Trivalent Disulfide Unit-Masked System Efficiently Delivers Large Oligonucleotide.

Oligonucleotide drugs are shining in clinical therapeutics, but efficient and safe delivery systems severely limit their widespread use. A disulfide unit technology platform based on dynamic thiol exchange chemistry at the cell membrane has the potential for drug delivery. However, the alteration of the disulfide unit CSSC dihedral angle induced by different substituents directly affects the effectiveness of this technology and its stability. Previously, we constructed a trivalent low dihedral angle disulfide unit that can effectively promote the cellular uptake of small molecules. Here, we constructed a novel disulfide unit-masked oligonucleotide hybrid based on a low dihedral angle disulfide unit, motivated by prodrug design. Cellular imaging results showed that such a system exhibited superior cellular delivery efficiency than the commercial Lipo2000 without cytotoxicity. The thiol reagents significantly reduced its cellular uptake (57-74%), which proved to be endocytosis-independent. In addition, in vivo distribution experiments in mice showed that such systems can be rapidly distributed in liver tissues with a duration of action of more than 24 h, representing a potential means of silencing genes involved in the pathogenesis of liver-like diseases. In conclusion, this trivalent disulfide unit-masked system we constructed can effectively deliver large oligonucleotide drugs.

Therapeutic Antisense Oligonucleotides in Oncology: From Bench to Bedside.

Advancements in our comprehension of tumor biology and chemoresistance have spurred the development of treatments that precisely target specific molecules within the body. Despite the expanding landscape of therapeutic options, there persists a demand for innovative approaches to address unmet clinical needs. RNA therapeutics have emerged as a promising frontier in this realm, offering novel avenues for intervention such as RNA interference and the utilization of antisense oligonucleotides (ASOs). ASOs represent a versatile class of therapeutics capable of selectively targeting messenger RNAs (mRNAs) and silencing disease-associated proteins, thereby disrupting pathogenic processes at the molecular level. Recent advancements in chemical modification and carrier molecule design have significantly enhanced the stability, biodistribution, and intracellular uptake of ASOs, thereby bolstering their therapeutic potential. While ASO therapy holds promise across various disease domains, including oncology, coronary angioplasty, neurological disorders, viral, and parasitic diseases, our review manuscript focuses specifically on the application of ASOs in targeted cancer therapies. Through a comprehensive examination of the latest research findings and clinical developments, we delve into the intricacies of ASO-based approaches to cancer treatment, shedding light on their mechanisms of action, therapeutic efficacy, and prospects.

Design and synthesis of nucleotidyl lipids and their application in the targeted delivery of siG12D for pancreatic cancer therapy.

Nucleic acid drugs are attracting significant attention as prospective therapeutics. However, their efficacy is hindered by challenges in penetrating cell membranes and reaching target tissues, limiting their applications. Nucleotidyl lipids, with their specific intermolecular interactions such as H-bonding and π-π stacking, offer a promising solution as gene delivery vehicles. In this study, a novel series of nucleotide-based amphiphiles were synthesized. These lipid molecules possess the ability to self-assemble into spherical vesicles of appropriate size and zeta potential in aqueous solution. Furthermore, their complexes with oligonucleotides demonstrated favorable biocompatibility and exhibited antiproliferative effects against a broad range of cancer cells. Additionally, when combined with the cationic lipid CLD, these complexes displayed promising in vitro performance and in vivo efficacy. By incorporating DSPE-PEGylated cRGD into the formulation, targeted accumulation of siG12D in pancreatic cancer cells increased from approximately 6% to 18%, leading to effective treatment outcomes (intravenous administration, 1 mg/kg). This finding holds significant importance for the liposomal delivery of nucleic acid drugs to extrahepatic tissues.

Design, Synthesis, and Biochemical Analysis of a Molecule Designed to Enhance Endosomal Escape.

RNA therapeutics, including siRNAs, ASOs, and PMOs, have great potential to treat human disease. However, RNA therapeutics are too large, too charged, and/or too hydrophilic to cross the cellular membrane and are instead taken up into cells by endocytosis. Unfortunately, the vast majority of RNA therapeutics remain trapped inside endosomes (≥ 99%), which is the sole reason preventing their use to treat cancer, COVID, and other diseases. In contrast, enveloped viruses, such as influenza, also have an endosomal escape problem, but have evolved a highly efficient endosomal escape mechanism using trimeric hemagglutinin (HA) fusogenic protein. HA contains an outer hydrophilic domain (HA1) that masks an inner hydrophobic fusogenic/endosomal escape domain (HA2). Once inside endosomes, HA1 is shed to expose HA2 that, due to hydrophobicity, buries itself into the endosomal lipid bilayer, driving escape into the cytoplasm in a non-toxic fashion. To begin to address the RNA therapeutics rate-limiting endosomal escape problem, we report here a first step in the design and synthesis of a universal endosomal escape domain (uEED) that biomimics the enveloped virus escape mechanism. uEED contains an outer hydrophilic mask covalently attached to an inner hydrophobic escape domain. In plasma, uEED is inert and highly metabolically stable; however, when placed in endo/lysosomal conditions, uEED is activated by enzymatic removal of the hydrophilic mask, followed by self-immolation of the linker resulting in exposure of the hydrophobic indole ring domain in the absence of any hydrophilic tags. Thus, uEED is a synthetic biomimetic of the highly efficient viral endosomal escape mechanism.

Therapeutic Oligonucleotides: An Outlook on Chemical Strategies to Improve Endosomal Trafficking.

The potential of oligonucleotide therapeutics is undeniable as more than 15 drugs have been approved to treat various diseases in the liver, central nervous system (CNS), and muscles. However, achieving effective delivery of oligonucleotide therapeutics to specific tissues still remains a major challenge, limiting their widespread use. Chemical modifications play a crucial role to overcome biological barriers to enable efficient oligonucleotide delivery to the tissues/cells of interest. They provide oligonucleotide metabolic stability and confer favourable pharmacokinetic/pharmacodynamic properties. This review focuses on the various chemical approaches implicated in mitigating the delivery problem of oligonucleotides and their limitations. It highlights the importance of linkers in designing oligonucleotide conjugates and discusses their potential role in escaping the endosomal barrier, a bottleneck in the development of oligonucleotide therapeutics.

Targeting Signalling Pathways in Chronic Wound Healing.

Chronic wounds fail to achieve complete closure and are an economic burden to healthcare systems due to the limited treatment options and constant medical attention. Chronic wounds are characterised by dysregulated signalling pathways. Research has focused on naturally derived compounds, stem-cell-based therapy, small molecule drugs, oligonucleotide delivery nanoparticles, exosomes and peptide-based platforms. The phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT), Wingless-related integration (Wnt)/ß-catenin, transforming growth factor-ß (TGF-ß), nuclear factor erythroid 2-related factor 2 (Nrf2), Notch and hypoxia-inducible factor 1 (HIF-1) signalling pathways have critical roles in wound healing by modulating the inflammatory, proliferative and remodelling phases. Moreover, several regulators of the signalling pathways were demonstrated to be potential treatment targets. In this review, the current research on targeting signalling pathways under chronic wound conditions will be discussed together with implications for future studies.

Synthesis and physical and biological properties of 1,3-diaza-2-oxophenoxazine-conjugated oligonucleotides.

The artificial nucleobase 1,3-diaza-2-oxophenoxazine (tCO) and its derivative G-clamp strongly bind to guanine and, when incorporated into double-stranded DNA, significantly increase the stability of the latter. As the phenoxazine skeleton is a constituent of major pharmaceuticals, we hypothesized that oligonucleotides (ONs) containing phenoxazine bases would induce property changes related to intracellular uptake and migration in tissues. In this study, we designed and synthesized a novel G-clamp-linker antisense oligonucleotide (ASO) in which a G-clamp base with a flexible linker was introduced into the 5'-end of an ASO targeting mouse long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (mMALAT1). Compared to unconjugated ASO, the G-clamp-linker ASO induced significantly more effective knockdown of mMALAT1 in mouse skeletal muscle. The ASOs conjugated with 2'-deoxyribonucleotide(s) bearing a tCO nucleobase at the 5'-end exhibited a similar knockdown effect in skeletal muscle. Thus, it may be possible to improve therapeutic effects against skeletal muscle diseases, such as muscular dystrophy, by using ONs with incorporated phenoxazine nucleobases.

Improvement of Transfection with PepFects Using Organic and Inorganic Materials.

Cell-penetrating peptides (CPPs) are a promising non-viral vector for gene and drug delivery. CPPs exhibit high cell transfection, and are biocompatible. They can be also conjugated with organic and inorganic nanomaterials, such as magnetic nanoparticles (MNPs), graphene oxide (GO), metal-organic frameworks (MOFs), and chitosan. Nanomaterials offered a high specific surface area and provided relatively straightforward methods to be modified with biomolecules including CPPs and oligonucleotides (ONs). Novel nanomaterials conjugates with CPP/ONs complexes are therefore of interest for cell transfection with high efficiency. In this chapter, we described a summary of the non-viral vectors consisting of CPPs and nanomaterials. The book chapter also included a protocol to generate hybrid biomaterials consisting of CPPs and nanoparticles (NPs) for the delivery of oligonucleotides. The conjugation of NPs with CPPs serves as an effective platform for gene therapy with high cell transfection efficiency. The protocol is simple, offers high cell transfection compared to the CPPs-ONs complexes, and can be used for further improvements using external stimuli.

Delivery of Oligonucleotide Therapeutics: Chemical Modifications, Lipid Nanoparticles, and Extracellular Vesicles.

Oligonucleotides (ONs) comprise a rapidly growing class of therapeutics. In recent years, the list of FDA-approved ON therapies has rapidly expanded. ONs are small (15-30 bp) nucleotide-based therapeutics which are capable of targeting DNA and RNA as well as other biomolecules. ONs can be subdivided into several classes based on their chemical modifications and on the mechanisms of their target interactions. Historically, the largest hindrance to the widespread usage of ON therapeutics has been their inability to effectively internalize into cells and escape from endosomes to reach their molecular targets in the cytosol or nucleus. While cell uptake has been improved, "endosomal escape" remains a significant problem. There are a range of approaches to overcome this, and in this review, we focus on three: altering the chemical structure of the ONs, formulating synthetic, lipid-based nanoparticles to encapsulate the ONs, or biologically loading the ONs into extracellular vesicles. This review provides a background to the design and mode of action of existing FDA-approved ONs. It presents the most common ON classifications and chemical modifications from a fundamental scientific perspective and provides a roadmap of the cellular uptake pathways by which ONs are trafficked. Finally, this review delves into each of the above-mentioned approaches to ON delivery, highlighting the scientific principles behind each and covering recent advances.

Nucleic acid delivery for therapeutic applications.

Recent medical advances have exploited the ability to address a given disease at the underlying level of transcription and translation. These treatment paradigms utilize nucleic acids - including short interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), and messenger RNA (mRNA) - to achieve a desired outcome ranging from gene knockdown to induced expression of a selected target protein. Towards this end, numerous strategies for encapsulation or stabilization of various nucleic acid structures have been developed in order to achieve intracellular delivery. In this review, we discuss several therapeutic applications of nucleic acids directed towards specific diseases and tissues of interest, in particular highlighting recent technologies which have reached late-stage clinical trials and received FDA approval.

Novel Orthogonally Hydrocarbon-Modified Cell-Penetrating Peptide Nanoparticles Mediate Efficient Delivery of Splice-Switching Antisense Oligonucleotides In Vitro and In Vivo.

Splice-switching therapy with splice-switching oligonucleotides (SSOs) has recently proven to be a clinically applicable strategy for the treatment of several mis-splice disorders. Despite this, wider application of SSOs is severely limited by the inherently poor bioavailability of SSO-based therapeutic compounds. Cell-penetrating peptides (CPPs) are a class of drug delivery systems (DDSs) that have recently gained considerable attention for improving the uptake of various oligonucleotide (ON)-based compounds, including SSOs. One strategy that has been successfully applied to develop effective CPP vectors is the introduction of various lipid modifications into the peptide. Here, we repurpose hydrocarbon-modified amino acids used in peptide stapling for the orthogonal introduction of hydrophobic modifications into the CPP structure during peptide synthesis. Our data show that α,α-disubstituted alkenyl-alanines can be successfully utilized to introduce hydrophobic modifications into CPPs to improve their ability to formulate SSOs into nanoparticles (NPs), and to mediate high delivery efficacy and tolerability both in vitro and in vivo. Conclusively, our results offer a new flexible approach for the sequence-specific introduction of hydrophobicity into the structure of CPPs and for improving their delivery properties.

Designing biomaterials for the delivery of RNA therapeutics to stimulate bone healing.

Ribonucleic acids (small interfering RNA, microRNA, and messenger RNA) have been emerging as a promising new class of therapeutics for bone regeneration. So far, however, research has mostly focused on stability and complexation of these oligonucleotides for systemic delivery. By comparison, delivery of RNA nanocomplexes from biomaterial carriers can facilitate a spatiotemporally controlled local delivery of osteogenic oligonucleotides. This review provides an overview of the state-of-the-art in the design of biomaterials which allow for temporal and spatial control over RNA delivery. We correlate this concept of spatiotemporally controlled RNA delivery to the most relevant events that govern bone regeneration to evaluate to which extent tuning of release kinetics is required. In addition, inspired by the physiological principles of bone regeneration, potential new RNA targets are presented. Finally, considerations for clinical translation and upscaled production are summarized to stimulate the design of clinically relevant RNA-releasing biomaterials.

Structural tuning of oligonucleotides for enhanced blood circulation properties of unit polyion complexes prepared from two-branched poly(ethylene glycol)-block-poly(l-lysine).

Downsizing nanocarriers is a promising strategy for systemically targeting fibrotic cancers, such as pancreatic cancer, owing to enhanced tissue permeability. We recently developed a small oligonucleotide nanocarrier called a unit polyion complex (uPIC) using a single oligonucleotide molecule and one or two molecule(s) of two-branched poly(ethylene glycol)-b-poly(l-lysine) (bPEG-PLys). The uPIC is a dynamic polyion-pair equilibrated with free bPEG-PLys, and thus, is highly stabilized in the presence of excess amounts of free bPEG-PLys in the bloodstream. However, the dynamic polyion-pairing behavior of uPICs needs to be further investigated for longevity in the bloodstream, especially under lower amounts of free bPEG-PLys. Herein, the polyion-pairing behavior of uPICs was investigated by highlighting oligonucleotide stability and negative charge number. To this end, small interfering RNA (siRNA) and antisense oligonucleotides (ASO) were chemically modified to acquire nuclease resistance, and the ASO was hybridized with complementary RNA (cRNA) to form a hetero-duplex oligonucleotide (HDO) with twice the negative charges. While all oligonucleotides similarly formed sub-20 nm-sized uPICs from a single oligonucleotide molecule, the association number of bPEG-PLys (ANbPEG-PLys) in uPICs varied based on the negative charge number of oligonucleotides (N-), that is, ANbPEG-PLys = ~2 at N- = ~40 (i.e., siRNA and HDO) and ANbPEG-PLys = ~1 at N- = 20 (i.e., ASO), presumably because of the balanced charge neutralization between the oligonucleotide and bPEG-PLys with a positive charge number (N+) of ~20. Ultimately, the uPICs prepared from the chemically modified oligonucleotide with higher negative charges showed considerably longer blood retention than those from the control oligonucleotides without chemical modifications or with lower negative charges. The difference in the blood circulation properties of uPICs was more pronounced under lower amounts of free bPEG-PLys. These results demonstrate that the chemical modification and higher negative charge in oligonucleotides facilitated the polyion-pairing between the oligonucleotide and bPEG-PLys under harsh biological conditions, facilitating enhanced blood circulation of uPICs.

Synthesis and Application of Peptide-siRNA Nanoparticles from Disulfide-Constrained Cyclic Amphipathic Peptides for the Functional Delivery of Therapeutic Oligonucleotides to the Lung.

The potential of RNAi therapies has been largely impeded by the inherent challenges in the functional delivery of siRNA to cells. Herein, we describe protocols for the synthesis and characterization of novel peptide-siRNA nanoparticles prepared from disulfide-constrained amphipathic peptides complexed with siRNA as promising siRNA delivery vectors. We also describe protocols for the application of these nanoparticles to the in vitro and in vivo delivery of siRNA to lung cells for the functional knockdown of lung proteins.

Role of pharmacokinetic consideration for the development of drug delivery systems: A historical overview.

Drug delivery system is defined as a system or technology to achieve optimum therapeutic effects of drugs through precise control of their movements in the body. In order to optimize function of drug delivery systems aiming at targeting, their whole-body distribution profiles should be systematically evaluated and analyzed, where pharmacokinetic analysis based on the clearance concepts plays important role. Organ perfusion experiments combined with statistical moment analysis further supply detailed information on drug disposition at organ and cellular levels. Based on general relationship between physicochemical properties and distribution profile, macromolecular prodrugs or polymer conjugates of proteins are rationally designed and further introduction of ligand structure brings cell-specific delivery for them. These approaches are also applicable for particulate carriers such as liposomes and offer various opportunities for biological drugs such as nucleic acid drugs for their delivery. Mechanistic approach for dermal absorption analysis based on physiological skin model offers another opportunity in rational design of drug delivery. Potential of drug delivery technology in future medicines such as cell therapy and nanomaterial platform application is further discussed in relation to pharmacokinetic consideration.

Evaluation of Cell-Penetrating Peptide Delivery of Antisense Oligonucleotides for Therapeutic Efficacy in Spinal Muscular Atrophy.

Antisense oligonucleotides (ASOs) are a widely used form of gene therapy, which is translatable to multiple disorders. A major obstacle for ASO efficacy is its bioavailability for in vivo and in vitro studies. To overcome this challenge we use cell-penetrating peptides (CPPs) for systemic delivery of ASOs. One of the most advanced clinical uses of ASOs is for the treatment of spinal muscular atrophy (SMA). In this chapter, we describe the techniques used for in vitro screening and analysing in vivo biodistribution of CPP-conjugated ASOs targeting the survival motor neuron 2, SMN2, the dose-dependent modifying gene for SMA.

Oligo-guanidyl targeted bioconjugates forming rod shaped polyplexes as a new nanoplatform for oligonucleotide delivery.

Novel bioconjugates (Agm6-M-PEG-FA) for active oligonucleotide (ON) delivery have been developed by conjugating a cationic oligo-guanidyl star-like shaped "head" (Agm6-M) to a polymeric "tail" (PEG) terminating with folic acid (FA) as targeting agent or methoxy group (Agm6-M-PEG-FA and Agm6-M-PEG-OCH3, respectively). Gel electrophoresis showed that the bioconjugates completely associated with ONs at 3 nitrogen/phosphate (N/P) ratio. Studies performed with folate receptor (FR)-overexpressing HeLa cells, showed that optimal cell up-take was obtained with the 75:25 w/w Agm6-M-PEG-OCH3:Agm6-M-PEG-FA mixture. Dynamic light scattering and transmission electron microscopy showed that the polyplexes had size <80 nm with narrow polydispersity and rod-shaped morphology. The polyplexes were stable for several hours in plasma while ON was released in the presence of heparin concentration 16-times higher than the physiological one. The polyplexes displayed negligible cytotoxicity, hemolysis and low pro-inflammatory TNF-α release. Studies performed with FR-overexpressing HeLa and MDA-MB-231 cells using siRac1 revealed that the folated polyplexes caused significantly higher gene silencing (86.1 ±â€¯9.6%) and inhibition of cell migration (40%) than the non-folated polyplexes obtained with Agm6-M-PEG-OCH3 only. Although cytofluorimetric analyses showed similar cell uptake for both folated and non-folated polyplexes, confocal, TEM and competition studies showed that the folated polyplexes were taken-up by lysosome escaping caveolin-mediated pathway with final polyplex localization within cytosol, while non-folated polyplexes were preferentially taken-up via clathrin-mediated pathway to localize in the lysosomes. Finally, preliminary in vivo studies carried out in mice revealed that the folated polyplexes dispose in the tumor mass.

Light-Triggered Cellular Delivery of Oligonucleotides.

The major challenge in the therapeutic applicability of oligonucleotide-based drugs is the development of efficient and safe delivery systems. The carriers should be non-toxic and stable in vivo, but interact with the target cells and release the loaded oligonucleotides intracellularly. We approached this challenge by developing a light-triggered liposomal delivery system for oligonucleotides based on a non-cationic and thermosensitive liposome with indocyanine green (ICG) as photosensitizer. The liposomes had efficient release properties, as 90% of the encapsulated oligonucleotides were released after 1-minute light exposure. Cell studies using an enhanced green fluorescent protein (EGFP)-based splicing assay with HeLa cells showed light-activated transfection with up to 70%⁻80% efficacy. Moreover, free ICG and oligonucleotides in solution transfected cells upon light induction with similar efficacy as the liposomal system. The light-triggered delivery induced moderate cytotoxicity (25%⁻35% reduction in cell viability) 1⁻2 days after transfection, but the cell growth returned to control levels in 4 days. In conclusion, the ICG-based light-triggered delivery is a promising method for oligonucleotides, and it can be used as a platform for further optimization and development.

Polyethylenimine-based Formulations for Delivery of Oligonucleotides.

Polyethyleneimine (PEI) is well-known as a non-viral gene delivery vector, especially for oligonucleotide delivery. However, its clinical applications are significantly limited due to its high cationic charge, lack of specificity, and interaction with the proteins and nontarget cells in the biological fluids, resulting in high cytotoxicity, poor stability and low transfection efficiency for oligonucleotides transporting. It has been shown that the molecular weight (MW) of PEI, degree of branching, N/P ratio, buffer capacity, oligonucleotide structure, culture medium pH, serum, presence or absence of and method of preparation make a significant difference in the cytoxicity, stability, and transfection efficiency for the PEI-based oligonucleotides delivery systems. Ligands, hydrophobic, hydrophilic, and amphiphilic modification of PEI have been investigated to reduce the cytoxicity and improve the stability, the transfection efficiency, and therapeutic effect. Moreover, various intelligent modifications of PEI, such as pH-responsive (hydrazone bond) and redox sensitive linkers (disulfide bond) can control oligonucleotides release and have attracted much attention. In general, more efficient oligonucleotide delivery can be achieved by the introduction of modifications to PEI and by optimization of parameters of PEI or PEI-based formulations.

Cationic Oligospermine-Oligonucleotide Conjugates Provide Carrier-free Splice Switching in Monolayer Cells and Spheroids.

We report the evaluation of 18-mer 2'-O-methyl-modified ribose oligonucleotides with a full-length phosphorothioate backbone chemically conjugated at the 5' end to the oligospermine units (Sn-: n = 5, 15, 20, 25, and 30 [number of spermine units]) as splice switching oligonucleotides (SSOs). These conjugates contain, in their structure, covalently linked oligocation moieties, making them capable of penetrating cells without transfection vector. In cell culture, we observed efficient cytoplasmic and nuclear delivery of fluorescein-labeled S20-SSO by fluorescent microscopy. The SSO conjugates containing more than 15 spermine units induced significant carrier-free exon skipping at nanomolar concentration in the absence and in the presence of serum. With an increasing number of spermine units, the conjugates became slightly toxic but more active. Advantages of these molecules were particularly demonstrated in three-dimensional (3D) cell culture (multicellular tumor spheroids [MCTSs]) that mimics living tissues. Whereas vector-complexed SSOs displayed a drastically reduced splice switching in MCTS compared with the assay in monolayer culture, an efficient exon skipping without significant toxicity was observed with oligospermine-grafted SSOs (S15- and S20-SSOs) transfected without vector. It was shown, by flow cytometry and confocal microscopy, that the fluorescein-labeled S20-SSO was freely diffusing and penetrating the innermost cells of MCTS, whereas the vector-complexed SSO penetrated only the cells of the spheroid's outer layer.