[Show abstract][Hide abstract] ABSTRACT: A lipid/calcium/phosphate (LCP) nanoparticle (NP) formulation (particle diameter ∼25 nm) with superior siRNA delivery efficiency was developed and reported previously. Here, we describe the successful formulation of (111)In into LCP for SPECT/CT imaging. Imaging and biodistribution studies showed that, polyethylene glycol grafted (111)In-LCP preferentially accumulated in the lymph nodes at ∼70% ID/g in both C57BL/6 and nude mice when the improved surface coating method was used. Both the liver and spleen accumulated only ∼25% ID/g. Larger LCP (diameter ∼67 nm) was less lymphotropic. These results indicate that 25 nm LCP was able to penetrate into tissues, enter the lymphatic system, and accumulate in the lymph nodes via lymphatic drainage due to 1) small size, 2) a well-PEGylated lipid surface, and 3) a slightly negative surface charge. The capability of intravenously injected (111)In-LCP to visualize an enlarged, tumor-loaded sentinel lymph node was demonstrated using a 4T1 breast cancer lymph node metastasis model. Systemic gene delivery to the lymph nodes after IV injection was demonstrated by the expression of red fluorescent protein cDNA. The potential of using LCP for lymphatic drug delivery is discussed.
[Show abstract][Hide abstract] ABSTRACT: To address the question of how cells respond to the possible Ca2+ toxicity caused by the release of Ca2+ into the cytoplasm by LCP nanoparticles , a series of in vitro and in vivo studies using Ca2+ pump inhibitors were conducted. The results indicated that two major Ca2+ pumps on the plasma membrane and the mitochondrial membrane, respectively, were able to rapidly respond to the elevated cytosolic Ca2+ concentration and prevent Ca2+-induced apoptosis or necrosis. However, exposure to specific inhibitors of calcium pumps would cause LCP-treated H460 cells to undergo necrosis both in vitro and in vivo. These results demonstrated that the Ca2+ delivered by LCP was not toxic to cells when the cells contain functional Ca2+ pumps.
[Show abstract][Hide abstract] ABSTRACT: Immunotherapy has shown the potential to become an essential component of the successful treatment of various malignancies. In many cases, such as in melanoma, however, induction of a potent and specific T-cell response against the endogenous antigen or self-antigen still remains a major challenge. To induce a potent MHC I-restricted cytotoxic T-lymphocyte (CTL) response, cytosol delivery of an exogenous antigen into dendritic cells is preferred, if not required. Lipid-Calcium-Phosphate (LCP) nanoparticles represent a new class of intracellular delivery systems for impermeable drugs. We are interested in exploring the potential of LCP NPs for use as a peptide vaccine delivery system for cancer therapy. To increase the encapsulation of Trp2 peptide into the calcium phosphate precipitate core of LCP, two phosphor-serine residues were added to the N-terminal of the peptide (p-Trp2). CpG ODN was also co-encapsulated with p-Trp2 as an adjuvant. The NPs were further modified with mannose to enhance and prolong the cargo deposit into the lymph nodes (LNs), which ensured persistent antigen loading and stimulation. Compared with free Trp2 peptide/CpG, vaccination with LCP encapsulating p-Trp2 and CpG resulted in superior inhibition of tumor growth in both B16F10 subcutaneous and lung metastasis models. An IFN-γ production assay and in vivo CTL response study revealed that the improved efficacy was a result of a Trp2-specific immune response. Thus, encapsulation of phospho-peptide antigens into LCP may be a promising strategy for enhancing the immunogenicity of poorly immunogenic self-antigens for cancer therapy.
[Show abstract][Hide abstract] ABSTRACT: PURPOSE: The biodistribution of Lipid/Calcium/Phosphate (LCP) nanoparticles (NPs) in tumor-bearing mice was investigated using fluorescence imaging. A quantitative validation of this method was done by (3)H and (111)In labeling of the nanoparticles. METHODS: The biodistribution of LCP NPs containing oligonucleotides was investigated using three different probes: Texas-Red labeled oligonucleotides, (3)H-labeled oligonucleotides, and (111)In-labled calcium phosphate. RESULTS: A discrepancy was found between the radioactivity and the fluorescence signals. Signals from (3)H and (111)In exhibited very similar distribution patterns, suggesting that liver and spleen were the major accumulation sites. However, fluorescence imaging indicated that tumor accumulation was predominant. We further confirmed that the fluorescence signals in both liver and spleen were greatly attenuated compared with those in the tumor due to the intrinsic tissue absorption and scattering. Near-infrared (NIR) dye Cy5.5 also suffered from the same problem, in that the quantitative data from whole organs was dramatically affected by absorption and scattering properties of the tissue. CONCLUSIONS: Careful attention must be paid to the quantification and interpretation of fluorescence imaging measurements when comparing different tissues.
Pharmaceutical Research 07/2012; 29(12). DOI:10.1007/s11095-012-0818-1 · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A lipid coated calcium phosphate (LCP) nanoparticle (NP) formulation was developed for efficient delivery of small interfering RNA (siRNA) to a xenograft tumor model by intravenous administration. Based on the previous formulation, liposome-polycation-DNA (LPD), which was a DNA-protamine complex wrapped by cationic liposome followed by post-insertion of PEG, LCP was similar to LPD NP except that the core was replaced by a biodegradable nano-sized calcium phosphate precipitate prepared by using water-in-oil micro-emulsions in which siRNA was entrapped. We hypothesized that after entering the cells, LCP would de-assemble at low pH in the endosome, which would cause endosome swelling and bursting to release the entrapped siRNA. Such a mechanism was demonstrated by the increase of intracellular Ca(2+) concentration as shown by using a calcium specific dye Fura-2. The LCP NP was further modified by post-insertion of polyethylene glycol (PEG) with or without anisamide, a sigma-1 receptor ligand for systemic administration. Luciferase siRNA was used to evaluate the gene silencing effect in H-460 cells which were stably transduced with a luciferase gene. The anisamide modified LCP NP silenced about 70% and 50% of luciferase activity for the tumor cells in culture and those grown in a xenograft model, respectively. The untargeted NP showed a very low silencing effect. The new formulation improved the in vitro silencing effect 3-4 folds compared to the previous LPD formulation, but had a negligible immunotoxicity.
[Show abstract][Hide abstract] ABSTRACT: Lipid-based nanoparticle technology has developed from chemical drug carrier into an efficient multifunctional siRNA tumor targeting delivery system. In this review, we start with an overview of the lipid-based nanomedicine history and the two classes of lipidic vectors for DNA or siRNA delivery. Then we discuss the features of lipid-based nanomedicine that lead to effective tumor targeting and the principles behind. We also discuss nanoparticle surface modification, classes of tumor targeting ligands, and other state-of-the-art strategies for enhancing endosome release primarily focused on lipid-based systems. At the end, we show that multifunctional self-assembled lipid-based nanoparticles could also be versatile delivery vehicles for cancer molecular imaging probes.
[Show abstract][Hide abstract] ABSTRACT: RNAi technology has brought a new category of treatments for various diseases including genetic diseases, viral diseases, and cancer. Despite the great versatility of RNAi that can down regulate almost any protein in the cells, the delicate and precise machinery used for silencing is the same. The major challenge indeed for RNAi-based therapy is the delivery system. In this review, we start with the uniqueness and mechanism of RNAi machinery and the utility of RNAi in therapeutics. Then we discuss the challenges in systemic siRNA delivery by dividing them into two categories-kinetic and physical barriers. At the end, we discuss different strategies to overcome these barriers, especially focusing on the step of endosome escape. Toxicity issues and current successful examples for lipid-based delivery are also included in the review.