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Currently, one of the most significant and rapidly growing unmet medical challenges is the treatment of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). This challenge encompasses the imperative development of efficacious therapeutic agents and overcoming the intricacies of the blood-brain barrier for successful drug delivery. Here we focus on the delivery aspect with particular emphasis on cell-penetrating peptides (CPPs), widely used in basic and translational research as they enhance drug delivery to challenging targets such as tissue and cellular compartments and thus increase therapeutic efficacy. The combination of CPPs with nanomaterials such as nanoparticles (NPs) improves the performance, accuracy, and stability of drug delivery and enables higher drug loads. Our review presents and discusses research that utilizes CPPs, either alone or in conjugation with NPs, to mitigate the pathogenic effects of neurodegenerative diseases with particular reference to AD and PD.

Establishment of multiple novel mechanisms and applications of cell-penetrating peptides (CPP) has been demonstrated, leading to novel drug delivery systems. Here, I present a brief introduction to the CPP area together with the selected recent achievements in the delivery of nucleic acids.

The low bioavailability and high toxicity of plasmid DNA (pDNA)-based therapeutics pose challenges for their in vivo application. Extracellular vesicles (EVs) have great potential to overcome these limitations, as they are biocompatible native cargo carriers. Various methods for loading pDNA into EVs, including electroporation, sonication, and co-incubation, have been previously investigated, but their success has been questionable. In this study, we report a unique method for loading EVs with pDNA through transient transfection using cell-penetrating peptides (CPPs). With this method, we found a 104-fold increase in the expression levels of the luciferase reporter protein in recipient cells compared to the untreated cells. These data point to the high transfection efficacy and bioavailability of the delivered encapsulated nucleic acid. Furthermore, the in vivo experimental data indicate that the use of pDNA-loaded EVs as native delivery vehicles reduces the toxic effects associated with traditional nucleic acid (NA) delivery and treatment.

The progress that has been made in computer science positioned in silico studies as an important and well-recognized methodology in the drug discovery and development process. It has numerous advantages in terms of costs and also plays a huge impact on the way the research is conducted since it can limit the use of animal models leading to more sustainable research. Currently, human trials are already being partly replaced by in silico trials. EMA and FDA are both endorsing these studies and have been providing webinars and guidance to support them. For instance, PBPK modeling studies are being used to gather data on drug interactions with other drugs and are also being used to support clinical and regulatory requirements for the pediatric population, pregnant women, and personalized medicine. This trend evokes the need to understand the role of in silico studies in vaccines, considering the importance that these products achieved during the pandemic and their promising hope in oncology. Vaccines are safer than other current oncology treatments. There is a huge variety of strategies for developing a cancer vaccine, and some of the points that should be considered when designing the vaccine technology are the following: delivery platforms (peptides, lipid-based carriers, polymers, dendritic cells, viral vectors, etc.), adjuvants (to boost and promote inflammation at the delivery site, facilitating immune cell recruitment and activation), choice of the targeted antigen, the timing of vaccination, the manipulation of the tumor environment, and the combination with other treatments that might cause additive or even synergistic anti-tumor effects. These and many other points should be put together to outline the best vaccine design. The aim of this article is to perform a review and comprehensive analysis of the role of in silico studies to support the development of and design of vaccines in the field of oncology and infectious diseases. The authors intend to perform a

Cell penetrating peptides (CPPs) can be broadly defined as relatively short synthetic, protein derived or chimeric peptides. Their most remarkable property is their ability to cross cell barriers and facilitate the translocation of cargo, such as drugs, nucleic acids, peptides, small molecules, dyes, and many others across the plasma membrane. Over the years there have been several approaches used, adapted, and developed for the evaluation of CPP efficacies as delivery systems, with the fluorophore attachment as the most widely used approach. It has become progressively evident, that the evaluation method, in order to lead to successful outcome, should concede with the specialties of the delivery. For characterization and assessment of CPP-cargo a combination of research tools of chemistry, physics, molecular biology, engineering, and other fields have been applied. In this review, we summarize the diverse, in silico, in vitro and in vivo approaches used for evaluation and characterization of CPP-based cargo delivery systems.

In this introductory chapter, we first define cell-penetrating peptides (CPPs), give short overview of CPP history and discuss several aspects of CPP classification. Next section is devoted to the mechanism of CPP penetration into the cells, where direct and endocytic internalization of CPP is explained. Kinetics of internalization is discussed more extensively, since this topic is not discussed in other chapters of this book. At the end of this section some features of the thermodynamics of CPP interaction with the membrane is also presented. Finally, we present different cargoes that can be transferred into the cells by CPPs and briefly discuss the effect of cargo on the rate and efficiency of penetration into the cells.

Development of nucleic acid delivery systems requires the use of adequate tissue analysis methods, especially when aiming tissue-targeted drug delivery. In this chapter, a protocol is presented for analyzing a reporter signal from the lung tissue. Because lung is an important target tissue from the clinical point of view, yet represents a challenge from the histological point of view, this protocol can be used in any lung-targeting drug delivery project.

The efficacy of transfection reagents and nanoparticles is often assessed by measuring levels of expressed reporter protein. Fluorescence and luminescence based assays provide sensitive, quantifiable and repeatable approaches. The genes expressing reporter protein can be integrated into the cells to create stable reporter cell lines or can be expressed from a transfected plasmid. Green fluorescent protein, luciferase, and secreted alkaline phosphatase are well-established reporters with versatile applications. Monitoring changes in live cells during and after transfection offer opportunities to reveal related mechanisms, efficacy, and bottlenecks of transfection. In this chapter, we describe the experimental setup and considerations for in vitro screening of delivery vectors. This can further be extended to measurements in reporter cell lines.

Cell-penetrating peptides (CPPs) are a promising non-viral vector for gene and drug delivery. CPPs exhibit high cell transfection, and are biocompatible. They can be also conjugated with organic and inorganic nanomaterials, such as magnetic nanoparticles (MNPs), graphene oxide (GO), metal-organic frameworks (MOFs), and chitosan. Nanomaterials offered a high specific surface area and provided relatively straightforward methods to be modified with biomolecules including CPPs and oligonucleotides (ONs). Novel nanomaterials conjugates with CPP/ONs complexes are therefore of interest for cell transfection with high efficiency. In this chapter, we described a summary of the non-viral vectors consisting of CPPs and nanomaterials. The book chapter also included a protocol to generate hybrid biomaterials consisting of CPPs and nanoparticles (NPs) for the delivery of oligonucleotides. The conjugation of NPs with CPPs serves as an effective platform for gene therapy with high cell transfection efficiency. The protocol is simple, offers high cell transfection compared to the CPPs-ONs complexes, and can be used for further improvements using external stimuli.

The efficacy of nanoparticle drugs necessitates the high bioactivity of constituents, but the distribution of the nanoparticles in organisms is mostly determined by their physical properties. Therefore, generation of stable particles with strictly defined characteristics is highly essential. Here we describe a formulation protocol of stable and homogenous CPP/pDNA nanoparticles for in vivo applications.

Mitochondria represent an important drug target for many phatology, including neurodegeneration, metabolic disease, heart failure, ischemia-reperfusion injury, and cancer. Mitochondrial dysfunctions are caused by mutation in mitochondrial DNA or in nuclear genes encoding mitochondrial proteins. Cell-penetrating peptides (CPPs) have been employed to overcome biological barriers, target this organelle, and therapeuticaly restore mitochondrial functions. Here, we describe recent methods used to deliver oligonucleotides targeting mitochondrial protein by using mitochondrial penetrating peptides. In particular, we highlight recent advances of formulated peptides/oligonucleotides nanocomplexes as a proof-of-principle for pharmaceutical form of peptide-based therapeutics.

Gene editing is increasing its popularity day by day especially as an essential tool for the research. It is based on two recombination mechanisms in mammalian cells: nonhomologous end-joining (NHEJ) and homology-directed repair (HDR). The first one can be used to silence a specific gene or a portion of it and the second one to insert new DNA, in presence of a donor template, in a targeted position in the genome. In order to exploit one of these two mechanisms, three major targeted nucleases have been developed: zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein). The last one seems to be the most promising tool among the others for gene editing. By using the properties and versatility of the Cell Penetrating Peptide (CPP) PepFect14, we developed a protocol to deliver a plasmid encoding for CRISPR-Cas9 and Green Fluorescent Protein (GFP) in BHM cell line expressing luciferase (Bomirsky Hamster Melanoma pLuc). Aiming to knocking down the luciferase gene in the cell line and to expressing GFP. Having two fast and easy read-outs of the plasmid’s activity at the same time. Furthermore, by labeling the CRISPR plasmid with Cy5 it is possible to have a visual confirmation of the cellular uptake of the pDNA/CPP complex, via fluorescent microscopy, as described.

PepFect14 is a cell-penetrating peptide (CPP) derived from stearylated transportan-10 (strearil-TP10) with which it shares the stearic acid residue on C′ terminus and the amino acid sequence except for lysines that in PepFect14 are substituted with ornithines. Being non-proteinogenic amino acids, ornithines make PepFect14 less sensitive to serum proteases and due to its positive charges the CPP can form complexes with negatively charged cargos, such as splice correcting oligonucleotides (SCOs), plasmid DNA (pDNA), and proteins. It has been reported that PepFect14/SCO complexes enter the cells mainly through endocytosis, in particular: macopinocitosys and caveolae-mediated endocytosis through the interaction with two receptors of the scavenger receptors class A family (SCARAs). PepFect14 and its complexes trigger the chaperone-mediated autophagy response involving the heat shock protein family (HSP70) whose inhibition leads to an increase of PepFect14 transfection efficacy. Exploiting the interaction between HSP70 and PepFect14 and their ability to form nanoparticle. HSP70 has been delivered in Bomirsky Hamster Melanoma cells (BHM) using PepFect14 of which a protocol is described at the end of this chapter.

Biophysical studies have a very high impact on the understanding of internalization, molecular mechanisms, interactions, and localization of CPPs and CPP/cargo conjugates in live cells or in vivo. Biophysical studies are often first carried out in test-tube set-ups or in vitro, leading to the complicated in vivo systems. This review describes recent studies of CPP internalization, mechanisms, and localization. The multiple methods in these studies reveal different novel and important aspects and define the rules for CPP mechanisms, hopefully leading to their improved applicability to novel and safe therapies.

Introduction Cell-penetrating peptide (CPP) technologies represent an important strategy to address drug delivery to specific intracellular compartments by covalent conjugation to targeting sequences, potentially enabling strategies to combat most diseases. Areas covered This updated review article provides an overview of current intracellular organelle targeting by CPP. The targeting strategies of CPP and CPP/cargo complexes to specific cells or intracellular organelles are summarized, and the review provides an update on the current data for their pharmacological and therapeutical applications. Expert opinion Targeted drug delivery is moving from the level of tissue or specific pathogenic cell to the level of specific organelle that is the target of the drug, an important aspect in drug design and development. Organelle-targeted drug delivery results in improved efficacy, ability to control mode of action, reduction of undesired toxicities and side effects, and the possibility to overcome drug resistance mechanisms.

This chapter describes the use of cell-penetrating peptides (CPPs) as carriers for transepithelial delivery of therapeutic peptides. Assessment of transepithelial peptide permeation and the mechanisms of action that permeability enhancing drug carriers exert on the epithelium requires subtle sample preparation and analysis by orthogonal methods. Here, the preparation and use of CPP-insulin physical mixture samples including the quantification of insulin by enzyme-linked immunosorbent assay (ELISA) is described. In addition, effects of CPPs on the epithelium and its barrier properties immediately upon exposure and after a recovery period are evaluated by epithelial cell viability, transepithelial electrical resistance, immunostaining of the tight junction associated zonula occludens (ZO-1) protein, and actin cytoskeleton staining.