The zebrafish: a preclinical screening model for the optimization of nanomedicine formulations

Nanomedicines are a valuable option to achieve drug accumulation specifically in diseased cells or tissues and therefore reduce side effects. Since the introduction of the revolutionary concept called the magic bullet for such sophisticated treatments more than 100 years ago, a lot of hope and expectations were placed into the field of nanoparticulate drug delivery. Initial forms of nanoparticles such as liposomes were described and extensively characterized which finally resulted in the FDA approval of the first cancer nanomedicine, namely Doxil, in 1996. This early success fueled the already gold-rush like atmosphere and resulted in a huge amount of time and money invested in nanomedicine research and development. As it is often the case for such a much-noticed field of medicinal research, the number of approved and clinically applied nanomedicines was not able to keep up with the unrealistic expectations resulting from the exponential increase of nanomedicine related publications. This triggered a lot of criticism questioning basic principles such as the enhanced permeability and retention effect or even the general use of nanomedicines. Despite the fact that the raised points are legitimate to a certain degree, the field of nanomedicine is far away from suffering from a general crisis, underlined by the steadily (but slowly) increasing number of approved formulations. Nevertheless, it cannot be denied that nanomedicine development is a cumbersome process suffering from a lot of drop-outs during very early phases of clinical trials. Among other things, this is due to the fact that formulation design and optimization is mainly based on in vitro studies, which are not able to fully mimic complex biological conditions. Moreover, only a selected number of formulations can subsequently be assessed in rodent in vivo experiments, since such studies are expensive, time consuming and suffer from ethical concerns. Obviously, there is a huge gap between in vitro cell culture and rodent in vivo studies, which makes the selection of potentially successful nanomedicine formulations extremely difficult. In addition, this situation does not allow a thorough formulation design and optimization under complex biological conditions and hampers a detailed understanding of basic nanomedicine interactions with biological environments at a macromolecular level. Therefore, this PhD thesis aimed to introduce the zebrafish as a complementary and easy accessible in vivo model in order to bridge the gap between in vitro and rodent in vivo studies during nanomedicine development. In the first part (Chapters I-I to III-I), the current nanomedicine development process prior to rodent in vivo studies was reviewed and the zebrafish model was set-up, validated, and further characterized. Briefly, already described formulation effects on nanomedicine pharmacokinetics were reproduced and the predictive power of the zebrafish model system was verified. Thereby, a special focus was put on two main nanomedicine clearance mechanisms, namely phagocytosis by macrophages as a part of the mononuclear phagocytic system and scavenger receptors expressed on cells, which belong to the reticuloendothelial system. Based on the successful completion of the first part, the zebrafish model was used for the development of sophisticated nanoparticulate delivery systems (Chapter IV). For example, the optimal ligand density for an actively targeted nanoparticle was established in the zebrafish model and verified in a subsequent rodent biodistribution experiment. In addition, two different nanoparticle-enzyme systems were tested regarding their stability, biocompatibility, and functionality in this living biological system, i.e. zebrafish. During this thesis, general advantages of the zebrafish model such as large clutch size, optical transparency, availability of many transgenic lines, the possibility to screen a large number of formulations, and relatively low regulatory requirements became evident. All parameters were adapted to the purpose of nanomedicine formulation design and optimization. The promising findings will be further pursued in detailed follow-up studies regarding the development of an accurate and quantitative pharmacokinetic model, the elucidation of exact formulation dependent nanomedicine cell uptake and trafficking mechanisms under in vivo conditions, or to support the formulation design and optimization of nanomedicines for infectious diseases. Altogether, the presented zebrafish model showed to be a valuable and promising tool for several applications in the field of nanomedicine development and will hopefully foster the successful translation of further nanomedicines from bench to bedside

Bioinspired Molecular Factories with Architecture and In Vivo Functionalities as Cell Mimics

Despite huge need in the medical domain and significant development efforts, artificial cells to date have limited composition and functionality. Whereas some artificial cells have proven successful for producing therapeutics or performing in vitro specific reactions, they have not been investigated in vivo to determine whether they preserve their architecture and functionality while avoiding toxicity. Here we overcome these limitations and achieve customizable cell mimic - molecular factories (MFs) - by supplementing giant plasma membrane vesicles derived from donor cells with nanometer-sized artificial organelles (AOs). MFs inherit the donor cell's natural cytoplasm and membrane, while the AOs house reactive components and provide cell-like architecture and functionality. We demonstrate that reactions inside AOs take place in a close-to-nature environment due to the unprecedented level of complexity in the composition of the MFs. We further demonstrate that in a zebrafish vertebrate animal model these cell mimics showed no apparent toxicity and retained their integrity and function. The unique advantages of highly varied composition, multi-compartmentalized architecture, and preserved functionality in vivo open new biological avenues ranging from the study of bio-relevant processes in robust cell-like environments to the production of specific bioactive compounds

DNA-directed arrangement of soft synthetic compartments and their behavior in vitro and in vivo

DNA has been widely used as a key tether to promote self-organization of super-assemblies with emergent properties. However, control of this process is still challenging for compartment assemblies and to date the resulting assemblies have unstable membranes precluding in vitro and in vivo testing. Here we present our approach to overcome these limitations, by manipulating molecular factors such as compartment membrane composition and DNA surface density, thereby controlling the size and stability of the resulting DNA-linked compartment clusters. The soft, flexible character of the polymer membrane and low number of ssDNA remaining exposed after cluster formation determine the interaction of these clusters with the cell surface. These clusters exhibit in vivo stability and lack of toxicity in a zebrafish model. To display the breadth of therapeutic applications attainable with our system, we encapsulated the medically established enzyme laccase within the inner compartment and demonstrated its activity within the clustered compartments. Most importantly, these clusters can interact selectively with different cell lines, opening a new strategy to modify and expand cellular functions by attaching such pre-organized soft DNA-mediated compartment clusters on cell surfaces for cell engineering or therapeutic applications

Front cover - Cell Membrane Wrapping: Influence of Cell Membrane Wrapping on the Cell−Porous Silicon Nanoparticle Interactions (Adv. Healthcare Mater. 17/2020)

Biohybrid nanosystems represent the cutting‐edge research in biofunctionalization of micro‐ and nano‐systems. Their physicochemical properties bring along advantages in the circulation time, camouflaging from the phagocytes, and novel antigens. This is partially a result of the qualitative differences in the protein corona, and the preferential targeting and uptake in homologous cells. However, the effect of the cell membrane on the cellular endocytosis mechanisms and time has not been fully evaluated yet. Here, the effect is assessed by quantitative flow cytometry analysis on the endocytosis of hydrophilic, negatively charged porous silicon nanoparticles and on their membrane‐coated counterparts, in the presence of chemical inhibitors of different uptake pathways. Principal component analysis is used to analyze all the data and extrapolate patterns to highlight the cell‐specific differences in the endocytosis mechanisms. Furthermore, the differences in the composition of static protein corona between naked and coated particles are investigated together with how these differences affect the interaction with human macrophages. Overall, the presence of the cell membrane only influences the speed and the entity of nanoparticles association with the cells, while there is no direct effect on the endocytosis pathways, composition of protein corona, or any reduction in macrophage‐mediated uptake

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

Influence of Cell Membrane Wrapping on the Cell-Porous Silicon Nanoparticle Interactions

Biohybrid nanosystems represent the cutting-edge research in biofunctionalization of micro- and nano-systems. Their physicochemical properties bring along advantages in the circulation time, camouflaging from the phagocytes, and novel antigens. This is partially a result of the qualitative differences in the protein corona, and the preferential targeting and uptake in homologous cells. However, the effect of the cell membrane on the cellular endocytosis mechanisms and time has not been fully evaluated yet. Here, the effect is assessed by quantitative flow cytometry analysis on the endocytosis of hydrophilic, negatively charged porous silicon nanoparticles and on their membrane-coated counterparts, in the presence of chemical inhibitors of different uptake pathways. Principal component analysis is used to analyze all the data and extrapolate patterns to highlight the cell-specific differences in the endocytosis mechanisms. Furthermore, the differences in the composition of static protein corona between naked and coated particles are investigated together with how these differences affect the interaction with human macrophages. Overall, the presence of the cell membrane only influences the speed and the entity of nanoparticles association with the cells, while there is no direct effect on the endocytosis pathways, composition of protein corona, or any reduction in macrophage-mediated uptake

Optimasi Portofolio Resiko Menggunakan Model Markowitz MVO Dikaitkan dengan Keterbatasan Manusia dalam Memprediksi Masa Depan dalam Perspektif Al-Qur`an

Risk portfolio on modern finance has become increasingly technical, requiring the use of sophisticated mathematical tools in both research and practice. Since companies cannot insure themselves completely against risk, as human incompetence in predicting the future precisely that written in Al-Quran surah Luqman verse 34, they have to manage it to yield an optimal portfolio. The objective here is to minimize the variance among all portfolios, or alternatively, to maximize expected return among all portfolios that has at least a certain expected return. Furthermore, this study focuses on optimizing risk portfolio so called Markowitz MVO (Mean-Variance Optimization). Some theoretical frameworks for analysis are arithmetic mean, geometric mean, variance, covariance, linear programming, and quadratic programming. Moreover, finding a minimum variance portfolio produces a convex quadratic programming, that is minimizing the objective function ðð¥with constraintsð ð 𥠥 ðandð´ð¥ = ð. The outcome of this research is the solution of optimal risk portofolio in some investments that could be finished smoothly using MATLAB R2007b software together with its graphic analysis

Search for heavy resonances decaying to two Higgs bosons in final states containing four b quarks

A search is presented for narrow heavy resonances X decaying into pairs of Higgs bosons (H) in proton-proton collisions collected by the CMS experiment at the LHC at root s = 8 TeV. The data correspond to an integrated luminosity of 19.7 fb(-1). The search considers HH resonances with masses between 1 and 3 TeV, having final states of two b quark pairs. Each Higgs boson is produced with large momentum, and the hadronization products of the pair of b quarks can usually be reconstructed as single large jets. The background from multijet and t (t) over bar events is significantly reduced by applying requirements related to the flavor of the jet, its mass, and its substructure. The signal would be identified as a peak on top of the dijet invariant mass spectrum of the remaining background events. No evidence is observed for such a signal. Upper limits obtained at 95 confidence level for the product of the production cross section and branching fraction sigma(gg -> X) B(X -> HH -> b (b) over barb (b) over bar) range from 10 to 1.5 fb for the mass of X from 1.15 to 2.0 TeV, significantly extending previous searches. For a warped extra dimension theory with amass scale Lambda(R) = 1 TeV, the data exclude radion scalar masses between 1.15 and 1.55 TeV

Search for supersymmetry in events with one lepton and multiple jets in proton-proton collisions at root s=13 TeV

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