Extracellular Barriers Research Articles

Non-Viral Carriers for Nucleic Acids Delivery: Fundamentals and Current Applications.

Over the past decades, non-viral DNA and RNA delivery systems have been intensively studied as an alternative to viral vectors. Despite the most significant advantage over viruses, such as the lack of immunogenicity and cytotoxicity, the widespread use of non-viral carriers in clinical practice is still limited due to the insufficient efficacy associated with the difficulties of overcoming extracellular and intracellular barriers. Overcoming barriers by non-viral carriers is facilitated by their chemical structure, surface charge, as well as developed modifications. Currently, there are many different forms of non-viral carriers for various applications. This review aimed to summarize recent developments based on the essential requirements for non-viral carriers for gene therapy.

Oral delivery of RNAi for cancer therapy.

Cancer is a major health concern worldwide and is still in a continuous surge of seeking for effective treatments. Since the discovery of RNAi and their mechanism of action, it has shown promises in targeted therapy for various diseases including cancer. The ability of RNAi to selectively silence the carcinogenic gene makes them ideal as cancer therapeutics. Oral delivery is the ideal route of administration of drug administration because of its patients' compliance and convenience. However, orally administered RNAi, for instance, siRNA, must cross various extracellular and intracellular biological barriers before it reaches the site of action. It is very challenging and important to keep the siRNA stable until they reach to the targeted site. Harsh pH, thick mucus layer, and nuclease enzyme prevent siRNA to diffuse through the intestinal wall and thereby induce a therapeutic effect. After entering the cell, siRNA is subjected to lysosomal degradation. Over the years, various approaches have been taken into consideration to overcome these challenges for oral RNAi delivery. Therefore, understanding the challenges and recent development is crucial to offer a novel and advanced approach for oral RNAi delivery. Herein, we have summarized the delivery strategies for oral delivery RNAi and recent advancement towards the preclinical stages.

Safe and Effective Delivery of mRNA Using Modified PEI-Based Lipopolymers

Chemically modified mRNA (modRNA) has proven to be a versatile tool for the treatment of various cancers and infectious diseases due to recent technological advancements. However, a safe and effective delivery system to overcome the complex extracellular and intracellular barriers is required in order to achieve higher therapeutic efficacy and broaden clinical applications. Here, we explored All-Fect and Leu-Fect C as novel transfection reagents derived from lipopolymers, which demonstrated excellent biocompatibility, efficient delivery capabilities, and a robust ability to escape the lysosomes. These properties directly increase mRNA stability by preventing mRNA degradation by nucleases and simultaneously promote efficient gene translation in vitro and in vivo. The modRNA delivered with lipopolymer vectors sustained effective transfection in mouse hearts following direct intramyocardial injection, as well as in major organs (liver and spleen) after systemic administration. No observable immune reactions or systemic toxicity were detected following the systemic administration of lipopolymer-mRNA complexes to additional solid organs. This study identified commercial reagents for the effective delivery of modRNA and may help facilitate the advancement of gene-based interventions involving the safe and effective delivery of nucleic acid drug substances.

The Plant Fatty Acyl Reductases.

Fatty acyl reductase (FAR) is a crucial enzyme that catalyzes the NADPH-dependent reduction of fatty acyl-CoA or acyl-ACP substrates to primary fatty alcohols, which in turn acts as intermediate metabolites or metabolic end products to participate in the formation of plant extracellular lipid protective barriers (e.g., cuticular wax, sporopollenin, suberin, and taproot wax). FARs are widely present across plant evolution processes and play conserved roles during lipid synthesis. In this review, we provide a comprehensive view of FAR family enzymes, including phylogenetic analysis, conserved structural domains, substrate specificity, subcellular localization, tissue-specific expression patterns, their varied functions in lipid biosynthesis, and the regulation mechanism of FAR activity. Finally, we pose several questions to be addressed, such as the roles of FARs in tryphine, the interactions between transcription factors (TFs) and FARs in various environments, and the identification of post-transcriptional, translational, and post-translational regulators.

Ligand-modified nanocarriers for oral drug delivery: Challenges, rational design, and applications.

Ligand-modified nanocarriers (LMNCs) specific to their targets have attracted increasing interest for enhanced oral drug delivery in recent decades. Although the design of LMNCs for enhanced endocytosis and improved exposure of the loaded drugs through the oral route has received abundant attention, it remains unclear how the design influences their transcellular process, especially the key factors affecting their functions. This review discusses the extracellular and cellular barriers to orally administered LMNCs in the gastrointestinal (GI) tract and new discoveries regarding the GI protein corona and the sequential transport barriers that impede the preplanned movements of LMNCs after oral administration. Furthermore, innovative progress in considering key factors (including target selection, ligand properties, and other important factors) in the rational design of LMNCs for oral drug delivery is presented. In particular, some factors that endow LMNCs with efficient transcytosis rather than only endocytosis are highlighted. Finally, the prospects of orally administered LMNCs in disease therapy for the enhanced oral/local bioavailability of active pharmaceutical ingredients, as well as emerging delivery routes, such as lymphatic drug delivery and systemic location-specific drug release based on oral transcellular LMNCs, are discussed.

Nonviral nanoparticle gene delivery into the CNS for neurological disorders and brain cancer applications.

Nonviral nanoparticles have emerged as an attractive alternative to viral vectors for gene therapy applications, utilizing a range of lipid-based, polymeric, and inorganic materials. These materials can either encapsulate or be functionalized to bind nucleic acids and protect them from degradation. To effectively elicit changes to gene expression, the nanoparticle carrier needs to undergo a series of steps intracellularly, from interacting with the cellular membrane to facilitate cellular uptake to endosomal escape and nucleic acid release. Adjusting physiochemical properties of the nanoparticles, such as size, charge, and targeting ligands, can improve cellular uptake and ultimately gene delivery. Applications in the central nervous system (CNS; i.e., neurological diseases, brain cancers) face further extracellular barriers for a gene-carrying nanoparticle to surpass, with the most significant being the blood-brain barrier (BBB). Approaches to overcome these extracellular challenges to deliver nanoparticles into the CNS include systemic, intracerebroventricular, intrathecal, and intranasal administration. This review describes and compares different biomaterials for nonviral nanoparticle-mediated gene therapy to the CNS and explores challenges and recent preclinical and clinical developments in overcoming barriers to nanoparticle-mediated delivery to the brain. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease Therapeutic Approaches and Drug Discovery > Emerging Technologies Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Nucleic acid strategies for infectious disease treatments: The nanoparticle-based oral delivery route.

Therapies based on orally administrated nucleic acids have significant potential for the treatment of infectious diseases, including chronic inflammatory diseases such as inflammatory bowel disease (IBD)-associated with the gastrointestinal (GI) tract, and infectious and acute contagious diseases like coronavirus disease 2019 (COVID-19). This is because nucleic acids could precisely regulate susceptibility genes in regulating the pro- and anti-inflammatory cytokines expression related to the infections. Unfortunately, gene delivery remains a major hurdle due to multiple intracellular and extracellular barriers. This review thoroughly discusses the challenges of nanoparticle-based nucleic acid gene deliveries and strategies for overcoming delivery barriers to the inflammatory sites. Oral nucleic acid delivery case studies were also present as vital examples of applications in infectious diseases such as IBD and COVID-19.

Redox homeostasis modulation using theranostic AIE nanoparticles results in positive-feedback drug accumulation and enhanced drug penetration to combat drug-resistant cancer.

Drug-resistant cancers usually have multiple barriers to compromise the effect of therapies, of which multidrug-resistance (MDR) phenotype as the intracellular barrier and dense tumor matrix as the extracellular barrier, significantly contribute to the poor anticancer performance of current drug delivery systems (DDS). Here in this study, we fabricated a novel aggregation-induced emission (AIE)-active polymer capable of self-assembling into ultrasmall nanoparticles (∼20 ​nm) with D-alpha Tocopheryl Polyethylene Glycol Succinate (TPGS), for dual-encapsulating of doxorubicin (Dox) and sulforaphane (SFN) (AT/Dox/SFN). It revealed that redox homeostasis modulation of MDR cells (MCF-7/Adr) using AT/Dox/SFN can trigger mitochondria damage and ATP deficiency, which reverse the MDR phenotype of MCF-7/Adr cells to afford enhanced cellular uptake of both drug and DDS in a positive-feedback manner. The enhanced cellular drug accumulation further initiates the “neighboring effect” for improved drug penetration. Using this strategy, the growth of in vivo MCF-7/Adr tumors can be effectively inhibited at a low dosage (1/5) of doxorubicin (Dox) as compared to free Dox. In summary, we offer a new approach to overcome both the intracellular and extracellular barriers of drug-resistant cancers and elucidate the potential action mechanisms, which are beneficial for better cancer management.

Nanocarriers for delivery of siRNA as gene silencing mediator.

The term nanocarrier refers to sub-micrometric particles of less than 100 nm, designed to transport, distribute, and release nanotechnology-based drug delivery systems. siRNA therapy is a novel strategy that has great utility for a variety of treatments, however naked siRNA delivery has not been an effective strategy, resulting in the necessary use of nanocarriers for delivery. This review aims to highlight the versatility of carriers based on smart drug delivery systems. The nanocarriers based on nanoparticles as siRNA DDS have provided a set of very attractive advantages related to improved physicochemical properties, such as high surface-to-volume ratio, versatility to package siRNA, provide a dual function to both protect extracellular barriers that lead to elimination and overcome intracellular barriers limiting cytosolic delivery, and possible chemical modifications on the nanoparticle surface to improve stability and targeting. Lipid and polymeric nanocarriers have proven to be stable, biocompatible, and effective in vitro, further exploration of the development of new nanocarriers is needed to obtain safe and biocompatible tools for effective therapy.

Nanoparticles-Based Strategies to Improve the Delivery of Therapeutic Small Interfering RNA in Precision Oncology.

Small interfering RNA (siRNA) can selectively suppress the expression of disease-causing genes, holding great promise in the treatment of human diseases, including malignant cancers. In recent years, with the development of chemical modification and delivery technology, several siRNA-based therapeutic drugs have been approved for the treatment of non-cancerous liver diseases. Nevertheless, the clinical development of siRNA-based cancer therapeutics remains a major translational challenge. The main obstacles of siRNA therapeutics in oncology include both extracellular and intracellular barriers, such as instability under physiological conditions, insufficient tumor targeting and permeability (particularly for extrahepatic tumors), off-target effects, poor cellular uptake, and inefficient endosomal escape. The development of clinically suitable and effective siRNA delivery systems is expected to overcome these challenges. Herein, we mainly discuss recent strategies to improve the delivery and efficacy of therapeutic siRNA in cancer, including the application of non-viral nanoparticle-based carriers, the selection of target genes for therapeutic silencing, and the combination with other therapeutic modalities. In addition, we also provide an outlook on the ongoing challenges and possible future developments of siRNA-based cancer therapeutics during clinical translation.

Biotechnological Evolution of siRNA Molecules: From Bench Tool to the Refined Drug.

The depth and versatility of siRNA technologies enable their use in disease targets that are undruggable by small molecules or that seek to achieve a refined turn-off of the genes for any therapeutic area. Major extracellular barriers are enzymatic degradation of siRNAs by serum endonucleases and RNAases, renal clearance of the siRNA delivery system, the impermeability of biological membranes for siRNA, activation of the immune system, plasma protein sequestration, and capillary endothelium crossing. To overcome the intrinsic difficulties of the use of siRNA molecules, therapeutic applications require nanometric delivery carriers aiming to protect double-strands and deliver molecules to target cells. This review discusses the history of siRNAs, siRNA design, and delivery strategies, with a focus on progress made regarding siRNA molecules in clinical trials and how siRNA has become a valuable asset for biopharmaceutical companies.

A Space-Time Conversion Vehicle for Programmed Multi-Drugs Delivery into Pancreatic Tumor to Overcome Matrix and Reflux Barriers.

The numerous biological barriers, which limit pharmacotherapy of pancreatic carcinoma, including inadequate drug accumulation in the tumor environment, a dense extracellular matrix (ECM) and efficient drug‐efflux mechanisms, illustrate the requirement of multifunctional delivery systems to overcome the individual barriers at the right place at the right time. Herein, a space–time conversion vehicle based on covalent organic framework (COF)‐coated mesoporous silica nanospheres (MSN) with a sandwiched polyethyleneimine (PEI) layer (MPCP), is designed. The space‐specific drugs‐loaded vehicle (MGPPCLP) is obtained by separately incorporating a chemotherapeutic agent (gemcitabine, G) into the MSN core, a P glycoprotein inhibitor (LY 335979, P) into the PEI layer, and an extracellular matrix disruptor (losartan, L) into the COF shell. Thereafter, a programmed drug delivery is achieved via the ordered degradation from COF shell to MSN core. Sequential release of the individual drugs, synergized with a change of nanoparticle surface charge, contribute to an obvious extracellular matrix distraction, distinct drug efflux inhibition, and consequently enhance chemotherapeutic outcomes in pancreatic carcinoma. This MPCP‐based vehicle design suggests a robust space–time conversion strategy to achieve programmed multi‐drugs delivery and represents a new avenue to the treatment of pancreatic carcinoma by overcoming extracellular matrix and drug reflux barriers.

Nanotechnology-Assisted Cell Tracking.

The usefulness of nanoparticles (NPs) in the diagnostic and/or therapeutic sector is derived from their aptitude for navigating intra- and extracellular barriers successfully and to be spatiotemporally targeted. In this context, the optimization of NP delivery platforms is technologically related to the exploitation of the mechanisms involved in the NP–cell interaction. This review provides a detailed overview of the available technologies focusing on cell–NP interaction/detection by describing their applications in the fields of cancer and regenerative medicine. Specifically, a literature survey has been performed to analyze the key nanocarrier-impacting elements, such as NP typology and functionalization, the ability to tune cell interaction mechanisms under in vitro and in vivo conditions by framing, and at the same time, the imaging devices supporting NP delivery assessment, and consideration of their specificity and sensitivity. Although the large amount of literature information on the designs and applications of cell membrane-coated NPs has reached the extent at which it could be considered a mature branch of nanomedicine ready to be translated to the clinic, the technology applied to the biomimetic functionalization strategy of the design of NPs for directing cell labelling and intracellular retention appears less advanced. These approaches, if properly scaled up, will present diverse biomedical applications and make a positive impact on human health.

Modification of Lipid-Based Nanoparticles: An Efficient Delivery System for Nucleic Acid-Based Immunotherapy.

Lipid-based nanoparticles (LBNPs) are biocompatible and biodegradable vesicles that are considered to be one of the most efficient drug delivery platforms. Due to the prominent advantages, such as long circulation time, slow drug release, reduced toxicity, high transfection efficiency, and endosomal escape capacity, such synthetic nanoparticles have been widely used for carrying genetic therapeutics, particularly nucleic acids that can be applied in the treatment for various diseases, including congenital diseases, cancers, virus infections, and chronic inflammations. Despite great merits and multiple successful applications, many extracellular and intracellular barriers remain and greatly impair delivery efficacy and therapeutic outcomes. As such, the current state of knowledge and pitfalls regarding the gene delivery and construction of LBNPs will be initially summarized. In order to develop a new generation of LBNPs for improved delivery profiles and therapeutic effects, the modification strategies of LBNPs will be reviewed. On the basis of these developed modifications, the performance of LBNPs as therapeutic nanoplatforms have been greatly improved and extensively applied in immunotherapies, including infectious diseases and cancers. However, the therapeutic applications of LBNPs systems are still limited due to the undesirable endosomal escape, potential aggregation, and the inefficient encapsulation of therapeutics. Herein, we will review and discuss recent advances and remaining challenges in the development of LBNPs for nucleic acid-based immunotherapy.

Rationally designed cationic amphiphilic peptides for selective gene delivery to cancer cells

Gene therapy has gained increasing attention as an alternative to pharmacotherapy for treatment of various diseases. The extracellular and intracellular barriers to gene delivery necessitate the use of gene vectors which has led to the development of myriads of gene delivery systems. However, many of these gene delivery systems have pitfalls such as low biocompatibility, low loading efficiency, low transfection efficiency, lack of tissue selectivity and high production costs. Herein, we report the development of a new series of short cationic amphiphilic peptides with anticancer activity for selective delivery of small interfering RNA (siRNA) and antisense oligodeoxynucleotides (ODNs) to cancer cells. The peptides consist of alternating dyads of hydrophobic (isoleucine (I) or leucine (L)) and hydrophilic (arginine (R) or lysine (L)) amino acids. The peptides exhibited higher preference for transfection of HCT 116 colorectal cancer cells compared to human dermal fibroblasts (HDFs) and induced higher level of gene silencing in the cancer cells. The nucleic acid complexation and transfection efficiency of the peptides was a function of their secondary structure, their hydrophobicity and their C-terminal amino acid. The peptides containing L in their hydrophobic domain formed stronger complexes with siRNA and successfully delivered it to the cancer cells but were unable to release their cargo inside the cells and therefore could not induce any gene silencing. On the contrary, the peptides containing I in their hydrophobic domain were able to release their associated siRNA and induce considerable gene silencing in cancer cells. The peptides exhibited higher selectivity for colorectal cancer cells and induced less gene silencing in fibroblasts compared to the lipid-based commercial transfection reagent DharmaFECT™ 1. The results from this study can serve as a tool for rational design of new peptide-based gene vectors for high selective gene delivery to cancer cells.

Modulating intracellular pathways to improve non-viral delivery of RNA therapeutics.

RNA therapeutics (e.g. siRNA, oligonucleotides, mRNA, etc.) show great potential for the treatment of a myriad of diseases. However, to reach their site of action in the cytosol or nucleus of target cells, multiple intra- and extracellular barriers have to be surmounted. Several non-viral delivery systems, such as nanoparticles and conjugates, have been successfully developed to meet this requirement. Unfortunately, despite these clear advances, state-of-the-art delivery agents still suffer from relatively low intracellular delivery efficiencies. Notably, our current understanding of the intracellular delivery process is largely oversimplified. Gaining mechanistic insight into how RNA formulations are processed by cells will fuel rational design of the next generation of delivery carriers. In addition, identifying which intracellular pathways contribute to productive RNA delivery could provide opportunities to boost the delivery performance of existing nanoformulations. In this review, we discuss both established as well as emerging techniques that can be used to assess the impact of different intracellular barriers on RNA transfection performance. Next, we highlight how several modulators, including small molecules but also genetic perturbation technologies, can boost RNA delivery by intervening at differing stages of the intracellular delivery process, such as cellular uptake, intracellular trafficking, endosomal escape, autophagy and exocytosis.

A Review of Brain-Targeted Nonviral Gene-Based Therapies for the Treatment of Alzheimer's Disease.

Diseases of the central nervous system (CNS) are difficult to treat owing to the complexity of the brain and the presence of a natural blood-brain-barrier (BBB). Alzheimer's disease (AD) is one of the major progressive and currently incurable neurodegenerative disorders of the CNS, which accounts for 60-80% of cases of dementia. The pathophysiology of AD involves the accumulation of amyloid beta (Aβ) plaques and neurofibrillary tangles (NFTs) in the brain. Additionally, synaptic loss and imbalance of neuronal signaling molecules are characterized as important markers of AD. Existing treatments of AD help in the management of its symptoms and aim toward the maintenance of cognitive functions, behavior, and attenuation of gradual memory loss. Over the past decade, nonviral gene therapy has attracted increasing interest due to its various advantages over its viral counterparts. Moreover, advancements in nonviral gene technology have led to their increasing contributions in clinical trials. However, brain-targeted nonviral gene delivery vectors come across various extracellular and intracellular barriers, limiting their ability to transfer the therapeutic gene into the target cells. Chief barriers to nonviral gene therapy have been discussed briefly in this review. We have also highlighted the rapid advancement of several nonviral gene therapies for AD, which are broadly categorized into physical and chemical methods. These methods aim to modulate Aβ, beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1), apolipoprotein E, or neurotrophic factors' expression in the CNS. Overall, this review discusses challenges and recent advancements of nonviral gene therapy for AD.

3D Bioprinting of Miniaturized Tissues Embedded in Self‐Assembled Nanoparticle‐Based Fibrillar Platforms

AbstractThe creation of microphysiological systems like tissue and organ‐on‐chip for in vitro modeling of human physiology and diseases is gathering increasing interest. However, the platforms used to build these systems have limitations concerning implementation, automation, and cost‐effectiveness. Moreover, their typical plastic‐based housing materials are poor recreations of native tissue extracellular matrix (ECM) and barriers. Here, the controlled self‐assembly of plant‐derived cellulose nanocrystals (CNC) is combined with the concept of 3D bioprinting in suspension baths for the direct biofabrication of microphysiological systems embedded within an ECM mimetic fibrillar support material. The developed support CNC fluid gel allows exceptionally high‐resolution bioprinting of 3D constructs with arbitrary geometries and low restrictions of bioink choice. The further induction of CNC self‐assembly with biocompatible calcium ions results in a transparent biomimetic nanoscaled fibrillar matrix that allows hosting different compartmentalized cell types and perfusable channels, has tailored permeability for biomacromolecules diffusion and cellular crosstalk, and holds structural stability to support long‐term in vitro cell maturation. In summary, this xeno‐free nanoscale CNC fibrillar matrix allows the biofabrication of hierarchical living constructs, opening new opportunities not only for developing physiologically relevant 3D in vitro models but also for a wide range of applications in regenerative medicine.

Albumin Nanostructures for Nucleic Acid Delivery in Cancer: Current Trend, Emerging Issues, and Possible Solutions.

Simple SummaryNucleic acids are showing tremendous potential in cancer therapy. However, their successful delivery to tumor sites is still a challenge. Herein, we report on the use of albumin-based nanocarriers for the delivery of nucleic acids because of their biosafety, ease of surface modification, and tumor targeting. In addition, we discuss various surface modification strategies to improve the internalization, efficacy, and specific tumor targeting of the albumin nanocarriers. Cancer is one of the major health problems worldwide, and hence, suitable therapies with enhanced efficacy and reduced side effects are desired. Gene therapy, involving plasmids, small interfering RNAs, and antisense oligonucleotides have been showing promising potential in cancer therapy. In recent years, the preparation of various carriers for nucleic acid delivery to the tumor sites is gaining attention since intracellular and extracellular barriers impart major challenges in the delivery of naked nucleic acids. Albumin is a versatile protein being used widely for developing carriers for nucleic acids. It provides biocompatibility, tumor specificity, the possibility for surface modification, and reduces toxicity. In this review, the advantages of using nucleic acids in cancer therapy and the challenges associated with their delivery are presented. The focus of this article is on the different types of albumin nanocarriers, such as nanoparticles, polyplexes, and nanoconjugates, employed to overcome the limitations of the direct use of nucleic acids in vivo. This review also highlights various approaches for the modification of the surface of albumin to enhance its transfection efficiency and targeted delivery in the tumor sites.

CASPARIAN STRIP INTEGRITY FACTOR (CIF) family peptides - regulator of plant extracellular barriers

In multicellular organisms, water and most of the small molecules, such as nutrients, toxic substances, and signaling compounds, move freely through extracellular spaces, depending on their biochemical nature. To restrict the simple diffusion of small molecules, multicellular organisms have evolved extracellular barriers across specific tissue layers, such as tight junctions in the animal epithelium. Similar extracellular barriers are also generated in plants through the accumulation of hydrophobic chemicals, such as lignin or cutin, although the detailed molecular mechanisms underlying this process remain elusive. Here, I summarize recent advances in extracellular barrier formation in plants by focusing mainly on CASPARIAN STRIP INTEGRITY FACTOR (CIF) family peptides, which trigger the spatially precise deposition of designated cell wall components, enabling plants to establish transcellular barrier networks correctly. The genome of Arabidopsis thaliana, a model plant species, harbors five CIF genes, which encode propeptides which are processed into small secreted peptides of 21–24 amino acids. Sulfation of tyrosine residues in CIF peptides ensures their full bioactivity and high-affinity binding to their receptors SCHENGEN3/GASSHO1 (SGN3/GSO1) and GSO2 in vitro. Additionally, in vivo analysis shows that physical restriction of CIF peptide diffusion and the subcellular localization of a signaling module and expression patterns of a peptide processing enzyme specify the location of signal activation. Thus, the CIF peptide family provides fascinating models for understanding mature peptide biogenesis and spatially limited signal activation with small diffusive molecules.