bims-novged 2023-02-12 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2023‒02‒12
ten papers selected by
the Merkel lab
Ludwig-Maximilians University

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing.
Cholesterol-Amino-Phosphate (CAP) Derived Lipid Nanoparticles for Delivery of Self-Amplifying RNA and Restoration of Spermatogenesis in Infertile Mice.
In vitro transfection efficiencies of T-shaped spermine-based cationic lipids with identical and nonidentical tails under high serum conditions.
A Review of Different Types of Liposomes and Their Advancements as a Form of Gene Therapy Treatment for Breast Cancer.
Reversible stabilization of DNA/PEI complexes by reducible click-linkage between DNA and polymer. A new polyplex concept for lowering polymer quantity.
How does the polymer architecture and position of cationic charges affect cell viability?
RNA-DNA hybrid nano-materials for highly efficient and long lasting RNA interference effect.
Molecular Bottlebrushes for Immunostimulatory CpG ODN Delivery: Relationship among Cation Density, Complex Formation Ability, and Cytotoxicity.
Endothelial-specific targeting of RhoA signaling via CD31 antibody-conjugated nanoparticles.
Assessment of core-shell nanoparticles surface structure heterogeneity by SAXS contrast variation and ab initio modeling.

J Vis Exp. 2023 Jan 20.

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing.

Rakan El-Mayta, Marshall S Padilla, Margaret M Billingsley, Xuexiang Han, Michael J Mitchell.

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and Pfizer/BioNTech. These vaccines have demonstrated the efficacy of mRNA-LNP therapeutics and opened the door for future clinical applications. In mRNA-LNP systems, the LNPs serve as delivery platforms that protect the mRNA cargo from degradation by nucleases and mediate their intracellular delivery. The LNPs are typically composed of four components: an ionizable lipid, a phospholipid, cholesterol, and a lipid-anchored polyethylene glycol (PEG) conjugate (lipid-PEG). Here, LNPs encapsulating mRNA encoding firefly luciferase are formulated by microfluidic mixing of the organic phase containing LNP lipid components and the aqueous phase containing mRNA. These mRNA-LNPs are then tested in vitro to evaluate their transfection efficiency in HepG2 cells using a bioluminescent plate-based assay. Additionally, mRNA-LNPs are evaluated in vivo in C57BL/6 mice following an intravenous injection via the lateral tail vein. Whole-body bioluminescence imaging is performed by using an in vivo imaging system. Representative results are shown for the mRNA-LNP characteristics, their transfection efficiency in HepG2 cells, and the total luminescent flux in C57BL/6 mice.

DOI: https://doi.org/10.3791/64810
Adv Sci (Weinh). 2023 Feb 07. e2300188

Cholesterol-Amino-Phosphate (CAP) Derived Lipid Nanoparticles for Delivery of Self-Amplifying RNA and Restoration of Spermatogenesis in Infertile Mice.

Shi Du, Wenqing Li, Yuebao Zhang, Yonger Xue, Xucheng Hou, Jingyue Yan, Jeffrey Cheng, Binbin Deng, David W McComb, Jennifer Lin, Hong Zeng, Xiaolin Cheng, Darrell J Irvine, Ron Weiss, Yizhou Dong.

  Male infertility caused by genetic mutations is an important type of infertility. Currently, there is no reliable method in the clinic to address this medical need. The emergence of mRNA therapy provides a possible strategy for restoring mutant genes in the reproductive system. However, effective delivery of mRNA to spermatocytes remains a formidable challenge. Here a series of cholesterol-amino-phosphate (CAP) lipids are reported by integrating three bioactive moieties into a geometric structure, which is favorable for mRNA delivery. The results demonstrate that CAP-derived lipid nanoparticles (CAP LNPs) can deliver RNA including traditional mRNA and self-amplifying RNA (saRNA) encoding DNA Meiotic Recombinase 1 (Dmc1) protein in spermatocytes and treat male infertility caused by the Dmc1 gene mutation. Notably, the delivery efficiency of CAP LNPs is significantly higher than that of the MC3 and ALC-0315 LNPs, which is consistent with the design of CAP molecules. More importantly, a single injection of CAP LNPs-saRNA can produce Dmc1 protein for an extended period, which restores the spermatogenesis in the Dmc1 gene knockout mouse model. Overall, this study proves the concept of LNPs for the delivery of mRNA to spermatocytes, which provides a unique method to probe male infertility caused by the genetic mutation.

Keywords:  lipid nanoparticles; mRNA therapy; male infertility; self-amplifying RNA; spermatocytes delivery

DOI:  https://doi.org/10.1002/advs.202300188
Org Biomol Chem. 2023 Feb 10.

In vitro transfection efficiencies of T-shaped spermine-based cationic lipids with identical and nonidentical tails under high serum conditions.

Chopaka Thongbamrer, Purichaya Teerakantrakorn, Ussanee Nongpong, Nuttapon Apiratikul, Wanlapa Roobsoong, Nawapol Kunkeaw, Wang Nguitragool, Jetsumon Sattabongkot, Boon-Ek Yingyongnarongkul.

T-shaped spermine-based cationic lipids with identical and nonidentical hydrophobic tails having variable carbon lengths (from C10 to C18) were designed and synthesized. These lipids were characterized, and their structure-activity relationships were determined for DNA binding and transfection ability of these compounds when formulated as cationic liposomes. These liposomes were then applied as non-viral vectors to transfect HEK293T, HeLa, PC3, H460, HepG2, and Calu'3 cell lines with plasmid DNA encoding the green fluorescent protein. ST9, ST12 and ST13 with nonidentical tails could deliver DNA into HEK293T cells up to 60% under serum-free conditions. The lipid ST15 bearing nonidentical tails was found to be a potent gene transfer agent under 40% serum conditions in HEK293T and HeLa cells. Besides their low cytotoxicity, these lipoplexes also exhibited greater transfection efficiency than the commercially available transfection agent, Lipofectamine 3000.

DOI: https://doi.org/10.1039/d2ob02129c
Molecules. 2023 Feb 03. pii: 1498. [Epub ahead of print]28(3):

A Review of Different Types of Liposomes and Their Advancements as a Form of Gene Therapy Treatment for Breast Cancer.

Gloria Yi Wei Tseu, Khairul Azfar Kamaruzaman.

  Breast cancer incidence and mortality rates have increased exponentially during the last decade, particularly among female patients. Current therapies, including surgery and chemotherapy, have significant negative physical and mental impacts on patients. As a safer alternative, gene therapy utilising a therapeutic gene with the potential to treat various ailments is being considered. Delivery of the gene generally utilises viral vectors. However, immunological reactions and even mortality have been recorded as side effects. As a result, non-viral vectors, such as liposomes, a system composed of lipid bilayers formed into nanoparticles, are being studied. Liposomes have demonstrated tremendous potential due to their limitless ability to combine many functions into a system with desirable characteristics and functionality. This article discusses cationic, anionic, and neutral liposomes with their stability, cytotoxicity, transfection ability, cellular uptake, and limitation as a gene carrier suitable for gene therapy specifically for cancer. Due to the more practical approach of employing electrostatic contact with the negatively charged nucleic acid and the cell membrane for absorption purposes, cationic liposomes appear to be more suited for formulation for gene delivery and therapy for breast cancer treatment. As the other alternatives have numerous complicated additional modifications, attachments need to be made to achieve a functional gene therapy system for breast cancer treatment, which were also discussed in this review. This review aimed to increase understanding and build a viable breast cancer gene therapy treatment strategy.

Keywords:  breast cancer; gene therapy; liposome; nanoparticles

DOI:  https://doi.org/10.3390/molecules28031498
Gene Ther. 2023 Feb 09.

Reversible stabilization of DNA/PEI complexes by reducible click-linkage between DNA and polymer. A new polyplex concept for lowering polymer quantity.

Delphine Maze, Chantal Pichon, Patrick Midoux.

Nonviral transfection of mammalian cells can be performed with electrostatic complexes (polyplexes) between a plasmid DNA (pDNA) encoding a foreign gene and a cationic polymer. However, an excess of the cationic polymer is required for pDNA condensation and polyplexes formation, which generate in vivo toxicity. Here, we present a new concept of polyplexes preparation aiming to reduce the polymer quantity. pDNA was functionalized with 3,6,9-trioxaundecan-1- {4 - [(2-chloroethyl) ethylamino)] - benzylamino}, 11-azide, and polyethyleneimine (lPEI) with reducible dibenzocyclooctyl (SS-DBCO) groups allowing azide-alkyne cycloaddition between pDNA and lPEI after condensation. The size of polyplexes with DBCO-SS-lPEI was smaller than with lPEI due to a stronger DNA condensation thanks to linkages between polymer and pDNA preventing dissociation until disulfide bridges reduction. In vitro transfection showed that the amount of DBCO-SS-lPEI leading to the most efficient polyplexes was three times lower than lPEI. As expected, toxicity in mice was significantly reduced upon intravenous injection of DBCO-SS-lPEI polyplexes at doses where the lPEI polyplexes killed mice. This is probably due to the high stability of the DBCO-SS-lPEI polyplexes which prevented their aggregation in the pulmonary capillaries. Overall, this new concept of polyplexes with DBCO-SS-lPEI offering the possibility of administering higher doses of polyplexes than lPEI and their ability to pass the pulmonary barrier could be favorably exploited for transfection of distant organs or tissues, such as tumors.

DOI: https://doi.org/10.1038/s41434-023-00386-1
Polym Chem. 2023 Jan 17. 14(3): 303-317

How does the polymer architecture and position of cationic charges affect cell viability?

Joana S Correia, Sofía Mirón-Barroso, Charlotte Hutchings, Silvia Ottaviani, Birsen Somuncuoğlu, Leandro Castellano, Alexandra E Porter, Jonathan Krell, Theoni K Georgiou.

Polymer chemistry, composition and molar mass are factors that are known to affect cytotoxicity, however the influence of polymer architecture has not been investigated systematically. In this study the influence of the position of the cationic charges along the polymer chain on cytotoxicity was investigated while keeping constant the other polymer characteristics. Specifically, copolymers of various architectures, based on a cationic pH responsive monomer, 2-(dimethylamino)ethyl methacrylate (DMAEMA) and a non-ionic hydrophilic monomer, oligo(ethylene glycol)methyl ether methacrylate (OEGMA) were engineered and their toxicity towards a panel of cell lines investigated. Of the seven different polymer architectures examined, the block-like structures were less cytotoxic than statistical or gradient/tapered architectures. These findings will assist in developing future vectors for nucleic acid delivery.

DOI: https://doi.org/10.1039/d2py01012g
RSC Adv. 2023 Jan 18. 13(5): 3139-3146

RNA-DNA hybrid nano-materials for highly efficient and long lasting RNA interference effect.

Joung Sug Kim, Junghyun Park, Jang Hyeon Choi, Seungjae Kang, Nokyoung Park.

In attempts to effectively improve RNAi function, we herein report a new RNAi approach using X-shaped RDNA and Dgel (RNA interfering DNA hydrogel, Ri-Dgel). X-shaped RDNA is a 4 branched nanostructure which was composed of three dsDNA branches and one dsRNA branch, and the structure was made by annealing partially complementary ssDNAs and chimeric RNA-DNA oligonucleotides. Ri-Dgel was synthesized through the ligation of the X-shaped RDNAs using their palindromic sticky ends. In MDCK/GFP cells transfected with 1 μM of each format of siRNA, Ri-Dgel and X-RDNA, the intensity of GFP fluorescence was significantly reduced by 65% and 56%, respectively, while dsRNA which is a conventional siRNA format showed a relatively weak reduction intensity of 7% compared with a negative control. We also observed the decreased intensity of GFP fluorescence by approximately 59% in MDA-MB-231/GFP cells transfected with 5 nM Ri-Dgel. Furthermore, the Ri-Dgel showed persistent RNAi efficiency up to 6 days from the treatment. The use of Ri-Dgel to trigger RNAi resulted in enhanced efficacy and longer duration at lower concentration compared to traditional dsRNA implying the nanostructured DNA-RNA hybrid materials have great potential as a platform technology for RNAi-based therapy.

DOI: https://doi.org/10.1039/d2ra06249f
Biomacromolecules. 2023 Feb 10.

Molecular Bottlebrushes for Immunostimulatory CpG ODN Delivery: Relationship among Cation Density, Complex Formation Ability, and Cytotoxicity.

Shin Takano, Yusuke Miyashima, Shota Fujii, Kazuo Sakurai.

Artificially designed short single-stranded DNA sequences containing unmethylated CG (CpG ODNs) are agonists for toll-like receptor 9 (TLR9); thus, they have great potential as vaccine adjuvants for cancer immunotherapy and preventing infectious diseases. To deliver effectively CpG ODNs into cells bearing TLR9, nanoparticle polyion complexes of cationic polymers that are able to ingest multiple CpG ODN molecules have been developed; however, their structures and synthesized polycations are hard to control and bioincompatible, respectively. To solve these issues, we designed cationic molecular bottlebrushes (CMBs) with branches that are made from copolymers of 2-methacryloyloxyethyl phosphorylcholine and 2-methacryloyloxyethyl trimethylammonium chloride. Several instrumental methods were carried out to determine the structure of a CMB and its complex with CpG ODNs. The complexation did not change the overall shape of the original CMB, and the bound CpG ODNs were captured by the outer layer of the CMB. The moderation of cations was important to reduce toxicity and improve secretion of inflammatory cytokines.

DOI: https://doi.org/10.1021/acs.biomac.2c01348
J Pharmacol Exp Ther. 2023 Feb 06. pii: JPET-AR-2022-001384. [Epub ahead of print]

Endothelial-specific targeting of RhoA signaling via CD31 antibody-conjugated nanoparticles.

Behnaz Lahooti, Racheal G Akwii, Dhavalkumar Patel, Siavash ShahbaziNia, Margarita Lamprou, Mahboubeh Madadi, Thomas J Abbruscato, Aristotelis Astrinidis, Ulrich Bickel, Abraham Al-Ahmad, Nadezhda A German, George Mattheolabakis, Constantinos M Mikelis.

  Existing Vascular Endothelial Growth Factor (VEGF)-oriented anti-angiogenic approaches are known for their high potency. However, significant side effects associated with their use drive the need for novel anti-angiogenic strategies. The small GTPase RhoA is an established regulator of actin cytoskeletal dynamics. Previous studies have highlighted the impact of endothelial RhoA pathway on angiogenesis. Rho-associate kinase (ROCK), a direct RhoA effector, is potently inhibited by Fasudil, a clinically-relevant ROCK inhibitor. Here, we aimed to target the RhoA signaling in endothelial cells by generating Fasudil-encapsulated CD31-targeting liposomes as a potential antiangiogenic therapy. The liposomes presented desirable characteristics, preferential binding to CD31-expressing HEK293T cells and to endothelial cells, inhibited stress fiber formation and cytoskeletal-related morphometric parameters and inhibited in vitro angiogenic functions. Overall, this work shows that the nanodelivery-mediated endothelial targeting of RhoA signaling can offer a promising strategy for angiogenesis inhibition in vascular-related diseases. Significance Statement Systemic administration of anti-angiogenic therapeutics induces side effects unrelated to the treatment tissues. We, among others, have shown the impact of the RhoA signaling in the endothelial cells and their angiogenic functions. Here, to minimize potential toxicity, we generated CD31-targeting liposomes with encapsulated Fasudil, a clinically-relevant Rho kinase inhibitor and successfully targeted endothelial cells. In this proof-of-principle study, the efficient Fasudil delivery, its impact on the endothelial signaling, morphometric alterations and angiogenic functions verifies the benefits of site-targeted anti-angiogenic therapy.

Keywords:  Nanoparticles; endothelial cells; liposomes

DOI:  https://doi.org/10.1124/jpet.122.001384
Colloids Surf B Biointerfaces. 2023 Feb 02. pii: S0927-7765(23)00061-9. [Epub ahead of print]224 113183

Assessment of core-shell nanoparticles surface structure heterogeneity by SAXS contrast variation and ab initio modeling.

I S Vaskan, A T Prikhodko, M V Petoukhov, E V Shtykova, N V Bovin, A B Tuzikov, V A Oleinikov, A V Zalygin.

  For the biomedical applications of nanoparticles, the study of their structure is a major step towards understanding the mechanisms of their interaction with biological environment. Detailed structural analysis of particles' surface is vital for rational design of drug delivery systems. In particular, for core-shell or surface-modified nanoparticles surface structure can be described in terms of shell coating uniformity and shell thickness uniformity around the nanoparticle core. Taken together, these terms can be used to indicate degree of heterogeneity of nanoparticle surface structure. However, characterization of nanoparticle surface structure under physiological conditions is challenging due to limitations of experimental techniques. In this paper, we apply SAXS contrast variation combined with ab initio bead modeling for this purpose. Approach is based on the fact that nanoparticles under study are produced by self-assembly of phospholipid-conjugated molecules that possess moieties with significantly different electron densities enabling SAXS technique to be used to distinguish nanoparticle shell and study its structure. Ab initio single phase and ab initio multiphase modeling based on SAXS curve of nanoparticles in phosphate buffer solution allowed to reconstruct nanoparticle shell coating and assess its uniformity, while serial nanoparticle reconstructions from solutions with gradually increased solvent electron densities revealed relative shell coating thickness around nanoparticle core. Nanoparticle shell structure representation was verified by molecular dynamics simulation and derived full-atom nanoparticle shell structure showed good agreement with SAXS-derived representation. Obtained data indicate that studied nanoparticles exhibit highly heterogeneous surface structure.

Keywords:  Lipid; MD; Nanoparticles; SAXS; Structural modeling; Surface heterogeneity

DOI:  https://doi.org/10.1016/j.colsurfb.2023.113183