Kugelförmiges Pferd im Vakuum oder anwendbare Mizellen? Verhalten und Form von Nanopartikeln als Wirkstoffträger für Krebsmedikamente in reeller Blutumgebung
Zusammenfassung der Projektergebnisse
Various forms of cancer belong to major causes of death up to recent days since the possibilities of treatment are still limited in many cases. Despite recent advances in the cancer treatment, anticancer therapy still suffers from severe side effects that significantly decrease achievable effectiveness of the therapy. Macromolecular nanoparticles based on polymers for solid tumor targeting are a new type of drug delivery systems promising for the therapy of cancer. They allow the decrease of general toxicity and side effects of anticancer drugs. However, proteins present in human blood may interfere with the drug delivery process. We conducted a comprehensive study of self-assembled polymeric nanoparticles for the targeted delivery of cancer drugs in the living body, focusing on the fundamental question whether and in which way blood proteins bind to the nanoparticles. The polymer system chosen was based on N-(2-hydroxypropyl)methacrylamide with a small amount of cholesterol moieties (pHPMA-Chol), which ensure nanoparticle formation, and the cancer drug Doxorubicin (pHPMA-Chol-Dox). The most abundant protein in human blood, human serum albumin (HSA), was chosen as a first step towards real human blood. All investigations were carried out in phosphate buffer saline. Experiments were carried out at TU München, EMBL Hamburg and at the Institute of Macromolecular Chemistry in Prague. To identify a suitable system for the investigations, we studied the influence of the chain architecture (random or block copolymers) and the overall molar mass of the copolymers as well as the type of hydrophobic moiety on the critical micelle concentration and on the structure of the nanoparticles. These variations did not lead to substantial differences, and for the investigations, random copolymers of a molar mass of 30,000 g mol-1 with 2 mol% of cholesterol were chosen. However, the presence of Doxorubicin alters the behavior of the hydrodynamic radius with increasing HSA concentration, as evidenced using dynamic light scattering. Using synchrotron small-angle X-ray scattering (SAXS) on pHPMA-Chol or pHPMA-Chol-Dox in presence of HSA, it was found that the interaction is weak between the former and HSA, and not detectable between the latter and HSA. Isothermal titration calorimetry (ITC) confirmed that the binding between pHPMA-Chol and HSA is stronger than the one between pHPMA-Chol- Dox and HSA. This is due to the presence of Dox in the shell of the nanoparticles which hampers the binding of HSA to the cholesterol moieties located in the core of the NPs. Using small-angle neutron scattering with contrast matching showed that the structure of HSA changes only in presence of pHPMA-Chol, but not of pHPMA-Chol-Dox. A very important aspect of the project was the development of the methodology of anomalous small angle scattering (ASAXS) for macromolecular solutions. ASAXS is a powerful technique utilized to obtain distributions of specific atoms in material science and potentially extremely useful for soft matter and biological objects containing heavy atoms. However, this is technically extremely complicated because of very small useful signals, necessitating very precise measurements and calibrations to generate reliable data. Thanks to the work on this project, ASAXS methodology is now established at P12 and is offered to biological and soft matter projects by the German and international users of the beamline. Fluorescence labeling was used to characterize the binding between nanoparticles from pHPMA-Chol and HSA further. Using fluorescence-labeled pHPMA-Chol as a dye and suppressing the fluorescence of HSA using optical and life-time filters, the hydrodynamic radius of the NPs could be determined in the presence of HSA. Reasonable values were obtained, but no increase with HSA concentration could be detected. Therefore, the intrinsic fluorescence emission of HSA was exploited. Quenching of the intrinsic fluorescence of HSA by pHPMA-Chol confirmed that the complexation between HSA and cholesterol side groups is at the origin of the interaction, and a binding constant was obtained. These studies were complemented by screening investigations using several blood proteins of the Czech partner. Overall, our results indicate that the drug delivery using the pHPMA-based nanoparticles is not affected by HSA, i.e. it is a promising system for delivery of cancer drugs to humans.
Projektbezogene Publikationen (Auswahl)
- ATSAS 2.8: A Comprehensive Data Analysis Suite for Small-angle Scattering from Macromolecular Solutions. J. Appl. Crystallogr. 50, 1212-1225 (2017)
D. Franke, M. V. Petoukhov, P. V. Konarev, A. Panjkovich, A. Tuukkanen, H. D. T. Mertens, A. G. Kikhney, N. R. Hajizadeh, J. M. Franklin, C. M. Jeffries, D. I. Svergun
(Siehe online unter https://doi.org/10.1107/S1600576717007786) - Influence of Molar Mass, Dispersity, and Type and Location of Hydrophobic Side Chain Moieties on the Critical Micellar Concentration and Stability of Amphiphilic HPMA-based Polymer Drug Carriers. Colloid Polym. Sci. 295, 1313-1325 (2017)
S. K. Filippov, N. S. Vishnevetskaya, B.-J. Niebuur, E. Koziolová, E. A. Lomkova, P. Chytil, T. Etrych, C. M. Papadakis
(Siehe online unter https://doi.org/10.1007/s00396-017-4027-7) - Self-assembled Nanoparticles for Drug Delivery – Behavior in the Presence of Proteins. Experimental Report MLZ, Garching, June 2017
C. M. Papadakis, X. Zhang
- Binding of HSA to Macromolecular pHPMA Based Nanoparticles for Drug Delivery: An Investigation Using Fluorescence Methods. Langmuir 34, 7998–8006 (2018)
X. Zhang, P. Chytil, T. Etrych, W. Liu, L. Rodrigues, G. Winter, S. K. Filippov, C. M. Papadakis
(Siehe online unter https://doi.org/10.1021/acs.langmuir.8b01015) - Macromolecular pHPMA-Based Nanoparticles with Cholesterol for Solid Tumor Targeting: Behavior in HSA Protein Environment. Biomacromolecules 19, 470-480 (2018)
X. Zhang, B.-J. Niebuur, P. Chytil, T. Etrych, S. K. Filippov, A. Kikhney, D. C. F Wieland, D. I. Svergun, C.M. Papadakis
(Siehe online unter https://doi.org/10.1021/acs.biomac.7b01579) - Smaller Capillaries Improve the Small-angle X-ray Scattering Signal and Sample Consumption for Biomacromolecular Solutions, J. Synchrotron Radiat. 25, 1113-1122 (2018)
M. A. Schroer, C. E. Blanchet, A. Y. Gruzinov, M. A. Gräwert, M. E. Brennich, N. R. Hajizadeh, C. M. Jeffries, D. I. Svergun
(Siehe online unter https://doi.org/10.1107/S1600577518007907)
