Dominik Witzigmann

Nanomedicine in cancer therapy: challenges, opportunities, and clinical applications.

Cancer is a leading cause of death worldwide. Currently available therapies are inadequate and spur demand for improved technologies. Rapid growth in nanotechnology towards the development of nanomedicine products holds great promise to improve therapeutic strategies against cancer. Nanomedicine products represent an opportunity to achieve sophisticated targeting strategies and multi-functionality. They can improve the pharmacokinetic and pharmacodynamic profiles of conventional therapeutics and may thus optimize the efficacy of existing anti-cancer compounds. In this review, we discuss state-of-the-art nanoparticles and targeted systems that have been investigated in clinical studies. We emphasize the challenges faced in using nanomedicine products and translating them from a preclinical level to the clinical setting. Additionally, we cover aspects of nanocarrier engineering that may open up new opportunities for nanomedicine products in the clinic.

PEG-PCL-based nanomedicines: A biodegradable drug delivery system and its application

Abstract The lack of efficient therapeutic options for many severe disorders including cancer spurs demand for improved drug delivery technologies. Nanoscale drug delivery systems based on poly(ethylene glycol)‐poly(&egr;‐caprolactone) copolymers (PEG‐PCL) represent a strategy to implement therapies with enhanced drug accumulation at the site of action and decreased off‐target effects. In this review, we discuss state‐of‐the‐art nanomedicines based on PEG‐PCL that have been investigated in a preclinical setting. We summarize the various synthesis routes and different preparation methods used for the production of PEG‐PCL nanoparticles. Additionally, we review physico‐chemical properties including biodegradability, biocompatibility, and drug loading. Finally, we highlight recent therapeutic applications investigated in vitro and in vivo using advanced systems such as triggered release, multi‐component therapies, theranostics, or gene delivery systems. Graphical abstract Figure. No Caption available.

Variable asialoglycoprotein-receptor 1 expression in liver disease: Implications for therapeutic intervention

One of the most promising strategies for the treatment of liver diseases is targeted drug delivery via the asialoglycoprotein receptor (ASGPR). The success of this approach heavily depends on the ASGPR expression level on parenchymal liver cells. In this study, we assessed the mRNA and protein expression levels of the major receptor subunit, ASGR1, in hepatocytes both in vitro and in vivo.

Hepatocyte targeting using pegylated asialofetuin-conjugated liposomes

Abstract Background and purpose: The hepatocyte asialoglycoprotein receptor mediates uptake of desiaylated glycoproteins by receptor-mediated endocytosis. This work explores a hepatocyte-specific targeting strategy using asialofetuin (AF) covalently coupled to pegylated liposomes. Methods: AF was conjugated to the distal end of polyethylene glycol–functionalized phospholipids. Chemical modification of AF did not interfere with its receptor interaction. AF-liposomes had a size of less than 130 nm, were judged to be monodisperse and were labelled with fluorescent organic dyes or loaded with quantum dots. Results: In vitro, binding and cellular uptake of fluorescent AF-liposomes by HepG2 hepatocellular carcinoma cells were reduced at low temperature and could be competitively inhibited by an excess of unbound AF. Hepatocyte-specific targeting and internalization of AF-liposomes in vivo was confirmed in the rat and could be competitively inhibited by co-injection of unbound AF. In contrast, non-pegylated liposomes accumulated in cells of the reticuloendothelial system such as hepatic Kupffer cells and spleen after intravenous administration. Conclusion: We conclude that the use of AF-conjugated, pegylated liposomes is a promising strategy to avoid the reticuloendothelial system and specifically target hepatocytes via the asialoglycoprotein receptor in vitro as well as in vivo.

Biocompatible polymer-Peptide hybrid-based DNA nanoparticles for gene delivery.

Currently, research on polymers to be used as gene delivery systems is one of the most important directions in both polymer science and biomedicine. In this report, we describe a five-step procedure to synthesize a novel polymer-peptide hybrid system for gene transfection. The block copolymer based on the biocompatible polymer poly(2-methyl-2-oxazoline) (PMOXA) was combined with the biocleavable peptide block poly(aspartic acid) (PASP) and finally modified with diethylenetriamine (DET). PMOXA-b-PASP(DET) was produced in high yield and characterized by (1)H NMR and FT-IR. Our biopolymer complexed plasmid DNA (pDNA) efficiently, and highly uniform nanoparticles with a slightly negative zeta potential were produced. The polymer-peptide hybrid system was able to efficiently transfect HEK293 and HeLa cells with GFP pDNA in vitro. Unlike the commonly used polymer, 25 kDa branched poly(ethylenimine), our biopolymer had no adverse effects on cell growth and viability. In summary, the present work provides valuable information for the design of new polymer-peptide hybrid-based gene delivery systems with biocompatible and biodegradable properties.

Zebrafish as an early stage screening tool to study the systemic circulation of nanoparticulate drug delivery systems in vivo

&NA; Nanomedicines have gained much attention for the delivery of small molecules or nucleic acids as treatment options for many diseases. However, the transfer from experimental systems to in vivo applications remains a challenge since it is difficult to assess their circulation behavior in the body at an early stage of drug discovery. Thus, innovative and improved concepts are urgently needed to overcome this issue and to close the gap between empiric nanoparticle design, in vitro assessment, and first in vivo experiments using rodent animal models. This study was focused on the zebrafish as a vertebrate screening model to assess the circulation in blood and extravasation behavior of nanoparticulate drug delivery systems in vivo. To validate this novel approach, monodisperse preparations of fluorescently labeled liposomes with similar size and zeta potential were injected into transgenic zebrafish lines expressing green fluorescent protein in their vasculature. Phosphatidylcholine‐based lipids differed by fatty acid chain length and saturation. Circulation behavior and vascular distribution pattern were evaluated qualitatively and semi‐quantitatively using image analysis. Liposomes composed of lipids with lower transition temperature (<28 °C) as well as PEGylated liposomes showed longer circulation times and extravasation. In contrast, liposomes composed of lipids with transition temperatures > 28 °C bound to venous parts of the vasculature. This circulation patterns in the zebrafish model did correlate with published and experimental pharmacokinetic data from mice and rats. Our findings indicate that the zebrafish model is a useful vertebrate screening tool for nanoparticulate drug delivery systems to predict their in vivo circulation behavior with respect to systemic circulation time and exposure. Graphical abstract Figure. No caption available.

Formation of lipid and polymer based gold nanohybrids using a nanoreactor approach

Nanocarriers encapsulating gold nanoparticles (AuNPs) hold tremendous promise for numerous biomedical applications. So far only a few fabrication strategies have been investigated and efficient processes for the manufacturing of gold nanohybrids (AuNHybs) are still missing. We encapsulated a tetrachloroaurate/citrate mixture within nanocarriers and initiated the AuNP formation after self-assembly of the nanomaterial by a temperature shift. This nanoreactor approach was successfully combined with the film-rehydration, nanoprecipitation, or microfluidics method. Different nanomaterials were validated including phospholipids and copolymers and the process was optimized towards encapsulation efficiency and physico-chemical homogeneity of AuNHybs. Our nanoreactor technology is versatile, efficient, and highly reproducible. Dynamic light scattering and electron microscopy techniques confirmed that generated lipid and polymer based AuNHybs were of uniform size below 130 nm and contained a single AuNP. The AuNHyb solutions had a deep-red color and exhibited the specific surface plasmon absorption of AuNPs. The unique optical properties of AuNHybs were used to visualize cellular uptake of nanocarriers in vitro demonstrating the promising applicability of AuNHybs as a bioimaging tool.

PDMS-b-PMOXA polymersomes for hepatocyte targeting and assessment of toxicity

Graphical abstract Figure. No Caption available. Abstract Nanoparticles, such as polymersomes, can be directed to the hepatic asialoglycoprotein receptor to achieve targeted drug delivery. In this study, we prepared asialofetuin conjugated polymersomes based on the amphiphilic di‐block copolymer poly(dimethylsiloxane)‐b‐poly(2‐methyloxazoline) (PDMS‐b‐PMOXA). They had an average diameter of 150 nm and formed monodisperse vesicles. Drug encapsulation and sustained release was monitored using the hydrophilic model compound carboxyfluorescein. Asialoglycoprotein receptor specific uptake by HepG2 cells in vitro was energy dependent and could be competitively inhibited by the free targeting ligand. Mechanistic uptake studies revealed intracellular trafficking of asialofetuin conjugated polymersomes from early endosomes and to the lysosomal compartment. Polymersomes showed no toxicity in the MTT assay up to concentrations of 500 &mgr;g/mL. In addition, acute toxicity and tolerability of our PDMS‐b‐PMOXA polymersome formulations was assessed in vivo using zebrafish embryos as a vertebrate screening model. In conclusion, a hepatocyte specific drug delivery system was designed, which is safe and biocompatible and which can be used to implement liver‐specific targeting strategies.

Biomimetic artificial organelles with in vitro and in vivo activity triggered by reduction in microenvironment

Despite tremendous efforts to develop stimuli-responsive enzyme delivery systems, their efficacy has been mostly limited to in vitro applications. Here we introduce, by using an approach of combining biomolecules with artificial compartments, a biomimetic strategy to create artificial organelles (AOs) as cellular implants, with endogenous stimuli-triggered enzymatic activity. AOs are produced by inserting protein gates in the membrane of polymersomes containing horseradish peroxidase enzymes selected as a model for natures own enzymes involved in the redox homoeostasis. The inserted protein gates are engineered by attaching molecular caps to genetically modified channel porins in order to induce redox-responsive control of the molecular flow through the membrane. AOs preserve their structure and are activated by intracellular glutathione levels in vitro. Importantly, our biomimetic AOs are functional in vivo in zebrafish embryos, which demonstrates the feasibility of using AOs as cellular implants in living organisms. This opens new perspectives for patient-oriented protein therapy.The efficacy of stimuli-responsive enzyme delivery systems is usually limited to in vitro applications. Here the authors form artificial organelles by inserting stimuli-responsive protein gates in membranes of polymersomes loaded with enzymes and obtain a triggered functionality both in vitro and in vivo.

Oral delivery of vancomycin by tetraether lipid liposomes

&NA; Despite the outstanding progress in modern medicine, the oral delivery of peptide drugs is limited until today due to their instability in the gastrointestinal tract and low mucosa penetration. To overcome these hurdles, liposomes containing the specific tetraether lipid GCTE (glycerylcaldityltetraether lipid) were examined. For this purpose, the glycopeptide antibiotic vancomycin was used as model substance and liposomes were prepared by DAC (dual assymetric centrifugation). These liposomes showed a size and polydispersity index comparable to standard liposomes. A high encapsulation efficiency of 58.53 ± 1.76% of the peptide drug vancomycin could be obtained as detected by HPLC. FCS analysis showed that in average each liposome contains 30 molecules of vancomycin. TEM and Cryo‐EM micrographs verified the size and lamellarity of the liposomal formulations. Cytotoxicity tests in Caco‐2 cells showed no significant cytotoxicity for all liposomal concentrations tested, indicating the good tolerability of these formulations. Furthermore, the use of sucrose as lyoprotector enabled the long term storage of the liposomal formulation for at least three months. The potency of this drug delivery system could be proven in an animal model using Wistar rats. One hour after oral application, 4.82 ± 0.56% of the administered dose of vancomycin could be found in the blood as detected by immunoassay measurements. This transport did also not affect the integrity of the peptide as verified by immunoassay measurements. In combination with long term storage stability, this formulation appears to be a promising delivery system for oral application of peptide drugs. Graphical abstract Figure. No caption available.