Article

Ultrasound-activated particles as CRISPR/Cas9 delivery system for androgenic alopecia therapy

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Abstract

Compared to a plasmid, viral, and other delivery systems, direct Cas9/sgRNA protein delivery has several advantages such as low off-targeting effects and non-integration, but it still has limitations due to low transfer efficiency. As such, the CRISPR/Cas9 system is being developed in combination with nano-carrier technology to enhance delivery efficiency and biocompatibility. We designed a microbubble-nanoliposomal particle as a Cas9/sgRNA riboprotein complex carrier, which effectively facilitates local delivery to a specific site when agitated by ultrasound activation. In practice, we successfully transferred the protein constructs into dermal papilla cells in the hair follicle of androgenic alopecia animals by microbubble cavitation induced sonoporation of our particle. The delivered Cas9/sgRNA recognized and edited specifically the target gene with high efficiency in vitro and in vivo, thus recovering hair growth. We demonstrated the topical application of ultrasound-activated nanoparticles for androgenic alopecia therapy through the suppression of SRD5A2 protein production by CRISPR-based genomic editing.

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... 18 Ultrasound Similar to heat and light control, the application of ultrasound to control the release of payloads from carriers has attracted increasing attention (Fig. 7). 149,150 The key to using this technology is the carrier design. Many nanomotors that can convert chemical fuels or external energy into mechanical motion have attractive characteristics, [151][152][153] including propelled motion and cargo transportation, enabling them to be implemented in biosensing and drug delivery. ...
... MB-NL can significantly promote local delivery to target sites under ultrasound activation (Fig. 7). 150 Ryu et al. designed an ultrasoundactivated microbubble-conjugated nanoliposome (MB-NL) system to deliver Cas9/sgRNA and conduct androgenic alopecia therapy. 150 MB cavitation-induced sonoporation of the carrier particle can boost the delivery efficiency of the Cas9/sgRNA complexes to the dermal papilla cell (DPC) under high acoustical wave ultrasound frequency (1-5 MHz). ...
... 150 Ryu et al. designed an ultrasoundactivated microbubble-conjugated nanoliposome (MB-NL) system to deliver Cas9/sgRNA and conduct androgenic alopecia therapy. 150 MB cavitation-induced sonoporation of the carrier particle can boost the delivery efficiency of the Cas9/sgRNA complexes to the dermal papilla cell (DPC) under high acoustical wave ultrasound frequency (1-5 MHz). 150 The MB-NL and Cas9/ sgRNA@AuNW systems improve the local delivery of gene editing tools via ultrasound stimulation, with unique advantages such as safe transportation at specific sites. ...
Article
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The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) gene editing technology, as a revolutionary breakthrough in genetic engineering, offers a promising platform to improve the treatment of various genetic and infectious diseases because of its simple design and powerful ability to edit different loci simultaneously. However, failure to conduct precise gene editing in specific tissues or cells within a certain time may result in undesirable consequences, such as serious off-target effects, representing a critical challenge for the clinical translation of the technology. Recently, some emerging strategies using genetic regulation, chemical and physical strategies to regulate the activity of CRISPR/Cas9 have shown promising results in the improvement of spatiotemporal controllability. Herein, in this review, we first summarize the latest progress of these advanced strategies involving cell-specific promoters, small-molecule activation and inhibition, bioresponsive delivery carriers, and optical/thermal/ultrasonic/magnetic activation. Next, we highlight the advantages and disadvantages of various strategies and discuss their obstacles and limitations in clinical translation. Finally, we propose viewpoints on directions that can be explored to further improve the spatiotemporal operability of CRISPR/Cas9.
... Androgenetic alopecia (AGA), as the most familiar type of hair loss, which possesses the characteristic of progressive thinning and loss of scalp hair, affects up to 50% of Caucasian men [1][2][3]. Perifollicular vascularization is essential for hair growth, as hair growth initiates inside the avascular hair follicles (HFs) and relies on perifollicular vascularization to satisfy the growing nutritional requirements of HFs [4][5][6]. However, the angiogenic genes are down-regulated in the balding scalp of AGA patients, and the vascular endothelial growth factor (VEGF), a vital mediator for boosting microvascular permeability and angiogenesis, is insufficient in the HFs of AGA patients [7,8]. ...
... CM) group, the CM-MNs group, and the minoxidil group. An AGA mouse model was established through daily topical application of testosterone solution as previously reported [3,30]. Testosterone (0.5%, w/v) was reconstituted in ethanol (50%, v/ v) and was applied daily to the depilated skin (0.1 mL/ cm 2 ) in all groups for 28 consecutive days after depilation. ...
Article
Androgenetic alopecia (AGA), the most prevalent type of hair loss in clinic, is induced partly by insufficient perifollicular vascularization. Here we designed a dissolvable microneedles (MNs) patch that was loaded with conditioned media (CM) derived from hypoxia-pretreated mesenchymal stem cells, which contained elevated HIF-1α. The CM-integrated MNs patch (designated as CM-MNs) can puncture the stratum corneum and deliver the pro-angiogenic factors directly into skin in a one-step and minimally invasive manner. Meanwhile, the administration of CM-MNs induced a certain mechanical stimulation on the skin, which can also promote neovascularization. With the combined effects of the pro-angiogenic factors in CM and the mechanical stimulation induced by MNs, CM-MNs successfully boosted perifollicular vascularization, and activated hair follicle stem cells, thereby inducing notably faster hair regeneration at a lower administration frequency on AGA mouse model compared with minoxidil. Furthermore, we proved that the inhibition of perifollicular angiogenesis can restrain the awakening of hair follicle stem cells, elucidating the tight correlation between perifollicular angiogenesis and the activation of hair follicle stem cells. The innovative integration of CM and MNs holds great promise for clinical AGA treatment and indicates that boosting angiogenesis around hair follicles is an effective strategy against AGA.
... Two recent studies have investigated how the principles of sonoporation can be used to increase hair follicle growth [75,76]. Liao et al. focused on the potential of inhibiting bacteria and allergies on the scalp with the use of lysozyme-shelled microbubbles, finding that ultrasound application significantly enhanced hair growth rates in mice [75]. ...
... Liao et al. focused on the potential of inhibiting bacteria and allergies on the scalp with the use of lysozyme-shelled microbubbles, finding that ultrasound application significantly enhanced hair growth rates in mice [75]. Ryu et al. designed a microbubble-nanoliposomal particle to act as a Cas9/sgRNA riboprotein complex carrier and found that the protein constructs were successfully transferred into dermal papilla cells, effectively treating the mice with androgenic alopecia [76]. The results of this study demonstrated that the external application of ultrasound allowed the treatment to diffuse deeper and that the riboprotein complex carrier experienced a high efficiency of recognition and gene editing, with an on-target effect of about 70%. ...
Article
Research on the capability of non-viral gene delivery systems to induce tissue regeneration is a continued effort as the current use of viral vectors can present with significant limitations. Despite initially showing lower gene transfection and gene expression efficiencies, non-viral delivery methods continue to be optimized to match that of their viral counterparts. Ultrasound-mediated gene transfer, referred to as sonoporation, occurs by the induction of transient membrane permeabilization and has been found to significantly increase the uptake and expression of DNA in cells across many organ systems. In addition, it offers a more favorable safety profile compared to other non-viral delivery methods. Studies have shown that microbubble-enhanced sonoporation can elicit significant tissue regeneration in both ectopic and disease models, including bone and vascular tissue regeneration. Despite this, no clinical trials on the use of sonoporation for tissue regeneration have been conducted, although current clinical trials using sonoporation for other indications suggest that the method is safe for use in the clinical setting. In this review, we describe the pre-clinical studies conducted thus far on the use of sonoporation for tissue regeneration. Further, the various techniques used to increase the effectiveness and duration of sonoporation-induced gene transfer, as well as the obstacles that may be currently hindering clinical translation, are explored.
... Recently, Ryu and co-workers [154] reported a very interesting study on the combined use of the CRISPR9/Cas9 system with NPs, to treat AGA in mice (Figure 6). The researchers developed nanoliposomes as a carrier for the Cas9/sgRNA riboprotein complex, as a way to target the delivery to dermal papilla cells in the hair follicle of a rat model of AGA (Figure 6A). ...
... Bu (bulge), HG (hair germ), and DP (dermal papilla) presented, and white arrow is indicated the portion of intimidated DP; (F) Comparison of black hair contrast intensity; (G) H&E staining of harvested mouse skin after 7 thweek of treatment. Reproduced with permission from reference [154]. Copyright Elsevier (2020). ...
Article
Hair care and treatment has evolved significantly through the years as new formulations are continuously being explored in an attempt to meet the demand in cosmetic and medicinal fields. While standard hair care procedures include hair washing, aimed at hair cleansing and maintenance, as well as hair dyeing and bleaching formulations for hair embellishment, modern hair treatments are mainly focused on circumventing hair loss conditions, strengthening hair follicle properties and treat hair infestations. In this regard, active compounds (ACs) included in hair cosmetic formulations include a vast array of hair cleansing and hair dye molecules, and typical hair treatments include anti-hair loss ACs (e.g. minoxidil and finasteride) and anti-lice ACs (e.g. permethrin). However, several challenges still persist, as conventional AC formulations exhibit sub-optimal performance and some may present toxicity issues, calling for an improved design of formulations regarding both efficacy and safety. More recently, nano-based strategies encompassing nanomaterials have emerged as promising tailored approaches to improve the performance of ACs incorporated into hair cosmetics and treatment formulations. The interest in using these nanomaterials is based on account of their ability to: (1) increase stability, safety and biocompatibility of ACs; (2) maximize hair affinity, contact and retention, acting as versatile biointerfaces; (3) enable the controlled release of ACs in both hair and scalp, serving as prolonged AC reservoirs; besides offering (4) hair follicle targeting features attending to the possibility of surface tunability. This review covers the breakthrough of nanomaterials for hair cosmetics and hair treatment, focusing on organic nanomaterials (polymer-based and lipid-based nanoparticles) and inorganic nanomaterials (nanosheets, nanotubes and inorganic nanoparticles), as well as their applications, highlighting their potential as innovative multifunctional nanomaterials towards maximized hair care and treatment. Statement of significance This manuscript is focused on reviewing the nanotechnological strategies investigated for hair care and treatment so far. While conventional formulations exhibit sub-optimal performance and some may present toxicity issues, the selection of improved and suitable nanodelivery systems is of utmost relevance to ensure a proper active ingredient release in both hair and scalp, maximize hair affinity, contact and retention, and provide hair follicle targeting features, warranting stability, efficacy and safety. This innovative manuscript highlights the advantages of nanotechnology-based approaches, particularly as tunable and versatile biointerfaces, and their applications as innovative multifunctional nanomaterials towards maximized hair care and treatment.
... Recently, Ryu and co-workers [154] reported a very interesting study on the combined use of the CRISPR9/Cas9 system with NPs, to treat AGA in mice ( Fig. 6 ). The researchers developed nanoliposomes as a carrier for the Cas9/sgRNA riboprotein complex, as a way to target the delivery to dermal papilla cells in the hair follicle of a rat model of AGA ( Fig. 6 A ). ...
... Bu (bulge), HG (hair germ), and DP (dermal papilla) presented, and white arrow is indicated the portion of intimidated DP; (F) Comparison of black hair contrast intensity; (G) H&E staining of harvested mouse skin after 7 th -week of treatment. Reproduced with permission from reference[154] . Copyright Elsevier (2020). ...
... It was shown that GFP gene expression has been reduced by 80% through knockout [75]. Recently, a microbubble-nanoliposomal particle has been developed as a carrier for Cas9/gRNA riboprotein complex; if stimulated by ultrasound waves, it can facilitate the effective delivery of the cargo at a specific site [76]. The researchers successfully transferred Cas9/sgRNA into the hair follicle dermal papilla cells of androgenic alopecia animals. ...
... The researchers successfully transferred Cas9/sgRNA into the hair follicle dermal papilla cells of androgenic alopecia animals. Cas9/gRNA transfer followed by ultrasound activation causes recovery of hair growth by suppression of SRD5A2 protein production through the CRISPR gene editing system [76]. Overall, such ultrasound-responsive NPs shown great perspective in CRISPR/Cas9 delivery into different diseased cell types such as cancerous and syndrome cells. ...
Article
Full-text available
The innovative research in genome editing domains such as CRISPR-Cas technology has enabled genetic engineers to manipulate the genomes of living organisms effectively in order to develop the next generation of therapeutic tools. This technique has started the new era of “genome surgery”. Despite these advances, the barriers of CRISPR-Cas9 techniques in clinical applications include efficient delivery of CRISPR/Cas9 and risk of off-target effects. Various types of viral and non-viral vectors are designed to deliver the CRISPR/Cas9 machinery into the desired cell. These methods still suffer difficulties such as immune response, lack of specificity, and efficiency. The extracellular and intracellular environments of cells and tissues differ in pH, redox species, enzyme activity, and light sensitivity. Recently, smart nanoparticles have been synthesized for CRISPR/Cas9 delivery to cells based on endogenous (pH, enzyme, redox specie, ATP) and exogenous (magnetic, ultrasound, temperature, light) stimulus signals. These methodologies can leverage genome editing through biological signals found within disease cells with less off-target effects. Here, we review the recent advances in stimulus-based smart nanoparticles to deliver the CRISPR/Cas9 machinery into the desired cell. This review article will provide extensive information to cautiously utilize smart nanoparticles for basic biomedical applications and therapeutic genome editing.
... The researchers directed the genetic editor to the SRD5A2 gene using a combination of nanoliposomal particles as a vehicle and sonoporation as an 'activator' of particles, for delivery. Under the action of cavitation, the particles burst and delivered plasmid vectors directly to the cells of the hair follicles [51]. Phototransfection. ...
Article
Gene editing with programmable nucleases opens new perspectives in important practice areas, such as healthcare and agriculture. The most challenging problem for the safe and effective therapeutic use of gene editing technologies is the proper delivery and expression of gene editors in cells and tissues of different organisms. Virus-based and nonviral systems can be used for the successful delivery of gene editors. Here we have reviewed structural elements of nonviral DNA- and RNA-based expression vectors for gene editing and delivery methods in vitro and in vivo.
... In 2020, Ryu et al. developed ultrasound-activated microbubble (MB)-conjugated with nanoliposome particles (NL; a type of LNP) for the delivery of the Cas9-sgRNA RNP complex (MB-NL(Cas9-sgRNA)) to treat androgenic alopecia in vivo in a mouse model through disruption of the Srd5a2 gene [180]. The MB-NL(Cas9-sgRNA) complex was fabricated through supramolecular interactions and covalent conjugation (Fig. 3D). ...
Article
Since its mechanism discovery in 2012 and the first application for mammalian genome editing in 2013, the CRISPR-Cas9 has revolutionized the genome engineering field and created countless opportunities in both basic science and translational medicine. The first clinical trial of CRISPR therapeutics was initiated in 2016, which employed ex vivo CRISPR-Cas9 edited PD-1 knockout T cells for the treatment of non-small cell lung cancer. So far there have been dozens of clinical trials registered on ClinicalTrials.gov in regard to using the CRISPR-Cas9 genome editing as the main intervention for therapeutic applications; however, most of these studies use ex vivo genome editing approach, and only a few apply the in vivo editing strategy. Compared to ex vivo editing, in vivo genome editing bypasses tedious procedures related to cell isolation, maintenance, selection, and transplantation. It is also applicable to a wide range of diseases and disorders. The main obstacles to the successful translation of in vivo therapeutic genome editing include the lack of safe and efficient delivery system and safety concerns resulting from the off-target effects. In this review, we highlight the therapeutic applications of in vivo genome editing mediated by the CRISPR-Cas9 system. Following a brief introduction of the history, biology, and functionality of CRISPR-Cas9, we showcase a series of exemplary studies in regard to the design and implementation of in vivo genome editing systems that target the brain, inner ear, eye, heart, liver, lung, muscle, skin, immune system, and tumor. Current challenges and opportunities in the field of CRISPR-enabled therapeutic in vivo genome editing are also discussed.
... However, its low control leads to low transfection efficiency, low success rate and high equipment requirements [54]. Ryu et al. constructed microbubble nano-liposome particles as Cas9/sgRNA nucleoprotein complex carriers and successfully transferred the protein complex into dermal papilla cells of hair follicles in male bald animals under ultrasonic activation The research validated the inhibitory effect of CRISPR/Cas9 on SRD5A2 in vivo and in vitro which finally restored hair growth [55]. To deliver CRISPR plasmids, Dong et al. developed a dual ultrasonic/magnetic responsive microdrop that can effectively deliver plasmids to cancer cells, which could be a potential strategy for clinical application of ultrasonic methods for cancer treatment [56]. ...
Article
Full-text available
CRISPR/Cas9 is a revolutionary genome editing technology with the tremendous advantages such as precisely targeting/shearing ability, low cost and convenient operation, becoming an efficient and indispensable tool in biological research. As a disruptive technique, CRISPR/Cas9 genome editing has a great potential to realize a future breakthrough in the clinical bone and cartilage repairing as well. This review highlights the research status of CRISPR/Cas9 system in bone and cartilage repair, illustrates its mechanism for promoting osteogenesis and chondrogenesis, and explores the development tendency of CRISPR/Cas9 in bone and cartilage repair to overcome the current limitations.
... By this, microbubbles rupture and generate transient pores on the blood wall and cell membrane that allow the entry of the target cargo into cells [115]. For instance, Ryu et al. applied the ultrasound-activated particles as CRISPR-Cas9 DNA plasmid delivery for disrupting the steroid type II 5-alpha-reductase (SRD5A2) gene, which encodes an enzyme involved in male pattern baldness, into dermal papilla cells of a mouse model to recover hair growth [116]. In another study, the ultrasound microbubble-mediated CRISPR-Cas9 plasmid delivery was shown to knockout the epidermal growth factor receptor 2 (C-erbB-2) gene, a proto-oncogene associated with breast cancer, in human endometrial cancer (HEC)-1A cells [117]. ...
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The discovery of clustered regularly interspaced short palindromic repeats (CRISPR) genome editing technology opened the door to provide a versatile approach for treating multiple diseases. Promising results have been shown in numerous pre-clinical studies and clinical trials. However, a safe and effective method to deliver genome-editing components is still a key challenge for in vivo genome editing therapy. Adeno-associated virus (AAV) is one of the most commonly used vector systems to date, but immunogenicity against capsid, liver toxicity at high dose, and potential genotoxicity caused by off-target mutagenesis and genomic integration remain unsolved. Recently developed transient delivery systems, such as virus-like particle (VLP) and lipid nanoparticle (LNP), may solve some of the issues. This review summarizes existing in vivo delivery systems and possible solutions to overcome their limitations. Also, we highlight the ongoing clinical trials for in vivo genome editing therapy and recently developed genome editing tools for their potential applications.
... The establishment of the AGA mouse model reported by Ryu was followed with slight modification. 56 In brief, the testosterone solution (0.2%, w/v) was prepared in ethanol solution (50%, v/v). Mice in all groups were topically applied with testosterone solution (0.1 mL/ cm 2 ) on the depilated area once a day for 28 consecutive days. ...
... The production of the protein in the bacterial hosts might be expensive and the endotoxin contamination could be considered as an additional obstacle for their large-scale production. Various non-viral carriers have been used to transfer such platforms into the target cells including gold NPs, graphene oxide, carboxylated branched poly (β-amino ester) NPs, β-cyclodextrin-conjugated low-molecular-weight PEI, microbubble-nanoliposomal particles, pH-responsive silica-metal-organic framework (SMOF) hybrid NPs consisting of both silica and zeolitic imidazole framework (ZIF) as well as cell-penetrating peptides and DNA nanoclews [319][320][321][322][323][324][325][326][327][328][329][330][331]. ...
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Gene therapy by expression constructs or down-regulation of certain genes has shown great potential for the treatment of various diseases. The wide clinical application of nucleic acid materials dependents on the development of biocompatible gene carriers. There are enormous various compounds widely investigated to be used as non-viral gene carriers including lipids, polymers, carbon materials, and inorganic structures. In this review, we will discuss the recent discoveries on non-viral gene delivery systems. We will also highlight the in vivo gene delivery mediated by non-viral vectors to treat cancer in different tissue and organs including brain, breast, lung, liver, stomach, and prostate. Finally, we will delineate the state-of-the-art and promising perspective of in vivo gene editing using non-viral nano-vectors.
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CRISPR/Cas9‐based gene therapy and photodynamic therapy both show promise for cancer treatment but still have their drawbacks limited by tumor microenvironment and long treatment duration. Herein, CRISPR/Cas9 genome editing and photodynamic strategy for a synergistic anti‐tumor therapeutic modality is merged. Chlorophyll (Chl) extracted from natural green vegetables is encapsulated in Pluronic F127 (F127) micelles and Histidine‐tagged Cas9 can be effectively chelated onto micelles via metal coordination by simple incubation, affording Cas9‐Chl@F127 micelles. Mg2+ acts as an enzyme cofactor to correlatively enhance Cas9 gene‐editing activity. Upon laser irradiation, Chl as an effective photosensitizer generates reactive oxygen species (ROS) to kill tumor cells. Meanwhile, CRISPR/Cas9, mediated by dual deliberately designed gRNAs of APE1 and NRF2, can reprogram the tumor microenvironment by increasing the intracellular oxygen accumulation and impairing the oxidative defense system of tumor cells. Cas9‐Chl@F127 micelles can responsively release Cas9 in the presence of abundant ATP or low pH in tumor cells. In a murine tumor model, Cas9‐Chl@F127 complexed with dual gRNAs including APE1 and NRF2 significantly inhibits the tumor growth. Taken together, Cas9‐Chl@F127 micelles, representing the first Chl‐based green biomaterial for the delivery of Cas9, show great promise for the synergistic anti‐tumor treatment by PDT and gene editing. A new CRISPR‐Cas9 delivery system is developed using naturally existing chlorophyll‐based micelles via metal coordination. Magnesium ion is not only used as a bridge to chelate Cas9 and chlorophyll, enabling synergistic therapy of photodynamic therapy and genome editing, but also acts as an enzyme cofactor to significantly increase the catalytic activity of Cas9.
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The need for effective hair loss treatments has fostered research and the emergence of several biotechnology companies. Pharmacological approaches, although competitive, have been surpassed by cell-based therapies, which remain clinically immature. But are the current efforts enough for the hairy goal, or will additional strategies be required? Keywords hair loss; androgenic alopecia; therapies; biotechs
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Clustered regularly interspaced short palindromic repeats (CRISPR)-associated systems have revolutionized traditional gene-editing tools and are a significant tool for ameliorating gene defects. Characterized by high target specificity, extraordinary efficiency, and cost-effectiveness, CRISPR/Cas systems have displayed tremendous potential for genetic manipulation in almost any organism and cell type. Despite their numerous advantages, however, CRISPR/Cas systems have some inherent limitations, such as off-target effects, unsatisfactory efficiency of delivery, and unwanted adverse effects, thereby resulting in a desire to explore approaches to address these issues. Strategies for improving the efficiency of CRISPR/Cas-induced mutations, such as reducing off-target effects, improving the design and modification of sgRNA, optimizing the editing time and the temperature, choice of delivery system, and enrichment of sgRNA, are comprehensively described in this review. Additionally, several newly emerging approaches, including the use of Cas variants, anti-CRISPR proteins, and mutant enrichment, are discussed in detail. Furthermore, the authors provide a deep analysis of the current challenges in the utilization of CRISPR/Cas systems and the future applications of CRISPR/Cas systems in various scenarios. This review not only serves as a reference for improving the maturity of CRISPR/Cas systems but also supplies practical guidance for expanding the applicability of this technology.
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CRISPR-Cas9 as a powerful gene-editing tool has tremendous potential for the treatment of genetic diseases. Herein, we report a new mesoporous nanoflower-like delivery nanoplatform termed Cas9-NF by crosslinking Cas9 and polymeric micelles that can accomplish efficient intracellular delivery and controlled release of Cas9 in response to reductive microenvironment in tumor cells. The flower morphology was flexibly tunable by the protein concentration and different types of crosslinkers. Cas9 protein, embedded between polymeric micelles and protected by Cas9-NF, remained stable even under extreme pH conditions. Responsive cleavage of crosslinkers in tumor cells, leads to the traceless release of Cas9 for efficient gene knockout in nucleus. This crosslinked nanoparticle exhibited excellent capability of downregulating oncogene expression and inhibiting tumor growth in a murine tumor model. Taken together, these findings pave a new pathway towards the applications of the protein-micelle crosslinked nanoflower for protein delivery that warrants further investigations for gene regulation and cancer treatment. This article is protected by copyright. All rights reserved
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Recently, the combination of cosmetology and microneedle therapy has made great progress. Compared with direct injection, microneedles not only have functions of targeted delivery and high bioavailability, but also have characteristics of painless, mild and convenient. Also, it is beneficial to users' self-administration. Therefore, microneedles are more inclined to be treated by patients with cosmetic needs. Since microneedles were first applied in the cosmetic field in 2005, new products have emerged one after another, and the application of microneedles in wrinkle-removing, scar-treating, whitening, fat-reducing and other aspects has been greatly improved. Meanwhile, the composition of microneedles is a key factor affecting the preparation process, drug loading/release capacity, stability and final effect of microneedles. This paper reviews the commonly used matrix materials, loading drugs and preparation methods of cosmetic microneedles from the perspective of materials, in order to provide basic reference for the preparation of cosmetic microneedles. It is vital to detail the representative applications of microneedles in cosmetology. In conclusion, as a new tool, microneedle system shows great promise in cosmetic application.
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Photothermal therapy (PTT) is a promising strategy for the treatment of advanced malignant neoplasm. However, the anti-tumor efficacy by PTT alone is insufficient to control tumor growth and metastasis. Here, we report a multifunctional nanotherapeutic system exerting a combined PTT and immunotherapy to synergistically enhance the therapeutic effect on melanoma. In particular, we selected the semiconductor nanomaterial copper sulfide (CuS), which served not only as a near-infrared (NIR) light-triggered photothermal converter for tumor hyperthermia but as a basic carrier to modify Cas9 ribonucleoprotein targeting PTPN2 on its surface. Efficient PTPN2 depletion was observed after the treatment of [email protected] nanoparticles, which caused the accumulation of intratumoral infiltrating CD8 T lymphocytes in tumor-bearing mice and upregulated the expression levels of IFN-ᵧ and TNF-α in tumor tissue, thus sensitizing tumors to immunotherapy. In addition, the effect worked synergistically with tumor ablation and immunogenic cell death (ICD) induced by PTT to amplify anti-tumor efficacy. Taken together, this exogenously controlled method provides a simple and effective treatment option for advanced malignant neoplasm.
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Cellular senescence refers to a state of irreversible arrest of cell proliferation in response to various forms of cellular stress. It is known that the accumulation of senescent cells is a hallmark of aging, and mounting evidence has shown that the chronic accumulation of senescent cells is a significant contributor to various deleterious age‐related pathologies. To limit the detrimental impacts of cellular senescence, there has been growing interest in targeted delivery of therapeutics to senescent cells to treat age‐related pathologies and promote healthy aging. Two popular strategies include the elimination of senescent cells using senolytic drugs, and rejuvenation of senescent cells. To that end, it is integral that the delivery of senolytics, senomorphics or rejuvenating biomolecules to senescent cells are highly selective to enhance delivery efficacy and safety. However, there is little understanding of how senescence‐associated biophysical changes such as cellular size and stiffness can be exploited for targeted therapeutics delivery. In this review, the biomolecular and biophysical markers of senescence along with senescence models and emerging therapeutics are first described. This review then focuses on how biophysical properties can be exploited for targeted therapeutics delivery, using approaches like nanoparticles, electroporation, sonoporation, photoporation and high aspect‐ratio nanostructures to senescent cells. The chronic accumulation of senescent cells is a significant contributor to many age‐related diseases. Cell specificity is integral to ensure therapeutic safety and efficacy for optimal clinical translation during delivery of therapeutics to eliminate or rejuvenate senescent cells. This review discusses how biophysical changes in senescent cells can be exploited for targeted therapeutics delivery.
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The potential of the cluster regularly interspaced short palindromic repeat (CRISPR)‐associated protein 9 (Cas9)‐based therapeutic genome editing is severely hampered by the difficulties in precise regulation of the in vivo activity of the CRISPR‐Cas9 system. Herein, sono‐controllable and reactive oxygen species (ROS)‐sensitive sonosensitizer‐integrated metal–organic frameworks (MOFs), denoted as P/M@CasMTH1, are developed for augmented sonodynamic therapy (SDT) efficacy using the genome‐editing technology. P/M@CasMTH1 nanoparticles comprise singlet oxygen (1O2)‐generating MOF structures anchored with CRISPR‐Cas9 systems via 1O2‐cleavable linkers, which serve not only as a delivery vector of CRISPR‐Cas9 targeting MTH1, but also as a sonoregulator to spatiotemporally activate the genome editing. P/M@CasMTH1 escapes from the lysosomes, harvests the ultrasound (US) energy and converts it into abundant 1O2 to induce SDT. The generated ROS subsequently trigger cleavage of ROS‐responsive thioether bonds, thus inducing controllable release of the CRISPR‐Cas9 system and initiation of genome editing. The genomic disruption of MTH1 conspicuously augments the therapeutic efficacy of SDT by destroying the self‐defense system in tumor cells, thereby causing cellular apoptosis and tumor suppression. This therapeutic strategy for synergistic MTH1 disruption and abundant 1O2 generation provides a paradigm for augmenting SDT efficacy based on the emerging nanomedicine‐enabled genome‐editing technology. This work not only pioneers a novel avenue to circumvent the resistance of tumor cells in conventional sonodynamic therapy, but also achieves targeted delivery and controllable release of the cluster regularly interspaced short palindromic repeat‐associated protein system.
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Ultrasonic technologies show great promise for diagnostic imaging and drug delivery in theranostic applications. The development of functional and molecular ultrasound imaging is based on the technical breakthrough of high frame–rate ultrasound. The evolution of shear wave elastography, high-frequency ultrasound imaging, ultrasound contrast imaging, and super-resolution blood flow imaging are described in this review. Recently, the therapeutic potential of the interaction of ultrasound with microbubble cavitation or droplet vaporization has become recognized. Microbubbles and phase-change droplets not only provide effective contrast media, but also show great therapeutic potential. Interaction with ultrasound induces unique and distinguishable biophysical features in microbubbles and droplets that promote drug loading and delivery. In particular, this approach demonstrates potential for central nervous system applications. Here, we systemically review the technological developments of theranostic ultrasound including novel ultrasound imaging techniques, the synergetic use of ultrasound with microbubbles and droplets, and microbubble/droplet drug-loading strategies for anticancer applications and disease modulation. These advancements have transformed ultrasound from a purely diagnostic utility into a promising theranostic tool.
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Manipulation of CRISPR delivery for stimuli‐responsive gene editing is crucial for cancer therapeutics through maximizing efficacy and minimizing side‐effects. However, realizing controlled gene editing for synergistic combination therapy remains a key challenge. Here, a near‐infrared (NIR) light‐triggered thermo‐responsive copper sulfide (CuS) multifunctional nanotherapeutic platform is constructed to achieve controlled release of CRISPR‐Cas9 ribonucleoprotein (RNP) and doxorubicin for tumor synergistic combination therapy involving in gene therapy, mild‐photothermal therapy (PTT), and chemotherapy. The semiconductor CuS serves as a “photothermal converter” and can stably convert NIR light (808 nm) into local thermal effect to provide photothermal stimulation. The double‐strand formed between CuS nanoparticle‐linked DNA fragments and single‐guide RNA is employed as a controlled element in response to photothermal stimulation for controlled gene editing and drug release. Hsp90α, one subunit of heat shock protein 90 (Hsp90), is targeted by Cas9 RNP to reduce tumor heat tolerance for enhanced mild‐PTT effects (≈43 °C). Significant synergistic therapy efficacy can be observed by twice NIR light irradiation both in vitro and in vivo, compared to PTT alone. Overall, this exogenously controlled method provides a versatile strategy for controlled gene editing and drug release with potentially synergistic combination therapy. A novel thermo‐responsive nanotherapeutic platform named CuS‐RNP/DOX@PEI is established, which is triggered by 808 nm near‐infrared light irradiation. This exogenously controlled method can be used in combination tumor therapy, including gene‐therapy, mild‐photothermal therapy, and chemotherapy, which provides a versatile strategy for controlled gene editing and drug release for synergistic therapy with maximizing efficacy and minimizing side‐effects.
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The CRISPR-Cas9 system is a powerful tool for genome editing, which can potentially lead to new therapies for genetic diseases. To date, various viral and non-viral delivery systems have been developed for the delivery of CRISPR-Cas9 in vivo. However, spatially and temporally controlled genome editing is needed to enhance the specificity in organs/tissues and minimize the off-target effects of editing. In this review, we summarize the state-of-the-art non-viral vectors that exploit external stimuli (i.e., light, magnetic field, and ultrasound) for spatially and temporally controlled genome editing and their in vitro and in vivo applications.
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Ultrasound‐targeted microbubble destruction (UTMD) mediates gene transfection with high biosafety and thus has been promising toward treatment of type 1 diabetes. However, the potential application of UTMD in type 2 diabetes (T2D) is still limited, due to the lack of systematic design and dynamic monitoring. Herein, an efficient gene delivery system is constructed by plasmid deoxyribonucleic acid (DNA) encoding glucagon‐like peptide 1 (GLP‐1) in ultrasound‐induced microbubbles, toward treatment of T2D in macaque. The as designed UTMD afforded enhancement of cell membrane penetration and GLP‐1 expression in macaque, which is characterized by ultrasound‐guided biopsy to monitor the dynamic process of islet cells for 6 months. Also, improvement of pancreatic beta cell regeneration, and regulation of plasma glucose in macaque with T2D is achieved. The approach would serve as promising alternatives for the treatment of T2D. An efficient gene delivery system is constructed using plasmid encoding glucagon‐like peptide 1 (GLP‐1) in ultrasound‐induced microbubbles, toward treatment of type 2 diabetes (T2D). Such therapy is mediated by ultrasound‐targeted microbubble destruction (UTMD), affording 1) enhancement of cell membrane penetration and GLP‐1 expression, 2) improvement of pancreatic beta‐cell regeneration, and 3) regulation of plasma glucose in macaque with T2D.
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Background Whilst traditional strategies to increase transfection efficiency of non-viral systems aimed at modifying the vector or the polyplexes/lipoplexes, biomaterial-mediated gene delivery has recently sparked increased interest. This review aims at discussing biomaterial properties and unravelling underlying mechanisms of action, for biomaterial-mediated gene delivery. DNA internalisation and cytoplasmic transport are initially discussed. DNA immobilisation, encapsulation and surface-mediated gene delivery (SMD), the role of extracellular matrix (ECM) and topographical cues, biomaterial stiffness and mechanical stimulation are finally outlined. Main text Endocytic pathways and mechanisms to escape the lysosomal network are highly variable. They depend on cell and DNA complex types but can be diverted using appropriate biomaterials. 3D scaffolds are generally fabricated via DNA immobilisation or encapsulation. Degradation rate and interaction with the vector affect temporal patterns of DNA release and transgene expression. In SMD, DNA is instead coated on 2D surfaces. SMD allows the incorporation of topographical cues, which, by inducing cytoskeletal re-arrangements, modulate DNA endocytosis. Incorporation of ECM mimetics allows cell type-specific transfection, whereas in spite of discordances in terms of optimal loading regimens, it is recognised that mechanical loading facilitates gene transfection. Finally, stiffer 2D substrates enhance DNA internalisation, whereas in 3D scaffolds, the role of stiffness is still dubious. Conclusion Although it is recognised that biomaterials allow the creation of tailored non-viral gene delivery systems, there still are many outstanding questions. A better characterisation of endocytic pathways would allow the diversion of cell adhesion processes and cytoskeletal dynamics, in order to increase cellular transfection. Further research on optimal biomaterial mechanical properties, cell ligand density and loading regimens is limited by the fact that such parameters influence a plethora of other different processes (e.g. cellular adhesion, spreading, migration, infiltration, and proliferation, DNA diffusion and release) which may in turn modulate gene delivery. Only a better understanding of these processes may allow the creation of novel robust engineered systems, potentially opening up a whole new area of biomaterial-guided gene delivery for non-viral systems.
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Improvements in the understanding of human genetics and its roles in disease development and prevention have led to an increased interest in therapeutic genome editing via the use of engineered nucleases. Various approaches have been explored in the past focusing on the development of an effective and safe system for sequence-specific editing. Compared to earlier nucleases such as zinc finger nuclease and transcription activator-like effector nuclease, the relatively low cost and ease of producing clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas9) systems have made therapeutic genome editing significantly more feasible. CRISPR/Cas9 genome editing has shown great potential to correct genetic mutations implicated in monogenic diseases and to eradicate latent or chronic viral infections in preclinical studies. Several CRISPR/Cas9-based therapeutics have reached the clinical stage, including treatments for inherited red blood cell disorders and Leber Congenital Amaurosis 10, as well as CRISPR/Cas9-edited T cells designed to target and destroy cancer cells. Further advances in therapeutic genome editing will rely on a safe and more efficient method of in vivo CRISPR/Cas9 delivery and improved efficiency of homology-directed repair for site-specific gene insertion or replacement. While other reviews have focused on one or two aspects of CRISPR/Cas9 genome editing, this review aims to provide a summary of the mechanisms of genome editing, the reasons for the emerging interest in CRISPR/Cas9 compared to other engineered nucleases, the current progress in developing CRISPR/Cas9 delivery systems, and the current preclinical and clinical applications of CRISPR/Cas9 genome editing.
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Gene therapy provides a promising strategy for curing monogenetic disorders and complex diseases. However, there are challenges associated with the use of viral delivery vectors. The advent of nanomedicine represents a quantum leap in the application of gene therapy. Recent advances in stimulus‐responsive nonviral nanocarriers indicate that they are efficient delivery systems for loading and unloading of therapeutic nucleic acids. Some nanocarriers are responsive to cues derived from the internal environment, such as changes in pH, redox potential, enzyme activity, reactive oxygen species, adenosine triphosphate, and hypoxia. Others are responsive to external stimulations, including temperature gradients, light irradiation, ultrasonic energy, and magnetic field. Multiple stimuli‐responsive strategies have also been investigated recently for experimental gene therapy.
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Familial hypercholesterolemia (FH), with high LDL (low-density lipoprotein) cholesterol levels, is due to inherited mutations in genes, such as low-density lipoprotein receptor (LDLR). Development of therapeutic strategies for FH, which causes atherosclerosis and cardiovascular disease, is urgently needed. Methods: Mice with low-density lipoprotein receptor (Ldlr) deletion (Ldlr-/- mice) were used as an FH model. Ldlr mRNA was encapsulated into exosomes by forced expression of Ldlr in the donor AML12 (alpha mouse liver) cells, and the resultant exosomes were denoted as ExoLdlr. In vivo distribution of exosomes was analyzed by fluorescence labeling and imaging. The delivery efficiency of Ldlr mRNA was analyzed by qPCR and Western blotting. Therapeutic effects of ExoLdlr were examined in Ldlr-/- mice by blood lipids and Oil Red O staining. Results: The encapsulated mRNA was stable and could be translated into functional protein in the recipient cells. Following tail vein injection, exosomes were mainly delivered into the liver, producing abundant LDLR protein, resembling the endogenous expression profile in the wild-type mouse. Compared with control exosomes, ExoLdlr treatment significantly decreased lipid deposition in the liver and lowered the serum LDL-cholesterol level. Significantly, the number and size of atherosclerotic plaques and inflammation were reduced in the ExoLdlr-treated mice. Conclusions: We have shown that exosome-mediated Ldlr mRNA delivery effectively restored receptor expression, treating the disorders in the Ldlr-/- mouse. Our study provided a new therapeutic approach for the treatment of FH patients and managing atherosclerosis.
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The prokaryotic CRISPR–Cas systems could be applied as revolutionized genome editing tool in live cells of various species to modify, visualize and identify definite sequences of DNA and RNA. CRISPR–Cas could edit the genome by homology-directed repair and non-homologous end joining mechanisms. Furthermore, DNA-targeting modification by CRISPR–Cas methodology provides opportunity for diagnosis, therapy and the genetic disorders investigation. Here, we summarized delivery systems employed for CRISPR–Cas9 for genome editing. Then preclinical studies of the CRISPR-Cas9-based therapeutics will be discussed considering the associated challenges and developments in its translation to clinic for cancer therapy.
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Gene delivery vectors possess critical roles in effective genome editing. In this study, a multiple functional vector for encapsulating CRISPR/Cas9 plasmid was designed to knock out PPM1D gene and prevent cancer malignancy. The plasmid was complexed with a KALA peptide with the capability of endosomal escape and histones for nuclear transportation and then decorated by hyaluronic acid (HA) and AS1411-incorporated hyaluronic acid (AHA) targeting CD44 and nucleolin overexpressed in cancer cells to form AHA/HA/KALA/histone/plasmid nanoparticles. The constructed multifunctional plasmid delivery system with the cancer targeting specificity can realize efficient genome editing for PPM1D knockout and thus dramatically downregulate PPM1D expression in targeted malignant cells. More importantly, PPM1D knockout results in upregulation of p21 and p-p38 as well as downregulation of cyclin D1, MMP9, CYR61, and vimentin. The edited cancer cells exhibit suppressed proliferation, migration, and invasion, indicating the successful reversal of tumor malignancy.
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CRISPR/Cas9 genome editing has gained rapidly increasing attentions in recent years, however, the translation of this biotechnology into therapy has been hindered by efficient delivery of CRISPR/Cas9 materials into target cells. Direct delivery of CRISPR/Cas9 system as a ribonucleoprotein (RNP) complex consisting of Cas9 protein and single guide RNA (sgRNA) has emerged as a powerful and widespread method for genome editing due to its advantages of transient genome editing and reduced off-target effects. In this review, we summarized the current Cas9 RNP delivery systems including physical approaches and synthetic carriers. The mechanisms and beneficial roles of these strategies in intracellular Cas9 RNP delivery were reviewed. Examples in the development of stimuli-responsive and targeted carriers for RNP delivery are highlighted. Finally, the challenges of current Cas9 RNP delivery systems and perspectives in rational design of next generation materials for this promising field will be discussed. © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.
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Skin diseases are the fourth leading non-fatal skin conditions that act as a burden and affect the world economy globally. This condition affects the quality of a patient's life and has a pronounced impact on both their physical and mental state. Treatment of these skin conditions with conventional approaches shows a lack of efficacy, long treatment duration, recurrence of conditions, systemic side effects, etc., due to improper drug delivery. However, these pitfalls can be overcome with the applications of nanomedicine-based approaches that provide efficient site-specific drug delivery at the target site. These nanomedicine-based strategies are evolved as potential treatment opportunities in the form of nanocarriers such as polymeric and lipidic nanocarriers, nanoemulsions along with emerging others viz. carbon nanotubes for dermatological treatment. The current review focuses on challenges faced by the existing conventional treatments along with the topical therapeutic perspective of nanocarriers in treating various skin diseases. A total of 213 articles have been reviewed and the application of different nanocarriers in treating various skin diseases has been explained in detail through case studies of previously published research works. The toxicity related aspects of nanocarriers are also discussed.
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CRISPR/Cas9-based genome editing has quickly emerged as a powerful breakthrough technology for use in diverse settings across biomedical research and therapeutic development. Recent efforts toward understanding gene modification methods in vitro have led to substantial improvements in ex vivo genome editing efficiency. Because disease targets for genomic correction are often localized in specific organs, realization of the full potential of genomic medicines will require delivery of CRISPR/Cas9 systems targeting specific tissues and cells directly in vivo. In this Perspective, we focus on progress toward in vivo delivery of CRISPR/Cas components. Viral and nonviral delivery systems are both promising for gene editing in diverse tissues via local injection and systemic injection. We describe the various viral vectors and synthetic nonviral materials used for in vivo gene editing and applications to research and therapeutic models, and summarize opportunities and progress to date for both methods. We also discuss challenges for viral delivery, including overcoming limited packaging capacity, immunogenicity associated with multiple dosing, and the potential for off-target effects, and nonviral delivery, including efforts to increase efficacy and to expand utility of nonviral carriers for use in extrahepatic tissues and cancer. Looking ahead, additional advances in the safety and efficiency of viral and nonviral delivery systems for tissue- and cell-type-specific gene editing will be required to enable broad clinical translation. We provide a summary of current delivery systems used for in vivo genome editing, organized with respect to route of administration, and highlight immediate opportunities for biomedical research and applications. Furthermore, we discuss current challenges for in vivo delivery of CRISPR/Cas9 systems to guide the development of future therapies.
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Today, about 50% of men and 15-30% of women suffer from hair loss as well as the associated psychological impact. Drug therapy, especially through topical administration, is the main treatment strategy for stimulating hair regrowth. However, challenges exist due to the skin barrier that hinders drug penetration. To this end, many efforts have been made to enhance drug penetration efficiency. This review focuses on the advancement of the transdermal drug delivery strategies for hair loss therapy reported in the last five years, especially those via nanoformulations for topical administration and microneedles for transdermal delivery. In addition, physical or chemical penetration enhancers are also introduced, which are often applied with the drug delivery systems to achieve a synergy effect.
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The development of new modalities for high-efficiency intracellular drug delivery is a priority for a number of disease areas. One such area is urinary tract infection (UTI), which is one of the most common infectious diseases globally and which imposes an immense economic and healthcare burden. Common uropathogenic bacteria have been shown to invade the urothelial wall during acute UTI, forming latent intracellular reservoirs that can evade antimicrobials and the immune response. This behaviour likely facilitates the high recurrence rates after oral antibiotic treatments, which are not able to penetrate the bladder wall and accumulate to an effective concentration. Meanwhile, oral antibiotics may also exacerbate antimicrobial resistance and cause systemic side effects. Using a human urothelial organoid model, we tested the ability of novel ultrasound-activated lipid microbubbles to deliver drugs into the cytoplasm of apical cells. The gas-filled lipid microbubbles were decorated with liposomes containing the non-cell-permeant antibiotic gentamicin and a fluorescent marker. The microbubble suspension was added to buffer at the apical surface of the bladder model before being exposed to ultrasound (1.1 MHz, 2.5 Mpa, 5500 cycles at 20 ms pulse duration) for 20 seconds. Our results show that ultrasound-activated intracellular delivery using microbubbles was over 16 times greater than the control group and twice that achieved by liposomes that were not associated with microbubbles. Moreover, no cell damage was detected. Together, our data show that ultrasound-activated microbubbles can safely deliver high concentrations of drugs into urothelial cells, and have the potential to be a more efficacious alternative to traditional oral antibiotic regimes for UTI. This modality of intracellular drug delivery may prove useful in other clinical indications, such as cancer and gene therapy, where such penetration would aid in treatment.
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Sonoporation mediated by microbubbles has being extensively studied as a promising technique to facilitate gene/drug delivery to cells. Previous studies mainly explored the membrane-level responses to sonoporation. To provide in-depth understanding on this process, various sonoporation-induced cellular responses (e.g., membrane permeabilization and cytoskeleton disassembly) generated at different impact parameters (e.g., acoustic driving pressure and microbubble-cell distances) were systemically investigated in the present work. HeLa cells, whose α-tubulin cytoskeleton was labeled by incorporation of a green fluorescence protein (GFP)-α-tubulin fusion protein, were exposed to a single ultrasound pulse (1 MHz, 20 cycles) in the presence of microbubbles. Intracellular transport via sonoporation was assessed in real time using propidium iodide and the disassembly of α-tubulin cytoskeleton was observed by fluorescence microscope. Meanwhile, the dynamics of an interacting bubble-cell pair was theoretically simulated by boundary element method. Both the experimental observations and numerical simulations showed that, by increasing the acoustic pressure or reducing the bubble-cell distance, intensified deformation could be induced in the cellular membrane, which could result in enhanced intracellular delivery and cytoskeleton disassembly. The current results suggest that more tailored therapeutic strategies could be designed for ultrasound gene/drug delivery by adopting optimal bubble-cell distances and/or better controlling incident acoustic energy.
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Direct cellular delivery of CRISPR/Cas9 complexes is of great significance for genome editing and other recently developed applications, such as gene expression regulation and RNA/DNA imaging. Here, we first constructed a graphene oxide (GO)-polyethylene glycol (PEG)-polyethylenimine (PEI) nanocarrier for the delivery of high-molecular-weight Cas9/single-guide RNA (sgRNA) complexes for endocytosis, endosomal escape, nuclear entry, and gene editing. The results demonstrate that the nanocarrier can be used successfully for efficient gene editing in human AGS cells with an efficiency of ~39 %. The results also show that this nanocarrier can protect sgRNA from enzymatic degradation, thus exhibiting extremely high stability, which is critical for future in vivo applications. Thus, this GO-mediated Cas9/sgRNA delivery system has potential as a new approach for biomedical research and targeted gene engineering applications.
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Due to its simplicity, versatility, and high efficiency, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology has emerged as one of the most promising approaches for treatment of a variety of genetic diseases, including human cancers. However, further translation of CRISPR/Cas9 for cancer gene therapy requires development of safe approaches for efficient, highly specific delivery of both Cas9 and single guide RNA to tumors. Here, novel core–shell nanostructure, liposome-templated hydrogel nanoparticles (LHNPs) that are optimized for efficient codelivery of Cas9 protein and nucleic acids is reported. It is demonstrated that, when coupled with the minicircle DNA technology, LHNPs deliver CRISPR/Cas9 with efficiency greater than commercial agent Lipofectamine 2000 in cell culture and can be engineered for targeted inhibition of genes in tumors, including tumors the brain. When CRISPR/Cas9 targeting a model therapeutic gene, polo-like kinase 1 (PLK1), is delivered, LHNPs effectively inhibit tumor growth and improve tumor-bearing mouse survival. The results suggest LHNPs as versatile CRISPR/Cas9-delivery tool that can be adapted for experimentally studying the biology of cancer as well as for clinically translating cancer gene therapy.
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Cpf1, a type V CRISPR effector, recognizes a thymidine-rich protospacer-adjacent motif and induces cohesive double-stranded breaks at the target site guided by a single CRISPR RNA (crRNA). Here we show that Cpf1 can be used as a tool for DNA-free editing of plant genomes. We describe the delivery of recombinant Cpf1 proteins with in vitro transcribed or chemically synthesized target-specific crRNAs into protoplasts isolated from soybean and wild tobacco. Designed crRNAs are unique and do not have similar sequences (≤3 mismatches) in the entire soybean reference genome. Targeted deep sequencing analyses show that mutations are successfully induced in FAD2 paralogues in soybean and AOC in wild tobacco. Unlike SpCas9, Cpf1 mainly induces various nucleotide deletions at target sites. No significant mutations are detected at potential off-target sites in the soybean genome. These results demonstrate that Cpf1–crRNA complex is an effective DNA-free genome-editing tool for plant genome editing.
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Significance The prokaryotic CRISPR/Cas9 system has recently been applied in genome editing in mammalian cells with the potential to bring curative therapies to patients with genetic diseases. However, efficient in vivo delivery of this machinery remains challenging for most tissue types. We now developed a method to locally deliver Cas9/sgRNA ribonucleoproteins into the skin of postnatal mice, which was used to correct genetic defects in skin stem cells of postnatal recessive dystrophic epidermolysis bullosa (RDEB) mice. Our study provides proof-of-principle evidence that Cas9/sgRNA ribonucleoprotein-based gene therapies can be applied to restore collagen VII protein function in postnatal RDEB mice, suggesting that the Cas9/sgRNA ribonucleoprotein-based gene therapy may offer curative treatment for RDEB and other genetic disorders.
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Gas (SF6)-filled microbubbles (MBs) were prepared by emulsion and solvent-evaporation method. The prepared MBs were further conjugated with doxorubicin (Dox)-loaded nano-sized liposome and peptide ligands to interleukin-4 receptor (IL4R) for targeting brain tumor cells. The final MB-liposome (Dox)-IL4R targeting peptide ligand [MB-Lipo (Dox)-IL4RTP] had a spherical structure with the mean size of 1,500 nm. The MB-Lipo (Dox)‑IL4RTP exhibited cellular uptake in U87MG brain tumor cells (a brain tumor cell line expressing strongly IL4R) with frequency ultrasound energy suggesting that MB-Lipo (Dox)‑IL4RTP provided effective targeting ability for brain tumor cells. In addition, WST-1 assay results showed that MB-Lipo (Dox)‑IL4RTP inhibited the proliferation of U87MG cells IL4R‑dependently. This was confirmed by western blotting of γH2AX, phospho (Ser15)-p53, p53 and p21 which are signal transduction proteins involved in DNA damage response and cell cycle arrest. Taken together, these results indicate that MB-Lipo (Dox)-IL4RTP represents a promising ultrasonic contrast agent for tumor-targeting ultrasonic imaging.
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Significance The therapeutic potential of protein-based genome editing is dependent on the delivery of proteins to appropriate intracellular targets. Here we report that combining bioreducible lipid nanoparticles and negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives the self-assembly of nanoparticles for potent protein delivery and genome editing. The design of bioreducible lipids facilitates the degradation of nanoparticles inside cells in response to the reductive intracellular environment, enhancing the endosome escape of protein. In addition, modulation of protein charge through either genetic fusion of supercharged protein or complexation of Cas9 with its inherently anionic sgRNA allows highly efficient protein delivery and effective genome editing in mammalian cells and functional recombinase delivery in the rodent brain.
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All cancers have multiple mutations that can largely be grouped into certain classes depending on the function of the gene in which they lie and these include oncogenic changes that enhance cellular proliferation, loss of function of tumor suppressors that regulate cell growth potential and induction of metabolic enzymes that confer resistance to chemotherapeutic agents. Thus the ability to correct such mutations is an important goal in cancer treatment. Recent research has led to the developments of reagents which specifically target nucleotide sequences within the cellular genome and these have a huge potential for expanding our anticancer armamentarium. One such a reagent is the clustered regulatory interspaced short palindromic repeat (CRISPR)-associated 9 (Cas9) system, a powerful, highly specific and adaptable tool that provides unparalleled control for editing the cellular genome. In this short review, we discuss the potential of CRISPR/Cas9 against human cancers and the current difficulties in translating this for novel therapeutic approaches.
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We developed an in vivo library-on-library methodology to simultaneously assess single guide RNA (sgRNA) activity across ∼1,400 genomic loci. Assaying across multiple human cell types and end-processing enzymes as well as two Cas9 orthologs, we unraveled underlying nucleotide sequence and epigenetic parameters. Our results and software (http://crispr.med.harvard.edu/sgRNAScorer) enable improved design of reagents, shed light on mechanisms of genome targeting, and provide a generalizable framework to study nucleic acid-nucleic acid interactions and biochemistry in high throughput.
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Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.
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Theranostic agents present a promising clinical approach for cancer detection and treatment. We herein introduce a microbubble and liposome complex (MB-Lipo) developed for ultrasound (US) imaging and activation. The MB-Lipo particles have a hybrid structure consisting of a MB complexed with multiple Lipos. The MB components are used to generate high echo signals in US imaging, while the Lipos serve as a versatile carrier of therapeutic materials. MB-Lipo allows high contrast US imaging of tumor sites. More importantly, the application of high acoustic pressure bursts MBs, which releases therapeutic Lipos and further enhances their intracellular delivery through sonoporation effect. Both imaging and drug release could thus be achieved by a single US modality, enabling in situ treatment guided by real-time imaging. The MB-Lipo system was applied to specifically deliver anti-cancer drug and genes to tumor cells, which showed enhanced therapeutic effect. We also demonstrate the clinical potential of MB-Lipo by imaging and treating tumor in vivo.
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RNA-guided engineered nucleases (RGENs) derived from the prokaryotic adaptive immune system known as CRISPR (clustered, regularly interspaced, short palindromic repeat)/Cas (CRISPR-associated) enable genome editing in human cell lines, animals, and plants but are limited by off-target effects and unwanted integration of DNA segments derived from plasmids encoding Cas9 and guide RNA at both on-target and off-target sites in the genome. Here, we deliver purified recombinant Cas9 protein and guide RNA into cultured human cells including hard-to-transfect fibroblasts and pluripotent stem cells. RGEN ribonucleoproteins (RNPs) induce site-specific mutations at frequencies of up to 79%, while reducing off-target mutations associated with plasmid transfection at off-target sites that differ by one or two nucleotides from on-target sites. RGEN RNPs cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells, reducing off-target effects. Furthermore, RNP delivery is less stressful to human embryonic stem cells, producing at least two-fold more colonies than does plasmid transfection.
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Background Finasteride is known to improve urinary symptoms in men with benign prostatic hyperplasia, but the extent to which the benefit is sustained and whether finasteride reduces the incidence of related events, including the need for surgery and the development of acute urinary retention, are not known. Methods In this double-blind, randomized, placebo-controlled trial, we studied 3040 men with moderate-to-severe urinary symptoms and enlarged prostate glands who were treated daily with 5 mg of finasteride or placebo for four years. Symptom scores (on a scale of 1 to 34), urinary flow rates, and the occurrence of outcome events were assessed every four months in 3016 men. Prostate volume was measured in a subgroup of the men. Complete data on outcomes were available for 2760 men. Results During the four-year study period, 152 of the 1503 men in the placebo group (10 percent) and 69 of the 1513 men in the finasteride group (5 percent) underwent surgery for benign prostatic hyperplasia (reduction in ris...
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Autocrine and paracrine factors are produced by balding dermal papilla (DP) cells following dihydrotestosterone (DHT)-driven alterations and are believed to be key factors involved in male pattern baldness. Herein we report that the IL-6 is upregulated in balding DP cells compared with non-balding DP cells. IL-6 was upregulated 3  hours after 10-100  nM DHT treatment, and ELISA showed that IL-6 was secreted from balding DP cells in response to DHT. IL-6 receptor (IL-6R) and glycoprotein 130 (gp130) were expressed in follicular keratinocytes, including matrix cells. Recombinant human IL-6 (rhIL-6) inhibited hair shaft elongation and suppressed proliferation of matrix cells in cultured human hair follicles. Moreover, rhIL-6 injection into the hypodermis of mice during anagen caused premature onset of catagen. Taken together, our data strongly suggest that DHT-inducible IL-6 inhibits hair growth as a paracrine mediator from the DP.
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Hair follicles (HFs) undergo cyclic bouts of degeneration, rest, and regeneration. During rest (telogen), the hair germ (HG) appears as a small cell cluster between the slow-cycling bulge and dermal papilla (DP). Here we show that HG cells are derived from bulge stem cells (SCs) but become responsive quicker to DP-promoting signals. In vitro, HG cells also proliferate sooner but display shorter-lived potential than bulge cells. Molecularly, they more closely resemble activated bulge rather than transit-amplifying (matrix) cells. Transcriptional profiling reveals precocious activity of both HG and DP in late telogen, accompanied by Wnt signaling in HG and elevated FGFs and BMP inhibitors in DP. FGFs and BMP inhibitors participate with Wnts in exerting selective and potent stimuli to the HG both in vivo and in vitro. Our findings suggest a model where HG cells fuel initial steps in hair regeneration, while the bulge is the engine maintaining the process.
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Testosterone and its 5 alpha-reduced derivative 5 alpha-dihydrotestosterone exert different actions in the male during embryogenesis and in postnatal life. Nevertheless the two hormones bind to the same intracellular androgen receptor, and genetic and endocrinological studies in the Tfm mouse suggest that the actions of both hormones are mediated by this single receptor. Previous studies indicate that dihydrotestosterone binds more tightly to the androgen receptor but that the Bmax of binding of the two hormones is the same. To determine whether these differences in binding parameters could explain the mechanism by which the two hormones exert different physiological actions via the same receptor, we introduced a plasmid encoding the androgen receptor cDNA and a reporter plasmid encoding MMTV-CAT into Chinese hamster ovary cells. These cells do not express endogenous androgen receptor and do not convert testosterone to dihydrotestosterone. Therefore, it was possible to examine the relation between the concentration of each of the steroids and reporter gene expression. Both hormones enhanced CAT activity, but dihydrotestosterone was approximately 10 times as potent (half maximal of 0.018 nM) as testosterone (half maximal of 0.2 nM); the maximal activity achieved was the same for the two androgens. These findings are nearly identical to the apparent Kd values for the interaction of the two hormones with the androgen receptor. Although testosterone and dihydrotestosterone may influence the expression of other genes differently, these findings are compatible with a model system in which the differential effects can be explained as a consequence of different binding affinities to the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)
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Finasteride is known to improve urinary symptoms in men with benign prostatic hyperplasia, but the extent to which the benefit is sustained and whether finasteride reduces the incidence of related events, including the need for surgery and the development of acute urinary retention, is not known. In this double-blind, randomized, placebo-controlled trial, we studied 3040 men with moderate-to-severe urinary symptoms and enlarged prostate glands who were treated daily with 5 mg of finasteride or placebo for four years. Symptom scores (on a scale of 1 to 34), urinary flow rates, and the occurrence of outcome events were assessed every four months in 3016 men. Prostate volume was measured in a subgroup of the men. Complete data on outcomes were available for 2760 men. During the four-year study period, 152 of the 1503 men in the placebo group (10 percent) and 69 of the 1513 men in the finasteride group (5 percent) underwent surgery for benign prostatic hyperplasia (reduction in risk with finasteride, 55 percent; 95 percent confidence interval, 41 to 65 percent). Acute urinary retention developed in 99 men (7 percent) in the placebo group and 42 men (3 percent) in the finasteride group (reduction in risk with finasteride, 57 percent; 95 percent confidence interval, 40 to 69 percent). Among the men who completed the study, the mean decreases in the symptom score were 3.3 in the finasteride group and 1.3 in the placebo group (P<0.001). Treatment with finasteride also significantly improved urinary flow rates and reduced prostate volume (P<0.001). Among men with symptoms of urinary obstruction and prostatic enlargement, treatment with finasteride for four years reduces symptoms and prostate volume, increases the urinary flow rate, and reduces the risk of surgery and acute urinary retention.
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Our understanding of the aetiology of androgenetic alopecia (AGA) has substantially increased in recent years. As a result, several treatment modalities have been tried with promising results especially in early stages of AGA. However, as far as has been ascertained, there is no comprehensive study comparing the efficacy of these agents alone and in combination with each other. One hundered male patients with AGA of Hamilton grades II to IV were enrolled in an open, randomized, parallel-group study, designed to evaluate and compare the efficacy of oral finasteride (1 mg per day), topical 2% minoxidil solution and topical 2% ketoconazole shampoo alone and in combination. They were randomized into four groups. Group I (30 patients) was administered oral finasteride, Group II (36 patients) was given a combination of finasteride and topical minoxidil, Group III (24 patients) applied minoxidil alone and Group IV (10 patients) was administered finasteride with topical ketoconazole. Treatment efficacy was assessed on the basis of patient and physician assessment scores and global photographic review during the study period of one year. At the end of one year, hair growth was observed in all the groups with best results recorded with a combination of finasteride and minoxidil (Group II) followed by groups IV, I and III. Subjects receiving finasteride alone or in combination with minoxidil or ketoconazole showed statistically significant improvement (p<0.05) over minoxidil only recipients. No signifcant side-effects related to the drugs were observed. In conclusion, it is inferred that the therapeutic efficacy is enhanced by combining the two drugs acting on different aetiological aspects of AGA.
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A new acoustically-active delivery vehicle was developed by conjugating liposomes and microbubbles, using the high affinity interaction between avidin and biotin. Binding between microbubbles and liposomes, each containing 5% DSPE-PEG2kBiotin, was highly dependent on avidin concentration and observed above an avidin concentration of 10 nM. With an optimized avidin and liposome concentration, we measured and calculated as high as 1000 to 10,000 liposomes with average diameters of 200 and 100 nm, respectively, attached to each microbubble. Replacing avidin with neutravidin resulted in 3-fold higher binding, approaching the calculated saturation level. High-speed photography of this new drug delivery vehicle demonstrated that the liposome-bearing microbubbles oscillate in response to an acoustic pulse in a manner similar to microbubble contrast agents. Additionally, microbubbles carrying liposomes could be spatially concentrated on a monolayer of PC-3 cells at the focal point of ultrasound beam. As a result of cell-vehicle contact, the liposomes fused with the cells and internalization of NBD-cholesterol occurred shortly after incubation at 37 degrees C, with internalization of NBD-cholesterol substantially enhanced in the acoustic focus.
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Ultrasound contrast agents, in the form of gas-filled microbubbles, are becoming popular in perfusion monitoring; they are employed as molecular imaging agents. Microbubbles are manufactured from biocompatible materials, they can be injected intravenously, and some are approved for clinical use. Microbubbles can be destroyed by ultrasound irradiation. This destruction phenomenon can be applied to targeted drug delivery and enhancement of drug action. The ultrasonic field can be focused at the target tissues and organs; thus, selectivity of the treatment can be improved, reducing undesirable side effects. Microbubbles enhance ultrasound energy deposition in the tissues and serve as cavitation nuclei, increasing intracellular drug delivery. DNA delivery and successful tissue transfection are observed in the areas of the body where ultrasound is applied after intravascular administration of microbubbles and plasmid DNA. Accelerated blood clot dissolution in the areas of insonation by cooperative action of thrombolytic agents and microbubbles is demonstrated in several clinical trials.
  • J.-Y Ryu
J.-Y. Ryu, et al. Biomaterials 232 (2020) 119736