Article

Pre-administration of PepFect6-microRNA-146a nanocomplexes inhibits inflammatory responses in keratinocytes and in a mouse model of irritant contact dermatitis

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Abstract

The skin is a difficult to access tissue for efficient delivery of large and/or charged macromolecules, including therapeutic DNA and RNA oligonucleotides. Cell-penetrating peptide PepFect6 (PF6) has been shown to be suitable transport vehicle for siRNAs in cell culture and systemically in vivo in mice. miR-146a is known as anti-inflammatory miRNA that inhibits multiple factors from the nuclear factor (NF)-κB pathway in various cell types, including keratinocytes. In this study, PF6 was shown to form unimodal nanocomplexes with miR-146a mimic that entered into human primary keratinocytes, where miR-146a inhibited the expression of its direct targets from the NF-κB pathway and the genes known to be activated by NF-κB, C-C motif ligand (CCL)5 and interleukin (IL)-8. The transfection of miR-146a mimic with PF6 was more efficient in sub-confluent keratinocyte cultures, affected keratinocyte proliferation less and had similar effect on cell viability when compared with a lipid based agent. Subcutaneous pre-administration of PF6-miR-146a nanocomplexes attenuated ear-swelling and reduced the expression of pro-inflammatory cytokines and chemokines IL-6, CCL11, CCL24 and C-X-C motif ligand 1 (CXCL1) in a mouse model of irritant contact dermatitis. Our data demonstrates that PF6-miR-146a nanoparticles might have potential in the development of therapeutics to target inflammatory skin diseases.

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... A member of this family, PF14, is an amphipathic CPP designed based on PepFect3, a transportan10 peptide modified with stearic acid at the N-terminus. PF14 has been widely used for the delivery of different NA cargoes, such as SCO [18], siRNA [19], miRNA [20,21], plasmid DNA [22,23], and mRNA [24], in both in vitro and in vivo applications [18,20]. When mixed in an aqueous milieu, PF14 can form colloidal nanoparticles (NPs) with NA cargo through electrostatic and hydrophobic interactions between the carrier and payload [18,25,26]. ...
... respective in vitro assays. Furthermore, we assessed the efficacy of potent PF14 novel analogs in delivering of mimics of anti-inflammatory miR-146a [20,21] into primary human keratinocytes. Finally, we tested the most efficient analog from in the in vitro screening, PF14-Lys, for in vivo delivery of miR-146a mimics in a mouse model of irritant contact dermatitis (ICD). ...
... To induce ICD, a topical application of 0.2 % 12-myristate 13-acetate (PMA) in acetone was applied 1 h post-injection. Ear thickness was measured at three different locations using a digital caliper (Mitutoyo, Tokyo, Japan), as previously described [21]. After 24 h from the injection, mice were euthanized with CO 2 , and ears were harvested for further analysis. ...
... One promising class of carriers, which we have shown to be tolerated well by multiple human primary cell types are cellpenetrating peptides (CPPs) [5,[12][13][14]. To prove the applicability of a particular set of CPPs in delivery of miRNAs in cell cultures and in vivo, we have used self-forming nanocomplexes of PepFect (PF) 14 or NickFect (NF) type of CPPs, NF70, NF71, NF72 and mimicking oligo for a well-characterized anti-inflammatory miR-146a [5,13,14]. ...
... One promising class of carriers, which we have shown to be tolerated well by multiple human primary cell types are cellpenetrating peptides (CPPs) [5,[12][13][14]. To prove the applicability of a particular set of CPPs in delivery of miRNAs in cell cultures and in vivo, we have used self-forming nanocomplexes of PepFect (PF) 14 or NickFect (NF) type of CPPs, NF70, NF71, NF72 and mimicking oligo for a well-characterized anti-inflammatory miR-146a [5,13,14]. PF14 was chosen because of its efficiency and enhanced capacity to release the cargo from the complex in delivery of siRNAs shown in previous studies [15,16]. As compared to its predecessor CPP, TP10, PF14 contains multiple ornithines instead of lysines enabling stronger binding to nucleic acids, and N-terminal stearyl, which ensures more efficient release from the endosome [16]. ...
... As our focus is inflammatory diseases affecting epithelium, such as atopic dermatitis and asthma, we use normal human epithelial keratinocytes (NHEKs), normal human bronchial epithelial (NHBE) cells and monocyte-derived dendritic cells (moDCs) as test cell cultures and corresponding mouse models [5,[12][13][14]. We specifically are focused on inflamed epithelium, as in the case of inflammation, the epithelial barrier is damaged and tight junctions between the cells are opened making the tissue more accessible to CPP-miRNA nanocomplexes [18,19]. ...
Chapter
microRNAs (miRNAs) have capacity to modulate numerous biological processes and therefore synthetic oligonucleotides mimicking or inhibiting particular miRNA have potential in the development of novel types of therapeutics. We have elaborated several methods for safe and efficient overexpression of miRNAs using self-forming nanocomplexes of PepFect or NickFect type of cell-penetrating peptides (CPPs) and oligonucleotides mimicking well-characterized anti-inflammatory miR-146a. We focus on chronic inflammatory diseases affecting epithelium, such as atopic dermatitis and asthma harnessing respective cell cultures and mouse models. Here we provide protocols for miRNA transfection into primary human keratinocytes, primary human bronchial epithelial cells, and human monocyte-derived dendritic cells using CPPs PepFect14, NickFect70, NickFect71, and NickFect72. In addition, we provide protocols for application of CPP-miRNA nanocomplexes in in vivo mouse models of irritant contact dermatitis and allergic airway inflammation.
... 31,32 In nanocomplexes with a cell-penetrating peptide, miR-146 was able to enter human keratinocytes and exert suppression of direct targets. 46 Subcutaneous administration of these nanocomplexes led to a decreased ear swelling and reduced expression of proinflammatory factors in a mouse model of irritant contact dermatitis, thereby showing the potential suitability of miR-146 as a therapeutic agent for inflammatory skin diseases. 46 By contrast with miR-146, miR-155 has proinflammatory properties. ...
... 46 Subcutaneous administration of these nanocomplexes led to a decreased ear swelling and reduced expression of proinflammatory factors in a mouse model of irritant contact dermatitis, thereby showing the potential suitability of miR-146 as a therapeutic agent for inflammatory skin diseases. 46 By contrast with miR-146, miR-155 has proinflammatory properties. By targeting, and consequently downregulating CTLA4, an inhibitor of T-cell activation, miR-155 enhances proliferation of activated CD4+ T cells. ...
... 51 All four overexpressed miRNAs in allergic skin also were found to be overexpressed in the skin specimens of dogs with demodicosis and dermatophytosis when compared to healthy skin in this study. This, together with the upregulated expression in other skin diseases, such as psoriasis, 39,44,45,49 irritant contact dermatitis 40,46 or hidradenitis suppurativa, 52 suggests a general role for these miRNAs in inflammatory skin responses. Increased expression of miR-142, miR-146a, miR-155 and miR-21 seems to be a characteristic of inflammatory skin conditions in general, rather than a cAD-specific expression pattern. ...
Article
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Background MicroRNAs (miRNA) are short, single‐stranded RNA molecules that regulate gene expression in a post‐transcriptional manner. Their expression is proposed to be tissue‐specific and alterations in miRNA expression have been detected in many diseases. Objective To compare miRNA expression in the skin of healthy Labrador and golden retrievers, and those with allergic and nonallergic dermatitis. Methods and materials Formalin‐fixed and paraffin‐embedded (FFPE) skin specimens from seven healthy Labrador and golden retrievers, and seven dogs with allergic skin disease were collected. A further mixed nonallergic inflammation group consisted of samples from five dogs with fungal infection, demodicosis and mast cell tumours. Total RNA was extracted and miRNA primer assays for 18 target miRNAs (miR‐142, miR‐363, miR‐18b, miR‐451, miR‐146a, miR‐124, miR‐409, miR‐193b, miR‐223, miR‐215, miR‐155, miR‐423a, miR‐143, miR‐1839, miR‐21, miR‐34b, miR‐146b and miR‐202) were performed, with RNU6‐2 and SNORD95 as miRNAs for normalisation. The selection of miRNAs for investigation was based on reported data and a pilot study evaluating miRNA extraction from FFPE tissue specimens. Results In the two dogs with mast cell tumours, miRNA expression was undetermined for most miRNAs, so both were excluded from analysis. Although there were differences in the miRNA expression between healthy and inflamed skin, allergic and nonallergic inflammation showed similar expression patterns. Conclusion and clinical relevance Although the number of included dogs was small, based on this study, none of the evaluated miRNAs allowed differentiation of allergic dermatitis from other inflammatory skin diseases in retriever dogs.
... Cell-penetrating peptides (CPPs) are a broad group of short peptides with membrane translocation activity, which have been successfully applied in vitro and in vivo to deliver potentially therapeutic molecules, including DNA, RNA, siRNA [19] and recently also miRNA mimics [20,21] into different cell types with minimal toxicity. CPPs condense the negatively charged NAs into self-forming non-covalent complexes through electrostatic interactions and thereby successfully deliver NAs into the cells. ...
... Peptides were incubated with miRNA mimic at a 17:1 CPP:miRNA molar ratio (MR) in MQ-water in 1/10 of final treatment volume at room temperature (RT) for 1 h to form CPP:miRNA complexes; mixed with media and added to the cells after removing the old growth media as previously described [20]. Transfections were performed at 30 nM or 100 nM (miRIDIAN microRNA Mimic Negative Control #1 and miRI-DIAN microRNA hsa-miR-146a mimic; all from Thermo Fisher Scientific, USA) concentration of miRNA mimics in KCs and DCs, respectively, for 48 h or 24 h. ...
... Next, all selected CPPs (Table 1) were used to deliver miR-146a and the control mimic into KCs and mRNA levels of target genes for miR-146a as well as related chemokines [20] were measured to assess the effect. Liposomal reagent LF2000 (Fig. 2) and naked mimic (Supplementary Fig. S2) were used as controls, CPP:miRNA MR ratio was 17:1. ...
Article
MicroRNAs (miRNAs) are post-transcriptional gene expression regulators with potential therapeutic applications. miR-146a is a negative regulator of inflammatory processes in both tissue-resident and specialized immune cells and may therefore have therapeutic effect in inflammatory skin diseases. PepFect (PF) and NickFect (NF) type of cell-penetrating peptides (CPPs) have previously been shown to deliver miRNA mimics and/or siRNAs into cell cultures and in vivo. Here, we first demonstrate that selected PF- and NF-type of CPPs support delivery of fluorescent labelled miRNA mimics into keratinocytes (KCs) and dendritic cells (DCs). Second, we show that both PF- and NF-miR-146a nanocomplexes were equally effective in KCs, while NFs were more efficient in DCs as assessed by downregulation of miR-146a-influenced genes. None of miRNA nanocomplexes with the tested CPPs influenced the viability of KCs and DCs nor caused activation of DCs according to CD86 and CD83 markers. Transmission electron microscopy analysis with Nanogold-labelled miR-146a mimics and assessment of endocytic trafficking pathways revealed endocytosis as an active route of delivery in both KCs and DCs for all tested CPPs. However, consistent with the higher efficiency, NF-delivered miR-146a was detected more often outside endosomes in DCs. Finally, pre-injection of NF71:miR-146a nanocomplexes was confirmed to suppress inflammatory responses in a mouse model of irritant contact dermatitis as shown by reduced ear swelling response and downregulation of pro-inflammatory cytokines, including IL-6, IL-1β, IL-33 and TNF-α. In conclusion, NF71 efficiently delivers miRNA mimics into KCs as well as DCs, and therefore may have advantage in therapeutic delivery of miRNAs in case of inflammatory skin diseases.
... In human primary keratinocytes, miR-146a inhibits the expression of many proinflammatory factors, including CCL5 and IL-8 ( Meisgen et al., 2014;Rebane et al., 2014). miR-146a has been shown to act as anti-inflammatory miRNA in mouse models of atopic dermatitis ( Rebane et al., 2014), irritant contact dermatitis ( Urgard et al., 2016), and psoriasis ( Srivastava et al., 2017). The single nucleotide polymorphism (SNP) rs2910164 located in the miR-146a precursor has been suggested to be associated with psoriasis in Han Chinese population ( Zhang et al., 2014) and among Caucasians ( Srivastava et al., 2017). ...
... Although we did not find reverse correlation of miR-146a/b and analyzed targets in psoriatic skin, there is strong evidence that the endogenous level of miR-146a is sufficient for the suppression of inflammatory responses and that local administration of miR-146a mimics has an anti-inflammatory effect in the skin. Accordingly, 146a À/À mice developed more severe disease in mouse models of atopic dermatitis and psoriasis, whereas preinjection of miR-146a alleviated the inflammation in mouse models of irritant contact dermatitis and psoriasis ( Rebane et al., 2014;Srivastava et al., 2017;Urgard et al., 2016). As miR-146a/b have a similar expression level in vivo in human and mouse skin, miR-146a/ b together might have a remarkably stronger influence than studies performed with miR-146a À/À mice demonstrate. ...
... In conclusion, the capacity of miR-146a/b simultaneously to inhibit inflammatory responses, activation-induced cell death, and proliferation of keratinocytes and fibroblasts suggests that miR-146a/b are mainly pacifying miRNAs that contribute to the skin homeostasis and controlling of inflammatory responses in both healthy and diseased skin. In addition, our results are in line with recently published in vivo mouse studies ( Srivastava et al., 2017;Urgard et al., 2016) suggesting that overexpression or local administration of miR-146a/b mimics may have potential for the treatment of psoriasis. ...
Article
miR-146a inhibits inflammatory responses in human keratinocytes and in different mouse models of skin inflammation. Little is known about the role of miR-146b in the skin. In the present study, we confirmed the increased expression of miR-146a and miR-146b (miR-146a/b) in lesional skin of psoriasis patients. The expression of miR-146a was about 2-fold higher than that of miR-146b in healthy human skin and it was more strongly induced by stimulation of pro-inflammatory cytokines in keratinocytes and fibroblasts. miR-146a/b target genes regulating inflammatory responses or proliferation were altered in the skin of psoriasis patients, among which FERMT1 was verified as direct target of miR-146a. In silico analysis of genome-wide data from >4,000 psoriasis cases and >8,000 controls confirmed a moderate association between psoriasis and genetic variants in miR-146a gene. Transfection of miR-146a/b suppressed and inhibition enhanced keratinocyte proliferation and the expression of psoriasis-related target genes. Enhanced expression of miR-146a/b-influenced genes was detected in cultured keratinocytes from miR-146a-/- and skin fibroblasts from miR-146a-/- and miR-146b-/- mice stimulated with psoriasis-associated cytokines as compared to wild type mice. Our results indicate that besides miR-146a, miR-146b is expressed and might be capable of modulation of inflammatory responses and keratinocyte proliferation in psoriatic skin.
... When used for transfection of miR-146a mimics, PF6 demonstrated superior efcacy in keratinized cell cultures compared to lipid-based reagents, with minimal impact on cell proliferation and similar efects on cell viability. In a mouse model of irritant contact dermatitis, subcutaneous administration of PF6-miR-146a nanocomplexes signifcantly reduced ear edema and decreased the expression of proinfammatory cytokines and chemokines, including IL-6, CCL11, CCL24, and CXCL1 [120]. Tese fndings suggest that PF6-miR-146a nanoparticles hold promise as therapeutic interventions for infammatory skin conditions. ...
Article
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Atopic dermatitis (AD) is a chronic inflammatory skin disorder affecting approximately 20% of children and 10% of adults. While previous studies have linked AD to allergen exposure, disruption of the skin barrier, and Type 2 immune responses, the precise pathophysiology of AD remains elusive, significantly limiting the effectiveness of current treatments. Noncoding RNAs (ncRNAs), a diverse group of transcripts that do not encode proteins and account for at least 98% of the human genome, are implicated in numerous physiological and pathological processes. A growing body of evidence underscores the pivotal role of ncRNAs in the pathogenesis and progression of AD. This review offers a detailed synthesis of the latest insights into the involvement of ncRNAs in AD, as well as their potential as diagnostic biomarkers and therapeutic targets.
... These challenges are being overcome through various delivery systems or carriers that have been utilized to enhance topical miRNA delivery in to target cells [ Table 5]. [94][95][96][97][98][99][100][101][102] ...
Article
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Engagement of microribonucleic acids (miRNA) in the regulation of cutaneous cellular health and diseases is a rapidly advancing niche in dermatology basic research. miRNAs have been identified to play a key role in the pathogenesis of various cutaneous inflammatory, autoimmune and neoplastic conditions, among others. In addition, their purported role as therapeutic targets and biomarkers in diseased conditions harbours exciting news for the approaching years in clinical research. The current review outlines the possible translational role of miRNA in skin health and diseases (encompassing pathogenesis, diagnosis, biomarkers and therapy) from bench to bedside.
... As in the case of inflammation delivery conditions change, we used the mouse model of ICD to generate an inflammatory environment. The particular model was chosen as a short "proof-of-concept" model that we had previously used successfully to test anti-inflammatory effect of miRNA nanoparticles formed with PF6 or NictFect71 (Urgard et al., 2016;Carreras-Badosa et al., 2020). Currently, inflammatory skin diseases are treated either with various topical treatments, systemic steroids, or biologicals. ...
Article
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mRNA-based vaccines and candidate therapeutics have great potential in various medical fields. For the delivery of mRNA into target cells and tissues, lipid formulations are often employed. However, this approach could cause the activation of immune responses, making it unsuitable for the treatment of inflammatory conditions. Therefore, alternative delivery systems are highly demanded. In this study, we evaluated the transport efficiency and characteristics of cell-penetrating peptide PepFect14 (PF14) and mRNA nanoparticles in the presence of different additives. Our results show that all PF14-mRNA formulations entered cultured cells, while calcium chloride enhanced the transport and production of the encoded protein in HeLa and HaCaT cell lines, and polysorbate 80 did so in primary human keratinocytes. All formulations had similar physical properties and did not remarkably affect cell viability. By selectively blocking endocytosis pathways, we show that PF14-mRNA nanoparticles primarily entered HeLa cells via macropinocytosis and HaCaT cells via both macropinocytosis and clathrin-mediated endocytosis, while none of the blockers significantly affected the delivery into primary keratinocytes. Finally, subcutaneous injection of PF14-mRNA nanoparticles before inducing mouse irritant contact dermatitis resulted in the expression of a reporter protein without provoking harmful immune responses in the skin. Together, our findings suggest that PF14-mRNA nanoparticles have the potential for developing mRNA-based therapeutics for treating inflammatory skin conditions.
... The impact of formulation on ncRNA persistence in skin has also been demonstrated by Ma et al., 2014, where siRNA at the administration site following subcutaneous injection was observed for up to 7 days when formulated with chitosan, while siRNA alone persisted for less than one day. Intradermal (Srivastava et al., 2017) or subcutaneous (Urgard et al., 2016) injection of formulated miRNAs has also demonstrated the ability to modify inflammatory responses at the target site. Regarding non-injection routes of administration, Desmet et al. (2016) used a liposomal encapsulation method which allowed delivery of siRNA to the epidermis in human skin samples. ...
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JT03522700 OFDE This document, as well as any data and map included herein, are without prejudice to the status of or sovereignty over any territory, to the delimitation of international frontiers and boundaries and to the name of any territory, city or area.
... It carries a positive charge of +5 that enables the formation of non-covalent complexes with NAs, which are internalised by cells via scavenger receptor-mediated endocytosis [38][39][40]. Peptides from the PepFect family have shown high efficiency in the delivery of SCO [39,41], siRNA [42], miRNA [43][44][45][46], and other types of NA molecules into mammalian cells both in vitro and in vivo and have been extensively studied by our group for several years [39,40,47,48]. ...
Article
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Cell-penetrating peptides (CPPs) are highly promising transfection agents that can deliver various compounds into living cells, including nucleic acids (NAs). Positively charged CPPs can form non-covalent complexes with negatively charged NAs, enabling simple and time-efficient nanoparticle preparation. However, as CPPs have substantially different chemical and physical properties, their complexation with the cargo and characteristics of the resulting nanoparticles largely depends on the properties of the surrounding environment, i.e., solution. Here, we show that the solvent used for the initial dissolving of a CPP determines the properties of the resulting CPP particles formed in an aqueous solution, including the activity and toxicity of the CPP–NA complexes. Using different biophysical methods such as dynamic light scattering (DLS), atomic force microscopy (AFM), transmission and scanning electron microscopy (TEM and SEM), we show that PepFect14 (PF14), a cationic amphipathic CPP, forms spherical particles of uniform size when dissolved in organic solvents, such as ethanol and DMSO. Water-dissolved PF14, however, tends to form micelles and non-uniform aggregates. When dissolved in organic solvents, PF14 retains its α-helical conformation and biological activity in cell culture conditions without any increase in cytotoxicity. Altogether, our results indicate that by using a solvent that matches the chemical nature of the CPP, the properties of the peptide–cargo particles can be tuned in the desired way. This can be of critical importance for in vivo applications, where CPP particles that are too large, non-uniform, or prone to aggregation may induce severe consequences.
... The skin-permeation of bioactives can be improved by using CPPs as penetration enhancers [151]. Urgard et al. [152] used the CPP PepFect6 to form a nanocomplex with miR-146a for treating irritant dermatitis. The nanocomplex exhibited a spherical and homogeneous particle distribution with an average diameter of 30-50 nm. ...
Article
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Skin inflammation occurs due to immune dysregulation because of internal disorders, infections, and allergic reactions. The inflammation of the skin is a major sign of chronic autoimmune inflammatory diseases, such as psoriasis, atopic dermatitis (AD), and lupus erythematosus. Although there are many therapies for treating these cutaneous inflammation diseases, their recurrence rates are high due to incomplete resolution. MicroRNA (miRNA) plays a critical role in skin inflammation by regulating the expression of protein-coding genes at the posttranscriptional level during pathogenesis and homeostasis maintenance. Some miRNAs possess anti-inflammatory features, which are beneficial for mitigating the inflammatory response. miRNAs that are reduced in inflammatory skin diseases can be supplied transiently using miRNA mimics and agomir. miRNA-based therapies that can target multiple genes in a given pathway are potential candidates for the treatment of skin inflammation. This review article offers an overview of the function of miRNA in skin inflammation regulation, with a focus on psoriasis, AD, and cutaneous wounds. Some bioactive molecules can target and modulate miRNAs to achieve the objective of inflammation suppression. This review also reports the anti-inflammatory efficacy of these molecules through modulating miRNA expression. The main limitations of miRNA-based therapies are rapid biodegradation and poor skin and cell penetration. Consideration was given to improving these drawbacks using the approaches of cell-penetrating peptides (CPPs), nanocarriers, exosomes, and low-frequency ultrasound. A formulation design for successful miRNA delivery into skin and target cells is also described in this review. The possible use of miRNAs as biomarkers and therapeutic modalities could open a novel opportunity for the diagnosis and treatment of inflammation-associated skin diseases.
... Moreover, because molecular imaging is such a powerful tool for visualization and quantification of pathological processes, such as cancer, Yang and colleagues recently demonstrated that a CPP, PepFect6, could also be used in complex with a radioactively-labeled AMO designed to target the oncomiR, miR-21, to successfully image miR-21 expression in A549 lung adenocarcinoma xenografts, thus demonstrating a promising method for the noninvasive imaging of miRNA expression levels in vivo [172]. Additionally, although not examples of cancer-related applications, peptides such as LMWP and PepFect6 have also been shown to successfully deliver miRNA mimics, including miR-29b, to stem cells to promote osteoblastic differentiation [173] and miR-146a (a known anti-inflammatory miRNA) to inhibit inflammatory responses in a murine model of irritant contact dermatitis [174], respectively. Lastly, regarding our own studies, we have also reported that CPPs can be effective carriers of therapeutic small ncRNAs, particularly siRNAs, which are similar to miRNAs in function in that they both can induce post-transcriptional gene silencing, but differ in that siRNAs typically inhibit the expression of a single mRNA target, whereas miRNAs normally regulate the expression of multiple mRNA targets [43]. ...
Article
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Three decades have passed from the initial discovery of a microRNA (miRNA) in Caenorhabditis elegans to our current understanding that miRNAs play essential roles in regulating fundamental physiological processes and that their dysregulation can lead to many human pathologies, including cancer. In effect, restoration of miRNA expression or downregulation of aberrantly expressed miRNAs using miRNA mimics or anti-miRNA inhibitors (anti-miRs/antimiRs), respectively, continues to show therapeutic potential for the treatment of cancer. Although the manipulation of miRNA expression presents a promising therapeutic strategy for cancer treatment, it is predominantly reliant on nucleic acid-based molecules for their application, which introduces an array of hurdles, with respect to in vivo delivery. Because naked nucleic acids are quickly degraded and/or removed from the body, they require delivery vectors that can help overcome the many barriers presented upon their administration into the bloodstream. As such, in this review, we discuss the strengths and weaknesses of the current state-of-the-art delivery systems, encompassing viral- and nonviral-based systems, with a specific focus on nonviral nanotechnology-based miRNA delivery platforms, including lipid-, polymer-, inorganic-, and extracellular vesicle-based delivery strategies. Moreover, we also shed light on peptide carriers as an emerging technology that shows great promise in being a highly efficacious delivery platform for miRNA-based cancer therapeutics.
... Nanocomplexes PepFect6-microRNA-146a Low expression of pro-inflammatory cytokines and chemokines IL-6, CCL11, and CCL24 in an animal model of irritant contact dermatitis [154] Vitiligo Nanodispersion TyRp-1 siRNA The developed formulation exhibited a higher rate of cellular uptake and presented an impressive (>80%) TyRP-1 target knockdown [155] Skin cancer Micelles siRNA Enhanced efficacy in retarding the progression and relapse of skin melanoma through the enhanced apoptosis effect both in vitro & in vivo [156] Liposome with NaChol edge activator BRAF siRNA Significant suppression of Tie2 mRNA expression. [157] Liposome TRP2 DNA (plasmid) Enhanced TRP2-specific immunity induced by a plasmid DNA cancer vaccine. ...
Article
Various skin problems are affected significantly by a combination of factors that include genetics, environmental factors, particular infections, and hitherto unexplained variables. The structure and function of key cellular networks may be altered by a single mutation, leading to significant phenotypic changes and the development of abnormal skin disorders in affected individuals. In recent years, there has been a great deal of discussion about the huge medicinal potential of nucleic acids. Although nucleic acids may be delivered to the desired location, there are a number of difficulties that must be solved before they can be used in clinical trials. Providing a comprehensive overview of numerous facts that highlight a range of skin-related autoimmune illnesses, as well as the use of dermal nanomedicine as a powerful tool for personalized therapy in the context of nuclear acid delivery, is our primary purpose in this paper. The most recent emerging trends and up-to-date metadata on various perspectives of dermal nanomedicines in different autoimmune diseases and related skin conditions as potential candidates for nucleic acid delivery have been gathered from a variety of databases that have been searched for potentially existing research to obtain the most recent emerging trends and up-to-date metadata. Existing research indicates that nanomedicines integrating nucleic acid, including liposomes, dendrimers, polymeric micelles, and other forms of nucleic acid delivery, have significant promise in the pharmaceutical industry. The current investigation focuses on the delivery of nucleic acid medications through the dermal route, as well as the therapeutic applications of these agents. Furthermore, the current state of nanomedicines has been discussed, as well as their potential and use.
... PF14 was first synthesized in 2011 by Ezzat and colleagues [26] and binds its cargos non-covalently, forming nanoparticles that infiltrate into cells mostly by inducing receptor-mediated endocytosis [26,28,29]. Members of PepFect family have shown great efficiency in transfecting siRNA [30,31], miRNA [7,[32][33][34] and SCO [28,35], and PF14-SCO nanoparticles have been studied by our group already for some years [28,29,36,37]. ...
Preprint
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Cell-penetrating peptides (CPPs) are promising tools for transfection of various substances, including nucleic acids, into cells. The aim of current work was to search for novel safe and effective approaches for enhancing transfection efficiency of nanoparticles formed of CPP and splice-correcting oligonucleotide (SCO) without increasing the concentration of peptide. We analyzed an effect of inclusion of calcium and magnesium ions into nanoparticles on CPP-mediated transfection in cell culture. We also studied the mechanism of such transfection as well as its efficiency, applicability in case of different cell lines, nucleic acid types and peptides, and possible limitations. We discovered a strong positive effect of these ions on transfection efficiency of SCO, that translated to enhanced synthesis of functional reporter protein. We observed significant changes in intracellular distribution and trafficking of nanoparticles formed with addition of the ions, without increasing cytotoxicity. We propose a novel strategy of preparing CPP-oligonucleotide nanoparticles with enhanced efficiency and, thus, higher therapeutic potential. Our discovery may be translated to primary cell cultures and, possibly, in vivo studies, in the aim to increase CPP-mediated transfection efficiency and likelihood of using CPPs in clinics.
... Further analysis showed that MPG and its mutant MPG ΔNLS delivered miR-122 mimic effectively regulated cholesterol levels in mouse hepatocyte, although adjustment of the miR-122 levels in cells treated with MPG ΔNLS /miR-122 complexes was higher than of MPG/miR-122 group with the same molar ratio. PF6 [112] was also shown to condense miR-146a mimic into nanocomplexes, entering human primary keratinocytes to inhibit the expression of target genes involved in the NF-κB pathway. Consequently, subcutaneous preadministration in a mouse model of irritant contact dermatitis demonstrated that PF6/miR-146a nanocomplexes attenuated ear-swelling and reduced the expression of pro-inflammatory cytokines and chemokines IL-6, CCL11, CCL24, and CXCL1. ...
Article
The safety issues like immunogenicity and unacceptable cancer risk of viral vectors for DNA/mRNA vaccine delivery necessitate the development of non-viral vectors with no toxicity. Among the non-viral strategies, cell-penetrating peptides (CPPs) have been a topic of interest recently because of their ability to cross plasma membranes and facilitate nucleic acids delivery both in vivo and in vitro. In addition to the application in the field of gene vaccine and gene therapy, CPPs based nucleic acids delivery have been proved by its potential application like gene editing, RNA-sequencing, and imaging. Here, we focus on summarizing the recent applications and progress of CPPs-mediated nucleic acids delivery and discuss the current problems and solutions in this field.
... The serum levels of IgE and cytokines were also significantly reduced by PEP-1-FK506BP. In another study by Urgard et al., PepFect6 (PF6) is used to deliver miR-146a mimics to inhibit the NF-κB pathway in the skin tissue [23]. PF6-CPP formed a nanocomplex with miR-146a mimics and was delivered to human primary keratinocytes to suppress the expression of genes in NF-κB downstream pathway. ...
Chapter
About 30 years ago, the discovery of CPP improved the therapeutic approach to treat diseases and extended the range of potential targets to intracellular molecules. There are potential drug candidates for FDA approval based on active studies in basic research, preclinical, and clinical trials. Various attempts by CPP application to control the diseases such as allergy, autoimmunity, cancer, and infection demonstrated a strategy to make a new drug pipeline for successful discovery of a biologic drug for immune modulation. However, there are still no CPP-based drug candidates for immune-related diseases in the clinical stage. To control immune responses successfully, not only increasing delivery efficiency of CPPs but also selecting potential target cells and cargoes could be important issues. In particular, as it becomes possible to control intracellular targets, efforts to find various novel potential target are being attempted. In this chapter, we focused on CPP-based approaches to treat diseases through modulation of immune responses and discussed for perspectives on future direction of the research for successful application of CPP technology to immune modulation and disease therapy in clinical trial.
... Among highly promising approaches is the application of cell-penetrating peptides (CPPs) [5][6][7] as efficient carrier vectors for the delivery of different functional nucleic acid (NA) molecules, e.g., plasmid DNA, mRNA, siRNA, miRNA, splicing correcting oligonucleotides, etc. [8]. Once transfected, these NAs can modulate the flow of genetic information in the cells and, consequently, alter cellular responses in target sites [9]. CPPs can condense NA by electrostatic complexing after simple mixing step and effectively transport compacted NA into the cells, mostly by harnessing various endocytic pathways. ...
Article
Full-text available
Nucleic acid molecules can be transferred into cells to alter gene expression and, thus, alleviate certain pathological conditions. Cell-penetrating peptides (CPPs) are vectors that can be used for transfecting nucleic acids as well as many other compounds. CPPs associate nucleic acids non-covalently, forming stable nanoparticles and providing efficient transfection of cells in vitro. However, in vivo, expected efficiency is achieved only in rare cases. One of the reasons for this discrepancy is the formation of protein corona around nanoparticles, once they are exposed to a biological environment, e.g., blood stream. In this study, we compared protein corona of CPP-nucleic acid nanoparticles formed in the presence of bovine, murine and human serum. We used Western blot and mass-spectrometry to identify the major constituents of protein corona forming around nanoparticles, showing that proteins involved in transport, haemostasis and complement system are its major components. We investigated physical features of nanoparticles and measured their biological efficiency in splice-correction assay. We showed that protein corona constituents might alter the fate of nanoparticles in vivo, e.g., by subjecting them to phagocytosis. We demonstrated that composition of protein corona of nanoparticles is species-specific that leads to dissimilar transfection efficiency and should be considered while developing delivery systems for nucleic acids.
... 71,72 We previously demonstrated the capacity of CPP-miR-146a noncovalent nanocomplexes to suppress inflammatory responses in cell cultures and a mouse model of irritant contact dermatitis. 73 To our knowledge, we report for the first time that intranasal administration of CPP-miRNA nanocomplexes is efficient for delivering miRNA mimics into mouse airways. ...
Article
Graphical Abstract miR-146a has anti-inflammatory properties in human bronchial epithelial cells and mouse airways during rhinovirus infection and in case of allergic inflammation. Particular cell penetrating peptide(CPP)-miR-146a nanocomplexes have therapeutic potential for targeting of airway inflammation.
... 71,72 We previously demonstrated the capacity of CPP-miR-146a noncovalent nanocomplexes to suppress inflammatory responses in cell cultures and a mouse model of irritant contact dermatitis. 73 To our knowledge, we report for the first time that intranasal administration of CPP-miRNA nanocomplexes is efficient for delivering miRNA mimics into mouse airways. ...
Article
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Rhinovirus (RV) infections are associated with asthma exacerbations. MicroRNA-146a and microRNA-146b (miR-146a/b) are anti-inflammatory miRNAs that suppress signaling through the nuclear factor kappa B (NF-κB) pathway and inhibit pro-inflammatory chemokine production in primary human bronchial epithelial cells (HBECs). In the current study, we aimed to explore whether miR-146a/b could regulate cellular responses to RVs in HBECs and airways during RV-induced asthma exacerbation. We demonstrated that expression of miR-146a/b and pro-inflammatory chemokines was increased in HBECs and mouse airways during RV infection. However, transfection with cell-penetrating peptide (CPP)-miR-146a nanocomplexes before infection with RV significantly reduced the expression of the pro-inflammatory chemokines CCL5, IL-8 and CXCL1, increased interferon-λ production, and attenuated infection with the green fluorescent protein (GFP)-expressing RV-A16 in HBECs. Concordantly, compared to wild-type (wt) mice, Mir146a/b−/− mice exhibited more severe airway neutrophilia and increased T helper (Th)1 and Th17 cell infiltration in response to RV-A1b infection and a stronger Th17 response with a less prominent Th2 response in house dust mite extract (HDM)-induced allergic airway inflammation and RV-induced exacerbation models. Interestingly, intranasal administration of CPP-miR-146a nanocomplexes reduced HDM-induced allergic airway inflammation without a significant effect on the Th2/Th1/Th17 balance in wild-type mice. In conclusion, the overexpression of miR-146a has a strong anti-inflammatory effect on RV infection in HBECs and a mouse model of allergic airway inflammation, while a lack of miR-146a/b leads to attenuated type 2 cell responses in mouse models of allergic airway inflammation and RV-induced exacerbation of allergic airway inflammation. Furthermore, our data indicate that the application of CPP-miR-146a nanocomplexes has therapeutic potential for targeting airway inflammation.
... Elevated serum level of IgE and proliferation of Th2 lymphocytes in skin lesions are the main characteristics of AD [264,265]. miRNAs such as miR-146a play role in AD pathogenesis [265,266] It was observed that exogenous miR-146a reduced the inflammation in AD experimental model, through targeting the IRAK1, TRAF6, and C -C motif chemokine ligand 5 (CCL5) [267] (Fig. 2). Rebane et al. demonstrated that miR-146a expression significantly increased in keratinocytes and skin tissue of patients with AD (Table 2). ...
Article
MicroRNAs (miRNAs) are single-strand endogenous and non-coding RNA molecules with a length of about 22 nucleotides, which regulate genes expression, through modulating the translation and stability of their target mRNAs. miR-146a is one of the most studied miRNAs, due to its central role in immune system homeostasis and control of the innate and acquired immune responses. Accordingly, abnormal expression or function of miR-146a results in the incidence and progression of immune and non-immune inflammatory diseases. Its deregulated expression pattern and inefficient function have been reported in a wide spectrum of these illnesses. Based on the existing evidence, this miRNA qualifies as an ideal biomarker for diagnosis, prognosis, and activity evaluation of immune and non-immune inflammatory disorders. Moreover, much attention has recently been paid to therapeutic potential of miR-146a and several researchers have assessed the effects of different drugs on expression and function of this miRNA at diverse experimental, animal, besides human levels, reporting motivating results in the treatment of the diseases. Here, in this comprehensive review, we provide an overview of miR-146a role in the pathogenesis and progression of several immune and non-immune inflammatory diseases such as Rheumatoid arthritis, Systemic lupus erythematosus, Inflammatory bowel disease, Multiple sclerosis, Psoriasis, Graves' disease, Atherosclerosis, Hepatitis, Chronic obstructive pulmonary disease, etc., discuss about its eligibility for being a desirable biomarker for these disorders, and also highlight its therapeutic potential. Understanding these mechanisms underlies the selecting and designing the proper therapeutic targets and medications, which eventually facilitate the treatment process.
... The risk can be overcome by maximizing delivery to the target organ and minimizing off-target effects. Topical delivery is one of the easiest strategies for targeted delivery and skin disorders can be greatly ameliorated using targeted miRNA therapy [55,121,122]. Thus, this review highlights the association between miRNAs and factors involved in epidermal barrier integrity, including regulatory factors and structural constituents associated with keratinocyte differentiation and proliferation, desquamation of corneodesmosomes, and skin lipids. ...
Article
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MicroRNAs (miRNAs), which mostly cause target gene silencing via transcriptional repression and degradation of target mRNAs, regulate a plethora of cellular activities, such as cell growth, differentiation, development, and apoptosis. In the case of skin keratinocytes, the role of miRNA in epidermal barrier integrity has been identified. Based on the impact of key genetic and environmental factors on the integrity and maintenance of skin barrier, the association of miRNAs within epidermal cell differentiation and proliferation, cell–cell adhesion, and skin lipids is reviewed. The critical role of miRNAs in the epidermal barrier extends the use of miRNAs for control of relevant skin diseases such as atopic dermatitis, ichthyoses, and psoriasis via miRNA-based technologies. Most of the relevant miRNAs have been associated with keratinocyte differentiation and proliferation. Few studies have investigated the association of miRNAs with structural proteins of corneocytes and cornified envelopes, cell–cell adhesion, and skin lipids. Further studies investigating the association between regulatory and structural components of epidermal barrier and miRNAs are needed to elucidate the role of miRNAs in epidermal barrier integrity and their clinical implications.
... The ear oedema essay was performed according to a previously reported method [27,28] with slight modifications in Kunming mice. After a 1-h pre-treatment, 20 lL DMB was topically applied on the right ear (10 lL each on the anterior and posterior surfaces). ...
Article
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Mulberry silkworm cocoon (MSC) carbonisata has been used for the treatment of inflammatory diseases for hundreds of years; however, after years of research efforts, little information is available on its anti-inflammatory components and underlying mechanism. We developed novel carbon dots (CDs) derived from MSC carbonisata (MSC-CDs), for the first time, with an average diameter of 2.26–9.35 nm and a quantum yield (QY) of 6.32%. The MSC-CDs were prepared using a modified pyrolysis method, and no further modification and external surface passivation agent was required. With abundant surface groups, MSC-CDs showed distinct solubility and bioactivity. In this study, we innovatively used three classical experimental models of inflammation to evaluate the anti-inflammatory bioactivity of MSC-CDs. The results indicated that MSC-CDs exhibited marked anti-inflammatory bioactivity which was likely mediated by inhibition of the expression of interleukin-6 and tumour necrosis factor-α. These results suggest that MSC-CDs possess a remarkable anti-inflammatory property, which provides evidence to support further investigation of the considerable potential and effective material basis of this traditional Chinese medicine.
... Increased expression of IL-8, CXCL1, IL-4R and IRF1 in patients with neutrophilic asthma and enhanced IL-33 in samples from eosinophilic patients confirmed phenotypic and endotypic similarity of these subgroups. As previous studies have shown miR-146a capacity to suppress IL-8 and CXCL1 in other cell types [28,42], our results suggest that the lower expression of miR-146a may be linked to enhanced production of these chemokines by the airway epithelial cells in asthma. Although there was a negative association between the miR-146a levels and the number of neutrophils in BALF fluid, additional linear regression analysis did not reveal any association between relative miR-146a expression and mRNA levels of studied indirect and direct genes (data not shown). ...
Article
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Background: The role of miRNAs in the pathogenesis and determining the phenotypes of asthma is not fully elucidated. miR-146a has been previously shown to suppress inflammatory responses in different cells. In this study, we investigated the functions of miR-146a in human bronchial epithelial cells (HBECs) in association with neutrophilic, eosinophilic, and paucigranulocytic phenotypes of asthma. Methods: Bronchial brushing specimens and brochial mucosal biopsy samples were collected from adult patients with asthma and from age- and gender-matched non-asthmatic individuals. The expression of miR-146a in bronchial brushing specimens, bronchial biopsy tissue sections or cultured primary bronchial epithelial cells was analyzed by RT-qPCR or by in situ hybridization. The expression of direct and indirect miR-146a target genes was determined by RT-qPCR or ELISA. The migration of neutrophils was studied by neutrophil chemotaxis assay and flow cytometry. For statistical analysis, unpaired two-way Student's t test, one-way ANOVA or linear regression analysis were used. Results: Reduced expression of miR-146a was found in bronchial brushing specimens from asthma patients as compared to non-asthmatics and irrespective of the phenotype of asthma. In the same samples, the neutrophil attracting chemokines IL-8 and CXCL1 showed increased expression in patients with neutrophilic asthma and increased IL-33 expression was found in patients with eosinophilic asthma. Linear regression analysis revealed a significant negative association between the expression of miR-146a in bronchial brushings and neutrophil cell counts in bronchoalveolar lavage fluid of patients with asthma. In bronchial biopsy specimens, the level of miR-146a was highest in the epithelium as determined with in situ hybridization. In primary conventional HBEC culture, the expression of miR-146a was induced in response to the stimulation with IL-17A, TNF-α, and IL-4. The mRNA expression and secretion of IL-8 and CXCL1 was inhibited in both stimulated and unstimulated HBECs transfected with miR-146a mimics. Supernatants from HBECs transfected with miR-146a had reduced capability of supporting neutrophil migration in neutrophil chemotaxis assay. Conclusion: Our results suggest that decreased level of miR-146a in HBECs from patients with asthma may contribute to the development of neutrophilic phenotype of asthma.
... Moreover, the dosage administered was also much lower than the dose of miRNA agomir administered via the blood. 50,51 Chronic neuroinflammation is generally considered to be a downstream factor in the pathology of AD and is closely related to the activation of glial cells. 52 However, the clinical and experimental evidence in studies targeting the inflammation-related pathway as a potential therapy for AD remains controversial. ...
Article
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Alzheimer's disease (AD) is the most common cause of dementia and cannot be cured. The etiology and pathogenesis of AD is still not fully understood, the genetics is considered to be one of the most important factors for AD onset, and the identified susceptible genes could provide clues to the AD mechanism and also be the potential targets. MicroRNA-146a-5p (miR-146a) is well known in the regulation of the inflammatory response, and the functional SNP of miR-146a was associated with AD risk. In this study, using a noninvasive nasal administration, we discovered that a miR-146a agomir (M146AG) rescued cognitive impairment in the APP/PS1 transgenic mouse and alleviated the overall pathological process in the AD mouse model, including neuroinflammation, glia activation, Aβ deposit, and tau phosphorylation in hippocampi. Furthermore, the transcriptional analysis revealed that besides the effect of neuroinflammation, M146AG may serve as a multi-potency target for intervention in AD. In addition, Srsf6 was identified as a target of miR-146a, which may play a role in AD progression. In conclusion, our study supports that the nasal-to-brain pathway is efficient and operable for the brain administration of microRNAs (miRNAs), and that miR-146a may be a new potential target for AD treatment.
... and stratum corneum for intracellular localization [112,113]. Another group tested efficiency of several CPPs (PF6, PF14 and CADY) to deliver MIR-146a (an anti-inflammatory miRNA) into human primary keratinocytes [114]. 24 hours after transfection, a strong signal was observed with PF6 and CADY-mediated dy547-labelled miRNAs whereas the expression of miR-146 target genes was more significantly interfered in the former. ...
Article
The selective infiltration of cell membranes and tissue barriers often blocks the entry of most active molecules. This natural defense mechanism prevents the invasion of exogenous substances and limits the therapeutic value of most available molecules. Therefore, it is particularly important to find appropriate ways of membrane translocation and therapeutic agent delivery to its target site. Cell penetrating peptides (CPPs) are a group of short peptides harnessed in this condition, possessing a significant capacity for membrane transduction and could be exploited to transfer various biologically active cargoes into the cells. Since their discovery, CPPs have been employed for delivery of a wide variety of therapeutic molecules to treat various disorders including cranial nerve involvement, ocular inflammation, myocardial ischemia, dermatosis and cancer. The promising results of CPPs-derived therapeutics in various tumor models demonstrated a potential and worthwhile scope of CPPs in chemotherapy. This review describes the detailed description of CPPs and CPPs-assisted molecular delivery against various tissues and organs disorders. An emphasis is focused on summarizing the novel insights and achievements of CPPs in surmounting the natural membrane barriers during the last 5 years.
... Transfections were performed with siPORT™ NeoFX™ (Life Technologies) according to the manufacturer's instructions or using cell-penetrating peptide PepFect14 as described. 26 Stimulations were performed at final concentrations of 10 ng/mL for IL-1β, 25 ng/mL for HB-EGF, 20 ng/ mL for IL-22, 40 ng/mL for IL-4, and 10 ng/mL for IL-17A (Peprotech). ...
Article
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Background miR‐10a‐5p has been shown to regulate cancer cell proliferation and invasiveness and endothelial cell inflammatory responses. The function of miR‐10a‐5p in the skin has not been previously studied. The aim of the current study was to examine miR‐10a‐5p expression, regulation, and function in keratinocytes (KCs) in association with atopic dermatitis (AD). Methods The expression of miR‐10a‐5p and its target genes was analyzed using RT‐qPCR, mRNA array analysis, in situ hybridization, and immunofluorescence. The transfection of miRNA mimics, cell cycle distribution analysis, and luciferase assays was used to study miR‐10a‐5p functions in human primary KCs. Results miR‐10a‐5p was found to be upregulated in lesional skin from patients with AD and in proliferating KCs. Array and pathway analysis of IL‐1β‐stimulated KCs revealed that miR‐10a‐5p inhibited many genes that affect cell cycle progression and only a few inflammation‐related genes. Accordingly, fewer cells in S‐phase and reduced proliferation were detected as characteristics of miR‐10a‐5p‐transfected KCs. The influence of miR‐10a‐5p on cell proliferation was also evident in KCs induced by AD‐related cytokines, including IL‐4, IL‐17, and IL‐1β, as measured by the capacity to strongly suppress the expression of the proliferation marker Ki‐67. Among AD‐related putative direct target genes, we verified hyaluronan synthase 3, a damage‐associated positive regulator of KC migration and proliferation, as a direct target of miR‐10a‐5p. Conclusions miR‐10a‐5p inhibits KC proliferation and directly targets hyaluronan synthase 3 and thereby may modulate AD‐associated processes in the skin.
... During the optimization, we counted cells prior to the flow cytometric analysis and noted a trend of collecting less cells from siRNA/PF14-treated samples than from untreated samples. This indicates that siRNA (30 nM) and/or PF14 affect the cell viability (Fig. 2c) [27]. To further optimize protocol and increase survival rates, we performed dosage studies, where siRNA and PF14 concentration was lowered proportionally. ...
Article
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Background Human embryonic stem (hES) cells serve as an invaluable tool for research and future medicine, but their transfection often leads to unwanted side effects as the method itself may induce differentiation. On the other hand, RNA interference (RNAi)-based targeted gene silencing is a quick, cost-effective, and easy-to-perform method to address questions regarding the function of genes, especially when hypomorphic knockdowns are needed. Therefore, effective transfection method with minimal side effects is essential for applying RNAi to hES cells. Here, we report a highly promising approach for targeted gene silencing in hES cells with siRNA complexed with cell-penetrating peptide PepFect 14 (PF14). This strategy provides researchers with efficient tool for unraveling the functions of genes or addressing the differentiation of pluripotent stem cells. Methods We present a method for delivery of siRNA into hES cells with cell-penetrating peptide PF14. Accordingly, hES cells were transfected in ROCK inhibitor containing medium for 24 h right after EDTA passaging as small cell clumps. Fluorescently labeled siRNA and siRNAs targeting OCT4 or beta-2-microglobulin (B2M) mRNA sequences were used to evaluate the efficiency of transfection and silencing. Analyses were performed at various time points by flow cytometry, RT-qPCR, and immunofluorescence microscopy. Results Effective downregulation of OCT4 in 70% of treated hES cells at protein level was achieved, along with 90% reduction at mRNA level in bulk population of cells. The applicability of this low-cost and easy-to-perform method was confirmed by inducing silencing of another target not associated with hES cell pluripotency (B2M). Furthermore, we discovered that downregulation of OCT4 induces neuroectodermal differentiation accompanied by reduced expression of B2M during early stage of this lineage. Conclusions The results demonstrate PF14 as a promising tool for studying gene function and regulatory networks in hES cells by using RNAi. Electronic supplementary material The online version of this article (10.1186/s13287-019-1144-x) contains supplementary material, which is available to authorized users.
... Both TEM profiles are quite similar to what we observed for other nanoparticle forming CPPs such as RICK 11 or Pepfect14. 37 Considering experimental differences between DLS (solvated sample) and TEM imaging (dried sample), both observed size distributions are in agreement for both WRAP nanoparticles. ...
Article
Delivery of small interfering RNA (siRNA) as a therapeutic tool is a hurdle due to critical obstacles such as the cellular barrier, the negative charges of the siRNA molecule and its instability in serum. Several siRNA delivery systems have been constructed using cell-penetrating peptides (CPPs) since the CPPs have shown a high potential for oligonucleotide delivery into the cells, especially by forming nanoparticles. In this study, we have developed a new family of short (15mer or 16mer) tryptophan-(W) and arginine-(R) rich Amphipathic Peptides (WRAP) able to form stable nanoparticles and to enroll siRNA molecules into cells. The lead peptides, WRAP1 and WRAP5, form defined nanoparticles smaller than 100 nm as characterized by biophysical methods. Furthermore, they have several benefits as oligonucleotide delivery tools such as the rapid encapsulation of the siRNA, the efficient siRNA delivery in several cell types and the high gene silencing activity, even in the presence of serum. In conclusion, we have designed a new family of CPPs specifically dedicated for siRNA delivery trough nanoparticle formation. Our results indicate that the WRAP family has significant potential for the safe, efficient, and rapid delivery of siRNA for diverse applications.
... It is possible that the reduction in the edema formation in mice ears and leukocyte migration occurs by its ability to reduce IL-1β and MIP-2 levels. The multiple cellular interactions through inflammatory mediators release can explain the diversity of inflammatory pathways involved in ICD pathogenesis, such as the transcriptional factor NF-κB (Han et al., 2007;Urgard et al., 2016). In this sense, we used the ICD model croton oilinduced since it activates several inflammatory pathways including the NF-κB, a transcription factor responsible by the production of pro-inflammatory cytokines (Siddiqui et al., 2016). ...
Article
Ethnopharmacological relevance: Inflammatory skin diseases treatments currently used cause adverse effects. Nasturtium officinale (watercress) is used popularly as an anti-inflammatory. However, until now, no study proved its effectiveness as a topical treatment to inflammatory skin diseases. The topical anti-inflammatory activity of N. officinale crude extract leaves (NoE) on an irritant contact dermatitis (ICD) model croton oil-induced in mice was investigated. Materials and methods: ICD models were induced by a single (1mg/ear; acute) or repeated (0.4mg/ear; chronic; 9 days total) croton oil application. NoE and dexamethasone solutions' (diluted in acetone; 20µL/ear) or NoE gel, dexamethasone gel and base gel (15mg/ear) were topically applied immediately after croton oil application. The NoE topical anti-inflammatory effect was evaluated for inflammatory parameters (ear edema, inflammatory cells infiltration, and inflammatory cytokines levels). NoE topical anti-inflammatory mechanism (NF-κB pathway and effect glucocorticoid-like) were assessed by western blot and ear edema analyses, respectively. UHPLC-MS/MS chromatography, gels accelerated stability and preliminary study of adverse effects was also performed. Results: UHPLC-MS/MS of the NoE revealed the presence of coumaric acid, rutin, and ferrulic. NoE gels stability study showed no relevant changes at low temperatures. NoE, dexamethasone, NoE gel and dexamethasone gel inhibited the ear edema croton oil-induced by 82±6% (1mg/ear), 99±1% (0.1mg/ear), 81±8% (3%) and 70±6% (0.5%) for the acute model, and 49±7% (1mg/ear), 80±4% (0.1mg/ear), 41±8% (3%) and 46±14% (0.5%) for the chronic model, respectively. The same treatments also reduced the inflammatory cells infiltration by 62±3% (1mg/ear), 97±2% (0.1mg/ear), 60±3% (3%) and 66±6% (0.5%) for the acute model, respectively, and 25±8% (1mg/ear) to NoE and 83±13% to dexamethasone to the chronic model. NoE and NoE gel reduced the pro-inflammatory cytokines levels (acute ICD model) by 62±5% and 71±3% (MIP-2) and 32±3% and 44±4% (IL-1β), while dexamethasone solution's and gel reduced by 79±7% and 44±4% to MIP-2 and 98±2% and 83±9% to IL-1β, respectively. NoE' and dexamethasone' solutions inhibited the reduction of IkB-α protein expression induced by croton oil by 100% and 80±14%, respectively. Besides, the mifepristone (glucocorticoid receptor antagonist) pre-treatment prevented the topical anti-edematogenic effect of NoE' and dexamethasone' solutions by 61±5% to NoE and 78±16% to dexamethasone. The repeated topical application of NoE did not cause adverse effects. Conclusion: Our results suggest the N.officinale use in the cutaneous inflammatory process treatment and demonstrate the NoE potential to develop a promising topical anti-inflammatory agent to treat inflammatory disorders.
... In the second approach mentioned -releasing siRNAs to downregulate miRNAs expression-, a clear candidate may be miR-146a, because it facilitates bacterial proliferation via the IRAK-1/TRAF-6 pathway, negatively regulates TNF-α and promotes mycobacterial survival in macrophages through suppression of NO production. Interestingly, nanoparticles loaded with miR-146 have been used to inhibit inflammation in keratinocytes ( Urgard et al., 2016) and human dental pulp cells ( Liu et al., 2016) and to inhibit kidney fibrosis (Morishita et al., 2015). This suggests that both methodologies - direct delivery and secondary molecular induction-can be complementary approaches in nanoparticle-mediated delivery of miRNAs. ...
Article
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Tuberculosis (TB) is one of the most fatal infectious diseases and a leading cause of mortality, with 95% of these deaths occurring in developing countries. The causative agent, Mycobacterium tuberculosis (Mtb), has a well-established ability to circumvent the host’s immune system for its intracellular survival. microRNAs (miRNAs) are small, non-coding RNAs having an important function at the post-transcriptional level and are involved in shaping immunity by regulating the repertoire of genes expressed in immune cells. It has been established in recent studies that the innate immune response against TB is significantly regulated by miRNAs. Moreover, differential expression of miRNA in Mtb infection can reflect the disease progression and may help distinguish between active and latent TB infection (LTBI). These findings encouraged the application of miRNAs as potential biomarkers. Similarly, active participation of miRNAs in modulation of autophagy and apoptosis responses against Mtb opens an exciting avenue for the exploitation of miRNAs as host directed therapy (HDT) against TB. Nanoparticles mediated delivery of miRNAs to treat various diseases has been reported and this technology has a great potential to be used in TB. In reality, this exploitation of miRNAs as biomarkers and in HDT is still in its infancy stage, and more studies using animal models mimicking human TB are advocated to assess the role of miRNAs as biomarkers and therapeutic targets. In this review, we attempt to summarize the recent advancements in the role of miRNAs in TB as immune modulator, miRNAs’ capability to distinguish between active and latent TB and, finally, usage of miRNAs as therapeutic targets against TB.
... PepFect6 is an analogue of transportan 10 designed with an N-terminal stearyl moiety that enhances membrane insertion and four trifluoromethylquinoline derivatives to promote endosomal escape [142,152]. Recently, subcutaneous injection of PF6 CPP complexed with a miR-146a mimic was shown to suppress inflammatory gene expression in a mouse model of irritant contact dermatitis [153]. There is arguably a case, therefore, for merging PF6 with SPACE peptide, R7 or TD-1 in order to combine the oligonucleotide-binding capacity of PF6 with the ability of the SPPs to cross the SC into the viable epidermis. ...
Article
There remains an unmet need dermatological need for innovative topical agents that achieve better long term outcomes with fewer side effects. Modulation of the expression and activity of microRNA (miRNAs) represents an emerging translational framework for the development of such innovative therapies because changes in the expression of one miRNA can have wide-ranging effects on diverse cellular processes associated with disease. In this short review, the roles of miRNA in epidermal development, psoriasis, cutaneous squamous cell carcinoma and re-epithelisation are highlighted. Consideration is given to the delivery of oligonucleotides that mimic or inhibit miRNA function using vehicles such as cell penetrating peptides, spherical nucleic acids, deformable liposomes and liquid crystalline nanodispersions. Formulation of miRNA-directed oligonucleotides with such skin-penetrating epidermal agents will drive the development of RNA-based cutaneous therapeutics for deployment as primary or adjuvant therapies for epidermal disorders.
... In the case of miR-146a, it was reported that the NF-jB pro-inflammatory signaling pathway was disrupted by miR-146a targeting chemokine ligand 5 (CCL5). It was then concluded that both miRNAs had the potential to be developed into therapeutics to combat atopic dermatitis, and efforts are already underway for an miR-146a-based application [109]. ...
Article
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The advent of RNA interference (RNAi) technology has profoundly impacted molecular biology research and medicine but has also advanced the field of skin care. Both effector molecules of RNAi, short-interfering RNA molecules and microRNAs (miRNAs), have been explored for their relative impact and utility for treating a variety of skin conditions. These post-transcriptional RNA regulatory molecules down-modulate protein expression through targeting of the 3′ untranslated regions of messenger RNAs, leading to their degradation or repression through sequestration. As researchers hunt for genetic linkages to skin diseases, miRNA regulators have emerged as key players in the biology of keratinocytes, fibroblasts, melanocytes, and other cells of the skin. Herein, we attempt to coalesce the current efforts to combat various skin disorders and diseases through the development of miRNA-based technologies.
... We could hypothesize that RICK:siRNA nanoparticles were formed in the same manner: first the formation of ~20 nm spheres composed of RICK:siRNA (R = 20) which self-associate in a "beads necklace"-like manner to nanoparticles of ~100 nm. Such assembly could be also compared to the "large branching agglomerates" described for the parental peptide CADY in the presence of miRNA [50]. ...
Article
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Background Small interfering RNAs (siRNAs) are powerful tools to control gene expression. However, due to their poor cellular permeability and stability, their therapeutic development requires a specific delivery system. Among them, cell-penetrating peptides (CPP) have been shown to transfer efficiently siRNA inside the cells. Recently we developed amphipathic peptides able to self-assemble with siRNAs as peptide-based nanoparticles and to transfect them into cells. However, despite the great potential of these drug delivery systems, most of them display a low resistance to proteases. ResultsHere, we report the development and characterization of a new CPP named RICK corresponding to the retro-inverso form of the CADY-K peptide. We show that RICK conserves the main biophysical features of its L-parental homologue and keeps the ability to associate with siRNA in stable peptide-based nanoparticles. Moreover the RICK:siRNA self-assembly prevents siRNA degradation and induces inhibition of gene expression. Conclusions This new approach consists in a promising strategy for future in vivo application, especially for targeted anticancer treatment (e.g. knock-down of cell cycle proteins).Open image in new windowGraphical abstractRICK-based nanoparticles: RICK peptides and siRNA self-assemble in peptide-based nanoparticles to penetrate into the cells and to induce target protein knock-down.
Chapter
In this Chapter, the targeting strategies of CPP and CPP/cargo complexes to specific cells or intracellular organelles are summarized, and their potential for pharmacological or clinical use is briefly discussed.
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Here, the brief summary of therapeutic developments and current situation with applications of CPPs is presented, hopefully leading to multiple CPP based drugs in near future.
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This Chapter summarizes the methods for CPP-conjugated oligonucleotide-based agents with the potential to use as the therapeutic drugs. The methods for gene silencing (or inhibiting specific genes) using antisense ONs (ASO) and RNA interference (RNAi) are summarized.
Article
RNA therapeutics offer great potential to transform the biomedical landscape, encompassing the treatment of hereditary conditions and the development of better vaccines. However, the delivery of RNAs into the cell is hampered, among others, by poor endosomal escape. This major hurdle is often tackled using special lipids, polymers, or protein-based delivery vectors. In this review, we will focus on the most prominent peptide- and protein-based endosomal escape strategies with focus on RNA drugs. We discuss cell penetrating peptides, which are still incorporated into novel transfection systems today to promote endosomal escape. However, direct evidence for enhanced endosomal escape by the action of such peptides is missing and their transfection efficiency, even in permissive cell culture conditions, is rather low. Endosomal escape by the help of pore forming proteins or phospholipases, on the other hand, allowed to generate more efficient transfection systems. These are, however, often hampered by considerable toxicity and immunogenicity. We conclude that the perfect enhancer of endosomal escape has yet to be devised. To increase the chances of success, any new transfection system should be tested under relevant conditions and guided by assays that allow direct quantification of endosomal escape.
Article
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Cell-penetrating peptides (CPPs) are promising tools for the transfection of various substances, including nucleic acids, into cells. The aim of the current work was to search for novel safe and effective approaches for enhancing transfection efficiency of nanoparticles formed from CPP and splice-correcting oligonucleotide (SCO) without increasing the concentration of peptide. We analyzed the effect of inclusion of calcium and magnesium ions into nanoparticles on CPP-mediated transfection in cell culture. We also studied the mechanism of such transfection as well as its efficiency, applicability in case of different cell lines, nucleic acid types and peptides, and possible limitations. We discovered a strong positive effect of these ions on transfection efficiency of SCO, that translated to enhanced synthesis of functional reporter protein. We observed significant changes in intracellular distribution and trafficking of nanoparticles formed by the addition of the ions, without increasing cytotoxicity. We propose a novel strategy for preparing CPP-oligonucleotide nanoparticles with enhanced efficiency and, thus, higher therapeutic potential. Our discovery may be translated to primary cell cultures and, possibly, in vivo studies, with the aim of increasing CPP-mediated transfection efficiency and the likelihood of using CPPs in clinics.
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Cell-Penetrating Peptides (CPP) are valuable tools capable of crossing the plasma membrane to deliver therapeutic cargo inside cells. Small interfering RNAs (siRNA) are double-stranded RNA molecules capable of silencing the expression of a specific protein triggering the RNA interference (RNAi) pathway, but they are unable to cross the plasma membrane and have a short half-life in the bloodstream. In this overview, we assessed the many different approaches used and developed in the last two decades to deliver siRNA through the plasma membrane through different CPPs sorted according to three different loading strategies: covalent conjugation, complex formation, and CPP-decorated (functionalized) nanocomplexes. Each of these strategies has pros and cons, but it appears the latter two are the most commonly reported and emerging as the most promising strategies due to their simplicity of synthesis, use, and versatility. Recent progress with siRNA delivered by CPPs seems to focus on targeted delivery to reduce side effects and amount of drugs used, and it appears to be among the most promising use for CPPs in future clinical applications.
Article
Most oligonucleotides fail to enter a cell and cannot escape from endosomes after endocytosis because of their negative charge and large molecular weight. More efficient cellular delivery of oligonucleotides should be developed for the widespread implementation of antisense imaging. The purpose of this study was to construct a novel antisense nanoprobe, 99mTc-labeled anti-miRNA oligonucleotides/cell-penetrating peptide PepFect6 (99mTc-AMO/PF6), and to evaluate its efficacy for imaging the miRNA-21 expression in A549 lung adenocarcinoma xenografts. Naked AMO and commercial Lipofectamine 2000-based nanoparticles (AMO/LIP) were used for comparison. The cellular delivery efficiency of AMO/PF6 was first investigated by laser confocal scanning microscopy using Cy5.5-labeled probes and further validated by in vivo fluorescence imaging. Then, the probes were labeled with 99mTc via hydrazinonicotinamide (HYNIC). The cytotoxicity assay, cellular uptake, and retention kinetics of the probes were evaluated in vitro. The biodistribution of the probes was investigated in A549 lung cancer xenografts, and SPECT imaging was performed in vivo. AMO/PF6 showed lower cytotoxicity than AMO/LIP (P < 0.05) but showed no significant difference with naked AMO. Fluorescence microscopy demonstrated more extensive and scattered signal distribution inside the A549 cells by AMO/PF6 than AMO/LIP. The labeling efficiency of 99mTc-AMO/PF6 was 72.6 ± 1.42%, and the specific activity was 11.6 ± 0.13 MBq/ng. The cellular uptake of 99mTc-PF6/AMO peaked at 12 h, with the uptake of 11.24 ± 0.12 mol/cell × 10-16, and the cellular retention of 99mTc-AMO/PF6 was 3.92 ± 0.15 mol/cell × 10-16 at 12 h after interrupted incubation. AMO/PF6 showed higher cellular uptake and retention than naked AMO and AMO/LIP. The biodistribution study showed that the tumor had the highest radioactivity accumulation, with the uptake ratio of tumor/muscle (T/M) increasing from 14.59 ± 0.67 to 21.76 ± 0.98 between 1 and 6 h after injection, followed by the uptake in the kidneys and the liver. The results of in vivo fluorescence and SPECT imaging were consistent with the results of the biodistribution. The tumor was visualized at 6 h after injection of AMO/PF6 with the highest T/M ratio among these probes (P < 0.05). PF6 improves cellular delivery of antisense oligonucleotides via noncovalent nanoparticles. 99mTc-AMO/PF6 shows favorable imaging properties and is promising for miRNAs imaging in vivo.
Article
In past 10 years, microRNAs (miRNAs) have gained scientific attention due to their importance in the pathophysiology of allergic diseases and their potential as biomarkers in liquid biopsies. They act as master post-transcriptional regulators that control most cellular processes. As one miRNA can target several mRNAs, often within the same pathway, dysregulated expression of miRNAs may alter particular cellular responses and contribute, or lead, to the development of various diseases. In this review, we give an overview of the current research on miRNAs in allergic diseases, including atopic dermatitis, allergic rhinitis, and asthma. Specifically, we discuss how individual miRNAs function in the regulation of immune responses in epithelial cells and specialized immune cells in response to different environmental factors and respiratory viruses. In addition, we review insights obtained from experiments with murine models of allergic airway and skin inflammation and offer an overview of studies focusing on miRNA discovery using profiling techniques and bioinformatic modeling of the network effect of multiple miRNAs. In conclusion, we highlight the importance of research into miRNA function in allergy and asthma to improve our knowledge of the molecular mechanisms involved in the pathogenesis of this heterogeneous group of diseases.
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In past 10 years, microRNAs (miRNAs) have gained scientific attention due to their importance in the pathophysiology of allergic diseases and their potential as biomarkers in liquid biopsies. They act as master post‐transcriptional regulators that control most cellular processes. As one miRNA can target several mRNAs, often within the same pathway, dysregulated expression of miRNAs may alter particular cellular responses and contribute, or lead, to the development of various diseases. In this review, we give an overview of the current research on miRNAs in allergic diseases, including atopic dermatitis, allergic rhinitis, and asthma. Specifically, we discuss how individual miRNAs function in the regulation of immune responses in epithelial cells and specialized immune cells in response to different environmental factors and respiratory viruses. In addition, we review insights obtained from experiments with murine models of allergic airway and skin inflammation and offer an overview of studies focusing on miRNA discovery using profiling techniques and bioinformatic modeling of the network effect of multiple miRNAs. In conclusion, we highlight the importance of research into miRNA function in allergy and asthma to improve our knowledge of the molecular mechanisms involved in the pathogenesis of this heterogeneous group of diseases.
Preprint
2020.11.20- This publication has now been accepted and published in Allergy. This article has been accepted for publication and undergone full peer review but has not been through the proofreading process, which may lead to differences between this version and the Version of Record.
Chapter
Cationic peptides designed for cellular delivery of nucleic acid molecules form noncovalent nanocomplexes with negatively charged oligonucleotides (ON). The electrostatically associated complexes are further compacted by hydrophobic interactions yielding nanoparticles (NP) of homogeneous shape and size that are efficiently taken up by cells. The shape and size of NP often correlate with the biological activity of delivered ON inside cells; and the stability and accessibility of NP in biological fluids govern its circulation in organism and the cellular uptake. Therefore, here we provide protocols for characterizing the shape and size and surface charge of peptide/ON NP by negative staining transmission electron microscopy (TEM) and dynamic light scattering (DLS) respectively, and analysis of NP stability against proteolytic degradation.
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Autosomal dominantly inherited genetic disorders such as corneal dystrophies are amenable to allele-specific gene silencing with small interfering RNA (siRNA). siRNA delivered to the cornea by injection, although effective, is not suitable for a frequent long-term treatment regimen, whereas topical delivery of siRNA to the cornea is hampered by the eye surface’s protective mechanisms. Herein we describe an attractive and innovative alternative for topical application using cell-penetrating peptide derivatives capable of complexing siRNA non-covalently and delivering them into the cornea. Through a rational design approach, we modified derivatives of a cell-penetrating peptide, peptide for ocular delivery (POD), already proved to diffuse into the corneal layers. These POD derivatives were able to form siRNA-peptide complexes (polyplexes) of size and ζ-potential similar to those reported able to undergo cellular internalization. Successful cytoplasmic release and gene silencing in vitro was obtained when an endosomal disruptor, chloroquine, was added. A palmitoylated-POD, displaying the best delivery properties, was covalently functionalized with trifluoromethylquinoline, an analog of chloroquine. This modified POD, named trifluoromethylquinoline-palmitoyl-POD (QN-Palm-POD), when complexed with siRNA and topically applied to the eye in vivo, resulted in up to 30% knockdown of luciferase reporter gene expression in the corneal epithelium. The methods developed within represent a valid standardized approach that is ideal for screening of a range of delivery formulations. Keywords: siRNA, cornea, peptide, CPP, delivery
Chapter
This chapter will summarize the methods for functionalization used in CPP research. Due to the very wide field of CPP applications as well as the involvement of CPPs in multiple biochemical pathways, the methods are also multiple. Basically, most of the methods of chemistry, biophysics, biochemistry, cell signaling, molecular biology, imaging etc., has been used to understand the action of CPPs. Hence, here we try to describe briefly the most widely used methods with highest impact for CPP research. It seems that it is reasonable to classify the CPP methods into non-functional and functional, based on the raised questions when applied.
Chapter
Biological or therapeutic targeting could be defined as the mechanism(s) by which a biological cargo (drug) is transported to its proper destination, in case of a patient to specific parts of the body, such as diseased tissue.
Chapter
Cell penetrating peptides as well as their applications have been extensively studied and multiple exciting reports about the topics emerge daily; this book hopefully is the proof for this statement. However, CPPs are not yet on the list of drugs for a patient therapy today and we can talk about the therapeutic potential of CPPs only. I believe that the CPP based drugs will be available in near future, however, several hurdles should be overcome before that. Below, the brief summary of therapeutic developments and current situation with applications of CPPs will be presented.
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The microRNA (miRNA) microRNA-34a (miR-34a) regulates a number of genes involved in cell cycle control and is therefore considered to have a high therapeutic potential. MiR-34a expression is often downregulated in cancer cells and its restoration has been shown to exert a tumor-suppressive effect. However, effective and safe delivery of synthetic miRNA analogs into cancer cells remains a challenge. The aim of this study was to evaluate cell-penetrating peptide PepFect (PF)14 as a carrier for delivery of miR-34a-5p into human primary prostate carcinoma-1 (PPC-1) cells. Using microarray expression analysis, we identified a total of 3,283 (1,744 upregulated and 1,539 downregulated) differentially expressed genes in PF14:miR-34a-5p-transfected cells. In comparison, miR-34a-5p delivery with the commercially available lipid-based reagent siPORT-NeoFX (siPORT) had less robust effects on differential expression and affected fewer genes significantly (90 upregulated and 91 downregulated genes). Functional annotation revealed significant enrichment for downregulated genes in processes and pathways associated with the cell cycle and proliferation regulation in PF14:miR-34a-5p-transfected cells. Five genes (ARHGAP1, AXL, CDC25A, FOSL1, and PDGFRA) were identified and validated as relevant quantitative real-time polymerase chain reaction-based markers of miR-34a-5p transfection efficiency. Our experiments revealed novel potential miR-34a-5p targets and demonstrate that PF14 is a reliable transfection reagent for miRNA mimics characterized by high efficiency and low toxicity relative to lipid-based reagents.
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Nearly 30 years ago, certain small, relatively nontoxic peptides were discovered to be capable of traversing the cell membrane. These cell-penetrating peptides, as they are now called, have been shown to not only be capable of crossing the cell membrane themselves but can also carry many different therapeutic agents into cells, including small molecules, plasmid DNA, siRNA, therapeutic proteins, viruses, imaging agents, and other various nanoparticles. Many cell-penetrating peptides have been derived from natural proteins, but several other cell-penetrating peptides have been developed that are either chimeric or completely synthetic. How cell-penetrating peptides are internalized into cells has been a topic of debate, with some peptides seemingly entering cells through an endocytic mechanism and others by directly penetrating the cell membrane. Although the entry mechanism is still not entirely understood, it seems to be dependent on the peptide type, the peptide concentration, the cargo the peptide transports, and the cell type tested. With new intracellular disease targets being discovered, cell-penetrating peptides offer an exciting approach for delivering drugs to these intracellular targets. There are hundreds of cell-penetrating peptides being studied for drug delivery, and ongoing studies are demonstrating their success both in vitro and in vivo. Copyright © 2015. Published by Elsevier Inc.
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NF-κB is constitutively activated in psoriatic epidermis. However, how activated NF-κB promotes keratinocyte hyperproliferation in psoriasis is largely unknown. Here we report that the NF-κB activation triggered by inflammatory cytokines induces the transcription of microRNA (miRNA) miR-31, one of the most dynamic miRNAs identified in the skin of psoriatic patients and mouse models. The genetic deficiency of miR-31 in keratinocytes inhibits their hyperproliferation, decreases acanthosis and reduces the disease severity in psoriasis mouse models. Furthermore, protein phosphatase 6 (ppp6c), a negative regulator that restricts the G1 to S phase progression, is diminished in human psoriatic epidermis and is directly targeted by miR-31. The inhibition of ppp6c is functionally important for miR-31-mediated biological effects. Moreover, NF-κB activation inhibits ppp6c expression directly through the induction of miR-31, and enhances keratinocyte proliferation. Thus, our data identify NF-κB-induced miR-31 and its target, ppp6c, as critical factors for the hyperproliferation of epidermis in psoriasis.
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Introduction: Macromolecular therapeutics, including enzymes, transcription factors, siRNAs, peptides and large synthetic molecules, can potentially be used to treat human diseases by targeting intracellular molecular pathways and modulating biological responses. However, large macromolecules have no ability to enter cells and require delivery vehicles. Protein transduction domains (PTDs), also known as cell-penetrating peptides (CPPs), are a diverse class of peptides that can deliver macromolecules into cells. Areas covered: In this review, we cover the uptake and usage of arginine-rich PTDs/CPPs (TAT-PTD, Penetratin/Antp and 8R). We review the endocytosis-mediated uptake of these peptides and highlight three important steps: i) cell association; ii) internalization and iii) endosomal escape. We also discuss the array of different cargos that have been delivered by cationic PTDs/CPPs as well as cellular processes and biological responses that have been modulated. Expert opinion: PTDs/CPPs have shown great potential to deliver otherwise undeliverable macromolecular therapeutics into cells for experimentation in cell culture and in animal disease models in vivo. Moreover, over 25 clinical trials have been performed predominantly using the TAT-PTD. However, more work is still needed. Endosomal escape and target-cell specificity remain two of the major future challenges.
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MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that have crucial roles in regulating gene expression. Increasing evidence supports a role for miRNAs in many human diseases, including cancer and autoimmune disorders. The function of miRNAs can be efficiently and specifically inhibited by chemically modified antisense oligonucleotides, supporting their potential as targets for the development of novel therapies for several diseases. In this Review we summarize our current knowledge of the design and performance of chemically modified miRNA-targeting antisense oligonucleotides, discuss various in vivo delivery strategies and analyse ongoing challenges to ensure the specificity and efficacy of therapeutic oligonucleotides in vivo. Finally, we review current progress on the clinical development of miRNA-targeting therapeutics.
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Although eosinophils have been detected in several human skin diseases in the vicinity of basophils, how eosinophils infiltrate the skin and the role of eosinophils in the development of skin inflammation have yet to be examined. Using murine irritant contact dermatitis (ICD) as a model, we sought to clarify the roles of eosinophils in ICD and the underlying mechanism of eosinophil infiltration of the skin. We induced croton oil-induced ICD in eosinophil-deficient ΔdblGATA mice with or without a reactive oxygen species (ROS) inhibitor. We performed cocultivation with fibroblasts and bone marrow-derived basophils and evaluated eosinophil migration using a chemotaxis assay. ICD responses were significantly attenuated in the absence of eosinophils or by treatment with the ROS inhibitor. ROS was produced abundantly by eosinophils, and both basophils and eosinophils were detected in human and murine ICD skin lesions. In coculture experiments, basophils attracted eosinophils, especially in the presence of fibroblasts. Moreover, basophils produced IL-4 and TNF-α in contact with fibroblasts and promoted the expression of eotaxin/CCL11 from fibroblasts in vitro. Eosinophils mediated the development of murine ICD, possibly through ROS production. Recruitment of eosinophils into the skin was induced by basophils in cooperation with fibroblasts. Our findings introduce the novel concept that basophils promote the recruitment of eosinophils into the skin through fibroblasts in the development of skin inflammation.
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Keratinocytes represent the first line of defense against pathogens in the skin and have important roles in initiating and regulating inflammation during infection and autoimmunity. Here we investigated the role of miR-146a in the regulation of the innate immune response of keratinocytes. Toll-like receptor 2 (TLR2) stimulation of primary human keratinocytes resulted in an NF-κB- and mitogen-activated protein kinase-dependent upregulation of miR-146a expression, which was surprisingly long lasting, contrasting with the rapid and transient induction of inflammatory mediators. Overexpression of miR-146a significantly suppressed the production of IL-8, CCL20, and tumor necrosis factor-α, which functionally suppressed the chemotactic attraction of neutrophils by keratinocytes. Inhibition of endogenous miR-146a induced the production of inflammatory mediators even in nonstimulated keratinocytes, and potentiated the effect of TLR2 stimulation. Transcriptomic profiling revealed that miR-146a suppresses the expression of a large number of immune-related genes in keratinocytes. MiR-146a downregulated interleukin-1 receptor-associated kinase 1 and TNF receptor-associated factor 6, two key adapter molecules downstream of TLR signaling, and suppressed NF-κB promoter-binding activity as shown by promoter luciferase experiments. Together, these data identify miR-146a as a regulatory element in keratinocyte innate immunity, which prevents the production of inflammatory mediators under homeostatic conditions and serves as a potent negative feedback regulator after TLR2 stimulation.Journal of Investigative Dermatology advance online publication, 27 March 2014; doi:10.1038/jid.2014.89.
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Cell-penetrating peptide-mediated delivery of phosphorodiamidate morpholino oligomers (PMOs) has shown great promise for exon-skipping therapy of Duchenne Muscular Dystrophy (DMD). Pip6a-PMO, a recently developed conjugate, is particularly efficient in a murine DMD model, although mechanisms responsible for its increased biological activity have not been studied. Here, we evaluate the cellular trafficking and the biological activity of Pip6a-PMO in skeletal muscle cells and primary cardiomyocytes. Our results indicate that Pip6a-PMO is taken up in the skeletal muscle cells by an energy- and caveolae-mediated endocytosis. Interestingly, its cellular distribution is different in undifferentiated and differentiated skeletal muscle cells (vesicular versus nuclear). Likewise, Pip6a-PMO mainly accumulates in cytoplasmic vesicles in primary cardiomyocytes, in which clathrin-mediated endocytosis seems to be the pre-dominant uptake pathway. These differences in cellular trafficking correspond well with the exon-skipping data, with higher activity in myotubes than in myoblasts or cardiomyocytes. These differences in cellular trafficking thus provide a possible mechanistic explanation for the variations in exon-skipping activity and restoration of dystrophin protein in heart muscle compared with skeletal muscle tissues in DMD models. Overall, Pip6a-PMO appears as the most efficient conjugate to date (low nanomolar EC50), even if limitations remain from endosomal escape.
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The first cancer-targeted microRNA (miRNA) drug - MRX34, a liposome-based miR-34 mimic - entered Phase I clinical trials in patients with advanced hepatocellular carcinoma in April 2013, and miRNA therapeutics are attracting special attention from both academia and biotechnology companies. Although miRNAs are the most studied non-coding RNAs (ncRNAs) to date, the importance of long non-coding RNAs (lncRNAs) is increasingly being recognized. Here, we summarize the roles of miRNAs and lncRNAs in cancer, with a focus on the recently identified novel mechanisms of action, and discuss the current strategies in designing ncRNA-targeting therapeutics, as well as the associated challenges.
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RNA interference (RNAi) has broad potential as a therapeutic to reversibly silence any gene. To achieve the clinical potential of RNAi, delivery materials are required to transport short interfering RNA (siRNA) to the site of action in the cells of target tissues. This Review provides an introduction to the biological challenges that siRNA delivery materials aim to overcome, as well as a discussion of the way that the most effective and clinically advanced classes of siRNA delivery systems, including lipid nanoparticles and siRNA conjugates, are designed to surmount these challenges. The systems that we discuss are diverse in their approaches to the delivery problem, and provide valuable insight to guide the design of future siRNA delivery materials.
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Basal cell carcinoma (BCC) of the skin represents the most common malignancy in humans. MicroRNAs (miRNAs), small regulatory RNAs with pleiotropic function, are commonly misregulated in cancer. Here we identify miR-203, a miRNA abundantly and preferentially expressed in skin, to be downregulated in BCCs. We show that activation of the Hedgehog (HH) pathway, critically involved in the pathogenesis of BCCs, as well as the EGFR/MEK/ERK/c-JUN signaling pathway suppresses miR-203. We identify c-JUN, a key effector of the HH pathway, as a novel direct target for miR-203 in vivo. Further supporting the role of miR-203 as a tumor suppressor, in vivo delivery of miR-203 mimics in a BCC mouse model results in the reduction of tumor growth. Our results identify a regulatory circuit involving miR-203 and c-JUN, which provides functional control over basal cell proliferation and differentiation. We propose that miR-203 functions as a 'bona fide' tumor suppressor in BCC, whose suppressed expression contributes to oncogenic transformation via derepression of multiple stemness- and proliferation-related genes, and its overexpression could be of therapeutic value.
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While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential.
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RNA interference constitutes a powerful tool for biological studies, but has also become one of the most challenging therapeutic strategies. However, small interfering RNA (siRNA)-based strategies suffer from their poor delivery and biodistribution. Cell-penetrating peptides (CPPs) have been shown to improve the intracellular delivery of various biologically active molecules into living cells and have more recently been applied to siRNA delivery. To improve cellular uptake of siRNA into challenging cell lines, we have designed a secondary amphipathic peptide (CADY) of 20 residues combining aromatic tryptophan and cationic arginine residues. CADY adopts a helical conformation within cell membranes, thereby exposing charged residues on one side, and Trp groups that favor cellular uptake on the other. We show that CADY forms stable complexes with siRNA, thereby increasing their stability and improving their delivery into a wide variety of cell lines, including suspension and primary cell lines. CADY-mediated delivery of subnanomolar concentrations of siRNA leads to significant knockdown of the target gene at both the mRNA and protein levels. Moreover, we demonstrate that CADY is not toxic and enters cells through a mechanism which is independent of the major endosomal pathway. Given its biological properties, we propose that CADY-based technology will have a significant effect on the development of fundamental and therapeutic siRNA-based applications.
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MicroRNAs (miRNAs) are approximately 22-nucleotide-long noncoding RNAs that normally function by suppressing translation and destabilizing messenger RNAs bearing complementary target sequences. Some miRNAs are expressed in a cell- or tissue-specific manner and may contribute to the establishment and/or maintenance of cellular identity. Recent studies indicate that tissue-specific miRNAs may function at multiple hierarchical levels of gene regulatory networks, from targeting hundreds of effector genes incompatible with the differentiated state to controlling the levels of global regulators of transcription and alternative pre-mRNA splicing. This multilevel regulation may allow individual miRNAs to profoundly affect the gene expression program of differentiated cells.
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BACKGROUND: Chronic skin inflammation in atopic dermatitis (AD) is associated with elevated expression of proinflammatory genes and activation of innate immune responses in keratinocytes. microRNAs (miRNAs) are short, single-stranded RNA molecules that silence genes via the degradation of target mRNAs or inhibition of translation. OBJECTIVE: The aim of this study was to investigate the role of miR-146a in skin inflammation in AD. METHODS: RNA and protein expression was analyzed using miRNA and mRNA arrays, RT-quantitative PCR, Western blotting, and immunonohistochemistry. Transfection of miR-146a precursors and inhibitors into human primary keratinocytes, luciferase assays, and MC903-dependent mouse model of AD were used to study miR-146a function. RESULTS: We show that miR-146a expression is increased in keratinocytes and chronic lesional skin of patients with AD. miR-146a inhibited the expression of numerous proinflammatory factors, including IFN-γ-inducible and AD-associated genes CCL5, CCL8, and ubiquitin D (UBD) in human primary keratinocytes stimulated with IFN-γ, TNF-α, or IL-1β. In a mouse model of AD, miR-146a-deficient mice developed stronger inflammation characterized by increased accumulation of infiltrating cells in the dermis, elevated expression of IFN-γ, CCL5, CCL8, and UBD in the skin, and IFN-γ, IL-1β, and UBD in draining lymph nodes. Both tissue culture and in vivo experiments in mice demonstrated th