Personalized anti-cancer medicine is boosted by the recent development of molecular diagnostics and molecularly targeted drugs requiring rapid and efficient ligation routes. Here, we present a novel approach to synthetize a conjugate able to act simultaneously as an imaging and as a chemotherapeutic agent by coupling functional peptides employing solid phase peptide synthesis technologies. Development and the first synthesis of a fluorescent dye with similarity in the polymethine part of the Cy7 molecule whose indolenine-N residues were substituted with a propylene linker are described. Methylating agent temozolomide is functionalized with a tetrazine as a diene component whereas Cy7-cell penetrating peptide conjugate acts as a dienophilic reaction partner for the inverse Diels-Alder click chemistry-mediated ligation route yielding a theranostic conjugate, 3-mercapto-propionic-cyclohexenyl-Cy7-bis-temozolomide-bromide-cell penetrating peptide. Synthesis route described here may facilitate targeted delivery of the therapeutic compound to achieve sufficient local concentrations at the target site or tissue. Its versatility allows a choice of adequate imaging tags applicable in e.g. PET, SPECT, CT, near-infrared imaging, and therapeutic substances including cytotoxic agents. Imaging tags and therapeutics may be simultaneously bound to the conjugate applying click chemistry. Theranostic compound presented here offers a solid basis for a further improvement of cancer management in a precise, patient-specific manner.

Disastrous effects of tobacco consumption on health remain in the focus of preclinical and clinical research. Exposures to tobacco smoke are closely connected to acute and chronic smoke lung dysfunctions and the effects are not restricted to airway inflammation processes. The facts that smoke exposure result in aberrant gene expression and changes in cellular phenotype resulting in clinical patterns. Here we pyrolyzed constituents of tobacco smoke, induced a stress response in human embryonic lung (HEL) cells, which respond with an altered expression of a broad spectrum of genes. We systematically analyzed the genetic expression, using the microarray-technology. After exposure of HEL cells to alkaline or acidic extracts of pyrolyzed smoke, already 2h after exposition the most affected genes (HMOX1, CYP1B1, ID3, and ID2) were rapidly up-regulated, whereas after 24 hours the genes were almost down-regulated. Using DAVID bioinformatics we detected annotation clusters with significant enrichment scores allowing insight into pharmacological processes and molecular functions. In the alkaline and acidic probes in a ratio 24 h versus 2h we identified annotation clusters with enrichment scores (between 5.77 and 2.89) representing upregulated genes. A negative transcription control leads to the conclusion that a "loss of function" can be possible. STRING tools give insight into the functional network of the gene products of affected genes. Noticeable is the fact that the detected genes with late response encode predicted proteins with unknown function (LOC100134504, LOC645157, LOC653156). With Blast2GO we generated and analysis and graphs of reliable functions.

Advances in imaging diagnostics using magnetic resonance tomography (MRT), positron emission tomography (PET) and fluorescence imaging including near infrared (NIR) imaging methods are facilitated by constant improvement of the concepts of peptide synthesis. Feasible patient-specific theranostic platforms in the personalized medicine are particularly dependent on efficient and clinically applicable peptide constructs. The role of peptides in the interrelations between the structure and function of proteins is widely investigated, especially by using computer-assisted methods. Nowadays the solid phase synthesis (SPPS) chemistry emerges as a key technology and is considered as a promising methodology to design peptides for the investigation of molecular pharmacological processes at the transcriptional level. SPPS syntheses could be carried out in core facilities producing peptides for large-scale scientific implementations as presented here.

The highly organized DNA architecture inside of the nuclei of cells is accepted in the scientific world. In the human genome about 3 billion nucleotides are organized as chromatin in the cell nucleus. In general, they are involved in gene regulation and transcription by histone modification. Small chromosomes are localized in a central nuclear position whereas the large chromosomes are peripherally positioned. In our experiments we inserted fusion proteins consisting of a component of the nuclear lamina (lamin B1) and also histone H2A, both combined with the light inducible fluorescence protein KillerRed (KRED). After activation, KRED generates reactive oxygen species (ROS) producing toxic effects and may cause cell death. We analyzed the spatial damage distribution in the chromatin after illumination of the cells with visible light. The extent of DNA damage was strongly dependent on its localization inside of nuclei. The ROS activity allowed to gain information about the location of genes and their functions via sequencing and data base analysis of the double strand breaks of the isolated DNA. A connection between the damaged gene sequences and some diseases was found.

Here, we review the development of prospective image processing systems in molecular diagnostics and of pharmacologically active ingredients for patient-specific therapeutic approaches. These projects require not only high demands on quality, safety, and specificity but also, rapid, efficient and irreversible ligation routes during the synthesis of future pharmaceuticals. The Diels-Alder ligation reaction with inverse electron demand (DAR(inv)) is an eligible technology not restricted to medical applications, but valuable in selective modification by functionalization of polymers, organic and inorganic surfaces and micro arrays. Additionally, the DARinv technology is considered as an attractive strategy-platform for efficient syntheses of promising pharmacologically active components and derivatives of natural molecules with an optimized therapeutic index. We like to encourage scientists working with the brilliant concept of Sharpless. s "Click chemistry", to intensify their research with this valuable DARinv technology able to open the door for regioselective, stereospecific, and bioorthogonal exigent syntheses of substances of highest quality inconceivable so far.

In contrast to the problematic health and economic effects of acute and chronic smoke exposure on lung function and airway inflammation, there are still few data dealing with the effects of smoking. Smoke exposure can result in aberrant cell growth. In our experiments, pyrolyzed components of cigarettes have been shown to induce a strong stress response in cultured cells. We used human embryonic lung (HEL) cells, which respond with an altered expression of a broad spectrum of genes. Therefore we performed a systematic analysis of the genetic expression behaviour, using the established whole genome microarray-technology which should be able to reveal the cellular effects. With these data we aim to generate a qualitative spectrum of cellular stress response activity. It is noticeable that after cells. exposure to pyrolyzed tobacco smoke components the products of the most affected genes, e.g. ID1, inhibitor of DNA binding, are up-regulated as a rapid response after 2 h with a factor 3.8 and RPS2, ribosomal protein S2, is down regulated to nearly 50 % after 24 hours. In databases they are documented as still uncharacterized and hypothetical proteins. The DDIT4 gene, encoding the DNAdamage-inducible transcript 4, associated with regulation and development of DNA processes after damage by ionizing radiation and in p53 mediated apoptotic processes, is up-regulated. The exposure leads to a rapid cellular stress response of genes like induction of the ID1, ID2, and ID3 genes, located on different chromosomes, already after two hours. They interact normally with DNA binding proteins under heterodimer formation and are considered as negative regulators of transcription. After 24 hours, a return back to normal was not observed and the genes remained stably down-regulated. The suppression of the GADD45B gene which is involved in the cell cycle regulation and after DNA damage a cell cycle arrest is mediated by the gene product. The C14orf4 gene (IRF2BPL) suggests a bifunctional role in transcription control as a promotor. s activator as well as a repressor. Recent data indicate a prominent role of this gene transcript in the control of female reproductive function. On balance, the role of the predominant amount of affected genes is focused on cellular stress response and DNA metabolism.

The personalized medicine, also documented as “individualized medicine”, is an effective and therapeutic approach. It is designed to treat the disease of the individual patient whose precise differential gene expression profile is well known. The trend in the biomedical and biophysical research shows important consequences for the pharmaceutical drug and diagnostics research. It requires a high variability in the design and safety of target-specific pharmacologically active molecules and diagnostic components for imaging of metabolic processes. A key technology which may fulfill the highest demands during synthesis of these individual drugs and diagnostics is the solid phase synthesis which is congenial to automated manufacturing. Additionally the choice of tools like resins and reagents is pivotal to synthesize drugs and diagnostics in high quality and yields. Here we demonstrate the solid phase synthesis effects dependent on the choice of resin and of the deprotection agent.

Progress in genomics and proteomics attended to the door for better understanding the recent rapid expanding complex research field of metabolomics. This trend in biomedical research increasingly focuses to the development of patient-specific therapeutic approaches with higher efficiency and sustainability. Simultaneously undesired adverse reactions are avoided. In parallel, the development of molecules for molecular imaging is required not only for the imaging of morphological structures but also for the imaging of metabolic processes like the aberrant expression of the cysteine protease cathepsin B (CtsB) gene and the activity of the resulting product associated with metastasis and invasiveness of malign tumors. Finally the objective is to merge imaging and therapy at the same level. The design of molecules which fulfil these responsibilities is pivotal and requires proper chemical methodologies. In this context our modified solid phase peptide chemistry using temperature shifts during synthesis is considered as an appropriate technology. We generated highly variable conjugates which consist of molecules useful as diagnostically and therapeutically active molecules. As an example the modular PNA products with the complementary sequence to the CtsB mRNA and additionally with a cathepsin B cleavage site had been prepared as functional modules for distinction of cell lines with different CtsB gene expression. After ligation to the modular peptide-based BioShuttle carrier, which was utilized to facilitate the delivery of the functional modules into the cells' cytoplasm, the modules were scrutinized.

With the increase in molecular diagnostics and patient-specific therapeutic approaches, the delivery and targeting of imaging molecules and pharmacologically active agents gain increasing importance.The ideal delivery system does not exist yet.The realization of two features is indispensable: first, a locally high concentration of target-specific diagnostic and therapeutic molecules; second, the broad development of effective and safe carrier systems.Here we characterize the transport properties of the peptide-based BioShuttle transporter using FFM and CLSM methods.The modular design of BioShuttle-based formulations results in a multi-faceted field of applications, also as a theranostic tool.

Red fluorescent proteins can generate reactive oxygen species (ROS) if their fluorochrome is stimulated e.g. by visible light illumination. ROS compounds have very reactive, highly toxic properties leading to cell damage which results in cell killing. In this context, the toxicity of the various red fluorochromes KillerRed, DsRed2, mCherry, and mRFP expressed in Escherichia coli bacteria was tested after illumination with white light. The toxic effect was determined by measurement of the colony forming ability 24h after transfection and illumination. KillerRed was found to be the most harmful, followed by mRFP and DsRed2 while bacteria expressing mCherry and controls without fluorescent proteins survived after application of identical illumination doses. Their application and a possible bactericide role is discussed.

Innovative and personalized therapeutic approaches result from the identification and control of individual aberrantly expressed genes at the transcriptional and post-transcriptional level.Therefore, it is of high interest to establish diagnostic, therapeutic and theranostic strategies at these levels.In the present study, we used the Diels-Alder Reaction with inverse electron demand (DAR inv ) click chemistry to prepare a series of cyclic RGD-BioShuttle constructs.These constructs carry the near-infrared (NIR) imaging agent Cy7 and the chemotherapeutic agent temozolomide (TMZ).We evaluated their uptake by and their efficacy against integrin α v β 3 -expressing MCF7 human breast carcinoma cells.In addition, using a mouse phantom, we analyzed the suitability of this targeted theranostic agent for NIR optical imaging.We observed that the cyclic RGD-based carriers containing TMZ and/or Cy7 were effectively taken up by α v β 3 -expressing cells, that they were more effective than free TMZ in inducing cell death, and that they could be quantitatively visualized using NIR fluorescence imaging.Therefore, these targeted theranostic agents are considered to be highly suitable systems for improving disease diagnosis and therapy.

In the field of diagnostics manifold methodologies exist to image morphological structures of tissues. Magnetic Resonance Imaging (MRI) is considered as an excellent tool to investigate detailed anatomical informations. Additionally, the MR got the ability to image metabolic processes and can act in the high field MRI as a new intracellular contrast agent which based on the BioShuttle carrier variant GdSc2N@ C-80n called Gd-cluster@-BioShuttle. As a cargo PNA-building blocks like the amide's backbone were attached to the carrier module after functionalization with the Gd-cluster@ by the DAR(inv)-mediated "Click Chemistry". Here we demonstrate shortly the synthesis and we like to approach this research subject from different angles including the development of tools for better monitoring of therapeutic interventions.

Fluorescent proteins (FPs) are established tools for new applications, not-restricted to the cell biological research. They could also be ideal in surgery enhancing the precision to differentiate between the target tissue and the surrounding healthy tissue. FPs like the KillerRed (KRED), used here, can be activated by excitation with visible day-light for emitting active electrons which produce reactive oxygen species (ROS) resulting in photokilling processes. It is a given that the extent of the KRED's cell toxicity depends on its subcellular localization. Evidences are documented that the nuclear lamina as well as especially the chromatin are critical targets for KRED-mediated ROS-based DNA damaging. Here we investigated the damaging effects of the KRED protein fused to the nuclear lamina and to the histone H2A DNA-binding protein. We detected a frequency of DNA strand breaks, dependent first on the illumination time, and second on the spatial distance between the localization at the chromatin and the site of ROS production. As a consequence we could identify defined DNA bands with 200, 400 and (600) bps as most prominent degradation products, presumably representing an internucleosomal DNA cleavage induced by KRED. These findings are not restricted to the detection of programmed cell death processes in the therapeutic field like PDT, but they can also contribute to a better understanding of the structure-function relations in the epigenomic world.

In the near future personalized medicine with nucleic acids will play a key role in molecular diagnostics and therapy, which require new properties of the nucleic acids, like stability against enzymatic degradation. Here we demonstrate that the replacement of nucleobases with PNA by functional molecules harbouring either a dienophile or a diene reactivity is feasible and confers all new options for functionalization. These newly developed derivatives allow independent multi-ligations of multi-faceted components by use of the inverse Diels Alder technology. The high chemical stability and the ease of synthesis qualify these polyamide building blocks as favourites for intracellular delivery and targeting applications. This allows local drug concentrations sufficient for imaging and therapy and simultaneously a reduction of the application doses. It is important to point out that this technology is not restricted to ligation of medicament material; it is also a candidate to develop new and highly efficient active compounds for a “sustainable pharmacy”.