bims-novged 2021-09-12 papers

on Non-viral vectors for gene delivery

Issue of 2021–09–12
eleven papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Cationic Liposome Decorated with Cyclic RGD Peptide for Targeted Delivery of anti-STAT3 siRNA to Melanoma Cancer Cells.
Increasing Angiogenesis Factors in Hypoxic Diabetic Wound Conditions by siRNA Delivery: Additive Effect of LbL-Gold Nanocarriers and Desloratadine-Induced Lysosomal Escape.
Tuning the Immunostimulation Properties of Cationic Lipid Nanocarriers for Nucleic Acid Delivery.
A design approach for layer-by-layer surface-mediated siRNA delivery.
Functional Polyglycidol-Based Block Copolymers for DNA Complexation.
Self-assembled polymeric micelle as a novel mRNA delivery carrier.
Nanoparticles-assisted delivery of antiviral-siRNA as inhalable treatment for human respiratory viruses: A candidate approach against SARS-COV-2.
Macrophage reprogramming into a pro-healing phenotype by siRNA delivered with LBL assembled nanocomplexes for wound healing applications.
Delivery of Oligonucleotide Therapeutics: Chemical Modifications, Lipid Nanoparticles, and Extracellular Vesicles.
Calcium phosphate-polymeric nanoparticle system for co-delivery of microRNA-21 inhibitor and doxorubicin.
Polycation-Mediated Transfection: Mechanisms of Internalization and Intracellular Trafficking.

J Drug Target. 2021 Sep 06. 1-34

Cationic Liposome Decorated with Cyclic RGD Peptide for Targeted Delivery of anti-STAT3 siRNA to Melanoma Cancer Cells.

Ehsan Khabazian, Faezeh Vakhshiteh, Parisa Norouzi, Yousef Fatahi, Rassoul Dinarvand, Fatemeh Atyabi.

  Gene therapy is regarded as a valuable strategy for efficient cancer treatment. However, the design of effective delivery systems that can deliver gene materials such as siRNA specifically to the tumour tissues plays a pivotal role in cancer therapy. For this reason, a targeted cationic liposome for melanoma treatment was developed. This system consists of cyclic RGD peptide conjugated to DSPE-PEG2000, cholesterol, DOTAP, and DSPC as cationic and neutral lipids, respectively. Cyclic RGD was selected based on speculation that cyclic RGD would effectively transport anti-signal transducer and activator of transcription 3 (STAT3) siRNA into melanoma cell via integrin receptors. The prepared liposomes provided excellent stability against electrolyte and serum nucleases. Targeted liposomes remarkably exhibited higher cellular internalisation in comparison with the non-targeted system in flow cytometry and confocal microscopy. Furthermore, incorporating peptide on the surface of liposomes resulted in considerably high cytotoxicity, a 2.1-times raise in apoptosis induction, and a significantly enhanced STAT3 gene suppression as compared with the corresponding non-targeted formulation on B16F10 murine melanoma cells. Whole-body imaging confirmed the more significant tumour accumulation of targeted liposomes in B16F10 melanoma xenograft tumour-bearing mice. Consequently, c-RGD peptide modified liposome suggests a promising option for specific siRNA delivery into melanoma cells.

Keywords:  Gene delivery carrier; Melanoma; STAT3 Transcription Factor; c-RGD peptide; cationic liposome; siRNA

DOI:  https://doi.org/10.1080/1061186X.2021.1973481
Int J Mol Sci. 2021 Aug 26. pii: 9216. [Epub ahead of print]22(17):

Increasing Angiogenesis Factors in Hypoxic Diabetic Wound Conditions by siRNA Delivery: Additive Effect of LbL-Gold Nanocarriers and Desloratadine-Induced Lysosomal Escape.

Elnaz Shaabani, Maryam Sharifiaghdam, Joris Lammens, Herlinde De Keersmaecker, Chris Vervaet, Thomas De Beer, Elahe Motevaseli, Mohammad Hossein Ghahremani, Parvin Mansouri, Stefaan De Smedt, Koen Raemdonck, Reza Faridi-Majidi, Kevin Braeckmans, Juan C Fraire.

  Impaired wound healing in people with diabetes has multifactorial causes, with insufficient neovascularization being one of the most important. Hypoxia-inducible factor-1 (HIF-1) plays a central role in the hypoxia-induced response by activating angiogenesis factors. As its activity is under precise regulatory control of prolyl-hydroxylase domain 2 (PHD-2), downregulation of PHD-2 by small interfering RNA (siRNA) could stabilize HIF-1α and, therefore, upregulate the expression of pro-angiogenic factors as well. Intracellular delivery of siRNA can be achieved with nanocarriers that must fulfill several requirements, including high stability, low toxicity, and high transfection efficiency. Here, we designed and compared the performance of layer-by-layer self-assembled siRNA-loaded gold nanoparticles with two different outer layers-Chitosan (AuNP@CS) and Poly L-arginine (AuNP@PLA). Although both formulations have exactly the same core, we find that a PLA outer layer improves the endosomal escape of siRNA, and therefore, transfection efficiency, after endocytic uptake in NIH-3T3 cells. Furthermore, we found that endosomal escape of AuNP@PLA could be improved further when cells were additionally treated with desloratadine, thus outperforming commercial reagents such as Lipofectamine® and jetPRIME®. AuNP@PLA in combination with desloratadine was proven to induce PHD-2 silencing in fibroblasts, allowing upregulation of pro-angiogenic pathways. This finding in an in vitro context constitutes a first step towards improving diabetic wound healing with siRNA therapy.

Keywords:  angiogenesis; cationic amphiphilic drugs; diabetic wound healing; gene delivery; gold nanoparticles; hypoxia; layer-by-layer

DOI:  https://doi.org/10.3390/ijms22179216
Front Immunol. 2021 ;12 722411

Tuning the Immunostimulation Properties of Cationic Lipid Nanocarriers for Nucleic Acid Delivery.

Arindam K Dey, Adrien Nougarède, Flora Clément, Carole Fournier, Evelyne Jouvin-Marche, Marie Escudé, Dorothée Jary, Fabrice P Navarro, Patrice N Marche.

  Nonviral systems, such as lipid nanoparticles, have emerged as reliable methods to enable nucleic acid intracellular delivery. The use of cationic lipids in various formulations of lipid nanoparticles enables the formation of complexes with nucleic acid cargo and facilitates their uptake by target cells. However, due to their small size and highly charged nature, these nanocarrier systems can interact in vivo with antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages. As this might prove to be a safety concern for developing therapies based on lipid nanocarriers, we sought to understand how they could affect the physiology of APCs. In the present study, we investigate the cellular and metabolic response of primary macrophages or DCs exposed to the neutral or cationic variant of the same lipid nanoparticle formulation. We demonstrate that macrophages are the cells affected most significantly and that the cationic nanocarrier has a substantial impact on their physiology, depending on the positive surface charge. Our study provides a first model explaining the impact of charged lipid materials on immune cells and demonstrates that the primary adverse effects observed can be prevented by fine-tuning the load of nucleic acid cargo. Finally, we bring rationale to calibrate the nucleic acid load of cationic lipid nanocarriers depending on whether immunostimulation is desirable with the intended therapeutic application, for instance, gene delivery or messenger RNA vaccines.

Keywords:  antigen presenting cells; immunotoxicity assessment; nanostructured lipid carrier; nucleic acid delivery; surface charge (zeta potential)

DOI:  https://doi.org/10.3389/fimmu.2021.722411
Acta Biomater. 2021 Sep 01. pii: S1742-7061(21)00575-4. [Epub ahead of print]

A design approach for layer-by-layer surface-mediated siRNA delivery.

Jonathan J Chou, Adam G Berger, Sasan Jalili-Firoozinezhad, Paula T Hammond.

  The ability to coat scaffolds and wound dressings with therapeutic short interfering RNA (siRNA) holds much potential for applications in wound healing, cancer treatment, and regenerative medicine. Layer-by-layer (LbL) technology is an effective method to formulate polyelectrolyte thin films for local delivery of siRNA; however, the formation and efficacy of LbL coatings as drug delivery systems are highly contingent on the assembly conditions. Here, we investigate the effects of LbL assembly parameters on film composition and consequent siRNA-mediated gene knockdown efficiency in vitro. Films comprising poly(β-amino ester) (PBAE) and siRNA were built on polyglactin 910 (Vicryl) sutures consisting of poly(10% L-lactide, 90% glycolide). A fractional factorial design was employed, varying the following LbL assembly conditions: pH, ionic strength, PBAE concentration, and siRNA concentration. Effects of these parameters on PBAE loading, siRNA loading, their respective weight ratios, and in vitro siRNA-mediated knockdown were elucidated. The parameter effects were leveraged to create a rationally designed set of solution conditions that was predicted to give effective siRNA-mediated knockdown, but not included in any of the original experimental conditions. This level of knockdown with our rationally designed loading conditions (47%) is comparable to previous formulations from our lab while being simpler in construction and requiring fewer film layers, which could save time and cost in manufacturing. This study highlights the importance of LbL solution conditions in the preparation of surface-mediated siRNA delivery systems and presents an adaptable methodology for extending these electrostatically-assembled coatings to the delivery of other therapeutic nucleic acids. STATEMENT OF SIGNIFICANCE: : Short interfering RNA (siRNA) therapeutics are powerful tools to silence aberrant gene expression in the diseased state; however, the clinical utility of these therapies relies on effective controlled delivery approaches. Electrostatic self-assembly through the Layer-by-layer (LbL) process enables direct release from surfaces, but this method is highly dependent upon the specific solution conditions used. Here, we use a fractional factorial design to illustrate how these assembly conditions impact composition of siRNA-eluting LbL thin films. We then elucidate how these properties mediate in vitro transfection efficacy. Ultimately, this work presents a significant step towards understanding how optimization of assembly conditions for surface-mediated LbL delivery can promote transfection efficacy while reducing the processing and material required.

Keywords:  Layer-by-layer; PBAE; delivery; design of experiment; siRNA

DOI:  https://doi.org/10.1016/j.actbio.2021.08.042
Int J Mol Sci. 2021 Sep 04. pii: 9606. [Epub ahead of print]22(17):

Functional Polyglycidol-Based Block Copolymers for DNA Complexation.

Radostina Kalinova, Miroslava Valchanova, Ivaylo Dimitrov, Sevdalina Turmanova, Iva Ugrinova, Maria Petrova, Zlatina Vlahova, Stanislav Rangelov.

  Gene therapy is an attractive therapeutic method for the treatment of genetic disorders for which the efficient delivery of nucleic acids into a target cell is critical. The present study is aimed at evaluating the potential of copolymers based on linear polyglycidol to act as carriers of nucleic acids. Functional copolymers with linear polyglycidol as a non-ionic hydrophilic block and a second block bearing amine hydrochloride pendant groups were prepared using previously synthesized poly(allyl glycidyl ether)-b-polyglycidol block copolymers as precursors. The amine functionalities were introduced via highly efficient radical addition of 2-aminoethanethiol hydrochloride to the alkene side groups. The modified copolymers formed loose aggregates with strongly positive surface charge in aqueous media, stabilized by the presence of dodecyl residues at the end of the copolymer structures and the hydrogen-bonding interactions in polyglycidol segments. The copolymer aggregates were able to condense DNA into stable and compact nanosized polyplex particles through electrostatic interactions. The copolymers and the corresponding polyplexes showed low to moderate cytotoxicity on a panel of human cancer cell lines. The cell internalization evaluation demonstrated the capability of the polyplexes to successfully deliver DNA into the cancer cells.

Keywords:  DNA complexation; cationic copolymers; cell internalization; cytotoxicity; gene delivery; non-viral vectors; polyglycidol copolymers; polyplex formation

DOI:  https://doi.org/10.3390/ijms22179606
J Control Release. 2021 Sep 02. pii: S0168-3659(21)00475-2. [Epub ahead of print]338 537-547

Self-assembled polymeric micelle as a novel mRNA delivery carrier.

Jin Ren, Yiming Cao, Lei Li, Xin Wang, Haitao Lu, Jing Yang, Shengqi Wang.

  mRNA-based therapy has been evaluated in preclinical and clinical studies for the treatment of a wide variety of disease such as cancer immunotherapies and infectious disease vaccines. However, it remains challenging to development safe and efficient delivery system. Here, we have designed a novel self-assembled polymeric micelle based on vitamin E succinate modified polyethyleneimine copolymer (PVES) to delivery mRNA. In vitro, PVES could transfect mRNA into multiple cell lines such as HEK-293T, HeLa and Vero and the transfection efficiencies were much higher than PEI 25 k. In addition, the cytotoxicity of PVES was much lower than PEI 25 k. Furthermore, mice administered intramuscularly with PVES/SARS-CoV-2 mRNA vaccine induced potent antibody response and show no obvious toxicity. These results demonstrated the potential of PVES as a safe and effective delivery carrier for mRNA.

Keywords:  Immune response; Polymeric micelle; mRNA therapy

DOI:  https://doi.org/10.1016/j.jconrel.2021.08.061
Nano Sel. 2020 Oct 12.

Nanoparticles-assisted delivery of antiviral-siRNA as inhalable treatment for human respiratory viruses: A candidate approach against SARS-COV-2.

Ata Ullah, Javaria Qazi, Lutfur Rahman, Antonios G Kanaras, Waheed S Khan, Irshad Hussain, Asma Rehman.

  The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has challenged healthcare structures across the globe. Although a few therapies are approved by FDA, the search for better treatment options is continuously on rise. Clinical management includes infection prevention and supportive care such as supplemental oxygen and mechanical ventilatory support. Given the urgent nature of the pandemic and the number of companies and researchers developing COVID-19 related therapies, FDA has created an emergency program to move potential treatments with already approved drugs to patients as quickly as possible in parallel to the development of new drugs that must first pass the clinical trials. In this manuscript, we have reviewed the available literature on the use of sequence-specific degradation of viral genome using short-interfering RNA (siRNA) suggesting it as a possible treatment against SARS-CoV-2. Delivery of siRNA can be promoted by the use of FDA approved lipids, polymers or lipid-polymer hybrids. These nanoparticulate systems can be engineered to exhibit increased targetability and formulated as inhalable aerosols.

Keywords:  Corona pandemic; SARS‐CoV‐2; human respiratory viruses; lipid nanoparticles; multifunctional nanocarriers; siRNA delivery

DOI:  https://doi.org/10.1002/nano.202000125
Nanoscale. 2021 Sep 10.

Macrophage reprogramming into a pro-healing phenotype by siRNA delivered with LBL assembled nanocomplexes for wound healing applications.

Maryam Sharifiaghdam, Elnaz Shaabani, Zeynab Sharifiaghdam, Herlinde De Keersmaecker, Bart Lucas, Joris Lammens, Hossein Ghanbari, Ladan Teimoori-Toolabi, Chris Vervaet, Thomas De Beer, Reza Faridi-Majidi, Stefaan C De Smedt, Kevin Braeckmans, Juan C Fraire.

Excessive inflammatory responses in wounds are characterized by the presence of high levels of pro-inflammatory M1 macrophages rather than pro-healing M2 macrophages, which leads to delayed wound healing. Macrophage reprogramming from the M1 to M2 phenotype through knockdown of interferon regulatory factor 5 (irf5) has emerged as a possible therapeutic strategy. While downregulation of irf5 could be achieved by siRNA, it very much depends on successful intracellular delivery by suitable siRNA carriers. Here, we report on highly stable selenium-based layer-by-layer (LBL) nanocomplexes (NCs) for siRNA delivery with polyethyleneimine (PEI-LBL-NCs) as the final polymer layer. PEI-LBL-NCs showed good protection of siRNA with only 40% siRNA release in a buffer of pH = 8.5 after 72 h or in simulated wound fluid after 4 h. PEI-LBL-NCs also proved to be able to transfect RAW 264.7 cells with irf5-siRNA, resulting in successful reprogramming to the M2 phenotype as evidenced by a 3.4 and 2.6 times decrease in NOS-2 and TNF-α mRNA expression levels, respectively. Moreover, irf5-siRNA transfected cells exhibited a 2.5 times increase of the healing mediator Arg-1 and a 64% increase in expression of the M2 cell surface marker CD206+. Incubation of fibroblast cells with conditioned medium isolated from irf5-siRNA transfected RAW 264.7 cells resulted in accelerated wound healing in an in vitro scratch assay. These results show that irf5-siRNA loaded PEI-LBL-NCs are a promising therapeutic approach to tune macrophage polarization for improved wound healing.

DOI: https://doi.org/10.1039/d1nr03830c
ACS Nano. 2021 Sep 10.

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

Jeremy P Bost, Hanna Barriga, Margaret N Holme, Audrey Gallud, Marco Maugeri, Dhanu Gupta, Taavi Lehto, Hadi Valadi, Elin K Esbjörner, Molly M Stevens, Samir El-Andaloussi.

  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.

Keywords:  RNA therapeutics; cellular uptake; endosomal escape; extracellular vesicles; intracellular trafficking; lipid nanoparticles; oligonucleotide; oligonucleotide delivery

DOI:  https://doi.org/10.1021/acsnano.1c05099
Colloids Surf B Biointerfaces. 2021 Aug 24. pii: S0927-7765(21)00505-1. [Epub ahead of print]208 112061

Calcium phosphate-polymeric nanoparticle system for co-delivery of microRNA-21 inhibitor and doxorubicin.

Vishnu Sriram, Joo-Youp Lee.

  Targeted combination therapy has shown promise to achieve maximum therapeutic efficacy by overcoming drug resistance. MicroRNA-21 (miR-21) is frequently overexpressed in various cancer types including breast and non-small cell lung cancer and its functions can be inhibited by miR inhibitor (miR-21i). A combination of miR-21i and a chemo drug, doxorubicin (Dox), can provide synergistic effects. Here, we developed a calcium phosphate (CaP)-coated nanoparticle (NP) formulation to co-deliver miR-21i along with Dox. This NP design can be used to deliver the two agents with different physiochemical properties. The NP formulation was optimized for particle size, polydispersity, Dox loading, and miR-21i loading. The NP formulation was confirmed to downregulate miR-21 levels and upregulate tumor suppressor gene levels. The cytotoxic efficacy of the combined miR-21i and Dox-containing NPs was found to be higher than that of Dox. Therefore, the CaP-coated hybrid lipid-polymeric NPs hold potential for the delivery of miR-21i and Dox.

Keywords:  Calcium phosphate; Co-delivery; Combination therapy; Doxorubicin; Polymeric nanoparticles; microRNA-21 inhibitor

DOI:  https://doi.org/10.1016/j.colsurfb.2021.112061
Biomacromolecules. 2021 Sep 09.

Polycation-Mediated Transfection: Mechanisms of Internalization and Intracellular Trafficking.

Bryn D Monnery.

Polyplex-mediated gene transfection is now in its' fourth decade of serious research, but the promise of polyplex-mediated gene therapy has yet to fully materialize. Only approximately one in a million applied plasmids actually expresses. A large part of this is due to an incomplete understanding of the mechanism of polyplex transfection. There is an assumption that internalization must follow a canonical mechanism of receptor mediated endocytosis. Herein, we present arguments that untargeted (and most targeted) polyplexes do not utilize these routes. By incorporating knowledge of syndecan-polyplex interactions, we can show that syndecans are the "target" for polyplexes. Further, it is known that free polycations (which disrupt cell-membranes by acid-catalyzed hydrolysis of phospholipid esters) are necessary for (untargeted) endocytosis. This can be incorporated into the model to produce a novel mechanism of endocytosis, which fits the observed phenomenology. After membrane translocation, polyplex containing vesicles reach the endosome after diffusing through the actin mesh below the cell membrane. From there, they are acidified and trafficked toward the lysosome. Some polyplexes are capable of escaping the endosome and unpacking, while others are not. Herein, it is argued that for some polycations, as acidification proceeds the polyplexes excluding free polycations, which disrupt the endosomal membrane by acid-catalyzed hydrolysis, allowing the polyplex to escape. The polyplex's internal charge ratio is now insufficient for stability and it releases plasmids which diffuse to the nucleus. A small proportion of these plasmids diffuse through the nuclear pore complex (NPC), with aggregation being the major cause of loss. Those plasmids that have diffused through the NPC will also aggregate, and this appears to be the reason such a small proportion of nuclear plasmids express mRNA. Thus, the structural features which promote unpacking in the endosome and allow for endosomal escape can be determined, and better polycations can be designed.

DOI: https://doi.org/10.1021/acs.biomac.1c00697