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.

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.

A Second Life for MAP, a Model Amphipathic Peptide.

Cell-penetrating peptides (CPP) have been shown to be efficient in the transport of cargoes into the cells, namely siRNA and DNA, proteins and peptides, and in some cases, small therapeutics. These peptides have emerged as a solution to increase drug concentrations in different tissues and various cell types, therefore having a relevant therapeutic relevance which led to clinical trials. One of them, MAP, is a model amphipathic peptide with an α-helical conformation and both hydrophilic and hydrophobic residues in opposite sides of the helix. It is composed of a mixture of alanines, leucines, and lysines (KLALKLALKALKAALKLA). The CPP MAP has the ability to translocate oligonucleotides, peptides and small proteins. However, taking advantage of its unique properties, in recent years innovative concepts were developed, such as in silico studies of modelling with receptors, coupling and repurposing drugs in the central nervous system and oncology, or involving the construction of dual-drug delivery systems using nanoparticles. In addition to designs of MAP-linked vehicles and strategies to achieve highly effective yet less toxic chemotherapy, this review will be focused on unique molecular structure and how it determines its cellular activity, and also intends to address the most recent and frankly motivating issues for the future.

Editorial on Special Issue "Precision Delivery of Drugs and Imaging Agents with Peptides".

Homing peptides and cell-penetrating peptides allow for systemic targeting of diseased tissues and/or efficient intracellular delivery of payloads [...].

Tissue Analysis of Lung-Targeted Delivery of siRNA and Plasmid DNA.

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.

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.

Design and Synthesis of a Peptide-Based Glioma-Targeted Drug Delivery Vector gHope2.

In the current chapter, we are detailing the synthesis path of a tumor-targeted, CPP-functionalized chemotherapeutic drug, as well as in vitro validation of the targeting and cell penetrating functionalities of the construct. The design of targeted drug delivery vehicle is based on a new glioma-specific homing peptide that has been conjugated to doxorubicin. Further functionalization with an 18-amino acid cell penetrating peptide pVEC was achieved, a CPP that was chosen because of its high cell penetrating efficacy and low toxicity. The three elements were combined into one drug delivery construct gHope2, and its tumor-homing and cell penetrating activity was demonstrated in human glioma cell line U87.

Status update in the use of cell-penetrating peptides for the delivery of macromolecular therapeutics.

INTRODUCTION: In this review, recent developments and applications with cell-penetrating peptides (CPP) are discussed. CPPs are widely used tools for the delivery of various macromolecular therapeutics, such as proteins and nucleic acids. AREAS COVERED: The current review focuses on recent important advances and reports that demonstrate high clinical and translational potential. Most important clinical developments have occurred with the CPP-drug conjugate approaches that target various protein-protein interactions, and these have been highlighted subsequently. Most of the applications are targeting cancer, but recently, noteworthy advances have taken place in the field of antisense oligonucleotides and muscular dystrophies, lung targeting, and trans-BBB targeting. EXPERT OPINION: Successful applications and clinical development with the drug conjugate approaches are discussed. On the other hand, the reasons of why the nanoparticle approaches are not as far in development are analyzed.

Effective lung-targeted RNAi in mice with peptide-based delivery of nucleic acid.

We have previously developed efficient peptide-based nucleic acid delivery vectors PF14 and NF55, where we have shown that these vectors preferentially transfect lung tissue upon systemic administration with the nucleic acid. In the current work, we have explored the utilization and potential of these vectors for the lung-targeted gene therapy. Accordingly, we assessed the efficacy of these peptides in (i) two different lung disease models - acute lung inflammation and asthma in mice and (ii) using two different nucleic acid cargos - siRNA and pDNA encoding shRNA. Using RNAi against cytokine TNFα, we showed efficient anti-inflammatory effects in both disease models and observed decreased disease symptoms. Our results highlight the potential of our transfection vectors for lung gene therapy.

Recent CPP-based applications in medicine.

Introduction: Cell-penetrating peptides (CPP) offer versatile tools for the field of drug delivery and development of macromolecular therapeutics. These tools have matured into applications that allow the use of proteins, nucleic acid, peptides, imaging agents, and low molecular weight drugs as therapeutic entities. Areas covered: The progress of the field is discussed in the current review, with the examples from a recent couple of years, in the utilization of CPPs in medicine. Specific focus is on the research articles that include applications that have direct translational value. Progress with protein mimicry and its recent leap in medicine is discussed with special attention, but also achievements with nanoformulation and drug targeting is presented. Expert opinion: The opinion section discusses some of the limitations and shortcomings, proposing the areas where more effort should be invested in order to increase the translational value of the current preclinical research.

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.

The future of peptides in cancer treatment.

Peptides hold great potential for the cancer therapy and diagnostics. In the current review, the main and most influential areas of peptide cancer therapeutics are overviewed. These include the development of anticancer peptides, use of peptides for drug delivery, and cancer targeting.

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.

Formulation of Stable and Homogeneous Cell-Penetrating Peptide NF55 Nanoparticles for Efficient Gene Delivery In Vivo.

Although advances in genomics and experimental gene therapy have opened new possibilities for treating otherwise incurable diseases, the transduction of nucleic acids into the cells and delivery in vivo remain challenging. The high molecular weight and anionic nature of nucleic acids require their packing into nanoparticles for the delivery. The efficacy of nanoparticle drugs necessitates the high bioactivity of constituents, but their distribution in organisms is mostly governed by the physical properties of nanoparticles, and therefore, generation of stable particles with strictly defined characteristics is highly essential. Using previously designed efficient cell-penetrating peptide NF55, we searched for strategies enabling control over the nanoparticle formation and properties to further improve transfection efficacy. The size of the NF55/pDNA nanoparticles correlates with the concentration of its constituents at the beginning of assembly, but characteristics of nanoparticles measured by DLS do not reliably predict the applicability of particles in in vivo studies. We introduce a new formulation approach called cryo-concentration, where we acquired stable and homogeneous nanoparticles for administration in vivo. The cryo-concentrated NF55/pDNA nanoparticles exhibit several advantages over standard formulation: They have long shelf-life and do not aggregate after reconstitution, have excellent stability against enzymatic degradation, and show significantly higher bioactivity in vivo.

Effective in vivo gene delivery with reduced toxicity, achieved by charge and fatty acid -modified cell penetrating peptide.

Non-viral gene delivery systems have gained considerable attention as a promising alternative to viral delivery to treat diseases associated with aberrant gene expression. However, regardless of extensive research, only a little is known about the parameters that underline in vivo use of the nanoparticle-based delivery vectors. The modest efficacy and low safety of non-viral delivery are the two central issues that need to be addressed. We have previously characterized an efficient cell penetrating peptide, PF14, for in vivo applications. In the current work, we first develop an optimized formulation of PF14/pDNA nanocomplexes, which allows removal of the side-effects without compromising the bioefficacy in vivo. Secondly, based on the physicochemical complex formation studies and biological efficacy assessments, we develop a series of PF14 modifications with altered charge and fatty acid content. We show that with an optimal combination of overall charge and hydrophobicity in the peptide backbone, in vivo gene delivery can be augmented. Further combined with the safe formulation, systemic gene delivery lacking any side effects can be achieved.

Magnetic Nanoparticle Assisted Self-assembly of Cell Penetrating Peptides-Oligonucleotides Complexes for Gene Delivery.

Magnetic nanoparticles (MNPs, Fe3O4) incorporated into the complexes of cell penetrating peptides (CPPs)-oligonucleotides (ONs) promoted the cell transfection for plasmid transfection, splice correction, and gene silencing efficiencies. Six types of cell penetrating peptides (CPPs; PeptFect220 (denoted PF220), PF221, PF222, PF223, PF224 and PF14) and three types of gene therapeutic agents (plasmid (pGL3), splicing correcting oligonucleotides (SCO), and small interfering RNA (siRNA) were investigated. Magnetic nanoparticles incorporated into the complexes of CPPs-pGL3, CPPs-SCO, and CPPs-siRNA showed high cell biocompatibility and efficiently transfected the investigated cells with pGL3, SCO, and siRNA, respectively. Gene transfer vectors formed among PF14, SCO, and MNPs (PF14-SCO-MNPs) showed a superior transfection efficiency (up to 4-fold) compared to the noncovalent PF14-SCO complex, which was previously reported with a higher efficiency compared to commercial vector called Lipofectamine™2000. The high transfection efficiency of the new complexes (CPPs-SCO-MNPs) may be attributed to the morphology, low cytotoxicity, and the synergistic effect of MNPs and CPPs. PF14-pDNA-MNPs is an efficient complex for in vivo gene delivery upon systemic administration. The conjugation of CPPs-ONs with inorganic magnetic nanoparticles (Fe3O4) may open new venues for selective and efficient gene therapy.

Optimization of in vivo DNA delivery with NickFect peptide vectors.

As the field of gene therapy progresses, an increasingly urgent need has arisen for efficient and non-toxic vectors for the in vivo delivery of nucleic acids. Cell-penetrating peptides (CPP) are very efficient transfection reagents in vitro, however, their application in vivo needs improvement. To enhance in vivo transfection we designed various CPPs based on previous knowledge of internalization studies and physiochemical properties of NickFect (NF) nanoparticles. We show that increment of the helicity of these Transportan10 analogues improves the transfection efficiency. We rationally design by modifying the net charge and the helicity of the CPP a novel amphipathic α-helical peptide NF55 for in vivo application. NF55 condenses DNA into stable nanoparticles that are resistant to protease degradation, promotes endosomal escape, and transfects the majority of cells in a large cell population. We demonstrate that NF55 mediates DNA delivery in vivo with gene induction efficiency that is comparable to commercial transfection reagents. In addition to gene induction in healthy mice, NF55/DNA nanoparticles showed promising tumor transfection in various mouse tumor models, including an intracranial glioblastoma model. The efficiency of NF55 to convey DNA specifically into tumor tissue increased even further after coupling a PEG2000 to the peptide via a disulphide-bond. Furthermore, a solid formulation of NF55/DNA displayed an excellent stability profile without additives or special storage conditions. Together, its high transfection efficacy and stability profile make NF55 an excellent vector for the delivery of DNA in vivo.