Benedict Law

Cell penetrating peptide tethered bi-ligand liposomes for delivery to brain in vivo: Biodistribution and transfection.

Targeted nano-particulate systems hold extraordinary potential for delivery of therapeutics across blood brain barrier (BBB). In this work, we investigated the potential of novel bi-ligand (transferrin-poly-l-arginine) liposomal vector for delivery of desired gene to brain, in vivo. The in vivo evaluation of the delivery vectors is essential for clinical translation. We followed an innovative approach of combining transferrin receptor targeting with enhanced cell penetration to design liposomal vectors for improving the transport of molecules into brain. The biodistribution profile of 1, 1-dioctadecyl-3,3,3,3-tetramethyl-indocarbocyanine iodide(DiR)-labeled liposomes was evaluated in adult rats after single intravenous injection at dose of 15.2μmoles of phospholipids/kg body weight. We demonstrated that bi-ligand liposomes accumulated in rat brain at significantly (p<0.05) higher concentrations as compared to the single-ligand (transferrin) or plain liposomes. In addition, the bi-ligand liposomes resulted in increased expression of β-galactosidase(β-gal) plasmid in rat brain tissue in comparison to the single-ligand liposomes. Histological examination of the transfected tissues did not show any signs of tissue necrosis or inflammation. Hemolysis assay further authenticated the biocompatibility of bi-ligand liposomes in blood up to 600 nmoles of phospholipids/1.4×10(7) erythrocytes. The findings of this study provide important and detailed information regarding the distribution of bi-ligand liposomes in vivo and accentuate their ability to demonstrate improved brain penetration and transfection potential over single-ligand liposomes.

Proteolysis: A Biological Process Adapted in Drug Delivery, Therapy, and Imaging

In many diseases, protease expressions are found deregulated when compared with them at the healthy states. The unique ability to hydrolyze protein amide bonds has made those deregulated proteases attractive biological triggers in drug development. Proteolysis has been widely applied in pro-drug design to achieve favorable pharmacokinetics. Controlled drug delivery systems are also reported by incorporating protease-sensitive motifs onto bio-, inorganic-, or organic- materials. In addition, protease responsive molecular probes are developed for in vitro bioanalysis and in vivo diagnostic imaging. This review focuses on various proteolysis-dependent approaches to drug delivery, therapy, and imaging. References are selected to illustrate the concepts and demonstrate the potentials of these enzyme-responsive strategies.

A mitochondrial targeted fusion peptide exhibits remarkable cytotoxicity

A potent cytotoxic peptide (r7-kla) was synthesized by incorporating a mitochondrial membrane disrupting peptide, kla (klaklakklaklak), with a cell-penetrating domain, r7 (rrrrrrr). The IC50 of r7-kla (3.54 ± 0.11 μmol/L) was more than two orders of magnitude lower than that of kla. r7-kla induced cell death in both in vitro and in vivo environments, and showed rapid kinetics. Within minutes, the morphologic changes in cells and mitochondrial leakage were apparent by microscopy and was consistent with rapid apoptosis. Our results suggested that r7-kla is an apoptosis inducer and can be potentially used as an antitumor agent, especially when combined with the appropriate systemic delivery systems. [Mol Cancer Ther 2006;5(8):1944–9]

Development of biocompatible polymeric nanoparticles for in vivo NIR and FRET imaging.

The majority of near-infrared (NIR) fluorophores are organic molecules that show significant overlap between the excitation and emission spectra and therefore exhibit high fluorescence backgrounds during in vivo imaging. Recently, cyanine dyes with a large Stokes shift have shown great promise for NIR imaging but often undergo rapid photodegradation and nonspecific protein adsorption. Alternatively, fluorescence resonance energy transfer (FRET) is a promising technique to generate a larger gap between the excitation and emission maxima and thus can reduce the background signal. Here, we report the rational design of FRET-based polymeric nanoparticles for NIR and FRET imaging. The particles were assembled from diblock copolymers of poly(d,l-lactic-co-glycolic acid) and maleimide-activated poly(ethylene glycol), which were also encapsulated with both the donor (1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine) and acceptor (1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine) fluorophores. Because of their extreme hydrophobicity, thousands of fluorophores could be encapsulated inside a single particle without causing leakage. FRET resulted in a large Stokes shift (>100 nm) of the emission maxima, and the transfer efficiency could be fine-tuned by further adjusting the doping ratio of the donor and acceptor fluorophores. The optimized formulation was less than 100 nm in size, brighter than quantum dots, stable in biological media, and demonstrated similar biodistribution to most nanomaterials. Additional animal phantom studies demonstrated that the FRET imaging platform developed could have far-reaching applications in optical imaging.

Release of Liposomal Contents by Cell-Secreted Matrix Metalloproteinase-9

Liposomes have been widely used as a drug delivery vehicle, and currently, more than 10 liposomal formulations are approved by the Food and Drug Administration for clinical use. However, upon targeting, the release of the liposome-encapsulated contents is usually slow. We have recently demonstrated that contents from appropriately formulated liposomes can be rapidly released by the cancer-associated enzyme matrix metalloproteinase-9 (MMP-9). Herein, we report our detailed studies to optimize the liposomal formulations. By properly selecting the lipopeptide, the major lipid component, and their relative amounts, we demonstrate that the contents are rapidly released in the presence of cancer-associated levels of recombinant human MMP-9. We observed that the degree of lipid mismatch between the lipopepides and the major lipid component profoundly affects the release profiles from the liposomes. By utilizing the optimized liposomal formulations, we also demonstrate that cancer cells (HT-29) which secrete low levels of MMP-9 failed to release a significant amount of the liposomal contents. Metastatic cancer cells (MCF7) secreting high levels of the enzyme rapidly release the encapsulated contents from the liposomes.

Polymeric Nanoparticles with Sequential and Multiple FRET Cascade Mechanisms for Multicolor and Multiplexed Imaging

The ability to map multiple biomarkers at the same time has far-reaching biomedical and diagnostic applications. Here, a series of biocompatible poly(D,L-lactic-co-glycolic acid) and polyethylene glycol particles for multicolor and multiplexed imaging are reported. More than 30 particle formulations that exhibit distinct emission signatures (ranging from the visible to NIR wavelength region) are designed and synthesized. These particles are encapsulated with combinations of carbocyanine-based fluorophores DiO, Dil, DiD, and DiR, and are characterized as <100 nm in size and brighter than commercial quantum dots. A particle formulation is identified that simultaneously emits fluorescence at three different wavelengths upon a single excitation at 485 nm via sequential and multiple FRET cascade events for multicolor imaging. Three other particles that display maximum fluorescence intensities at 570, 672, or 777 nm for multiplexed imaging are also identified. These particles are individually conjugated with specific (Herceptin or IgG2A11 antibody) or nonspecific (heptaarginine) ligands for targeting and, thus, could be applied to differentiate different cancer cells from a cell mixture according to the expressions of cell-surface human epidermal growth factor receptor 2 and the receptor for advanced glycation endproducts. Using an animal model subcutaneously implanted with the particles, it is further demonstrated that the developed platform could be useful for in vivo multiplexed imaging.

In-vivo imaging of tumor associated urokinase-type plasminogen activator activity

The ability to image tumor associated protease in vivo has biological and clinical implications. In the present study, we describe the development and validation of a urokinase-type plasminogen activator (uPA) sensitive fluorescence imaging probe. The activation of our probe is highly specific to uPA in both enzymatic and cellular-based assays. In two distinct in-vivo tumor models (human colon adenocarcinoma HT-29 and human fibrosarcoma HT-1080), the observed fluorescence changes correlate well with tumor associated uPA activity. The signal intensities of the tumors are about three-fold higher in animals with probe injections. Our results suggest a direct detection method for uPA activity in vivo and the approach can be used for monitoring tumor growth and development.

A short circulating peptide nanofiber as a carrier for tumoral delivery.

UNLABELLEDnThe cellular interactions and in vivo distribution of the nanomaterials are known to be strongly influenced by their physiochemical properties. Here, we investigated and compared the biocompatibility, pharmacokinetics, and biodistribution of previously reported peptide-based nanofiber (NFP), with commercially available nanomaterials. The NFP was a 2-dimensional (2D) structure with an extremely narrow width (4 nm) and a controllable length (50 to 400 nm). NFP was found to be non-toxic, hemocompatible, and with a minimum uptake by macrophages. In vivo studies further demonstrated that NFP could be delivered to the tumor site more effectively, and within a very shorter period of time, than spherical nanoparticles. Importantly, the undelivered NFP was rapidly eliminated by renal clearance and, thus, avoiding its accumulation in the spleen or liver. Overall, our data suggested a new paradigm in drug delivery via using a short circulating NFP, rather than a long circulating 3D nanoparticle, as a delivery cargo.nnnFROM THE CLINICAL EDITORnIn this study, the role of small peptide-based nanocarriers is investigated for tumor-specific delivery, reporting excellent targeting properties and a favorable toxicity profile.

Design and synthesis of a near-infrared fluorescent nanofiber precursor for detecting cell-secreted urokinase activity

Abnormal proteolysis is often observed during disease progression. Up-regulation of certain tumor-associated proteases such as urokinase plasminogen activator (uPA) can be a hallmark of malignant transformation. Here we report the design and synthesis of a near-infrared nanofiber precursor (NIR-NFP) for detecting uPA activity. NIR-NFP, which is optically silent in its native state, is composed of multiple self-assembled peptide units (PEG(54)-BK(NIR664)SGRSANA-kldlkldlkldl-CONH(2)). On uPA activation, NIR-NFP releases peptide fragments (PEG(54)-BK(NIR664)SGR-CONH(2)) that contribute to a significant fluorescence amplification at 684nm. NIR-NFP was able to detect cell-secreted uPA from human cancer cells (SKBR-3, PANC-1, MCF-7, SKOV-3, MDA-MB-231, PC-3, and HT-1080) expressing various levels of uPA. Fluorescence changes were uPA dependent, as confirmed with both Western blot analysis and enzyme activity assay. Our data suggest that an optimized preparation may be useful for imaging uPA activity in vivo.

Docosahexaenoic acid (DHA) sensitizes brain tumor cells to etoposide-induced apoptosis.

In this study, we investigated whether DHA, a nutritionally important n-3 unsaturated fatty acid, modulated the sensitivity of brain tumor cells to the anticancer drug, etoposide (VP16). Medulloblastoma (MB) cell lines, Daoy and D283, and glioblastoma (GBM) cell lines, U138 and U87, were exposed to DHA or VP16 alone or in combination. The effects on cell proliferation and the induction of apoptosis were determined by using MTS and Hoechest 33342/PI double staining. U87 and U138 cells were found to be insensitive to the addition of DHA and VP16, whereas the two MB cell lines showed high sensitivity. DHA or VP16 alone showed little effect on cell proliferation or death in either the MB or GBM cell lines, but pretreatment with DHA enhanced the responsiveness to VP16 in the MB cell lines. To understand the mechanisms of combined DHA and VP16 on MB cells, pathway specific oligo array analyses were performed to dissect possible signaling pathways involved. The addition of DHA and VP16, in comparison to VP16 added alone, resulted in marked suppression in the expression of several genes involved in DNA damage repair, cell proliferation, survival, invasion, and angiogenesis, including PRKDC, Survivin, PIK3R1, MAPK14, NFκB1, NFκBIA, BCL2, CD44, and MAT1. These results suggest (1) that the effects of DHA and VP16 in brain tumor cells are mediated in part by the down regulation of events involved in DNA repair and the PI3K/MAPK signaling pathways and (2) that brain tumors genotypically mimicked by MB cells may benefit from therapies combining DHA with VP16.