Matthias Wacker

Novel photosensitizer-protein nanoparticles for photodynamic therapy: photophysical characterization and in vitro investigations.

In this work two types of pheophorbide-HSA (Pheo-HSA) nanoparticles, PHSA40 and PHSA100, were prepared and their photophysical and photosensitizing properties were investigated. Due to intramolecular interactions the singlet oxygen quantum yield of PHSA40 and PHSA100 is very low (less than 0.1). Intracellular uptake and phototoxicity of pheophorbide a as well as of the Pheo-HSA nanoparticles were studied in Jurkat cells. The HSA nanoparticles do not influence the amount of dye accumulation in cells. After 24h incubation, PHSA40 and PHSA100 showed a higher phototoxicity than Pheo. The reason for this behavior is an efficient nanoparticle decomposition in the cellular lysosomes. The process of drug release during incubation of cells with Pheo-HSA nanoparticles was illustrated by fluorescence lifetime imaging (FLIM) and confocal laser scanning microscopy (CLSM). The final phototoxicity of Pheo-HSA is at the same scale as induced by free Pheo. The drug release ability of HSA nanoparticles shows the possibility to use such formulations as drug carriers in PDT treatment. Therefore, this work constructs a standard for further investigation and optimization of photosensitizer-HSA drug carrier system.

Nanocarriers for intravenous injection—The long hard road to the market

Nanodispersed drug delivery systems for the intravenous injection have successfully overcome the hurdle of drug approval in the European Union and the United States. Although there is a need for highly advanced nanocarrier devices they have not been the result of a rational formulation design but were developed as stand-alone products in a long chain of case-by-case studies. This review focuses on aspects in development, composition, and manufacture of these innovative dosage forms that are relevant for the translation into new drug products.

Targeted human serum albumin nanoparticles for specific uptake in EGFR-Expressing colon carcinoma cells

UNLABELLED The specific application and transport of drugs into malignant tissue is a critical point during diagnosis and therapy. Nanoparticles are known as excellent drug carrier systems and offer the possibility of surface modification with targeting ligands, leading to a specific accumulation in the targeted tissue. First, the specificity of such a carrier system has to be proven. In this study, cetuximab-modified nanoparticles based on biodegradable human serum albumin (HSA) are investigated regarding their cellular binding and intracellular accumulation. Different EGFR-expressing colon carcinoma cells were used to test possible cytotoxic potential, specific binding and intracellular accumulation. A specific accumulation targeting the EGFR could be shown. These results emphasize that cetuximab-modified HSA-nanoparticles are a promising carrier system for later drug transport. To our knowledge, this is the first study investigating the specific accumulation of HSA nanoparticles into different EGFR-expressing colon carcinoma cells. FROM THE CLINICAL EDITOR In this study, cetuximab-modified nanoparticles based on human serum albumin (HSA) are investigated regarding their cellular binding and intracellular accumulation. The results suggest that these nanoparticles are a promising carrier system for EGFR overexpressing colon carcinoma cells.

Photosensitizer loaded HSA nanoparticles. I: Preparation and photophysical properties

Photodynamic therapy (PDT) is a promising option in the treatment of cancer. Efficient photosensitizers are available but many of them have insufficient physico-chemical properties for parenteral application. We have established nanoparticles consisting of human serum albumin (HSA) as a drug carrier system for 5,10,15,20-tetrakis(m-hydroxyphenyl)porphyrine (mTHPP) and 5,10,15,20-tertrakis(m-hydroxyphenyl)chlorin (mTHPC), two well-known photosensitizers. Nanoparticle loading was performed in water/ethanol mixtures in the presence of dissolved HSA acting as solubilizer for photosensitizers. The HSA concentration was optimized to exclude precipitation in the nanoparticle suspension and to increase binding to nanoparticles. Additionally, the influence of pH and incubation time on drug adsorption was investigated. A freeze drying method was established for mTHPC loaded nanoparticles and the storage stability of the freeze dried formulation was tested. PDT related photophysical parameters of drug loaded HSA nanoparticles, especially singlet oxygen generation, are presented. Both preparations were able to generate singlet oxygen with low quantum yield. In contrast, efficient singlet oxygen generation was obtained when Jurkat cells were incubated with mTHPP and mTHPC loaded HSA nanoparticles. This indicates that the photosensitizer molecules were successfully released from the nanoparticles that were taken up by the cells. Therefore, the efficiency of HSA nanoparticles as drug carriers for photosensitizers was proven under in vitro conditions.

A toolbox for the upscaling of ethanolic human serum albumin (HSA) desolvation.

Nanoparticles consisting of human serum albumin (HSA) play an emerging role in the development of new drug delivery systems. Many of these protein-based colloidal carriers are prepared by the well-known desolvation technique, which has shown to be a robust and reproducible method for the laboratory-scale production of HSA nanoparticles. The aim of the present study was to upscale the ethanolic desolvation process utilizing the paddle stirring systems Nanopaddle I and II in combination with a HPLC pump in order to find the optimal conditions for the controlled desolvation of up to 2000 mg of the protein. For characterization of the HSA nanoparticles particle size, zeta potential as a function of the pH, polydispersity index and particle content were investigated. The particle content was determined by microgravimetry and by a turbidimetry to allow optimized in-process control for the novel desolvation technique. Furthermore the sedimentation coefficient was measured by analytical ultracentrifugation (AUC) to gain deeper insight into the size distribution of the nanoparticles. The formed nanocarriers were freeze dryed to achieve a solid preparation for long-term storage and further processing. Particles ranging in size between 251.2 ± 27.0 and 234.1 ± 1.5 nm and with a polydispersity index below 0.2 were achieved.

Photophysical evaluation of mTHPC-loaded HSA nanoparticles as novel PDT delivery systems

Controlled drug release is one of the main goals of recent developments in drug carrier systems. In this work human serum albumin (HSA) nanoparticles as carriers for 5-, 10-, 15-, 20-Tetrakis (3-hydroxyphenyl)-chlorin (mTHPC) were investigated. The photophysical properties of mTHPC-HSA nanoparticles in dependence of loading ratio and level of HSA cross-linking were determined. Further the drug release after uptake by Jurkat cells and in vitro singlet oxygen kinetics were examined. The loading ratio of the mTHPC-HSA nanoparticles turned out to be of major importance for the PDT relevant electronic parameters in solution. Therefore, only HSA nanoparticles with low mTHPC-loading ratio generate singlet oxygen in D(2)O. However, after cellular uptake all mTHPC-HSA samples generate singlet oxygen in Jurkat cells, but the decomposition rate depends on the level of HSA cross-linking.

Targeting by cmHsp70.1-antibody coated and survivin miRNA plasmid loaded nanoparticles to radiosensitize glioblastoma cells.

Nanoparticles (NP) as carriers for anti-cancer drugs have shown great promise. Specific targeting of NP to malignant cells, however, remains an unsolved problem. Conjugation of antibodies specific for tumor membrane antigens to NP represents one approach to improve specificity and to increase therapeutic efficacy. In the present study, for the first time a novel membrane heat shock protein (Hsp70)-specific antibody (cmHsp70.1) was coupled to human serum albumin (HSA) NP, loaded with microRNA (miRNA) plasmids to target the inhibitor of apoptosis protein survivin. The physicochemical properties of monodisperse miRNA-loaded NP showed a diameter of 180 nm to 220 nm, a plasmid incorporation of more than 95% and a surface binding capacity of the antibody of 70-80%. Antibody-conjugated NP displayed an increased cellular uptake in U87MG and LN229 glioblastoma cells compared to isotype control antibody, PEG-coupled controls and peripheral blood lymphocytes (PBL). Survivin expression was significantly reduced in cells treated with the Hsp70-miRNA-NP as compared to non-conjugated NP. Hsp70-miRNA-NP enhanced radiation-induced increase in caspase 3/7 activity and decrease in clonogenic cell survival. In summary, cmHsp70.1 miRNA-NP comprise an enhanced tumor cell uptake and increased therapeutic efficacy of radiation therapy in vitro and provide the basis for the development of antibody-based advanced carrier systems for a tumor cell specific targeting.

Assessing the drug release from nanoparticles: Overcoming the shortcomings of dialysis by using novel optical techniques and a mathematical model.

The aim of the present investigation was to develop a reliable method which can be applied to the measurement of in vitro drug release from nanocarriers. Since the limited membrane transport is one major obstacle to the assessment of drug release with dialysis techniques, the determination of this parameter was our objective. Therefore, a novel drug release automatic monitoring system (DREAMS) was designed to conduct continuous measurements during the dialysis process. Moreover, a mathematical model was used for evaluation of the experimental data. This combination of mathematical and analytical tools enabled the quantification of the total amount of free drug in the system. Eudragit(®) RS 100 nanoparticles loaded with the model compound 5,10,15,20-tetrakis(m-hydroxypheny)chlorin (mTHPC) were investigated and the drug release was continuously monitored by using a fluorescence spectrometer that is part of the setup. Free drug and drug-loaded nanoparticles were tested to discriminate between the two formulations. In addition, two types of membranes composed of different materials were evaluated and the kinetics of membrane transport was determined. The data obtained from the apparatus were further treated by a mathematical model, which yielded distinguishable release profiles between samples of different compositions. The method offers a promising option for release testing of nanoparticles.

The role of recent nanotechnology in enhancing the efficacy of radiation therapy

Radiation therapy is one of the most commonly used non-surgical interventions in tumor treatment and is often combined with other modalities to enhance its efficacy. Despite recent advances in radiation oncology, treatment responses, however, vary considerably between individual patients. A variety of approaches have been developed to enhance radiation response or to counteract resistance to ionizing radiation. Among them, a relatively novel class of radiation sensitizers comprises nanoparticles (NPs) which are highly efficient and selective systems in the nanometer range. NPs can either encapsulate radiation sensitizing agents, thereby protecting them from degradation, or sensitize cancer cells to ionizing radiation via their physicochemical properties, e.g. high Z number. Moreover, they can be chemically modified for active molecular targeting and the imaging of tumors. In this review we will focus on recent developments in nanotechnology, different classes and modifications of NPs and their radiation sensitizing properties.

Photosensitizer loaded HSA nanoparticles II: In vitro investigations

The photosensitizing efficiency of human serum albumin (HSA) nanoparticles loaded with the photosensitizers meta-tetra(hydroxy-phenyl)-chlorin (mTHPC) and meta-tetra(hydroxy-phenyl)-porphyrin (mTHPP) was investigated in vitro. The endocytotic intracellular uptake, and the time dependent drug release caused by nanoparticle decomposition of the PS loaded HSA nanoparticles were studied on Jurkat cells in suspension. The photoxicity as well as the intracellular singlet oxygen ((1)O(2)) generation were investigated in dependence on the incubation time. The obtained results show that HSA nanoparticles are promising carriers for the clinical used mTHPC (Foscan). After release the ((1)O(2)) generation as well as the phototoxicity are more efficient compared with mTHPC applied without the HSA nanoparticles.