies were: approximately 50% for HEK-293 cells targeted with lipoplexes containing pEGFP-N1 and engrafted with T2, and 30-40% for HepG2 cells targeted with lipoplexes engrafted with a peptide specific for the VEGF receptor Flt-1. CONCLUSIONS: The results show that DODAP-containing lipoplexes incorporating NTA(3)-DTDA enable the engraftment of targeting molecules and the effective targeting of pDNA to cells in serum-containing media, resulting in efficient transgene expression. The strategy may provide a convenient approach for targeting pDNA to cells in vivo in therapeutic applications.

Convenient targeting of stealth siRNA-lipoplexes to cells with chelator lipid-anchored molecules.

A major obstacle for the use of siRNAs as novel therapeutics is the requirement for functional delivery to specific cells in vivo. siRNA delivery by cationic agents is generally non-specific and a convenient targeting strategy has been lacking. This work explored the potential for using the chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA(3)-DTDA) with neutral stealth liposomes to target siRNA to cells. A novel method for incorporating siRNAs into lipoplexes was developed which utilised helper lipids and the ionisable lipid 1,2-dioleoyl-3-dimethylammonium-propane (DODAP). This approach results in an efficient (>50%) incorporation of siRNA into lipoplexes, which when incorporated with Ni-NTA(3)-DTDA and engrafted with a His-tagged form of murine CD4 can target siRNA to murine A20 B cells, in vitro. Also, siRNA-lipoplexes engrafted with His-tagged peptides that target receptors on HEK-293 cells, or the receptor for tumour necrosis factor alpha expressed on the murine dendritic cell line DC2.4, could target siRNA and silence the expression of enhanced green fluorescence protein (EGFP). siRNA-lipoplexes produced by this method are approximately 240 nm dia, exhibit low zeta-potential (-1 mV), and target cells in serum-containing media. The results show that NTA(3)-DTDA can be used to target siRNA-lipoplexes to cells, and could provide a convenient approach for targeting siRNA to cells in vivo for therapeutic applications.

Targeted liposomal delivery of TLR9 ligands activates spontaneous antitumor immunity in an autochthonous cancer model.

Accessibility of tumors for highly effective local treatment represents a major challenge for anticancer therapy. Immunostimulatory oligodeoxynucleotides (ODN) with CpG motifs are ligands of TLR9, which prime spontaneous antitumor immunity, but are less effective when applied systemically. We therefore developed a liposome-based agent for selective delivery of CpG-ODN into the tumor environment. A peptide that specifically targets angiogenic endothelial cells in a transgenic tumor model for islet cell carcinogenesis was engrafted into CpG-ODN containing liposomes. Intravenous injection of these liposomes resulted in specific accumulation around tumor vessels, increased uptake by tumor-resident macrophages, and retention over time. In contrast, nontargeted liposomes did not localize to the tumor vasculature. Consequently, only vascular targeting of CpG-ODN liposomes provoked a marked inflammatory response at vessel walls with enhanced CD8(+) and CD4(+) T cell infiltration and, importantly, activation of spontaneous, tumor-specific cytotoxicity. In a therapeutic setting, 40% of tumor-bearing, transgenic mice survived beyond week 45 after systemic administration of vascular-directed CpG-ODN liposomes. In contrast, control mice survived up to 30 wk. Therapeutic efficacy was further improved by increasing the frequency of tumor-specific effector cells through adoptive transfers. NK cells and CD8(+) T cells were major effectors which induced tumor cell death and acted in conjunction with antivascular effects. Thus, tumor homing with CpG-ODN-loaded liposomes is as potent as direct injection of free CpG-ODN and has the potential to overcome some major limitations of conventional CpG-ODN monotherapy.