- Email: [email protected]
Inhibition of influenza virus activity by newly designed multivalent glycoarchitectures
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Abstracts / Journal of Controlled Release 148 (2010) e112–e124
Result and discussion DOX-liposome loaded microbubbles were prepared by attaching Doxil-like liposomes to the surface of lipid microbubbles via avidinbiotin interaction (Fig. 1). Melanoma cells were exposed to DOX-liposomes and DOXliposome loaded microbubbles without and with ultrasound exposure (USE). Exposure of the cells to DOX-liposome loaded microbubbles resulted in a much higher tumor cell killing efficiency than exposure to DOX-liposomes (Fig. 2). We did not see an outspoken improvement of cell killing by DOX-liposomes when ultrasound was applied.
created cell membrane pores. The in vitro results shown in this article indicate that DOX-liposome-loaded microbubbles can be a very interesting tool to obtain an efficient ultrasound-controlled DOX delivery in vivo. Acknowledgements I.L. is supported by the Fund for Scientific Research–Flanders (Belgium). The financial support of this institution is acknowledged with gratitude. The financial support of the European Union (via the FP7 projects Arise and Sonodrugs) is acknowledged. References [1] I. Lentacker, S.C. De Smedt, N.N. Sanders, Drug loaded microbubble design for ultrasound triggered delivery, Soft Matter 5 (11) (2009) 2161–2170. [2] I. Lentacker, B. Geers, J. Demeester, S.C. De Smedt, N.N. Sanders, Design and evaluation of doxorubicin containing microbubbles: cytotoxicity and mechanisms involved, Molecular Therapy 18 (1) (2010) 101–108.
doi:10.1016/j.jconrel.2010.07.085
Inhibition of influenza virus activity by newly designed multivalent glycoarchitectures
Fig. 2.
We also studied the intracellular localization of DOX after 4 h incubation time with DOX-liposomes or DOX-liposome loaded microbubbles and ultrasound. The DOX was almost exclusively present in the nuclei of cells treated with DOX-liposome loaded microbubbles, whereas the DOX was found in both, cytoplasm and nucleus of cells treated with DOX-liposomes (Fig. 3). After a short incubation time of only 15 min, only the cells treated with DOXloaded microbubbles and ultrasound showed DOX uptake. Additional experiments revealed that at least two different mechanisms are responsible for the high tumor cell killing efficiency of DOX-liposome loaded microbubbles after USE. During implosion of the microbubbles, liposomes are damaged and free DOX is released, which is immediately taken up through cell membrane perforations.
Ilona Papp1,⁎, Christian Sieben2, Adam L. Sisson3, Andreas Herrmann2, Rainer Haag1 1 Institut für Chemie und Biochemie, Freie Universität Berlin, Takustr. 3, D-14195 Berlin, Germany 2 Institut für Biologie/Biophysik, Humboldt-Universität zu Berlin, Invalidenstraße 43, 10115 Berlin, Germany 3 Berlin-Brandenburg Center for Regenerative Therapies, Augustenburger Platz 1, 13353 Berlin, Germany ⁎Corresponding author. E-mail: [email protected]. Abstract summary Influenza virus utilizes its envelope protein hemagglutinin (HA) to bind sialic acid (SA) residues of glycoproteins on the host cell plasma membrane [1]. The affinity of a single HA–SA pair is low (103 M− 1) compared to the overall affinity between virus and cell surface caused by multiple simultaneous interactions. Multivalency is ubiquitous in biology and can dramatically enhance affinities [2]. We use synthetic polymers with multiple SA moieties to investigate their inhibitory effect on virus binding and fusion. Introduction Many multivalent systems were introduced to inhibit influenza virus adhesion [3]. Some use natural scaffolds like proteins or liposomes [4–5]. Alternatively, synthetic polymeric scaffolds may be employed such as Whitesides' polyacrylamides [6] or Landers' polyamidoamines (PAMAM) [7]. Creating inhibitors that are not toxic/irritating to tissues and water-soluble still remains problematic. To circumvent these problems we coupled multiple SA residues to polyglycerols and nanogels, which are highly water soluble and biocompatible [8–10]. The aim was to address questions related to how particle size and multivalency affects biological processes and to gain insights into the molecular mechanism.
Fig. 3.
Conclusion It is possible to attach biotinylated doxil-like liposomes to the microbubble wall. The tumor cell killing efficiency of these liposomes is highly increased after microbubble coupling and ultrasound exposure. Ultrasonic treatment leads to the release of free doxorubicin which can subsequently enter the tumor cells trough the
Result and discussion This project deals with the evaluation of different architectures for multivalent interactions. Hyperbranched polyglycerols (HPGs), which are globular, multivalent, facilely prepared dendrimer analogues, are excellent biocompatible substrates for numerous applications [11– 12]. However, their synthesis is limited to afford particles of approx. 10 nm in diameter (Mn up to 1MDa). Through miniemulsion polymerization [13] well-defined polyglycerol nanogels (nPG) can
Abstracts / Journal of Controlled Release 148 (2010) e112–e124
e115
2
be prepared up to 100 nm in diameter [14]. Nanoparticles can be formed bearing unreacted epoxide units for further functionalization by controlling reaction time (number of remaining epoxides per glycerol unit can be determined by NMR) [14]. By addition of NaN3, azido nPGs are formed, which are quantitatively converted to triazole-derivates, following copper catalyzed click-chemistry. In the present study, we describe the conjugation of SA to HPGs and to newly developed nanogels, which are based on HPGs but exceed them in size up to 5–10 fold. Further, we present the binding efficacies of these glycoarchitectures on influenza virus and show that our inhibitors decrease the viral activity of different strains. Since SA is the major ligand for the viral surface protein HA we investigated the steps of viral replication that involve HA. Both multivalent architectures show a high inhibitory effect. The impact is also strongly dependent on the size of the multivalent inhibitor and on the degree of functionalization. Monomeric SA had no effect.
Université européenne de Bretagne, France Université de Bretagne Occidentale, I3S (Institut de Synergie des Sciences et de la Santé) – INSERM U613, CHU Morvan, 2 Avenue Foch, F-29609 Brest Cedex, France ⁎Corresponding author. E-mail: [email protected].
Conclusion A very simple approach to the preparation of SA-functionalized polyglycerol based NPs of previously unreported size (45–75 nm diameter) has been developed. The inhibitory activity of polymeric compounds was drastically increased with nanoparticle size. The monomeric form of sialic acid has no effect on the viral adhesion between 400 nM and 4 μM. In contrast pre-incubation of influenza virus with sialic acid functionalized HPG and nPGs leads to a strong reduction of fluorescence signal per erythrocyte. Current research is ongoing to optimize the inhibitor by changing the ligand properties (eg. size, length/flexibility of side chain, terminal sugar units) and further investigation into the mechanism of multivalent inhibition.
Introduction Archaeosomes are made with unusual membrane lipids of Archaebacteria which can survive under harsh environments [4]. Typical archaeal membrane core lipids (Fig. 1) are characterized by tetraether-type macrocyclic components formed by two substituted chains linked at both ends to a sn-2-glycerol unit. This lipid has got a structure particular in comparison with the classical lipids.
Acknowledgements This study has been carried with financial support from the Fonds der Chemischen Industrie (VCI Stipendium to I. Papp), as well as from collaborative research center ‘SFB 765’. References [1] J.J. Skehel, D.C. Wiley, Annu. Rev. Biochem. 69 (2000) 531. [2] M. Mammen, S.-K. Choi, G.M. Whitesides, Angew. Chem. Int. Ed. 37 (1998) 2754. [3] I. Carlescu, D. Scutaru, M. Popa, C. Uglea, Med. Chem. Res. 18 (2009) 477. [4] T.J. Pritchett, J.C.J. Paulson, Biol. Chem. 264 (1989) 9850. [5] W. Spevak, J.O. Nagy, D.H. Charych, M.E. Schaefer, J.H. Gilbert, M.D. Bednarski, J. Am. Chem. Soc. 115 (1993) 1146. [6] M. Mammen, G. Dahmann, G.M. Whitesides, J. Med. Chem. 38 (1995) 4179. [7] J.J. Landers, Z. Cao, I. Lee, L.T. Piehler, P.P. Myc, A. Myc, T. Hamouda, A.T. Galecki, J. R. Baker jr., J. Infect. Dis. 186 (2002) 1222. [8] R.K. Kainthan, D.E. Brooks, Biomaterials 28 (2007) 4779. [9] R.K. Kainthan, M. Gnanamani, M. Ganguli, T. Ghosh, D.E. Brooks, S. Maiti, J.N. Kizhakkedathu, Biomaterials 27 (2006) 5377. [10] R.K. Kainthan, S.R. Hester, E. Levin, D.V. Devine, D.E. Brooks, Biomaterials 28 (2007) 4581. [11] H. Tuerk, A. Shukla, P.C.A. Rodrigues, H. Rehage, R. Haag, Chem. Eur. J. 13 (2007) 4187. [12] H. Frey, R. Haag, Rev. Mol. Biotech. 90 (2002) 257. [13] K. Landfester, Top. Curr. Chem. 227 (2003) 75. [14] A.L. Sisson, D. Steinhilber, T. Rossow, P. Welker, K. Licha, R. Haag, Angew Chem. Int. Ed. 48 (41) (2009) 7540.
doi:10.1016/j.jconrel.2010.07.086
Folate PEGylated archaeal lipids: Cell targeting and drug delivery Yvan Portier1,2,⁎, Céline Lainé1,2, Loïc Lemiègre1,2, Tristan Montier1,3, Sandrine Cammas-Marion1,2, Pierre Lehn1,3, Thierry Benvegnu1,2 1 Ecole Nationale Supérieure de Chimie de Rennes, Equipe COS (Chimie Organique et Supramoléculaire) – UMR 6226, Campus de Beaulieu, 263 Avenue du Général Leclerc, CS 50837, F-35708 Rennes Cedex 7, France
3
Abstract summary Archaeosomes are of interest for potential biotechnological applications such as biomaterial delivery. Indeed, the archaeal lipid analogues improve the stability of delivery systems, including oral administration [1,2]. We developed new archaeal lipid analogues equipped with cell targeting ligands such as folic acid (FA) [3]. In vitro transfection efficiencies of formulations including these lipids were evaluated on several cell lines, and have shown good efficiencies at neutral charge ratios and a clear ligand mediated cell internalisation.
Fig. 1. Typical structure of thermoacidophilic archaeal membrane lipids.
To develop new supramolecular structures for encapsulation or delivery systems, it became thus interesting to combine these stabilizing effects with 1) sterical stabilisation ability by functionalizing the terminal end with poly(ethylene glycol) chains (PEG), 2) active targeting properties by adding ligands such as folic acid (FA) [5] or sugar [3]. Hereby, we designed and synthesized diether and tetraether-PEGfolate derivatives (Fig. 2).
Fig. 2. Synthetic diether and tetraether-PEG-folate derivatives.
Result and discussion The size of the vesicles formed from these synthetic amphiphile lipids was determined by dynamic light scattering. In vitro transfection efficiencies of formulations including this new co-lipids and a glycine betaine derived cationic lipid were evaluated using luciferase reporter gene and the ligand/receptor internalisation was evaluated by competitive inhibition with free folic acid. The results reveal that our new FA-PEGlipids afforded efficient transfection efficacies from a comparable to a higher level than lipofectamine [5]. Furthermore, the pre-saturation of the folate receptor with free folic acid clearly evidences that the internalisation of the lipoplexes is mediated by a folate/receptor interaction. Conclusion The third generation-type lipids developed here are promising colipids for gene delivery systems since they permit to use neutral
Abstracts / Journal of Controlled Release 148 (2010) e112–e124
Result and discussion DOX-liposome loaded microbubbles were prepared by attaching Doxil-like liposomes to the surface of lipid microbubbles via avidinbiotin interaction (Fig. 1). Melanoma cells were exposed to DOX-liposomes and DOXliposome loaded microbubbles without and with ultrasound exposure (USE). Exposure of the cells to DOX-liposome loaded microbubbles resulted in a much higher tumor cell killing efficiency than exposure to DOX-liposomes (Fig. 2). We did not see an outspoken improvement of cell killing by DOX-liposomes when ultrasound was applied.
created cell membrane pores. The in vitro results shown in this article indicate that DOX-liposome-loaded microbubbles can be a very interesting tool to obtain an efficient ultrasound-controlled DOX delivery in vivo. Acknowledgements I.L. is supported by the Fund for Scientific Research–Flanders (Belgium). The financial support of this institution is acknowledged with gratitude. The financial support of the European Union (via the FP7 projects Arise and Sonodrugs) is acknowledged. References [1] I. Lentacker, S.C. De Smedt, N.N. Sanders, Drug loaded microbubble design for ultrasound triggered delivery, Soft Matter 5 (11) (2009) 2161–2170. [2] I. Lentacker, B. Geers, J. Demeester, S.C. De Smedt, N.N. Sanders, Design and evaluation of doxorubicin containing microbubbles: cytotoxicity and mechanisms involved, Molecular Therapy 18 (1) (2010) 101–108.
doi:10.1016/j.jconrel.2010.07.085
Inhibition of influenza virus activity by newly designed multivalent glycoarchitectures
Fig. 2.
We also studied the intracellular localization of DOX after 4 h incubation time with DOX-liposomes or DOX-liposome loaded microbubbles and ultrasound. The DOX was almost exclusively present in the nuclei of cells treated with DOX-liposome loaded microbubbles, whereas the DOX was found in both, cytoplasm and nucleus of cells treated with DOX-liposomes (Fig. 3). After a short incubation time of only 15 min, only the cells treated with DOXloaded microbubbles and ultrasound showed DOX uptake. Additional experiments revealed that at least two different mechanisms are responsible for the high tumor cell killing efficiency of DOX-liposome loaded microbubbles after USE. During implosion of the microbubbles, liposomes are damaged and free DOX is released, which is immediately taken up through cell membrane perforations.
Ilona Papp1,⁎, Christian Sieben2, Adam L. Sisson3, Andreas Herrmann2, Rainer Haag1 1 Institut für Chemie und Biochemie, Freie Universität Berlin, Takustr. 3, D-14195 Berlin, Germany 2 Institut für Biologie/Biophysik, Humboldt-Universität zu Berlin, Invalidenstraße 43, 10115 Berlin, Germany 3 Berlin-Brandenburg Center for Regenerative Therapies, Augustenburger Platz 1, 13353 Berlin, Germany ⁎Corresponding author. E-mail: [email protected]. Abstract summary Influenza virus utilizes its envelope protein hemagglutinin (HA) to bind sialic acid (SA) residues of glycoproteins on the host cell plasma membrane [1]. The affinity of a single HA–SA pair is low (103 M− 1) compared to the overall affinity between virus and cell surface caused by multiple simultaneous interactions. Multivalency is ubiquitous in biology and can dramatically enhance affinities [2]. We use synthetic polymers with multiple SA moieties to investigate their inhibitory effect on virus binding and fusion. Introduction Many multivalent systems were introduced to inhibit influenza virus adhesion [3]. Some use natural scaffolds like proteins or liposomes [4–5]. Alternatively, synthetic polymeric scaffolds may be employed such as Whitesides' polyacrylamides [6] or Landers' polyamidoamines (PAMAM) [7]. Creating inhibitors that are not toxic/irritating to tissues and water-soluble still remains problematic. To circumvent these problems we coupled multiple SA residues to polyglycerols and nanogels, which are highly water soluble and biocompatible [8–10]. The aim was to address questions related to how particle size and multivalency affects biological processes and to gain insights into the molecular mechanism.
Fig. 3.
Conclusion It is possible to attach biotinylated doxil-like liposomes to the microbubble wall. The tumor cell killing efficiency of these liposomes is highly increased after microbubble coupling and ultrasound exposure. Ultrasonic treatment leads to the release of free doxorubicin which can subsequently enter the tumor cells trough the
Result and discussion This project deals with the evaluation of different architectures for multivalent interactions. Hyperbranched polyglycerols (HPGs), which are globular, multivalent, facilely prepared dendrimer analogues, are excellent biocompatible substrates for numerous applications [11– 12]. However, their synthesis is limited to afford particles of approx. 10 nm in diameter (Mn up to 1MDa). Through miniemulsion polymerization [13] well-defined polyglycerol nanogels (nPG) can
Abstracts / Journal of Controlled Release 148 (2010) e112–e124
e115
2
be prepared up to 100 nm in diameter [14]. Nanoparticles can be formed bearing unreacted epoxide units for further functionalization by controlling reaction time (number of remaining epoxides per glycerol unit can be determined by NMR) [14]. By addition of NaN3, azido nPGs are formed, which are quantitatively converted to triazole-derivates, following copper catalyzed click-chemistry. In the present study, we describe the conjugation of SA to HPGs and to newly developed nanogels, which are based on HPGs but exceed them in size up to 5–10 fold. Further, we present the binding efficacies of these glycoarchitectures on influenza virus and show that our inhibitors decrease the viral activity of different strains. Since SA is the major ligand for the viral surface protein HA we investigated the steps of viral replication that involve HA. Both multivalent architectures show a high inhibitory effect. The impact is also strongly dependent on the size of the multivalent inhibitor and on the degree of functionalization. Monomeric SA had no effect.
Université européenne de Bretagne, France Université de Bretagne Occidentale, I3S (Institut de Synergie des Sciences et de la Santé) – INSERM U613, CHU Morvan, 2 Avenue Foch, F-29609 Brest Cedex, France ⁎Corresponding author. E-mail: [email protected].
Conclusion A very simple approach to the preparation of SA-functionalized polyglycerol based NPs of previously unreported size (45–75 nm diameter) has been developed. The inhibitory activity of polymeric compounds was drastically increased with nanoparticle size. The monomeric form of sialic acid has no effect on the viral adhesion between 400 nM and 4 μM. In contrast pre-incubation of influenza virus with sialic acid functionalized HPG and nPGs leads to a strong reduction of fluorescence signal per erythrocyte. Current research is ongoing to optimize the inhibitor by changing the ligand properties (eg. size, length/flexibility of side chain, terminal sugar units) and further investigation into the mechanism of multivalent inhibition.
Introduction Archaeosomes are made with unusual membrane lipids of Archaebacteria which can survive under harsh environments [4]. Typical archaeal membrane core lipids (Fig. 1) are characterized by tetraether-type macrocyclic components formed by two substituted chains linked at both ends to a sn-2-glycerol unit. This lipid has got a structure particular in comparison with the classical lipids.
Acknowledgements This study has been carried with financial support from the Fonds der Chemischen Industrie (VCI Stipendium to I. Papp), as well as from collaborative research center ‘SFB 765’. References [1] J.J. Skehel, D.C. Wiley, Annu. Rev. Biochem. 69 (2000) 531. [2] M. Mammen, S.-K. Choi, G.M. Whitesides, Angew. Chem. Int. Ed. 37 (1998) 2754. [3] I. Carlescu, D. Scutaru, M. Popa, C. Uglea, Med. Chem. Res. 18 (2009) 477. [4] T.J. Pritchett, J.C.J. Paulson, Biol. Chem. 264 (1989) 9850. [5] W. Spevak, J.O. Nagy, D.H. Charych, M.E. Schaefer, J.H. Gilbert, M.D. Bednarski, J. Am. Chem. Soc. 115 (1993) 1146. [6] M. Mammen, G. Dahmann, G.M. Whitesides, J. Med. Chem. 38 (1995) 4179. [7] J.J. Landers, Z. Cao, I. Lee, L.T. Piehler, P.P. Myc, A. Myc, T. Hamouda, A.T. Galecki, J. R. Baker jr., J. Infect. Dis. 186 (2002) 1222. [8] R.K. Kainthan, D.E. Brooks, Biomaterials 28 (2007) 4779. [9] R.K. Kainthan, M. Gnanamani, M. Ganguli, T. Ghosh, D.E. Brooks, S. Maiti, J.N. Kizhakkedathu, Biomaterials 27 (2006) 5377. [10] R.K. Kainthan, S.R. Hester, E. Levin, D.V. Devine, D.E. Brooks, Biomaterials 28 (2007) 4581. [11] H. Tuerk, A. Shukla, P.C.A. Rodrigues, H. Rehage, R. Haag, Chem. Eur. J. 13 (2007) 4187. [12] H. Frey, R. Haag, Rev. Mol. Biotech. 90 (2002) 257. [13] K. Landfester, Top. Curr. Chem. 227 (2003) 75. [14] A.L. Sisson, D. Steinhilber, T. Rossow, P. Welker, K. Licha, R. Haag, Angew Chem. Int. Ed. 48 (41) (2009) 7540.
doi:10.1016/j.jconrel.2010.07.086
Folate PEGylated archaeal lipids: Cell targeting and drug delivery Yvan Portier1,2,⁎, Céline Lainé1,2, Loïc Lemiègre1,2, Tristan Montier1,3, Sandrine Cammas-Marion1,2, Pierre Lehn1,3, Thierry Benvegnu1,2 1 Ecole Nationale Supérieure de Chimie de Rennes, Equipe COS (Chimie Organique et Supramoléculaire) – UMR 6226, Campus de Beaulieu, 263 Avenue du Général Leclerc, CS 50837, F-35708 Rennes Cedex 7, France
3
Abstract summary Archaeosomes are of interest for potential biotechnological applications such as biomaterial delivery. Indeed, the archaeal lipid analogues improve the stability of delivery systems, including oral administration [1,2]. We developed new archaeal lipid analogues equipped with cell targeting ligands such as folic acid (FA) [3]. In vitro transfection efficiencies of formulations including these lipids were evaluated on several cell lines, and have shown good efficiencies at neutral charge ratios and a clear ligand mediated cell internalisation.
Fig. 1. Typical structure of thermoacidophilic archaeal membrane lipids.
To develop new supramolecular structures for encapsulation or delivery systems, it became thus interesting to combine these stabilizing effects with 1) sterical stabilisation ability by functionalizing the terminal end with poly(ethylene glycol) chains (PEG), 2) active targeting properties by adding ligands such as folic acid (FA) [5] or sugar [3]. Hereby, we designed and synthesized diether and tetraether-PEGfolate derivatives (Fig. 2).
Fig. 2. Synthetic diether and tetraether-PEG-folate derivatives.
Result and discussion The size of the vesicles formed from these synthetic amphiphile lipids was determined by dynamic light scattering. In vitro transfection efficiencies of formulations including this new co-lipids and a glycine betaine derived cationic lipid were evaluated using luciferase reporter gene and the ligand/receptor internalisation was evaluated by competitive inhibition with free folic acid. The results reveal that our new FA-PEGlipids afforded efficient transfection efficacies from a comparable to a higher level than lipofectamine [5]. Furthermore, the pre-saturation of the folate receptor with free folic acid clearly evidences that the internalisation of the lipoplexes is mediated by a folate/receptor interaction. Conclusion The third generation-type lipids developed here are promising colipids for gene delivery systems since they permit to use neutral