bims-novged 2023-11-12 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2023–11–12
eleven papers selected by
the Merkel lab, Ludwig-Maximilians University

Cationic amino-acid functionalized polymethacrylamide vectors for siRNA transfection based on modification of poly(2-isopropenyl-2-oxazoline).
Enhancing Endosomal Escape and Gene Regulation Activity for Spherical Nucleic Acids.
Combination of Backbone Rigidity and Richness in Aryl Structures Enables Direct Membrane Translocation of Polymer Scaffolds for Efficient Gene Delivery.
Simultaneous dendritic cells targeting and effective endosomal escape enhance sialic acid-modified mRNA vaccine efficacy and reduce side effects.
Computational fluid dynamics modeling of aerosol particle transport through lung airway mucosa.
Cholesterol-Conjugated Supramolecular Multimeric siRNAs: Effect of siRNA Length on Accumulation and Silencing In Vitro and In Vivo.
Transmucosal Delivery of Nasal Nanovaccines Enhancing Mucosal and Systemic Immunity.
Flow cytometry-based quantification of genome editing efficiency in human cell lines using the L1CAM gene.
Reconstituted dry powder formulations of ZnO-adjuvanted ovalbumin induce equivalent antigen specific antibodies but lower T cell responses than ovalbumin adjuvanted with Alhydrogel® or cationic adjuvant formulation 01 (CAF®01).
Engineering of targeting antioxidant polypeptide nanopolyplexes for the treatment of acute lung injury.
A Proof-of-Concept Preparation of Lipid-Plasmid DNA Particles Using Novel Extrusion-Based 3D-Printing Technology, SMART.

J Control Release. 2023 Nov 05. pii: S0168-3659(23)00718-6. [Epub ahead of print]

Cationic amino-acid functionalized polymethacrylamide vectors for siRNA transfection based on modification of poly(2-isopropenyl-2-oxazoline).

Florica Adriana Jerca, Cristina Muntean, Katrien Remaut, Valentin Victor Jerca, Koen Raemdonck, Richard Hoogenboom.

  Poly(2-isopropenyl-2-oxazoline) (PiPOx) is a functional polymer showing great potential for the development of smart biomaterials. The straightforward synthesis and post-polymerization functionalization of PiPOx offers many opportunities for tailoring the properties of the polymer towards biomaterials. In this study we report for the first time PiPOx-based cationic charged polymethacrylamides with amino acid side chains that can complex siRNA and promote transfection in vitro. Therefore, PiPOx was fully modified via ring opening addition reactions with the carboxylic acid groups of a series of N-Boc-L-amino acids and their reaction kinetics were investigated. Based on the determined kinetic constants, another series of PiPOx-based copolymers with balanced hydrophilic/hydrophobic content of N-Boc-L-amino acids were obtained via one-pot modification reaction with two different N-Boc-L-amino acids. The N-Boc protected homopolymers and related copolymers were deprotected to obtain (co)polymers with the targeted side chain cationic charged units. The (co)polymers' structures were fully investigated via FT-IR and 1H NMR spectroscopy, size exclusion chromatography (SEC), and TGA-DSC-MS analysis. The polarimetry measurements revealed that the homopolymers retain their chiroptical properties after post-modification, and a sign inversion is noticed from (L) N-Boc-protected analogues to (D) for the TFA cationic charged homopolymers. Generally, cationically charged homopolymers with hydrophilic amino acids on the side chain showed efficient complexation of siRNA, but poor transfection while cationic copolymers having both tryptophan and valine or proline side chains revealed moderate siRNA binding, high transfection efficiency (> 90% of the cells) and potent gene silencing with IC50 values down to 5.5 nM. Particularly, these cationic copolymers showed higher gene silencing potency as compared to the commercial JetPRIME® reference, without reducing cell viability in the concentration range used for transfection, making this a very interesting system for in vitro siRNA transfection.

Keywords:  Poly(oxazoline)s; Polymethacrylamide; Polyplexes; RNA; Transfection

DOI:  https://doi.org/10.1016/j.jconrel.2023.11.001
Small. 2023 Nov 06. e2306902

Enhancing Endosomal Escape and Gene Regulation Activity for Spherical Nucleic Acids.

Jungsoo Park, Michael Evangelopoulos, Matthew Kuo Vasher, Sergej Kudruk, Namrata Ramani, Vinzenz Mayer, Alexander Carlos Solivan, Andrew Lee, Chad Alexander Mirkin.

  The therapeutic potential of small interfering RNAs (siRNAs) is limited by their poor stability and low cellular uptake. When formulated as spherical nucleic acids (SNAs), siRNAs are resistant to nuclease degradation and enter cells without transfection agents with enhanced activity compared to their linear counterparts; however, the gene silencing activity of SNAs is limited by endosomal entrapment, a problem that impacts many siRNA-based nanoparticle constructs. To increase cytosolic delivery, SNAs are formulated using calcium chloride (CaCl2 ) instead of the conventionally used sodium chloride (NaCl). The divalent calcium (Ca2+ ) ions remain associated with the multivalent SNA and have a higher affinity for SNAs compared to their linear counterparts. Importantly, confocal microscopy studies show a 22% decrease in the accumulation of CaCl2 -salted SNAs within the late endosomes compared to NaCl-salted SNAs, indicating increased cytosolic delivery. Consistent with this finding, CaCl2 -salted SNAs comprised of siRNA and antisense DNA all exhibit enhanced gene silencing activity (up to 20-fold), compared to NaCl-salted SNAs regardless of sequence or cell line (U87-MG and SK-OV-3) studied. Moreover, CaCl2 -salted SNA-based forced intercalation probes show improved cytosolic mRNA detection.

Keywords:  PLGA nanoparticles; calcium chloride; gene regulation; siRNAs (small interfering RNAs); spherical nucleic acids

DOI:  https://doi.org/10.1002/smll.202306902
Biomacromolecules. 2023 Nov 09.

Combination of Backbone Rigidity and Richness in Aryl Structures Enables Direct Membrane Translocation of Polymer Scaffolds for Efficient Gene Delivery.

Ying Liu, Leyue Zhou, Xiang Xu, Zehong Cheng, Yajie Chen, Xue-Ao Mei, Nan Zheng, Chunhui Zhang, Yugang Bai.

The development of cell-penetrating polymers with endocytosis-independent cell uptake pathways has emerged as a prominent strategy to enhance the transfection efficiency. Inspired by the rigid α-helical structure that endows polypeptides with cell-penetrating ability, we propose that a rigid backbone can facilitate the corresponding polymer vector's performance in gene delivery by bypassing the difficult endosomal escape process. Meanwhile, the installation of aromatic domains, as a way to promote gene transfection efficiency, is employed through the construction of a poly(benzyl ether) (PBE)-based scaffold in this work. We demonstrate that the direct membrane translocation capability of the synthesized PBE contributes to its enhanced transfection performance and excellent biocompatibility profile, rendering the imidazolium-functionalized PBE scaffold with higher activity and biocompatibility. Molecular details of the PBE-lipid interaction are also revealed in molecular dynamics simulations, indicating the important roles of individual structural elements on the polymeric scaffold in the membrane penetration process.

DOI: https://doi.org/10.1021/acs.biomac.3c00682
J Control Release. 2023 Nov 08. pii: S0168-3659(23)00725-3. [Epub ahead of print]

Simultaneous dendritic cells targeting and effective endosomal escape enhance sialic acid-modified mRNA vaccine efficacy and reduce side effects.

Xueying Tang, Jiashuo Zhang, Dezhi Sui, Qiongfen Yang, Tianyu Wang, Zihan Xu, Xiaoya Li, Xin Gao, Xinyang Yan, Xinrong Liu, Yanzhi Song, Yihui Deng.

  mRNA vaccines are attractive prospects for the development of DC-targeted vaccines; however, no clinical success has been realized because, currently, it is difficult to simultaneously achieve DC targeting and efficient endosomal/lysosomal escape. Herein, we developed a sialic acid (SA)-modified mRNA vaccine that simultaneously achieved both. The SA modification promoted DCs uptake of lipid nanoparticles (LNPs) by 2 times, >90% of SA-modified LNPs rapidly escaped from early endosomes (EEs), avoided entering lysosomes, achieved mRNA simultaneously translated in ribosomes distributed in the cytoplasm and endoplasmic reticulum (ER), significantly improved the transfection efficiency of mRNA LNPs in DCs. Additionally, we applied cleavable PEG-lipids in mRNA vaccines for the first time and found this conducive to cellular uptake and DC targeting. In summary, SA-modified mRNA vaccines targeted DCs efficiently, and showed significantly higher EEs/lysosomal escape efficiency (90% vs 50%), superior tumor treatment effect, and lower side effects than commercially formulated mRNA vaccines.

Keywords:  Dendritic cells; Sialic acid; Tumor immunotherapy; mRNA vaccine

DOI:  https://doi.org/10.1016/j.jconrel.2023.11.008
Comput Chem Eng. 2023 Nov;pii: 108458. [Epub ahead of print]179

Computational fluid dynamics modeling of aerosol particle transport through lung airway mucosa.

Blake A Bartlett, Yu Feng, Catherine A Fromen, Ashlee N Ford Versypt.

  Delivery of aerosols to the lung can treat various lung diseases. However, the conducting airways are coated by a protective mucus layer with complex properties that make this form of delivery difficult. Mucus is a non-Newtonian fluid and is cleared from the lungs over time by ciliated cells. Further, its gel-like structure hinders the diffusion of particles through it. Any aerosolized treatment of lung diseases must penetrate the mucosal barrier. Using computational fluid dynamics, a model of the airway mucus and periciliary layer was constructed to simulate the transport of impacted aerosol particles. The model predicts the dosage fraction of particles of a certain size that penetrate the mucus and reach the underlying tissue, as well as the distance downstream of the dosage site where tissue concentration is maximized. Reactions that may occur in the mucus are also considered, with simulated data for the interaction of a model virus and an antibody.

Keywords:  Diffusion; Inhaled particles; Lung-aerosol dynamics; Mucociliary clearance; Nanoparticles

DOI:  https://doi.org/10.1016/j.compchemeng.2023.108458
Nucleic Acid Ther. 2023 Nov 09.

Cholesterol-Conjugated Supramolecular Multimeric siRNAs: Effect of siRNA Length on Accumulation and Silencing In Vitro and In Vivo.

Ivan V Chernikov, Ul'yana A Ponomareva, Mariya I Meschaninova, Irina K Bachkova, Anna A Teterina, Daniil V Gladkikh, Innokenty A Savin, Valentin V Vlassov, Marina A Zenkova, Elena L Chernolovskaya.

  Conjugation of small interfering RNA (siRNA) with lipophilic molecules is one of the most promising approaches for delivering siRNA in vivo. The rate of molecular weight-dependent siRNA renal clearance is critical for the efficiency of this process. In this study, we prepared cholesterol-containing supramolecular complexes containing from three to eight antisense strands and examined their accumulation and silencing activity in vitro and in vivo. We have shown for the first time that such complexes with 2'F, 2'OMe, and LNA modifications exhibit interfering activity both in carrier-mediated and carrier-free modes. Silencing data from a xenograft tumor model show that 4 days after intravenous injection of cholesterol-containing monomers and supramolecular trimers, the levels of MDR1 mRNA in the tumor decreased by 85% and 68%, respectively. The in vivo accumulation data demonstrated that the formation of supramolecular structures with three or four antisense strands enhanced their accumulation in the liver. After addition of two PS modifications at the ends of antisense strands, 47% and 67% reductions of Ttr mRNA levels in the liver tissue were detected 7 days after administration of monomers and supramolecular trimers, respectively. Thus, we have obtained a new type of RNAi inducer that is convenient for synthesis and provides opportunities for modifications.

Keywords:  MDR1; TTR; biodistribution; chemically modified siRNA; cholesterol conjugate; supramolecular siRNA

DOI:  https://doi.org/10.1089/nat.2023.0051
Nano Lett. 2023 Nov 09.

Transmucosal Delivery of Nasal Nanovaccines Enhancing Mucosal and Systemic Immunity.

Yuchun Xu, Xiaoying Yan, Ting Wei, Minming Chen, Jiafei Zhu, Juxin Gao, Bo Liu, Wenjun Zhu, Zhuang Liu.

  Intranasal vaccines can induce protective immune responses at the mucosa surface entrance, preventing the invasion of respiratory pathogens. However, the nasal barrier remains a major challenge in the development of intranasal vaccines. Herein, a transmucosal nanovaccine based on cationic fluorocarbon modified chitosan (FCS) is developed to induce mucosal immunity. In our system, FCS can self-assemble with the model antigen ovalbumin and TLR9 agonist CpG, effectively promoting the maturation and cross-presentation of dendritic cells. More importantly, it can enhance the production of secretory immunoglobin A (sIgA) at mucosal surfaces for those intranasally vaccinated mice, which in the meantime showed effective production of immunoglobulin G (IgG) systemically. As a proof-of-concept study, such a mucosal vaccine inhibits ovalbumin-expressing B16-OVA melanoma, especially its lung metastases. Our work presents a unique intranasal delivery system to deliver antigen across mucosal epithelia and promote mucosal and systemic immunity.

Keywords:  chitosan nanocomplexes; intranasal vaccine; mucosal immunity; mucosal penetration; sIgA

DOI:  https://doi.org/10.1021/acs.nanolett.3c03419
PLoS One. 2023 ;18(11): e0294146

Flow cytometry-based quantification of genome editing efficiency in human cell lines using the L1CAM gene.

Muhammad Nazmul Hasan, Toshinori Hyodo, Mrityunjoy Biswas, Md Lutfur Rahman, Yuko Mihara, Sivasundaram Karnan, Akinobu Ota, Shinobu Tsuzuki, Yoshitaka Hosokawa, Hiroyuki Konishi.

CRISPR/Cas9 is a powerful genome editing system that has remarkably facilitated gene knockout and targeted knock-in. To accelerate the practical use of CRISPR/Cas9, however, it remains crucial to improve the efficiency, precision, and specificity of genome editing, particularly targeted knock-in, achieved with this system. To improve genome editing efficiency, researchers should first have a molecular assay that allows sensitive monitoring of genome editing events with simple procedures. In the current study, we demonstrate that genome editing events occurring in L1CAM, an X-chromosome gene encoding a cell surface protein, can be readily monitored using flow cytometry (FCM) in multiple human cell lines including neuroblastoma cell lines. The abrogation of L1CAM was efficiently achieved using Cas9 nucleases which disrupt exons encoding the L1CAM extracellular domain, and was easily detected by FCM using anti-L1CAM antibodies. Notably, L1CAM-abrogated cells could be quantified by FCM in four days after transfection with a Cas9 nuclease, which is much faster than an established assay based on the PIGA gene. In addition, the L1CAM-based assay allowed us to measure the efficiency of targeted knock-in (correction of L1CAM mutations) accomplished through different strategies, including a Cas9 nuclease-mediated method, tandem paired nicking, and prime editing. Our L1CAM-based assay using FCM enables rapid and sensitive quantification of genome editing efficiencies and will thereby help researchers improve genome editing technologies.

DOI: https://doi.org/10.1371/journal.pone.0294146
Int J Pharm. 2023 Nov 04. pii: S0378-5173(23)01002-5. [Epub ahead of print] 123581

Reconstituted dry powder formulations of ZnO-adjuvanted ovalbumin induce equivalent antigen specific antibodies but lower T cell responses than ovalbumin adjuvanted with Alhydrogel® or cationic adjuvant formulation 01 (CAF®01).

Marie Hellfritzsch, Dennis Christensen, Camilla Foged, Regina Scherließ, Aneesh Thakur.

  Most licensed human vaccines are based on liquid dosage forms but have poor storage stability and require continuous and expensive cold-chain storage. In contrast, the use of solid vaccine dosage forms, produced by for example spray drying, extends shelf life and eliminates the need for a cold chain. Zinc oxide (ZnO)-based nanoparticles display immunomodulatory properties, but their adjuvant effect as a dry powder formulation is unknown. Here, we show that reconstituted dry powder formulations of ZnO particles containing the model antigen ovalbumin (OVA) induce antigen specific CD8+ T cells and humoral responses. By systematically varying the ratio between ZnO and mannitol during spray drying, we manufactured dry powder formulations of OVA-containing ZnO particles that displayed: (i) a spherical or wrinkled surface morphology, (ii) an aerodynamic diameter and particle size distribution optimal for deep lung deposition, and (iii) aerosolization properties suitable for lung delivery. Reconstituted dry powder formulations of ZnO particles were well-tolerated by Calu-3 lung epithelial cells. Furthermore, almost equivalent OVA-specific serum antibody responses were stimulated by reconstituted ZnO particles, OVA adjuvanted with Alhydrogel®, and OVA adjuvanted with the cationic adjuvant formulation 01 (CAF®01). However, reconstituted dry powder ZnO particles and OVA adjuvanted with Alhydrogel® induced significantly lower OVA-specific CD8+CD44+ T-cell responses in the spleen than OVA adjuvanted with CAF®01. Similarly, reconstituted dry powder ZnO particles activated significantly lower percentages of follicular helper T cells and germinal center B cells in the draining lymph nodes than OVA adjuvanted with CAF®01. Overall, our results show that reconstituted dry powder formulations of ZnO nanoparticles can induce antigen-specific antibodies and can be used in vaccines to enhance antigen specific humoral immune responses against subunit protein antigens.

Keywords:  Alum; CAF®01; CD8(+) T cell response; Zinc oxide (ZnO) nanoparticles; adjuvant; spray drying

DOI:  https://doi.org/10.1016/j.ijpharm.2023.123581
Int J Biol Macromol. 2023 Nov 06. pii: S0141-8130(23)04771-2. [Epub ahead of print] 127872

Engineering of targeting antioxidant polypeptide nanopolyplexes for the treatment of acute lung injury.

Yi-Xuan Li, Ya-Ting Bao, Jing-Bo Hu.

  The pathogenesis of acute lung injury (ALI) involves various mechanisms, such as oxidative stress, inflammation, and epithelial cell apoptosis. However, current drug therapies face limitations due to issues like systemic distribution, drug degradation in vivo, and hydrophobicity. To address these challenges, we developed a pH-responsive nano-drug delivery system for delivering antioxidant peptides to treat ALI. In this study, we utilized low molecular weight chitosan (LMWC) and hyaluronic acid (HA) as carrier materials. LMWC carries a positive charge, while HA carries a negative charge. By stirring the two together, the electrostatic adsorption between LMWC and HA yielded aggregated drug carriers. To specifically target the antioxidant drug WNWAD to lung lesions and enhance therapeutic outcomes for ALI, we created a targeted drug delivery system known as HA/LMWC@WNWAD (NPs) through a 12-h stirring process. In our research, we characterized the particle size and drug release of NPs. Additionally, we assessed the targeting ability of NPs. Lastly, we evaluated the improvement of lung injury at the cellular and animal levels to investigate the therapeutic mechanism of this drug targeting delivery system.

Keywords:  Acute lung injury; Nanoparticles; pH- responsive release

DOI:  https://doi.org/10.1016/j.ijbiomac.2023.127872
Mol Pharm. 2023 Nov 06.

A Proof-of-Concept Preparation of Lipid-Plasmid DNA Particles Using Novel Extrusion-Based 3D-Printing Technology, SMART.

Jaidev Chakka, Mohammed Maniruzzaman.

  Gene therapy is a promising approach with delivery of mRNA, small interference RNA, and plasmid DNA to elicit a therapeutic action in vitro using cationic or ionizable lipid nanoparticles. In the present study, a novel extrusion-based Sprayed Multi Adsorbed-droplet Reposing Technology (SMART) developed in-house was employed for the preparation, characterization, and transfection abilities of the green fluorescence protein (GFP) plasmid DNA in cancer cells in vitro. The results showed 100% encapsulation of pDNA (GFP) in LNPs of around 150 nm (N/P 5) indicating that the processes developed using SMART technology are consistent and can be utilized for commercial applications.

Keywords:  GFP; SMART; lipid nanoparticles; plasmid DNA; transfection; transient expression

DOI:  https://doi.org/10.1021/acs.molpharmaceut.3c00783