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Cell-penetrating peptides: Tools for intracellular delivery of therapeutics
Abstract
The main problem of therapeutic efficiency lies in the crossing of cellular membranes. Therefore, significant effort is being made to develop agents which can cross these barriers and deliver therapeutic agents into cellular compartments. In recent years, a large amount of data on the use of peptides as delivery agents has accumulated. Several groups have published the first positive results using peptides for the delivery of therapeutic agents in relevant animal models. These peptides, called cell-penetrating peptides (CPPs), are short peptides (fewer than 30 residues) with a net positive charge and acting in a receptor- and energy-independent manner. Here, we give an extensive review of peptide-mediated delivery systems and discuss their applications, with particular focus on the mechanisms leading to cellular internalization.
... Then, in 2005, a pharmaceutical company, Nastech Pharmaceutical Company Inc., unaware of its existence, discovered a peptide, PN159, with the same sequence as a MAP, and demonstrated incredible cellular membrane interactions [33][34][35]. The structure of a MAP allows positioning of hydrophilic and hydrophobic residues on opposite sides, which, in the right conditions, can adopt a stable α-helix structure, getting close to lipidic membrane in temperatures between 25 and 55 °C [36,37], and even a β-structure with increased pressure and concentration [7,38]. In addition, MAPs are characterized by an overall positive net charge of five [39] and are very stable at physiological conditions associated with slow blood clearance (half-life above 72 h) in in vivo studies [40]. ...
... Initial studies indicated that amphipathic structure was crucial for its high interaction and penetration of cellular membranes. [36,41,42] Further ahead, new studies with different MAP derivatives demonstrated that both amphipathic and non-amphipathic peptides were able to transgress cell membranes within a few minutes [12,[43][44][45]. A study demonstrated that a reduction of four amino acid residues in MAPs, on both the N (KLAL) and C terminal end (LKLA), resulted in a substantial reduction of cell uptake, showing One CPP that has demonstrated this possibility for dual functionality is the model amphipathic peptide (MAP-KLALKLALKALKAALKLA). ...
... Then, in 2005, a pharmaceutical company, Nastech Pharmaceutical Company Inc., unaware of its existence, discovered a peptide, PN159, with the same sequence as a MAP, and demonstrated incredible cellular membrane interactions [33][34][35]. The structure of a MAP allows positioning of hydrophilic and hydrophobic residues on opposite sides, which, in the right conditions, can adopt a stable α-helix structure, getting close to lipidic membrane in temperatures between 25 and 55 • C [36,37], and even a β-structure with increased pressure and concentration [7,38]. In addition, MAPs are characterized by an overall positive net charge of five [39] and are very stable at physiological conditions associated with slow blood clearance (half-life above 72 h) in in vivo studies [40]. ...
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.
... Majority of the CPPs possess basic amino acids (arginines and/or lysines), through which these interact with cell membranes and help in the internalization of CPPs (Endoh & Ohtsuki 2009). The experiments on Tat and penetratin revealed that the role of positive charges is crucial for translocation (Deshayes et al. 2005). After studying various cationic polypeptides, CPPs with polyarginines were found to be more efficient than other cationic polypeptides like polyhistidines and polylysines and among polyarginine peptides, Arg7 and Arg9 have been the most widely used for in vitro and in vivo delivery (Deshayes et al. 2005). ...
... The experiments on Tat and penetratin revealed that the role of positive charges is crucial for translocation (Deshayes et al. 2005). After studying various cationic polypeptides, CPPs with polyarginines were found to be more efficient than other cationic polypeptides like polyhistidines and polylysines and among polyarginine peptides, Arg7 and Arg9 have been the most widely used for in vitro and in vivo delivery (Deshayes et al. 2005). ...
... The amphipathic peptides possess hydrophobic (polar) and hydrophilic (non-polar) domains. The amphipathic property of CPPs may arise from either primary or secondary structure (Deshayes et al. 2005). Primary amphipathic peptides consists of the sequential assembly of a domain of hydrophobic residues with a domain of hydrophilic residues while secondary amphipathic peptides are produced by conformational state that allows positioning of hydrophobic and hydrophilic residues on opposite sides of the molecule (Deshayes et al. 2005). ...
... These peptides are called cell penetrating peptides (CPPs) and are usually small cationic peptides with fewer than 20 amino acids, mostly consisting of Lysines and Arginines [18,19]. Because of their ability to transport drugs through cell membranes, CPPs are of interest for the drug delivery community [20][21][22]. ...
... Activation of the Nrf2 transcriptional system, a transcription enhancer of a variety of endogenous antioxidants, and detoxifying genes has been demonstrated as a viable method for the remediation of oxidative radicals by various investigators [33,[54][55][56]. Peptidergic drugs have the potential as a therapeutic alternative to improve recovery in TBI patients [57]. Nrf2 cell-penetrating peptides can activate the antioxidant genes by sustained stabilization and increased nuclear translocation of Nrf2. ...
In this review, we discuss the possibility and feasibility of nuclear factor erythroid 2-related factor 2 (Nrf2) as a therapeutic target to minimize the devastating effects of a brain injury. To complete this review, comprehensive literature searches were conducted in MEDLINE, PubMed, Embase, and PsycINFO databases for English scientific peer-reviewed articles through December 2022. This short review addressed the different sources of oxidative stress and its effects on blood-brain barrier (BBB) dysfunction, mitochondrial damage, and changes in a variety of inflammatory molecules associated with central nervous system (CNS) injury. At last, we explained the potential efficacy of the Nrf2 transcription factor in reducing oxidative stress-mediated secondary damages after a CNS injury. The role of CPUY192018, an inhibitor of Nrf2-Keap1 protein-protein interaction in protecting the injured brain cells is given as evidence of Nrf2’s role in activating antioxidant genes. Overall, the scope of Nrf2 in developing therapeutic interventions for a variety of pathophysiological conditions associated with CNS injury-induced free radical/inflammatory signaling is acknowledged. Nrf2 has a widespread application in basic and clinical neuroscience for understanding and treating free radical/inflammatory signaling disorders, including neurological diseases. The development of innovative therapeutic strategies using Nrf2-inducing agents can be applied to reduce the complications of TBI before advancing it to posttraumatic stress disorder (PTSD).
... Cationic cell-penetrating peptides (CPPs) have been used to carry macromolecules, including plasmids, proteins, peptides and, more recently, siRNA, across membranes into cells in vitro and in vivo (93)(94)(95)(96)(97). CPPs are small arginine-rich peptides that include HIV-1 Tat (98-100), Penetratin™ from the antennapedia protein (101), transportan (a hybrid derived from galanin and mastoparan) (102) and polyarginine-synthetic peptides (103)(104)(105)(106)(107)(108). These arginine-rich peptides range from 8 to 30 amino acids in length and interact with negatively charged glycosaminoglycans on the cell surface (109,110). ...
... The cell penetrating peptide (CPP), are short peptides with positive charge, contains synthetic and natural CPP, protein transduction domains (PTDs) and membrane-translocating sequences (Wadia and Dowdy, 2002). These CPPs and the drugs they transport are internalized by cells through the endocytic pathway (Deshayes et al., 2005). Therapeutic peptides in DKD mainly include insulin, GLP-1 and analogs, which are administrated parenterally. ...
Diabetic kidney disease (DKD) is the main complication in diabetes mellitus (DM) and the main cause of end-stage kidney disease worldwide. However, sodium glucose cotransporter 2 (SGLT2) inhibition, glucagon-like peptide-1 (GLP-1) receptor agonist, mineralocorticoid receptor antagonists and endothelin receptor A inhibition have yielded promising effects in DKD, a great part of patients inevitably continue to progress to uremia. Newly effective therapeutic options are urgently needed to postpone DKD progression. Recently, accumulating evidence suggests that targeted drug delivery strategies, such as macromolecular carriers, nanoparticles, liposomes and so on, can enhance the drug efficacy and reduce the undesired side effects, which will be a milestone treatment in the management of DKD. The aim of this article is to summarize the current knowledge of targeted drug delivery strategies and select the optimal renal targeting strategy to provide new therapies for DKD.
... This improved antigen uptake, processing, and presentation by APCs increase both humoral and cellular immune responses. Without the aid of membrane proteins, CPPs can pass through the membrane semipermeable barrier and enter the cell interior noninvasively (Deshayes et al., 2005), but however, the delivery method of the CPPs and their cargo is not well known. Energy-independent ("direct penetration") and endocytotic pathways have been proposed as the two prominent types of CPP absorption processes which can be influenced by the type and size of the CPP and cargo. ...
One of the main obstacles to most medication administrations (such as the vaccine constructs) is the cellular membrane’s inadequate permeability, which reduces their efficiency. Cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) are well-known as potent biological nanocarriers to overcome this natural barrier, and to deliver membrane-impermeable substances into cells. The physicochemical properties of CPPs, the attached cargo, concentration, and cell type substantially influence the internalization mechanism. Although the exact mechanism of cellular uptake and the following processing of CPPs are still uncertain; but however, they can facilitate intracellular transfer through both endocytic and non-endocytic pathways. Improved endosomal escape efficiency, selective cell targeting, and improved uptake, processing, and presentation of antigen by antigen-presenting cells (APCs) have been reported by CPPs. Different in vitro and in vivo investigations using CPP conjugates show their potential as therapeutic agents in various medical areas such as infectious and non-infectious disorders. Effective treatments for a variety of diseases may be provided by vaccines that can cooperatively stimulate T cell-mediated immunity (T helper cell activity or cytotoxic T cell function), and immunologic memory. Delivery of antigen epitopes to APCs, and generation of a potent immune response is essential for an efficacious vaccine that can be facilitated by CPPs. The current review describes the delivery of numerous vaccine components by various CPPs and their immunostimulatory properties.
... CPPs have been exploited for intracellular delivery of various nucleic acids, including oligomers, plasmid DNA 10 , siRNA 11 , and DNA 12,13 . They have also been applied as delivery systems for several drugs, ranging from nanoparticles to therapeutic proteins [14][15][16] . ...
The skin has a protective barrier against the external environment, making the transdermal delivery of active macromolecules very difficult. Cell-penetrating peptides (CPPs) have been accepted as useful delivery tools owing to their high transduction efficiency and low cytotoxicity. In this study, we evaluated the hydrophobic peptide, macromolecule transduction domain 1067 (MTD 1067) as a CPP for the transdermal delivery of protein cargoes of various sizes, including growth hormone-releasing hexapeptide-6 (GHRP-6), a truncated form of insulin-like growth factor-I (des(1-3)IGF-I), and platelet-derived growth factor BB (PDGF-BB). The MTD 1067-conjugated GHRP-6 (MTD-GHRP-6) was chemically synthesized, whereas the MTD 1067-conjugated des(1-3)IGF-I and PDGF-BB proteins (MTD-des(1-3)IGF-I and MTD-PDGF-BB) were generated as recombinant proteins. All the MTD 1067-conjugated cargoes exhibited biological activities identical or improved when compared to those of the original cargoes. The analysis of confocal microscopy images showed that MTD-GHRP-6, MTD-des(1-3)IGF-I, and MTD-PDGF-BB were detected at 4.4-, 18.8-, and 32.9-times higher levels in the dermis, respectively, compared to the control group without MTD. Furthermore, the MTD 1067-conjugated cargoes did not show cytotoxicity. Altogether, our data demonstrate the potential of MTD 1067 conjugation in developing functional macromolecules for cosmetics and drugs with enhanced transdermal permeability.
... N-Methylpurine DNA Glycosylase or MPG (GALFLGFLGAAGSTMGAWSQPKKKRKV) peptide is a great example of amphiphilic CPP. The hydrophobic stretch of this amphiphilic CPP (underlined) adopts a transient β-sheet structure creating a temporary channel in the cell membrane allowing the peptide-nucleic acid complex to internalize [143,144]. Amphiphilic α-helical peptide like penetratin (RQIKIWFQNRRMKWKK) generally remains unstructured in an aqueous solution but tends to adopt an α-helical conformation while interacting with cell membrane [84]. Majority of CPP internalizes via endosomal pathway and are accumulated inside endosome. ...
Nanotherapy has emerged as an improved anticancer therapeutic strategy to circumvent the harmful side effects of chemotherapy. It has been proven to be beneficial to offer multiple advantages, including their capacity to carry different therapeutic agents, longer circulation time and increased therapeutic index with reduced toxicity. Over time, nanotherapy evolved in terms of their designing strategies like geometry, size, composition or chemistry to circumvent the biological barriers. Multifunctional nanoscale materials are widely used as molecular transporter for delivering therapeutics and imaging agents. Nanomedicine involving multi-component chemotherapeutic drug-based combination therapy has been found to be an improved promising approach to increase the efficacy of cancer treatment. Next-generation nanomedicine has also utilized and combined immunotherapy to increase its therapeutic efficacy. It helps in targeting tumor immune response sparing the healthy systemic immune function. In this review, we have summarized the progress of nanotechnology in terms of nanoparticle designing and targeting cancer. We have also discussed its further applications in combination therapy and cancer immunotherapy. Integrating patient-specific proteomics and biomarker based information and harnessing clinically safe nanotechnology, the development of precision nanomedicine could revolutionize the effective cancer therapy.
... Furthermore, the cell-penetrating peptides containing short chains of amino acid residues (5-30 residues) also can penetrate the BBB via electrostatic interactions with the exposed plasma membrane [147,148]. These also act as transporters that mediate the transport of small macromolecules across the cell membrane [149]. These cellpenetrating peptides are divided into three categories i.e., natural (Tatderived peptides), synthetic (polyarginine), and chimeric peptides (transportan). ...
In recent years, unique physicochemical properties of amphiphilic block copolymers have been utilized to design the polymeric micelles for brain-specific delivery of drugs, proteins, peptides, and genes. Their unique properties such as nano-size, charge-switching ability, stimuli-responsive cargo release, flexible structure, and self-assembly enable them to overcome limitations of conventional dosage forms that include rapid drug release, drug efflux, and poor brain bioavailability, and poor stability. These limitations hinder their therapeutic efficacy in treating brain diseases. Their ease of functionalization and enhanced penetration and retention effect make them suitable nanocarriers for the diagnosis of various brain diseases. In this context, the present manuscript provides an insight into the progress made in the functionalization of micelles such as the incorporation of stimuli-sensitive moieties in copolymers, conjugation of cargo molecules with the core-forming block via responsive smart linkers, and conjugation of active ligands and imaging moieties with the corona forming block for brain targeting and imaging. Further, the review also expounds on the role of polymeric micelles in delivering neurotherapeutic to the brain. Some patents related to polymeric micelles formulated for brain delivery are also enlisted.
... In addition to nanoparticles, cell penetrating peptides (CPPs) can also aid in the delivery of donor DNA into cells [29,30]. CPPs are short peptides of no more than 30 hydrophobic amino acids [31]. ...
Background
The genetic engineering of crops has enhanced productivity in the face of climate change and a growing global population by conferring desirable genetic traits, including the enhancement of biotic and abiotic stress tolerance, to improve agriculture. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system has been found to be a promising technology for genomic editing. Protoplasts are often utilized for the development of genetically modified plants through in vitro integration of a recombinant DNA fragment into the plant genome. We targeted the citrus Nonexpressor of Pathogenesis-Related 3 ( CsNPR3 ) gene, a negative regulator of systemic acquired resistance (SAR) that governs the proteasome-mediated degradation of NPR1 and developed a genome editing technique targeting citrus protoplast DNA to produce stable genome-edited citrus plants.
Results
Here, we determined the best cationic lipid nanoparticles to deliver donor DNA and described a protocol using Lipofectamine™ LTX Reagent with PLUS Reagent to mediate DNA delivery into citrus protoplasts. A Cas9 construct containing a gRNA targeting the CsNPR3 gene was transfected into citrus protoplasts using the cationic lipid transfection agent Lipofectamine with or without polyethylene glycol (PEG, MW 6000). The optimal transfection efficiency for the encapsulation was 30% in Lipofectamine, 51% in Lipofectamine with PEG, and 2% with PEG only. Additionally, plasmid encapsulation in Lipofectamine resulted in the highest cell viability percentage (45%) compared with PEG. Nine edited plants were obtained and identified based on the T7EI assay and Sanger sequencing. The developed edited lines exhibited downregulation of CsNPR3 expression and upregulation of CsPR1 .
Conclusions
Our results demonstrate that utilization of the cationic lipid-based transfection agent Lipofectamine is a viable option for the successful delivery of donor DNA and subsequent successful genome editing in citrus.
... One of the most common and effective methods for delivering genetic material into cells is using cell penetrating peptides (CPPs) (Deshayes et al., 2005;Geng et al., 2020;Guidotti et al., 2017;Heitz et al., 2009;Kato et al., 2016). Covalent attachment of CPPs to gene delivery systems and electrostatic complexes of nucleic acid with CPPs have been studied (Khan et al., 2020;Xu et al., 2019). ...
Cell penetrating peptides (CPPs) have been used as alternative delivery vectors to translocate therapeutic cargo molecules across cell membranes. One example of CPPs is the dTAT peptide, which has shown great promise in the design of highly efficient and low-cytotoxic gene vectors when condensed via “soft” calcium cross links. Here, we investigated the effect of fluorination on the formulation of dTAT complexes and explored their potential for pDNA delivery to cells. Fluorinated dTAT complexes achieve excellent gene transfection efficacy compared to fluorinated PEI polyplexes in A549, HeLa, and MCF-7 cell lines. Furthermore, the fluorinated dTAT complexes exhibit excellent serum resistance, high gene transfection efficacy even in 10% FBS medium, and no detectable cytotoxicity on transfected cells. The optimum NaF concentration (14 mM) resulted in an over 1000-fold enhancement in dTAT complexes (N/P 33) transfection efficiency. According to these findings, fluorination seems to be a potential strategy for creating gene vectors without requiring complex syntheses.
... Despite intensive studies, the uptake mechanisms for cellpenetrating peptides remain ambiguous and largely varied due to their complicated nature [72][73][74] . Small molecule inhibitors specific for each endocytic pathway have been routinely utilized to interrogate the cellular uptake mechanisms of many transporters [75][76][77][78] . ...
We report the discovery of a facile peptide macrocyclization and stapling strategy based on a fluorine thiol displacement reaction (FTDR), which renders a class of peptide analogues with enhanced stability, affinity, cellular uptake, and inhibition of cancer cells. This approach enabled selective modification of the orthogonal fluoroacetamide side chains in unprotected peptides in the presence of intrinsic cysteines. The identified benzenedimethanethiol linker greatly promoted the alpha helicity of a variety of peptide substrates, as corroborated by molecular dynamics simulations. The cellular uptake of benzenedimethanethiol stapled peptides appeared to be universally enhanced compared to the classic ring-closing metathesis (RCM) stapled peptides. Pilot mechanism studies suggested that the uptake of FTDR-stapled peptides may involve multiple endocytosis pathways in a distinct pattern in comparison to peptides stapled by RCM. Consistent with the improved cell permeability, the FTDR-stapled lead Axin and p53 peptide analogues demonstrated enhanced inhibition of cancer cells over the RCM-stapled analogues and the unstapled peptides.
... CPP are short sequences of amino-acids that facilitate trans-membrane transport through a variety of endocytic pathways, without the requirement of specific targeting receptors [8]. Moreover, the primary and tertiary structure of the CPP can be manipulated to facilitate intra-cellular delivery of various therapeutic cargo [9]. The most promising CPPs possess cationic, basic, amphipathic and fusogenic properties. ...
Background
Gold nanoparticles (AuNP) are effective radiosensitisers, however, successful clinical translation has been impeded by short systemic circulation times and poor internalisation efficiency. This work examines the potential of RALA, a short amphipathic peptide, to enhance the uptake efficiency of negatively charged AuNPs in tumour cells, detailing the subsequent impact of AuNP internalisation on tumour cell radiation sensitivity.
Results
RALA/Au nanoparticles were formed by optimising the ratio of RALA to citrate capped AuNPs, with assembly occurring through electrostatic interactions. Physical nanoparticle characteristics were determined by UV–vis spectroscopy and dynamic light scattering. Nano-complexes successfully formed at w:w ratios > 20:1 (20 µg RALA:1 µg AuNP) yielding positively charged nanoparticles, sized < 110 nm with PDI values < 0.52. ICP-MS demonstrated that RALA enhanced AuNP internalisation by more than threefold in both PC-3 and DU145 prostate cancer cell models, without causing significant toxicity. Importantly, all RALA-AuNP formulations significantly increased prostate cancer cell radiosensitivity. This effect was greatest using the 25:1 RALA-AuNP formulation, producing a dose enhancement effect (DEF) of 1.54 in PC3 cells. Using clinical radiation energies (6 MV) RALA-AuNP also significantly augmented radiation sensitivity. Mechanistic studies support RALA-AuNP nuclear accumulation resulting in increased DNA damage yields.
Conclusions
This is the first study to demonstrate meaningful radiosensitisation using low microgram AuNP treatment concentrations. This effect was achieved using RALA, providing functional evidence to support our previous imaging study indicating RALA-AuNP nuclear accumulation.
Graphic abstract
... Such a simple and charge free nature facilitated conversion of the tetrapeptide into clinical compounds. Clinical molecules (Figure 1), still retain a largely peptide scaffold and carry a net positive charge that was thought to be favorable for membrane penetration and water solubility 168 . ...
Protein-protein interactions (PPIs) are macromolecular contacts critical in physiological processes and they play a role in both normal cellular processes and disease pathogenesis. Targeting PPIs represents a new but poorly explored therapeutic strategy. In this thesis, I have investigated peptide and nonpeptide approaches to two separate PPIs. Repressor activator protein 1 (RAP1) is a binding partner of telomeric repeat-binding factor 2 (TRF2) for the regulation of telomere function. A previous study has suggested that RAP1 recruits IκB kinases (IKK) to NF-κB complex phosphorylate p65 S536 and activates NF-κB pathway. Knocking down RAP1 sensitizes breast cancer cells apoptosis via NF-κB suppression. We employed a structure-based design strategy to develop a series of triazole-stapled α-helical peptides based upon the TRF2 sequence to block both RAP1/TRF2 and RAP1/IKK interactions. The most potent peptide synthesized binds to RAP1 with a Ki value of 7 nM, and is 400-fold more potent than the initial TRF2 peptide. Cellular studies show that an optimized peptide dose-dependently down-regulates p65 phosphorylation and inhibits cell growth in the HeLa and MDA-MB-157 cancer cell lines. This study generates the first-in-class inhibitor targeting RAP1, which can be used as a pharmacological tool to study NF-κB signaling in certain type of cancer. Bromodomain and extra terminal (BET) proteins, including BRD2, BRD3 and BRD4, are epigenetic readers and play a key role in regulation of gene transcription by binding to acetylated lysine residues on histone tails. Small-molecule inhibitors of BET have therapeutic potential for the treatment of human cancers and other diseases. We have carried out structure-based design, synthesis and evaluation of γ-carboline-containing compounds as a new class of small molecule BET inhibitors. Our most potent inhibitor (compound 18, RX-37) binds to BET bromodomain proteins (BRD2, BRD3 and BRD4) with Ki values of 3.2-24.7 nM and demonstrates high selectivity over other bromodomain proteins. RX-37 potently and selectively inhibits cell growth in human acute leukemia cell lines harboring rearranged mixed lineage leukemia 1 gene. Further modification of RX-37 yielded RX-201 which shows high oral bioavailability and in vivo efficacy in mice. This study has introduced a new chemical scaffold with potential therapeutic value in clinic.
... C ELL penetrating peptides (CPPs) are small permeable proteins ranging from 5-30 amino acid residues that act as workhorses in the life cycles of living cells [1]. These peptides are naturally nontoxic and cationic and perform critical functions in the delivery of various pharmacologically active macromolecules, including proteins, DNA fragments, green fluorescent protein and other nanomaterials, to the intracellular space [2][3][4]. ...
Cell-penetrating peptides (CPPs) are special kind of peptides capable of carrying variety of bioactive molecules such as genetic materials, short interfering RNA and nanoparticles into cell. In recent era, research on CPP has gained substantial interest from researchers to analyze its biological mechanisms for safe drug delivery agents and therapeutic application. Identifying CPP through traditional methods is extremely slow, overpriced and laborious, particularly due to large volume of unannotated peptide sequences accumulating in World Bank repository. To date; numerous computational methods have been developed, however, the available machine-learning tools cannot distinguish the CPPs and their uptake efficiency. This study aiming to develop two-layer deep learning framework, named DeepCPPred for identifying both CPPs in the first-phase and uptake efficiency peptides in the second-phase. The predictor first uses the four types of descriptors that cover the evolutionary, energy estimation, reduced sequence and amino-acid contact information. Then the extracted features are optimized through elastic net algorithm and fed into cascade deep-forest for building the final CPP model. The proposed method achieved 99.45% overall accuracy on benchmark dataset in the first-layer and 95.43% accuracy in the second-layer using 5-fold cross-validation test. Thus, our proposed bioinformatics tool surpassed all the existing state-of-the-art sequence-based CPP approach.
... The non-covalent complex formation of CPPs with ONs was an obvious choice on first sight due to the availability of cationic CPPs and anionic ONs, enabling the formation of efficient and stable aggregates or even nanoparticles, possibly yielding efficient transfections. The first CPPs for non-covalent ON complexation were stearoyl-R9, GALA, KALA, MPG, Pep-1, CADY [81][82][83][84][85][86][87][88], Chol-R9 [89], stearoyl-(RXR)4 [90] and others. ...
In the most recent 25–30 years, multiple novel mechanisms and applications of cell-penetrating peptides (CPP) have been demonstrated, leading to novel drug delivery systems. In this review, I present a brief introduction to the CPP area with selected recent achievements. This is followed by a nostalgic journey into the research in my own laboratories, which lead to multiple CPPs, starting from transportan and paving a way to CPP-based therapeutic developments in the delivery of bio-functional materials, such as peptides, proteins, vaccines, oligonucleotides and small molecules, etc.
... Based on their individual properties and depending on their interaction with the therapeutic agent, a classification can be implemented. Our own working group [89] and several other authors presented detailed reviews on CPPs, their classification and internalization mechanisms [90][91][92]. Briefly, to distinguish the CPPs, two main classes regarding the binding strategies are mostly used. ...
Macromolecular biomolecules are currently dethroning classical small molecule therapeutics because of their improved targeting and delivery properties. Protamine-a small polycationic peptides represent a promising candidate. In nature, it binds and protects DNA against degradation during spermatogenesis due to electrostatic interactions between the negatively charged DNA-phosphate backbone and the positively charged protamine. Researchers are mimicking this technique to develop innovative nanopharmaceutical drug delivery systems, incorporating protamine as a carrier for biologically active components such as DNA or RNA. The first part of this review highlights ongoing investigations in the field of protamine-associated nanotechnology, discussing the self-assembling manufacturing process and nanoparticle engineering. Immune-modulating properties of protamine are those that lead to the second key part, which is protamine in novel vaccine technologies. Protamine-based RNA delivery systems in vaccines (some belong to the new class of mRNA-vaccines) against infectious disease and their use in cancer treatment are reviewed, and we provide an update on the current state of latest developments with protamine as pharmaceutical excipient for vaccines.
... Based on their individual properties and depending on their interaction with the therapeutic agent, a classification can be implemented. Our own working group [89] and several other authors presented detailed reviews on CPPs, their classification and internalization mechanisms [90][91][92]. Briefly, to distinguish the CPPs, two main classes regarding the binding strategies are mostly used. ...
In our modern days, macromolecular biomolecules are dethroning classical small molecule therapeutics because of improved targeting and delivery properties. Protamine – a small polycationic peptide represents such a promising candidate. In nature, it binds and protects DNA against degradation during spermatogenesis due to electrostatic interaction between the negatively charged DNA-Phosphate backbone and the positively charged protamine. Researchers are mimicking this technique in order to develop innovative nanopharmaceutical drug delivery systems, incorporating protamine as carrier for biologically active components such as DNA or RNA. The first key part of this review highlights ongoing investigation in the field of protamine-associated nanotechnology, discussing the self-assembling manufacturing process and nanoparticle engineering. Immune-modulating properties of protamine are referred which lead to the second key part protamine in novel vaccine technologies. Protamine-based RNA delivery systems in vaccines (some of them belong to the new class of mRNA-vaccines) against infectious disease and their use in cancer treatment are reviewed and an update on the current state of latest developments with protamine as pharmaceutical excipient for vaccines is given.
... For example, PEG conjugation was applied to proteins before it was used to improve the delivery of small molecules (Box 1), and high-throughput screening techniques first developed to optimize small-molecule therapies have helped to identify microenvironment-modulating materials for live-cell delivery (as exemplified by the clinical use of immune-modulating polymers for SIG-001, an extended cell therapy depot that secretes human factor VIII for the treatment of haemophilia A). Environmental modifiers have also been repurposed across classes of therapeutic, including cytokines for the modification of the tumour microenvironment in cell-based immunotherapies, antibiotics for the engraftment of living biological therapeutics and chemically synthesized cell-penetrating peptides for improving the intracellular delivery of nucleic acids and small molecules 214 . Moreover, advancements made in the context of one class of therapeutic have facilitated the clinical translation of therapeutics belonging to other classes. ...
Drug delivery technologies have enabled the development of many pharmaceutical products that improve patient health by enhancing the delivery of a therapeutic to its target site, minimizing off-target accumulation and facilitating patient compliance. As therapeutic modalities expanded beyond small molecules to include nucleic acids, peptides, proteins and antibodies, drug delivery technologies were adapted to address the challenges that emerged. In this Review Article, we discuss seminal approaches that led to the development of successful therapeutic products involving small molecules and macromolecules, identify three drug delivery paradigms that form the basis of contemporary drug delivery and discuss how they have aided the initial clinical successes of each class of therapeutic. We also outline how the paradigms will contribute to the delivery of live-cell therapies. This Review Article discusses how delivery challenges associated with small molecules, nucleic acids, peptides, proteins and cells led to the development of commercial products and are now informing the delivery of live-cell therapeutics.
... The 50S ribosomal protein L7/L12 act as an immunoadjuvant for dendritic-cell based immunotherapy and it is capable of inducing dendritic-cell maturation [135]. The sequential vaccine diagram is presented in Fig. 4. A major problem of vaccine proteins is the crossing of cellular membranes [136]. However, the Cell-penetrating peptides (CPPs) are short peptides (fewer than 30 residues) and that are able to cross biological membranes without clear toxicity and facilitate the intracellular delivery of a variety of substances into cellular compartments and also capable of enhancing the immunogenic properties [137]. ...
Hantaviruses are an emerging zoonotic group of rodent-borne viruses that are having serious implications on global public health due to the increase in outbreaks. Since there is no permanent cure, there is increasing interest in developing a vaccine against the hantavirus. This research aimed to design a robust cross-protective subunit vaccine using a novel immunoinformatics approach. After careful evaluation, the best predicted cytotoxic & helper T-cell and B-cell epitopes from nucleocapsid proteins, glycoproteins, RdRp proteins, and non-structural proteins were considered as potential vaccine candidates. Among the four generated vaccine models with different adjuvant, the model with toll-like receptor-4 (TLR-4) agonist adjuvant was selected because of its high antigenicity, non-allergenicity, and structural quality. The selected model was 654 amino acids long and had a molecular weight of 70.5 kDa, which characterizes the construct as a good antigenic vaccine candidate. The prediction of the conformational B-lymphocyte (CBL) epitope secured its ability to induce the humoral response. Thereafter, disulfide engineering improved vaccine stability. Afterwards, the molecular docking confirmed a good binding affinity of −1292 kj/mol with considered immune receptor TLR-4 and the dynamics simulation showed high stability of the vaccine-receptor complex. Later, the in silico cloning confirmed the better expression of the constructed vaccine protein in E. coli K12. Finally, in in silico immune simulation, significantly high levels of immunoglobulin M (IgM), immunoglobulin G1 (IgG1), cytotoxic & helper T lymphocyte (CTL & HTL) populations, and numerous cytokines such as interferon-γ (IFN-γ), interleukin-2 (IL-2) etc. were found as coherence with actual immune response and also showed faster antigen clearance for repeated exposures. Nonetheless, experimental validation can demonstrate the safety and cross-protective ability of the proposed vaccine to fight against hantavirus infection.
... We therefore synthesized a variant modified with an N-terminal TAT sequence to enhance its permeability (compound 49, see Table 1 and Supplemental Figure 4). [24] Compound 49 was first tested in the FP assay and found to be slightly more active than its parent compound 33. The TAT sequence alone (compound 50, see Table 1) was found to weakly inhibit the interaction in the FP assay albeit at an IC 50 three orders of magnitude higher than 49. ...
Potent bicyclic peptide inhibitors of the RbAp48‐MTA1 interaction were developed by structure based stepwise optimization of the cyclization linker. The strategy exemplifies design of peptide derived inhibitors of protein–protein interactions involving large surface areas.
Abstract
The scaffolding protein RbAp48 is part of several epigenetic regulation complexes and is overexpressed in a variety of cancers. In order to develop tool compounds for the study of RbAp48 function, we have developed peptide inhibitors targeting the protein–protein interaction interface between RbAp48 and the scaffold protein MTA1. Based on a MTA1‐derived linear peptide with low micromolar affinity and informed by crystallographic analysis, a bicyclic peptide was developed that inhibits the RbAp48/MTA1 interaction with a very low nanomolar KD value of 8.56 nM, and which showed appreciable stability against cellular proteases. Design included exchange of a polar amide cyclization strategy to hydrophobic aromatic linkers enabling mono‐ and bicyclization by means of cysteine alkylation, which improved affinity by direct interaction of the linkers with a hydrophobic residue on RbAp48. Our results demonstrate that stepwise evolution of a structure‐based design is a suitable strategy for inhibitor development targeting PPIs.
... Despite intensive studies, the uptake mechanisms for cell-penetrating peptides remain ambiguous and largely varied due to their complicated nature. [54][55][56] Small molecule inhibitors specific for each endocytic pathway have been routinely utilized to interrogate the cellular uptake mechanisms of many transporters. [57][58][59][60] For example, nystatin as a sterol-binding agent was used to selectively block caveolin-dependent endocytosis, 60 while chlorpromazine can selectively inhibit clathrin-mediated endocytosis. ...
Stapled peptides serve as a powerful tool for probing protein-protein interactions, but its application has been largely impeded by the limited cellular uptake. Here we report the discovery of a facile peptide macrocyclization and stapling strategy based on a fluorine thiol displacement reaction (FTDR), which renders a class of peptide analogues with enhanced stability, affinity, and cell permeability. This new approach enabled selective modification of the orthogonal fluoroacetamide side chains in unprotected peptides, with the identified 1,3-benzenedimethanethiol linker promoting alpha helicity of a variety of peptide substrates, as corroborated by molecular dynamics simulations. The cellular uptake of these stapled peptides was universally enhanced compared to the classic ring-closing metathesis (RCM) stapled peptides. Pilot mechanism studies suggested that the uptake of FTDR-stapled peptides may involve multiple endocytosis pathways. Consistent with the improved cell permeability, the FTDR-stapled lead Axin analogues demonstrated better inhibition of cancer cell growth than the RCM-stapled analogues.
Graphical Abstract
... Currently, there is much need to understand how the CPP-cargo complexes traverse lipid bilayers, and to identify the physicochemical variables that most effectively regulate the CPPeffect. [11][12][13][14] The cellular intake of a CPP-cargo complex may occur by endocytocis, by direct translocation of the cell membrane, or through a combination of these two mechanisms. [15][16][17][18][19] However, in all cases, the complex must eventually diffuse across a lipid bilayer in order to reach the cytoplasm. ...
The CPP-effect makes reference to the process by which the membrane translocation rate of a cargo is enhanced by chemical functionalization with cell-penetrating peptides (CPPs). In this work we combine a simple kinetic model with free-energy calculations to explore the energetic basis of the CPP-effect. Two polyglicines are selected as model hydrophilic cargoes, and nona-arginine as a prototypical CPP. We assess the cargo carrying efficiency of nona-arginine by comparing the adsorption and insertion energies of the cargoes, the cargo-free CPPs, and the CPP–cargo complexes, into lipid membranes of varying composition. We also analyze the effect of modifying the type and concentration of anionic lipids, and the implication of these factors on the translocation rate of the CPP–cargo complex. Of particular interest is the evaluation of the catalytic role of palmitic acid (palmitate) as a promoter of the CPP-effect. We also analyse the influence of the size of the cargo on the membrane adsorption and insertion energies. Our results show that the efficiency of nona-arginine as a transmembrane carrier of simple hydrophilic molecules is modulated by the size of the cargo, and is strongly enhanced by increasing the concentration of anionic lipids and of ionized fatty acids in the membrane.
... We therefore synthesized a variant modified with an N-terminal TAT sequence to enhance its permeability (compound 49, see Table 1 and Supplemental Fig. 4). [24] Compound 49 was first tested in the FP assay and found to be slightly more active than its parent compound 33. The TAT sequence alone (compound 50, see Table 1) was found to weakly inhibit the interaction in the FP assay albeit at an IC50 three orders of magnitude higher than 49. ...
The scaffolding protein RbAp48 is part of several epigenetic regulation complexes and is overexpressed in a variety of cancers. In order to develop tool compounds for the study of RbAp48 function, we have developed peptide inhibitors targeting the protein-protein interaction interface between RbAp48 and the scaffold protein MTA1. Based on a MTA1-derived linear peptide with low micromolar affinity and informed by crystallographic analysis, a bicyclic peptide was developed that inhibits the RbAp48/MTA1 interaction with a very low nanomolar KD value of 8.56 nM, and which showed appreciable stability against cellular proteases. Design included exchange of a polar amide cyclization strategy to hydrophobic aromatic linkers enabling mono- and bicyclization by means of cysteine alkylation, which improved affinity by direct interaction of the linkers with a hydrophobic residue on RbAp48. Our results demonstrate that stepwise evolution of a structure-based design is a suitable strategy for inhibitor development targeting PPIs.
... This method has been widely used in examining antimicrobial peptide-membrane interactions. 12,20,27,28 In our simulations, a harmonic potential with a force constant of 500 kJ/mol/nm 2 was applied between the centre of mass of the polymyxin molecule and the membrane along the Z-axis, 29 which allowed the polymyxin molecule to penetrate the bacterial OM smoothly and achieve the unique conformational transition consistent with previous experimental observations. 14,16 For each OM-polymyxin interaction system, three independent simulation replicates were performed to examine the reproducibility of the simulation results. ...
Background:
MDR bacteria represent an urgent threat to human health globally. Polymyxins are a last-line therapy against life-threatening Gram-negative 'superbugs', including Acinetobacter baumannii. Polymyxins exert antimicrobial activity primarily via permeabilizing the bacterial outer membrane (OM); however, the mechanism of interaction between polymyxins and the OM remains unclear at the atomic level.
Methods:
We constructed a lipid A-based OM model of A. baumannii using quantitative membrane lipidomics data and employed all-atom molecular dynamics simulations with umbrella sampling techniques to elucidate the structure-interaction relationship and thermodynamics governing the penetration of polymyxins [B1 and E1 (i.e. colistin A) representing the two clinically used polymyxins] into the OM.
Results:
Polymyxin B1 and colistin A bound to the A. baumannii OM by the initial electrostatic interactions between the Dab residues of polymyxins and the phosphates of lipid A, competitively displacing the cations from the headgroup region of the OM. Both polymyxin B1 and colistin A formed a unique folded conformation upon approaching the hydrophobic centre of the OM, consistent with previous experimental observations. Polymyxin penetration induced reorientation of the headgroups of the OM lipids near the penetration site and caused local membrane disorganization, thereby significantly increasing membrane permeability and promoting the subsequent penetration of polymyxin molecules into the OM and periplasmic space.
Conclusions:
The thermodynamics governing the penetration of polymyxins through the outer leaflet of the A. baumannii OM were examined and novel structure-interaction relationship information was obtained at the atomic and membrane level. Our findings will facilitate the discovery of novel polymyxins against MDR Gram-negative pathogens.
... FITC-labeled peptide PROTACs demonstrated cell penetration in T47D and MCF-7 breast cancer cell lines using immunofluorescence and the PROTAC also triggered ERα degradation with a DC 50 < 20μM based on immunoblot analysis. Methods that involve conjugating short penetrating polyarginine sequences have been used to improve the cell permeability of biologics [195]. Lu et al. applied this strategy to develop peptide-based PROTACs targeting the aggregation prone Tau protein. ...
... In this respect, crossing of the blood-brain barrier (BBB) for drugs acting on neurodegenerative disorders is even more complicated [2]. Cell-penetrating peptides (CPPs) can reach membranes alone or in combination with a cargo both in vitro and in vivo and thus represent a promising strategy for scientists working in the area of drug development [3][4][5][6][7][8][9][10][11][12]. Advantages of CPPs include low cytotoxicity, ability to permeate into a range of cell types, dose-dependent efficiency, without restrictions regarding the size or the type of cargo, ability to assist in the delivery of covalently or noncovalently conjugated cargoes, biocompatibility, ease of synthesis, small size, reduced side effects and small administration doses. ...
Cell-penetrating peptides present huge biomedical applications in a variety of pathologies, thanks to their ability to penetrate membranes and carry a variety of cargoes inside cells. Progress in peptide synthesis has produced a greater availability of virtually any synthetic peptide, increasing their attractiveness. Most molecules when associated to a cell-penetrating peptides can be delivered into a cell, however, understanding of the critical factors influencing the uptake mechanism is of paramount importance to construct nanoplatforms for effective delivery in vitro and in vivo in medical applications. Focus is now on the state-of-art of the mechanisms enabling therapeutics/diagnostics to reach the site target of their activities, and in support of scientists developing platforms for drug delivery and personalized therapies.
Peptide‐drug conjugates (PDCs) are a promising class of drug delivery systems that utilize covalently conjugated carrier peptides with therapeutic agents. PDCs offer several advantages over traditional drug delivery systems, including enhanced target engagement, improved bioavailability, and increased cell permeability. However, the development of efficient transcellular peptides capable of effectively transporting drugs across biological barriers remains an unmet need. In this study, we employ physicochemical criteria based on cell penetrating peptides (CPPs) to design transcellular peptides derived from an antimicrobial peptides (AMPs) library. Among those the statistically designed transcellular peptides (SDTs), SDT7 exhibits higher skin permeability, faster kinetics, and improved cell permeability in human keratinocyte cells compared to the control peptide. Subsequently, we find that 6‐Paradol (PAR) exhibited inhibitory activity against phosphodiesterase 4 (PDE4), which can be utilized for an anti‐inflammatory PDC. The transcellular PDC (SDT7‐conjugated with PAR, named as TM5) is evaluated in mouse models of psoriasis, exhibiting superior therapeutic efficacy compared to PAR alone. These findings highlight the potential of transcellular peptide‐drug conjugates (TDCs) as a promising approach for the treatment of inflammatory skin disorders.
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Possibilities for classification of CPPs based on their type of origin, biochemical properties, and physico-chemical properties are presented together with examples. Eight different and arbitrary levels for classification are presented, due to their partly overlapping properties.
Peptides are of great importance in the areas of science because they can act as drug carriers and their therapeutic effect and their ability to generate an immune response. As culturing of viral agents for drugs and vaccine development is harmful, therefore, peptide-based drugs and vaccines have achieved great importance. Large immunoglobulins cannot pass the plasma membrane, so peptides are used to study this interaction because of their small size. Peptides with substituted amino acid sequences are also stable in blood serum, which makes them significant for drug development. Peptides with substituted amino acid sequences are stable in blood serum hence, their stability, small size, easy screening, cost-effectiveness, ease of administration and particularity (target specificity) make them effective to be used in pharmaceutical companies. Mostly branched peptides are used for the development of drugs because they are not prone to be degraded by proteolytic enzymes. In peptide-based vaccines, protein acts as the main constituent from which the main componentthat causes the infection is deleted by recombinant DNA technology, and these peptides act as antigens to stimulate the immune response. Self-assembled peptides have the main role in the delivery of drugs and vaccine molecules inside the living cells because they may also assemble into nano technological structures to improve their efficiency. This review focuses on the characteristics of peptides that make them effective to develop drugs and vaccines. Different peptides like synthetic peptides, antimicrobial peptides, signal peptides, carrier peptides, and their role against various viral, pathogenic, and microbial diseases and in cosmetics are described briefly.
Background:
To have a better and longer effect, botulinum neurotoxin (BoNT) is injected several times in a treatment course, which could increase side effects and cost. Some of the most cutting-edge strategies being investigated for proteins to their physiologic targets involve the reformulation of BoNT based on peptide-based delivery systems. For this purpose, cell-penetrating peptides (CPPs) are of particular interest because of their capacity to cross the biological membranes.
Objectives:
A short and simple CPP sequence was used as a carrier to create nanocomplex particles from BoNT/A, with the purpose of increasing toxin entrapment by target cells, reducing diffusion, and increasing the duration of the effect.
Method:
CPP-BoNT/A nanocomplexes were formed by polyelectrolyte complex (PEC) method, considering the anionic structure of botulinum toxin and the cationic CPP sequence. The cellular toxicity, and absorption profile of the complex nanoparticles were evaluated, and the digit abduction score (DAS) was used to assess the local muscle weakening efficacy of BoNT/A and CPP-BoNT/A.
Results:
The provided optimized polyelectrolyte complex nanoparticles had a 244 ± 20 nm particle size and 0.28 ± 0.04 PdI. In cellular toxicity, CPP-BoNT/A nanocomplexes as extended-release formulations of BoNT/A showed that nanocomplexes had a more toxic effect than BoNT/A. Furthermore, the comparison of weakening effectiveness on muscle was done among nanoparticles and free toxin on mice based on the digit abduction score (DAS) method, and nanocomplexes had a slower onset effect and a longer duration of action than toxin.
Conclusion:
Using PEC method allowed us to form nanocomplex from proteins, and peptides without a covalent bond and harsh conditions. The muscle-weakening effect of toxin in CPP-BoNT/A nanocomplexes showed acceptable efficacy and extended-release pattern.
Nanotechnology has revolutionized cancer diagnosis and therapy through targeted drug delivery. Advances in protein engineering and materials science have led to the development of nanocarriers (NCs), which have helped overcome the challenges faced during conventional cancer treatment. These nanocarriers serve as an efficient transport module for drugs. Nano-drug delivery has emerged as a promising technology that results in early detection and better treatment of various cancers. The approved nanoparticles currently used in cancer treatment strategies include liposomes, dendrimers, polyplexes, solid lipid nano-carriers, etc. These nanocarriers shall provide a quick, safe, and cost-effective method in cancer therapy and management. This book contains thirteen chapters focusing on the treatment of various cancers, i.e., lung, breast, cervical, ovarian, colon, prostate, head and neck, etc., through nanocarrier drugs. The chapters include reviews on a wide variety of advanced data from existing and ongoing clinical research and major regulatory considerations.
The free-energy profile of a compound is an essential measurement in evaluating the membrane permeation process by means of theoretical methods. Computationally, molecular dynamics (MD) simulation allows the free-energy profile calculation. However, MD simulations frequently fail to sample membrane permeation because they are rare events induced in longer timescales than the accessible timescale of MD, leading to an insufficient conformational search to calculate an incorrect free-energy profile. To achieve a sufficient conformational search, several enhanced sampling methods have been developed and elucidated the membrane permeation process. In addition to these enhanced sampling methods, we proposed a simple yet powerful free-energy calculation of a compound for the membrane permeation process based on originally rare-event sampling methods developed by us. Our methods have a weak dependency on external biases and their optimizations to promote the membrane permeation process. Based on distributed computing, our methods only require the selection of initial structures and their conformational resampling, whereas the enhanced sampling methods may be required to adjust external biases. Furthermore, our methods efficiently search membrane permeation processes with simple scripts without modifying any MD program. As demonstrations, we calculated the free-energy profiles of seven linear compounds for their membrane permeation based on a hybrid conformational search using two rare-event sampling methods, that is, (1) parallel cascade selection MD (PaCS-MD) and (2) outlier flooding method (OFLOOD), combined with a Markov state model (MSM) construction. In the first step, PaCS-MD generated initial membrane permeation paths of a compound. In the second step, OFLOOD expanded the unsearched conformational area around the initial paths, allowing for a broad conformational search. Finally, the trajectories were employed to construct reliable MSMs, enabling correct free-energy profile calculations. Furthermore, we estimated the membrane permeability coefficients of all compounds by constructing the reliable MSMs for their membrane permeation. In conclusion, the calculated coefficients were qualitatively correlated with the experimental measurements (correlation coefficient (R2) = 0.8689), indicating that the hybrid conformational search successfully calculated the free-energy profiles and membrane permeability coefficients of the seven compounds.
Proteins are a set of unique biomacromolecules harboring a wide variety of three‐dimensional (3D) structure. We may consider each of them as nanomachines working in different biological processes. Not only the structural and functional variation, the conformational dynamics of bidomain or multidomain proteins offer opportunity to design tuneable nanostructures. Protein‐based nanostructure designing and its application in different fields of biological, chemical, and medical sciences have become a lucrative field of research. Designing such nanostructures requires knowledge in the areas of chemistry, biology, computational modeling, and primarily about the 3D structure of protein. In this chapter, we have discussed several aspects of protein and peptide‐based nanostructures where the basic of protein structure has been mingled with the recent advancements in protein‐based nanostructure designing.
In the present scenario, the role of the peptide as a targeting moiety is enhanced in different types of cancer treatment which have biological actions based on their properties of penetration of the cell (cell-penetrating peptide), by conjugating the peptide with the drug studied (peptide–drug conjugates), or by using a target-specific peptide (cell-targeting peptide). These are used in the drug delivery of different biological molecules including nucleic acid, therapeutic drugs, liposomes, imaging molecules, nanomolecules, and large plasmids such as therapeutic driver molecules. The mechanism of the transport/internalization is endocytosis. This chapter describes different types of CPP/CPP-cargo, cell-targeting peptides, and peptide–drug conjugates for effective targeting of breast cancer treatment.
Technologies for enabling RNAi therapeutics discusses the advancements made in the field of diverse types of delivery vehicles, with a special emphasis on peptide-based delivery vehicles. Delivery is one of the key hurdles for enabling RNAi therapeutics. Here we have also discussed the application of RNAi therapeutics mainly in cancer treatment.
Abstract
RNA interference (RNAi) is a primitive evolutionary mechanism developed to escape incorporation of foreign genetic material. siRNA has been instrumental in achieving the therapeutic potential of RNAi by theoretically silencing any gene of interest in a reversible and sequence-specific manner. Extrinsically administered siRNA generally needs a delivery vehicle to span across different physiological barriers and load into the RISC complex in the cytoplasm in its functional form to show its efficacy. This review discusses the designing principles and examples of different classes of delivery vehicles that have proved to be efficient in RNAi therapeutics. We also briefly discuss the role of RNAi therapeutics in genetic and rare diseases, epigenetic modifications, immunomodulation and combination modality to inch closer in creating a personalized therapy for metastatic cancer. At the end, we present, strategies and look into the opportunities to develop efficient delivery vehicles for RNAi which can be translated into clinics.
The positively‐charged peptide antp derived from Antennapedia transcription protein is demonstrated to mediate the liposome translocation across the cell membrane. In the current investigation, we prepared a stable liposomal doxorubicin (Dox) formulation and targeted it with the antp peptide from 0 to 200 ligand/liposome. These antp‐containing liposomes were investigated in terms of physical stability on storage in the refrigerator and upon incubation in blood. Also, other features like cell binding, uptake, biodistribution, and treatment efficiency were evaluated in C26 colon carcinoma BALB/c mice. The Antp in liposomes resulted in enhanced particle growth with the development of the enormously large liposomes from 2000 nm to 6000 nm. Upon incubation in blood, these large liposomes were removed. The antp also enhanced the cell binding affinity and cell uptake rate of the liposomes and resulted in the restriction of the cancer cell proliferation, but it failed to improve the chemotherapeutic property of the Dox‐liposome. The i.v. injection of antp‐liposomes (15 mg Dox/kg) caused severe body weight loss and early death incidence due to probably increased toxicity. The antp targeting offered no advantage to the Dox‐liposome in the delivery of Dox to the tumor, and failed to enhance the treatment efficiency of the liposomes.
Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium that caused 1.5 million fatalities globally in 2018. New strains of Mtb resistant to all known classes of antibiotics pose a global healthcare problem. In this work we have conjugated novel indole-3-acetic acid-based DNA primase/gyrase inhibitor with cell-penetrating peptide via cleavable and non-cleavable bonds. For non-cleavable linkage, inhibitor was conjugated with peptide via an amide bond to the N-terminus, whereas a cleavable linkage was obtained by conjugating the inhibitor through a disulfide bond. We performed the conjugation of the inhibitor either directly on a solid surface, or by using solution-phase chemistry. M. smegmatis (non-pathogenic model of Mtb) was used to determine the minimal inhibitory concentration (MIC) of the synthetic conjugates. Conjugates were found more active as compared to free inhibitor molecules. Strikingly, the conjugate also impair the development of biofilm, showing a therapeutic potential against infections caused by both planktonic and sessile forms of mycobacterium species.
Liposomes are amphipathic lipidic supramolecular aggregates that are able to encapsulate and carry molecules of both hydrophilic and hydrophobic nature. They have been widely used as in vivo drug delivery systems for some time because they offer features such as synthetic flexibility, biodegradability, biocompatibility, low immunogenicity, and negligible toxicity. In recent years, the chemical modification of liposomes has paved the way to the development of smart liposome-based drug delivery systems, which are characterized by even more tunable and disease-directed features. In this review, we highlight the different types of chemical modification introduced to date, with a particular focus on internal stimuli-responsive liposomes and prodrug activation.
Peptide-based vaccine development represents a highly promising strategy for preventing Group A Streptococcus (GAS) infection. However, these vaccines need to be administered with the help of a delivery system and/or immune adjuvant. Cell-penetrating peptides (CPPs) have been used as a powerful tool for delivering various therapeutic agents, including peptides, as they can overcome the permeability barrier of cell membranes. Here, we used CPPs to deliver our lead lipopeptide-based vaccine (LCP-1). CPPs were anchored through a spacer to LCP-1-bearing multilamellar and unilamellar liposomes and administered to Swiss outbred mice. Tat47–57 conjugated to two palmitic acids via a (Gly)6 spacer (to form a liposome-anchoring moiety) was the most efficient system for triggering immune responses when combined with multilamellar liposomes bearing LCP-1. The immunostimulatory potential of a variety of other CPPs was examined following intranasal administration in mice. Among them, LCP-1/liposomes/Tat47–57 and LCP-1/liposomes/KALA induced the highest antibody titers. The antibodies produced showed high opsonic activity against clinically isolated GAS strains D3840 and GC2 203. The use of the CPP-liposome delivery system is a promising strategy for liposome-based GAS vaccine development.
Vaccination with small antigens, such as proteins, peptides, or nucleic acids, is used to activate the immune system and trigger the protective immune responses against a pathogen. Currently, nanovaccines are undergoing development instead of conventional vaccines. The size of nanovaccines is in the range of 10–500 nm, which enables them to be readily taken up by cells and exhibit improved safety profiles. However, low-level immune responses, as the removal of redundant pathogens, trigger counter-effective activation of the immune system invalidly and present a challenging obstacle to antigen recognition and its uptake via antigen-presenting cells (APCs). In addition, toxicity can be substantial. To overcome these problems, a variety of cell-penetrating peptide (CPP)-mediated vaccine delivery systems based on nanotechnology have been proposed, most of which are designed to improve the stability of antigens in vivo and their delivery into immune cells. CPPs are particularly attractive components of antigen delivery. Thus, the unique translocation property of CPPs ensures that they remain an attractive carrier with the capacity to deliver cargo in an efficient manner for the application of drugs, gene transfer, protein, and DNA/RNA vaccination delivery. CPP-mediated nanovaccines can enhance antigen uptake, processing, and presentation by APCs, which are the fundamental steps in initiating an immune response. This review describes the different types of CPP-based nanovaccines delivery strategies.
In the field of nanotherapeutics, gaining cellular entry into the cytoplasm of the target cell continues to be an ultimate challenge. There are many physicochemical factors such as charge, size and molecular weight of the molecules and delivery vehicles, which restrict their cellular entry. Hence, to dodge such situations, a class of short peptides called cell-penetrating peptides (CPPs) was brought into use. CPPs can effectively interact with the cell membrane and can assist in achieving the desired intracellular entry. Such strategy is majorly employed in the field of cancer therapy and diagnosis, but now it is also used for other purposes such as evaluation of atherosclerotic plaques, determination of thrombin levels and HIV therapy. Thus, the current review expounds on each of these mentioned aspects. Further, the review briefly summarizes the basic know-how of CPPs, their utility as therapeutic molecules, their use in cancer therapy, tumor imaging and their assistance to nanocarriers in improving their membrane penetrability. The review also discusses the challenges faced with CPPs pertaining to their stability and also mentions the strategies to overcome them. Thus, in a nutshell, this review will assist in understanding how CPPs can present novel possibilities for resolving the conventional issues faced with the present-day nanotherapeutics.
Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main common features required for this activity resulted from the interactions of CPPs with the plasma membrane (lipids) and more particularly with components of the extracellular matrix of the membrane itself (heparan sulphate). Indeed, independent of the direct translocation or the endocytosis-dependent internalization, lipid bilayers are involved in the internalization process both at the level of the plasma membrane and at the level of intracellular traffic (endosomal vesicles). In this article, we present a detailed protocol describing the different steps of a large unilamellar vesicles (LUVs) formulation, purification, characterization, and application in fluorescence leakage assay in order to detect possible CPP-membrane destabilization/interaction and to address their role in the internalization mechanism. LUVs with a lipid composition reflecting the plasma membrane content are generated in order to encapsulate both a fluorescent dye and a quencher. The addition of peptides in the extravesicular medium and the induction of peptide-membrane interactions on the LUVs might thus induce in a dose-dependent manner a significant increase in fluorescence revealing a leakage. Examples are provided here with the recently developed tryptophan (W)-and arginine (R)-rich Amphipathic Peptides (WRAPs), which showed a rapid and efficient siRNA delivery in various cell lines. Finally, the nature of these interactions and the affinity for lipids are discussed to understand and to improve the membrane translocation and/ or the endosomal escape.
The design of a non-viral gene delivery system that can release a functional nucleic acid at the intracellular destination site is an exciting but also challenging proposition. The ideal gene delivery vector must be non-toxic, non-immunogenic, overcome extra- and intra-cellular barriers, protect the nucleic acid cargo from degradation with stability over a range of temperatures. A new 15 amino acid linear peptide termed CHAT was designed in this study with the goal of delivering DNA with high efficiency into cells in vitro and tissues in vivo. Rational design involved incorporation of key amino acids including arginine for nucleic acid complexation and cellular uptake, tryptophan to enhance hydrophobic interaction with cell membranes, histidine to facilitate endosomal escape and cysteine for stability and controlled cargo release. Six linear peptides were synthesised with strategic sequences and amino acid substitutions. Data demonstrated that all six peptides complexed pDNA to produce cationic nanoparticles less than 200 nm in diameter, but not all peptides resulted in successful transfection; indicating the influence of peptide design for endosomal escape. Peptide 4, now termed CHAT, was non-cytotoxic, traversed the plasma membrane of breast and prostate cancer cell lines, and elicited reporter-gene expression following intra-tumoural and intravenous delivery in vivo. CHAT presents an exciting new peptide for the delivery of nucleic acid therapeutics.
Aberrant gene expression is a hallmark of acute leukemias. However, therapeutic strategies for its blockade are generally lacking, largely due to the pharmacologic challenges of drugging transcription factors. MYB-driven gene trans-activation with CREB-binding protein (CBP)/P300 is required for the initiation and maintenance of a variety of acute lymphoblastic and myeloid leukemias, including refractory MLL-rearranged leukemias. Using structure-guided molecular design, we developed a prototypical peptidomimetic inhibitor MYBMIM that interferes with the assembly of the molecular MYB:CBP/P300 complex at micromolar concentrations and rapidly accumulates in the nuclei of AML cells. We found that treatment of AML cells with MYBMIM, led to the displacement of the MYB:CBP/P300 complex in cells, displacement of MYB from oncogenic enhancers and promoters enriched for MYB binding sites, and downregulation of MYB-dependent gene expression, including of MYC and BCL2 oncogenes. Both human MLL-rearranged and non-rearranged AML cells, underwent mitochondrial apoptosis in response to MYBMIM treatment, which could be partially rescued by ectopic expression of BCL2 . We observed that MYBMIM treatment impeded leukemia growth and extended survival of immunodeficient mice engrafted with primary patient-derived MLL-rearranged leukemia cells. These findings emphasize the exquisite dependence of human AML on MYB:CBP/P300 transcriptional dysregulation, and establish a pharmacologic approach for its therapeutic blockade.
Efficient gene transfer is necessary for advanced biotechnologies ranging from gene therapy to synthetic biology. Peptide nanoparticles provide suitable packaging systems promoting targeted gene expression or silencing. Though these systems have yet to match the transfection efficacy of viruses, they are typically devoid of drawbacks characteristic of virus-based vectors, including insertional mutagenesis, low packaging capacities, and strong immune responses. Given the promise nanoparticle formulations hold for gene delivery, methods of their preparation and accurate analysis of their physicochemical and biological properties become indispensable for progress toward systems that seek to outperform viral vectors. Herein, we report a comprehensive protocol for the preparation and characterization of archetypal peptide nanoparticles resulting from nonspecific and noncovalent complexation with RNA and DNA.
The 60-amino acid long homeodomain of Antennapedia crosses biological membranes by an energy-independent mechanism, a phenomenon abolished by directed mutagenesis within the polypeptide C-terminal region. This finding led us to study the internalization of several chemically synthesized peptides derived from the third helix of the homeodomain. We report here that a polypeptide of 16 amino acids in length corresponding to the third helix of the homeodomain deleted of its N-terminal glutamate is still capable of translocating through the membrane. A longer peptide of 20 amino acids also translocates, whereas shorter peptides (15 amino acids) are not internalized by the cells. As is also the case for the entire homeodomain, the 20- and 16-amino acid long peptides are internalized at 4 degrees C, suggesting an energy-independent mechanism of translocation not involving classical endocytosis. The two translocated peptides can be recovered, intact, within the cells, strongly suggesting that they are not targeted to the lysosomal compartment. Finally, substitution of two tryptophans by two phenylalanines strongly diminishes translocation, raising the possibility that the internalization of the third helix is not solely based on its general hydrophobicity.
Several nonviral vectors including linear polyethylenimine(L-PEI) confer a pronounced lung tropism to plasmid DNA when injected into the mouse tail vein in a nonionic solution.
We have optimized this route by injecting 50 µg DNA with excess L-PEI (PEI nitrogen/DNA phosphate=10) in a large volume of 5% glucose (0.4 ml). In these conditions, 1–5% of lung cells were transfected (corresponding to 2 ng luciferase/mg protein), the other organs remaining essentially refractory to transfection (1–10 pg luciferase/mg protein).β-Galactosidase histochemistry confirmed alveolar cells, including pneumocytes, to be the main target, thus leading to the puzzling observation that the lung microvasculature must be permeable to cationic L-PEI/DNA particles of ca 60 nm. A smaller injected volume, premixing of the complexes with autologous mouse serum, as well as removal of excess free L-PEI, all severely decreased transgene expression in the lung. Arterial or portal vein delivery did not increase transgene expression in other organs.
These observations suggest that effective lung transfection primarily depends on the injection conditions: the large nonionic glucose bolus prevents aggregation as well as mixing of the cationic complexes and excess free L-PEI with blood. This may favour vascular leakage in the region where the vasculature is dense and fragile, i.e. around the lung alveoli. Cationic particles can thus reach the epithelium from the basolateral side where their receptors (heparan sulphate proteoglycans) are abundant. Copyright
Many systemically effective drugs such as cyclosporin A are ineffective
topically because of their poor penetration into skin. To surmount this problem,
we conjugated a heptamer of arginine to cyclosporin A through a pH-sensitive
linker to produce R7−CsA. In contrast to unmodified cyclosporin A, which
fails to penetrate skin, topically applied R7−CsA was efficiently transported
into cells in mouse and human skin. R7−CsA reached dermal T lymphocytes
and inhibited cutaneous inflammation. These data establish a general strategy
for enhancing delivery of poorly absorbed drugs across tissue barriers and
provide a new topical approach to the treatment of inflammatory skin disorders.
The amyloid precursor protein (APP) is a transmembrane protein expressed in several cell types. In the nervous system, APP is expressed by glial and neuronal cells, and several lines of evidence suggest that it plays a role in normal and pathological phenomena. To address the question of the actual function of APP in normal developing neurons, we undertook a study aimed at blocking APP expression using antisense oligonucleotides. Oligonucleotide internalization was achieved by linking them to a vector peptide that translocates through biological membranes. This original technique, which is very efficient and gives direct access to the cell cytosol and nucleus, allowed us to work with extracellular oligonucleotide concentrations between 40 and 200 nM. Internalization of antisense oligonucleotides overlapping the origin of translation resulted in a marked but transient decrease in APP neosynthesis that was not observed with the vector peptide alone, or with sense oligonucleotides. Although transient, the decrease in APP neosynthesis was sufficient to provoke a distinct decrease in axon and dendrite outgrowth by embryonic cortical neurons developing in vitro. The latter decrease was not accompanied by changes in the spreading of the cell bodies. A single exposure to coupled antisense oligonucleotides at the onset of the culture was sufficient to produce significant morphological effects 6, 18, and 24 h later, but by 42 h, there were no remaining significant morphologic changes. This report thus demonstrates that amyloid precursor protein plays an important function in the morphological differentiation of cortical neurons in primary culture.
Defined branched peptides (loligomers) incorporating cytoplasmic translocation signals, nuclear localization sequences, and fluorescent probes were designed and synthesized to demonstrate the feasibility and simplicity of creating novel classes of intracellular vehicles. Loligomers containing all the above signals were rapidly internalized by Chinese hamster ovary (CHO) cells and accumulated in their nucleus. At 4 degrees C, the interaction of peptide constructs with CHO cells was limited to membrane association. Loligomers entered cells at higher temperatures by adsorptive endocytosis. Inhibitors of ATP synthesis affected cytoplasmic import only weakly but abolished nuclear uptake. The peptide signals guided both cytoplasmic and nuclear localization events. The properties exhibited by loligomers suggest a strategy for the facile design of "guided" classes of intracellular agents.
The Tat protein of human immunodeficiency virus 1 (HIV-1) can enter cells efficiently when added exogenously in tissue culture. To assess if Tat can carry other molecules into cells, we chemically cross-linked Tat peptides (residues 1-72 or 37-72) to beta-galactosidase, horseradish peroxidase, RNase A, and domain III of Pseudomonas exotoxin A (PE) and monitored uptake colorimetrically or by cytotoxicity. The Tat chimeras were effective on all cell types tested, with staining showing uptake into all cells in each experiment. In mice, treatment with Tat-beta-galactosidase chimeras resulted in delivery to several tissues, with high levels in heart, liver, and spleen, low-to-moderate levels in lung and skeletal muscle, and little or no activity in kidney and brain. The primary target within these tissues was the cells surrounding the blood vessels, suggesting endothelial cells, Kupffer cells, and/or splenic macrophages. Tat-mediated uptake may allow the therapeutic delivery of macromolecules previously thought to be impermeable to living cells.
We previously showed that the downregulation of Cu/Zn superoxide dismutase (SOD1) activity in PC12 cells by exposure to an appropriate antisense oligonucleotide causes their apoptotic death. In this report, we used this model to examine the pathways by which SOD1 downregulation leads to death and to compare these pathways with those responsible for death caused by withdrawal of trophic support. To improve delivery of the SOD1 antisense oligonucleotide, we coupled it to a carrier "vector" peptide homologous to the third helix of the Drosophila Antennapedia homeodomain. This caused not only efficient cellular uptake even in the presence of serum, but also inhibition of SOD1 activity and promotion of apoptosis at 100-fold lower concentrations of oligonucleotide. Death induced by SOD1 downregulation appeared to require the reaction of superoxide with nitric oxide (NO) to form peroxynitrite. In support of this, inhibitors of NO synthase, the enzyme responsible for NO synthesis, blocked death in our experiments, whereas NO generators and donors accelerated cell death. N-Acetylcysteine and chlorophenylthiol cAMP, which rescue PC12 cells and neurons from the withdrawal of nerve growth factor and other forms of trophic support, did not protect PC12 cells from SOD1 downregulation. In contrast, overexpression of bcl-2, which also rescues these cells form loss of trophic support, was equally effective in saving the cells in the SOD1 downregulation paradigm. Taken together with past findings, such observations suggest that SOD1 downregulation and withdrawal of trophic support trigger apoptosis via distinct initial mechanisms but may utilize a common final pathway to bring about death. Our findings may be relevant to the causes and potential amelioration of neuronal degenerative disorders caused by impaired regulation of cellular levels of NO and superoxide.
We have recently reported that a 16-amino acid long polypeptide corresponding to the third helix of the DNA binding domain (homeodomain) of Antennapedia, a Drosophila transcription factor, is internalized by cells in culture (Derossi, D., Joliot, A. H., Chassaing, G., and Prochiantz, A.(1994) J. Biol. Chem. 269, 10444-10450). The capture of the homeodomain and of its third helix at temperatures below 10 degrees C raised the problem of the mechanism of internalization. The present demonstration, that a reverse helix and a helix composed of D-enantiomers still translocate across biological membranes at 4 and 37 degrees C strongly suggests that the third helix of the homeodomain is internalized by a receptor-independent mechanism. The finding that introducing 1 or 3 prolines in the structure does not hamper internalization also demonstrates that the alpha-helical structure is not necessary. The data presented are compatible with a translocation process based on the establishment of direct interactions with the membrane phospholipids. The third helix of the homeodomain has been used successfully to address biologically active substances to the cytoplasm and nucleus of cells in culture (Théodore, L., Derossi, D., Chassaing, G., Llirbat, B., Kubes, M., Jordan, P., Chneiweiss, H., Godement, P., and Prochiantz, A.(1995) J. Neurosci. 15, 7158-7167). Therefore, in addition to their physiological implications (Prochiantz, A., and Théodore, L.(1995) BioEssays 17, 39-45), the present results open the way to the molecular design of cellular vectors.
To develop a nonviral gene delivery system for treatment of diseases, our strategy is to construct DNA complexes with short synthetic peptides that mimic the functions of viral proteins. We have designed and synthesized two peptides which emulate viral functions - a DNA condensing agent, YKAK(8)WK, and an amphipathic, pH-dependent endosomal releasing agent, GLFEALLELLESLWELLLEA. The active gene delivery complex was constructed step-wise through a spontaneous self-assembly process involving oppositely charged, electrostatic interactions. To assemble DNA-peptide complexes with different overall net charges, only the negative charges of DNA phosphate, the positive charges of the 10 epsilon-amino groups of YKAK(8)WK and the negative charges of the 5 gamma-carboxyl groups of GLFEALLELLESLWELLLEA were considered. In the first step, negatively charged DNA was rapidly-mixed with an excess of YKAK(8)WK to form positively charged DNA-YKAK(8)WK complexes, which gave little gene transfer. In the second step and to form the active complex,the cationic DNA complex was rapidly mixed with spontaneously incorporated through electrostatic interactions. Transfection using these complexes of CMV-luc, YKAK(8)WK and GLFEALLELLESLWELLLEA gave high-levels of gene expression in a variety of cell lines. These simple DNA complexes, which contain only three molecularly defined components, have general utility for gene delivery and can replace viral vectors and cationic lipids for some applications in gene therapy.
Tat is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1). Several studies have shown that exogenous Tat protein was able to translocate through the plasma membrane and to reach the nucleus to transactivate the viral genome. A region of the Tat protein centered on a cluster of basic amino acids has been assigned to this translocation activity. Recent data have demonstrated that chemical coupling of a Tat-derived peptide (extending from residues 37 to 72) to several proteins allowed their functional internalization into several cell lines or tissues. A part of this same domain can be folded in an alpha-helix structure with amphipathic characteristics. Such helical structures have been considered as key determinants for the uptake of several enveloped viruses by fusion or endocytosis. In the present study, we have delineated the main determinants required for Tat translocation within this sequence by synthesizing several peptides covering the Tat domain from residues 37 to 60. Unexpectedly, the domain extending from amino acid 37 to 47, which corresponds to the alpha-helix structure, is not required for cellular uptake and for nuclear translocation. Peptide internalization was assessed by direct labeling with fluorescein or by indirect immunofluorescence using a monoclonal antibody directed against the Tat basic cluster. Both approaches established that all peptides containing the basic domain are taken up by cells within less than 5 min at concentrations as low as 100 nM. In contrast, a peptide with a full alpha-helix but with a truncated basic amino acid cluster is not taken up by cells. The internalization process does not involve an endocytic pathway, as no inhibition of the uptake was observed at 4 degrees C. Similar observations have been reported for a basic amino acid-rich peptide derived from the Antennapedia homeodomain (1). Short peptides allowing efficient translocation through the plasma membrane could be useful vectors for the intracellular delivery of various non-permeant drugs including antisense oligonucleotides and peptides of pharmacological interest.
The development of antisense and gene therapy has focused mainly on improving methods for oligonucleotide and gene delivery into cells. In the present work, we describe a potent new strategy for oligonucleotide delivery based on the use of a short peptide vector, termed MPG (27 residues), which contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain derived from the nuclear localization sequence of SV40 T-antigen. The formation of peptide vector/oligonucleotide complexes was investigated by measuring changes in intrinsic tryptophan fluorescence of peptide and of mansyl-labelled oligonucleotides. MPG exhibits relatively high affinity for both single- and double-stranded DNA in a nanomolar range. Based on both intrinsic and extrinsic fluorescence titrations, it appears that the main binding between MPG and oligonucleotides occurs through electrostatic interactions, which involve the basic-residues of the peptide vector. Further peptide/peptide interactions also occur, leading to a higher MPG/oligonucleotide ratio (in the region of 20/1), which suggests that oligonucleotides are most likely coated with several molecules of MPG. Premixed complexes of peptide vector with single or double stranded oligonucleotides are delivered into cultured mammalian cells in less than 1 h with relatively high efficiency (90%). This new strategy of oligonucleotide delivery into cultured cells based on a peptide vector offers several advantages compared to other commonly used approaches of delivery including efficiency, stability and absence of cytotoxicity. The interaction with MPG strongly increases both the stability of the oligonucleotide to nuclease and crossing of the plasma membrane. The mechanism of cell delivery of oligonucleotides by MPG does not follow the endosomal pathway, which explains the rapid and efficient delivery of oligonucleotides in the nucleus. As such, we propose this peptide vector as a powerful tool for potential development in gene and antisense therapy.
Peptide nucleic acids (PNAs) form stable and tight complexes with complementary DNA and/or RNA and would be promising antisense reagents if their cellular delivery could be improved. We show that a 21-mer PNA, complementary to the human galanin receptor type 1 mRNA, coupled to the cellular transporter peptides, transportan or pAntennapedia(43-58), is efficiently taken up into Bowes cells where they block the expression of galanin receptors. In rat, the intrathecal administration of the peptide-PNA construct results in a decrease in galanin binding in the dorsal horn. The decrease in binding results in the inability of galanin to inhibit the C fibers stimulation-induced facilitation of the rat flexor reflex, demonstrating that peptide-PNA constructs act in vivo to suppress expression of functional galanin receptors.
The herpes simplex virus protein VP22 exhibits the unusual property of intercellular transport whereby after being synthesised in a subpopulation of cells, in which it is largely cytoplasmic, the protein is transported to adjacent cells where it accumulates mainly in the nucleus. Here we examine the transport of a fusion protein consisting of VP22 linked to the green fluorescent protein (GFP). Intercellular transport, nuclear accumulation and chromatin binding of VP22-GFP could be detected by intrinsic GFP fluorescence in fixed cells. However, while the cytoplasmic localisation of VP22-GFP could be detected in live cells actively synthesising the protein, we were unable to detect intercellular transport by intrinsic GFP fluorescence in livecells, indicating that the levels of transported protein may be below those required for live detection, or that GFP fluorescence was quenched. The use of antibody to GFP was more sensitive than intrinsic GFP fluorescence and allowed ready detection of transport and nuclear accumulation of VP22-GFP. Intercellular transport was also confirmed in coplating experiments. Consistent with previous results showing a requirement for the C-terminus of VP22 in transport of the native protein, a fusion protein consisting of GFP linked to the N-terminal 1-192 residues of VP22 failed to transport between cells. The results support the proposal that VP22 has the ability to transport cargo proteins between cells and that it has significant potential in the field of gene therapy.
We demonstrate that fusion proteins consisting of the herpes simplex virus (HSV) transport protein VP22 linked in frame to HSV thymidine kinase (tk) retain the ability to be transported between cells. In vivo radiolabelling experiments and in vitro assays show that the fusion proteins also retain tk activity. When transfected COS cells, acting as a source of the VP22-tk chimera, were co-plated on to gap junction-negative neuroblastoma cells, ganciclovir treatment induced efficient cell death in the recipient neuroblastoma cell monolayer. No such effect was observed with COS cells transfected with tk alone. Tumours established in mice with neuroblastoma cell lines expressing VP22-tk regressed upon administration of ganciclovir. Furthermore tumours established from 50:50 mixtures of VP22-tk transduced and nontransduced cells also regressed while no significant effect was observed in similar experiments with cells transduced with tk alone. VP22 mediated transport may thus have application in a clinical setting to amplify delivery of the target protein in enzyme-prodrug protocols.
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We have shown previously that a peptide, MPG, derived from the hydrophobic fusion peptide of HIV-1 gp41 and the hydrophilic
nuclear localisation sequence of SV40 large T antigen, can be used as a powerful tool for the delivery of oligonucleotides
into cultured cells. Now we extend the potential of MPG to the delivery of nucleic acids into cultured cells. In vitro, MPG interacts strongly with nucleic acids, most likely forming a peptide cage around them, which stabilises and protects
them from degradation in cell culture media. MPG is non-cytotoxic, insensitive to serum and efficiently delivers plasmids
into several different cell lines in only 1 h. Moreover, MPG enables complete expression of the gene products encoded by the
plasmids it delivers into cultured cells. Finally, we have investigated the potential of MPG as an efficient delivery agent
for gene therapy, by attempting to deliver antisense nucleic acids targeting an essential cell cycle gene. MPG efficiently
delivered a plasmid expressing the full-length antisense cDNA of human cdc25C, which consequently successfully reduced cdc25C
expression levels and promoted a block to cell cycle progression. Based on our results, we conclude that MPG is a potent delivery
agent for the generalised delivery of nucleic acids as well as of oligonucleotides into cultured cells and believe that its
contribution to the development of new gene therapy strategies could be of prime interest.
SSeCKS, first isolated as a G1→S inhibitor that is downregulated in src- and ras-transformed cells, is a major cytoskeleton-associated PKC substrate with tumor suppressor and kinase-scaffolding activities.
Previous attempts at constitutive expression resulted in cell variants with truncated ectopic SSeCKS products. Here, we show
that tetracycline-regulated SSeCKS expression in NIH 3T3 cells induces G1 arrest marked by extracellular signal-regulated kinase 2-dependent decreases in cyclin D1 expression and pRb phosphorylation.
Unexpectedly, the forced reexpression of cyclin D1 failed to rescue SSeCKS-induced G1 arrest. Confocal microscopy analysis revealed cytoplasmic colocalization of cyclin D1 with SSeCKS. Because the SSeCKS gene
encodes two potential cyclin-binding motifs (CY) flanking major in vivo protein kinase C (PKC) phosphorylation sites (Ser507/515), we addressed whether SSeCKS encodes a phosphorylation-dependent cyclin scaffolding function. Bacterially expressed SSeCKS-CY
bound cyclins D1 and E, whereas K→S mutations within either CY motif ablated binding. Activation of PKC in vivo caused a rapid
translocation of cyclin D1 to the nucleus. Cell permeable, penetratin-linked peptides encoding wild-type SSeCKS-CY, but not
K→S or phospho-Ser507/515 variants, released cyclin D1 from its cytoplasmic sequestration and induced higher saturation density in cyclin D1-overexpressor
cells or rat embryo fibroblasts. Our data suggest that SSeCKS controls G1→S progression by regulating the expression and localization of cyclin D1. These data suggest that downregulation of SSeCKS
in tumor cells removes gating checkpoints for saturation density, an effect that may promote contact independence.
The detection of intracellular cell-penetrating peptides (CPPs) poses several obstacles - for example, the difficulty of distinguishing membrane-bound CPP from intracellularly localized CPP. In this chapter we focus on methods used in quantifying the efficiency of CPP internalization, as well as the quantification of cargoes delivered by CPPs.
Background The major advantage in choosing non-viral vectors such as cationic polymers for in vitro and in vivo transfection is their higher biosafety than viral ones. Among the cationic polymers, polyethylenimines (PEIs) are promising molecules for gene delivery to a variety of cells. Efficient transfection of primary endothelial cells using PEIs could be regarded as an interesting strategy of treatment in some ischemic cardiovascular diseases.
Internalization of exogenous macromolecules by live cells provides a powerful approach for studying cellular functions. Understanding the mechanism of transfer from the extracellular milieu to the cytoplasm and nucleus could also contribute to the development of new therapeutic approaches. This article summarizes the unexpected properties of penetratins, a class of peptides with translocating properties and capable of carrying hydrophilic compounds across the plasma membrane. This unique system allows direct targeting of oligopeptides and oligonucleotides to the cytoplasm and nucleus, is non-cell-type specific and highly efficient, and therefore has several applications of potential cell-biology and clinical interest.
For N-terminally fluoresceine labelled KLALKLALKALKAALKLA-NH2 (I) extensive incorporation into aortic endothelial cells (AEC) was found, resulting in an about 500-fold enrichment within the cell interior. The cellular uptake was not influenced by energy depletion and only partly by lowering the temperature to 0° C. Using double-D-amino acid analogs of this pepfide, possessing a graduate propensity to adopt an α-helical structure, clear dependency of the peptide incorporation upon ampnipathicity could be demonstrated.
Delivery of therapeutic proteins into tissues and across the blood-brain barrier is severely limited by the size and biochemical
properties of the proteins. Here it is shown that intraperitoneal injection of the 120-kilodalton β-galactosidase protein,
fused to the protein transduction domain from the human immunodeficiency virus TAT protein, results in delivery of the biologically
active fusion protein to all tissues in mice, including the brain. These results open new possibilities for direct delivery
of proteins into patients in the context of protein therapy, as well as for epigenetic experimentation with model organisms.
This chapter describes a new strategy for the delivery of full-length proteins and peptides into mammalian cells based on a short amphipathic peptide carrier, Pep-1. This peptide carrier allows the delivery of several distinct proteins and peptides into different cell lines in a fully biologically active form, without the need for prior chemical covalent coupling or denaturation steps. Pep-1/macromolecule particle is favored as soon as it has crossed the cell membrane. This peptide-based protein delivery strategy presents several advantages, including rapid delivery of proteins into cells with very high efficiency, stability in physiological buffers, lack of toxicity, and lack of sensitivity to serum. Detection of fluorescently labeled antibodies in cells requires a large amount of antibody to be delivered. Depending on the sensitivity required to detect antibodies, it may be necessary to increase the amount of Pep-1 and antibody used. The advantages of the Pep-1 technology are directly associated with the mechanism through which this carrier promotes the delivery of proteins and peptides into cells.
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Several proteins can traverse biological membranes through protein transduction. Small sections of these proteins (10–16 residues long) are responsible for this. Linking these domains covalently to compounds, peptides, antisense peptide nucleic acids or 40-nm iron beads, or as in-frame fusions with full-length proteins, lets them enter any cell type in a receptor- and transporter-independent fashion. Moreover, several of these fusions, introduced into mice, were delivered to all tissues, even crossing the blood–brain barrier. These domains thus might let us address new questions and even help in the treatment of human disease.
Certain short peptides, which are able to translocate across cell membranes with a low lytic activity, can be useful as carriers (vectors) for hydrophilic molecules. We have studied three such cell penetrating peptides: pAntp (‘penetratin’), pIsl and transportan. pAntp and pIsl originate from the third helix of homeodomain proteins (Antennapedia and Isl-1, respectively). Transportan is a synthetic chimera (galanin and mastoparan). The peptides in the presence of various phospholipid vesicles (neutral and charged) and SDS micelles have been characterized by spectroscopic methods (fluorescence, EPR and CD). The dynamics of pAntp were monitored using an N-terminal spin label. In aqueous solution, the CD spectra of the three peptides show secondary structures dominated by random coil. With phospholipid vesicles, neutral as well as negatively charged, transportan gives up to 60% α-helix. pAntp and pIsl bind significantly only to negatively charged vesicles with an induction of around 60% β-sheet-like secondary structure. With all three peptides, SDS micelles stabilize a high degree of α-helical structure. We conclude that the exact nature of any secondary structure induced by the membrane model systems is not directly correlated with the common transport property of these translocating peptides.
It has been shown that human calcitonin (hCT) is absorbed through the nasal mucosa when administered together with promoters like sodium glycocholate (SGC) or dihydrofusinate. The aim of this study was to compare the clinical and metabolic effect of intranasal (in) and intramuscular (im) hCT in patients with osteoporosis or with Paget's disease of bones. Fifteen women with postmenopausal or with senile osteoporosis entered a randomized six months trial with in hCT (plus SGC) or with im hCT 100 U on alternate days. Six women in each group were treated for 2 months, and only four women in each group continued treatment for an additional 4 months period. In hCT, but not im hCT, reduced subjective pain, while urinary cAMP increased to a similar extent in the 2 groups. Other metabolic indexes and bone mineral content (BMC) were unchanged, no new fractures took place, and side effects were fewer with in than with im hCT. To confirm the analgesic effect of in hCT, twelve patients with Paget's disease of bone were randomly treated for 20 days with in or im hCT 100 U/day: during the short period of treatment, pain was reduced by in, not by im hCT, and urinary cAMP excretion similarly increased in the two groups of patients.
While developing an assay to measure the activity of the tat protein from human immunodeficiency virus 1 (HIV-1), we discovered that the purified protein could be taken up by cells growing in tissue culture and subsequently trans-activate the viral promoter. Trans-activation is dramatically increased by a variety of lysosomotrophic agents. For example, trans-activation can be detected at tat concentrations as low as 1 nM in the presence of chloroquine. Experiments using radioactive protein show that tat becomes localized to the nucleus after uptake and suggest that chloroquine protects tat from proteolytic degradation. These results raise the possibility that, under some conditions, tat might act as a viral growth factor to stimulate viral replication in latently infected cells or alter expression of cellular genes.
A 30-residue amphipathic peptide was designed to interact with uncharged bilayers in a pH-dependent fashion. This was achieved by a pH-induced random coil-alpha-helical transition, exposing a hydrophobic face in the peptide. The repeat unit of the peptide, glutamic acid-alanine-leucine-alanine (GALA), positioned glutamic acid residues on the same face of the helix, and at pH 7.5, charge repulsion between aligned Glu destabilized the helix. A tryptophan was included at the N-terminal as a fluorescence probe. The rate and extent of peptide-induced leakage of contents from large, unilamellar vesicles composed of egg phosphatidylcholine were dependent on pH. At pH 5.0 with a lipid/peptide mole ratio of 500/1, 100% leakage of vesicle contents occurred within 1 min. However, no leakage of vesicle contents occurred at pH 7.5. Circular dichroism measurements indicated that the molar ellipticity at 222 nm changed from about -4000 deg cm2 dmol-1 at pH 7.6 to -11,500 deg cm2 dmol-1 at pH 5.1, indicating a substantial increase in helical content as the pH was reduced. Changes in molar ellipticity were most significant over the same pH range where a maximum change in the extent and rate of leakage occurred. The tryptophan fluorescence emission spectra and the circular dichroism spectra of the peptide, in the presence of lipid, suggest that GALA did not associate with the bilayer at neutral pH. A change in the circular dichroism spectrum and a blue shift of the maximum of the tryptophan fluorescence emission spectra at pH 5.0, in the presence of lipid, indicated an association of GALA with the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)
Various substances are absorbed by the mucosa of the respiratory system, including vasopressin, luteinising hormone releasing hormone, insulin, and glucagon. We have evaluated the absorption of another polypeptide hormone, calcitonin, through the nasal mucosas of normal subjects.
We show that the HSV-1 structural protein VP22 has the remarkable property of intercellular transport, which is so efficient that following expression in a subpopulation the protein spreads to every cell in a monolayer, where it concentrates in the nucleus and binds chromatin. VP22 movement was observed both after delivery of DNA by transfection or microinjection and during virus infection. Moreover, we demonstrate that VP22 trafficking occurs via a nonclassical Golgi-independent mechanism. Sensitivity to cytochalasin D treatment suggests that VP22 utilizes a novel trafficking pathway that involves the actin cytoskeleton. In addition, we demonstrate intercellular transport of a VP22 fusion protein after endogenous synthesis or exogenous application, indicating that VP22 may have potential in the field of protein delivery.
We have designed a cationic amphipathic peptide, KALA (WEAKLAKALAKALAKHLAKALAKALKACEA), that binds to DNA, destabilizes membranes, and mediates DNA transfection. KALA undergoes a pH-dependent random coil to amphipathic alpha-helical conformational change as the pH is increased from 5.0 to 7.5. One face displays hydrophobic leucine residues, and the opposite face displays hydrophilic lysine residues. KALA-mediated release of entrapped aqueous contents from neutral and negatively charged liposomes increases with increasing helical content. KALA binds to oligonucleotides or plasmid DNA and retards their migration in gel electrophoresis. It displaces 50% of ethidium bromide from DNA at a charge ratio (+/-) of 0.9/1. In cultured cells, KALA assists oligonucleotide nuclear delivery when complexes are prepared at a 10/1 (+/-) charge ratio. KALA/DNA (10/1)(+/-) complexes mediate transfection of a variety of cell lines. The KALA sequence provides a starting point for a family of peptides that incorporate other functions to improve DNA delivery systems.
Cationic peptide, poly-arginine, promotes transfection efficiency under a proper concentration. Using fluorescence microscopy, we observed the structure of the DNA carrier-complex in solution directly. Under low concentration of poly-arginine, small complexes were observed with fluorescence images like a blight light spot. This small complex (sizes are c.a. 1 approximately 2 microns order) had no transfection ability. As the concentration of poly-arginine became high, the complex grew into a big one whose size reached to a degree of 10 microns. Transfection was succeeded under that condition. The relationship between transfection efficiency and higher-order structure of the DNA carrier-complex was discussed.
We report the solid-phase synthesis by the Fmoc strategy of a peptide containing a cysteamide group at its C-terminus. This peptide was subjected to further modifications including the linkage of fluorophores, namely lucifer yellow and coumarin respectively, at the C- and/or N-terminals. After incubation with living cultured cells these two probes were localized and it is concluded that the post-synthesis modifications can strongly modify the localization of the peptide.
Two peptides designed for drug delivery were generated by the combination of a signal peptide with a nuclear localization sequence and are shown to facilitate the cellular internalization of small molecules which are covalently linked to these peptides. In order to understand the mechanism of internalization, the conformations of the peptides were investigated through different approaches both in solution and in membrane-mimicking environments. These peptides are highly versatile and adopt different conformational states depending on their environment. While in a disordered form in water, they adopt an alpha-helical structure in TFE and in the presence of micelles of SDS or DPC. The structured domain encompasses the hydrophobic part of the peptides, whereas the charged C-termini remain unstructured. In contrast, in the presence of lipids and whatever the nature of the phosphate headgroup, the two peptides mainly adopt an antiparallel beta-sheet form and embed in the lipidic cores. This result suggests that the beta-sheet is responsible for the translocation through the cellular membranes but also questions the conformational state of signal peptides when associated to hydrophilic sequences.
Transportan is a 27 amino acid-long peptide containing 12 functional amino acids from the amino terminus of the neuropeptide galanin and mastoparan in the carboxyl terminus, connected via a lysine. Transportan is a cell-penetrating peptide as judged by indirect immunofluorescence using N epsilon13-biotinyl-transportan. The internalization of biotinyl-transportan is energy independent and takes place efficiently at 37 degrees, 4 degrees, and 0 degrees C. Cellular uptake of transportan is probably not mediated by endocytosis, since it cannot be blocked by treating the cells with phenylarsine oxide or hyperosmolar sucrose solution and is nonsaturable. The kinetics of internalization was studied with the aid of the 125I-labeled peptide. At 37 degrees C, the maximal intracellular concentration is reached in about 20 min. The internalized transportan is protected from trypsin. The cell-penetrating ability of transportan is not restricted by cell type, but seems to be a general feature of this peptide. In Bowes' melanoma cells, transportan first localizes in the outer membrane and cytoplasmatic membrane structures. This is followed by a redistribution into the nuclear membrane and uptake into the nuclei where transportan concentrates in distinct substructures, probably the nucleoli.
Peptides containing a hydrophobic motif associated with a nuclear localization signal separated by various linkers were synthesized in solid phase. The hydrophobic sequence corresponds either to a signal peptide sequence or to a fragment of the fusion peptide of GP41 while the hydrophilic sequence is that of a nuclear localization signal. The C-termini of these peptides bear a cysteamide group that was linked to a fluorescent probe. This allowed the cellular localization of the probe to be determined as a function of the peptide sequences. The labeled peptides were then incubated with fibroblasts. Using N-biotinylated derivatives we confirmed by indirect immunofluorescence that the observed localizations corresponds to those of the peptides. The presence of a linker appears to play a role in the cellular localization. One of these peptides was successfully used to target fluorescent oligodeoxynucleotides into living cells demonstrating improved cell delivery of peptide-oligodeoxynucleotide conjugates.
The herpes simplex virus type 1 (HSV-1) virion protein VP22 exhibits the remarkable property of intercellular trafficking whereby the protein spreads from the cell in which it is synthesized to many surrounding cells. In addition to having implications for protein trafficking mechanisms, this function of VP22 might be exploited to overcome a major hurdle in gene therapy, i.e., efficient delivery of genes and gene products. We show that chimeric polypeptides, consisting of VP22 linked to the entire p53 protein, retain their ability to spread between cells and accumulate in recipient cell nuclei. Furthermore the p53-VP22 chimeric protein efficiently induces apoptosis in p53 negative human osteosarcoma cells resulting in a widespread cytotoxic effect. The intercellular delivery of functional p53-VP22 fusion protein is likely to prove beneficial in therapeutic strategies based on restoration of p53 function. These results, demonstrating intracellular transport of large functional proteins, indicate that VP22 delivery may have applications in gene therapy.
The peptide SP-NLS (Ac-Met-Gly-Leu-Gly-Leu-His-Leu-Leu-Leu-Ala10-Ala-Ala-Leu-Gln-Gly- Ala -Lys-Lys-Lys-Arg20-Lys-Val-NH-CH2-CH2-SH) is composed of a hydrophobic signal sequence (SP, Met-1 to Ala-16) followed by a polycationic nuclear localization sequence (NLS, Lys-17 to Val-22) terminated by a cysteamide group. Designed to act as drug carrier this primary amphipathic peptide proved cytotoxic and bactericidal when used at high concentrations, probably by inducing the formation of ion channels. In this work, we show that indeed SP-NLS exhibits a pore-forming activity when incorporated into planar lipid bilayers and Xenopus laevis oocyte plasma membranes, with conductance values of 25 pS in 0.1 M NaCl. In both membranes, the insertion of the peptide was voltage-triggered whereas the induced conductances proved almost voltage-independent. Moreover, SP-NLS ion channels were selective for monovalent cations (K+>Na+>Li+>tetraethylammonium+>choline+). The ion channel activity of this type of peptides thus provides some insight on their toxicity but also on the mechanism involved for their membrane crossing process.
Evidence that multiple, probably non-endocytic mechanisms are involved in the uptake into mammalian cells of the alpha-helical amphipathic model peptide FLUOS-KLALKLALKALKAALKLA-NH2 (I) is presented. Extensive cellular uptake of N-terminally GC-elongated derivatives of I, conjugated by disufide bridges to differently charged peptides, indicated that I-like model peptides might serve as vectors for intracellular delivery of polar bioactive compounds. The mode of the cellular internalization of I comprising energy-, temperature-, pH- and ion-dependent as well as -independent processes suggests analogy to that displayed by small unstructured peptides reported previously (Oehlke et al., Biochim. Biophys. Acta 1330 (1997) 50-60). The uptake behavior of I also showed analogy to that of several protein-derived helical peptide sequences, recently found to be capable of efficiently carrying tagged oligonucleotides and peptides directly into the cytosol of mammalian cells (Derossi et al., J. Biol. Chem. 269 (1994) 10444-10450; Lin et al., J. Biol. Chem. 270 (1995) 14255-14258; Fawell et al., Proc. Natl. Acad. Sci. USA 91 (1994) 664-668; Chaloin et al., Biochemistry 36 (1997) 11179-11187; Vives et al., J. Biol. Chem., 272 (1997) 16010-16017).
We studied the mechanisms involved in the translocation of human calcitonin (hCT) through excised bovine nasal mucosa (net mucosal-to-serosal permeability approximately 10(-)5 cm s-1). To determine structural requirements for the suggested vesicular internalization two carboxyfluorescein-labeled (fl) hCT fragments, the C-terminal fragment [Nalpha-fl]hCT(9-32) and the N-terminal fragment [Lys(fl)18]hCT(1-24) were synthesized. In presence of the endocytosis inhibitor cytochalasin D mucosal-to-serosal and serosal-to-mucosal hCT permeabilities were equal. Pathway visualization by confocal laser scanning microscopy showed punctated fluorescence indicating vesicular internalization of both hCT and [Nalpha-fl]hCT(9-32). In contrast, the N-terminal fragment lacking the beta-sheet forming C-terminus (25-32) was not internalized. Circular dichroism showed that, when interacting with neutral and negatively charged liposomes, hCT adopts beta-sheet conformation. In a concentrated aqueous solution, beta-sheet formation induces hCT self-assembly and fibrillation. High partitioning of hCT into lipid bilayer membranes was reflected by an apparent partition coefficient log D(pH 7.4) = 2.5 (liposome-buffer equilibrium dialysis). We propose that the high lipid partitioning and beta-sheet formation result in C-terminus-restricted supramolecular self-assembly of hCT and [Nalpha-fl]hCT(9-32) in lipid membranes. Vesicular internalization is suggested to be associated with self-assembly induced perturbation of the lipid bilayer. Condensed hCT self-assemblies may explain the high capacity of net mucosal-to-serosal hCT permeation, which compares favorably with the low transport capacity of receptor-mediated endocytosis.
The structure of the cell-permeable alpha-helical amphipathic model peptide FLUOS-KLALKLALKALKAALKLA-NH2 (I) was modified stepwise with respect to its helix parameters hydrophobicity, hydrophobic moment and hydrophilic face as well as molecular size and charge. Cellular uptake and membrane destabilizing activity of the resulting peptides were studied using aortic endothelial cells and HPLC combined with CLSM. With the exceptions that a reduction of molecule size below 16 amino acid residues and the introduction of a negative net charge abolished uptake, none of the investigated structural parameters proved to be essential for the passage of these peptides across the plasma membrane. Membrane toxicity also showed no correlation to any of the parameters investigated and could be detected only at concentrations higher than 2 microM. These results implicate helical amphipathicity as the only essential structural requirement for the entry of such peptides into the cell interior, in accord with earlier studies. The pivotal role of helical amphipathicity was confirmed by uptake results obtained with two further pairs of amphipathic/non-amphipathic 18-mer peptides with different primary structure, net charge and helix parameters from I. The amphipathic counterparts were internalized into the cells to a comparable extent as I, whereas no cellular uptake could be detected for the non-amphipathic analogues. The mode of uptake remains unclear and involves both temperature-sensitive and -insensitive processes, indicating non-endocytic contributions.
Loligomers are peptide-based intracellular vehicles able to penetrate cells and self-localize into distinct cellular compartments. Loligomers can be rapidly assembled by automated solid-phase approaches and were designed to act as nonviral, nonlipophilic intracellular shuttles. One nucleus-directed loligomer, termed loligomer 4, was evaluated for its ability to function as a transfection agent. Loligomer 4 readily associates with plasmids to form noncovalent complexes. The migration of loligomer 4-plasmid complexes into cells was monitored by flow cytometry and fluorescence microscopy. Populations of plasmids labeled with 7-AAD exist either free or in association with loligomer 4 inside cells and are visible throughout the cytosol and nucleus of chinese hamster ovary (CHO) cells. Loligomer 4-plasmid complexes were not cytotoxic to cells and were readily imported by most cells (>70%). CHO cells were transfected with complexes of loligomer 4 and plasmids harboring luciferase, green fluorescent protein or beta-galactosidase reporter genes. The transfection efficiency of loligomer 4-plasmid DNA complexes was greater when cells were maintained as suspensions instead of monolayers. Transfections could be performed with cells suspended in serum-containing medium. The observed levels of transfection, however, were modest with 5-10% of CHO cells expressing either a green fluorescent protein or the enzyme beta-galactosidase. Loligomers have recently been observed in vesicular compartments [Singh, D., Kiarash, R., Kawamura, K, LaCasse, E. C., and Gariépy, J. (1998) Biochemistry 37, 5798-5809] and differences between levels of cellular import and transfection efficiency may well reflect the need to optimize the release of loligomers and their complexes from these compartments in future designs. In summary, loligomer 4 behaves as a stable, soluble and effective transfection agent. These results demonstrate the feasibility of designing loligomers able to act as intracellular guided agents aimed at gene transfer applications.
Photodynamic therapy (PDT) is a targeted treatment modality where photosensitizers accumulate into cells and are selectively activated by light leading to the production of toxic species and cell death. Focusing the action of photosensitizers to a unique intracellular target may enhance their cytotoxicity. In this study, we demonstrate that the routing of the porphyrin-based photosensitizer chlorin e(6), to the nucleus of cells can significantly alter its toxicity profile. The cellular localization of chlorin e(6) was achieved by coupling the chromophore during solid-phase synthesis to a nucleus-directed linear peptide (Ce6-peptide) or a branched peptide (Ce6-loligomer) composed of eight identical arms displaying the sequence of the Ce6-peptide. These constructs incorporated signals guiding their cytoplasmic uptake and nuclear localization. Ce6-peptide and Ce6-loligomer displayed an enhanced photodynamic activity compared to unconjugated chlorin e(6), lowering the observed CD(50) values for CHO and RIF-1 cells by 1 or more orders of magnitude. The intracellular accumulation of Ce6-peptide and Ce6-loligomer was assessed by electron and confocal microscopy as well as by flow cytometry. Constructs were internalized by cells within an hour and by 6 h, the release of active oxygen species could be observed within the nucleus of cells pretreated with Ce6-loligomer. These results highlight the utility of designing peptides as vehicles for regulating the intracellular distribution of photosensitizers such as chlorin e(6) in order to maximize their efficacy in PDT.
The in vivo Tat–β-gal studies in mice show that protein transduction holds a tremendous amount of potential for manipulation of model organisms and protein therapy; however, this needs to be tempered until several important questions are answered. First, Tat–β-gal is 120 kDa and requires assembly into a homotetramer before it is active, although it has a high specific activity and therefore, probably requires a low level of protein to detect in tissue sections. The administration of additional Tat fusion proteins to model organisms that generate phenotypic changes is necessary to get a sense of how much potential in vivo protein transduction really holds.Second, the immunogenicity of transduced proteins is a significant and currently unknown concern. Although future protein therapies will probably comprise proteins that are ∼99% of human origin, the PTD might be antigenic or other aspects of the delivery might initiate an immune response. Several variations of PTDs have been described and therefore an immune response to one PTD might be engineered by alteration of anchor residues or TCR recognition motifs on the PTD to reduce or avoid major histocompatibility complex (MHC) presentation. However, what does the immune system do when all antigen-presenting cells (including macrophages, and T and B cells) are presenting the same foreign antigen? This presents a unique unanswered immunological question. One qualitative piece of evidence does exist; we injected mice once a day for 14 consecutive days with Tat–GFP protein and we failed to see any gross signs of a severe immune response. This observation also suggests that continuous transduction of proteins into all cells of a mouse, including into the brain, are not detrimental to viability.Lastly, transduced proteins appear to be cleared from the body not by conventional mechanisms, but based on the half-life of the protein. Therefore, the half-life of the PTD fusion proteins could be extended or shortened depending on the specific application. The most exciting aspect of this technology is the previously unheard-of ability to address specific mutations and deletions of certain disease-causing genes. We look forward to being able to restore tumour suppressor function or interfere with various oncogenic pathways using this technology in the coming years.
The penetratin peptide, a 16 amino acid sequence extracted from Antennapedia homeodomain, is able to translocate across a neural cell membrane through an unknown mechanism, most likely a non-specific interaction with membrane lipids. Beyond its potential application as vector targeting small hydrophilic molecules and enabling them to reach a cell nucleus, this observation raises intriguing questions concerning the physico-chemistry of peptide-lipid interactions. Here we present a study of the role of lipid surface pressure and head charge on the mechanism of interaction. This was performed using optical techniques: surface infrared spectroscopy and ellipsometry, applied to a monolayer of phospholipids deposited at the air-water interface. Determination of the structure and orientation of peptides and lipids (separately or together) evidenced that electrostatic rather than amphiphilic interactions determine the peptide adsorption and its action on lipids.
Novel analogues of the cell-penetrating peptides penetratin and transportan were synthesized. The distribution of the biotin-labeled peptides in Bowes melanoma cell line has been investigated by indirect fluorescence with fluorescein-streptavidin detection. The time course of uptake of (125)I-labeled transportan analogues has been characterized in the same cell line. Molecular modeling was used to analyze the penetration and the orientation of molecules in a simulated biological membrane. The results, both from molecular modeling and fluorescence studies, imply that penetratin and transportan do not enter the cells by related mechanisms and that they do not belong to the same family of translocating peptides.
The potential use of polypeptides and oligonucleotides for therapeutical purposes has been questioned because of their inherently poor cellular uptake. However, the 16-mer oligopeptide penetratin, derived from the homeodomain of Antennapedia, has been reported to enter cells readily via a non-endocytotic and receptor- and transporter-independent pathway, even when conjugated to large hydrophilic molecules. We here present the first study where penetratin is shown to traverse a pure lipid bilayer. The results support the idea that the uptake mechanism involves only the interaction of the peptide with the membrane lipids. Furthermore, we conclude that the translocation does not involve pore formation.