- Email: [email protected]
Nanosuspensions of chemically modified saponins: Reduction of hemolytic side effects and potential tool in drug targeting strategy
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Abstracts / Journal of Controlled Release 148 (2010) e112–e124
Experimental methods For active targeting, the polymer was modified with PDP (pyridyldithio propionate) on the PEG block. This way, PDP surface modified micelles could be formed, on which a SATA modified anti-EGFR nanobody was conjugated via disulfide bonding by mixing and reacting at room temperature overnight. For doxorubicin covalent entrapment, 20 mg/ml of polymer was dissolved in ammonium acetate buffer, and KPS (90 μl, 30 mg/ml in buffer), TEMED (50 μl, 120 mg/ml in buffer) and DOX-MA (30 mg/ml in methanol) were subsequently added, followed by rapid heating at 50 °C, to result in micelle formation. The DOX loading and release were determined by HPLC. Finally to obtain information about the efficacy of the formulation, the cell viability of ovarian carcinoma cells was monitored using a WST assay, after 72 h incubation at 37 °C with free DOX, DOX-MA, and DOX loaded micelles.
Conclusion A novel delivery system was developed, which can covalently entrap doxorubicin through a biodegradable linker that releases the drug preferentially in an acidic environment, and which can be targeted via the conjugation of an anti-EGFR nanobody on the micellar surface.
Results and discussion To ensure tumor cell recognition and uptake of the corecrosslinked micelles, an anti-EGFR EGa1 nanobody was attached on the surface of fluorescently labeled empty micelles using disulfide bonds, and the cell association was studied in cancer cells overexpressing EGFR. The conjugation was successful; cell association experiments in EGFR expressing cancer cells (A431) showed increased association of the nanobody micelles compared to empty micelles, which also increased with concentration (Fig. 2). It was shown that around 60% of the methacrylated doxorubicin added was covalently attached to the core of the micelles (without nanobodies on the surface), while the free drug that remained was less than 5%. The micelles released the entire drug payload within approximately 24 h incubation at pH 5 (where hydrolysis of the hydrazone bond occurs), while release at pH 7.4 was less than 10% (Fig. 3).
References
Acknowledgements The authors would like to thank Karel Ulbrich and his team (institute of Macromolecular chemistry, Prague) for providing the doxorubicin derivative, Amir Varkouhi (Utrecht University) for his help with the cytotoxicity experiments, as well as Sabrina Oliveira and Roy Meel (both Utrecht University) for providing the EGa1 nanobody.
[1] O. Soga, et al., J. Control. Release 103 (2005) 341–353. [2] C.J.F. Rijcken, et al., J. Control. Release 124 (2007) 144–153. [3] P.M. van Hasselt, et al., J. Control. Release 133 (2009) 161–168. [4] M. Talelli, et al., Langmuir 25 (2009) 2060–2067. [5] T. Etrych, et al., J. Control. Release 103 (2005) 341–353.
doi:10.1016/j.jconrel.2010.07.092
Nanosuspensions of chemically modified saponins: Reduction of hemolytic side effects and potential tool in drug targeting strategy H. Van de Ven1,⁎, L. Van Dyck1, W. Weyenberg1, L. Maes2, A. Ludwig1 1 Laboratory of Pharmaceutical Technology and Biopharmacy, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium 2 Laboratory of Microbiology, Parasitology and Hygiene, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium ⁎Corresponding author. E-mail: [email protected]. Abstract summary Pro-drug design was explored as a potential tool in drug targeting strategies. Saponins with structural similarity to the antileishmanial triterpene saponin maesabalide III were acetylated in order to obtain the hydrophobic derivative with reduced hemolytic activity. Nanosuspensions with Zave within the size range for passive targeting of phagocytic cells were produced by the emulsion-solvent evaporation technique.
Fig. 3. Release of doxorubicin from the polymeric micelles tailored by the pH.
Finally, in vitro cytotoxicity experiments in tumor cells showed no toxicity for empty micelles (data not shown), while DOX-loaded micelles showed similar toxicity as free DOX (Fig. 4), indicating possible cell internalization and release in the acidic lysosomes.
Fig. 4. % viability of ovarian carcinoma cells after 72 h incubation with free doxorubicin (DOX), its derivative (DOX-MA), as well as DOX loaded micelles.
Introduction The intracellular location of microorganisms such as Leishmania species limits the efficacy of currently used drugs. A rewarding strategy consists of entrapping drugs in drug delivery systems (DDS) such as liposomes, polymeric nanoparticles and nanosuspensions with capacity for selective distribution in phagocytic cells [1,2]. Pharmaceutical research focuses more and more on the latter because of its various advantages such as high drug loading, commercial feasibility and low cost [1,3]. Nanosuspensions can be prepared by top-down processes such as media milling and high pressure homogenization, or by bottom-up techniques involving controlled drug precipitation from a solution [3,4]. The optimum particle size of nanosuspensions for parenteral application depends on the desired biopharmaceutical properties. Rapid clearance by the phagocytic cells of liver, spleen and bone marrow is achieved in the case of particles >150 nm [5,6]. Nanosuspensions can only deliver hydrophobic drugs [1]. However, this hurdle can be tackled by chemically modifying the compound and subsequent nanomodification of the lipophilic derivative [7]. In this study, nanosuspensions of the acetylated saponin aescin were prepared as proof of concept for a DDS with the antileishmanial triterpene saponin maesabalide III [8]. Moreover, membrane-toxic
Abstracts / Journal of Controlled Release 148 (2010) e112–e124
properties and thus hemolytic effects should be absent for the chemically modified saponin. Experimental methods Aescin, which is structurally very similar to maesabalide III, was chosen as model molecule. The saponin was acetylated using acetic anhydride and iodine as an acetyl transfer catalyst [9] (Fig. 1).
e123
relevance. The twisted response surface for zeta potential (Fig. 3) indicates the interaction between sonication time and O:W ratio. The size of the effect of this two-way interaction is 4.1 mV (p≤ 0.001). The acetylated aescin and nanosuspension thereof exhibited reduced hemolytic activity in comparison to the unmodified saponin (Fig. 4). Hemolysis increased by prolonging the incubation time at 37 °C from 30 to 120 min, probably due to partial deacetylation of the pro-drug.
Fig. 1. Reaction scheme of aescin acetylation.
Nanosuspensions containing 0.7% (w/v) of acetylated aescin and polyvinyl alcohol (30–70 kDa; 71.4% w/w to compound) as stabilizer were prepared by solvent-antisolvent precipitation or emulsion-solvent evaporation. The controlled drug precipitation from an organic solution or nano-emulsification was attained by sonication at 20–22 W on ice. Organic solvents were removed, depending on their water-miscibility, by means of cross-flow filtration or evaporation at room temperature. Mean particle size Zave and zeta potential of the nanosuspensions were determined by Photon Correlation Spectroscopy (PCS) and Electrophoretic Light Scattering (ELS) with a Zetasizer 3000 (Malvern Instruments, UK). The emulsion-solvent evaporation method was optimized using a 23 full factorial design with center point. The hemolysis test was carried out as described in previous report [10]. For the experiments with the acetylated aescin and the nanosuspension concentrations are expressed as equivalent concentration of aescin. Result and discussion The acetyl derivative of aescin was synthesized with a yield ≥97%. The obtained compound was readily soluble in DMSO, DMF and DCM. Zave of nanosuspensions prepared by solvent-antisolvent precipitation was in the range 150–380 nm, zeta potential was negative as a consequence of the ionization of the acid group (Fig. 2).
Fig. 3. Response surface of Zave and zeta potential of nanosuspensions prepared by emulsion–solvent evaporation.
Fig. 4. Hemolytic activity of aescin, the acetyl derivative and nanosuspension after 30 and 120 min of incubation at 37 °C.
Conclusion Nanosuspensions of a chemically modified saponin were prepared by solvent–antisolvent precipitation and emulsion–solvent evaporation. The latter was preferred for the production of nanosuspensions with Zave > 150 nm and low PI. The acetylated aescin and nanosuspension thereof exhibited reduced hemolytic activity in comparison to the unmodified saponin. References
Fig. 2. Influence of organic solvent on Zave and zeta potential of nanosuspensions prepared by the solvent-antisolvent precipitation.
The effects and interaction effects of 3 preparation parameters of the emulsion-solvent evaporation on particle size and net charge were studied using a 23 full factorial design. As indicated by the 3D surface plot (Fig. 3), Zave was significantly (p < 0.05) influenced by the parameters sonication time and O:W ratio. By increasing the sonication time from 2 to 5 min, Zave decreased with 95 nm. Likewise, the polydispersity index (PI) decreased with 0.21. Thus, prolonging the duration of energy input gives rise to more homogeneously dispersed nano-emulsions with smaller droplets. An O:W ratio of 2:50 instead of 4:50 leads to an increase of Zave of 12%. This can be explained by the higher dynamic viscosity of the more concentrated organic solution in the case of formulations with a lower O:W ratio. Sonication time as well as evaporation time had a significant influence on zeta potential, the effects, however, were of minor practical
[1] A.A. Date, M.D. Joshi, V.B. Patravale, Parasitic diseases: liposomes and polymeric nanoparticles versus lipid nanoparticles, Adv. Drug Deliv. Rev. 59 (2007) 505–521. [2] E. Briones, C.I. Colino, J.M. Lanao, Delivery systems to increase the selectivity of antibiotics in phagocytic cells, J. Control. Release 125 (2008) 210–227. [3] A.A. Date, V.B. Patravale, Current strategies for engineering drug nanoparticles, Curr. Opin. Colloid Interface Sci. 9 (2004) 222–235. [4] S. Verma, Y. Lan, R. Gokhale, D.J. Burgess, Quality by design approach to understand the process of nanosuspension preparation, Int. J. Pharm. 377 (2009) 185–198. [5] K. Peters, S. Leitzke, J.E. Diederichs, K. Borner, et al., Preparation of a clofazimine nanosuspension for intravenous use and evaluation of its therapeutic efficacy in murine Mycobacterium avium infection, J. Antimicrob. Chemother. 45 (2000) 77–83. [6] J. Wong, A. Brugger, A. Khare, M. Chaubal, et al., Suspensions for intravenous (IV) injection: a review of development, preclinical and clinical aspects, Adv. Drug Deliv. Rev. 60 (2008) 939–954. [7] F. Hyafil, J.-C. Cornily, J.E. Feig, R. Gordon, et al., Noninvasive detection of macrophages using a nanoparticulate contrast agent for computed tomography, Nat. Med. 13 (2007) 636–641. [8] L. Maes, N. Germonprez, L. Quirijnen, L. van Puyvelde, et al., Comparative activities of the triterpene saponin maesabalide III and liposomal amphotericin B (AmBisome) against Leishmania donovani in hamsters, Antimicrob. Agents Chemotherap. 48 (2004) 2056–2060. [9] J.P. Malkinson, R.A. Falconer, I. Toth, Synthesis of C-terminal glycopeptides from resin-bound glycosyl azides via a modified Staudinger reaction, J. Org. Chem. 65 (2000) 5249–5252. [10] J.B. Sindambiwe, M. Calomme, S. Geerts, L. Pieters, et al., Evaluation of biological activities of triterpenoid saponins from Maesa lanceolata, J. Nat. Prod. 61 (1998) 585–590.
doi:10.1016/j.jconrel.2010.07.093
Abstracts / Journal of Controlled Release 148 (2010) e112–e124
Experimental methods For active targeting, the polymer was modified with PDP (pyridyldithio propionate) on the PEG block. This way, PDP surface modified micelles could be formed, on which a SATA modified anti-EGFR nanobody was conjugated via disulfide bonding by mixing and reacting at room temperature overnight. For doxorubicin covalent entrapment, 20 mg/ml of polymer was dissolved in ammonium acetate buffer, and KPS (90 μl, 30 mg/ml in buffer), TEMED (50 μl, 120 mg/ml in buffer) and DOX-MA (30 mg/ml in methanol) were subsequently added, followed by rapid heating at 50 °C, to result in micelle formation. The DOX loading and release were determined by HPLC. Finally to obtain information about the efficacy of the formulation, the cell viability of ovarian carcinoma cells was monitored using a WST assay, after 72 h incubation at 37 °C with free DOX, DOX-MA, and DOX loaded micelles.
Conclusion A novel delivery system was developed, which can covalently entrap doxorubicin through a biodegradable linker that releases the drug preferentially in an acidic environment, and which can be targeted via the conjugation of an anti-EGFR nanobody on the micellar surface.
Results and discussion To ensure tumor cell recognition and uptake of the corecrosslinked micelles, an anti-EGFR EGa1 nanobody was attached on the surface of fluorescently labeled empty micelles using disulfide bonds, and the cell association was studied in cancer cells overexpressing EGFR. The conjugation was successful; cell association experiments in EGFR expressing cancer cells (A431) showed increased association of the nanobody micelles compared to empty micelles, which also increased with concentration (Fig. 2). It was shown that around 60% of the methacrylated doxorubicin added was covalently attached to the core of the micelles (without nanobodies on the surface), while the free drug that remained was less than 5%. The micelles released the entire drug payload within approximately 24 h incubation at pH 5 (where hydrolysis of the hydrazone bond occurs), while release at pH 7.4 was less than 10% (Fig. 3).
References
Acknowledgements The authors would like to thank Karel Ulbrich and his team (institute of Macromolecular chemistry, Prague) for providing the doxorubicin derivative, Amir Varkouhi (Utrecht University) for his help with the cytotoxicity experiments, as well as Sabrina Oliveira and Roy Meel (both Utrecht University) for providing the EGa1 nanobody.
[1] O. Soga, et al., J. Control. Release 103 (2005) 341–353. [2] C.J.F. Rijcken, et al., J. Control. Release 124 (2007) 144–153. [3] P.M. van Hasselt, et al., J. Control. Release 133 (2009) 161–168. [4] M. Talelli, et al., Langmuir 25 (2009) 2060–2067. [5] T. Etrych, et al., J. Control. Release 103 (2005) 341–353.
doi:10.1016/j.jconrel.2010.07.092
Nanosuspensions of chemically modified saponins: Reduction of hemolytic side effects and potential tool in drug targeting strategy H. Van de Ven1,⁎, L. Van Dyck1, W. Weyenberg1, L. Maes2, A. Ludwig1 1 Laboratory of Pharmaceutical Technology and Biopharmacy, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium 2 Laboratory of Microbiology, Parasitology and Hygiene, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium ⁎Corresponding author. E-mail: [email protected]. Abstract summary Pro-drug design was explored as a potential tool in drug targeting strategies. Saponins with structural similarity to the antileishmanial triterpene saponin maesabalide III were acetylated in order to obtain the hydrophobic derivative with reduced hemolytic activity. Nanosuspensions with Zave within the size range for passive targeting of phagocytic cells were produced by the emulsion-solvent evaporation technique.
Fig. 3. Release of doxorubicin from the polymeric micelles tailored by the pH.
Finally, in vitro cytotoxicity experiments in tumor cells showed no toxicity for empty micelles (data not shown), while DOX-loaded micelles showed similar toxicity as free DOX (Fig. 4), indicating possible cell internalization and release in the acidic lysosomes.
Fig. 4. % viability of ovarian carcinoma cells after 72 h incubation with free doxorubicin (DOX), its derivative (DOX-MA), as well as DOX loaded micelles.
Introduction The intracellular location of microorganisms such as Leishmania species limits the efficacy of currently used drugs. A rewarding strategy consists of entrapping drugs in drug delivery systems (DDS) such as liposomes, polymeric nanoparticles and nanosuspensions with capacity for selective distribution in phagocytic cells [1,2]. Pharmaceutical research focuses more and more on the latter because of its various advantages such as high drug loading, commercial feasibility and low cost [1,3]. Nanosuspensions can be prepared by top-down processes such as media milling and high pressure homogenization, or by bottom-up techniques involving controlled drug precipitation from a solution [3,4]. The optimum particle size of nanosuspensions for parenteral application depends on the desired biopharmaceutical properties. Rapid clearance by the phagocytic cells of liver, spleen and bone marrow is achieved in the case of particles >150 nm [5,6]. Nanosuspensions can only deliver hydrophobic drugs [1]. However, this hurdle can be tackled by chemically modifying the compound and subsequent nanomodification of the lipophilic derivative [7]. In this study, nanosuspensions of the acetylated saponin aescin were prepared as proof of concept for a DDS with the antileishmanial triterpene saponin maesabalide III [8]. Moreover, membrane-toxic
Abstracts / Journal of Controlled Release 148 (2010) e112–e124
properties and thus hemolytic effects should be absent for the chemically modified saponin. Experimental methods Aescin, which is structurally very similar to maesabalide III, was chosen as model molecule. The saponin was acetylated using acetic anhydride and iodine as an acetyl transfer catalyst [9] (Fig. 1).
e123
relevance. The twisted response surface for zeta potential (Fig. 3) indicates the interaction between sonication time and O:W ratio. The size of the effect of this two-way interaction is 4.1 mV (p≤ 0.001). The acetylated aescin and nanosuspension thereof exhibited reduced hemolytic activity in comparison to the unmodified saponin (Fig. 4). Hemolysis increased by prolonging the incubation time at 37 °C from 30 to 120 min, probably due to partial deacetylation of the pro-drug.
Fig. 1. Reaction scheme of aescin acetylation.
Nanosuspensions containing 0.7% (w/v) of acetylated aescin and polyvinyl alcohol (30–70 kDa; 71.4% w/w to compound) as stabilizer were prepared by solvent-antisolvent precipitation or emulsion-solvent evaporation. The controlled drug precipitation from an organic solution or nano-emulsification was attained by sonication at 20–22 W on ice. Organic solvents were removed, depending on their water-miscibility, by means of cross-flow filtration or evaporation at room temperature. Mean particle size Zave and zeta potential of the nanosuspensions were determined by Photon Correlation Spectroscopy (PCS) and Electrophoretic Light Scattering (ELS) with a Zetasizer 3000 (Malvern Instruments, UK). The emulsion-solvent evaporation method was optimized using a 23 full factorial design with center point. The hemolysis test was carried out as described in previous report [10]. For the experiments with the acetylated aescin and the nanosuspension concentrations are expressed as equivalent concentration of aescin. Result and discussion The acetyl derivative of aescin was synthesized with a yield ≥97%. The obtained compound was readily soluble in DMSO, DMF and DCM. Zave of nanosuspensions prepared by solvent-antisolvent precipitation was in the range 150–380 nm, zeta potential was negative as a consequence of the ionization of the acid group (Fig. 2).
Fig. 3. Response surface of Zave and zeta potential of nanosuspensions prepared by emulsion–solvent evaporation.
Fig. 4. Hemolytic activity of aescin, the acetyl derivative and nanosuspension after 30 and 120 min of incubation at 37 °C.
Conclusion Nanosuspensions of a chemically modified saponin were prepared by solvent–antisolvent precipitation and emulsion–solvent evaporation. The latter was preferred for the production of nanosuspensions with Zave > 150 nm and low PI. The acetylated aescin and nanosuspension thereof exhibited reduced hemolytic activity in comparison to the unmodified saponin. References
Fig. 2. Influence of organic solvent on Zave and zeta potential of nanosuspensions prepared by the solvent-antisolvent precipitation.
The effects and interaction effects of 3 preparation parameters of the emulsion-solvent evaporation on particle size and net charge were studied using a 23 full factorial design. As indicated by the 3D surface plot (Fig. 3), Zave was significantly (p < 0.05) influenced by the parameters sonication time and O:W ratio. By increasing the sonication time from 2 to 5 min, Zave decreased with 95 nm. Likewise, the polydispersity index (PI) decreased with 0.21. Thus, prolonging the duration of energy input gives rise to more homogeneously dispersed nano-emulsions with smaller droplets. An O:W ratio of 2:50 instead of 4:50 leads to an increase of Zave of 12%. This can be explained by the higher dynamic viscosity of the more concentrated organic solution in the case of formulations with a lower O:W ratio. Sonication time as well as evaporation time had a significant influence on zeta potential, the effects, however, were of minor practical
[1] A.A. Date, M.D. Joshi, V.B. Patravale, Parasitic diseases: liposomes and polymeric nanoparticles versus lipid nanoparticles, Adv. Drug Deliv. Rev. 59 (2007) 505–521. [2] E. Briones, C.I. Colino, J.M. Lanao, Delivery systems to increase the selectivity of antibiotics in phagocytic cells, J. Control. Release 125 (2008) 210–227. [3] A.A. Date, V.B. Patravale, Current strategies for engineering drug nanoparticles, Curr. Opin. Colloid Interface Sci. 9 (2004) 222–235. [4] S. Verma, Y. Lan, R. Gokhale, D.J. Burgess, Quality by design approach to understand the process of nanosuspension preparation, Int. J. Pharm. 377 (2009) 185–198. [5] K. Peters, S. Leitzke, J.E. Diederichs, K. Borner, et al., Preparation of a clofazimine nanosuspension for intravenous use and evaluation of its therapeutic efficacy in murine Mycobacterium avium infection, J. Antimicrob. Chemother. 45 (2000) 77–83. [6] J. Wong, A. Brugger, A. Khare, M. Chaubal, et al., Suspensions for intravenous (IV) injection: a review of development, preclinical and clinical aspects, Adv. Drug Deliv. Rev. 60 (2008) 939–954. [7] F. Hyafil, J.-C. Cornily, J.E. Feig, R. Gordon, et al., Noninvasive detection of macrophages using a nanoparticulate contrast agent for computed tomography, Nat. Med. 13 (2007) 636–641. [8] L. Maes, N. Germonprez, L. Quirijnen, L. van Puyvelde, et al., Comparative activities of the triterpene saponin maesabalide III and liposomal amphotericin B (AmBisome) against Leishmania donovani in hamsters, Antimicrob. Agents Chemotherap. 48 (2004) 2056–2060. [9] J.P. Malkinson, R.A. Falconer, I. Toth, Synthesis of C-terminal glycopeptides from resin-bound glycosyl azides via a modified Staudinger reaction, J. Org. Chem. 65 (2000) 5249–5252. [10] J.B. Sindambiwe, M. Calomme, S. Geerts, L. Pieters, et al., Evaluation of biological activities of triterpenoid saponins from Maesa lanceolata, J. Nat. Prod. 61 (1998) 585–590.
doi:10.1016/j.jconrel.2010.07.093