Carriers For Delivery Of Nucleic Acids Research Articles

Efficient in vitro and in vivo transfection of self-amplifying mRNA with linear poly(propylenimine) and poly(ethylenimine-propylenimine) random copolymers as non-viral carriers.

Messenger RNA (mRNA) based vaccines have been introduced worldwide to combat the Covid-19 pandemic. These vaccines consist of non-amplifying mRNA formulated in lipid nanoparticles (LNPs). Consequently, LNPs are considered benchmark non-viral carriers for nucleic acid delivery. However, the formulation and manufacturing of these mRNA-LNP nanoparticles are expensive and time-consuming. Therefore, we used self-amplifying mRNA (saRNA) and synthesized novel polymers as alternative non-viral carrier platform to LNPs, which enable a simple, rapid, one-pot formulation of saRNA-polyplexes. Our novel polymer-based carrier platform consists of randomly concatenated ethylenimine and propylenimine comonomers, resulting in linear, poly(ethylenimine-ran-propylenimine) (L-PEIx-ran-PPIy) copolymers with controllable degrees of polymerization. Here we demonstrate in multiple cell lines, that our saRNA-polyplexes show comparable to higher in vitro saRNA transfection efficiencies and higher cell viabilities compared to formulations with Lipofectamine MessengerMAX™ (LFMM), a commercial, lipid-based carrier considered to be the in vitro gold standard carrier. This is especially true for our in vitro best performing saRNA-polyplexes with N/P 5, which are characterised with a size below 100 nm, a positive zeta potential, a near 100% encapsulation efficiency, a high retention capacity and the ability to protect the saRNA from degradation mediated by RNase A. Furthermore, an ex vivo hemolysis assay with pig red blood cells demonstrated that the saRNA-polyplexes exhibit negligible hemolytic activity. Finally, a bioluminescence-based in vivo study was performed over a 35-day period, and showed that the polymers result in a higher and prolonged bioluminescent signal compared to naked saRNA and L-PEI based polyplexes. Moreover, the polymers show different expression profiles compared to those of LNPs, with one of our new polymers (L-PPI250) demonstrating a higher sustained expression for at least 35 days after injection.

Development of Tris‐(triazolyl)‐s–triazine Linker‐Based Cationic Amphiphiles: Stability and Gene Delivery Applications

AbstractThe ability to readily be designed, manufactured, and described has made cationic lipids one of the highly adaptable vehicles for the transfer of DNA, RNA, and numerous additional therapeutic compounds. In this article, we synthesized a variety of Tris‐(triazolyl)‐s‐triazine linker‐based cationic lipids with different types of anchoring groups, and assessed the transfection efficacy. Specifically, two identical tails like either two alpha‐tocopherol domains or cholesterol domains or aliphatic chains (saturated), and on the other hand, two different tails, alpha‐tocopherol domain and cholesterol domain or alpha‐tocopherol domain and aliphatic chain (saturated) were employed as hydrophobic motifs to synthesize the cationic lipids. The stability, size, and surface charge of liposomes were performed by dynamic light scattering technique. Cytotoxicity study on cell survival showed that the synthesized lipids are less hazardous to the cells despite the fact that cationic lipids have large molecular structure. The lipid having di alpha‐tocopherol domains demonstrated phenomenal β‐galactosidase protein expression and enhanced green fluorescence expression in the hepatocellular carcinoma cell line. Findings from the physico‐chemical and biological evaluations the developed lipids are compatible in various forms while maintaining low cytotoxicity and efficient carriers for nucleic acid delivery.

Control of cell penetration enhancer shielding and endosomal escape-kinetics crucial for efficient and biocompatible siRNA delivery

Although cationic liposomes are efficient carriers for nucleic acid delivery, their toxicity often hampers the clinical translation. Polyethylene glycol (PEG) coating has been largely used to improve their stability and reduce toxicity. Nevertheless, it has been found to decrease the transfection process. In order to exploit the advantages of cationic liposomes and PEG decoration for nucleic acid delivery, liposomes decorated with tetraArg-[G-1]-distearoyl glycerol (Arg4-DAG) dendronic oligo-cationic lipid enhancer (OCE) and PEG-lipid have been investigated. Non decorated or OCE-decorated lipoplexes (OCEfree-LPX and OCE-LPX, respectively) were obtained by lipid film hydration using oligonucleotide (ON) solutions. PEG and OCE/PEG decorated lipoplexes (PEG-OCEfree-LPX and PEG-OCE-LPX, respectively) were obtained by post-insertion of 2 or 5 kDa PEG-DSPE on preformed lipoplexes. The OCE decoration yielded lipoplexes with size of about 240 nm, 84% loading efficiency at 10 N/P ratio, ten times higher than OCEfree-LPX, and prevented the ON release when incubated with physiological heparin concentration or with plasma. The PEG decoration reduced the zeta potential, enhanced the lipoplex stability in serum and decreased both hemolysis and cytotoxicity, while it did not affect the lipoplex size and ON loading. With respect to OCEfree-LPX, the OCE-LPX remarkably associated with cells and were taken up by different cancer cell lines (HeLa and MDA-MB-231). Interestingly, 2 or 5 kDa PEG decoration did not reduce either the cell interaction or the cell up-take of the cationic lipoplexes. With siRNA as a payload, OCE enabled efficient internalization, but endosomal release was hampered. Post-transfection treatment with the lysosomotropic drug chloroquine allowed to identify the optimal time point for endosomal escape. Chloroquine treatment after 12 to 20 h of LPX pre-incubation enabled siRNA mediated target knockdown indicating that this is the time window of endo-lysosomal processing. This indicates that OCE can protect siRNA from lysosomal degradation for up to 20 h, as shown by these rescue experiments.

Molecular Chameleon Carriers for Nucleic Acid Delivery: The Sweet Spot between Lipoplexes and Polyplexes.

Taking advantage of effective intracellular delivery mechanisms of both cationizable lipids and polymers, highly potent double pH-responsive nucleic acid carriers are generated by combining at least two lipo amino fatty acids (LAFs) as hydrophobic cationizable motifs with hydrophilic cationizable aminoethylene units into novel sequence-defined molecules. The pH-dependent tunable polarity of the LAF is successfully implemented by inserting a central tertiary amine, which disrupts the hydrophobic character once protonated, resulting in pH-dependent structural and physical changes. This "molecular chameleon character" turns out to be advantageous for dynamic nucleic acid delivery via lipopolyplexes. By screening different topologies (blocks, bundles, T-shapes, U-shapes), LAF types, and LAF/aminoethylene ratios, highly potent pDNA, mRNA, and siRNA carriers are identified, which are up to several 100-fold more efficient than previous carrier generations and characterized by very fast transfection kinetics. mRNA lipopolyplexes maintain high transfection activity in cell culture even in the presence of ≥90% serum at an ultra-low mRNA dose of 3 picogram (≈2 nanoparticles/cell), and thus are comparable in potency to viral nanoparticles. Importantly, they show great in vivo performance with high expression levels especially in spleen, tumor, lungs, and liver upon intravenous administration of 1-3µg luciferase-encoding mRNA in mice.

Engineering polyphenol-based carriers for nucleic acid delivery.

Gene therapy, an effective medical intervention strategy, is increasingly employed in basic research and clinical practice for promising and unique therapeutic effects for diseases treatment, such as cardiovascular disorders, cancer, neurological pathologies, infectious diseases, and wound healing. However, naked DNA/RNA is readily hydrolyzed by nucleic acid degrading enzymes in the extracellular environment and degraded by lysosomes during intracellular physiological conditions, thus gene transfer must cross complex cellular and tissue barriers to deliver genetic materials into targeted cells and drive efficient activation or inhibition of the proteins. At present, the lack of safe, highly efficient, and non-immunogenic drug carriers is the main drawback of gene therapy. Considering the dense hydroxyl groups on the benzene rings in natural polyphenols that exert a strong affinity to various nucleic acids via hydrogen bonding and hydrophobic interactions, polyphenol-based carriers are promising anchors for gene delivery in which polyphenols serve as the primary building blocks. In this review, the recent progress in polyphenol-assisted gene delivery was summarized, which provided an easily accessible reference for the design of future polyphenol-based gene delivery vectors. Nucleic acids discussed in this review include DNA, short interfering RNAs (siRNA), microRNA (miRNA), double-strand RNA (dsRNA), and messenger RNA (mRNA).

Lipidoid nanoparticles increase ATP uptake into hypoxic brain endothelial cells

Lipidoid nanoparticles (LNPs) are clinically successful carriers for nucleic acid delivery to liver and muscle targets. The ability of LNPs to load and deliver small molecule drugs has not been reported yet. We propose that the delivery of adenosine triphosphate (ATP) to brain endothelial cells (BECs) lining the blood–brain barrier may increase cellular energetics of the injured BECs. We formulated and studied the physicochemical characteristics of ATP-loaded LNPs using the C12-200 ionizable cationic lipid and other helper lipids. Polyethylene glycol-dimyristoyl glycerol (PEG-DMG), one of the helper lipids, played a crucial role in maintaining colloidal stability of LNPs over time whereas the inclusion of both ATP and PEG-DMG maintained the colloidal stability of LNPs in the presence of serum proteins. ATP-LNPs formulated with PEG-DMG resulted in a 7.7- and 6.6- fold increased uptake of ATP into normoxic and hypoxic BECs, respectively. Altogether, our results demonstrate the potential of LNPs as a novel carrier for the delivery of small molecular mass actives to BECs—a CNS target.

Rational design of engineered H-ferritin nanoparticles with improved siRNA delivery efficacy across an in vitro model of the mouse BBB.

Gene therapy holds tremendous potential for the treatment of incurable brain diseases including Alzheimer's disease (AD), stroke, glioma, and Parkinson's disease. The main challenge is the lack of effective gene delivery systems traversing the blood-brain barrier (BBB), due to the complex microvessels present in the brain which restrict substances from the circulating blood passing through. Recently, increasing efforts have been made to develop promising gene carriers for brain-related disease therapies. One such development is the self-assembled heavy chain ferritin (HFn) nanoparticles (NPs). HFn NPs have a unique hollow spherical structure that can encapsulate nucleic acid drugs (NADs) and specifically bind to cancer cells and BBB endothelial cells (BBB ECs) via interactions with the transferrin receptor 1 (TfR1) overexpressed on their surfaces, which increases uptake through the BBB. However, the gene-loading capacity of HFn is restricted by its limited interior volume and negatively charged inner surface; therefore, these drawbacks have prompted the demand for strategies to remould the structure of HFn. In this work, we analyzed the three-dimensional (3D) structure of HFn using Chimera software (v 1.14) and developed a class of internally cationic HFn variants (HFn+ NPs) through arginine mutation on the lumenal surface of HFn. These HFn+ NPs presented powerful electrostatic forces in their cavities, and exhibited higher gene encapsulation efficacy than naive HFn. The top-performing candidate, HFn2, effectively delivered siRNA to glioma cells after traversing the BBB and achieved the highest silencing efficacy among HFn+ NPs. Overall, our findings demonstrate that HFn+ NPs obtained by this genetic engineering method provide critical insights into the future development of nucleic acid delivery carriers with BBB-crossing ability.

Systematic Investigation of Biocompatible Cationic Polymeric Nucleic Acid Carriers for Immunotherapy of Hepatocellular Carcinoma.

Simple SummaryImmunotherapy, including adaptive and innate immunotherapy, exhibits promising future for the treatment of hepatocellular carcinoma. As a common tool for immunotherapy, the safe and efficient gene delivery turns to be especially important. Biocompatible polymers are a category of promising materials used in gene delivery, while there still lacks a comprehensive review article to discuss the updates on multiple disciplines covering biocompatible polymers, gene therapy, tumor immune microenvironment, and immunotherapy. This review is well-integrated with biocompatible polymers, nonviral gene therapy, and cancer immunotherapy. Our investigation will provide different perspective for the scientists focusing on the domains of biomaterials, gene therapy, and oncologists to move their research work forward.Hepatocellular carcinoma (HCC) is the third-largest cause of cancer death worldwide, while immunotherapy is rapidly being developed to fight HCC with great potential. Nucleic acid drugs are the most important modulators in HCC immunotherapy. To boost the efficacy of therapeutics and amplify the efficiency of genetic materials, biocompatible polymers are commonly used. However, under the strong need of a summary for current developments of biocompatible polymeric nucleic acid carriers for immunotherapy of HCC, there is rare review article specific to this topic to our best knowledge. In this article, we will discuss the current progress of immunotherapy for HCC, biocompatible cationic polymers (BCPs) as nucleic acid carriers used (or potential) to fight HCC, the roles of biocompatible polymeric carriers for nucleic acid delivery, and nucleic acid delivery by biocompatible polymers for immunotherapy. At the end, we will conclude the review and discuss future perspectives. This article discusses biocompatible polymeric nucleic acid carriers for immunotherapy of HCC from multidiscipline perspectives and provides a new insight in this domain. We believe this review will be interesting to polymer chemists, pharmacists, clinic doctors, and PhD students in related disciplines.

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.

Polysaccharide–Peptide Conjugates: A Versatile Material Platform for Biomedical Applications

AbstractNatural polymers, such as polysaccharides and proteins, have been widely studied for numerous biomedical applications because of their bioactivity, natural origin, and biodegradability. To address the different needs of a broad spectrum of biomedical applications, natural polymers are modified with or conjugated to small molecular compounds, synthetic and natural polymers, and inorganic nanomaterials and surfaces. Among these hybrid materials, polysaccharide–peptide conjugates have drawn much attention due to their design flexibility, biocompatibility, tunable degradability, and structure similarity to naturally occurring glycoproteins. In the past 20 years, polysaccharide–peptide conjugates have demonstrated the promising potential to address many long‐standing medical challenges. Thus, the design, conjugation chemistry and method, and biomedical applications of polysaccharide–peptide conjugates are summarized and discussed in this review. The conjugation techniques are reviewed from the view of the chemical reactions. Meanwhile, some inspiring examples of polysaccharide–peptide conjugates in biomedical applications, including drug delivery systems, nucleic acid delivery carriers, tissue engineering, and antimicrobial applications, are highlighted. Moreover, the outlook on the challenges and demands of polysaccharide–peptide conjugates is also elaborated.

The Multifaceted Histidine-Based Carriers for Nucleic Acid Delivery: Advances and Challenges.

Histidines incorporated into carriers of nucleic acids may enhance the extracellular stability of the nanoparticle, yet aid in the intracellular disruption of the nanoparticle, enabling the release of the nucleic acid. Moreover, protonation of histidines in the endosomes may result in endosomal swelling with subsequent lysis. These properties of histidine are based on its five-member imidazole ring in which the two nitrogen atoms may form hydrogen bonds or act as a base in acidic environments. A wide variety of carriers have integrated histidines or histidine-rich domains, which include peptides, polyethylenimine, polysaccharides, platform delivery systems, viral phages, mesoporous silica particles, and liposomes. Histidine-rich carriers have played key roles in our understanding of the stability of nanocarriers and the escape of the nucleic acids from endosomes. These carriers show great promise and offer marked potential in delivering plasmids, siRNA, and mRNA to their intracellular targets.

Degradable branched polycationic systems for nucleic acid delivery.

Nowadays, nucleic acid-based therapy has become a promising way for the treatment of various malignant diseases like cancers. However, the process of nucleic acid delivery is hampered by several extracellular and intracellular obstacles. To overcome these obstacles, many nucleic acid delivery carriers have been developed to achieve improved safety and enhanced gene transfection performance. Among these carriers, degradable branched polycations have attracted much attention due to their good responsive degradability and impressive transfection performances. In this review, we mainly summarized the redox- and pH-responsive degradable branched polycationic systems used for nucleic acid delivery. For responsive degradable branched polycationic systems, the preparation methods were introduced, where amino-epoxy ring-opening reaction as a novel step growth approach was mainly presented. The properties of redox- and pH-responsive degradable branched polycationic systems were subsequently introduced and highlighted. We hope this brief review will motivate the delicate design of responsive degradable branched polycationic systems for potential clinical applications. This article is categorized under: Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures.

Lipid-Modified Aminoglycosides for mRNA Delivery to the Liver.

Cationic lipid nanoparticles (LNPs) are widely used as carriers for delivery of nucleic acids. Most synthetic routes toward cationic lipids have derived from simple amine cores. Greater chemical diversity can be obtained through starting with natural products containing basic nitrogen atoms, which offers routes to more complex molecules. Natural building blocks are not extensively explored, such as aminoglycosides, which are both structurally and functionally interesting for developing new carriers for nucleic acid delivery. Herein, cationic lipid-modified aminoglycosides (CLAs) are explored as a family of vehicles for messenger RNA (mRNA) delivery. CLAs are synthesized from natural existing aminoglycosides coupling with alkyl epoxides and acrylates. The top hit (GT-EP10) is able to deliver Luc mRNA to C57BL/6 mice at a dose of 0.05 mg kg-1 to achieve a 107 average luminescence intensity in the liver. The Lox-Stop-Lox tdTomato mouse model is used to further demonstrate that this efficient mRNA delivery system can be potentially used for gene editing. Successful delivery of human erythropoietin mRNA shows that CLA-based LNPs have promising opportunities for delivery of therapeutic nucleic acids in the future.

Alkylation enhances biocompatibility and siRNA delivery efficiency of cationic curdlan nanoparticles

Cationic curdlan derivatives are a class of promising carriers for nucleic acid delivery including short interfering RNA (siRNA). While our previous studies demonstrated the siRNA delivery efficiency of aminated curdlan derivatives, the associated cytotoxicity issue remained unsolved. To investigate the effects of alkylation on the toxicity as well as the transfection efficiency, we conjugated short alkyl chains to 6-amino-6-deoxy-curdlan (6AC-100). The cytotoxicity of alkylated 6AC-100 derivatives (denote CuVa polymers) decreased with the increase of the degree of substitution (DS). CuVa3, with the highest DS, showed a 50% decreased cytotoxicity compared to 6AC-100 to 6AC-100 at a concentration of 140 μg/mL. The CuVa polymers readily complexed with siRNA to form nanoparticles, and induced significant knockdown of a disease related gene (STAT3) in mouse melanoma cell line B16. However, B16 cells transfected with siSTAT3 complexed to CuVa3 showed the highest phenotypic changes. These findings suggest that CuVa polymers have significantly enhanced biocompatibility and may be a promising delivery system for delivery of therapeutic siRNAs.

Polymeric Carriers for Nucleic Acid Delivery: Current Designs and Future Directions.

Within the last two decades, a series of novel therapeutic nucleic acids entered research and clinical evaluation. Their differences both in biophysical properties as well as in mode and site of biological action provide polymer-based carriers with new delivery challenges. Recent tailor-made designs of polymeric carriers are reviewed that were optimized for nucleic acid cargos such as plasmid DNA, siRNA, and micro RNA, mRNA, or genome-modifying nucleic acids. The specific requirements for the various therapeutic cargos are discussed. Future directions include dynamic bioresponsive polymers as components of nanomachines, multifunctional sequence-defined carriers for evolution-based selective optimization, and organic-inorganic multicomponent nanoassemblies.

Improved synthesis and characterization of cholesteryl oleate-loaded cationic solid lipid nanoparticles with high transfection efficiency for gene therapy applications

The development of new nanoparticle formulations that are capable of high transfection efficiency without toxicity is essential to provide new tools for gene therapy. However, the issues of complex, poorly reproducible manufacturing methods, and low efficiencies during in vivo testing have prevented translation to the clinic. We have previously reported the use of cholesteryl oleate as a novel excipient for solid lipid nanoparticles (SLNs) for the development of highly efficient and nontoxic nucleic acid delivery carriers. Here, we performed an extensive characterization of this novel formulation to make the scale up under Good Manufacturing Practice (GMP) possible. We also describe the complete physicochemical and biological characterization of cholesteryl oleate-loaded SLNs to ensure the reproducibility of this formula and the preservation of its characteristics before and after the lyophilization process. We defined the best manufacturing method and studied the influence of some parameters on the obtained nanoparticles using the Quality by Design (ICH Q8) guideline to obtain cholesteryl oleate-loaded SLNs that remain stable during storage and guarantee in vitro nucleic acid delivery efficacy. Our results indicate that this improved formulation is suitable for gene therapy with the possibility of scale-up the manufacturing of nanoparticles under GMP conditions.

Synthesis of Bioreducible Polycations with Controlled Topologies.

Bioreducible polycations, which possess disulfide linkages in the backbone, have emerged as promising nucleic acid delivery carriers due to their high stability in extracellular physiological condition and bioreduction-triggered release of the genetic material. Further benefits of bioreducible polycations include decreased cytotoxicity due to intracellular reducing environment in the cytoplasm that contains high levels of reducing molecules such as glutathione. Here, we describe the synthesis of bioreducible polycations with emphasis on methods to control their topology.

Efficient encapsulation and release of RNA molecules from gelatin-based nanoparticles

The design of synthetic carriers for nucleic acid delivery has become a research field of increasing interest. Studies on the delivery of DNA have brought up a variety of gene delivery vehicles. Recent studies in our group have demonstrated the preparation of new gelatin-based nanoparticles for the sustainable intracellular DNA delivery. Furthermore, the more recently emerged strategy by the intracellular delivery of RNA takes benefit from existing expertise in DNA transfer. In this work, the preparation and physicochemical characterization of new nucleic acid-based particles for the sustainable RNA delivery have been demonstrated. Gelatin (either high or low gel strength) and protamine sulfate have been selected to form particles by interaction of oppositely charged compounds. Particles in the absence of RNA (binary system) and in the presence of RNA (ternary system) have been prepared. The physicochemical characterization (particle size, polydispersity index, degree of RNA entrapment and RNA binding efficiency) has been evaluated as a function of the nature of the RNA derivative (acid form, diethylaminoethanol salt or core form) from torula yeast. The pH-dependent response of nanoparticles co-incubated in buffers at defined pHs that mimic late endo-lysosomal environment has demonstrated that the nanoparticles tend to destabilize and RNA can be successfully released as a consequence of changes in the intracellular pHs. Among the different systems, gelatin B (RNA)-PS nanoparticles using RNA acid form from torula yeast has proved to be the best system, from an effective and economic point of view.

Smart polymeric nanocarriers for small nucleic acid delivery.

In recent decades, nucleic acid therapeutics have been attracting much attention because of their potential as treatments for various diseases that require high target specificity. Nevertheless, the number of approved nucleic acid drugs is very limited due to substantially poor bioavailability, thereby generating the need for nucleic acid delivery carriers. A variety of delivery formulations have been reported so far. This review particularly describes polymer-based delivery carriers for systemic administration, highlighting the potential of biological environment-responsive chemistry for overcoming the biological barriers to nucleic acid delivery.