Enhancing Cellular Uptake of Native Proteins through Bio-Orthogonal Conjugation with Chemically Synthesized Cell-Penetrating Peptides.

The potential for native proteins to serve as a platform for biocompatible, targeted, and personalized therapeutics in the context of genetic and metabolic disorders is vast. Nevertheless, their clinical application encounters challenges, particularly in overcoming biological barriers and addressing the complexities involved in engineering transmembrane permeability. This study is dedicated to the development of a multifunctional nanoentity in which a model therapeutic protein is covalently linked to a cell-penetrating peptide, NickFect 55, with the objective of enhancing its intracellular delivery. Successful binding of the nanoentity fragments was achieved through the utilization of an intein-mediated protein-trans splicing reaction. Our research demonstrates that the fully assembled nanoentity-containing protein was effectively internalized by the cells, underscoring the potential of this approach in overcoming barriers associated with protein-based therapeutics for the treatment of genetic disorders.

A Method for Using Cell-Penetrating Peptides for Loading Plasmid DNA into Secreted Extracellular Vesicles.

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.

Modification of the Linker Amino Acid in the Cell-Penetrating Peptide NickFect55 Leads to Enhanced pDNA Transfection for In Vivo Applications.

Despite numerous efforts over the last three decades, nucleic acid-based therapeutics still lack delivery platforms in the clinical stage. Cell-penetrating peptides (CPPs) may offer solutions as potential delivery vectors. We have previously shown that designing a "kinked" structure in the peptide backbone resulted in a CPP with efficient in vitro transfection properties. Further optimization of the charge distribution in the C-terminal part of the peptide led to potent in vivo activity with the resultant CPP NickFect55 (NF55). Currently, the impact of the linker amino acid was further investigated in the CPP NF55, with the aim to discover potential transfection reagents for in vivo application. Taking into account the expression of the delivered reporter in the lung tissue of mice, and the cell transfection in the human lung adenocarcinoma cell line, the new peptides NF55-Dap and NF55-Dab* have a high potential for delivering nucleic acid-based therapeutics to treat lung associated diseases, such as adenocarcinoma.

The Development of Cell-Penetrating Peptides for Efficient and Selective In Vivo Expression of mRNA in Spleen Tissue.

mRNA-based therapeutics are presently one of the nucleic acid-based therapeutics with a high potential for extraordinary success as preventive vaccines. Current applications with mRNA therapeutics rely on lipid nanoparticle (LNP) mediated delivery of nucleic acids. In order to achieve the transition from preventive to therapeutic vaccines, there is a challenge of delivering the mRNA into non-hepatic tissues, especially into lymphoid tissues such as the spleen and lymph nodes. In this work, we characterize new cell-penetrating peptides NF424 and NF436 that exhibit preferential delivery of mRNA into the spleen after a single i.v. injection, without the use of any active targeting mechanisms. We show that between the spleen, liver, and the lungs, >95% of mRNA expression arises in the spleen tissue and the majority of expression occurs in the dendritic cells. The cell-penetrating peptides NF424 and NF436 represent promising candidates for cancer immunotherapeutic applications with tumor antigens.

Aggregation Limiting Cell-Penetrating Peptides Derived from Protein Signal Sequences.

Alzheimer's disease (AD) is the most common neurodegenerative disease (ND) and the leading cause of dementia. It is characterized by non-linear, genetic-driven pathophysiological dynamics with high heterogeneity in the biological alterations and the causes of the disease. One of the hallmarks of the AD is the progression of plaques of aggregated amyloid-ß (Aß) or neurofibrillary tangles of Tau. Currently there is no efficient treatment for the AD. Nevertheless, several breakthroughs in revealing the mechanisms behind progression of the AD have led to the discovery of possible therapeutic targets. Some of these include the reduction in inflammation in the brain, and, although highly debated, limiting of the aggregation of the Aß. In this work we show that similarly to the Neural cell adhesion molecule 1 (NCAM1) signal sequence, other Aß interacting protein sequences, especially derived from Transthyretin, can be used successfully to reduce or target the amyloid aggregation/aggregates in vitro. The modified signal peptides with cell-penetrating properties reduce the Aß aggregation and are predicted to have anti-inflammatory properties. Furthermore, we show that by expressing the Aß-EGFP fusion protein, we can efficiently assess the potential for reduction in aggregation, and the CPP properties of peptides in mammalian cells.

Approaches for evaluation of novel CPP-based cargo delivery systems.

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.

Predicting Transiently Expressed Protein Yields: Comparison of Transfection Methods in CHO and HEK293.

Therapeutic proteins are currently at the apex of innovation in pharmaceutical medicine. However, their industrial production is technically challenging and improved methods for transient transfection of mammalian cell cultures are necessary. We aimed to find a fast, microliter-scale transfection assay that allows the prediction of protein expression in the transient production settings. We used an array of lipid, polymeric and cell-penetrating peptide transfection reagents, and compared their performance in various high throughput transfection assays to their performance in protein (antibody) expression in professional protein-producer cell lines. First, we show that some of the most frequently used microliter-scale transfection efficacy assays fail to predict performance in the protein production in milliliter and liter scale settings. We found that CHO suspension culture post-transfection EGFP(+) population and SEAP quantitation correlate with large-scale protein production, whereas the adhesion culture assays and transfection of pLuc are non-predictive. Second, we demonstrated that cell-penetrating peptide-based transfection achieves significantly higher protein yields compared to PEI and lipoplex methods in both CHO and HEK293 producer cell lines. In this work we demonstrate a CPP-based transient protein expression approach that significantly outperformed the current industry standard workhorse method of PEI.

Endpoint and Kinetic Approaches for Assessing Transfection Efficacy in Mammalian Cell Culture.

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 Peptide and siRNA-Mediated Therapeutic Effects on Endometriosis and Cancer In Vitro Models.

Gene therapy is a powerful tool for the development of new treatment strategies for various conditions, by aiming to transport biologically active nucleic acids into diseased cells. To achieve that goal, we used highly potential delivery vectors, cell-penetrating peptides (CPPs), as oligonucleotide carriers for the development of a therapeutic approach for endometriosis and cancer. Despite marked differences, both of these conditions still exhibit similarities, like excessive, uncoordinated, and autonomous cellular proliferation and invasion, accompanied by overlapping gene expression patterns. Thus, in the current study, we investigated the therapeutic effects of CPP and siRNA nanoparticles using in vitro models of benign endometriosis and malignant glioblastoma. We demonstrated that CPPs PepFect6 and NickFect70 are highly effective in transfecting cell lines, primary cell cultures, and three-dimensional spheroids. CPP nanoparticles are capable of inducing siRNA-specific knockdown of therapeutic genes, ribonucleotide reductase subunit M2 (RRM2), and vascular endothelial growth factor (VEGF), which results in the reduction of in vitro cellular proliferation, invasion, and migration. In addition, we proved that it is possible to achieve synergistic suppression of endometriosis cellular proliferation and invasion by combining gene therapy and hormonal treatment approaches by co-administering CPP/siRNA nanoparticles together with the endometriosis-drug danazol. We suggest a novel target, RRM2, for endometriosis therapy and as a proof-of-concept, we propose a CPP-mediated gene therapy approach for endometriosis and cancer.

Cell-Penetrating Peptides Predicted From CASC3, AKIP1, and AHRR Proteins.

Peptides can be used as research tools and for diagnostic or therapeutic applications. Peptides, alongside small molecules and antibodies, are used and are gaining further interest as protein-protein interaction (PPI) modulators. Peptides have high target specificity and high affinity, but, unlike small molecule modulators, they are not able to cross the cell membranes to reach their intracellular targets. To overcome this limitation, the special property of the cell-penetrating peptides (CPPs) could benefit their cause. CPPs are a class of peptides that can enter the cells and with them also deliver the attached cargoes. Today, with the advancement of in silico prediction tools and the availability of protein databases, designing new and multifunctional peptides that are able to reach intracellular targets and inhibit certain cellular processes in a very specific manner is reachable. Although there are several efficient CPP sequences already known, the discovery of new CPPs is crucial for the development of efficient delivery methods for both biotechnological and therapeutic applications. In this work, we chose 10 human nuclear proteins from which we predicted new potential CPP sequences by using three different CPP predictors: cell-penetrating peptide prediction tool, CellPPD, and SkipCPP-Pred. From each protein, one predicted CPP sequence was synthesized and its internalization into cells was assessed. Out of the tested sequences, three peptides displayed features characteristic to CPPs. These peptides and also the predicted peptide sequences could be used to design and modify new CPPs. In this work, we show that we can use protein sequences as input for generating new peptides with cell internalization properties. Three new CPPs, AHRR8-24, CASC3251-264, and AKIP127-37, can be further used for the delivery of other cargoes or designed into multifunctional peptides with capability of internalizing cells.

Approaches for the discovery of new cell-penetrating peptides.

Introduction: The capability of cell-penetrating peptides (CPP), also known as protein transduction domains (PTD), to enter into cells possibly with an attached cargo, makes their application as delivery vectors or as direct therapeutics compelling. They are generally biocompatible, nontoxic, and easy to synthesize and modify. Three decades after the discovery of the first CPPs, ~2,000 CPP sequences have been identified, and many more predicted. Nevertheless, the field has a strong commitment to authenticate new, more efficient, and specific CPPs.Areas covered: Although a scattering of CPPs have been found by chance, various systematic approaches have been developed and refined over the years to directly aid the identification and depiction of new peptide-based delivery vectors or therapeutics. Here, the authors give an overview of CPPs, and review various approaches of discovering new ones. An emphasis is placed on in silico methods, as these have advanced rapidly in recent years.Expert opinion: Although there are many known CPPs, there is a need to find more efficient and specific CPPs. Several approaches are used to identify such sequences. The success of these approaches depends on the advancement of others and the successful prediction of CPP sequences relies on experimental data.

Amyloid-like Self-Assembly of a Hydrophobic Cell-Penetrating Peptide and Its Use as a Carrier for Nucleic Acids.

Cell-penetrating peptides (CPPs) are a topical subject potentially exploitable for creating nanotherapeutics for the delivery of bioactive loads. These compounds are often classified into three major categories according to their physicochemical characteristics: cationic, amphiphilic, and hydrophobic. Among them, the group of hydrophobic CPPs has received increasing attention in recent years due to toxicity concerns posed by highly cationic CPPs. The hexapeptide PFVYLI (P, proline; F, phenylalanine; V, valine; Y, tyrosine; L, leucine; and I, isoleucine), a fragment derived from the C-terminal portion of α1-antitrypsin, is a prototypal example of hydrophobic CPP. This sequence shows reduced cytotoxicity and a capacity of nuclear localization, and its small size readily hints at its suitability as a building block to construct nanostructured materials. In this study, we examine the self-assembling properties of PFVYLI and investigate its ability to form noncovalent complexes with nucleic acids. By using a combination of biophysical tools including synchrotron small-angle X-ray scattering and atomic force microscopy-based infrared spectroscopy, we discovered that this CPP self-assembles into discrete nanofibrils with remarkable amyloidogenic features. Over the course of days, these fibrils coalesce into rodlike crystals that easily reach the micrometer range. Despite lacking cationic residues in the composition, PFVYLI forms noncovalent complexes with nucleic acids that retain ß-sheet pairing found in amyloid aggregates. In vitro vectorization experiments performed with double-stranded DNA fragments indicate that complexes promote the internalization of nucleic acids, revealing that tropism toward cell membranes is preserved upon complexation. On the other hand, transfection assays with splice-correction oligonucleotides (SCOs) for luciferase expression show limited bioactivity across a narrow concentration window, suggesting that the propensity to form amyloidogenic aggregates may trigger endosomal entrapment. We anticipate that the findings presented here open perspectives for using this archetypical hydrophobic CPP in the fabrication of nanostructured scaffolds, which potentially integrate properties of amyloids and translocation capabilities of CPPs.

Characterization of Peptide-Oligonucleotide Complexes Using Electron Microscopy, Dynamic Light Scattering, and Protease Resistance Assay.

Cationic peptides designed for cellular delivery of nucleic acid molecules form noncovalent nanocomplexes with negatively charged oligonucleotides (ON). The electrostatically associated complexes are further compacted by hydrophobic interactions yielding nanoparticles (NP) of homogeneous shape and size that are efficiently taken up by cells. The shape and size of NP often correlate with the biological activity of delivered ON inside cells; and the stability and accessibility of NP in biological fluids govern its circulation in organism and the cellular uptake. Therefore, here we provide protocols for characterizing the shape and size and surface charge of peptide/ON NP by negative staining transmission electron microscopy (TEM) and dynamic light scattering (DLS) respectively, and analysis of NP stability against proteolytic degradation.

Enhancement of siRNA transfection by the optimization of fatty acid length and histidine content in the CPP.

Extracellular synthetic nucleic acids, such as siRNAs, are unable to reach their intended targets efficiently. Therefore, delivery methods such as cell-penetrating peptides (CPP), which increase their transport, could enhance the potency of siRNA as therapeutic agents. The CPP NickFect55 (NF55) is an efficient peptide-based delivery vector, which has been previously used to deliver plasmid DNA into cells in vivo. To achieve higher intracellular delivery and bioactivity from the delivered cargo, we designed a series of histidine-containing peptides by optimizing pH-sensitivity, net charge, hydrophobicity, and charge distribution in the sequence of the CPP NF55. In the current work, we have applied a strategy where we have replaced amino acids in the C-terminus of the peptide in order to distribute hydrophobic and hydrophilic amino acids into distinct regions along the alpha-helical projection, including histidine amino acids into the sequence at the N-terminus, and optimizing the N-terminal fatty acid modification to suit the specific peptide sequence. We tested the CPPs based on the transfection efficacy, CPP/siRNA complex stability, and the properties of the CPPs, such as hemolytic activity, buffering capability and cell toxicity. As a result, we have introduced a new peptide with a completely redesigned N-terminus that displays adaptive response to its physical environment. This peptide - NickFect70 (NF70) - efficiently condenses siRNA, protects the cargo against degradation and effectively mediates target gene knockdown both in mammalian cell culture and in vivo, in a mouse model.

Tumor gene therapy by systemic delivery of plasmid DNA with cell-penetrating peptides.

Gene therapy is a prospective strategy for treating cancer. However, finding efficient and tumor-specific gene delivery vectors remains an issue. Tumor responsive cell-penetrating peptide (CPP) PepFect144 (PF144) has previously been shown to deliver reporter gene encoding plasmid DNA specifically into tumors upon systemic administration, but its capability to reduce tumor growth has not yet been evaluated. Here, we study the potential of PF144-based anti-angiogenic gene delivery to inhibit tumor growth by silencing vascular endothelial growth factor (VEGF) expression in tumors. This approach led to the inhibition of tumor growth in both the HT1080 fibrosarcoma model and orthotopic 4T1 breast tumor model. We additionally saw that the addition of αvß3 integrin targeting did not further improve the tumor sensitive CPPs. Our results suggest that activatable cell-penetrating peptide PF144 is a promising nonviral plasmid DNA delivery vector for cancer treatment.