Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 10 de 10
Filter
Add more filters










Publication year range
1.
Biomedicines ; 9(8)2021 Aug 19.
Article in English | MEDLINE | ID: mdl-34440250

ABSTRACT

Splice-switching therapy with splice-switching oligonucleotides (SSOs) has recently proven to be a clinically applicable strategy for the treatment of several mis-splice disorders. Despite this, wider application of SSOs is severely limited by the inherently poor bioavailability of SSO-based therapeutic compounds. Cell-penetrating peptides (CPPs) are a class of drug delivery systems (DDSs) that have recently gained considerable attention for improving the uptake of various oligonucleotide (ON)-based compounds, including SSOs. One strategy that has been successfully applied to develop effective CPP vectors is the introduction of various lipid modifications into the peptide. Here, we repurpose hydrocarbon-modified amino acids used in peptide stapling for the orthogonal introduction of hydrophobic modifications into the CPP structure during peptide synthesis. Our data show that α,α-disubstituted alkenyl-alanines can be successfully utilized to introduce hydrophobic modifications into CPPs to improve their ability to formulate SSOs into nanoparticles (NPs), and to mediate high delivery efficacy and tolerability both in vitro and in vivo. Conclusively, our results offer a new flexible approach for the sequence-specific introduction of hydrophobicity into the structure of CPPs and for improving their delivery properties.

2.
Pharmaceutics ; 13(6)2021 Jun 14.
Article in English | MEDLINE | ID: mdl-34198625

ABSTRACT

The toolbox for genetic engineering has quickly evolved from CRISPR/Cas9 to a myriad of different gene editors, each with promising properties and enormous clinical potential. However, a major challenge remains: delivering the CRISPR machinery to the nucleus of recipient cells in a nontoxic and efficient manner. In this article, we repurpose an RNA-delivering cell-penetrating peptide, PepFect14 (PF14), to deliver Cas9 ribonucleoprotein (RNP). The RNP-CPP complex achieved high editing rates, e.g., up to 80% in HEK293T cells, while being active at low nanomolar ranges without any apparent signs of toxicity. The editing efficiency was similar to or better compared to the commercially available reagents RNAiMAX and CRISPRMax. The efficiency was thoroughly evaluated in reporter cells and wild-type cells by restriction enzyme digest and next-generation sequencing. Furthermore, the CPP-Cas9-RNP complexes were demonstrated to withstand storage at different conditions, including freeze-thaw cycles and freeze-drying, without a loss in editing efficiency. This CPP-based delivery strategy complements existing technologies and further opens up new opportunities for Cas9 RNP delivery, which can likely be extended to other gene editors in the future.

3.
iScience ; 24(5): 102436, 2021 May 21.
Article in English | MEDLINE | ID: mdl-33997707

ABSTRACT

Ethanol (EtOH) abuse induces significant mortality and morbidity worldwide because of detrimental effects on brain function. Defining the contribution of astrocytes to this malfunction is imperative to understanding the overall EtOH effects due to their role in homeostasis and EtOH-seeking behaviors. Using a highly controllable in vitro system, we identify chemical signaling mechanisms through which acute EtOH exposure induces a modulatory feedback loop between neurons and astrocytes. Neuronally-derived purinergic signaling primed a subpopulation of astrocytes to respond to subsequent acute EtOH exposures (SEastrocytes: signal enhanced astrocytes) with greater calcium signal strength. Generation of SEastrocytes arose from astrocytic hemichannel-derived ATP and accumulation of its metabolite adenosine within the astrocyte microenvironment to modulate adenylyl cyclase and phospholipase C activity. These results highlight an important role of astrocytes in shaping the overall physiological responsiveness to EtOH and emphasize the unique plasticity of astrocytes to adapt to single and multiple exposures of EtOH.

4.
J Mater Chem B ; 8(47): 10825-10836, 2020 12 21.
Article in English | MEDLINE | ID: mdl-33174901

ABSTRACT

Cell-penetrating peptides are a promising therapeutic strategy for a wide variety of degenerative diseases, ageing, and cancer. Among the multitude of cell-penetrating peptides, PepFect14 has been preferentially used in our laboratory for oligonucleotide delivery into cells and in vivo mouse models. However, this activity has mainly been reported towards cytoplasm and nuclei, while the mentioned disorders have been linked to mitochondrial defects. Here, we report a library generated from a combinatorial covalent fusion of a mitochondrial-penetrating peptide, mtCPP1, and PepFect14 in order to deliver therapeutic biomolecules to influence mitochondrial protein expression. The non-covalent complexation of these peptides with oligonucleotides resulted in nano-complexes affecting biological functions in the cytoplasm and on mitochondria. This delivery system proved to efficiently target mitochondrial genes, providing a framework for the development of mitochondrial peptide-based oligonucleotide technologies with the potential to be used as a treatment for patients with mitochondrial disorders.


Subject(s)
Cell-Penetrating Peptides/metabolism , Drug Delivery Systems/methods , Intracellular Fluid/metabolism , Mitochondrial Proteins/metabolism , Oligonucleotides, Antisense/metabolism , RNA, Messenger/metabolism , Animals , Cell-Penetrating Peptides/administration & dosage , Cell-Penetrating Peptides/genetics , Cytoplasm/drug effects , Cytoplasm/genetics , Cytoplasm/metabolism , HeLa Cells , Humans , Intracellular Fluid/drug effects , Membrane Potential, Mitochondrial/drug effects , Membrane Potential, Mitochondrial/physiology , Mice , Mitochondrial Proteins/genetics , Oligonucleotides, Antisense/administration & dosage , Oligonucleotides, Antisense/genetics , RNA, Messenger/genetics
5.
Biochim Biophys Acta Biomembr ; 1860(2): 491-504, 2018 Feb.
Article in English | MEDLINE | ID: mdl-28962904

ABSTRACT

Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane.


Subject(s)
Cell Membrane/metabolism , Cell-Penetrating Peptides/metabolism , Protein Aggregates , Spectrometry, Fluorescence/methods , Amino Acid Sequence , Animals , Biological Transport , Carbocyanines/chemistry , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/genetics , Hydrophobic and Hydrophilic Interactions , Lipopeptides/chemistry , Lipopeptides/genetics , Lipopeptides/metabolism , Microscopy, Fluorescence , PC12 Cells , Protein Binding , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Rhodamines/chemistry
6.
Sci Rep ; 7(1): 17056, 2017 12 06.
Article in English | MEDLINE | ID: mdl-29213085

ABSTRACT

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.


Subject(s)
Cell-Penetrating Peptides/genetics , Fatty Acids/chemistry , Lipopeptides/genetics , Transfection/methods , Animals , CHO Cells , Cell-Penetrating Peptides/chemistry , Cricetinae , Cricetulus , Dynamic Light Scattering , Female , Fluorescent Dyes/chemistry , Lipopeptides/chemistry , Lung/metabolism , Mice , Mice, Inbred BALB C , Nanoparticles/chemistry
7.
Bioconjug Chem ; 28(3): 782-792, 2017 03 15.
Article in English | MEDLINE | ID: mdl-28209057

ABSTRACT

Modifying cell-penetrating peptides (CPPs) with fatty acids has long been used to improve peptide-mediated nucleic acid delivery. In this study we have revisited this phenomenon with a systematic approach where we developed a structure-activity relationship to describe the role of the acyl chain length in the transfection process. For that we took a well-studied CPP, PepFect14, as the basis and varied its N-terminal acyl chain length from 2 to 22 carbons. To evaluate the delivery efficiency, the peptides were noncovalently complexed with a splice-correcting oligonucleotide (SCO) and tested in HeLa pLuc705 reporter cell line. Our results demonstrate that biological splice-correction activity emerges from acyl chain of 12 carbons and increases linearly with each additional carbon. To assess the underlying factors regarding how the transfection efficacy of these complexes is dependent on hydrophobicity, we used an array of different methods. For the functionally active peptides (C12-22) there was no apparent difference in their physicochemical properties, including complex formation efficiency, hydrodynamic size, and zeta potential. Moreover, membrane activity studies with peptides and their complexes with SCOs confirmed that the toxicity of the complexes at higher molar ratios is mainly caused by the free fraction of the peptide which is not incorporated into the peptide/oligonucleotide complexes. Finally, we show that the increase in splice-correcting activity correlates with the ability of the complexes to associate with the cells. Collectively these studies lay the ground work for how to design highly efficient CPPs and how to optimize their oligonucleotide complexes for lowest toxicity without losing efficiency.


Subject(s)
Cell-Penetrating Peptides/chemistry , Fatty Acids/chemistry , Lipopeptides/chemistry , Oligonucleotides/administration & dosage , Transfection/methods , Acylation , Amino Acid Sequence , Animals , Cattle , HeLa Cells , Humans , Hydrophobic and Hydrophilic Interactions , Oligonucleotides/genetics
8.
J Control Release ; 209: 238-47, 2015 Jul 10.
Article in English | MEDLINE | ID: mdl-25935707

ABSTRACT

Gene therapy has great potential to treat a range of different diseases, such as cancer. For that therapeutic gene can be inserted into a plasmid vector and delivered specifically to tumor cells. The most frequently used applications utilize lipoplex and polyplex approaches where DNA is non-covalently condensed into nanoparticles. However, lack of in vivo efficacy is the major concern that hinders translation of such gene therapeutic applications into clinics. In this work we introduce a novel method for in vivo delivery of plasmid DNA (pDNA) and efficient tumor-specific gene induction using intravenous (i.v) administration route. To achieve this, we utilize a cell penetrating peptide (CPP), PepFect14 (PF14), double functionalized with polyethylene glycol (PEG) and a matrix metalloprotease (MMP) substrate. We show that this delivery vector effectively forms nanoparticles, where the condensed CPP and pDNA are shielded by the PEG, in an MMP-reversible manner. Administration of the complexes results in efficient induction of gene expression specifically in tumors, avoiding normal tissues. This strategy is a potent gene delivery platform that can be used for tumor-specific induction of a therapeutic gene.


Subject(s)
Cell-Penetrating Peptides/administration & dosage , Gene Transfer Techniques , Lipopeptides/administration & dosage , Matrix Metalloproteinase 2/metabolism , Neoplasms/genetics , Polyethylene Glycols/administration & dosage , Animals , CHO Cells , Cell Line , Cell Line, Tumor , Cell-Penetrating Peptides/chemistry , Cricetinae , Cricetulus , DNA/administration & dosage , DNA/chemistry , Gene Expression , Genes, Reporter , Humans , Lipopeptides/chemistry , Luciferases/genetics , Luciferases/metabolism , Mice , Nanoparticles/chemistry , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Plasmids , Polyethylene Glycols/chemistry , Tumor Burden/drug effects
9.
Biomol Concepts ; 5(6): 479-88, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25429600

ABSTRACT

Peptides and peptide-cargo complexes have been used for drug delivery and gene therapy. One of the most used delivery vectors are cell-penetrating peptides, due to their ability to be taken up by a variety of cell types and deliver a large variety of cargoes through the cell membrane with low cytotoxicity. In vitro and in vivo studies have shown their possibility and full effectiveness to deliver oligonucleotides, plasmid DNA, small interfering RNAs, antibodies, and drugs. We report in this review some of the latest strategies for peptide-mediated delivery of nucleic acids. It focuses on peptide-based vectors for therapeutic molecules and on nucleic acid delivery. In addition, we discuss recent applications and clinical trials.


Subject(s)
Cell-Penetrating Peptides/chemistry , Drug Delivery Systems , Genetic Vectors , Animals , Cell-Penetrating Peptides/metabolism , Clinical Trials as Topic , Humans , Nucleic Acids/administration & dosage , Oligonucleotides/administration & dosage
10.
Mol Pharm ; 10(1): 199-210, 2013 Jan 07.
Article in English | MEDLINE | ID: mdl-23186360

ABSTRACT

The successful applicability of gene therapy approaches will heavily rely on the development of efficient and safe nonviral gene delivery vectors, for example, cell-penetrating peptides (CPPs). CPPs can condense oligonucleotides and plasmid DNA (pDNA) into nanoparticles, thus allowing the transfection of genetic material into cells. However, despite few promising attempts, CPP-mediated pDNA delivery has been relatively inefficient due to the unfavorable nanoparticle characteristics or the nanoparticle entrapment to endocytic compartments. In many cases, both of these drawbacks could be alleviated by modifying CPPs with a stearic acid residue, as demonstrated in the delivery of both the pDNA and the short oligonucleotides. In this study, PepFect14 (PF14) peptide, previously used for the transport of shorter oligonucleotides, is demonstrated to be suited also for the delivery of pDNA. It is shown that PF14 forms stable nanoparticles with pDNA with a negative surface charge and size of around 130-170 nm. These nanoparticles facilitate efficient gene delivery and expression in a variety of regular adherent cell lines and also in difficult-to-transfect primary cells. Uptake studies indicate that PF14/pDNA nanoparticles are utilizing class A scavenger receptors (SCARA) and caveolae-mediated endocytosis as the main route for cellular internalization. Conclusively, PF14 is an efficient nonviral vector for gene delivery.


Subject(s)
Cell-Penetrating Peptides/administration & dosage , Cell-Penetrating Peptides/genetics , Gene Transfer Techniques , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Lipopeptides/administration & dosage , Lipopeptides/genetics , Animals , CHO Cells , Cell Culture Techniques , Cell-Penetrating Peptides/metabolism , Cricetinae , DNA/genetics , Endocytosis/genetics , Genetic Vectors/metabolism , HEK293 Cells , Humans , Lipopeptides/metabolism , Nanoparticles/administration & dosage , Oligonucleotides/administration & dosage , Oligonucleotides/genetics , Oligonucleotides/metabolism , Particle Size , Plasmids/genetics , Plasmids/metabolism , Transfection/methods
SELECTION OF CITATIONS
SEARCH DETAIL
...