Targeted RNAi of BIRC5/Survivin Using Antibody-Conjugated Poly(Propylene Imine)-Based Polyplexes Inhibits Growth of PSCA-Positive Tumors

Delivery of siRNAs for the treatment of tumors critically depends on the development of efficient nucleic acid carrier systems. The complexation of dendritic polymers (dendrimers) results in nanoparticles, called dendriplexes, that protect siRNA from degradation and mediate non-specific cellular uptake of siRNA. However, large siRNA doses are required for in vivo use due to accumulation of the nanoparticles in sinks such as the lung, liver, and spleen. This suggests the exploration of targeted nanoparticles for enhancing tumor cell specificity and achieving higher siRNA levels in tumors. In this work, we report on the targeted delivery of a therapeutic siRNA specific for BIRC5/Survivin in vitro and in vivo to tumor cells expressing the surface marker prostate stem cell antigen (PSCA). For this, polyplexes consisting of single-chain antibody fragments specific for PSCA conjugated to siRNA/maltose-modified poly(propylene imine) dendriplexes were used. These polyplexes were endocytosed by PSCA-positive 293TPSCA/ffLuc and PC3PSCA cells and caused knockdown of reporter gene firefly luciferase and Survivin expression, respectively. In a therapeutic study in PC3PSCA xenograft-bearing mice, significant anti-tumor effects were observed upon systemic administration of the targeted polyplexes. This indicates superior anti-tumor efficacy when employing targeted delivery of Survivin-specific siRNA, based on the additive effects of siRNA-mediated Survivin knockdown in combination with scFv-mediated PSCA inhibition

Targeted delivery of TLR3 agonist to tumor cells with single chain antibody fragment-conjugated nanoparticles induces type I-interferon response and apoptosis

Application of Toll-like receptor (TLR) agonists is a promising approach to treat cancer. In particular, nucleic acid-based TLR agonists such as short ssRNA and dsRNA molecules, which activate endosomal TLRs, can be delivered to tumors by use of nanoparticle delivery systems. However, such delivery systems bear unspecific side effects and poor pharmacokinetics. To overcome these limitations we developed a system for targeted delivery of a 50 bp dsRNA TLR3 agonist (Riboxxol) to treat PSCA-positive tumor cells, which consists of neutravidin conjugated to mono-biotinylated dsRNA and to humanized mono-biotinylated anti-PSCA single chain antibody derivative scFv(h-AM1)-BAP. The assembly of the components resulted in the formation of nanoparticle-like immunoconjugates designated Rapid Inducer of Cellular Inflammation and Apoptosis (RICIA). Anti-PSCA-RICIA exclusively delivered Riboxxol to PSCA-positive tumor cells as well as subcutaneous tumors. Uptake of anti-PSCA-RICIA induced a type I-interferon response and apoptosis in HEK-Blue hTLR3/PSCA reporter cells and PSCA-positive HT1376 bladder cancer cells in vitro. No such effects were observed when using RICIA coupled to an unspecific control antibody or when using Riboxxol alone. Treatment of HT1376 xenografts in immune-deficient hosts with targeted delivery of TLR3 agonist did not induce adverse effects and only modestly inhibited tumor growth when compared to controls. These results suggest promising activation of innate immune response and apoptosis upon selective delivery of TLR3 agonists in tumor cells. Yet, further studies using syngeneic and orthotopic tumor models are needed to fully exploit the potential of RICIA immunoconjugates. © 2019, The Author(s)

Reconstruction and control of a time-dependent two-electron wave packet

The concerted motion of two or more bound electrons governs atomic1 and molecular2,3 non-equilibrium processes including chemical reactions, and hence there is much interest in developing a detailed understanding of such electron dynamics in the quantum regime. However, there is no exact solution for the quantumthree-body problem, and as a result even the minimal system of two active electrons and a nucleus is analytically intractable4. This makes experimental measurements of the dynamics of two bound and correlated electrons, as found in the helium atom, an attractive prospect.However, although the motion of single active electrons and holes has been observed with attosecond time resolution5-7, comparable experiments on two-electron motion have so far remained out of reach. Here we showthat a correlated two-electron wave packet can be reconstructed froma 1.2-femtosecondquantumbeatamong low-lying doubly excited states in helium.The beat appears in attosecond transient-absorption spectra5,7-9 measured with unprecedentedly high spectral resolution and in the presence of an intensity-tunable visible laser field.Wetune the coupling10-12 between the two low-lying quantum states by adjusting the visible laser intensity, and use the Fano resonance as a phase-sensitive quantum interferometer13 to achieve coherent control of the two correlated electrons. Given the excellent agreement with large-scalequantum-mechanical calculations for thehelium atom, we anticipate thatmultidimensional spectroscopy experiments of the type we report here will provide benchmark data for testing fundamental few-body quantumdynamics theory in more complex systems. Theymight also provide a route to the site-specificmeasurement and control of metastable electronic transition states that are at the heart of fundamental chemical reactionsWe thank E. Lindroth for calculating the dipole moment (2p2|r|sp2,3+), and also A. Voitkiv, Z.-H. Loh, and R. Moshammer for helpful discussions. We acknowledge financial support by the Max-Planck Research Group Program of the Max-Planck Gesellschaft (MPG) and the European COST Action CM1204 XLIC. L. A. and F. M. acknowledge computer time from the CCC-UAM and Mare Nostrum supercomputer centers and financial support by the European Research Council under the ERC Advanced Grant no. 290853 XCHEM, the Ministerio de Economía y Competitividad projects FIS2010-15127, FIS2013-42002-R and ERA-Chemistry PIM2010EEC-00751, and the European grant MC-ITN CORIN

Targeted delivery of TLR3 agonist to tumor cells with single chain antibody fragment-conjugated nanoparticles induces type I-interferon response and apoptosis

Abstract Application of Toll-like receptor (TLR) agonists is a promising approach to treat cancer. In particular, nucleic acid-based TLR agonists such as short ssRNA and dsRNA molecules, which activate endosomal TLRs, can be delivered to tumors by use of nanoparticle delivery systems. However, such delivery systems bear unspecific side effects and poor pharmacokinetics. To overcome these limitations we developed a system for targeted delivery of a 50 bp dsRNA TLR3 agonist (Riboxxol) to treat PSCA-positive tumor cells, which consists of neutravidin conjugated to mono-biotinylated dsRNA and to humanized mono-biotinylated anti-PSCA single chain antibody derivative scFv(h-AM1)-BAP. The assembly of the components resulted in the formation of nanoparticle-like immunoconjugates designated Rapid Inducer of Cellular Inflammation and Apoptosis (RICIA). Anti-PSCA-RICIA exclusively delivered Riboxxol to PSCA-positive tumor cells as well as subcutaneous tumors. Uptake of anti-PSCA-RICIA induced a type I-interferon response and apoptosis in HEK-BluehTLR3/PSCA reporter cells and PSCA-positive HT1376 bladder cancer cells in vitro. No such effects were observed when using RICIA coupled to an unspecific control antibody or when using Riboxxol alone. Treatment of HT1376 xenografts in immune-deficient hosts with targeted delivery of TLR3 agonist did not induce adverse effects and only modestly inhibited tumor growth when compared to controls. These results suggest promising activation of innate immune response and apoptosis upon selective delivery of TLR3 agonists in tumor cells. Yet, further studies using syngeneic and orthotopic tumor models are needed to fully exploit the potential of RICIA immunoconjugates

Targeted RNAi of BIRC5/Survivin Using Antibody-Conjugated Poly(Propylene Imine)-Based Polyplexes Inhibits Growth of PSCA-Positive Tumors

Delivery of siRNAs for the treatment of tumors critically depends on the development of efficient nucleic acid carrier systems. The complexation of dendritic polymers (dendrimers) results in nanoparticles, called dendriplexes, that protect siRNA from degradation and mediate non-specific cellular uptake of siRNA. However, large siRNA doses are required for in vivo use due to accumulation of the nanoparticles in sinks such as the lung, liver, and spleen. This suggests the exploration of targeted nanoparticles for enhancing tumor cell specificity and achieving higher siRNA levels in tumors. In this work, we report on the targeted delivery of a therapeutic siRNA specific for BIRC5/Survivin in vitro and in vivo to tumor cells expressing the surface marker prostate stem cell antigen (PSCA). For this, polyplexes consisting of single-chain antibody fragments specific for PSCA conjugated to siRNA/maltose-modified poly(propylene imine) dendriplexes were used. These polyplexes were endocytosed by PSCA-positive 293TPSCA/ffLuc and PC3PSCA cells and caused knockdown of reporter gene firefly luciferase and Survivin expression, respectively. In a therapeutic study in PC3PSCA xenograft-bearing mice, significant anti-tumor effects were observed upon systemic administration of the targeted polyplexes. This indicates superior anti-tumor efficacy when employing targeted delivery of Survivin-specific siRNA, based on the additive effects of siRNA-mediated Survivin knockdown in combination with scFv-mediated PSCA inhibition