Extracellular Barriers Research Articles

Lipid synthesis leads the way for invasive migration.

Invasive migration requires cells to break through extracellular matrix barriers, which is an energy-expensive process. In this issue, Park et al. (https://doi.org/10.1083/jcb.202402035) highlight the importance of biosynthesis of fatty acids, phospholipids, and isoprenoids in driving invasive migration of the Caenorhabditis elegans anchor cell through a basement membrane barrier during development.

Lipid-based Non-viral Vector: Promising Approach for Gene Delivery.

The present review aims to discuss various strategies to overcome intracellular and extracellular barriers involved in gene delivery as well as the advantages, challenges, and mechanisms of gene delivery using non-viral vectors. Additionally, patents, clinical studies, and various formulation approaches related to lipid-based carrier systems are discussed. Data were searched and collected from Google Scholar, ScienceDirect, Pubmed, and Springer. In this review, we have investigated the advantages of non-viral vectors over viral vectors. The advantage of using non-viral vectors are that they seek more attention in different fields. They play an important role in delivering the genetic materials. However, few nonviral vector-based carrier systems have been found in clinical settings. Challenges are developing more stable, site-specific gene delivery and conducting thorough safety assessments to minimize the undesired effects. In comparison to viral vectors, nonviral vector-based lipid nanocarriers have more advantages for gene delivery. Gene therapy research shows promise in addressing health concerns. Lipid-based nanocarriers can overcome intracellular and extracellular barriers, allowing efficient delivery of genetic materials. Non-viral vectors are more attractive due to their biocompatibility, ease of synthesis, and cost-effectiveness. They can deliver various nucleic acids and have improved gene delivery efficacy by avoiding degradation steps. Despite limited clinical use, many patents have been filed for mRNA vaccine delivery using non-viral vectors.

Combating bacterial biofilms and related drug resistance: Role of phyto-derived adjuvant and nanomaterials

The emergence of antimicrobial resistance (AMR) in clinical microbes has led to a search for novel antibiotics for combating bacterial infections. The treatment of bacterial infections becomes more challenging with the onset of biofilm formation. AMR is further accelerated by biofilm physiology and differential gene expression in bacteria with an inherent resistance to conventional antibiotics. In the search for innovative strategies to control the spread of AMR in clinical isolates, plant-derived therapeutic metabolites can be repurposed to control biofilm-associated drug resistance. Unlike antibiotics, designed to act on a single cellular process, phytochemicals can simultaneously target multiple cellular components. Furthermore, they can disrupt biofilm formation and inhibit quorum sensing, offering a comprehensive approach to combat bacterial infections. In bacterial biofilms, the first line of AMR is due to biofilms associated with the extracellular matrix, diffusion barriers, quorum sensing, and persister cells. These extracellular barriers can be overcome using phytochemical-based antibiotic adjuvants to increase the efficacy of antibiotic treatment and restrict the spread of AMR. Furthermore, phytochemicals can be used to target bacterial intracellular machinery such as DNA replication, protein synthesis, efflux pumps, and degrading enzymes. In parallel with pristine phytochemicals, phyto-derived nanomaterials have emerged as an effective means of fighting bacterial biofilms. These nanomaterials can be formulated to cross the biofilm barriers and function on cellular targets. This review focuses on the synergistic effects of phytochemicals and phyto-derived nanomaterials in controlling the progression of biofilm-related AMR. IT provides comprehensive insights into recent advancements and the underlying mechanisms of the use of phyto-derived adjuvants and nanomaterials.

Neural regeneration in the human central nervous system-from understanding the underlying mechanisms to developing treatments. Where do we stand today?

Mature neurons in the human central nervous system (CNS) fail to regenerate after injuries. This is a common denominator across different aetiologies, including multiple sclerosis, spinal cord injury and ischemic stroke. The lack of regeneration leads to permanent functional deficits with a substantial impact on patient quality of life, representing a significant socioeconomic burden worldwide. Great efforts have been made to decipher the responsible mechanisms and we now know that potent intra- and extracellular barriers prevent axonal repair. This knowledge has resulted in numerous clinical trials, aiming to promote neuroregeneration through different approaches. Here, we summarize the current understanding of the causes to the poor regeneration within the human CNS. We also review the results of the treatment attempts that have been translated into clinical trials so far.

Carbon-Based Nanostructures as Emerging Materials for Gene Delivery Applications.

Gene therapeutics are promising for treating diseases at the genetic level, with some already validated for clinical use. Recently, nanostructures have emerged for the targeted delivery of genetic material. Nanomaterials, exhibiting advantageous properties such as a high surface-to-volume ratio, biocompatibility, facile functionalization, substantial loading capacity, and tunable physicochemical characteristics, are recognized as non-viral vectors in gene therapy applications. Despite progress, current non-viral vectors exhibit notably low gene delivery efficiency. Progress in nanotechnology is essential to overcome extracellular and intracellular barriers in gene delivery. Specific nanostructures such as carbon nanotubes (CNTs), carbon quantum dots (CQDs), nanodiamonds (NDs), and similar carbon-based structures can accommodate diverse genetic materials such as plasmid DNA (pDNA), messenger RNA (mRNA), small interference RNA (siRNA), micro RNA (miRNA), and antisense oligonucleotides (AONs). To address challenges such as high toxicity and low transfection efficiency, advancements in the features of carbon-based nanostructures (CBNs) are imperative. This overview delves into three types of CBNs employed as vectors in drug/gene delivery systems, encompassing their synthesis methods, properties, and biomedical applications. Ultimately, we present insights into the opportunities and challenges within the captivating realm of gene delivery using CBNs.

Identification of Differentially Expressed mRNAs and miRNAs and Related Regulatory Networks in Cumulus Oophorus Complexes Associated with Fertilization.

Cumulus oophorus complexes (COCs) are the first extracellular barriers that sperm must pass through to fuse with oocytes, which have an important role in oocyte maturation and fertilization. However, little is known about the molecular mechanisms of COCs involved in fertilization. In this study, COCs were collected and then randomly divided into a test group that interacted with sperm and a control group that did not interact with sperm. Then, the total RNA was extracted; RNA transcriptome and small RNA libraries were prepared, sequenced, and analyzed. The results showed that 1283 differentially expressed genes (DEGs), including 560 upregulated and 723 downregulated genes. In addition, 57 differentially expressed miRNAs (DEMIs) with 35 upregulated and 22 downregulated were also detected. After the RNA-seq results were verified by RT-qPCR, 86 effective DEGs and 40 DEMIs were finally screened and a DEMI-DEG regulatory network was constructed. From this, the top ten hub target genes were HNF4A, SPN, WSCD1, TMEM239, SLC2A4, E2F2, SIAH3, ADORA3, PIK3R2, and GDNF, and they were all downregulated. The top ten hub DEMIs were miR-6876-5p, miR-877-3p, miR-6818-5p, miR-4690-3p, miR-6789-3p, miR-6837-5p, miR-6861-5p, miR-4421, miR-6501-5p, and miR-6875-3p, all of which were upregulated. The KEGG signaling pathway enrichment analysis showed that the effective DEGs were significantly enriched in the calcium, AMPK, and phospholipase D signaling pathways. Our study identified several DEGs and DEMIs and potential miRNA-mRNA regulatory pathways in COCs and these may contribute to fertilization. This study may provide novel insights into potential biomarkers for fertilization failure.

Recent Advances in Engineering Carriers for siRNA Delivery.

RNA interference (RNAi) technology has been a promising treatment strategy for combating intractable diseases. However, the applications of RNAi in clinical are hampered by extracellular and intracellular barriers. To overcome these barriers, various siRNA delivery systems have been developed in the past two decades. The first approved RNAi therapeutic, Patisiran (ONPATTRO) using lipids as the carrier, for the treatment of amyloidosis is one of the most important milestones. This has greatly encouraged researchers to work on creating new functional siRNA carriers. In this review, the recent advances in siRNA carriers consisting of lipids, polymers, and polymer-modified inorganic particles for cancer therapy are summarized. Representative examples are presented to show the structural design of the carriers in order to overcome the delivery hurdles associated with RNAi therapies. Finally, the existing challenges and future perspective for developing RNAi as a clinical modality will be discussed and proposed. It is believed that the addressed contributions in this review will promote the development of siRNA delivery systems for future clinical applications.

Ultrasound-Launched Targeted Nanoparticle Enhances Antibacterial Sonodynamic Therapy for Effective Eradication of Pseudomonas aeruginosa Biofilm

Abstract Infections caused by Pseudomonas aeruginosa biofilm significantly endanger human health worldwide. Biofilms are closely associated with antibiotic resistance because biofilms significantly undermine the efficacy of antibiotics. A novel ultrasound-launched targeted nanoparticle was developed to universally destroy biofilm, target bacteria, deliver antibiotics, and efficiently kill bacteria via ultrasonic cavitation and antibacterial sonodynamic therapy. The nanoparticle consisted of poly (lactic-co-glycolic acid) loading ciprofloxacin and perfluoropentane with a bacteria-targeted antibody installed on the nanoparticle for binding to specific bacteria. The nanoparticle exhibited a sensitive response to ultrasound and the rapid liquid-gas phase transition of perfluoropentane resulted in a cavitation effect that destroyed the extracellular polymeric substances of the biofilm and allowed deep penetration of the antibiotics. In addition, ciprofloxacin induced additional reactive oxygen species production under ultrasound stimulation, leading to an enhanced bactericidal effect and potent anti-infective activity in vivo. This study presents an effective strategy to tackle the extracellular polymeric substance barriers for overcoming antibiotic resistance and removing a biofilm.

Development of polypeptide-based materials toward messenger RNA delivery.

Messenger RNA (mRNA)-based therapeutic agents have demonstrated significant potential in recent times, particularly in the context of the COVID-19 pandemic outbreak. As a promising prophylactic and therapeutic strategy, polypeptide-based mRNA delivery systems attract significant interest because of their low cost, simple preparation, tuneable sizes and morphology, convenient large-scale production, biocompatibility, and biodegradability. In this review, we begin with a brief discussion of the synthesis of polypeptides, followed by a review of commonly used polypeptides in mRNA delivery, including classical polypeptides and cell-penetrating peptides. Then, the challenges against mRNA delivery, including extracellular, intracellular, and clinical barriers, are discussed in detail. Finally, we highlight a range of strategies for polypeptide-based mRNA delivery, offering valuable insights into the advancement of polypeptide-based mRNA carrier development.

Translational regulation of cell invasion through extracellular matrix-an emerging role for ribosomes.

Many developmental and physiological processes require cells to invade and migrate through extracellular matrix barriers. This specialized cellular behavior is also misregulated in many diseases, such as immune disorders and cancer. Cell invasive activity is driven by pro-invasive transcriptional networks that activate the expression of genes encoding numerous different proteins that expand and regulate the cytoskeleton, endomembrane system, cell adhesion, signaling pathways, and metabolic networks. While detailed mechanistic studies have uncovered crucial insights into pro-invasive transcriptional networks and the distinct cell biological attributes of invasive cells, less is known about how invasive cells modulate mRNA translation to meet the robust, dynamic, and unique protein production needs of cell invasion. In this review we outline known modes of translation regulation promoting cell invasion and focus on recent studies revealing elegant mechanisms that expand ribosome biogenesis within invasive cells to meet the increased protein production requirements to invade and migrate through extracellular matrix barriers.

Combinatorial development of nebulized mRNA delivery formulations for the lungs.

Inhaled delivery of mRNA has the potential to treat a wide variety of diseases. However, nebulized mRNA lipid nanoparticles (LNPs) face several unique challenges including stability during nebulization and penetration through both cellular and extracellular barriers. Here we develop a combinatorial approach addressing these barriers. First, we observe that LNP formulations can be stabilized to resist nebulization-induced aggregation by altering the nebulization buffer to increase the LNP charge during nebulization, and by the addition of a branched polymeric excipient. Next, we synthesize a combinatorial library of ionizable, degradable lipids using reductive amination, and evaluate their delivery potential using fully differentiated air-liquid interface cultured primary lung epithelial cells. The final combination of ionizable lipid, charge-stabilized formulation and stability-enhancing excipient yields a significant improvement in lung mRNA delivery over current state-of-the-art LNPs and polymeric nanoparticles.

Myddosome clustering in IL-1 receptor signaling regulates the formation of an NF-kB activating signalosome.

IL-1 receptor (IL-1R) signaling can activate thresholded invariant outputs and proportional outputs that scale with the amount of stimulation. Both responses require the Myddosome, a multiprotein complex. The Myddosome is required for polyubiquitin chain formation and NF-kB signaling. However, how these signals are spatially and temporally regulated to drive switch-like and proportional outcomes is not understood. During IL-1R signaling, Myddosomes dynamically reorganize into multi-Myddosome clusters at the cell membrane. Blockade of clustering using nanoscale extracellular barriers reduces NF-kB activation. Myddosomes function as scaffolds that assemble an NF-kB signalosome consisting of E3-ubiquitin ligases TRAF6 and LUBAC, K63/M1-linked polyubiquitin chains, phospho-IKK, and phospho-p65. This signalosome preferentially assembles at regions of high Myddosome density, which enhances the recruitment of TRAF6 and LUBAC. Extracellular barriers that restrict Myddosome clustering perturbed the recruitment of both ligases. We find that LUBAC was especially sensitive to clustering with 10-fold lower recruitment to single Myddosomes than clustered Myddosomes. These data reveal that the clustering behavior of Myddosomes provides a basis for digital and analog IL-1R signaling.

Polymeric-Micelle-Based Delivery Systems for Nucleic Acids.

Nucleic acids can modulate gene expression specifically. They are increasingly being utilized and show huge potential for the prevention or treatment of various diseases. However, the clinical translation of nucleic acids faces many challenges due to their rapid clearance after administration, low stability in physiological fluids and limited cellular uptake, which is associated with an inability to reach the intracellular target site and poor efficacy. For many years, tremendous efforts have been made to design appropriate delivery systems that enable the safe and effective delivery of nucleic acids at the target site to achieve high therapeutic outcomes. Among the different delivery platforms investigated, polymeric micelles have emerged as suitable delivery vehicles due to the versatility of their structures and the possibility to tailor their composition for overcoming extracellular and intracellular barriers, thus enhancing therapeutic efficacy. Many strategies, such as the addition of stimuli-sensitive groups or specific ligands, can be used to facilitate the delivery of various nucleic acids and improve targeting and accumulation at the site of action while protecting nucleic acids from degradation and promoting their cellular uptake. Furthermore, polymeric micelles can be used to deliver both chemotherapeutic drugs and nucleic acid therapeutics simultaneously to achieve synergistic combination treatment. This review focuses on the design approaches and current developments in polymeric micelles for the delivery of nucleic acids. The different preparation methods and characteristic features of polymeric micelles are covered. The current state of the art of polymeric micelles as carriers for nucleic acids is discussed while highlighting the delivery challenges of nucleic acids and how to overcome them and how to improve the safety and efficacy of nucleic acids after local or systemic administration.

Lipid-, Inorganic-, Polymer-, and DNA-Based Nanocarriers for Delivery of the CRISPR/Cas9 system.

The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (CRISPR/Cas9) system has been widely explored for the precise manipulation of target DNA and has enabled efficient genomic editing in cells. Recently, CRISPR/Cas9 has shown promising potential in biomedical applications, including disease treatment, transcriptional regulation and genome-wide screening. Despite these exciting achievements, efficient and controlled delivery of the CRISPR/Cas9 system has remained a critical obstacle to its further application. Herein, we elaborate on the three delivery forms of the CRISPR/Cas9 system, and discuss the composition, advantages and limitations of these forms. Then we provide a comprehensive overview of the carriers of the system, and focus on the nonviral nanocarriers in chemical methods that facilitate efficient and controlled delivery of the CRISPR/Cas9 system. Finally, we discuss the challenges and prospects of the delivery methods of the CRISPR/Cas9 system in depth, and propose strategies to address the intracellular and extracellular barriers to delivery in clinical applications.

Advances and Challenges of Stimuli-Responsive Nucleic Acids Delivery System in Gene Therapy.

Gene therapy has emerged as a powerful tool to treat various diseases, such as cardiovascular diseases, neurological diseases, ocular diseases and cancer diseases. In 2018, the FDA approved Patisiran (the siRNA therapeutic) for treating amyloidosis. Compared with traditional drugs, gene therapy can directly correct the disease-related genes at the genetic level, which guarantees a sustained effect. However, nucleic acids are unstable in circulation and have short half-lives. They cannot pass through biological membranes due to their high molecular weight and massive negative charges. To facilitate the delivery of nucleic acids, it is crucial to develop a suitable delivery strategy. The rapid development of delivery systems has brought light to the gene delivery field, which can overcome multiple extracellular and intracellular barriers that prevent the efficient delivery of nucleic acids. Moreover, the emergence of stimuli-responsive delivery systems has made it possible to control the release of nucleic acids in an intelligent manner and to precisely guide the therapeutic nucleic acids to the target site. Considering the unique properties of stimuli-responsive delivery systems, various stimuli-responsive nanocarriers have been developed. For example, taking advantage of the physiological variations of a tumor (pH, redox and enzymes), various biostimuli- or endogenous stimuli-responsive delivery systems have been fabricated to control the gene delivery processes in an intelligent manner. In addition, other external stimuli, such as light, magnetic fields and ultrasound, have also been employed to construct stimuli-responsive nanocarriers. Nevertheless, most stimuli-responsive delivery systems are in the preclinical stage, and some critical issues remain to be solved for advancing the clinical translation of these nanocarriers, such as the unsatisfactory transfection efficiency, safety issues, complexity of manufacturing and off-target effects. The purpose of this review is to elaborate the principles of stimuli-responsive nanocarriers and to emphasize the most influential advances of stimuli-responsive gene delivery systems. Current challenges of their clinical translation and corresponding solutions will also be highlighted, which will accelerate the translation of stimuli-responsive nanocarriers and advance the development of gene therapy.

Radiolabelled Extracellular Vesicles as Imaging Modalities for Precise Targeted Drug Delivery.

Extracellular vesicles (ECVs) have been abandoned as bio-inspired drug delivery systems (DDS) in the biomedical field. ECVs have a natural ability to cross over extracellular and intracellular barriers, making them superior to manufactured nanoparticles. Additionally, they have the ability to move beneficial biomolecules among far-flung bodily cells. These advantages and the accomplishment of favorable in vivo results convincingly show the value of ECVs in medication delivery. The usage of ECVs is constantly being improved, as it might be difficult to develop a consistent biochemical strategy that is in line with their useful clinical therapeutic uses. Extracellular vesicles (ECVs) have the potential to enhance the therapy of diseases. Imaging technologies, particularly radiolabelled imaging, have been exploited for non-invasive tracking to better understand their in vivo activity.

Phosphocholine-Functionalized Zwitterionic Highly Branched Poly(β-amino ester)s for Cytoplasmic Protein Delivery.

Proteins have tremendous potential for vaccine development and disease treatment, but multiple extracellular and intracellular biological barriers must be overcome before they can exert specific biological functions in the target tissue. The use of polymers as carriers would greatly improve their bioavailability and therapeutic efficiency. Nevertheless, effective protein packaging and cell membrane penetration without causing cytotoxicity is particularly challenging, due largely to the simultaneous distribution of positive and negative charges on protein surface. Here, phosphocholine-functionalized zwitterionic poly(β-amino ester)s, HPAE-D-(±), are developed for cytoplasmic protein delivery. The zwitterionic phosphocholine is capable of binding to both proteins and the cell membrane to facilitate protein packaging and nanoparticle cellular uptake. Compared to amine-functionalized HPAE-E-(+) and carboxylic acid-functionalized HPAE-C-(-), HPAE-D-(±) exhibits much higher cytoplasmic protein delivery efficiency and lower cytotoxicity. In addition, HPAE-D-(±) are readily degraded in aqueous solution. This strategy may be extended to other zwitterions and polymers, thus having profound implications for the development of safe and efficient protein delivery systems.

Light-Triggered Mechanical Disruption of Extracellular Barriers by Swarms of Enzyme-Powered Nanomotors for Enhanced Delivery.

Targeted drug delivery depends on the ability of nanocarriers to reach the target site, which requires the penetration of different biological barriers. Penetration is usually low and slow because of passive diffusion and steric hindrance. Nanomotors (NMs) have been suggested as the next generation of nanocarriers in drug delivery due to their autonomous motion and associated mixing hydrodynamics, especially when acting collectively as a swarm. Here, we explore the concept of enzyme-powered NMs designed as such that they can exert disruptive mechanical forces upon laser irradiation. The urease-powered motion and swarm behavior improve translational movement compared to passive diffusion of state-of-the-art nanocarriers, while optically triggered vapor nanobubbles can destroy biological barriers and reduce steric hindrance. We show that these motors, named Swarm 1, collectively displace through a microchannel blocked with type 1 collagen protein fibers (barrier model), accumulate onto the fibers, and disrupt them completely upon laser irradiation. We evaluate the disruption of the microenvironment induced by these NMs (Swarm 1) by quantifying the efficiency by which a second type of fluorescent NMs (Swarm 2) can move through the cleared microchannel and be taken up by HeLa cells at the other side of the channel. Experiments showed that the delivery efficiency of Swarm 2 NMs in a clean path was increased 12-fold in the presence of urea as fuel compared to when no fuel was added. When the path was blocked with the collagen fibers, delivery efficiency dropped considerably and only depicted a 10-fold enhancement after pretreatment of the collagen-filled channel with Swarm 1 NMs and laser irradiation. The synergistic effect of active motion (chemically propelled) and mechanical disruption (light-triggered nanobubbles) of a biological barrier represents a clear advantage for the improvement of therapies which currently fail due to inadequate passage of drug delivery carriers through biological barriers.

Inactivation of the lysine binding sites of human plasminogen (hPg) reveals novel structural requirements for the tight hPg conformation, M-protein binding, and rapid activation.

Accelerated activation of the human plasminogen zymogen (hPg) to two-chain active plasmin (hPm) is achieved following conformational changes induced by ligand-binding at the lysine-binding sites (LBSs) in four of the five hPg kringle domains. In this manner, pattern D skin-trophic strains of Group A streptococci (GAS), through the expression of surface plasminogen-binding M-protein (PAM), immobilize surface hPg, thereby enabling rapid hPg activation by GAS-secreted streptokinase (SK). Consequently, GAS enhances virulence by digesting extracellular and tight cellular junctional barriers using hPm activity. Many studies have demonstrated the singular importance of the kringle-2 domain of hPg (K2hPg) to PAM-binding using hPg fragments. Recently, we showed, using full-length hPg, that K2hPg is critical for PAM binding. However, these studies did not eliminate any modulatory effects of the non-K2hPg LBS on this interaction. Moreover, we sought to establish the significance of the intramolecular interaction between Asp219 of the LBS of K2hPg and its serine protease domain binding partner, Lys708, to conformational changes in hPg. In the current study, selective inactivation of the LBS of K1hPg, K4hPg, and K5hPg revealed that the LBS of these kringle domains are dispensable for hPg binding to PAM. However, the attendant conformational change upon inactivation of K4hPg LBS increased the affinity of hPg for PAM by an order of magnitude. This finding suggests that the native hPg conformation encloses PAM-binding exosites or sterically hinders access to K2hPg. While simultaneous inactivation of the LBS of K1hPg, K4hPg, and K5hPg inhibited hPg/SK association alongside hPg activation, the replacement of Lys708 generated a slight conformational change that optimally accelerated hPg activation. Thus, we accentuate disparate functions of hPg LBS and conclude, using intact proteins, that K2hPg plays a central role in regulating hPg activation.

Biomimetic cell membrane-coated nanocarriers for targeted siRNA delivery in cancer therapy.

Small interfering RNA (siRNA) therapeutics for cancer are a focus of increasing research interest. However, the major obstacle to their clinical application is the targeted delivery of siRNA to cancer cells at desirable levels. Cell membrane-coated nanocarriers have the advantage of combining the properties of both cell membranes and nanoparticles (NPs). In this review, we highlight the most common RNAi therapeutics and the extracellular and intracellular barriers to siRNA delivery. Moreover, we discuss clinical applications of different cell membrane-coated nanocarriers for targeted siRNA delivery, including cancer cell membranes (CCMs), platelet membranes, erythrocyte membranes, stem cell membranes, exosome membranes, and hybrid membranes. Taken together, biomimetic cell membrane-coated nanotechnology is a promising strategy for targeted siRNA delivery for cancer treatment.