Fluorescent Nanorods and Nanospheres for Real-Time In Vivo Probing of Nanoparticle Shape-Dependent Tumor Penetration

Shape dependent: Fluorescent quantum-dot-based nanospheres and nanorods with identical hydrodynamic size and surface properties but different aspect ratios were developed for real-time in vivo tumor imaging. The nanorods exhibited superior transport and distribution into mammary tumors in vivo versus nanospheres of similar plasma half-life.National Cancer Institute (U.S.) (Grant Number R01-CA126642)MIT-Harvard Center for Cancer Nanotechnology Excellence (1U54-CA119349)Massachusetts Institute of Technology. Dept. of Chemistry Instrumentation Facility (CHE-980806)Massachusetts Institute of Technology. Dept. of Chemistry Instrumentation Facility (DBI-9729592)ISN (W911NF-07-D-0004

Delivery of therapeutic shRNA and siRNA by Tat fusion peptide targeting bcr-abl fusion gene in Chronic Myeloid Leukemia cells

Gene silencing by RNA interference (RNAi) is a promising therapeutic approach for a wide variety of diseases for which the biological cause is known. The main challenge remains the ineffective RNAi delivery inside the cells. Non-viral gene delivery vectors have low immunogenicity compared to viral vectors, but are constrained by their reduced transfection efficiency. Silencing of the bcr-abl gene expression by RNAi confers therapeutic potential in Chronic Myeloid Leukemia (CML), but is limited by the cytotoxicity of the existing delivery methods. Here, we present evidence that the fusion between the cell penetrating peptide (CPP) HIV-Tat (49-57) and the membrane lytic peptide (LK15), Tat-LK15, mediates high transfection efficiency in delivering short hairpin RNA (shRNA) and small interfering RNA (siRNA) targeting the BCR-ABL oncoprotein in K562 CML cells. Our results show that shRNA complexes induce a more stable gene silencing of bcr-abl when compared to silencing mediated by siRNA complexes. In addition, silencing of the BCR-ABL oncoprotein by both shRNA and siRNA delivered by Tat-LK15 is more efficient and longer lasting than that achieved using Lipofectamine and more importantly without considerable cytotoxicity. In these terms Tat-LK15 can be an alternative to DNA/siRNA delivery in difficult-to-transfect leukemic cells. © 2010 Elsevier B.V

Improved Tat-mediated plasmid DNA transfer by fusion to LK15 peptide

The use of cell penetrating peptides (CPPs), such as Tat-derived peptide, to deliver DNA into cells is limited as evidenced by the low transfection efficiency of their DNA complexes. Here, we demonstrate that covalent attachment of membrane active peptide LK15 to Tat peptide improves gene transfer. Our results demonstrate that Tat peptide was able to form complexes with DNA, but their transfection efficiency was insufficient as assessed by luciferase assay. The attachment of LK15 to Tat significantly improved the physiochemical properties of the DNA complexes, rendered the complexes membrane active and enhanced the gene expression in HT29 and in HT1080 cultured cells. The enhanced transfection ability of Tat-LK15 compared to Tat is likely to be due mainly to the higher uptake of DNA. Finally, we evaluated the penetration and transfection ability of Tat and Tat-LK15 in multicellular tumour spheroids (MCTS) to mimic in vivo delivery to tumours. The results showed that the penetration and transfection ability of Tat and Tat-LK15/DNA complexes were limited to the rim of HT29 spheroids. Taken together, our data shows improvement in the transfection efficiency of Tat peptide by covalent attachment to LK15. Further advancements are needed before any potential applications in tissues as the penetration into the core of MCTS remains severely restricted. © 2010 Elsevier B.V

A Systems Approach for Tumor Pharmacokinetics

Recent advances in genome inspired target discovery, small molecule screens, development of biological and nanotechnology have led to the introduction of a myriad of new differently sized agents into the clinic. The differences in small and large molecule delivery are becoming increasingly important in combination therapies as well as the use of drugs that modify the physiology of tumors such as anti-angiogenic treatment. The complexity of targeting has led to the development of mathematical models to facilitate understanding, but unfortunately, these studies are often only applicable to a particular molecule, making pharmacokinetic comparisons difficult. Here we develop and describe a framework for categorizing primary pharmacokinetics of drugs in tumors. For modeling purposes, we define drugs not by their mechanism of action but rather their rate-limiting step of delivery. Our simulations account for variations in perfusion, vascularization, interstitial transport, and non-linear local binding and metabolism. Based on a comparison of the fundamental rates determining uptake, drugs were classified into four categories depending on whether uptake is limited by blood flow, extravasation, interstitial diffusion, or local binding and metabolism. Simulations comparing small molecule versus macromolecular drugs show a sharp difference in distribution, which has implications for multi-drug therapies. The tissue-level distribution differs widely in tumors for small molecules versus macromolecular biologic drugs, and this should be considered in the design of agents and treatments. An example using antibodies in mouse xenografts illustrates the different in vivo behavior. This type of transport analysis can be used to aid in model development, experimental data analysis, and imaging and therapeutic agent design.National Institutes of Health (U.S.) (grant T32 CA079443

Study of cellular delivery of siRNA and shRNA targeting bcr-abl in chronic myeloid leukemia using Tat derived peptide

Chronic Myeloid Leukemia is characterised by the formation of a fusion gene bcr-abl. The gene product BCR-ABL has deregulated tyrosine kinase activity that plays a direct role in the pathogenesis of the disease. Recently, use of siRNA in leukaemic cells has led to effective gene silencing of bcr-abl. Gene delivery systems like viral vectors, electroporation and lipid based vectors have showed varying efficiencies but are limited by their level of toxicity and immunogenicity. Developments in the field of Cell Penetrating Peptides have shown effective cellular uptake of nucleic acids and proteins by the CPPs in vitro and in vivo. Report from our lab has shown the use of CPP Tat along with membrane active peptide LK15 to improve the transfection efficiency of both Tat and LK15 peptides individually. Hence, this study will focus on the use of Tat-LK15 peptide to study the delivery of siRNA and shRNA plasmid in K562 cells and observe the BCR-ABL protein expression. Cellular uptake studies using Tat-LK15 based complexes of Cy5-labelled DNA and siRNA showed a concentration dependent uptake leading to increase in percentage transfected cells. Tat-LK15 based DNA complexes achieved 80% transfected cells (charge ratio of 2:1) while siRNA complexes resulted in a maximum of 60% (charge ratio of 3:1). However, Lipofectamine based DNA complexes did not show a concentration dependent increase in percentage transfected cells. Interestingly, Tat-LK15 based siRNA complexes showed a similar level of uptake and percentage transfected cells as that of Lipofectamine based siRNA complexes. Cellular uptake studies using confocal microscopy 4 hours post transfection, showed that when 1μg of DNA was transfected, the labelled DNA was primarily localised on the cell membrane. Interestingly, using 5μg of DNA led to increased intracellular localisation of the labelled DNA, but this observation was not made with Lipofectamine based complexes. The observation at 24 hours post transfection of Tat-LK15/labelled DNA complexes was of higher intensity when compared to that of Lipofectamine based DNA complexes. The reason for this is however not known. Interestingly, the cellular uptake profile using siRNA based complexes was different. At 4 hours post transfection, there was intracellular localisation of labelled siRNA. 24 hours post transfection, there was diffuse cytoplasmic localisation using lower concentration of siRNA whereas using higher concentration led to more high intensity punctate localisations within the cell. Similar observations were made for both Tat-LK15 and Lipofectamine based siRNA complexes.Gene silencing studies of Tat-LK15/shRNA plasmid complex resulted in 80% reduction in protein levels 96 hours post transfection for higher concentrations of shRNA plasmid treated. Similar level of reduction in BCR-ABL was observed with Lipofectamine based complex. Supporting evidence of reduction in mRNA levels was observed using qRT-PCR 48 hours post transfection. However, Tat-LK15/shRNA plasmid complexes led to around 80% of protein reduction 192 hours post transfection while Lipofectamine based complexes resulted in only 40% of protein reduction. Transfection using increasing concentrations of siRNA complexed to Tat-LK15 and Lipofectamine led to greater than 70% reduction in protein levels for most concentration ranges tested. This reduction in protein levels lasted only 48 hours post transfection. In conclusion, Tat-LK15 peptide could be used for shRNA plasmid and siRNA based delivery and could offer an efficient gene delivery model for studying RNAi.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Arginine to lysine mutations increase the aggregation stability of a single chain variable fragment through unfolded state interactions

Increased protein solubility is known to correlate with an increase in the proportion of lysine over arginine residues. Previous work has shown that the aggregation propensity of a single-chain variable fragment (scFv) does not correlate with its conformational stability or native-state protein-protein interactions. Here we test the hypothesis that aggregation is driven by the colloidal stability of partially unfolded states, studying the behaviour of scFv mutants harbouring single or multiple site-specific arginine/lysine mutations in denaturing buffers. In 6 M guanidine hydrochloride (GdmCl) or 8 M urea, repulsive protein-protein interactions were measured for the wild-type and lysine enriched (4RK) scFvs on account of weakened short-ranged attractions and increased excluded volume, in contrast to the arginine enriched (7KR) scFv which demonstrated strong reversible association. In 3 M GdmCl, the minimum concentration at which the scFvs were unfolded, the hydrodynamic radius of 4RK remained constant but increased for the wild-type and especially for 7KR. Individually swapping lysine back to arginine in 4KR indicated that the observed aggregation propensity of arginine in denaturing conditions was non-specific. Interestingly, one such swap generated a scFv with especially low aggregation rates under low/high ionic strength and denaturing buffers; molecular modelling identified hydrogen-bonding between the arginine side chain and main chain peptide groups, stabilising the structure. The arginine/lysine ratio is not routinely considered in biopharmaceutical scaffold design, or current amyloid prediction methods. This work therefore suggests a simple method to increase the stability of a biopharmaceutical protein against aggregation.</p

Comparison of IgG diffusion and extracellular matrix composition in rhabdomyosarcomas grown in mice versus in vitro as spheroids reveals the role of host stromal cells

The tumour extracellular matrix acts as a barrier to the delivery of therapeutic agents. To test the hypothesis that extracellular matrix composition governs the penetration rate of macromolecules in tumour tissue, we measured the diffusion coefficient of nonspecific IgG in three rhabdomyosarcoma subclones growing as multicellular spheroids in vitro or as subcutaneous tumours in dorsal windows in vivo. In subcutaneous tumours, the diffusion coefficient decreased with increasing content of collagen and sulphated glycosaminoglycans. When grown as multicellular spheroids, no differences in either extracellular matrix composition or diffusion coefficient were found. Comparison of in vitro vs in vivo results suggests an over-riding role of host stromal cells in extracellular matrix production subjected to modulation by tumour cells. Penetration of therapeutic macromolecules through tumour extracellular matrix might thus be largely determined by the host organ. Hence, caution must be exercised in extrapolating drug penetrability from spheroids and multilayer cellular sandwiches consisting of only tumour cells to tumours in vivo

3D simulations of early blood vessel formation

Blood vessel networks form by spontaneous aggregation of individual cells migrating toward vascularization sites (vasculogenesis). A successful theoretical model of two dimensional experimental vasculogenesis has been recently proposed, showing the relevance of percolation concepts and of cell cross-talk (chemotactic autocrine loop) to the understanding of this self-aggregation process. Here we study the natural 3D extension of the computational model proposed earlier, which is relevant for the investigation of the genuinely threedimensional process of vasculogenesis in vertebrate embryos. The computational model is based on a multidimensional Burgers equation coupled with a reaction diffusion equation for a chemotactic factor and a mass conservation law. The numerical approximation of the computational model is obtained by high order relaxed schemes. Space and time discretization are performed by using TVD schemes and, respectively, IMEX schemes. Due to the computational costs of realistic simulations, we have implemented the numerical algorithm on a cluster for parallel computation. Starting from initial conditions mimicking the experimentally observed ones, numerical simulations produce network-like structures qualitatively similar to those observed in the early stages of \emph{in vivo} vasculogenesis. We develop the computation of critical percolative indices as a robust measure of the network geometry as a first step towards the comparison of computational and experimental data.Comment: 25 pages, 9 figures Submitted to: JC