Synthesis of Cell-Penetrating Peptide Coated Silica Nanoparticles and Their Physicochemical and Biological Characterization.

The surface decoration of nanoparticles with cell-penetrating peptides (CPPs) represents a common technique for intracellular delivery of nanotherapeutics. Conjugate formation can be performed via covalent or non-covalent strategies. Here, we describe on the synthesis of silica nanoparticles, a well-known inorganic drug delivery vehicle type, and their surface modification with cell-penetrating peptides using sC18 and derivatives thereof. Moreover, physicochemical as well as biological characterization methods, including cellular uptake measurements, of particle-peptide conjugates are described.

Cell-permeable CaaX-peptides affect K-Ras downstream signaling and promote cell death in cancer cells.

Cysteine prenylation is a post-translational modification that is used by nature to control crucial biological functions of proteins, such as membrane trafficking, signal transduction, and apoptosis. It mainly occurs in eukaryotic proteins at a C-terminal CaaX box and is mediated by prenyltransferases. Since the discovery of prenylated proteins, various tools have been developed to study the mechanisms of prenyltransferases, as well as to visualize and to identify prenylated proteins. Herein, we introduce cell-permeable peptides bearing a C-terminal CaaX motif based on Ras sequences. We demonstrate that intracellular accumulation of those peptides in different cells is controlled by the presence of their CaaX motif and that they specifically interact with intracellular prenyltransferases. As proof of concept, we further highlight their utilization to alter downstream signaling of Ras proteins, particularly of K-Ras-4B, in pancreatic cancer cells. Application of this strategy holds great promise to better understand and regulate post-translational cysteine prenylation.

Interdependence of charge and secondary structure on cellular uptake of cell penetrating peptide functionalized silica nanoparticles.

The capability of cell-penetrating peptides (CPPs) to enable translocation of cargos across biological barriers shows promising pharmaceutical potential for the transport of drug molecules, as well as nanomaterials, into cells. Herein, we report on the optimization of a CPP, namely sC18, in terms of its translocation efficiency and investigate new CPPs regarding their interaction with silica nanoparticles (NPs). First, alanine scanning of sC18 yielded 16 cationic peptides from which two were selected for further studies. Whereas in the first case, a higher positive net charge and enhanced amphipathicity resulted in significantly higher internalization rates than sC18, the second one demonstrated reduced cellular uptake efficiencies and served as a control. We then attached these CPPs to silica nanoparticles of different sizes (50, 150 and 300 nm) via electrostatic interactions and could demonstrate that the secondary alpha-helical structure of the peptides was preserved. Following this, cellular uptake studies using HeLa cells showed that the tested CPP-NPs were successfully translocated into HeLa cells in a size-dependent manner. Moreover, depending on the CPP used, we realized differences in translocation efficiency, which were similar to what we had observed for the free peptides. All in all, we highlight the high potential of sequential fine-tuning of CPPs and provide novel insights into their interplay with inorganic biologically benign nanoparticles. Given the high cellular permeability of CPPs and their ability to translocate into a wide spectrum of cell types, our studies may stimulate future research of CPPs with inorganic nanocarrier surfaces.

Selective Capture and Purification of MicroRNAs and Intracellular Proteins through Antisense-vectorized Magnetic Nanobeads.

MicroRNAs (miRNAs) are small non-coding nucleotides playing a crucial role in posttranscriptional expression and regulation of target genes in nearly all kinds of cells. In this study, we demonstrate a reliable and efficient capture and purification of miRNAs and intracellular proteins using magnetic nanoparticles functionalized with antisense oligonucleotides. For this purpose, a tumor suppressor miRNA (miR-198), deregulated in several human cancer types, was chosen as the model oligonucleotide. Magnetite nanoparticles carrying the complementary sequence of miR-198 (miR-198 antisense) on their surface were delivered into cells and subsequently used for the extracellular transport of miRNA and proteins. The successful capture of miR-198 was demonstrated by isolating RNA from magnetic nanoparticles followed by real-time PCR quantification. Our experimental data showed that antisense-coated particles captured 5-fold higher amounts of miR-198 when compared to the control nanoparticles. Moreover, several proteins that could play a significant role in miR-198 biogenesis were found attached to miR-198 conjugated nanoparticles and analyzed by mass spectrometry. Our findings demonstrate that a purpose-driven vectorization of magnetic nanobeads with target-specific recognition ligands is highly efficient in selectively transporting miRNA and disease-relevant proteins out of cells and could become a reliable and useful tool for future diagnostic, therapeutic and analytical applications.

Recent advances of anti-cancer therapies including the use of cell-penetrating peptides.

Cancer is one of the major growing public health problems making the development of new anti-cancer treatment strategies still compulsory. Conventionally used chemotherapies are quite often associated with severe side effects. One reason is limited cell-permeability of the used drugs resulting in only poor overall bioavailability. During the last thirty years, cell-penetrating peptides (CPPs) have extensively been studied as efficient vehicles for several classes of cargos, and the development of novel therapeutic applications including CPPs has gained a major role in current cancer research. This review summarizes recent trends in CPP-mediated cargo delivery with a future impact on anti-cancer therapy.

Bifunctional peptide hybrids targeting the matrix of mitochondria.

The mitochondrial organelle is associated with many diseases, including diabetes, age-related neuro-degenerative diseases and cancer. Therefore, the effective delivery of drug molecules to mitochondria became increasingly important during the past years. Within this work, we designed and analyzed bifunctional hybrid peptides comprised of a mitochondrial targeting sequence (MTS) attached to a cell-penetrating peptide (CPP). Our results demonstrate that choice of the MTS must be carefully undertaken, since not every MTS that was selected was comparably capable to target mitochondria. In addition, we highlight the use of the CPP sC18 as necessary part of the hybrid construct, inducing not only cellular uptake, but likewise supporting sub-organelle uptake into the mitochondrial matrix. The herein designed cell-permeable mitochondrial targeting peptide was furthermore proven to enhance the intracellular uptake of the cytostatic drug chlorambucil, making it a powerful candidate for further studies in this important field.