Pharmacokinetics and Proceedings in Clinical Application of Nucleic Acid Therapeutics.

Oligonucleotide therapeutics are a novel promising class of drugs designed to specifically target either coding or non-coding RNA molecules to revolutionize treatment of various diseases. During preclinical development, investigations of the pharmacokinetic characteristics of these oligonucleotide-based drug candidates are essential. Oligonucleotides possess a long history of chemical modifications to enhance their stability and binding affinity, as well as reducing toxicity. Phosphorothioate backbone modifications of oligonucleotides were a hallmark of this development process that greatly enhanced plasma stability and protein binding of these agents. Modifications such as 2'-O-methylation further improved stability, while other modifications of the ribose, such as locked nucleic acid (LNA) modification, significantly increased binding affinity, potency, and tissue half-life. These attributes render oligonucleotide therapeutics able to regulate protein expression in both directions depending on the target RNA. Thus, a growing interest has emerged using these oligonucleotides in the treatment of neurodegenerative and cardiac disorders as well as cancer, since the deregulation of certain coding and non-coding RNAs plays a key role in the development of these diseases. Cutting edge research is being performed in the field of non-coding RNAs, identifying potential therapeutic targets, and developing novel oligonucleotide-based agents that outperform classical drugs. Some of these agents are either in clinical trials showing promising results or are already US Food and Drug Administration (FDA) approved, with more oligonucleotides being developed for therapeutic purposes. This is the advent of mechanism-based next-generation therapeutics for a wide range of diseases.

A cationic amphiphilic co-polymer as a carrier of nucleic acid nanoparticles (Nanps) for controlled gene silencing, immunostimulation, and biodistribution.

Programmable nucleic acid nanoparticles (NANPs) provide controlled coordination of therapeutic nucleic acids (TNAs) and other biological functionalities. Beyond multivalence, recent reports demonstrate that NANP technology can also elicit a specific immune response, adding another layer of customizability to this innovative approach. While the delivery of nucleic acids remains a challenge, new carriers are introduced and tested continuously. Polymeric platforms have proven to be efficient in shielding nucleic acid cargos from nuclease degradation while promoting their delivery and intracellular release. Here, we venture beyond the delivery of conventional TNAs and combine the stable cationic poly-(lactide-co-glycolide)-graft-polyethylenimine with functionalized NANPs. Furthermore, we compare several representative NANPs to assess how their overall structures influence their delivery with the same carrier. An extensive study of various formulations both in vitro and in vivo reveals differences in their immunostimulatory activity, gene silencing efficiency, and biodistribution, with fibrous NANPs advancing for TNA delivery.

Endosomal Escape and Cytosolic Penetration of Macromolecules Mediated by Synthetic Delivery Agents.

Cell delivery reagents often exploit the endocytic pathway as a route of cell entry. Once endocytosed, these reagents must overcome endosomal entrapment to ensure the release of their macromolecular cargo into the cytosol of cells. In this review, we describe several examples of prototypical synthetic reagents that are capable of endosomal escape and examine their mechanisms of action, their efficiencies, and their effects on cells. Although these delivery systems are chemically distinct, some commonalities in how they interact with cellular membranes can be inferred. This, in turn, sheds some light on the process of endosomal escape, and may help guide the development and optimization of next-generation delivery tools.

A Direct Comparison of in Vitro and in Vivo Nucleic Acid Delivery Mediated by Hundreds of Nanoparticles Reveals a Weak Correlation.

Endothelial cells and macrophages play active roles in disease and as a result are important targets for nucleic acid therapies. While thousands of chemically distinct lipid nanoparticles (LNPs) can be synthesized to deliver nucleic acids, studying more than a few LNPs in vivo is challenging. As a result, it is difficult to understand how nanoparticles target these cells in vivo. Using high throughput LNP barcoding, we quantified how well LNPs delivered DNA barcodes to endothelial cells and macrophages in vitro, as well as endothelial cells and macrophages isolated from the lung, heart, and bone marrow in vivo. We focused on two fundamental questions in drug delivery. First, does in vitro LNP delivery predict in vivo LNP delivery? By comparing how 281 LNPs delivered barcodes to endothelial cells and macrophages in vitro and in vivo, we found in vitro delivery did not predict in vivo delivery. Second, does LNP delivery change within the microenvironment of a tissue? We quantified how 85 LNPs delivered barcodes to eight splenic cell populations, and found that cell types derived from myeloid progenitors tended to be targeted by similar LNPs, relative to cell types derived from lymphoid progenitors. These data demonstrate that barcoded LNPs can elucidate fundamental questions about in vivo nanoparticle delivery.

Modifying a Commonly Expressed Endocytic Receptor Retargets Nanoparticles in Vivo.

Nanoparticles are often targeted to receptors expressed on specific cells, but few receptors are (i) highly expressed on one cell type and (ii) involved in endocytosis. One unexplored alternative is manipulating an endocytic gene expressed on multiple cell types; an ideal gene would inhibit delivery to cell type A more than cell type B, promoting delivery to cell type B. This would require a commonly expressed endocytic gene to alter nanoparticle delivery in a cell type-dependent manner in vivo; whether this can occur is unknown. Based on its microenvironmental regulation, we hypothesized Caveolin 1 (Cav1) would exert cell type-specific effects on nanoparticle delivery. Fluorescence was not sensitive enough to investigate this question, and as a result, we designed a platform named QUANT to study nanoparticle biodistribution. QUANT is 108× more sensitive than fluorescence and can be multiplexed. By measuring how 226 lipid nanoparticles (LNPs) delivered nucleic acids to multiple cell types in vivo in wild-type and Cav1 knockout mice, we found Cav1 altered delivery in a cell-type specific manner. Cav1 knockout did not alter LNP delivery to lung and kidney macrophages but substantially reduced LNP delivery to Kupffer cells, which are liver-resident macrophages. These data suggest caveolin-mediated endocytosis of nanomedicines by macrophages varies with tissue type. These results suggest manipulating receptors expressed on multiple cell types can tune drug delivery.

Methods for Evaluating the Stimuli-Responsive Delivery of Nucleic Acid and Gene Medicines.

In this review, we have summarized evaluation methods for the analysis of external stimuli-mediated nucleic acid and gene delivery. Prior to reviewing these evaluation methods, we describe various delivery processes of nucleic acid and gene medicines (small interfering RNA (siRNA), micro RNA, mRNA, plasmid DNA, etc.), which include interaction with blood components, bio-distribution, disposition in the target tissue, cell entry, intracellular trafficking, nuclear localization, and dissociation from the carriers. Next, we discuss the advantages of external stimuli-mediated nucleic acid and gene delivery. External stimuli enable us to effectively deliver nucleic acids and genes to targeted regions. Evaluation methods are required to elucidate the behaviors of nucleic acid and gene medicines in the body. Quantitative analyses of the bio-distribution and in situ disposition in perfused organs, as well as visualization of bio-distribution, transgene expression in the body, and intracellular trafficking of nucleic acid and gene medicines, are all useful in evaluating not only the efficacy and safety of delivery, but also serve as guidelines for the further development of nucleic acid and gene medicines by elucidating delivery problems. Progress in evaluation methods, including tissue optical clearing and super resolution microscopy, will help to better elucidate the in vivo fate of nucleic acid and gene medicines.

Mechanism for the endocytosis of spherical nucleic acid nanoparticle conjugates.

Intracellular delivery of nucleic acids as gene regulation agents typically requires the use of cationic carriers or viral vectors, yet issues related to cellular toxicity or immune responses hamper their attractiveness as therapeutic candidates. The discovery that spherical nucleic acids (SNAs), polyanionic structures comprised of densely packed, highly oriented oligonucleotides covalently attached to the surface of nanoparticles, can effectively enter more than 50 different cell types presents a potential strategy for overcoming the limitations of conventional transfection agents. Unfortunately, little is known about the mechanism of endocytosis of SNAs, including the pathway of entry and specific proteins involved. Here, we demonstrate that the rapid cellular uptake kinetics and intracellular transport of SNAs stem from the arrangement of oligonucleotides into a 3D architecture, which supports their targeting of class A scavenger receptors and endocytosis via a lipid-raft-dependent, caveolae-mediated pathway. These results reinforce the notion that SNAs can serve as therapeutic payloads and targeting structures to engage biological pathways not readily accessible with linear oligonucleotides.

The Sequence-Specific Cellular Uptake of Spherical Nucleic Acid Nanoparticle Conjugates.

The sequence-dependent cellular uptake of spherical nucleic acid nanoparticle conjugates (SNAs) is investigated. This process occurs by interaction with class A scavenger receptors (SR-A) and caveolae-mediated endocytosis. It is known that linear poly(guanine) (poly G) is a natural ligand for SR-A, and it has been proposed that interaction of poly G with SR-A is dependent on the formation of G-quadruplexes. Since G-rich oligonucleotides are known to interact strongly with SR-A, it is hypothesized that SNAs with higher G contents would be able to enter cells in larger amounts than SNAs composed of other nucleotides, and as such, cellular internalization of SNAs is measured as a function of constituent oligonucleotide sequence. Indeed, SNAs with enriched G content show the highest cellular uptake. Using this hypothesis, a small molecule (camptothecin) is chemically conjugated with SNAs to create drug-SNA conjugates and it is observed that poly G SNAs deliver the most camptothecin to cells and have the highest cytotoxicity in cancer cells. Our data elucidate important design considerations for enhancing the intracellular delivery of spherical nucleic acids.

[Existing problems and strategies in liposome-mediated nucleic acid delivery].

Liposome-mediated nucleic acid delivery has been a focus recently, but in the course of delivering nucleic acid, some hurdles seriously limit the nucleic acid exerting treatment effect. This review refers to a series of problems such as low blood stability, reticuloendothelial system absorption, the lower targeting of liposome and the restriction of endosomal escape which are suffered in liposome-mediated nucleic acid delivery; and gives a detail introduction of strategies such as PEGylation, ligand modification, photochemical internalization, the application of degradation liposome and membrane-lytic peptide, to overcome those problems.

Epidermal SR-A Complexes Are Lipid Raft Based and Promote Nucleic Acid Nanoparticle Uptake.

Scavenger receptors clear pathogens, transport lipid, and mediate polyanionic ligand uptake in macrophages, but their expression and role in the skin are poorly understood. Although the epidermal barrier typically excludes nucleic acid entry, topically applied, spherically arranged oligonucleotide nanoconjugates (spherical nucleic acids [SNAs]) penetrate mouse skin, three-dimensional (3D) skin equivalents, and human skin. We explored the mechanism of SNA uptake in normal human epidermal keratinocytes and 3D skin equivalents. Normal human epidermal keratinocytes and 3D raft treatment with SR-A inhibitors reduced SNA uptake by >80%. The human epidermis expresses SR-As SCARA3 and, to a lesser extent, MARCO. Simultaneous lentiviral knockdown of SCARA3 and MARCO reduced SNA uptake in normal human epidermal keratinocytes and 3D rafts after topical application, affirming a role for SR-As in SNA uptake and 3D raft penetration. Incubation of normal human epidermal keratinocytes at 4oC or with sodium azide prevented SNA uptake, suggesting active endocytosis. Endocytosis inhibitors, immunofluorescence, immunoprecipitation, and knockdown studies localized functional SR-As to FLOT-1-containing lipid rafts throughout the epidermis and CAV-1-containing rafts only in the upper epidermis. These studies suggest a central role for SR-A complexes in epidermal lipid rafts in mediating the uptake of nucleic acid‒laden nanoparticles.

Cationic dynamic covalent polymers for gene transfection.

Dynamic covalent polymers are materials formed by reversible covalent bonds and non-covalent interactions through an adaptive constitutional dynamic chemistry. The implementation of dynamic covalent polymers in gene delivery has recently emerged due to their responsive and adaptive features. Indeed, such an approach offers the alluring promise of discovering optimal delivery vectors self-fitted to their nucleic acid cargos and responsive to environmental changes (e.g. pH changes or the presence of a biomolecular target). This review will discuss more precisely the structural features of the molecular building blocks used so far, the architecture of the resulting dynamic covalent polymers from linear to 2D and 3D, and the covalent and supramolecular self-assembly processes at play in nucleic acid recognition and delivery, showcasing in particular the very few examples of adaptive self-assembly of dynamic covalent polymers templated by nucleic acids and responsive to the presence of biomolecular targets found in cell membranes that facilitate cell entry.

Nanomedicines for the delivery of glucocorticoids and nucleic acids as potential alternatives in the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that affects 0.5-1% of the world population. Current treatments include on one hand non-steroidal anti-inflammatory drugs and glucocorticoids (GCs) for treating pain and on the other hand disease-modifying anti-rheumatic drugs such as methotrexate, Janus kinase inhibitors or biologics such as antibodies targeting mainly cytokine expression. More recently, nucleic acids such as siRNA, miRNA, or anti-miRNA have shown strong potentialities for the treatment of RA. This review discusses the way nanomedicines can target GCs and nucleic acids to inflammatory sites, increase drug penetration within inflammatory cells, achieve better subcellular distribution and finally protect drugs against degradation. For GCs such a targeting effect would allow the treatment to be more effective at lower doses and to reduce the administration frequency as well as to induce much fewer side-effects. In the case of nucleic acids, particularly siRNA, knocking down proteins involved in RA, could importantly be facilitated using nanomedicines. Finally, the combination of both siRNA and GCs in the same carrier allowed for the same cell to target both the GCs receptor as well as any other signaling pathway involved in RA. Nanomedicines appear to be very promising for the delivery of conventional and novel drugs in RA therapeutics. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures.

Cancer-Targeting Nanoparticles for Combinatorial Nucleic Acid Delivery.

Nucleic acids are a promising type of therapeutic for the treatment of a wide range of conditions, including cancer, but they also pose many delivery challenges. For efficient and safe delivery to cancer cells, nucleic acids must generally be packaged into a vehicle, such as a nanoparticle, that will allow them to be taken up by the target cells and then released in the appropriate cellular compartment to function. As with other types of therapeutics, delivery vehicles for nucleic acids must also be designed to avoid unwanted side effects; thus, the ability of such carriers to target their cargo to cancer cells is crucial. Classes of nucleic acids, hurdles that must be overcome for effective intracellular delivery, types of nonviral nanomaterials used as delivery vehicles, and the different strategies that can be employed to target nucleic acid delivery specifically to tumor cells are discussed. Additonally, nanoparticle designs that facilitate multiplexed delivery of combinations of nucleic acids are reviewed.

A self-assembled, modular nucleic acid-based nanoscaffold for multivalent theranostic medicine.

Rationale: Within the field of personalized medicine there is an increasing focus on designing flexible, multifunctional drug delivery systems that combine high efficacy with minimal side effects, by tailoring treatment to the individual. Methods: We synthesized a chemically stabilized ~4 nm nucleic acid nanoscaffold, and characterized its assembly, stability and functional properties in vitro and in vivo. We tested its flexibility towards multifunctionalization by conjugating various biomolecules to the four modules of the system. The pharmacokinetics, targeting capability and bioimaging properties of the structure were investigated in mice. The role of avidity in targeted liver cell internalization was investigated by flow cytometry, confocal microscopy and in vivo by fluorescent scanning of the blood and organs of the animals. Results: We have developed a nanoscaffold that rapidly and with high efficiency can self-assemble four chemically conjugated functionalities into a stable, in vivo-applicable system with complete control of stoichiometry and site specificity. The circulation time of the nanoscaffold could be tuned by functionalization with various numbers of polyethylene glycol polymers or with albumin-binding fatty acids. Highly effective hepatocyte-specific internalization was achieved with increasing valencies of tri-antennary galactosamine (triGalNAc) in vitro and in vivo. Conclusion: With its facile functionalization, stoichiometric control, small size and high serum- and thermostability, the nanoscaffold presented here constitutes a novel and flexible platform technology for theranostics.

Framework nucleic acids as programmable carrier for transdermal drug delivery.

DNA nanostructures are promising drug carriers with their intrinsic biocompatibility, uniformity and versatility. However, rapid serum disintegration leads to low bioavailability at targeted sites following systemic administration, hindering their biomedical applications. Here we demonstrate transdermal delivery of framework nucleic acids (FNAs) through topical applications. By designing FNAs with distinct shapes and sizes, we interrogate their penetration on mice and human skin explant. Skin histology reveals size-dependent penetration, with FNAs ≤75 nm effectively reaching dermis layer. 17 nm-tetrahedral FNAs show greatest penetration to 350 µm from skin periphery. Importantly, structural integrity is maintained during the skin penetration. Employing a mouse melanoma model, topical application of doxorubicin-loaded FNAs accommodates ≥2-fold improvement in drug accumulation and tumor inhibition relative to topically-applied free doxorubicin, or doxorubicin loaded in liposomes and polymeric nanoparticles. Programmable penetration with minimal systemic biodistribution underlines FNA potential as localized transdermal drug delivery carriers.

Delivery of proteins and nucleic acids using a non-covalent peptide-based strategy.

The recent discovery of new potent therapeutic molecules which do not reach the clinic due to poor delivery and low bioavailability have made of delivery a key stone in therapeutic development. Several technologies have been designed to improve cellular uptake of therapeutic molecules, including cell-penetrating peptides (CPPs), which have been successfully applied for in vivo delivery of biomolecules and constitute very promising tools. Distinct families of CPPs have been described; some require chemical linkage between the drug and the carrier for cellular drug internalization while others like Pep-and MPG-families, form stable complexes with drugs depending on their chemical nature. Pep and MPG are short amphipathic peptides, which form stable nanoparticles with proteins and nucleic acids respectively. MPG and Pep based nanoparticles enter cells independently of the endosomal pathway and efficiently deliver cargoes in a fully biologically active form into a large variety of cell lines as well as in animal models. This review will focus on the mechanisms of non-covalent MPG and Pep-1 strategies and their applications in cultured cells and animal models.

Nucleic acid beacons for long-term real-time intracellular monitoring.

Real time intracellular monitoring of biological molecules inside living cells is important in many biomedical studies and reveals valuable information unobtainable by conventional molecular biology techniques. A variety of methods and molecular probes have been developed, but long term (from a few hours to days) intracellular monitoring with high sensitivity and selectivity is impossible and has not been accomplished. We have used locked nucleic acids (LNA) to engineer novel molecular beacons (MBs) for long-term intracellular monitoring. The LNA-MBs were made of a mixed LNA and DNA bases to have extremely high biostability. The new beacons were tested with MDA-MB-231 cancer cells and used effectively to monitor mRNA expression levels in real-time for 5-24 h. After 24 h inside living cells, the LNA-MBs were still functional, demonstrating a greatly enhanced stability enabling the measurement of intracellular gene expression over an extended period of time.

Establishment and validation of an ex vivo human cervical tissue model for local delivery studies.

The objective of this study was to establish and validate an ex vivo human cervical tissue model appropriate for transport studies of molecular and especially nucleic acid based drugs. For that purpose conditions had to be established for a standardized tissue handling and preparation following hysterectomy to allow an immediate experimental use of fresh tissue samples. Samples of the ectocervical, endocervical and the transition zone representing the entire cervix organ were characterized in Franz diffusion cells by the determination of the in vitro permeation of low and high molecular weight markers (propanolol, mannitol, dextran 4000, 10,000, 20,000 and 40,000Da). Additionally, the permeability of mannitol and dextran 4000 across fresh and frozen cervical tissue was compared. The apparent permeability coefficients (P(app)) of the various markers demonstrated (i) that with increasing molecular weight the marker permeability decreases, (ii) an upper permeability limit between 10,000 and 20,000Da, (iii) no significant difference of the permeability across the three cervical tissue zones, (iv) a statistically significant but effectively small variation of the permeability among different patient samples. A continuous difference of approximately two log values between the P(app) values of mannitol and dextran 4000 makes them suitable as an internal marker control pair for each biopsy. Moreover, the P(app) values of both markers across fresh and frozen tissue are comparable. According to the presented data we conclude that the human cervical tissue model has been well characterized and is therefore suitable for local delivery and permeation studies.

Oral nucleic acid therapy using multicompartmental delivery systems.

Nucleic acid-based therapeutics has the potential for treating numerous diseases by correcting abnormal expression of specific genes. Lack of safe and efficacious delivery strategies poses a major obstacle limiting clinical advancement of nucleic acid therapeutics. Oral route of drug administration has greater delivery challenges, because the administered genes or oligonucleotides have to bypass degrading environment of the gastrointestinal (GI) tract in addition to overcoming other cellular barriers preventing nucleic acid delivery. For efficient oral nucleic acid delivery, vector should be such that it can protect encapsulated material during transit through the GI tract, facilitate efficient uptake and intracellular trafficking at desired target sites, along with being safe and well tolerated. In this review, we have discussed multicompartmental systems for overcoming extracellular and intracellular barriers to oral delivery of nucleic acids. A nanoparticles-in-microsphere oral system-based multicompartmental system was developed and tested for in vivo gene and small interfering RNA delivery for treating colitis in mice. This system has shown efficient transgene expression or gene silencing when delivered orally along with favorable downstream anti-inflammatory effects, when tested in a mouse model of intestinal bowel disease. WIREs Nanomed Nanobiotechnol 2018, 10:e1478. doi: 10.1002/wnan.1478 This article is categorized under: Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Emerging Technologies.

[Development of a Novel Liposomal DDS by Manipulating Pharmacokinetics and Intracellular Trafficking for Drug Therapy and Nucleic Acid Medicine].

　Nucleic acid therapy is expected to be a next generation medicine. We recently developed a multifunctional envelope-type nano device (MEND) for use as a novel delivery system. The modification of polyethylene glycol (PEG), i.e., PEGylation, is useful for achieving the delivery of MENDs to tumors via an enhanced permeability and retention (EPR) effect. However, PEGylation strongly inhibits the cellular uptake and endosomal escape of MEND, which results in significant loss of action, and therefore lost effectiveness, of the cargo therapeutic. For successful nucleic acid delivery in cancer treatment, the crucial problem associated with the use of PEG, known as the "PEG dilemma", must be solved. In this review, we describe the development and application of MEND in overcoming the PEG dilemma based on manipulating both the pharmacokinetics and intracellular trafficking of cellular uptake and endosomal release using a cleavable PEG lipid, a pH-sensitive fusogenic peptide, and a pH-sensitive cationic lipid. We also developed dual-ligand liposomes with a controlled diameter of around 300 nm, then modified these with a specific ligand and a cell penetrating peptide designed to target the neovasculature of tumors. Dual-ligand liposomes could induce an anti-tumor effect in drug resistant tumors by delivering drugs to tumor blood vessels, rather than to the cancer cells themselves. Here, we review our recent efforts to develop a novel liposomal drug delivery system (DDS) by manipulating pharmacokinetics and intracellular trafficking for drug therapy and nucleic acid medicine.